download report - Istituto Pasteur
download report - Istituto Pasteur
download report - Istituto Pasteur
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
<strong>Istituto</strong> <strong>Pasteur</strong><br />
Fondazione Cenci Bolognetti<br />
Report of activity<br />
2009-2010
© 2011 - Sapienza-Università di Roma<br />
P.le Aldo Moro, 5 - 00185 Roma<br />
Edited by Lucia Ugo<br />
Photos by Giovanni Canitano<br />
www.istitutopasteur.it
Contents<br />
• Forward ..........................................................................................................................................................................<br />
by Paolo Amati, President, and Angela Santoni, Scientific Director<br />
• Boards and Staff .......................................................................................................................................................<br />
• Fellowships awarded in 2009 and 2010 ........................................................................................................<br />
Fellowships awarded for two years for training in foreign laboratories<br />
Fellowships awarded to students who had a two-year experience abroad<br />
Fellowships awarded for one year to students who have completed their PhD and are seeking a position<br />
Fellows working on the research programs of the Foundation<br />
• Seminars ........................................................................................................................................................................<br />
• Meetings and Conferences ..................................................................................................................................<br />
• Scientific Reports<br />
Research area 1: Molecular biology of microorganisms and viruses<br />
Alberto BOFFI, Bacterial globins as regulators of thiol redox equilibrium in bacteria ....................................<br />
Daniela DE BIASE, The acid resistance genes of Escherichia coli: does their complex transcriptional control<br />
hide novel biological roles? ........................................................................................................................................<br />
Alberto FAGGIONI, Epstein-Barr virus interactions with cellular microRNAs .................................................<br />
Paola LONDEI, Translational regulation: from the archaea to the eukarya ........................................................<br />
Research area 2: Pathogenic mechanisms of microbially associated diseases<br />
Guido ANTONELLI, Molecular characterization of viruses causing bronchiolitis and study of viral and<br />
host factors affecting Type I IFN antiviral response induced by respiratory viruses ...............................<br />
Andrea BELLELLI, A structural biology study of schistosomiasis .........................................................................<br />
Maria Lina BERNARDINI, Lipopolysaccharide and peptidoglycan adaptation to host as an immune evasion<br />
strategy of gram-negative pathogens ....................................................................................................................<br />
Bianca COLONNA, Involvement of sRNA molecules in the complex regulatory circuits of virulence gene<br />
expression in Shigella flexneri and in enteroinvasive E. coli ..............................................................................<br />
Anna Teresa PALAMARA, Redox mediated mechanisms involved in the influenza virus replication and in<br />
the pathogenesis of influenza associated diseases ...............................................................................................<br />
Paolo SARTI, Defense mechanisms against oxidative and nitrosative stress in pathogenic protozoa<br />
Maria Rosaria TORRISI, Role of the keratinocyte growth factor receptor on the molecular and cellular<br />
alterations induced by the expression of HPV16 E5 oncoprotein .................................................................<br />
Research area 3: Molecular genetics of eukaryotes<br />
Fiorentina ASCENZIONI, CFTR gene engineering by means of prokaryotic and eukaryotic artificial<br />
chromosome ..................................................................................................................................................................<br />
Irene BOZZONI, RNA-RNA and RNA-protein interactions: role of small non-coding RNAs in gene<br />
expression control .......................................................................................................................................................<br />
Emanuele CACCI, Role of metalloproteinases and their tissue inhibitors in the regulation of neurogenesis<br />
and gliogenesis from neural stem /progenitor cells ...........................................................................................<br />
Paola CAIAFA, Does PARylated PARP-1 introduce an epigenetic mark on chromatin? .................................<br />
Antonella DE JACO, Processing of the Neuroligins proteins and autism-related mutations ........................<br />
Maria D’ERME, Epigenetic modifications in neurodegenerative diseases ............................................................<br />
Lucia FABIANI, DNA replication mechanisms and genome stability in Saccharomyces cerevisiae ....................<br />
Claudio FALCONE, New tools in deciphering aging and apoptotic pathways .....................................................<br />
Laura FRONTALI, Mitochondrial studies and the use of simple eukaryotic models for the analysis of<br />
molecular and cellular aspects of human pathologies .......................................................................................<br />
Maurizio GATTI, The role of membrane trafficking during Drosophila cytokinesis .........................................<br />
III<br />
VII<br />
VIII<br />
IX<br />
XI<br />
XII<br />
3<br />
5<br />
7<br />
9<br />
13<br />
15<br />
17<br />
19<br />
21<br />
23<br />
25<br />
29<br />
33<br />
37<br />
39<br />
43<br />
45<br />
47<br />
49<br />
51<br />
53
Marco LUCARELLI, The interplay between epigenetics, cell cycle and homologous recombination in gene<br />
therapy by Small Fragment Homologous Replacement (SFHR) ....................................................................<br />
Rossella MAIONE, Interplay between myogenic factors and cell cycle control: regulation and role of the<br />
cdk inhibitor p57kip2 ..................................................................................................................................................<br />
Franco MANGIA, Molecular regulation of cell proliferation in early embryo blastomeres and neuronal<br />
precursors of the mouse ............................................................................................................................................<br />
Antonio MUSARO’, Study of the molecular and cellular mechanisms of sarcopenia: role of mIGF-1 and<br />
oxidative stress .............................................................................................................................................................<br />
Rodolfo NEGRI, Role of the COP9 signalosome in transcription modulation and chromatin organization<br />
in yeast and plants .......................................................................................................................................................<br />
Sergio PIMPINELLI, The biogenesis of piRNAs and their involvement in transposon silencing and<br />
heterochromatin formation in Drosophila ..............................................................................................................<br />
Sabrina SABATINI, To the root of organ growth: the control of root meristem activity in Arabidopsis ....<br />
Mario STEFANINI, Biological characterization and in vitro culture of spermatogonial stem cells ................<br />
Marco TRIPODI, Molecular mechanisms driving liver stem cell fate ....................................................................<br />
Giuseppe ZARDO, Identification of novel genetic and epigenetic targets in Leukemia by genome wide<br />
approaches .....................................................................................................................................................................<br />
Research area 4: Molecular recognition in biomolecules<br />
Fabio ALTIERI, Cellular response to oxidative stress: involvement of protein disulfide isomerases ............<br />
Maurizio BRUNORI, How proteins recognize their biochemical partners: ligand binding and folding<br />
pathways of PDZ domains .......................................................................................................................................<br />
Felice CERVONE, Plant innate immunity: cell wall-mediated signalling and recognition in plant defense<br />
Ernesto DI MAURO, Spontaneous formation and evolution of informational nucleic polymers ...................<br />
Martino Luigi DI SALVO, Synthesis of pyridoxal phosphate in the vitamin B6 salvage pathway and<br />
targeting of the cofactor to apo-enzymes .............................................................................................................<br />
Francesco GASPARRINI, Integrating advanced functional materials and chromatography-mass<br />
spectrometry to examine protein-ligand interactions ........................................................................................<br />
Cristina LIMATOLA, Molecular and functional approaches to investigate the physiopathological role of the<br />
chemokines and their receptors in the central nervous system .......................................................................<br />
Paola PAGGI, Neurone response to experimental injury and lack of dystrophin: a molecular, functional<br />
and structural study in autonomic ganglia in vivo and in vitro ........................................................................<br />
Maria SAVINO, Structural and superstructural features of human telomeric chromatin ...............................<br />
Research area 5: Cellular and molecular immunology<br />
Vincenzo BARNABA, Interplay amongst chronic immune activation, apoptosis, cross-presentation,<br />
immune-regulation, and autoimmunity .................................................................................................................<br />
Roberto FOA’, Potential role of miRNAS in IgM-mediated signal transduction in normal and neoplastic<br />
B cells ..............................................................................................................................................................................<br />
Rossella PAOLINI, Signalling events negatively regulating FcepsilonRI expression and mast cell<br />
functional responses ....................................................................................................................................................<br />
Enza PICCOLELLA, Analysis of the molecular mechanisms regulating FOXP3 gene and protein expression<br />
in TCR- and CD28-activated CD4+CD25-T cells and their influence on regulatory functions ............<br />
Angela SANTONI, Anti-tumor effector functios of NK cells in tumor microenviroment ...............................<br />
Isabella SCREPANTI, Dissection of Notch signaling-dependent pathways involved in the progression of<br />
T cell leukemia .............................................................................................................................................................<br />
Rosa SORRENTINO, The role of HLA-B27 in autoimmunity: from the genetics to the function .................<br />
Loretta TUOSTO, CD28 co-stimulatory molecule as a key regulator of T lymphocyte differentiation and<br />
survival: characterisation of the biochemical pathways and molecules coupling CD28 to NF-kB activation<br />
Elio ZIPARO, The role of Toll Like Receptors in immune responses to infections and in inflammationassociated<br />
pathologies of the male reproductive system ..................................................................................<br />
Research area 6: New antimicrobial and antiviral agents<br />
Donatella BARRA, Peptide effectors of innate immunity ........................................................................................<br />
Mariangela BIAVA, New pyrrole derivatives of BM 212: a new class of antimycobacterial agents. Design,<br />
synthesis, biological evaluation and study of their mode of action ...............................................................<br />
Roberto DI SANTO, Pyrrolyl diketo hexenoic acid derivatives as novel anti-HIV agents targeted to the<br />
ribonuclease H function of the HIV-1 reverse transcriptase enzyme ............................................................<br />
IV<br />
55<br />
57<br />
59<br />
61<br />
65<br />
67<br />
69<br />
71<br />
73<br />
75<br />
79<br />
81<br />
83<br />
85<br />
87<br />
89<br />
91<br />
93<br />
95<br />
101<br />
105<br />
107<br />
109<br />
111<br />
115<br />
117<br />
119<br />
121<br />
125<br />
129<br />
131
Antonello MAI, Design, synthesis and biological evaluation of small molecule epigenetic modulators: a<br />
novel approach for anticancer, antifungal, and antiviral chemotherapy ........................................................<br />
Elena MATTIA, Effects of resveratrol on Epstein Barr Virus latent and lytic phases of infection ..............<br />
Romano SILVESTRI, Drug design and synthesis of non-nucleoside inhibitors of both HIV-1 wild type and<br />
resistant mutant strains reverse transcriptase, and Coxsackie b4 virus .......................................................<br />
Research area 7: Biology of Malaria and other vector-borne diseases<br />
Alessandra DELLA TORRE e Vincenzo PETRARCA, Genetic and phenotypic characterization of species and<br />
“molecular forms” of the Anopheles gambiae complex (Diptera: Culicidae), afrotropical malaria vectors<br />
David MODIANO, Immune responses to malaria and autoimmune disorders: investigating common generegulatory<br />
networks ...................................................................................................................................................<br />
Anna TRAMONTANO, Computational Analysis of the gene products of the Plasmodium falciparum<br />
genome and their interaction with human proteins ...........................................................................................<br />
Start-up program<br />
Giuseppe LUPO, Specification and maintenance of retinal stem cells ..................................................................<br />
• Bibliography ...............................................................................................................................................................<br />
V<br />
135<br />
139<br />
141<br />
145<br />
147<br />
149<br />
153<br />
157
Forward<br />
The <strong>Istituto</strong> <strong>Pasteur</strong>-Fondazione Cenci Bolognetti is a private non-profit foundation which was<br />
established according to the terms of the bequest of Princess Beatrice Fiorenza Cenci Bolognetti to<br />
Sapienza University of Rome for the general purpose of encouraging scientific research in the area of<br />
pasteurian sciences and specifically for founding a <strong>Pasteur</strong> Institute in Rome. Since 1970 the Institute<br />
has been part of a global network of <strong>Pasteur</strong> Institutes known as Réseau International des Instituts<br />
<strong>Pasteur</strong> et Instituts Associés. The funding of its activity derives from the rent of three historical palaces<br />
in Rome and during the period of the present <strong>report</strong> of activity the <strong>Istituto</strong> <strong>Pasteur</strong>-Fondazione<br />
Cenci has been supported by the Fondazione Roma and the BNP Paribas BNL that are gratefully<br />
acknowledged.<br />
The research activity of the <strong>Istituto</strong> <strong>Pasteur</strong>-Fondazione Cenci Bolognetti is committed to<br />
studying the mechanisms that regulate the basic processes of life. This mission is pursued through<br />
the funding of research projects, the award of fellowships and the organization of scientific meetings<br />
and seminars.<br />
At a time when attention of granting agencies and science policy makers in Italy and Europe is largely<br />
focused towards application, it should not be forgotten that important breakthroughs in biology and<br />
medicine often originated from curiosity driven research. Starting from this premise, the <strong>Istituto</strong><br />
<strong>Pasteur</strong>-Fondazione Cenci Bolognetti has concentrated on the selection of applications exclusively<br />
on scientific merit, without bias for application. Our scientific programs reflect this philosophy. The<br />
research projects illustrated in this Report of activity, were selected by a peer review system based<br />
on highly qualified international referees, to whom we are very grateful. The general themes witness<br />
a modern interpretation of the sciences pasteuriennes, with adherence to the mission of the Foundation<br />
Training young researchers has always been a top priority of the Foundation, which is achieved by:<br />
i) providing fellowships to work abroad as well as at the Sapienza-University of Rome; ii) co-funding<br />
the international Ph.D. program in <strong>Pasteur</strong>ian Science at the Sapienza; and iii) financing fellowships for<br />
the first year after the PhD; iv) beginning in 2009, the funding of start-up grants for young brilliant<br />
researchers.<br />
The institute’s development plan has scheduled the construction of its own laboratories for the near<br />
future. These facilities will host leading researchers of all nationalities operating in the field of<br />
pasteurian sciences, with special reference to the molecular bases of human pathologies.<br />
The results obtained during the years 2009 and 2010, documented in this volume, witness the<br />
enthusiasm and productivity of the scientists involved.<br />
Paolo Amati<br />
President<br />
VII<br />
Angela Santoni<br />
Scientific Director
Boards and Staff<br />
Administrative Board<br />
The Board of Administration is presided over<br />
by a President appointed by the Rector of<br />
Sapienza-University of Rome chosen from a list<br />
of three names selected during a joint session of<br />
the board and the Scientific Council. Members<br />
of the board include a Scientific Director, four<br />
members of the Faculties of Pharmacy and<br />
Medicine, Natural Sciences, and Medicine and<br />
Dentistry. Other members are a legal expert<br />
designated by the University of Rome Board<br />
of Administration, three auditors nominated by<br />
the University, the Ministry of the Treasury,<br />
and the Ministry of Universities and Scientific<br />
Research.<br />
President<br />
Paolo Amati (Pharmacy and Medicine)<br />
Members<br />
Paolo Costantino (Natural Sciences), Alberto<br />
Faggioni (Medicine and Dentistry), David<br />
Modiano (Pharmacy and Medicine), Angela<br />
Santoni (Pharmacy and Medicine), Romano<br />
Silvestri (Pharmacy and Medicine)<br />
Secretary<br />
Emanuela Gloriani<br />
Administrative Expert<br />
Giuseppina Capaldo<br />
Auditors<br />
Anna Carmela Ferrante, Simona Ranalli,<br />
Carla Vassallo<br />
VIII<br />
Scientific Council<br />
The Scientific Council is a board of seven<br />
scholars in the field of the pasteurian sciences.<br />
Members are elected to a four-year term by the<br />
Faculties Natural Sciences, and Pharmacy and<br />
Medicine. The scientific director, appointed by<br />
the Scientific Council, is ex-officio a member<br />
of the Board of Administration. The Scientific<br />
Council attends to examine and co-ordinate the<br />
research programs as well as the several scientific<br />
activities.<br />
Scientific Director<br />
Angela Santoni (Pharmacy and Medicine)<br />
Members<br />
Ernesto Di Mauro (Natural Sciences), Laura<br />
Frontali (Natural Sciences), Anna Teresa<br />
Palamara (Pharmacy and Medicine), Anna<br />
Tramontano (Pharmacy and Medicine), Marco<br />
Tripodi (Pharmacy and Medicine), Carlo<br />
Turano (Pharmacy and Medicine)<br />
Secretariat<br />
Sarah Gainsforth, Maria Pia Lorenzoni,<br />
Lynda Romani, Nicoletta Silvestri, Lucia Ugo<br />
Consultants<br />
Tommaso De Dominicis (legal affairs), Anna<br />
Maria Pivetti (architectural supervision),<br />
Barbara Hell (financial affairs)
Fellowships awarded in 2009 and 2010<br />
Fellowships awarded for two years for<br />
training in foreign laboratories<br />
Fabio DI DOMENICO, at University of Kentucky,<br />
from Department of Biochemical Sciences<br />
Daniela DIMASTROGIOVANNI, at Department of<br />
Biochemistry, University of Cambridge, UK, from<br />
Department of Biochemical Sciences<br />
Michela ESPOSITO, at Institut de Génétique<br />
et Microbiologie, Université Paris XI, from<br />
Department of Biology and Biotechnologies «Charles<br />
Darwin»<br />
Roberto GAETANI, at Department of Cardiology,<br />
University Medical Center, Utrecht, NL, from<br />
Department of Molecular Medicine<br />
Isabelle JOURDAN, at Inserm, Université Pierre et<br />
Marie Curie, Paris, France, from Department of<br />
Anatomical, Histological, Forensic & Orthopaedic<br />
Sciences<br />
Marta MORETTI, at Division of Immunology and<br />
Inflammation, Imperia College, London, UK, from<br />
Department of Molecular Medicine<br />
Tommaso MOSCHETTI, at Department of<br />
Biochemistry, University of Cambridge, UK, from<br />
Department of Biochemical Sciences<br />
Giuseppe SCIUMÈ, at NIH/NIAMS, Bethesda, MD,<br />
USA, from Department of Molecular Medicine<br />
Piera SMERIGLIO, at ISERM, Université Pierre<br />
et Marie Curie, from Department of Anatomical,<br />
Histological, Forensic & Orthopaedic Sciences<br />
Fellowships awarded to students who had a<br />
two-year experience abroad<br />
Daniela CECCARELLI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”, from Université<br />
de Sherbrooke, Québec, Canada<br />
Antonio COLUCCIA, at Department of Medicinal<br />
Chemistry and Technologies, from School of<br />
Pharmacy, University of Cardiff, UK<br />
Valentina MANGANO, at Department of Public<br />
Health and Infectious Diseases, from MRC Centre<br />
for Genomics and Global Health, Oxford, UK<br />
Bich Lich NGUYEN, at Department of Cardiovalular<br />
Pathophysiology, Anesthesiology and General<br />
Surgery, from Cardiology Department of the Cedars-<br />
Sinai Medical Center, California, USA<br />
Fellowships awarded to students who have<br />
completed their PhD and are seeking a position<br />
Eleni ANASTASIADOU, at Department of<br />
Experimental Medicine<br />
IX<br />
Tonino ANELLI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Cristina CAPUANO, at Department of Molecular<br />
Medicine<br />
Isotta CHIMENTI, at Department of Molecular<br />
Medicine<br />
Alessandra GALATI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Francesca GASPARRINI, at Department of<br />
Molecular Medicine<br />
Luigi LEMBO FAZIO, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Adriana MAGNACCA, at Department of Biology<br />
and Biotechnologies “Charles Darwin”<br />
Marianna PELLONI, at Department of Molecular<br />
Medicine<br />
Marianna Nicoletta ROSSI, at Department of Cell<br />
Biotechnology and Hematology<br />
Fellows working on the research programs<br />
of the Foundation<br />
Salvatore ALFONSO, at Department of Medicinal<br />
Chemistry and Technologies<br />
Francesco ANGELUCCI, at Department of<br />
Biochemical Sciences<br />
Valentina ANNESE, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Chiara ARDICCIONI, at Department of Biochemical<br />
Sciences<br />
Pamela AVELLINO, at Department of Public Health<br />
and Infectious Diseases<br />
Enrico M. BATASSA, at Department of Cell<br />
Biotechnology and Hematology<br />
Nicoletta CALOSCI, at Department of Biochemical<br />
Sciences<br />
Cristina CAMPERIO, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Cristina CAPUANO, at Department of Molecular<br />
Medicine<br />
Maria Rosaria CARBONE, at Department of Cell<br />
Biotechnology and Hematology<br />
Nicoletta CASTIGLIONE, at Department of<br />
Biochemical Sciences<br />
Ignacio Celestino, at Department of Public Health<br />
and Infectious Diseases<br />
Marcella CESANA, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Anna CHAILYAN, at Department of Biochemical<br />
Sciences<br />
Isotta CHIMENTI, at Department of Molecular
Medicine<br />
Alberto CIOLFI, at Department of Cell<br />
Biotechnology and Hematology<br />
Ivan COLADARCI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Antonio COLUCCIA, at Department of Medicinal<br />
Chemistry and Technologies<br />
Domenico COZZETTO, at Department of<br />
Biochemical Sciences<br />
Simona CUGUSI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Cristina DE LUCA, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Francesca DE VITO, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Donatella D’ELISEO, at Department of Molecular<br />
Medicine<br />
Enea Gino DI DOMENICO, at Department of<br />
Biology and Biotechnologies “Charles Darwin”<br />
Maria Letizia DI MARTINO, at Department of<br />
Biology and Biotechnologies “Charles Darwin”<br />
Matteo DI PENTIMA, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Michela ESPOSITO, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Antonella FARINA, at Department of Experimental<br />
Medicine<br />
Fedra FRANCOCCI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Alessandra GALATI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Francesca GARIBALDI, at Department of Cell<br />
Biotechnology and Hematology<br />
Francesca GASPARRINI, at Department of<br />
Molecular Medicine<br />
Roberta GONNELLA, at Department of<br />
Experimental Medicine<br />
Caterina GRILLO, at Department of Biochemical<br />
Sciences<br />
Vittoria IONTA, at Department of Molecular<br />
Medicine<br />
Ilias KOUTRIS, at Department of Biochemical<br />
Sciences<br />
Loredana LE PERA, at Department of Biochemical<br />
Sciences<br />
Lanfranco LEO, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Rosalba LEPORE, at Department of Biochemical<br />
Sciences<br />
Fabrizio LOMBARDO, at Department of Public<br />
Health and Infectious Diseases<br />
Vincenzo LUCA, at Department of Biochemical<br />
Sciences<br />
Emiliano MANCINI, at Department of Public Health<br />
and Infectious Diseases<br />
X<br />
Paolo MARCATILI, at Department of Biochemical<br />
Sciences<br />
Roberto MATTIOLI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Emanuela MICHELI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Chiara MICOLONGHI, at Department of Biology<br />
and Biotechnologies “Charles Darwin”<br />
Vanessa MODESTI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Rosa MOLFETTA, at Department of Molecular<br />
Medicine<br />
Arianna MONTANARI, at Department of Biology<br />
and Biotechnologies “Charles Darwin”<br />
Linda Celeste MONTEMIGLIO, at Department of<br />
Biochemical Sciences<br />
Gaëlle NOEL, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Vanessa PALERMO, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Valeria Luciana PALUMBO, at Department of<br />
Biology and Biotechnologies “Charles Darwin”<br />
Claudia PELLACANI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Serena PERILLI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Francesco PISCITELLI, at Department of Medicinal<br />
Chemistry and Technologies<br />
Alessandra PORZIA, at Department of Molecular<br />
Medicine<br />
Pasqualina PUNZI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Wilhelmina RENSEN, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Cinzia RIZZO, at Department of Public Health and<br />
Infectious Diseases<br />
Dimitri RYKUNOV, at Department of Biochemical<br />
Sciences<br />
Maria Isabel SANTORI, at Department of Biology<br />
and Biotechnologies “Charles Darwin”<br />
Daniel SAVATIN, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Stefano SECHI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Riccardo SILIGATO, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Aise Ozlem TASTAN BISHOP, at Department of<br />
Biochemical Sciences<br />
Simona TROMBETTI, at Department of Molecular<br />
Medicine<br />
Lucia TUFANO, at Department of Biology and<br />
Biotechnologies “Charles Darwin”<br />
Elena ZANNI, at Department of Biology and<br />
Biotechnologies “Charles Darwin”
Seminars<br />
2009<br />
February 2 • Protein folding, misfolding and neurodegenerative<br />
diseases<br />
Prof. Mikael Oliveberg, University of Stockholm,<br />
Sweden<br />
March 16 • Final touches to nucleosome positioning<br />
sequence pattern<br />
March 23 • The earliest stages of (bio) molecular life<br />
Prof. Edward N. Trifonov, University of Haifa,<br />
Israel, and Masaryk University, Brno, Czech<br />
Republic<br />
April 22 • Inflammatory cytokine gene regulation<br />
through intrinsic RNA elements that activate the cell’s<br />
RNA-dependent protein kinase<br />
May 11 • How Ebola virus blocks the host antiviral<br />
response<br />
Prof. Raymond Kaempfer, Faculty of Medicine,<br />
The Hebrew University, Jerusalem, Israel<br />
May 4 • Challenging the delusion: Histone H2A.Bbd<br />
structure and function<br />
Prof. Juan Ausio, University of Victoria, Canada<br />
May 5 • Estimating an upper bound for transition path<br />
times in protein folding from single molecule photon<br />
trajectories<br />
Prof. William A. Eaton, Laboratory of Chemical<br />
Pysics, NIH, MD, USA<br />
June 19 • Understanding the regulation and specific<br />
inhibition of protein kinases using large scale structural<br />
comparison<br />
Prof. Stefan Knapp, Structural Genomics<br />
Consortium, Oxford University, UK<br />
June 19 • Vieving Neuronal Signaling by NMR<br />
Spectroscopy and X-ray Crystallography<br />
Prof. Mingjie Zhang, University of Hong Kong<br />
June 30 • Darwin e il Vaticano: Storia di una evoluzione<br />
Prof. Antonio Lazcano, Universidad Nacional<br />
Autónoma de México<br />
October 16 • Structural studies of antiviral lectins,<br />
potential ingredients of anti-HIV microbicides<br />
Prof. Alex Wlodawer, National Cancer Institute<br />
at Frederick, MD, USA<br />
XI<br />
November 19 • Molecular mechanisms of daily biological<br />
clock, the tale from a simple eukaryotic organism<br />
Prof. Yi Liu, University of Texas Southwestern<br />
Medical Center, Dallas, USA<br />
December 18 • Darwin’s Revolution: Natural<br />
Theology to Natural Selection<br />
Prof. Francisco J. Ayala, University of California,<br />
Irvine, USA<br />
2010<br />
March 30 • Folding-induced ion transfer in the ALSassociated<br />
protein SOD1<br />
Prof. Mikael Oliveberg, University of Stockholm,<br />
Sweden<br />
April 30 • Sex and Death in Bdelloid Rotifers<br />
Prof. Mathew Meselson, Harvard University,<br />
Cambridge, MA, USA<br />
May 20 • Control of protective immunity through<br />
dynamic refolding of mRNA<br />
May 27 • Making hemoglobin: why stress is good for you,<br />
a story of mRNA splicing<br />
Prof. Raymond Kaempfer, Faculty of Medicine,<br />
The Hebrew University, Jerusalem, Israel<br />
October 18 • Leucemia acuta mieloide con mutazioni<br />
di nucleofosmina (NPM1): dalla lesione genetica alla<br />
clinica<br />
Prof. Brunangelo Falini, Università degli Studi di<br />
Perugia, Italy<br />
October 25 • Regolazione della latenza nel virus di<br />
Epstein Barr. L’interessante caso di CTCF, una proteina<br />
multi-funzione<br />
Dr. Italo Tempera, Wistar Institute, University<br />
of Philadelphia, USA<br />
December 6 • Identification of new drugs targeting<br />
redox biochemistry for the control of Schistosomiasis<br />
Prof. David Williams, Rush University Medical<br />
Center, Chicago, USA
Meetings and Conferences organized or supported by the Foundation<br />
<strong>Istituto</strong> <strong>Pasteur</strong> Science Days<br />
Liver development and disease<br />
Rome, October 9-10, 2009<br />
• Organogenesis<br />
• Immunity and Immunopathology<br />
• Fibrosis and tumorigenesis<br />
Joint Workshop<br />
New frontiers in the pathogenesis and<br />
treatment of infectious diseases<br />
Rome, March 25, 2010<br />
In collaboration with the Stephan Angeloff Institute<br />
of Microbiology of Sofia<br />
European Day of Immunology<br />
Un amico sorprendente: il tuo sistema<br />
immunitario<br />
Rome, April 29, 2010<br />
Interactive study experience on the basic mechanisms<br />
of the immune response<br />
22° <strong>Pasteur</strong>-Weizmann Symposium – 35° Anniversary<br />
of <strong>Pasteur</strong>-Weizmann<br />
Stem cells: from basic biology to translational<br />
medicine<br />
Rome, June 7-9, 2010<br />
In collaboration with the Foundation<br />
• Muscle stem cells<br />
• Epithelial stem cells and tissue<br />
reprogramming<br />
• Embryo and postnatal stem cells<br />
• Embryonic stemm cells and induced<br />
pluripotency<br />
• Neuronal stem cells<br />
• Stem cell niches<br />
XII<br />
Mini-Workshop in<br />
Epigenetics<br />
Rome, September 10, 2010<br />
Associazione Genetica Italiana - Genetics School<br />
Lessons<br />
Le malattie genetiche: genetica, epigenetica<br />
e altro…<br />
Cortona, November 25-26, 2010<br />
with the contribution of the Foundation<br />
Lincei International Symposium on<br />
Protein structure and dynamics<br />
Rome, December 1-3, 2010<br />
with the contribution of the Foundation<br />
• Folding and binding<br />
• Dynamics and complexity of the<br />
conformational landscape<br />
• Single molecule biophysics
AREA 1<br />
Molecular<br />
biology of<br />
microorganisms<br />
and<br />
viruses
AREA 1<br />
Bacterial globins as regulators of thiol redox equilibrium in bacteria<br />
Principal investigator: Alberto Boffi<br />
Professor of Molecular Biology<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 49910990; Fax: (+39) 06 4440062<br />
alberto.boffi@uniroma1.it<br />
Participants:<br />
Alessandra Bonamore, Alberto Macone, research fellows;<br />
Andrea Ilari, CNR researcher; Paola Baiocco, post-doc fellow.<br />
Collaborations:<br />
Dipartimento di Chimica, Università di Firenze (Prof. Giulietta<br />
Smulevich), CPC Biotech srl, Napoli (Dr. Fabio Arenghi),<br />
Departamento de Química Inorgánica, Analítica y Química Física,<br />
Universidad de Buenos Aires (Dr. Leonardo Boechi).<br />
Report of activity<br />
The specific targets of the project concern the elucidation<br />
of the mechanism of heme based thiol oxidation<br />
in bacterial truncated globins (trHbs) and the<br />
discovery of novel pathways in thiol redox homeostasis<br />
in bacteria. The first point entails a complete<br />
biochemical characterization of the observed<br />
catalytic activity towards sulfide reactive species and<br />
model substrates such as cysteine and glutathione.<br />
The understanding of the kinetic properties of thiol<br />
oxidation is a prerequisite for the elucidation of<br />
the reaction mechanism. Spectroscopic techniques<br />
have been applied in order to characterize the reaction<br />
profiles as a function of standard parameters<br />
(pH, salt composition, reducing environment etc.,<br />
see methods). In particular, a complete resonance<br />
Raman spectroscopic characterization of the globins<br />
from Thermobifida fusca and Bacillus subtilis has been<br />
carried out both on native proteins and in selected<br />
active-site mutants (Droghetti et al., 2010; Nicoletti<br />
et al., 2010). The Raman studies brought out that<br />
these globins display the spectroscopic signature<br />
characteristics of heme peroxidases, with a strong<br />
electron donation from the heme iron to the distal<br />
ligand both in their ferrous and ferric adducts.<br />
X-ray crystallography, coupled to X-Ray absorption<br />
(EXAFS) and fluorescence (XANES) studies have<br />
ascribed the peroxidase character to an unusually<br />
3<br />
short bond distance between the iron atom and the<br />
proximal histidine residue (Arcovito et al., 2010). On<br />
these basis, a genuine peroxidase activity has also<br />
been described in the interactions of Thermobifida<br />
fusca truncated hemoglobin, and on a set of selected<br />
active site mutants, with a battery of typical peroxidase<br />
substrates (Torge et al., 2009). A very high specific<br />
peroxidase activity has been observed towards<br />
several substrates, different from those typical of<br />
vegetal peroxidases. However, the most significant<br />
observations in relation with bacterial biochemistry<br />
and physiology are centered on the unusually high<br />
affinity of both Thermobifida fusca and Bacillus subtilis<br />
trHb towards thiol containing derivatives. In<br />
particular, these proteins, when overexpressed in E.<br />
coli, are capable of sequestering virtually all hydrogen<br />
sulfide produced within the host metabolism<br />
and of oxidizing cysteine and other thiol containing<br />
compounds thus altering the thiol redox homeostasis<br />
and sulphur related metabolic pathways (Nicoletti et<br />
al., 2010). The nature of the heme-sulfide complexes<br />
has been further explored in spectroscopic experiments<br />
that allowed for the first time the assignment<br />
of the specific iron-sulfur stretching frequencies in<br />
a hemoprotein (Nicoletti et al., 2010). In parallel,<br />
molecular dynamics of sulfide motion inside the<br />
heme pocket has been investigated and highlighted<br />
a novel mechanism of ligand stabilization within<br />
the heme pocket, based on hydrogen bonding from<br />
a conserved tryptophan residue to the iron bound<br />
sulfur atom (Nicoletti et al., 2010). X-ray crystallographic<br />
studies are in progress in order to determine<br />
the geometry of thiol-globin complexes by means of<br />
crystal soaking studies and/or co-cristallization with<br />
non-reactive analogues (e.g. selenocysteine).<br />
The second target of the research concerns the identification<br />
of partner proteins involved in the complex<br />
physiological network of thiol homeostasis. At least<br />
in the case of Bacillus subtilis, the partner protein
Alberto Boffi<br />
YjbH has been identified already (Larsson et al.,<br />
2007). Bioinformatic methods have been applied in<br />
order to single out possible paralogues in actinomyces,<br />
including mycobacteria. The aim of the “in silico”<br />
search is to establish potential redox partners belonging<br />
to the thioredoxin superfamily and identify novel<br />
pathways within the network of thiol-disulfide oxidoreductases.<br />
On these basis, it is suggested that the<br />
globin may be able to catalyze disulfide bonds formation<br />
and thus act as a heme based peroxiredoxin. The<br />
first step towards the identification of a novel thiol<br />
redox pathway in Bacillus subtilis concerns the demonstration<br />
of the postulated interaction between the<br />
globin (yjbI) and the “disulfide isomerase-like” protein<br />
(yjbH) located upstream within the same operon. In<br />
this framework, the whole operon contaning yjbI and<br />
yjbH has been cloned both as a copy of the original<br />
operon from B. subtilis and as a synthetic gene, optimized<br />
for expression in E. coli. Overexpression of<br />
the whole operon allowed us to single out that trHb<br />
is capable of direct oxidation of the -CxxC- motif<br />
of YjbH thus regenerating active protein. YjbH has<br />
been previously demonstrated to be necessary for the<br />
proteolytic control of the global stress regulator Spx<br />
under disulfide stress and directly interacts with the<br />
global regulator Spx thus preventing its proteolitic<br />
degradation. It is envisaged that only the oxidized<br />
form of YjbH is able to perform such regulatory<br />
interaction and thus, trHb (YjbI) should restore the<br />
disulfide bond by directly interacting with YjbH. In<br />
order to clarify the nature of trHb-YjbH interaction,<br />
analytical ultracentifugation experiments are<br />
in progress under different experimental conditions<br />
(buffer composition, presence of reductants, absence<br />
of oxygen). Thereafter, X-ray crystallographic studies<br />
of the trHb-YjbH complex will be carried out. In<br />
parallel, a metabolomic study has started in order to<br />
verify whether trHb act by oxidizing the low molecular<br />
weight thiol containing species within the cytosol<br />
and thus modulates directly the thiol redox equilibrium.<br />
In this case, low molecular weight thiols have<br />
been monitored by LC/MS methods, using E. coli<br />
overexpressing YjbIH operon as model organism. As<br />
expected, a tenfold increase in cytosolic free cysteine<br />
4<br />
is observed with an unprecedented metabolic drift<br />
towards the synthesis of sulfur aminoacids. However,<br />
the final picture underlyning the role of the truncated<br />
globins within the thiol redox homeostasis is still far<br />
from complete. In our understanding, it is clear that<br />
trHbs act as redox switches in that they are capable<br />
of oxidizing disulfide bonds of the YjbH regulator<br />
which, in turn, governs the activity of the global<br />
stress regulator Spx by expiring its lifespan and<br />
quenching the transcription of genes associated with<br />
oxidative stress. In this framework, trHbs would be<br />
terminators of the redox stress response. However,<br />
trHbs are also capable of efficient sequestering of all<br />
free hydrogen sulfide present in the cell thus shifting<br />
the metabolism towards sulfur compound anabolism,<br />
a role that point to trHbs as active effectors in counterbalancing<br />
the oxidative stress through production<br />
of more complex thiol species. The solution of this<br />
apparently contrasting biological functions will be<br />
addressed in the second year of the project, by examining<br />
in detail the nature of the interactions of trHbs<br />
with their partner proteins.<br />
Publications<br />
Arcovito A, Bonamore A, Hazemann JL, Boffi A,<br />
D’Angelo P. Unusual proximal heme pocket geometry<br />
in the deoxygenated Thermobifida fusca: a combined<br />
spectroscopic investigation. Biophys Chem.<br />
2010, 147: 1-7. doi:10.1016/j.bpc.2009.11.006.<br />
Droghetti E, Nicoletti FP, Bonamore A, Boechi<br />
L, Pau AM, Estrin D, Boffi A, Smulevich G, Feis<br />
A. Heme pocket structural properties of a bacterial<br />
truncated hemoglobin from Thermobifida fusca.<br />
Biochemistry. 2010, 49: 10394-402. doi:10.1021/<br />
bi101452k.<br />
Nicoletti FP, Comandini A, Bonamore A, Boechi<br />
L, Boubeta FM, Feis A, Smulevich G, Boffi A.<br />
Sulfide binding properties of truncated hemoglobins.<br />
Biochemistry. 2010, 49: 2269-78. doi: 10.1021/<br />
bi901671d.
The acid resistance genes of Escherichia coli: does their complex<br />
transcriptional control hide novel biological roles?<br />
Principal investigator: Daniela De Biase<br />
Professor of Biochemistry<br />
Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche<br />
Sede di Latina<br />
Tel: (+39) 0773 1757212; Fax: (+39) 0773 1757254<br />
daniela.debiase@uniroma1.it<br />
Participants:<br />
Francesco Bossa, professor; Angela Tramonti, CNR<br />
researcher; Eugenia Pennacchietti, post-doc fellow; Daniela<br />
Bastianelli, PhD student; Denise Galzerano, Gaia Grassini,<br />
undergraduate students.<br />
Collaborations:<br />
Paul Scherrer Institute, Villigen PSI, Switzerland (Dr. Guido<br />
Capitani); Wageningen University and Research Centre,<br />
Wageningen, The Netherlands (Prof. Maurice C.R. Franssen);<br />
School of Biosciences University of Wales, Cardiff, UK (Prof.<br />
Robert A. John); CNRS-ENS de Cachan, Cachan, France (Dr.<br />
Sylvie Rimsky); Institute of Molecular Cell Biology, VU University<br />
Amsterdam, The Netherlands (Prof. Joen Luirink).<br />
Report of activity<br />
Our research project focusses on the analysis of the<br />
molecular bases that in Escherichia coli lead to acid<br />
resistance (AR), a necessary pre-requisite to breach<br />
the gastric barrier and gain access into the host gut.<br />
In particular we are investigating the regulation and<br />
the mechanism of action of the most efficient acid<br />
resistance system in E. coli, the glutamate-dependent<br />
acid resistance (GDAR) system, which is functional<br />
not only in commensal and pathogenic E. coli, but<br />
also in Shigella flexneri and Listeria monocytogenes. We<br />
aim to unveil the specific role played by the transcriptional<br />
regulators GadX and GadE, also with respect<br />
to the genome silencer H-NS, as well as the structurefunction<br />
relationships in the structural components of<br />
the GDAR system. Recent <strong>report</strong>s in the fields of<br />
quorum sensing and biofilm formation showed that<br />
some GDAR genes are switched on or off upon specific<br />
stimuli or growth conditions. Therefore we are<br />
performing experiments aimed at uncovering novel<br />
biological roles of the GDAR genes.<br />
The GDAR system requires the intracellular protonconsuming<br />
activity of two glutamate decarboxylase<br />
paralogs, GadA and GadB, as well as the antiport<br />
activity of an integral membrane protein, GadC, which<br />
5<br />
AREA 1<br />
exchanges glutamate for the decarboxylation product<br />
γ-aminobutyrate (GABA). The gadB and gadC structural<br />
genes are co-transcribed, while the gadA structural<br />
gene, which is 2.1 Mb from gadBC, can be either<br />
independently transcribed or co-transcribed with the<br />
downstream gadX gene. The gadA gene belongs to<br />
the so-called acid fitness island (AFI), a 15-kb genome<br />
region repressed by H-NS (the histone-like nucleoidstructuring<br />
protein) and controlled by RpoS, (the stationary<br />
phase σ factor of the RNA polymerase). The<br />
AFI contains 13 genes that encode 12 proteins and a<br />
small RNA (GadY); the genes are mainly organised<br />
into operons and the protein products of the AFI contribute<br />
to E. coli AR in different ways.<br />
The gadA and gadBC promoter regions are under<br />
the control of a very complex regulatory network<br />
that integrates multiple environmental signals and<br />
physiological adaptations. The essential regulator<br />
GadE is under the control of three different activation<br />
circuits. The GadX/GadW-dependent circuit,<br />
the main object of our investigation, involves the<br />
specific transcriptional regulators GadX, GadW and<br />
GadY, encoded by the corresponding genes in the<br />
AFI, and also the global regulators H-NS, CRP and<br />
RpoS. GadX and GadW are AraC-like transcriptional<br />
regulators, capable of up-regulating the gadA and<br />
gadBC structural genes by direct binding to a 42-bp<br />
sequence in their 5’ regulatory regions (Tramonti et<br />
al., J. Bacteriol. 2006, 188:8118-8127). Interestingly,<br />
also the 5’ regulatory regions of the AFI operons<br />
slp-yhiF, hdeAB-yhiD, hdeD, gadE-mtdEF and gadY<br />
are direct targets of these activators (Tramonti et<br />
al., Mol. Microbiol. 2008, 70:965-982). We generated<br />
a 42-bp GadX/GadW consensus sequence by aligning<br />
the six 42-bp long GadX/GadW-binding sites<br />
identified by DNAse I footprinting analyses. This<br />
sequence arises from the tandem arrangement of two<br />
21-bp sequences, in which nine positions (43%) are<br />
highly-to-strictly conserved.<br />
In the period 2009-2010 we carried out experiments<br />
aimed at fully characterizing the GadX regulon
Daniela De Biase<br />
(manuscript in preparation) and at analyzing GadB<br />
site-specific mutants to establish the sequence of<br />
steps that allow this enzyme to control its activity<br />
in the cell. We also set up the basis to investigate<br />
the GadX-H-NS interplay at different targets and<br />
produced GDAR genes deletion mutants to be used<br />
in motility and biofilm formation assays.<br />
Characterization of the GadX regulon<br />
To fully characterize the GadX regulon in the E. coli<br />
K12 strain MG1655, we used chromatin immunoprecipitation<br />
coupled with microarrays (ChIP on chip).<br />
Cultures expressing the 3xFlag-tagged GadX were<br />
grown in LB-MES, pH 5.5, to the stationary phase<br />
and then treated with formaldehyde to crosslink the<br />
DNA-protein complexes. Immunoprecipitation using<br />
anti-Flag antibodies yielded DNA-GadX complexes.<br />
The corresponding DNA fragments were analysed<br />
on microarrays specifically designed for Cy5 (or<br />
Cy3)-labelled DNA fragments obtained from ChIP<br />
experiments and consisting of 21321 60-mer oligonucleotide<br />
probes that match MG1655 sequences at<br />
intervals of 160 bp (OGT, UK). For each probe, the<br />
Cy5/Cy3 (or Cy3/Cy5) ratio was measured and this<br />
was plotted against the corresponding position on the<br />
E. coli MG1655 genome. More than 60 peaks in the<br />
fluorescence ratio are easily distinguishable from the<br />
background signal and therefore identify in vivo GadX<br />
binding sites. Some of the highest peaks are located in<br />
the genomic positions corresponding to known GadX<br />
targets, i.e. gadBC and AFI regulatory regions. More<br />
than 30 DNA fragments corresponding to the genomic<br />
regions encompassing the major peaks were analysed<br />
by EMSA in the presence of GadX. In most of<br />
the cases, we were able to reproduce in vitro (EMSA)<br />
the in vivo data (ChIP-chip). Some of the GadX targets<br />
were also selected for DNaseI footprinting analysis to<br />
improve the GadX consensus sequence.<br />
6<br />
Structure-function relationships in GadB<br />
GadB exhibits an acidic pH optimum and undergoes<br />
a spectroscopically detectable and strongly cooperative<br />
pH-dependent conformational change involving<br />
at least six protons. Crystallographic studies showed<br />
that at mildly alkaline pH GadB is inactive because all<br />
active sites are locked by the C-termini and that the<br />
340 nm absorbance is an aldamine formed by the pyridoxal<br />
5’-phosphate-Lys276 Schiff base with the distal<br />
nitrogen of His465, the penultimate residue in the<br />
GadB sequence (Gut et al., EMBO J. 2006, 25:2643-<br />
2651). We have shown that His465 has a massive<br />
influence on the equilibrium between active and<br />
inactive forms, the former being favoured when this<br />
residue is absent. His465 contributes with n ≈ 2.5 to<br />
the overall cooperativity of the system. The residual<br />
cooperativity (n ≈ 3) is associated with the conformational<br />
changes still occurring at the N-terminal<br />
ends, regardless of the mutation. His465, dispensable<br />
for the cooperativity that affects enzyme activity, is<br />
essential to include the conformational change of the<br />
N-termini into the cooperativity of the whole system.<br />
In the absence of His465, a 330-nm absorbing species<br />
appears, with fluorescence emission spectra more<br />
complex than model compounds and consisting of two<br />
maxima at 390 and 510 nm. Because His465 mutants<br />
are active at pH well above 5.7, we suggest that they<br />
might be employed for biotechnological applications.<br />
Publications<br />
Pennacchietti E, Lammens TM, Capitani G, Franssen<br />
MC, John RA, Bossa F, De Biase D. Mutation of<br />
His465 alters the pH-dependent spectroscopic properties<br />
of Escherichia coli glutamate decarboxylase<br />
and broadens the range of its activity toward more<br />
alkaline pH. J Biol Chem. 2009, 284: 31587-96. doi:<br />
10.1074/jbc.M109.049577.
Epstein-Barr virus interactions with cellular microRNAs<br />
Principal investigator: Alberto Faggioni<br />
Professor of General Pathology<br />
Dipartimento di Medicina Sperimentale<br />
Tel: (+39) 06 4461500; Fax: (+39) 06 4454820<br />
alberto.faggioni@uniroma1.it<br />
Participants:<br />
Antonio Angeloni, Pankaj Trivedi, professors; Roberta<br />
Santarelli, Mara Cirone, researchers; Eleni Anastasiadou,<br />
Antonella Farina, Roberta Gonnella, Marisa Granato,<br />
post-doc fellows; Francesco Boccellato, Paola Rosato, PhD<br />
students; Claudia Zompetta, technician.<br />
Report of activity<br />
Epstein-Barr virus (EBV) has the unique distinction of<br />
being the first human oncogenic virus. Its discovery<br />
caused a renewal of interest in the field of viral oncology<br />
which laid dormant for more than half a century after<br />
the initial discovery of avian Rous sarcoma virus (RSV)<br />
which Peyton Rous discovered in 1911. Initially found<br />
in Burkitt lymphoma (BL) biopsies by Epstein and Barr<br />
in 1964, EBV did not disappoint the expectation that it<br />
might have an important role in cancer development.<br />
Besides being tightly associated with several other<br />
human cancers like nasopharyngeal carcinoma (NPC),<br />
Hodgkin’s disease (HD), it is the only virus which<br />
transforms normal B lymphocytes into continuously<br />
growing lymphoblastoid cell lines (LCLs). The virus<br />
achieves this by virtue of its nine latent proteins out of<br />
which six are located in the nucleus of the infected cell<br />
and the other three are membrane proteins.<br />
The discovery of microRNA (miR) represents a novel<br />
paradigm in RNA based regulation of gene expression<br />
and their dysregulation has become a hallmark<br />
of many a tumor. The miRs participate in gene regulation<br />
of a broad spectrum of biological pathways<br />
through fine-tuning of protein expression levels.<br />
Their aberrant expression is a hallmark of a variety<br />
of diseases. The initial evidence of their involvement<br />
in cancer came in light by studies in chronic lymphocytic<br />
leukemia (CLL). It is estimated that more than<br />
50% human miRs are located to genomic regions<br />
like common breakpoints and fragile sites, involved<br />
in cancers. Some miRs are overexpressed in cancers<br />
in comparison to normal tissues, while others are<br />
7<br />
AREA 1<br />
downregulated. The former thus may be considered<br />
oncomiRs and the latter tumor supressor miRs.<br />
In virally associated cancers, the host-pathogen interaction<br />
could involve alteration in miR expression.<br />
Interestingly, EBV besides encoding its own miRs and<br />
can also influence cellular miRs. The initial evidence<br />
that this is so came from studies showing that EBV<br />
negative and EBV positive BLs differ in their miR<br />
expression signature (Kluiver et al., 2007). Moreover,<br />
among EBV positive BLs, it was shown that the miR<br />
profile of restricted latency expressing BLs (latency I)<br />
and broader latency expressors (latency III) is diverse.<br />
Given the importance on the one hand, of LMP1<br />
and EBNA2 in EBV driven transformation and on<br />
the other, the dysregulation of miRs in cancer, we<br />
asked how these viral proteins influence cellular miR<br />
expression. To this end, the following tasks were put<br />
forward in our original proposal: 1: miR profiling<br />
of tumor cell lines and tissues derived from different<br />
stages of B cell differentiation. 2: Expression of<br />
cellular miRs in LMP1 and EBNA2 transfected B<br />
lymphoma cells. 3: Differential regulation of miR-<br />
146a by EBNA2 and LMP1. 4: Regulation of TCL1<br />
by LMP1 through miR-29b. 5: miR-21 regulation<br />
by EBNA2. 6: Effect of miR-29b expression on EBV<br />
latency shift and 7: EBV regulated cellular miRs and<br />
their influence on Hedgehog signalling pathway.<br />
Microarray miR profile of EBV infected cells and<br />
compared it to the same cells transfected with either<br />
LMP1 or EBNA2 only was established (Anastasiadou<br />
et al., 2009, 2010; Imig et al., 2010). The progress on<br />
the first five tasks is the following: EBV encoded<br />
LMP1 is noted for its transforming potential. Yet, it<br />
also acts as a cytostatic and growth-relenting factor<br />
in Burkitt’s lymphoma (BL) cells. The underlying<br />
molecular mechanisms of the growth inhibitory<br />
property of LMP1 have remained largely unknown.<br />
In the 1st 2nd, 3rd and 4th task, we investigated<br />
how LMP1 negatively regulates a major oncogene,<br />
TCL1, in diffuse large B-cell lymphoma (DLBCL)<br />
and BL cells. MicroRNA (miR) profiling of LMP1
Alberto Faggioni<br />
transfectants showed that among others, miR-29b,<br />
is upregulated. LMP1 diminished TCL1 by inducing<br />
miR-29b through C-terminus activation region 1<br />
(CTAR1) and CTAR2. miR-29b locked nucleic acid<br />
(LNA) antisense oligonucleotide transfection into<br />
LMP1-expressing cells reduced miR-29b expression<br />
and consequently reconstituted TCL1, suggesting<br />
that LMP1 negatively regulates TCL1 through miR-<br />
29b upregulation (Anastasiadou et al., 2010). The<br />
miR-29b increase by LMP1 was due to an increase<br />
in the cluster pri-miR-29b1-a transcription, derived<br />
from human chromosome 7. Using pharmacological<br />
inhibitors, we found that p38 mitogen-activated protein<br />
kinase-activating function of LMP1 is important<br />
for this effect. The ability of LMP1 to negatively<br />
regulate TCL1 through miR-29b might underlie<br />
its B-cell lymphoma growth antagonistic property.<br />
As LMP1 is also important for B-cell transformation,<br />
we suggest that the functional dichotomy of<br />
this viral protein may depend on a combination of<br />
levels of its expression, lineage and differentiation of<br />
the target cells and regulation of miRs, which then<br />
directs the outcome of the cellular response.<br />
EBNA2 is indispensable for the capacity of the<br />
virus to transform B cells in vitro. In the 2nd, 3rd<br />
and 5 th task, we studied how it affects cellular miRs.<br />
Extensive miR profiling of the virus infected and<br />
EBNA2 transfected B lymphoma cells revealed<br />
that oncomiR miR-21 is positively regulated by<br />
this viral protein. Conversely, BL cell lines infected<br />
with EBNA2 lacking P3HR1 strain did not show<br />
any increase in miR-21. EBNA2 increased pAKT<br />
expression and this was directly correlated with<br />
increased miR-21. The primary miR-21 transcripts<br />
were unaltered by EBNA2 and this suggests that<br />
the increase in mature miR-21 is due to higher<br />
post-transcriptional processing. Interestingly,<br />
miR-146a, whose expression is known to be induced<br />
by LMP1 was severely downregulated by EBNA2<br />
in U2932 DLBCL cells. The primary miR-146a<br />
transcription and the promoter activity was hampered<br />
by EBNA2 whereas LMP1 had no such effect.<br />
Low miR-146a expression generally correlates with<br />
high IRAK1 and consequently higher type I interferon<br />
level. Indeed an elevated level of IRAK1 and<br />
type I interferon was found in EBNA2 transfectants.<br />
Further studies will be required to investigate<br />
precise molecular mechanisms how EBNA2 affects<br />
miR expression and in particular miR-21 and miR-<br />
146a. However the data suggest that EBNA2 might<br />
contribute to EBV induced B cell transformation<br />
by a positive regulation of oncomiR miR-21 and<br />
by interfering with the innate immunity through<br />
8<br />
regulating a key regulator of it, namely miR-146a<br />
(Rosato et al., submitted). Additionally, this miR<br />
has been attributed with oncosuppressive functions.<br />
Our data thus suggest that EBNA2 may promote<br />
B cell transformation by simultaneously increasing<br />
an oncomiR and reducing a miR with noted tumor<br />
suppressing functions.<br />
Publications<br />
Anastasiadou E, Boccellato F, Rosato P, Junker S,<br />
Winberg G, Frati L, Wade P, Faggioni A, Trivedi<br />
P. Epstein-Barr virus infection leads to phenotypic<br />
reversion of terminally differentiated B cells<br />
of malignant origin. Cancer Lett. 2009, 284: 165-174.<br />
doi: 10.1016/j.canlet.2009.04.025.<br />
Anastasiadou E, Boccellato F, Vincenti S, Rosato P,<br />
Bozzoni I, Frati L, Faggioni A, Presutti C, Trivedi<br />
P. Epstein-Barr virus encoded LMP1 downregulates<br />
TCL1 oncogene through miR-29b. Oncogene. 2010,<br />
29: 1316-28. doi: 10.1038/onc.2009.439.<br />
Imig J, Motsch N, Zhu J, Barth S, Okonievski<br />
M, Tinguely M, Kurrer M, Schraml P, Moch H,<br />
Faggioni A, Trivedi P, Meister G, Renner C, Grasser<br />
MG. microRNA profiling in EBV-associated B-cell<br />
lymphomas. Nucleic Acids Res. 2010. doi: 10.1093/<br />
nar/gkg1043.<br />
Heat map of global microarray profile of EBV infected<br />
and EBNA2 transfected U2932 DLBCL<br />
A: Two EBV infected clones of U2932 cells, U2932 EBVGFP cl-A<br />
(EBNA2 negative) and U2932 EBVGFP cl-B (EBNA2 positive) were<br />
compared for their miR profile. B: Overview of miR profiling of<br />
EBNA2 transfected U2932 cells. C: a comparison of principally<br />
altered miRs in U2932 EBVGFP cl-B and U2932 EBNA2 cl-2
Translational regulation: from the archaea to the eukarya<br />
Principal investigator: Paola Londei<br />
Professor of Applied Biology<br />
Dipartimento Biotecnologie Cellulari ed Ematologia<br />
Tel: (+39) 06 4940463; Fax: (+39) 06 4462891<br />
londei@bce.uniroma1.it<br />
Participants:<br />
Dario Benelli, research fellow; Antimo Naspi, Michela<br />
Pinzaglia, PhD students; Dorina Polinari, undergraduate<br />
student.<br />
Collaborations:<br />
University of Wien, Austria (Prof. Udo Blaesi); University J.W.<br />
Goethe Frankfurt, Germany (Prof. Joerg Soppa and Prof.<br />
Robert Tampè); CNRS Strasbourg, France (Dr. Bruno Klaholtz);<br />
Università Politecnica delle Marche, Ancona (Prof. Anna La<br />
Teana); Università di Camerino (Prof. Claudio Gualerzi).<br />
Report of activity<br />
Summary<br />
It is well known that the process of gene expression<br />
in Archaea has many features in common with that of<br />
Eukarya. This is especially true for the initiation step<br />
of translation, which in Archaea and in Eukarya has<br />
a similar high complexity. The project is specifically<br />
centred on investigating the function and mechanism<br />
of action in Archaea of two translation initiation factors,<br />
a/eIF2 and aIF6, shared by the Archaea and the<br />
Eukarya but absent in Bacteria. The eukaryal factors<br />
(eIF2 and eIF6) have important roles in regulating<br />
protein synthesis in both physiological and pathological<br />
situations, but several aspects of their function remain<br />
to be clarified. The function of a/eIF2 and aIF6 in<br />
Archaea is currently poorly understood; its elucidation<br />
will help to reconstruct the early evolution of these<br />
interesting proteins, thus gaining valuable insight into<br />
the onset of eukaryotic-type translational regulation.<br />
An important regulator of translation: a/e IF2<br />
In both Archaea and Eukarya, a/eIF2 is trimeric<br />
protein consisting of the α, β and γ subunits. It interacts<br />
with initiator tRNA (met-tRNAi) and delivers it<br />
to the ribosome. A mechanism of translational control<br />
essential and widespread in eukaryotic cells, but<br />
lacking in Bacteria, targets specifically eIF2, inac-<br />
9<br />
AREA 1<br />
tivating it by the phosphorylation of its α-subunit.<br />
Why the Archaea, as the Eukarya, should use a<br />
trimeric protein as the met-tRNAi binding factor is<br />
as yet unclear. To investigate this problem, we initially<br />
analyzed the modification pattern of the a/eIF2<br />
α-subunit in the archaeon Sulfolobus solfataricus. In<br />
vitro and in vivo studies with wild-type and mutated<br />
a/eIF2-α led to the conclusion that this protein is<br />
most probably not phosphorylated in archaea, suggesting<br />
a mechanism of translational control different<br />
from the eukaryal one.<br />
However, our studies have revealed an interesting and<br />
unexpected function of a/eIF2. Both trimeric a/eIF2<br />
and its isolated γ-subunit appear to interact with the 5’<br />
end of mRNAs, protecting them against exonuclease<br />
degradation. Strikingly, only mRNA 5’ ends carrying<br />
three phosphate groups are recognized by the factor,<br />
while mono-phosphorylated and dephosphorylated<br />
mRNAs are not. In living cells, the physiological<br />
ligand of mRNA 5’-3P end is probably the isolated a/<br />
eIF2 γ-subunit, which is 2-3 fold more abundant than<br />
the α and β ones in normal conditions and is further<br />
over-expressed under stress, e.g. heat- or cold-shock.<br />
This situation is reminiscent of eukaryotic capping,<br />
and may well be an evolutionary precursor of this<br />
critical feature of eukaryotic translation. Work is in<br />
progress to ascertain whether the IF2/mRNA interaction<br />
has also a role in promoting translation.<br />
In the last stage of the project we have extended<br />
our studies to the analysis of eIF2 modifications in<br />
certain human tumours. Specifically, we have found<br />
that in metastatic melanomas, but not primary ones,<br />
eIF2-α is hyperphosphorylated. Unexpectedly, we<br />
have also found that phosphor-IF2-α is relocated to<br />
the cell nucleus during the transition from primary to<br />
metastatic tumours, hinting to some unprecedented<br />
function of the factor in modulating gene expression<br />
of the metastatic cells. Work is in progress to identify<br />
the nuclear interactors of eIF2-α and to obtain<br />
insight into the nuclear function of the protein.
Paola Londei<br />
A still mysterious protein: IF6<br />
The factor known as aIF6 (archaea) and eIF6<br />
(eukarya) is a monomeric protein of about 25 kDa.<br />
Eukaryotic IF6 functions as a ribosome anti-association<br />
factors and probably has also a role in the<br />
biogenesis of 60S ribosomal subunits. To enter the<br />
translation cycle, 60S subunits must release eIF6; in<br />
mammals, this is triggered by the phosphorylation of<br />
the factor carried out by certain regulatory kinases.<br />
Recently, it has also been <strong>report</strong>ed that eIF6 depletion<br />
inhibits miRNA-induced gene silencing in both<br />
human cells and in nematodes. However, the mechanism<br />
of action of eIF6 remains elusive.<br />
Archaeal IF6 is highly homologous with eIF6<br />
in both sequence and structure. We have found<br />
that S. solfataricus aIF6, similar to its eukaryal<br />
counterpart, inhibits association of the ribosomal<br />
subunits. For the first time, we have mapped the<br />
ribosomal binding site of the factor, locating it in<br />
the middle of the 30S-interacting face of the large<br />
ribosomal subunit. Our model of the aIF6/50S<br />
complex reveals that the factor masks certain<br />
RNA and protein domains critical for bridging<br />
the two subunits. Work is in progress to unravel<br />
the mechanism whereby aIF6 dissociates from<br />
the 50S subunit. Unlike the eukaryal homologue,<br />
aIF6 is not phosphorylated, suggesting that the<br />
mechanism of release from ribosomes is not conserved.<br />
Our recent data indicate that the release<br />
of aIF6 from the ribosomes may entail a novel<br />
type of protein modification, possibly in associa-<br />
10<br />
tion with a ribosome-dependent GTPase activity,<br />
yet to be identified.<br />
Lastly, we have begun to study the involvement of<br />
eukaryal IF6 in cellular transformation, suggested by<br />
the fact that eIF6 is over-expressed in many spontaneous<br />
tumours. Our recent results have revealed that<br />
Notch-1, a receptor protein implicated in cell proliferation<br />
and survival, directly controls the expression<br />
of eIF6. We found that the eIF6 promoter contains<br />
two binding sites for RBP-jk, a downstream target<br />
of Notch signalling. Indeed, the transcription of a<br />
<strong>report</strong>er construct containing the eIF6 promoter was<br />
down-regulated when human leukaemia cell lines,<br />
having constitutively activated Notch signalling,<br />
were treated with Notch-1 inhibitors. Conversely,<br />
the transcription of the eIF6 <strong>report</strong>er construct was<br />
up-regulated upon over-expression of Notch-1 in<br />
NIH3T3 cells. Work is in progress to determine the<br />
physiological significance of these findings.<br />
Publications<br />
Benelli D, Londei P. Begin at the beginning: evolution<br />
of translational initiation. Res Microbiol. 2009,<br />
160: 493-501. doi:10.1016/j.resmic.2009.06.003.<br />
Benelli D, Marzi S, Mancone C, Alonzi T, la Teana<br />
A, Londei P. Function and ribosomal localization<br />
of aIF6, a translational regulator shared by archaea<br />
and eukarya. Nucleic Acids Res. 2009, 37: 256-67. doi:<br />
10.1093/nar/gkn959.
AREA 2<br />
Pathogenetic<br />
mechanisms<br />
of microbially<br />
associated<br />
diseases
Molecular characterization of viruses causing bronchiolitis and<br />
study of viral and host factors affecting Type I IFN antiviral<br />
response induced by respiratory viruses<br />
Principal investigator: Guido Antonelli<br />
Professor of Virology<br />
Dipartimento di Medicina Molecolare<br />
Tel: (+39) 06 4474122; Fax: (+39) 06 44741236<br />
guido.antonelli@uniroma1.it<br />
Participants:<br />
Ombretta Turriziani, Professor; Laura Antonelli, Fabio<br />
Midulla, Paola Papoff, Alessandra Pierangeli, Carolina<br />
Scagnolari, researchers; Carla Selvaggi, research fellow;<br />
Katia Monteleone, PhD student.<br />
Report of activity<br />
Bronchiolitis severity and subsequent episodes of<br />
wheezing in children are likely to be due to either<br />
viral factors (e.g. genotype and strain, viral load) or<br />
to many other, yet under recognized, host factors<br />
determining effectiveness of innate and acquired<br />
immune response. Most previous studies had been<br />
focused on respiratory syncytial virus (RSV), either<br />
in vitro or in patient groups; correlation of RSV<br />
genotypes or strains, and viral load with disease<br />
severity and recurrent wheezing is still controversial.<br />
Recently, increasing evidence suggested that<br />
rhinoviruses (RV) were important agents of bronchiolitis<br />
and represented the major risk factor in<br />
recurrent wheezing episodes in children. However,<br />
it is not yet clear whether viral bronchiolitis directly<br />
contributes to recurrent wheezing and asthma causation<br />
or infants are at risk for subsequent wheezing as<br />
a result of genetic conditions. Hence, we proposed<br />
to study viral species and strains involved in bronchiolitis,<br />
and the role of type I IFNs in antiviral<br />
response, either in patient samples or in cell lines,<br />
properly infected with respiratory viruses.<br />
In the first year of the project, we characterized<br />
viral infections, detecting 14 different viruses with<br />
a comprehensive panel of PCR-based reactions, in<br />
respiratory samples (pharyngeal swabs and nasal<br />
washings) from patients seeking medical care in<br />
Emergency Departments (ED). Patient signs and<br />
symptoms, laboratory results and clinical outcome<br />
were compared with virus results: no respiratory<br />
signs/symptoms could differentiate virus infected<br />
individuals (either infants, children or adult patients)<br />
13<br />
AREA 2<br />
from other cause of acute respiratory syndrome and<br />
clinical presentations were not useful to discriminate<br />
viral pathogens (Pierangeli et al., CMI 2010).<br />
Bronchiolitis (defined as a history of upper respiratory<br />
tract infection followed by acute onset of respiratory<br />
distress with cough, tachypnea, retraction<br />
and diffuse crackles on auscultation in infants within<br />
one year) was the more frequent clinical diagnosis<br />
overall in children attending ED; RSV single infection<br />
represented about 50% of bronchiolitis cases,<br />
in nearly 30% of bronchiolitis cases no virus was<br />
found, 10-15% of bronchiolitis were due to RV single<br />
infections, whereas other viruses or coinfections<br />
were detected in the remaining cases. Clinical data<br />
of enrolled patients confirmed that RSV is the pathogen<br />
with the highest severity of infection in children,<br />
with highest PICU admissions, even though total<br />
days of hospitalization were not significantly different<br />
from the other virus groups (“Virological and<br />
clinical characterizations of respiratory infections in<br />
children attending an emergency department” manuscript<br />
in preparation).<br />
To perform viral strain characterizations in respiratory<br />
samples from children with bronchiolitis, either<br />
using primers from published papers or designed for<br />
the project, we sequenced genomic region longer and<br />
less conserved than those used for virus detection in<br />
PCR tests: RSV G glycoprotein and RV 5’ UTR and<br />
VP4/VP2 coding region. Preliminary results from<br />
RV sequencing identified 16 RV cases encountered in<br />
the epidemic season 2009/10 as being RV-A N = 9,<br />
RV-B N = 1, RV-C N = 6, <strong>report</strong>ing for the first time,<br />
RV-C (the novel RV species identified in 2007) circulation<br />
in central Italy (manuscript in preparation).<br />
Moreover, viral genome determination by real-time<br />
PCR of RSV, influenza A and RV were performed<br />
in respiratory samples taken at admittance and at<br />
follow-up visits; preliminary results showed no<br />
clear association of viral loads with illness severity<br />
scores, but we are currently performing a thoughtful<br />
statistical analysis of the data, to evaluate different
Guido Antonelli<br />
contribution of viral genotypes and strains (e.g. RSV<br />
A or B, RV A, B and C).<br />
In the framework of the project, we studied cases<br />
of infants younger than one year hospitalised during<br />
several epidemic seasons for their first episode<br />
of acute bronchiolitis, to determine the incidence of<br />
severe bronchiolitis and factors predicting disease<br />
severity in previously healthy term infants. Groups<br />
of infants were assessed on a severity score: (0)<br />
required only conservative treatment; (1) needed<br />
intravenous fluids or oxygen treatment or both for<br />
less than 12 hours; (2) needed oxygen for more than<br />
12 hours without ventilatory support and intravenous<br />
fluids; (3) needed either mechanical ventilation<br />
or noninvasive respiratory support. Data analyzed<br />
included: specific viral infection, patient demographics<br />
and clinical data, chest radiographs. Younger<br />
age on admission (
A structural biology study of schistosomiasis<br />
Principal investigator: Andrea Bellelli<br />
Professor of Biochemistry<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 49910955; Fax: (+39) 06 4440062<br />
andrea.bellelli@uniroma1.it<br />
Participants:<br />
Maurizio Brunori, Adriana Erica Miele, professors;<br />
Francesco Angelucci, Ilyas Koutris, post-doc fellows;<br />
Daniela Dimastrogiovanni, Fulvio Saccoccia, PhD students;<br />
Giovanna Boumis, technician.<br />
Collaborations:<br />
Rush University, Chicago, IL, USA (Prof. David L. Williams,<br />
Dr. Ahmed A. Sayed); Sapienza Università di Roma (Prof.<br />
Alfredo di Nola, Dr. Massimiliano Anselmi); University of<br />
Lund, Sweden (Dr. Linn Petterson, Dr. Frida Pauly); <strong>Istituto</strong><br />
di Biologia Cellulare, CNR, Monterotondo, Roma (Prof. Donato<br />
Cioli, Prof. Livia Pica Mattoccia, Dr. Cristiana Valle).<br />
Report of activity<br />
Schistosomiasis is a parasitic disease affecting over<br />
200 million people in tropical and subtropical countries.<br />
The disease is currently treated with only one<br />
drug, Praziquantel; since the appearance of strains of<br />
Schistosoma mansoni displaying reduced sensitivity<br />
to Praziquantel has been <strong>report</strong>ed, the study of alternative<br />
drugs is important (Cioli et al., 2008).<br />
A possible drug target is the seleno-protein thioredoxin-glutathione<br />
reductase (TGR), a key flavoenzyme<br />
in the pathway of the parasite for detoxification<br />
of reactive oxygen species. The enzyme is a unique<br />
fusion of a glutaredoxin domain with a thioredoxin<br />
reductase domain, which contains a selenocysteine<br />
(Sec) as the penultimate amino acid. TGR bridges<br />
two detoxification pathways crucial for the parasite<br />
survival in the host’s organism: those based on<br />
glutathione and thioredoxin respectively. The parasite’s<br />
reduction system is very different from that<br />
of the host, which is based on two separated (even<br />
though evolutionarily related) enzymes, Thioredoxin<br />
Reductase and Glutathione Reductase respectively.<br />
SmTGR is a validated drug target since (i) selective<br />
inhibition of its biosynthesis by RNA interference kills<br />
the parasite in vitro; and (ii) inhibition of its function<br />
15<br />
AREA 2<br />
by Auranofin and other drugs kills the parasite both in<br />
vitro and in vivo (Kuntz et al., 2007).<br />
In the project “A structural biology study of schistosomiasis”<br />
we solved the crystal structure (at 2.2 Å resolution)<br />
of TGR from Schistosoma mansoni (SmTGR),<br />
deleted in the last two residues; our structure has been<br />
the first for a Thioredoxin Glutathione Reductase<br />
(see Angelucci et al., 2008). The structure reveals the<br />
peculiar architecture of this chimeric enzyme: the small<br />
Glutaredoxin (Grx) domain at the N-terminus is joined<br />
to the large thioredoxin reductase (TrxR) one via an<br />
extended complementary surface, involving residues<br />
not conserved in the Grx superfamily; the TR domain<br />
interacts with an identical partner via its C-terminal<br />
domain, forming a dimer with a twisted “W” shape.<br />
Although lacking the penultimate Selenocysteine residue<br />
(Sec), the enzyme is still able to reduce oxidized<br />
glutathione. These data update the interpretation of the<br />
interdomain communication in TGR enzymes.<br />
Auranofin (AF), a gold-containing compound already<br />
in clinical use as an anti-arthritic drug, has been<br />
shown to inhibit TGR and to substantially reduce<br />
worm burden in mice. Using X-ray crystallography<br />
we solved (at 2.5 Å resolution) the structure of wild<br />
type TGR incubated with AF (see Angelucci et al.,<br />
2009). The electron density maps show that the<br />
actual inhibitor is gold, released from AF. Gold is<br />
bound at three different sites not directly involving<br />
the C-terminal Sec residue; however, because the C<br />
terminus in the electron density maps is disordered,<br />
we cannot exclude the possibility that gold may also<br />
bind to Sec. To investigate the possible role of Sec in<br />
the inactivation kinetics, we tested the effect of AF<br />
on a model enzyme of the same superfamily, i.e. the<br />
naturally Sec-lacking glutathione reductase, and on<br />
truncated TGR. We demonstrated that the role of<br />
selenium in the onset of inhibition by AF is catalytic<br />
and can be mimicked by the external source of selenium<br />
benzeneselenol. Therefore, we proposed that<br />
Sec mediates the transfer of gold from its ligands in<br />
AF to the redox-active Cys couples of TGR.
Andrea Bellelli<br />
Moreover, with the aim of achieving a better understanding<br />
of the electron flow inside the enzyme<br />
macromolecule, we dissected the catalytic cycle of<br />
Schistosoma mansoni TGR by structural and functional<br />
analysis of the U597C mutant. Crystallographic<br />
data were obtained for (i) the oxidized form (at 1.9 Å<br />
resolution); (ii) the NADPH- and GSH-bound forms<br />
(2.3 and 1.9 Å, respectively); and (iii) a different<br />
crystal form of the (partially) reduced enzyme (3.1<br />
Å), showing the physiological dimer and the entire<br />
C terminus of one subunit. Whenever possible, we<br />
determined the rate constants for the interconversion<br />
between the different oxidation states of TGR<br />
by kinetic methods. By combining the crystallographic<br />
analysis with computer modeling, we were<br />
able to throw further light on the mechanism of<br />
action of S. mansoni TGR. In particular, we were<br />
able to model the putative functionally relevant<br />
conformational change of the C terminus after the<br />
transfer of reducing equivalents from NADPH to the<br />
redox sites of the enzyme (see Angelucci et al., 2010).<br />
Another enzyme belonging to the same metabolic<br />
pathway that has been indicated (but not yet validated)<br />
as a possible drug target is Glutathione peroxidase<br />
(Gpx). Glutathione peroxidases are a family<br />
of sulfur or selenium-dependent isozymes sharing<br />
the ability to reduce peroxides using the reducing<br />
equivalents provided by glutathione or possibly<br />
small proteins such as thioredoxin. The genome of<br />
Schistosoma mansoni encodes for a Gpx that, as<br />
for other frontline antioxidant enzymatic systems,<br />
is localized in the worm’s tegument, the outermost<br />
defense layer. We solved the first crystal structure at<br />
1.0 and 1.7 Å resolution of two recombinant SmGpx<br />
16<br />
variants, carrying the active site mutations Sec43Cys<br />
and Sec43Ser, respectively (see Dimastrogiovanni et<br />
al., 2010). The structures confirm that this enzyme<br />
belongs to the monomeric class 4 (phospholipid<br />
hydroperoxide) Gpx. In the case of the Sec to Cys<br />
mutant, the catalytic Cys residue is oxidized to sulfonic<br />
acid. By combining static crystallography with<br />
molecular dynamics simulations, we obtained insight<br />
into the substrate binding sites and the conformational<br />
changes relevant to catalysis, proposing a role<br />
for the unusual reactivity of the catalytic residue.<br />
Publications<br />
Angelucci F, Sayed AA, Williams DL, Boumis G,<br />
Brunori M, Dimastrogiovanni D, Miele AE, Pauly<br />
F, Bellelli A.Inhibition of Schistosoma mansoni thioredoxin-glutathione<br />
reductase by auranofin: structural<br />
and kinetic aspects. J Biol Chem. 2009, 284: 28977-85.<br />
doi: 10.1074/jbc.M109.020701.<br />
Angelucci F, Dimastrogiovanni D, Boumis G,<br />
Brunori M, Miele AE, Saccoccia F, Bellelli A.<br />
Mapping the catalytic cycle of Schistosoma mansoni<br />
thioredoxin glutathione reductase by X-ray crystallography.<br />
J Biol Chem. 2010, 285: 32557-67. doi:<br />
10.1074/jbc.M110.141960.<br />
Dimastrogiovanni D, Anselmi M, Miele AE, Boumis<br />
G, Petersson L, Angelucci F, Di Nola A, Brunori M,<br />
Bellelli A. Combining crystallography and molecular<br />
dynamics: the case of Schistosoma mansoni phospholipid<br />
glutathione peroxidase. Proteins. 2010, 78: 259-<br />
70. doi: 10.1002/prot.22536.
Lipopolysaccharide and peptidoglycan adaptation to host as an<br />
immune evasion strategy of gram-negative pathogens<br />
Principal investigator: Maria Lina Bernardini<br />
Researcher in Cellular Microbiology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49917850; Fax: (+39) 06 49917994<br />
marialina.bernardini@uniroma1.it<br />
Participants:<br />
Luigi Lembo-Fazio, post-doc fellow; Laura Curcurù, Gaëlle<br />
Noël, PhD students.<br />
Collaborations:<br />
Dipartimento di Immunologia, Trapianti e Malattie infettive,<br />
Fondazione S. Raffaele del Monte Tabor (Prof. Alessandra<br />
Bragonzi); Dipartimento di Chimica Organica e Biochimica,<br />
Università di Napoli “Federico II” (Prof. Antonio Molinaro);<br />
Dipartimento di Oncologia Sperimentale, <strong>Istituto</strong> Europeo di<br />
Oncologia, Milano (Prof. Maria Rescigno).<br />
Report of activity<br />
This study focuses on the molecular mechanisms<br />
underlining the adaptation to host of two pathogens,<br />
Pseudomonas aeruginosa (PA) and Shigella flexneri. P.<br />
aeruginosa (PA) is an opportunistic pathogen, which<br />
causes severe infections in immune-compromised<br />
individuals, as in the lung of patients suffering from<br />
cystic fibrosis (CF) (Lyczak et al., 2000). During<br />
the colonization of the host airways, PA undergoes<br />
genetic changes, which allow it to evade detection<br />
and clearance by the immune system and to establish<br />
a chronic infection (Gibson et al., 2003). S. flexneri is a<br />
Gram-negative enteroinvasive bacterium that causes<br />
shigellosis, a severe inflammatory dysentery, characterized<br />
by strong abdominal cramps, fever and stools<br />
containing blood and mucus (Schroeder & Hilbi,<br />
2008). During dissemination through the intestinal<br />
epithelium, bacterial components are released, which<br />
activate immune responses of the host, inducing<br />
proinflammatory cytokines and chemokines production<br />
(Phalipon & Sansonetti, 2007). The mammalian<br />
innate immune system detects the presence of microbial<br />
infection through germ-line encoded pattern<br />
recognition receptors (PRRs) (Kawai & Akira, 2009).<br />
These proteins are implicated in the recognition of a<br />
limited, but highly conserved, set of molecular structures<br />
that are produced by micro-organisms and are<br />
17<br />
AREA 2<br />
absent from the host cell, called pathogen associated<br />
molecular patterns (PAMPs). The activation of a<br />
PRR by its cognate PAMP induces pro-inflammatory<br />
signaling pathways such as that governed by<br />
NF-κB and MAPKs, leading to the clearance of the<br />
intruder. However, pathogens have evolved immune<br />
evasion strategies, including the alteration of their<br />
structures, in order to escape or to modulate the<br />
immune system recognition.<br />
The lypopolisaccharide (LPS) and peptidoglycan<br />
(PGN) of three sequential, clonal, PA clinical isolates<br />
from a CF patient were analyzed (Bragonzi et<br />
al., 2009). The AA2 strain was isolated at the initial<br />
stage of lung infection, while the strains AA43<br />
and AA44 during the chronic infection. Chemical<br />
analysis of AA2 and AA43 P. aeruginosa strain LPS<br />
demonstrated that these structures were composed<br />
essentially by tetra-, penta- and hexa-acylated species,<br />
but present at different percent concentrations.<br />
In contrast, the AA44 LPS of the other strain, AA44,<br />
isolated at the chronic stage of the disease, was constituted<br />
by hexa-acylated and hepta-acylated moieties.<br />
These findings are in accordance with previous<br />
studies, describing that P. aeruginosa synthesizes<br />
more highly acylated (hexa- and hepta-acylated) lipid<br />
A during the passage from acute to chronic infection<br />
(Ernst et al., 2007). The presence of the hepta-acylated<br />
lipid A in PA AA44 was supported by genetic<br />
data, indicating a mutation in pagL gene, encoding<br />
for a lipid A deacylase (Rutten et al., 2006). This<br />
observation is in accordance with results obtained<br />
with other pathogens, such as Salmonella spp., showing<br />
that microorganisms modify their LPS structure<br />
through deacylase activity in order to modulate lipid<br />
A recognition by innate immune receptor of the host<br />
(Kawasaki et al., 2004; Kawasaki & Manabe, 2010).<br />
On this basis, the biological activity of LPS was<br />
evaluated in different cell lines in vitro and in a mouse<br />
model in vivo. The LPS of the “chronic” strains AA43<br />
and AA44 displayed a reduced immunostimulatory<br />
activity compared to the LPS of AA2 in different
Maria Lina Bernardini<br />
cell models, including HEK293 cells expressing the<br />
LPS receptor TLR4, together with MD2 and CD14,<br />
the macrophage-like THP-1 cells and a CF airway<br />
epithelial IB3-1 cell line and in its isogenic non-CF<br />
C38 cells. In accordance with in vitro results, in vivo<br />
in a murine model of intranasal injection of LPS,<br />
neutrophil recruitment and cytokines (MIP-2, KC<br />
and IL-1β) release was major after stimulation with<br />
PA AA2 LPS than AA43 and AA44 strain LPS.<br />
As for LPS, the PGN structure of the three CF strains<br />
was elucidated, through extraction and muramidase<br />
digestion. HPLC analysis of PA PGN showed the<br />
same muramyl peptide species in the three strains<br />
albeit in different relative amount. HEK293 cells<br />
expressing Nod1 and CF IB3 cell line were stimulated<br />
with the PGN of the three clinical strains, and<br />
the inflammatory responses, i.e. NF-κB activity and<br />
IL-8 production, were evaluated. The results are in<br />
accordance with those obtained with LPS: in fact,<br />
PGN of the “chronic” strains displayed a reduced<br />
immunopotential, compared to the PGN of the strain<br />
from initial colonization. A recent study <strong>report</strong>ed<br />
that in the airways of cystic fibrosis patients the high<br />
levels of the inflammatory cytokine IL-17 prime CF<br />
epithelial cells to increase PRR receptors, and in<br />
particular Nod1, stressing the involvement of Nod1<br />
in PA infection (Roussel & Rousseau, 2009). These<br />
issues reinforce the interest to study the relationship<br />
between PGN of PA and Nod1 activity.<br />
Finally, the results about PA adaptation during the<br />
passage from acute to chronic infection, suggest that<br />
the pathogen promotes its survival into the host<br />
airways through PAMP modification. This strategy<br />
allows the bacterium to evade innate immune system<br />
recognition and to impair the homeostasis of the<br />
infected tissues.<br />
To identify parallel mechanisms in the pathogenesis<br />
of the highly inflammatory S. flexneri the<br />
relevant bacterial structures were analyzed during<br />
infection. It was demonstrated that approximately<br />
one-quarter of S. flexneri genes altered their<br />
expression profile, during infection of epithelial<br />
and macrophagic cell lines (Lucchini et al., 2005).<br />
On this basis, the LPS structure of intracellular<br />
bacteria was evaluated and compared to that of the<br />
bacteria grown in vitro. Chemical analysis showed<br />
the same O-chain structure of the LPS of the intracellular<br />
and the in vitro grown shigellae (Clément<br />
et al., 2003). The only difference resides on the<br />
core region of LPS, where a phosphate replaced<br />
a phosphoethanolamine. Surprisingly, the lipid<br />
A region of intracellular S. flexneri was underacylated,<br />
compared to the hexa-acylated lipid A of<br />
18<br />
the same in vitro grown strain. According to this<br />
structure, the LPS of the intracellular bacterium<br />
showed a lower capability to induce innate immune<br />
system activation in HEK293/TLR4-MD2-CD14<br />
cells, with respect to the in vitro maintained strain.<br />
This immune evasion strategy of the intracellular<br />
S. flexneri strain is in agreement with previous<br />
findings <strong>report</strong>ing that TLR4 is internalized from<br />
plasma membrane into early endosomes, after<br />
ligand binding (Kagan et al., 2008).<br />
In conclusion, these data showed parallel mechanisms<br />
of adaptation of two bacteria with different behavior<br />
into the host. The highly inflammatory pathogen S.<br />
flexneri and P. aeruginosa from lung chronic infection<br />
adapt their LPS in order to evade immune recognition<br />
by TLR4 receptor. However, while P. aeruginosa modifies<br />
its PAMP in a permanent manner, as indicated by<br />
pagL mutation, it is presumable that Shigella alters its<br />
LPS transiently, through expression profile adaptation.<br />
References<br />
- Bragonzi et al., 2009, Am J Respir Crit Care Med<br />
180:138-45.<br />
- Clément et al., 2003, J Biol Chem 278:47928-36.<br />
- Ernst et al., 2007, J Infect Dis 196:1088-92.<br />
- Gibson et al., 2003, Am J Respir Crit Care Med<br />
168:918-51.<br />
- Kagan et al. 2008, Nat Immunol 9:361-368.<br />
- Kawai T, Akira S. 2009, Int Immunol 21:317-337.<br />
- Kawasaki et al., 2004, J Biol Chem 279:20044-<br />
20048.<br />
- Kawasaki et al., 2010, J Bacteriol 192:5837-5840.<br />
- Lucchini et al., 2005, Infect Immun 73:88-102.<br />
- Lyczak et al., 2000, Microbes Infect 2:1051-1060.<br />
- Phalipon A, Sansonetti JP. 2007, Immunol Cell Biol<br />
85:119-129.<br />
- Roussel L, Rousseau S. 2010, Biochem Biophys Res<br />
Commun 391:505-509.<br />
- Rutten et al., 2006, Proc Natl Acad Sci USA<br />
103:7071-7076.<br />
- Schroeder GN, Hilbi H. 2008, Clin Microbiol Rev<br />
21:134-156.<br />
Publications<br />
Cigana C, Curcurù L, Leone MR, Ieranò T, Lorè<br />
NI, Bianconi I, Silipo A, Cozzolino F, Lanzetta R,<br />
Molinaro A, Bernardini ML, Bragonzi A. Pseudomonas<br />
aeruginosa exploits lipid A and muropeptides modification<br />
as a strategy to lower innate immunity during<br />
cystic fibrosis lung infection. PLoS One. 2009,<br />
4:e8439. doi: 10.1371/journal.pone.0008439.
Involvement of sRNA molecules in the complex regulatory<br />
circuits of virulence gene expression in Shigella flexneri and in<br />
enteroinvasive E. coli<br />
Principal investigator: Bianca Colonna<br />
Professor of General Microbiology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49917580, 06 49917582; Fax: (+39) 06 49917594<br />
bianca.colonna@uniroma.it<br />
Participants:<br />
Gianni Prosseda, researcher; Marialuisa Barbagallo, Rosaria<br />
Campilongo, Maria Letizia Di Martino, PhD students.<br />
Collaborations:<br />
Dipartimento di Biologia, Università di Roma-Tre (Prof.<br />
Mariassunta Casalino); Dipartimento di Biologia Molecolare,<br />
Cellulare e Animale, Università di Camerino (Dr. Maurizio<br />
Falconi); <strong>Istituto</strong> di Biologia e Patologia Molecolari, CNR, Roma<br />
(Dr. Gioacchino Micheli).<br />
Report of activity<br />
Shigella is a highly adapted human pathogen that<br />
causes bacillary dysentery, a severe enteric syndrome,<br />
mainly in the developing world. The pathogenicity<br />
mechanism of Shigella strains and enteroinvasive<br />
E. coli (EIEC) is based on their capacity<br />
to reach and invade colonic epithelial cells, leading<br />
to intracellular bacterial multiplication and spread<br />
to adjacent cells with consequent cell death and<br />
destruction of the colonic mucosa. The cellular<br />
pathogenesis and clinical presentation of shigellosis<br />
result from the complex action of a large number<br />
of bacterial virulence factors mainly located on a<br />
large virulence plasmid (pINV). The expression of<br />
pINV genes is controlled by multiple environmental<br />
stimuli through a regulatory cascade involving proteins<br />
encoded by both, pINV and the chromosome.<br />
Transcriptional activation of the invasivity operons<br />
in response to temperature, pH, and osmolarity<br />
conditions found in the human intestine requires<br />
two positively acting plasmid-encoded regulators,<br />
VirF and VirB. Among environmental stimuli, temperature<br />
is a crucial factor since transcription of the<br />
virulence genes is strongly repressed at 30°C by the<br />
chromosomal-encoded protein H-NS. Within the<br />
virF promoter, the nucleoid protein H-NS interacts<br />
- only at low temperature - with two sites spaced by<br />
an intrinsically curved DNA region. With increasing<br />
19<br />
AREA 2<br />
temperature a consequent decrease in DNA curvature<br />
and in H-NS binding occur, and at a temperature<br />
higher than 30°C the virF promoter becomes<br />
insensitive to H-NS repression, strongly suggesting<br />
that the environmentally induced structural collapse<br />
of the promoter’s intrinsic bend is a key event in the<br />
regulation of virulence gene expression.<br />
Recent observations from our group confirm that<br />
among the molecular strategies bacteria have set up<br />
to quickly match their transcriptional program to<br />
new environments, changes in sequence mediated<br />
DNA curvature play a crucial role. By analyzing the<br />
temperature dependent expression of a <strong>report</strong>er gene<br />
in a construct carrying a progressively decreasing<br />
bent sequence upstream from the promoter, we have<br />
shown that with decreasing temperature a narrow<br />
curvature range accounts for a significant enhancement<br />
of promoter activity. This strengthen the view<br />
that DNA-curvature-mediated regulation of gene<br />
expression is likely a strategy offering fine-tuning<br />
control possibilities and that, considering the widespread<br />
presence of curved sequences upstream from<br />
bacterial promoters, it may represent one of the most<br />
primitive forms of gene regulation.<br />
Recently small non-coding RNAs (sRNA or ncRNA)<br />
have emerged as major elements in bacterial cell regulation.<br />
Moreover, in several human bacterial pathogens<br />
sRNAs appear to be crucial for integrating<br />
environmental stimuli into outputs that are relevant<br />
for pathogenicity. Therefore, we decided to investigate<br />
the potential involvement of sRNA transcripts<br />
in the modulation of virulence gene expression in<br />
Shigella. By analysing the large intergenic region<br />
separating the virA and icsA genes, located on the<br />
virulence plasmid of S. flexneri, we identified a novel<br />
gene coding for an antisense RNA (RnaG). Mapping<br />
the 5’- and 3’-ends of RnaG reveals that this sRNA<br />
molecule is quite long (450 bases) and is transcribed<br />
from a promoter located within the sequence corresponding<br />
to the 5’-untranslated region of the icsA<br />
mRNA. The icsA gene encodes a structural protein
Bianca Colonna<br />
involved in the reorganization of the cytoskeleton<br />
upon penetration of bacteria into the host cells and<br />
its activity is crucial for the intra- and inter-spreading<br />
of the bacterial pathogen. In order to understand<br />
if and how RnaG is able to interfere with the expression<br />
of icsA, the promoters of icsA and RnaG were<br />
alternatively inactivated by introducing mutations in<br />
the corresponding -10 elements. As opposed to other<br />
sRNAs which affect the ability of the target mRNAs<br />
to be translated, we found that RnaG is capable to<br />
downregulate icsA transcription by means of two,<br />
not mutually esclusive mechanisms.<br />
First we showed that icsA and RnaG are subject<br />
to Transcriptional Interference (TI) regulation: the<br />
activity of the icsA promoter (P icsA ) is significantly<br />
reduced when a transcription process starts from the<br />
convergent RnaG promoter (P RnaG ). In this case TI is<br />
an asymmetric phenomenon as P RnaG acts as aggressive<br />
promoter reducing the expression of the weak<br />
one, P icsA . The extent of TI is higher in vitro than in<br />
vivo, suggesting that other factors besides promoter<br />
strength could contribute to the regulation of this<br />
genetic system. At the moment our results do not<br />
allow to figure out if the TI mechanism depends on<br />
the collision between the two RNA polymerases moving<br />
in opposite directions or on the removal of the<br />
promoter-bound complexes by the passage of RNA<br />
polymerases from the opposite promoter. We hope to<br />
obtain more detailed information about the TI mechanism<br />
by altering the arrangement of the P icsA and P RnaG<br />
sequences, as well as by introducing a transcriptional<br />
terminator between the two face-to-face promoters.<br />
In addition to TI, RnaG can also cause a direct<br />
repression of icsA transcription by acting primarily<br />
as antisense RNA targeting the icsA messenger.<br />
By combining computational predictions and data<br />
from chemical and enzymatic RNA probing we<br />
demonstrated that binding of RnaG to the target<br />
icsA mRNA alters the secondary structure of the<br />
untranslated region of the sense RNA. This alternate<br />
structure, which originates in the course of the transcriptional<br />
process but only in the presence of RnaG,<br />
gives rise to an intrinsic terminator in the nascent<br />
icsA messenger that likely leads to premature termination.<br />
The proposed model is further strengthened<br />
by the fact that mutations which destabilize the<br />
conserved stem structure of this intrinsic terminator,<br />
significantly affect the ability of RnaG to inhibit<br />
icsA transcription. This RnaG-mediated attenuation<br />
20<br />
process constitutes a very powerful strategy adopted<br />
by a sRNA molecule to modulate the expression of<br />
a crucial virulence gene at the transcriptional level.<br />
The expression of icsA is subject to a complex regulatory<br />
network that, besides being affected by RnaG,<br />
also depends on the coordinated action of two major<br />
players of the Shigella regulon, VirF and H-NS. We<br />
are currently investigating how the action of these<br />
regulatory factors is coordinated on the icsA promoter<br />
in order to obtain the full expression of the<br />
icsA only when the bacterium needs to move inside<br />
the host cell and towards the adjacent cells.<br />
Since current evidence indicates that regulation<br />
mediated by sRNA molecules appears to be more<br />
common than previously thought, we plan to perform<br />
genome-wide analyses to deeper understand<br />
the transcriptome of Shigella. This will allow us to<br />
identify new sRNAs molecules, with emphasis on<br />
those transcribed during peculiar environmental<br />
conditions, like high osmolarity or low oxygen availability,<br />
that more closely mimic the intracellular<br />
habitat of Shigella.<br />
Publications<br />
Rocco F, De Gregorio E, Colonna B, Di Nocera PP.<br />
Stenotrophomonas maltophilia genomes: a start-up<br />
comparison. Int J Med Microbiol. 2009, 299: 535-46.<br />
doi: 10.1016/j.ijmm.2009.05.004.<br />
Casalino M, Prosseda G, Barbagallo M, Iacobino A,<br />
Ceccarini P, Latella M C, Nicoletti M, Colonna B.<br />
Interference of the CadC regulator in the argininedependent<br />
acid resistance system of Shigella and<br />
enteroinvasive E. coli. Int J Med Microbiol. 2010, 300:<br />
289-95. doi:10.1016/j.ijmm.2009.10.008.<br />
Giangrossi M, Prosseda G, Tran CN, Brandi A,<br />
Colonna B, Falconi M. A novel antisense RNA regulates<br />
at transcriptional level the virulence gene icsA<br />
of Shigella flexneri. Nucleic Acids Res. 2010, 38: 3 362-<br />
75. doi: 10.1093/nar/gkq025.<br />
Prosseda G, Mazzola A, Di Martino M L, Tielker D,<br />
Micheli G, Colonna B. A temperature-induced narrow<br />
DNA curvature range sustains the maximum<br />
activity of a bacterial promoter in vitro. Biochemistry.<br />
2010, 49: 2778-85. doi: 10.1021/bi902003g.
Principal investigator: Anna teresa Palamara<br />
Professor of Microbiology<br />
Dipartimento di Sanità Pubblica e Malattie Infettive<br />
Tel: (+39) 06 4468622; Fax: (+39) 06 4468625<br />
annateresa.palamara@uniroma1.it<br />
21<br />
AREA 2<br />
Redox mediated mechanisms involved in the influenza virus<br />
replication and in the pathogenesis of influenza associated diseases<br />
Participants:<br />
Lucia Nencioni, Claudio Passariello, researchers; Rossella<br />
Sgarbanti, Donatella Amatore, post-doc fellows; Ignacio<br />
Celestino, PhD student.<br />
Collaborations:<br />
Dipartimento di Biologia, Università di Roma Tor Vergata (Prof.<br />
M. R. Ciriolo); <strong>Istituto</strong> di Biochimica “Giorgio Fornaini”,<br />
Università di Urbino (Prof. M. Magnani); Institute of Molecular<br />
Virology, University of Monaco (Prof. S. Ludwig).<br />
Report of activity<br />
Every year, influenza epidemics cause numerous<br />
deaths and millions of hospitalizations, but the<br />
most frightening effects are seen when new strains<br />
of the virus emerge from different species, causing<br />
worldwide outbreaks of infection. All the antiviral<br />
compounds developed against influenza virus target<br />
viral structures but unfortunately, the efficacy<br />
of these drugs is often limited by toxicity and the<br />
almost inevitable selection of drug-resistant viral<br />
mutants. Thus, the discovery of novel anti-influenza<br />
drugs targeting specific cell pathways essential for<br />
viral replication, could reduce the emergence of drug<br />
resistance and increase the effectiveness towards different<br />
viral strains.<br />
Influenza viruses use several strategies to manipulate<br />
host cell machinery to their advantage. Among<br />
these, the imbalance of intracellular redox state can<br />
play an important role in modulating the activity of<br />
several signal pathways (MAP kinases, ER oxidoreductases)<br />
involved in viral replication.<br />
The scientific activities of our group in 2008-2010<br />
were aimed at defining the mechanisms through<br />
which influenza virus activates redox regulated<br />
intracellular pathways and their potential role in<br />
regulating viral life cycle. Since secondary bacterial<br />
pneumonia are one of the primary cause of<br />
excess mortality associated with influenza A virus<br />
(IAV) infection we also investigated the potential<br />
involvement of redox-mediated mechanisms in the<br />
increased internalization of Staphylococcus aureus in<br />
influenza virus-infected cells. The obtained results<br />
are described in the following sections:<br />
Role of p38MAPK in influenza virus<br />
replication and viral induced apoptosis<br />
In cells, which are fully permissive to viral infection,<br />
virus-induced p38MAPK activation contributed to<br />
the phosphorylation of viral nucleoprotein (NP) that<br />
is an essential step for its export from the nucleus<br />
and virus assembly. Interestingly, p38MAPK-mediated<br />
NP phosphorylation was impaired in cells that<br />
are characterized by high levels of glutathione (GSH)<br />
and the expression of the antiapoptotic protein Bcl-<br />
2. In these cells Bcl-2 did not prevent infected cells<br />
from undergoing virally triggered apoptosis despite<br />
its negative impact on viral replication. The protein’s<br />
reduced antiapoptotic capacity was related to<br />
its phosphorylation by virally activated p38MAPK.<br />
Our data suggest that p38MAPK’s impact on the<br />
IAV life cycle and the apoptotic response of host<br />
cells to infection depend on whether or not the cells<br />
express Bcl-2, highlighting the possibility that the<br />
virus’ pathological effects are partly determined by<br />
the type of cell it targets. These results are <strong>report</strong>ed<br />
in the paper Nencioni et al., Journal of Biological<br />
Chemistry 2009.<br />
Role of virus-induced oxidative stress in the<br />
activation of intracellular signaling pathways<br />
On the basis of results described above, we wondered<br />
if the oxidative stress induced by IAV could play a<br />
role in the activation of p38MAPK. We found that<br />
few minutes after viral challenge, i.e., during the<br />
period of viral adsorption to the cells, IAV caused<br />
a significant but transient increase in the intracellular<br />
levels of reactive oxygen species (ROS).<br />
The ROS over-production was efficiently inhibited
Anna Teresa Palamara<br />
by NADPH-oxidase inhibitor diphenyl iodonium<br />
(DPI), thus suggesting a significant contribution<br />
of this enzyme to virus-induced oxidative stress.<br />
ROS increase paralleled with the phosphorylation<br />
of p38MAPK, a kinase involved in the regulation<br />
of viral replication, as well as in the induction of<br />
virus-induced inflammatory responses. p38MAPK<br />
activation was also inhibited by treatment with DPI,<br />
suggesting that virus-induced oxidative stress is<br />
involved in its activation.<br />
In the late steps of influenza virus life-cycle, we<br />
found a further increase in ROS production that was<br />
associated to a decrease in the content of GSH, the<br />
main intracellular antioxidant. This second round<br />
of oxidative stress is still related to an activation of<br />
p38MAPK. ROS over-production, p38MAPK activation<br />
and viral replication were efficiently inhibited<br />
by DPI, suggesting that the virus-induced oxidative<br />
stress plays a key role in regulating influenza viral<br />
replication. These results suggest that redox-regulated<br />
intracellular pathways might represent useful<br />
targets for new anti-influenza approaches. A paper<br />
<strong>report</strong>ing these results is in preparation (Amatore<br />
et al.).<br />
Definition of redox mediated pathways<br />
involved in hemagglutinin (HA) maturation<br />
Folding of influenza HA, a disulfide-rich viral glycoprotein,<br />
is an event strictly dependent on intracellular<br />
redox state. Recently our attention has been<br />
focused on the effect of GSH-C4, a GSH derivative<br />
with hydrophobic properties, on HA maturation<br />
process. In MDCK cells, the treatment with GSH-<br />
C4 decreased by 90-95% the replication of different<br />
human and avian strains of influenza viruses,<br />
without producing toxic effects on uninfected cells.<br />
This effect was associated to a specific block of HA<br />
folding: the viral protein remains in the endoplasmic<br />
reticulum as a reduced monomer instead of undergoing<br />
oligomerization and plasma-membrane insertion.<br />
HA maturation depends on the host-cell oxidoreductase,<br />
PDI, whose activity is probably facilitated<br />
by virus-induced glutathione depletion. GSH-C4induced<br />
correction of this depletion increases the<br />
presence of reduced PDI, thereby impairing its<br />
oxidizing ability. Host-cell glycoprotein expression<br />
and secretion in uninfected cells was unaffected,<br />
22<br />
suggesting that GSH-C4 acts only on glutathionedepleted<br />
cells. Antinfluenza activity of GSH-C4 was<br />
confirmed also in a murine model of lethal infection.<br />
Collectively, these results suggest that redox-sensitive<br />
host-cell pathways exploited specifically by<br />
the virus are promising targets for effective antiinfluenza<br />
strategies. A paper <strong>report</strong>ing these results<br />
has been under revision (Sgarbanti et al.).<br />
Mechanisms involved in the internalization<br />
of Staphylococcus aureus in influenza virusinfected<br />
cells<br />
We have investigated in vitro the cellular and molecular<br />
mechanisms underlying the synergism between<br />
S. aureus and IAV. Experimental data show a significant<br />
increase in the efficiency of internalisation<br />
of S. aureus into cultured pneumocytes. In particular<br />
we demonstrated that S. aureus bind the viral HA<br />
exposed at the surface of infected cells and that the<br />
affinity of this binding is enhanced by altered redox<br />
environment that the virus generate extracellularly.<br />
A paper <strong>report</strong>ing these results has been accepted for<br />
pubblication (Passariello et al., 2011)<br />
Selected publications<br />
Nencioni L, De Chiara G, Sgarbanti R, Amatore D,<br />
Aquilano K, Marcocci ME, Serafino A, Torcia M,<br />
Cozzolino F, Ciriolo MR, Garaci E, and Palamara<br />
AT. Bcl-2 expression and p38MAPK activity in cells<br />
infected with influenza A virus: impact on virally<br />
induced apoptosis and viral replication. J Biol Chem.<br />
2009, 284: 16004-15. doi: 10.1074/jbc.M900146200.<br />
De Chiara G, Marcocci ME, Civitelli L, Argnani<br />
R, Piacentini R, Ripoli C, Manservigi R, Grassi C,<br />
Garaci E, Palamara AT. APP processing induced by<br />
herpes simplex virus type 1 (HSV-1) yelds several<br />
APP fragments in human and rat neuronal cells.<br />
PLoS One. 2010, 5: e13989. doi:10.1371/journal.<br />
pone.0013989.<br />
Saladino R, Barontini M, Crucianelli M, Nencioni<br />
L, Sgarbanti R, Palamara AT. Current advances in<br />
Anti-influenza Therapy. Curr Med Chem. 2010, 17:<br />
2101-40.
Defense mechanisms against oxidative and nitrosative stress in<br />
pathogenic protozoa<br />
Principal investigator: Paolo Sarti<br />
Professor of Chemistry and Biochemistry<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 49910944; Fax: (+39) 06 4440062<br />
paolo.sarti@uniroma1.it<br />
Participants:<br />
Alessandro Giuffrè, CNR researcher; Elena Forte, researcher;<br />
Daniela Mastronicola, post-doc fellow; Fabrizio Testa, PhD<br />
student.<br />
Collaborations:<br />
Instituto de Tecnologia Química e Biológica, Universidade Nova<br />
de Lisboa, Portugal (Prof. Miguel Teixeira); Dipartimento di<br />
Scienze Biomediche, Università di Sassari (Prof. Pier Luigi Fiori);<br />
<strong>Istituto</strong> Nazionale per le Malattie Infettive “L. Spallanzani”, Roma<br />
(Dr. Leopoldo Paolo Pucillo).<br />
Report of Activity<br />
Introduction<br />
Our research has been mainly focused on the human<br />
protozoan parasite Giardia intestinalis, the causative<br />
agent of giardiasis, a very common human intestinal<br />
disease with an estimated incidence of 280 million<br />
cases per year worldwide. This air-sensitive anaerobic<br />
parasite, though lacking some of the conventional<br />
ROS-scavenging systems (such as catalase<br />
and superoxide dismutase), preferentially colonizes<br />
a fairly aerobic tract of the gut, i.e., the proximal<br />
small intestine. Parasite survival in the host is thus<br />
expected to rely on antioxidant defense systems,<br />
largely unknown as yet. Elucidating these defense<br />
systems at a molecular level, one of the goals of the<br />
current project, may thus provide the basis for pharmacological<br />
applications. In the recently sequenced<br />
genome of Giardia, a gene coding for a flavodiiron<br />
protein (FDP) was identified. FDPs are typically<br />
expressed in facultative anaerobic prokaryotes where<br />
they afford from oxidative and/or nitrosative stress.<br />
Based on an extensive structural and functional<br />
characterization of the recombinant Giardia FDP, we<br />
previously <strong>report</strong>ed that such a protein is an efficient<br />
H O-forming O -reductase, whose physiological role<br />
2 2<br />
may be to protect the parasite from O toxicity.<br />
2<br />
23<br />
AREA 2<br />
State of Art<br />
In the current project, the studies on Giardia FDP<br />
have been extended. Despite their remarkable<br />
structural similarity, unlike some of the homologous<br />
prokoryatic FDPs able to use also nitric oxide (NO)<br />
as the substrate, Giardia FDP is exclusively reactive<br />
towards O . The equilibrium redox properties of<br />
2<br />
the Giardia FDP cofactors (FMN and Fe-Fe) were<br />
investigated by potentiometry combined with EPR<br />
or UV/visible absorption spectroscopy. We <strong>report</strong>ed<br />
that the Giardia enzyme has virtually identical<br />
FMN<br />
reduction potentials for the FMN cofactor (E1 FMN = -66 mV and E = -83 mV) and considerably<br />
2<br />
up-shifted reduction potentials for the diiron center<br />
Fe-Fe Fe-Fe (E = +163 ± 20 mV and E2 = +2 ± 20 mV),<br />
1<br />
if compared with those measured for NO-consuming<br />
FDPs and similarly with those <strong>report</strong>ed for the FDP<br />
from the pathogenic protozoan Trichomonas vaginalis.<br />
These different redox properties are suggestive<br />
of subtle, yet unidentified, structural differences in<br />
protozoan FDPs that may account for the substrate<br />
selectivity.<br />
By immunoblotting analysis, the Giardia FDP was<br />
shown to be expressed at detected levels in the<br />
disease-causing cells of the parasite (the trophozoites),<br />
grown under standard anaerobic reducing<br />
conditions. Evidence was provided that, whereas<br />
nitrosative stress does not change the intracellular<br />
levels of FDP, after shortly (10 minutes) exposing<br />
the parasitic cells to ≥ 100 µM H O , the protein is<br />
2 2<br />
degraded and cellular O -consumption is severely<br />
2<br />
impaired, eventually causing cell death. The FDP<br />
degradation was shown to involve a cellular proteolytic<br />
pathway, sensitive to the proteasome-inhibitor<br />
MG132. Based on this notable susceptibility of<br />
Giardia to H O , we raised the hypothesis that the<br />
2 2<br />
peculiar localization of the parasite in the proximal<br />
small intestine (where microbial flora is much less<br />
abundant than in the large intestine) is a strategy to<br />
escape oxidative stress: the small intestine is indeed
Paolo Sarti<br />
endowed with a significantly higher redox buffering<br />
capacity, compared to the large intestine, thereby<br />
providing a more favorable microenvironment for<br />
Giardia proliferation.<br />
Nitric oxide (NO) is an antimicrobial agent produced<br />
together with ROS by the host immune<br />
system to control pathogens infections. In vitro,<br />
NO-stress has been <strong>report</strong>ed to have cytostatic,<br />
rather than cytotoxic effects on Giardia. This effect<br />
leads to the suggestion that Giardia is endowed with<br />
defense mechanisms against NO, though, as yet,<br />
none NO-detoxifying enzyme has been described for<br />
the parasite.<br />
In the genome of Giardia, a prokaryotic gene coding<br />
for a flavohemoglobin (FlavoHb) was recently<br />
identified. As far as our knowledge, this is the first<br />
flavohemoglobin identified from parasitic protozoa.<br />
We have purified and characterized the recombinant<br />
FlavoHb from Giardia. Under aerobic conditions and<br />
in the presence of NADH, it was shown that Giardia<br />
FlavoHb metabolizes NO with high efficacy (V max =<br />
116 ± 10 s -1 at [O 2 ] = 200 µM). The enzyme shows<br />
an apparent O 2 affinity (K m = 22 ± 7 µM) compatible<br />
with the physiological O 2 levels in the small intestine.<br />
In response to nitrosative stress, the expression<br />
of Giardia FlavoHb markedly increases (~ 8 fold) in<br />
the trophozoites, which thereby become able to efficiently<br />
metabolize NO under aerobic conditions. In<br />
24<br />
line with previous studies on bacterial FlavoHbs, by<br />
using NO-releasers, NO has been shown to enhance<br />
FlavoHb expression in Giardia. Based on these<br />
results, we propose that physiologically FlavoHb<br />
protects Giardia from NO toxicity.<br />
Publications<br />
Borisov VB, Forte E, Giuffrè A, Konstantinov A,<br />
Sarti P. Reaction of nitric oxide with the oxidized diheme<br />
and heme-copper oxygen-reducing centers of<br />
terminal oxidases: Different reaction pathways and<br />
end-products. J Inorg Biochem. 2009, 103: 1185-7.<br />
doi:10.1016/j.jinorgbio.2009.06.002.<br />
Vicente JB, Testa F, Mastronicola D, Forte E,<br />
Sarti P, Teixeira M, Giuffrè A. Redox properties<br />
of the oxygen-detoxifying flavodiiron protein<br />
from the human parasite Giardia intestinalis. Arch<br />
Biochem Biophys. 2009, 488: 9-13. doi: 10.1016/j.<br />
abb.2009.06.011.<br />
Mastronicola D, Testa F, Forte E, Bordi E, Pucillo<br />
L P, Sarti P, Giuffrè A. Flavohemoglobin and<br />
Nitric Oxide Detoxification in the Human Protozoan<br />
Parasite Giardia intestinalis. Biochem Biophys Res<br />
Commun. 2010, 399: 654-8. doi: 10.1016/j.<br />
bbrc.2010.07.137.
Principal investigator: Maria Rosaria torrisi<br />
Professor of General Pathology<br />
Dipartimento di Medicina Clinica e Molecolare<br />
Tel/Fax: (+39) 06 49979797<br />
Azienda Ospedaliera Sant’Andrea<br />
Tel: (+39) 06 33775502; Fax: (+39) 06 33775257<br />
mara.torrisi@uniroma1.it<br />
25<br />
AREA 2<br />
Role of the keratinocyte growth factor receptor on the molecular and<br />
cellular alterations induced by the expression of HPV16 E5 oncoprotein<br />
Participants:<br />
Maurizio Alimandi, Deborah French, Patrizia Mancini,<br />
Vincenzo Visco, professors; Francesca Belleudi, Salvatore<br />
Raffa, researchers; Laura Leone, post-doc fellow; Danilo<br />
Ranieri, Valeria Purpura, PhD students; Antonio Sabatucci,<br />
technician.<br />
Collaboration:<br />
Laboratorio di Virologia, <strong>Istituto</strong> Regina Elena IFO-IRE, Roma (Dr.<br />
Aldo Venuti).<br />
Report of activity<br />
The high-risk human papillomaviruses (HPVs) play<br />
key roles in the pathogenesis of cervical cancers. The<br />
E5 protein encoded by HPV type 16 is an oncoprotein<br />
which contributes to epithelial transformation<br />
and cooperates in tumor progression by deregulating<br />
cell growth, survival and differentiation through<br />
the modulation of growth factor receptors, such as<br />
the epidermal growth factor receptor (EGFR). The<br />
expression of the16E5 protein at the skin level in a<br />
transgenic mouse model induces epidermal hyperplasia,<br />
aberrant differentiation and skin tumors. Among<br />
the epithelial growth factors, the keratinocyte growth<br />
factor (KGF/FGF7) and the fibroblast growth factor<br />
10 (FGF10/KGF2) are major paracrine mediators of<br />
proliferation, differentiation, survival and migration<br />
of epithelial cells. Both KGF and FGF10 bind to and<br />
activate exclusively the keratinocyte growth factor<br />
receptor (KGFR/FGFR2b). The KGFR, in contrast<br />
to most of the growth factor receptors, appears to<br />
play an unique and unusual role in epithelial tissues,<br />
exerting a tumor suppressive function in vitro and in<br />
vivo. Interestingly, the KGFR/FGFR2b null-mice<br />
phenotype closely reminds that shown by the 16E5<br />
transgenic mice, characterized by a similar behaviour<br />
in skin carcinogenic model. Thus, KGFR and 16E5<br />
might be inversely correlated in their expression<br />
and might exert opposite and interplaying roles in<br />
skin homeostasis and tumorigenesis. With the aim<br />
to better elucidate the molecular events involved in<br />
the pathological effects induced by HPV infection,<br />
our research project attempted: a) to establish in<br />
vitro the correlation between the expression levels<br />
of 16E5 and those of KGFR and other epithelial<br />
RTKs and the keratinocyte growth, differentiation,<br />
survival, and transformation; b) to identify the<br />
mechanisms and signaling pathways controlling the<br />
possible effects of 16E5 expression in modulating the<br />
ligand-dependent KGFR activation and endocytosis.<br />
During the first year of the project, to investigate if<br />
16E5 expression would be able to modulate KGFR in<br />
vitro, we had used human cultured HaCaT keratinocytes<br />
expressing 16E5 under the control of dexamethasone-inducible<br />
promoter, as a model to study the 16E5<br />
effects on the receptor activation and modulation and on<br />
the related epithelial proliferation/differentiation. The<br />
results of that preliminary part of the study have shown<br />
KGFR transcriptional down-modulation induced by<br />
E5 expression. In the second and third year of the project<br />
this receptor down-modulation was confirmed in<br />
HaCaT cells singly or doubly transfected with pCIneo<br />
E5 (kindly provided by Dr. Venuti) or pCIneo KGFR;<br />
the E5-induced down-regulation was demonstrated also<br />
at the protein level by Western blot analysis. According<br />
to this KGFR down-modulation, we found that 16E5<br />
expression in human keratinocytes decreased the KGFinduced<br />
proliferation and differentation.<br />
Because the altered expression of transforming growth<br />
factor β (TGFβ) and of its receptor serine/threosine<br />
kinase TGF-βRII are also involved in the pathogenesis<br />
of cervical carcinomas, during this project we have analyzed<br />
also the possible relationship between HPV-16<br />
integration, E5 transcription and TGF-βRII modulation<br />
in the progression of cervical neoplasia. To this<br />
aim quantitative Real Time PCR was performed for<br />
the detection of HPV-16 E5 and TGF-βRII mRNA in<br />
Low-Grade Squamous Intraepithelial Lesions (L-SIL)<br />
and High-Grade Squamous Intraepithelial Lesions<br />
(H-SIL), all E6/E7 positive for HPV-16. We found that<br />
the transcript levels of TGF-βRII, compared to control<br />
samples, were down-regulated in all HSILs, while in
Maria Rosaria Torrisi<br />
LSILs we observed a clear decrease of the receptor<br />
expression only in relation to high levels of E5 mRNA,<br />
suggesting that E5 expression could play a transforming<br />
role in cervical lesions through down-modulation<br />
of TGF-βRII expression. These results have been<br />
confirmed by a series of in vitro experiments showing<br />
receptor down-regulation and deregulation of TGFβ<br />
signaling (Belleudi, French et al., in preparation).<br />
Since 16E5 is known to inhibit the EGFR endocytic<br />
degradative pathway and to enhance receptor recycling,<br />
with the aim to better elucidate the molecular<br />
events involved in the pathological effects induced by<br />
HPV infection we investigated if HPV16 E5 protein<br />
would be able: a) to interfere with the two alternative<br />
KGFR endocytic pathways and b) to affect the proliferative<br />
and differentiative response to the ligands. In<br />
fact, we have previously demonstrated that FGF10/<br />
KGF2 induces receptor recycling, whereas KGF/<br />
FGF7 stimulates receptor degradation (Belleudi et al.,<br />
Traffic 2007): since the recycling endocytic pathway<br />
followed by KGFR upon FGF10 stimulation correlates<br />
with the higher mitogenic activity exerted by this<br />
ligand on epithelial cells compared to KGF, suggesting<br />
that the two ligands may play different functional<br />
roles through the regulation of the receptor endocytic<br />
transport, our hypothesis was that 16E5 could play a<br />
similar role in targeting KGFR to the recycling route.<br />
To this aim we studied in detail the intracellular endocytic<br />
traffic followed by KGFR in HaCaT E5/KGFR<br />
cotrasfected cells. In particular, we focused our attention<br />
on the late steps of KGFR internalization to search<br />
for a possible differential sorting of receptors destined<br />
to recycling or degradation in the presence of E5. The<br />
KGFR endocytic intracellular transport was analyzed<br />
at immunofluorescence and confocal microscopy: we<br />
found that the expression of E5 inhibits the KGFinduced<br />
KGFR transport to the degradative pathway<br />
but does not affect the FGF10-induced receptor traffic<br />
through the recycling compartment. In addition, we<br />
wondered if, in the presence of 16E5, KGFR would<br />
recycle back to the plasma membrane upon KGF<br />
treatment: the results showed receptor reappearance at<br />
the plasma membrane upon KGF stimulation only in<br />
cells expressing the 16E5 protein, indicating that the<br />
receptor could be sorted to the slow indirect recycling<br />
pathway and implying intracellular traffic through the<br />
juxtanuclear compartment. Consistent with the 16E5<br />
effect on the KGFR traffic, parallel biochemical western<br />
blot analysis revealed that E5 protein expression<br />
was able to induce a block of KGF-dependent degradation<br />
(Belleudi et al., submitted).<br />
Our results lead to the interesting hypothesis that<br />
KGFR direct recycling is a default pathway, being<br />
activated only when the degradative ruote is blocked,<br />
while the indirect perinuclear recycling would be<br />
26<br />
inducible and regulated by a specific sorting machinery<br />
(Belleudi et al., 2009): thus we cannot exclude that<br />
E5 protein might be directly involved in the shifting<br />
of KGFR trafficking from degradation to recycling.<br />
Therefore, to investigate if 16E5 expression could<br />
redirect the KGF-induced endocytic trafficking from<br />
the degradative to the slow recycling pathway inducing<br />
an inefficient recruitment of the Grb2/c-Cbl complex<br />
and consequent ligand-dependent ubiquitination<br />
of the receptor, as occurring upon FGF10 treatment<br />
(Belleudi et al., 2007), we analyzed the phosphorylation<br />
level of tyrosine 196 in the KGFR substrate FRS2,<br />
which represent the principal docking site for Grb2,<br />
and we found that E5 expression correlates with a<br />
reduced phosphorylation of the FRS2 tyrosine 196.<br />
Thus, the KGFR alternative endocytic traffic triggered<br />
by the two ligands may provide a useful physiological<br />
model for further investigations directed to<br />
elucidate the distinct functions of the molecular components<br />
of the endosomal sorting complexes as well as<br />
the mechanisms controlling the endocytic traffic and<br />
intracellular receptor signaling. In addition, we propose<br />
that HPV16 E5 expression can be used as a model<br />
to analyze the endocytic indirect recycling pathway.<br />
Publications<br />
Belleudi F, Leone L, Maggio M, Torrisi MR. Hrs<br />
regulates the endocytic sorting of the fibroblast<br />
growth factor receptor 2b. Exp Cell Res. 2009, 315:<br />
2181-91. doi:10.1016/j.yexcr.2009.03.022.<br />
Visco V, Bava FA, d’Alessandro F, Cavallini M,<br />
Ziparo V, Torrisi MR. Human colon fibroblasts<br />
induce differentiation and proliferation of intestinal<br />
epithelial cells through the direct paracrine action of<br />
keratinocyte growth factor. J Cell Physiol. 2009, 220:<br />
204-13. doi: 10.1002/jcp.21752.<br />
Belleudi F, Cardinali G, Kovacs D, Picardo M, Torrisi<br />
MR. KGF promotes paracrine activation of the SCF/c-<br />
KIT axis from human keratinocytes to melanoma cells.<br />
Transl Oncol. 2010, 3: 80-90. doi: 10.1593/tlo.0919.<br />
Kovacs D, Cardinali G, Aspite N, Cota C, Luzi F,<br />
Bellei B, Briganti S, Amantea A, Torrisi MR, Picardo<br />
M. Role of fibroblast-derived growth factors in<br />
regulating hyperpigmentation of solar lentigo. Br J<br />
Dermatol. 2010, 163: 1020-27. doi: 10.1111/j.1365-<br />
2133.2010.09946.x.<br />
Santonico E, Belleudi F, Panni S, Torrisi MR,<br />
Cesareni G, Castagnoli L. Multiple modification<br />
and protein interaction signals drive the Ring finger<br />
protein 11 (RNF11) E3 ligase to the endosomal compartment.<br />
Oncogene. 2010, 29: 5604-18. doi:10.1038/<br />
onc.2010.294.
AREA 3<br />
Molecular<br />
genetics<br />
of<br />
eukaryotes
CFTR gene engineering by means of prokaryotic and eukaryotic<br />
artificial chromosome<br />
Principal investigator: Fiorentina Ascenzioni<br />
Professor of Microbiology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49917577/7614; Fax: (+39) 06 49917594<br />
fiorentina.ascenzioni@uniroma1.it<br />
Participants:<br />
Cristina Auriche, Enea Gino Di Domenico, Francesca<br />
Tilesi, post-doc fellows; Ivan Coladarci, graduate student.<br />
Collaborations:<br />
Dipartimento di Scienze Biomediche, Università di Foggia (Prof.<br />
Massimo Conese); Dipartimento di Biotecnologie Cellulari<br />
ed Ematologia, Sapienza-Università di Roma (Prof. Marco<br />
Lucarelli); Laboratorio di Genetica Molecolare, <strong>Istituto</strong> Giannina<br />
Gaslini, Genova (Prof. Olga Zegarra-Moran); <strong>Istituto</strong> di<br />
Neurobiologia e Medicina Molecolare, CNR, Roma (Prof. Ettore<br />
D’Ambrosio).<br />
Report of activity<br />
Cystic fibrosis (CF) is a recessive congenital disease<br />
that shows high incidence in Caucasian populations<br />
(ca. 1 in 2 000). CF is caused by mutations in the gene<br />
coding for CF transmembrane conductance regulator<br />
(CFTR; OMIM 602421), a Cl - channel expressed<br />
in the apical membrane of several epithelia. CFTR is<br />
the main pathway for Cl - secretion; therefore, normal<br />
CFTR activity is necessary for the maintenance of<br />
the level of the airway surface liquid. Indeed, CFTR<br />
loss-of-function leads not only to reduced Cl - secretion,<br />
but also to an increase in Na + and water absorption,<br />
probably because of a predominant activity of<br />
the epithelial Na + channel (ENaC). In vivo as well as<br />
in vitro models of well-differentiated CF airway epithelia<br />
have shown isotonic reduction of the surface<br />
liquid, and the consequent impaired movement of<br />
the overlying mucus; leading to increased adherence<br />
of micro-organisms such as Pseudomonas aeruginosa.<br />
Despite recent developments in the treatment of CF,<br />
there is no definitive cure for this disease, and the<br />
life expectancy for the patient with CF is around<br />
30-40 years. Since localization of the CFTR gene to<br />
the long arm of chromosome 7 in 1989, a huge effort<br />
has been put into the development of gene therapies<br />
for this life-threatening disease. To date, more than<br />
29<br />
AREA 3<br />
30 clinical gene therapy trials have been performed<br />
worldwide, although with only very limited success.<br />
As effective therapy by such in-vivo delivery of therapeutic<br />
DNA requires physiological levels of expression<br />
and long-term maintenance, it has been hypothesized<br />
that this can be achieved by using genomic<br />
fragments that contain all of the long-range controlling<br />
elements that will allow tissue-specific gene<br />
expression to a physiological level. Although CFTR<br />
expression was initially thought to be dictated by a<br />
“housekeeping” promoter, it appears that regulation<br />
of CFTR expression involves interactions between a<br />
number of different regulatory factors and elements.<br />
Indeed, cell-specific expression is determined by regulatory<br />
elements that are upstream and downstream<br />
of the coding region, and in various introns.<br />
The human CFTR locus was isolated in a YAC vector<br />
of about 320 kb in size (YAC 37AB12) by Anand<br />
and collaborators in 1991, and subsequently Manson<br />
and collaborators showed that this vector complemented<br />
CF defects in cftr-/- mice.<br />
From the YAC- to the BAC-CFTR<br />
The YAC37AB12 containing the intact CFTR<br />
locus, was converted into a circular BAC by using<br />
the retrofitting plasmid pNK-G418. Subsequently, to<br />
insert a selectable marker for mammalian cells into<br />
the circularized YAC, we performed a second DNA<br />
modification by replacing the region between -50 kb<br />
to -25 kb from the CFTR start codon, with a fragment<br />
containing markers for selection of mammalian<br />
cells (Zeocin) and for yeast cells (Phleomicin). Such<br />
substitution caused a size reduction of the circular<br />
YAC of about 20 kb, leading to a circular molecule<br />
smaller than 300 kb. Next, both circular YAC/BACs,<br />
cCFTR5A and cCFTRD12, were recovered into E.<br />
coli EC100 cell and analyzed by sequencing.<br />
Sequence analysis demonstrated that the cCFTR5A<br />
contained an insert running from -49,923 bp upstream<br />
the CFTR start codon (position 42245379 of the reference<br />
contig NT_00793.14) to +58,214 bp downstream
Fiorentina Ascenzioni<br />
the stop codon (position 42542218, NT_00793.14)<br />
(Fig. 1). The two vectors cCFTR5A and cCFTRD12<br />
showed the same 5’ and 3’ ends confirming that the<br />
pruning procedure occurred as expected, additionally<br />
we found that the 5’ upstream DNA removed in<br />
the cCFTRD12 vector corresponded to the region<br />
spanning from -25,696 bp to -49,788 bp (position<br />
42277897 to 42253805 of the reference contig<br />
NT_00793.14). Sequence analysis of all the introns,<br />
intron/exon junctions and the proximal 5’-flanking<br />
region of the CFTR locus resulted wild type and the<br />
studied polymorphisms were as follows: the repeats in<br />
intron 6a were (GATT) 7 ; the repeats in intron 8 were<br />
(TG) 11 T 7 ; the polymorphic site at the aminoacid 470<br />
was V470. Overall the BAC cCFTR5A, and its derivative<br />
cCFTRD12, contain the most common wild type<br />
CFTR haplotype in the Caucasian population.<br />
Next cCFTRD12 was transferred into Fisher Rat<br />
Thyroid (FRT) cells for functional analysis. The<br />
resulting clones were analyzed for the CFTR mRNA<br />
and the transepithelial Cl - current. The results<br />
obtained clearly showed CFTR mRNA in half of<br />
clones analysed (FRTcl2-7), although at different<br />
level, and CFTR Cl- currents in 5 out of the 6 CFTR<br />
mRNA positive clone. One of these (FRTcl7) was the<br />
best clone responding to the cAMP agonist with a<br />
short-circuit current (Isc) increase of about 18 µA/<br />
cm 2 (Fig. 2). Finally, by confocal microscopy analysis<br />
revealed that the CFTR protein was localized both<br />
to the apical membrane and to the cytoplasm.<br />
CFTR-bacteria interaction<br />
It was shown that CFTR functions as an internalizing<br />
receptor for P. aeruginosa by binding of amino<br />
acids 108-117 to the outer core LPS of P. aeruginosa.<br />
According to this finding, we hypothesized that<br />
CFTR expression and proper trafficking to the apical<br />
membrane of FRT cells should increase the rate<br />
of bacteria internalization. Infection assays with P.<br />
aeruginosa (ATCC 27853) clearly demonstrated<br />
that FRT cells expressing CFTR internalized much<br />
30<br />
more bacteria than FRT cells, additionally treatment<br />
of the cells with the CFTR inhibitor (CFTR Ihn172 )<br />
reduced the percentage of internalized bacteria in<br />
CFTR-expressing cells but not in parental FRT<br />
cells. These results not only confirm the role of<br />
CFTR as P. aeruginosa receptor but also suggest a<br />
possible role of CFTR activity in the survival of bacteria<br />
within the cells, soon after infection.<br />
Publications<br />
Di Domenico EG, Auriche C, Viscardi V, Longhese<br />
MP, Gilson E, Ascenzioni F. The Mec1p and Tel1p<br />
checkpoint kinases allow humanized yeast to tolerate<br />
chronic telomere dysfunctions by suppressing<br />
telomere fusions. DNA Rep. 2009, 8: 209-18. doi:<br />
10.1016/j.dnarep.2008.10.005.<br />
Tilesi F, Fradiani P, Socci V, Willems D, Ascenzioni<br />
F. Design and validation of siRNAs and shRNAs.<br />
Curr Opin Mol Ther. 2009, 11: 156-64.<br />
Auriche C, Di Domenico EG, Pierandrei S, Lucarelli<br />
M, Castellani S, Conese M, Melani R, Zegarra-Moran<br />
O, Ascenzioni F. CFTR expression and activity from<br />
the human CFTR locus in BAC vectors, with regulatory<br />
regions, isolated by a single-step procedure. Gene<br />
Ther. 2010, 17: 1341-54. doi:10.1038/gt.2010.89.<br />
Fradiani PA, Petrucca A, Ascenzioni F, Di Nucci<br />
G, Teggi A, Bilancini S, Cipriani P. Endocarditis<br />
caused by Lactobacillus jensenii in an immunocompetent<br />
patient. J Med Microbiol. 2010, 59: 607-9. doi:<br />
10.1099/jmm.0.017764-0.<br />
Rocchi L, Braz C, Cattani S, Ramalho A, Christan<br />
S, Edlinger M, Ascenzioni F, Laner A, Kraner S,<br />
Amaral M, Schindelhauer D. Escherichia coli-cloned<br />
CFTR loci relevant for human artificial chromosome<br />
therapy. Hum Gene Ther. 2010, 21: 1077-92.<br />
doi:10.1089/hum.2009.225.
31<br />
AREA 3<br />
Fig. 1 - Schematic representation of the contig NT_00793.14 (in the middle); the CFTR locus is flanked upstream by ASZ1 (ankyrin repeat,<br />
SAM and basic leucin zipper) and downstream by CTTNBP2 (cortactin binding protein 2). The arrows point to the positions 42245379 and<br />
42542218 delimitating the fragment cloned into the BACs; bent arrow represents the initiation site of translation. cCFTR5A (below the<br />
contig) contains the entire CFTR locus flanked upstream by 49,9 kb and downstream by 58,2 kb. cCFTRD12 (above the contig) is the same<br />
as cCFTR5A but the upstream region between position 42253805-42277897 (dashed line) was deleted.<br />
Fig. 2 - Short-circuit current recordings. Summary of CFTR-dependent short-circuit currents obtained with each clone. Data are means ±SE<br />
(n = 3). The negative control was parental FRT cells.
RNA-RNA and RNA-protein interactions: role of small non-coding<br />
RNAs in gene expression control<br />
Principal investigator: Irene Bozzoni<br />
Professor of Molecular Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912202; Fax: (+39) 06 49912500<br />
irene.bozzoni@uniroma1.it<br />
Participants:<br />
Carlo Presutti, professor; Elisa Caffarelli, Alessandro<br />
Fatica, researchers; Monica Ballarino, Davide Cacchiarelli,<br />
Erika Girardi, Tania Incitti, Pietro Laneve, Julie Martone,<br />
Mariangela Morlando, Tiziana Santini, post-doc fellows;<br />
Marcella Cesana, PhD student.<br />
Collaborations:<br />
Dipartimento di Medicina Molecolare, Sapienza-Università di<br />
Roma (Prof. Alberto Gulino); Scienze Anatomiche, Istologiche,<br />
Medico-Legali e dell’Apparato Locomotorio; Sapienza-Università<br />
di Roma (Prof. Antonio Musarò); TIGEM, Napoli (Prof. Alberto<br />
Auricchio); City of Hope, Duarte, USA (Prof. John Rossi).<br />
Report of activity<br />
The aim of this project was to exploit and further<br />
develop the high potential of RNA-based methodologies<br />
for the comprehension of the molecular basis of<br />
several processes of gene regulation and to apply this<br />
knowledge to the study and cure of several genetic<br />
disorders, such as cancer or inherited diseases.<br />
The project covers topics ranging from the biosynthesis<br />
of small-non coding RNAs to their function in<br />
normal and pathological conditions. In particular, we<br />
have focused on the study of the role that miRNAs<br />
play in biological processes as important as developmental<br />
control, cell proliferation and apoptosis.<br />
Accordingly, altered miRNA expression can have<br />
profound effects on human pathology, including<br />
cancer, making it crucial to understand how miRNA<br />
genes are regulated.<br />
In a parallel line of research, we have exploited the<br />
vast potential of different RNA activities (antisense<br />
and interference) for human therapy, based on an<br />
advanced understanding of the underlying mechanisms.<br />
To achieve these goals, integrated and multidisciplinary<br />
action was required including basic research<br />
and development, genetic approaches, as well as cell<br />
biology assessment.<br />
33<br />
AREA 3<br />
miRNA function<br />
miRNA in neuronal differentiation<br />
We previously showed that miR-9, which is almost<br />
exclusively expressed in the brain, is upregulated<br />
during in vitro neuronal differentiation, whereas it<br />
is downregulated in 50% of primary neuroblastoma<br />
(NB) tumours, suggesting its potential function as an<br />
oncosuppressor.<br />
In the last years we have characterized the transcriptional<br />
regulation of the miR-9-2 gene and found that,<br />
despite its localization inside an exon of a host-gene,<br />
it is expressed as an independent unit with the promoter<br />
located in the upstream intron. We also show<br />
that the repressor REST inhibits expression of the<br />
miR-9-2 promoter in undifferentiated neuroblastoma<br />
cells, whereas REST dismissal and simultaneous<br />
binding of the phosphorylated active form of CREB<br />
triggers transcription in differentiating cells. Finally,<br />
we have been able to highlight a regulatory feed-back<br />
mechanism in which the reciprocal action of miR-9<br />
and the antineurogenic transcription factor REST<br />
may be relevant for the maintenance of the neuronal<br />
differentiation program (Laneve et al., 2010).<br />
A high throughput microRNA expression profile<br />
was also performed in human primary medulloblastoma<br />
specimens. We identified specific micro-<br />
RNA expression patterns which distinguish medulloblastoma<br />
differing in histotypes (anaplastic, classic<br />
and desmoplastic), in molecular features (ErbB2 or<br />
c-Myc overexpressing tumors) and in disease-risk<br />
stratification (Ferretti et al., 2009). We found a<br />
limited number of miRNAs displaying differential<br />
expression between tumours and controls, indicating<br />
that specific miRNAs may represent biological markers<br />
that could be useful in predicting MB prognosis<br />
and further subdivision of patients.<br />
miRNA in muscle differentiation<br />
miRNA deregulation linked to pathology finds an<br />
interesting example in the Duchenne Muscular<br />
Dystrophy (DMD), characterized by the absence of
Irene Bozzoni<br />
dystrophin. We found that this deficiency at the sarcolemma<br />
delocalizes and downregulates Nitric Oxide<br />
Synthase (NOS) and that this alters S-nitrosylation<br />
of HDAC2 and its chromatin association. During<br />
this study we showed that the differential HDAC2<br />
nitrosylation state in Duchenne versus wild-type conditions<br />
deregulates the expression of a specific subset<br />
of microRNA genes (Cacchiarelli et al., 2010a).<br />
Several circuitries controlled by the identified micro-<br />
RNAs, such as the one linking miR-1 to the G6PD<br />
enzyme and the redox state of cell, or miR-29<br />
to extracellular proteins and the fibrotic process,<br />
explain some of the DMD pathogenetic traits. We<br />
also showed that, at variance with other myomiRs,<br />
miR-206 escapes from the dystrophin-nNOS control<br />
being produced in activated satellite cells before<br />
dystrophin expression; in these cells, it contributes<br />
to muscle regeneration through repression of the<br />
satellite specific factor, Pax7.<br />
We concluded that the pathway activated by dystrophin/nNOS<br />
controls several important circuitries<br />
increasing the robustness of the muscle differentiation<br />
programme.<br />
miRNAs in hematopoietic differentiation<br />
In the last years we have identified several miRNAs<br />
playing a crucial role in myeloid differentiation<br />
(Fazi et al., 2005; Rosa et al., 2007). Recently, the<br />
list has included the newly identified miR-342. We<br />
demonstrated that miR-342 and its host transcript<br />
are under the control of the same promoter: the<br />
transcriptional factor IRF-1 (interferon regulatory<br />
factor-1) is required for maintaining basal levels of<br />
miR-342, while IRF-9 and PU.1 are responsible for<br />
its responsivenes to RA. Notably, PU.1 and IRF-1<br />
play a crucial role in myeloid differentiation and<br />
PU1 is very often mutated in myeloid leukemia (De<br />
Marchis et al., 2009).<br />
With the aim of identifying the molecular circuitries<br />
controlled by miR-342 we have pursued the SILAC<br />
methodology on NB4 cells infected with a lentiviral<br />
vector expressing miR-342. 100 proteins have been<br />
identified whose expression appears to be controlled<br />
by miR-342 and among them approximately 15% are<br />
predicted to be its direct targets. Validation of these<br />
targets and the building of integrated circuitries connecting<br />
these different factors is currently undergoing.<br />
Use of RNA for the therapy of Duchenne<br />
Muscular Dystrophy<br />
The use of the mdx mouse as the animal model for<br />
the Duchenne Muscular Dystrophy has allowed us<br />
to demonstrate the effectiveness of the antisense<br />
34<br />
strategy in restoring the synthesis of dystrophin in<br />
vivo for the entire life of the animal. Therefore, this<br />
approach provided solid basis for a systemic use of<br />
AAV-mediated antisense-U1 snRNA expression for<br />
the therapeutic treatment of DMD.<br />
However, when thinking of possible intervention on<br />
human, it is very crucial to identify the most appropriate<br />
antisense sequences able to provide the highest<br />
possible skipping efficiency.<br />
In the course of the last year, we compared the<br />
exon 51 skipping activity of ten different antisense<br />
molecules, raised against splice junctions and/or<br />
exonic splicing enhancers, expressed as part of the<br />
U1 snRNA. The effectiveness of each construct<br />
was tested in human DMD myoblasts carrying<br />
the deletion of exons 48-50 which can be cured by<br />
skipping of exon 51. Our results showed that the<br />
highest skipping activity and dystrophin rescue is<br />
achieved upon expression of a U1 snRNA-derived<br />
antisense molecule targeting exon 51 splice sites<br />
in combination with an internal exon sequence.<br />
The efficacy of this molecule was further proven<br />
on an exon 45-50 deletion background, utilizing<br />
patient’s fibroblasts trans-differentiated into myoblasts.<br />
In this system, we showed that the selected<br />
antisense was able to produce 50% skipping of exon<br />
51. These data allowed us to select the most active<br />
molecule for the skipping of exon 51 that has been<br />
selected for entering human clinical trials (Incitti<br />
et al., 2010).<br />
Publications<br />
Ballarino M, Pagano F, Girardi E, Morlando M,<br />
Cacchiarelli D, Marchioni M, Proudfoot NJ, Bozzoni<br />
I. Coupled RNA processing and transcription of<br />
intergenic primary microRNAs. Mol Cell Biol. 2009,<br />
29: 5632-8. doi:10.1128/MCB.00664-09.<br />
De Marchis ML, Ballarino M, Salvatori B, Puzzolo<br />
MC, Bozzoni I, Fatica A. A new molecular network<br />
comprising PU.1, interferon regulatory factor proteins<br />
and miR-342 stimulates ATRA-mediated granulocytic<br />
differentiation of acute promyelocytic leukemia cells.<br />
Leukemia. 2009, 23: 856-62. doi:10.1038/leu.2008.372.<br />
Fatica A, Bozzoni I. Role of microRNAs in hematological<br />
malignancies. Expert Review Hematol. 2009, 2:<br />
415-23. doi: 10.1586/ehm.09.32.<br />
Ferretti E, De Smaele E, Po A, Di Marcotullio<br />
L, Tosi E, Espinola MS, Di Rocco C, Riccardi R,<br />
Giangaspero F, Farcomeni A, Nofroni I, Laneve P,
Gioia U, Caffarelli E, Bozzoni I, Screpanti I, Gulino<br />
A. MicroRNA profiling in human medulloblastoma.<br />
Int J Cancer. 2009, 124: 568-77. doi: 10.1002/ijc.23948.<br />
Puttini S, Lekka M, Dorchies OM, Saugy D, Incitti<br />
T, Ruegg UT, Bozzoni I, Kulik AJ, Mermod N.<br />
Gene-mediated restoration of normal myofiber elasticity<br />
in dystrophic muscles. Mol Ther. 2009, 17:<br />
19-25. doi: 10.1038/mt.2008.239.<br />
Anastasiadou E, Boccellato F, Vincenti S, Rosato P,<br />
Bozzoni I, Frati L, Faggioni A, Presutti C, Trivedi<br />
P. Epstein-Barr virus encoded LMP1 downregulates<br />
TCL1 oncogene through miR-29b. Oncogene. 2010,<br />
29: 1316-28. doi:10.1038/onc.2009.439.<br />
Cacchiarelli D, Martone J, Girardi E, Cesana M,<br />
Incitti T, Nicoletti C, Santini T, Sthandier O,<br />
Auricchio A, Musarò A, Bozzoni I. MicroRNAs<br />
involved in molecular circuitries relevant for the<br />
Duchenne muscular dystrophy pathogenesis are controlled<br />
by the dystrophin/nNOS pathway. Cell Metab.<br />
2010, 12: 341-51. doi: 10.1016/j.cmet.2010.07.008.<br />
Chiaretti S, Messina M, Tavolaro S, Zardo G, Elia L,<br />
Vitale A, Fatica A, Gorello P, Piciocchi A, Scappucci<br />
35<br />
AREA 3<br />
G, Bozzoni I, Fozza C, Candoni A, Guarini A, Foà<br />
R. Gene expression profiling identifies a subset of<br />
adult T-cell acute lymphoblastic leukemia (T-ALL)<br />
with myeloid-like gene features and overexpression<br />
of miR-223. Haematologica. 2010, 95: 1114-21. doi:<br />
10.3324/haematol.2009.015099.<br />
Incitti T, De Angelis FG, Cazzella V, Sthandier O,<br />
Pinnarò C, Legnini I, Bozzoni I. Exon skipping and<br />
Duchenne muscular dystrophy therapy: selection of<br />
the most active U1 snRNA-antisense able to induce<br />
dystrophin exon 51. Mol Ther. 2010, 18: 1675-82.<br />
doi:10.1038/mt.2010.123.<br />
Laneve P, Gioia U, Andriotto A, Moretti F, Bozzoni<br />
I, Caffarelli E. A minicircuitry involving REST and<br />
CREB controls miR-9-2 expression during human<br />
neuronal differentiation. Nucleic Acids Res. 2010, 38:<br />
6895-905. doi: 10.1093/nar/gkq604.<br />
Rinaldi A, Vincenti S, De Vito F, Bozzoni I, Oliverio<br />
A, Presutti C, Fragapane P, Mele A. Stress induces<br />
region specific alterations in microRNAs expression<br />
in mice. Behav Brain Res. 2010, 208: 265-9.<br />
doi:10.1016/j.bbr.2009.11.012.
Role of metalloproteinases and their tissue inhibitors in the<br />
regulation of neurogenesis and gliogenesis from neural stem/<br />
progenitor cells<br />
Principal investigator: Emanuele Cacci<br />
Researcher in Clinical Biochemistry and clinical Molecular Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912206; Fax: (+39) 06 49912351<br />
emanuele.cacci@uniroma1.it<br />
Participants:<br />
Stefano Biagioni, professor, Tonino Anelli, post-doc fellow;<br />
Pasquale Caramanica, PhD student.<br />
Collaborations:<br />
Università Carlo Bo, Urbino (Prof. Ferdinando Mannello, Dr.<br />
Gaetana A. Tonti); <strong>Istituto</strong> Superiore di Sanità, Roma (Dr.<br />
Luisa Minghetti, Dr. Maria Antonietta Ajmone-Cat); King’s<br />
College, London, UK (Prof. Noel Bukley).<br />
Report of activity<br />
Neural stem cells (NSC), which generate new neurons<br />
and glia under physiological and pathological<br />
conditions, persist in the adult brain. Regulation<br />
of adult neurogenesis involves a wide spectrum of<br />
signals from NSC microenvironment, the so-called<br />
neurogenic niche. As demonstrated by our and other<br />
groups, microglial cells are crucial components of the<br />
niche and they exert either beneficial or detrimental<br />
effects on neurogenesis, depending on their activation<br />
state and ranges of mediators produced.<br />
The identification of intrinsic and extrinsic factors<br />
implicated in the modulation of neurogenesis is crucial<br />
to define strategies to successfully manipulate<br />
the neurogenic and gliogenic potential of NSC.<br />
Among extrinsic factors, matrix metalloproteinases<br />
(MMPs) and their natural tissue inhibitors (TIMPs)<br />
are emerging as contributors to neurogenesis modulation.<br />
In addition, MMPs are multifaceted contributors<br />
to neuroinflammation. Particularly MMP-2 and<br />
MMP-9, have been extensively studied in the brain,<br />
where they are found expressed at low levels under<br />
physiological conditions and up-regulated in various<br />
pathological conditions. MMPs and TIMPs are<br />
expressed in NSCs and other brain cell types, including<br />
microglia. Noteworthy, MMPs and TIMPs regulate<br />
cytokine production in microglia in autocrine/<br />
paracrine manner and, vice versa, cytokines appear to<br />
37<br />
AREA 3<br />
be potent regulators of microglial MMP production.<br />
In light of these considerations, we started the present<br />
project with the following major aims:<br />
- Analysis of the possible roles of MMPs and<br />
TIMPs in the regulation of NSC properties.<br />
- Characterization of MMP and TIMP profile in<br />
NSCs.<br />
- Analysis of the molecular mechanisms regulating<br />
MMP and TIMP expression during NSC differentiation.<br />
- Analysis of inflammatory mediators (cytokines,<br />
MMPs, and TIMPs) produced in resting and activated<br />
microglia and their possible implications for<br />
neurogenesis and gliogenesis modulation.<br />
Results<br />
a) We first generated cell lines, which display NSC<br />
properties, from embryonic and adult mouse brain.<br />
We also isolated and extensively characterized several<br />
clonal lines from adult mouse brain. These lines<br />
express typical markers of NSCs (e.g. Pax6, Sox2,<br />
Nestin, Blbp, RC2) and retain self-renewal and multipotency<br />
(ability to differentiate into neurons, astrocytes<br />
and oligodendrocytes), two functional criteria<br />
to define stem cells. Once established suitable cellular<br />
models, we investigated the role of MMPs in the<br />
regulation of NSC properties. We used a broad spectrum<br />
MMP pharmacological inhibitor. The inhibitor<br />
increased NSC differentiation into neurons (almost<br />
doubling the percentage of neurons compared with<br />
control cultures) and reduced the generation of glial<br />
cells in a dose-dependent manner. The inhibitor did<br />
not affect NSC differentiation if added once the differentiation<br />
process was ongoing. From these and other<br />
data we conclude that: 1) MMPs modulate NSC commitment;<br />
2) The inhibitor treatment is an efficient<br />
and simple protocol for the in vitro generation of neurons<br />
from NSC. The generation of highly enriched<br />
neuronal (or glial) cultures is highly demanded, as<br />
it enables to dissect differentiation and maturation<br />
processes at both biochemical and molecular levels.
Emanuele Cacci<br />
b) Since MMP-2 and MMP-9 are the main MMPs<br />
in the brain, we speculated that the MMP inhibitor<br />
effects on NSCs were mediated through MMP-2<br />
and/or MMP-9 inhibition. To clarify this point we<br />
first investigated the expression profile of MMP-2<br />
and MMP-9 in NSCs. RT-PCR analysis and gel<br />
zymography showed that MMP-2 is expressed in<br />
NSCs and increases during differentiation, whereas<br />
MMP-9 is undetectable under our culture conditions.<br />
To directly assess if MMP-2 is important in<br />
the modulation of NSC differentiation we silenced<br />
these proteins by means of specific siRNAs. Our<br />
results indicate that MMP-2 is not the major controller<br />
of NSC differentiation into neurons and that<br />
other MMPs could be involved in the modulation of<br />
this process (manuscript in preparation).<br />
c) In this first year project we also addressed the<br />
molecular mechanisms involved in the regulation<br />
of NSC properties. We focused our attention on the<br />
RE1-silencing transcription factor (REST), a master<br />
regulator of neuronal genes that can induce neuronal<br />
differentiation in NSC. Our computational analysis<br />
and experimental data suggest that REST can modulate,<br />
among others, several genes of the MMP and<br />
TIMP families.<br />
To clarify which genes are transcriptionally repressed<br />
by REST we have used an adenovirus carrying the<br />
dominant negative of REST (DN:REST), or shRNA<br />
against REST. DN:REST comprises the REST DNA<br />
binding domain, but lacks the N and C termini; it<br />
thus derepresses transcription of REST target genes.<br />
NSCs were infected with DN:REST vector and with<br />
the corresponding control vector and after 48 h the<br />
cells were lysated and RNA extracted. Analysis of<br />
global gene expression changes by DNA microarray<br />
is currently ongoing (in collaboration with Prof.<br />
Bukley). In parallel, we immunoprecipitated chromatin<br />
extracted from DN:REST infected and control<br />
cells by using an anti-REST antibody. Preliminary<br />
data suggest REST enrichment at the RE1 elements<br />
of well known neuronal genes; Real time PCR analysis<br />
will be performed to demonstrate specific target<br />
gene occupancy by REST. These studies will help<br />
38<br />
to identify novel mechanisms for the regulation of<br />
genes known, or yet unexplored, as possible modulators<br />
of NSC properties.<br />
d) To identify released factors produced by microglia<br />
under different activation conditions (i.e. acute or<br />
chronic stimulation) and their consequences on neurogenesis,<br />
we characterized the expression profile of<br />
several pro- and anti-inflammatory cytokines. We<br />
focused our attention on IL1a and IL1b, abundantly<br />
produced under acute but not chronic activation, and<br />
found that IL1a and IL1b are important modulators<br />
of NSC differentiation (Ajmone-Cat et al., 2010).<br />
We aim to further characterize the expression profile<br />
of inflammatory mediators, including MMPs<br />
and TIMPs, under different activation conditions.<br />
We will test the effects of MMP inhibition on the<br />
functional phenotypes of activated microglia and in<br />
particular we will investigate if MMPs inhibition<br />
shapes activated microglia towards a pro- or antineurogenic<br />
phenotype.<br />
Perspective<br />
We aim to pursue the following goals: 1) To complete<br />
transcriptome and ChIp analysis to deepen<br />
our knowledge on the role of REST in NSC differentiation<br />
and in the regulation of specific genes,<br />
such as MMPs and TIMPs; 2) To understand the<br />
contribution of MMPs and TIMPs to the modulation<br />
of microglial activation and the consequences on<br />
the neurogenic and gliogenic processes. Altogether<br />
these experiments will contribute to a better comprehension<br />
of the events and cues that regulate brain<br />
repair mechanisms and will offer new opportunities<br />
to identify possible targets for the successful therapeutic<br />
manipulation of endogenous neurogenesis.<br />
Publications<br />
Ajmone-Cat MA, Cacci E, Ragazzoni Y, Minghetti<br />
L, Biagioni S. Pro-gliogenic effect of IL-1alpha in<br />
the differentiation of embryonic neural precursor<br />
cells in vitro. J Neurochem. 2010, 113: 1060-72. doi:<br />
10.1111/j.1471-4159.2010.06670.x.
Crosstalk between poly(ADP-ribosyl)ation and DNA methylation<br />
in the regulation of gene expression<br />
Principal investigator: Paola Caiafa<br />
Professor of Clinical Biochemistry and Molecular Biology<br />
Dipartimento di Biotecnologie Cellulari ed Ematologia<br />
Tel: (+39) 06 49976530; Fax: (+39) 06 44231961<br />
caiafa@bce.uniroma1.it<br />
Participants:<br />
Anna Reale, professor; Michele Zampieri, Tiziana<br />
Guastafierro, post-doc fellows; Maria Giulia Bacalini, PhD<br />
student.<br />
Collaborations:<br />
<strong>Istituto</strong> di Biologia e Patologia Molecolari, CNR, Roma (Dr.<br />
Claudio Passananti).<br />
Report of activity<br />
Our findings showed (1-2) that along the chain of<br />
events that induce DNA methylation-dependent<br />
chromatin condensation/decondensation, a postsynthetic<br />
modification, namely poly(ADP-ribosyl)<br />
ation (PARylation), participates in the establishment<br />
and maintenance of genome methylation pattern.<br />
To explain how PARP activity controls DNA<br />
methylation pattern we suggested a model (3) in<br />
which ADP-ribose polymers (PARs), present on<br />
PARylated PARP-1, interact noncovalently with<br />
Dnmt1, preventing its enzymatic activity. We found<br />
that Dnmt1 is able to bind long and branched PARs<br />
in a noncovalent way and PARs, either free or<br />
PARP-1 bound, inhibit human recombinant Dnmt1<br />
activity both in vitro and in vivo (3). In absence of<br />
PARylated PARP-1, Dnmt1 is free to methylate<br />
DNA; if, on the other hand, high levels of PARylated<br />
PARP-1 persist, Dnmt1 will be stably inhibited,<br />
preventing DNA methylation. Our new data supported<br />
this model and have now included CTCF as<br />
an important player in the PARP-1/Dnmt1 interplay<br />
(4). CTCF over-expression caused genomic<br />
hypomethylation. In vitro data confirmed that the<br />
inhibitory role on Dnmt1 was not due to CTCF,<br />
but to PARylated PARP-1. In fact, CTCF was per<br />
se capable of activating PARP-1 automodification<br />
and PARylated PARP-1 was, in turn, able to inhibit<br />
Dnmt1 activity, according to the above mentioned<br />
model. Considering all the above mentioned data, it<br />
39<br />
AREA 3<br />
was suggested that CTCF marks DNA regions for<br />
PARP-1 binding, and thus for maintaining CTCF<br />
target sequences in an unmethylated state. The<br />
maintenance of proper DNA methylation pattern on<br />
these sites determines the correct expression of the<br />
correlated genes, e.g. imprinted genes.<br />
By using PARG overexpression to deplete cells of<br />
PARs, we showed that the Dnmt1 gene promoter is<br />
protected from methylation by PARylated PARP-1,<br />
adding another dimension to the regulatory functions<br />
of PARylated PARP-1, through its control<br />
of DNA methylation pattern of housekeeping gene<br />
promoters. We demonstrated that the CpG island<br />
located in the promoter of Dnmt1 becomes methylated<br />
in absence of PARs (5). ChIP analyses, performed<br />
to verify if PARP-1 and/or PARs are involved in the<br />
control of DNA methylation pattern of Dnmt1 promoter,<br />
showed that only the fragment that we found<br />
methylated following PARG overexpression, was<br />
present in ChIP with both antibodies anti PARP-1<br />
and anti PARs, Figure 1. Therefore the PARylated<br />
form of PARP-1 sits on Dnmt1 promoter maintaining<br />
this region non methylated and this introduces<br />
PARylation as an epigenetic mark capable of preventing<br />
the methylation of some regions that need<br />
to maintain their unmethylated state in normal cells,<br />
e.g. some CpG islands.<br />
A model can be put forward: when the cells are depleted<br />
of PARylated PARP-1 and PARs, Dnmt1, not inhibited,<br />
is free to introduce new aberrant methyl groups<br />
onto DNA. Otherwise, PARP-1, already PARylated<br />
or when PARylated because of binding to CTCF, is<br />
capable of attracting Dnmt1, through the polymers<br />
present on its PARylated form and polymers, in turn,<br />
inhibit the Dnmt1 methyltransferase activity. These<br />
data raise the possibility that PARylated PARP-<br />
1 marks those DNA sequences that must remain<br />
unmethylated, thus playing a role in the epigenetic<br />
regulation of gene expression. The eventuality that<br />
PARP-1 activity could confer in trans the protection<br />
against aberrant DNA methylation to CpG islands of
Paola Caiafa<br />
housekeeping gene promoters is of great interest and<br />
it is also strongly supported by data on p16 gene (6).<br />
It could provide important information on one possible<br />
mechanism by which proper DNA methylation<br />
patterns, which determine the correct expression of<br />
correlated genes, is protected under physiological<br />
conditions.<br />
Our aim is to verify if PARylated PARP-1 introduces<br />
an epigenetic code on chromatin. The aim has been<br />
to verify if the interplay between PARylation and<br />
DNA methylation could be responsible for the maintenance<br />
of the methylation state of specific CTCF<br />
target sequences. To this purpose, we have been<br />
using as model some well characterised CTCF binding<br />
regions, i.e. the differentially methylated region<br />
(ICR) in the Igf2/H19 imprinted locus.<br />
Here the two genes are adjacent and share the same<br />
enhancer region but, while H19 is expressed only<br />
by the maternal allele, Igf2 is expressed only by<br />
the paternal one (7). The unmethylated ICR DNA<br />
region, localized between the two genes, exerts<br />
the function of insulator by acting as a boundary<br />
to limit the function of the enhancer region at the<br />
Igf2 promoter in the maternal allele. The insulator<br />
function of the ICR requires the binding of the<br />
CTCF protein that occurs only when the ICR is<br />
unmethylated. It has recently been demonstrated<br />
that also PARylation plays a role in imprinting<br />
control (10). In fact, a covalently PARylated form<br />
of CTCF, whose MW shifts from 130 to 180 kDa<br />
as a consequence of the modification, has been<br />
found to be bound to the ICR. Moreover, the<br />
evidence that the treatment of cells with competitive<br />
PARP inhibitors causes loss of imprinting at<br />
the Igf2/H19 locus together with almost 140<br />
CTCF target sites in the genome (10), strongly<br />
suggested that PARylation of CTCF is required<br />
in the control of imprinted gene expression. The<br />
notion of a functional link between PARylation<br />
and DNA methylation (1) prompted us to verify<br />
the hypothesis that these two epigenetic events do<br />
not act separately but work together in the control<br />
of imprinting and in particular that PARylation<br />
is required to maintain proper DNA methylation<br />
patterns at imprinted DNA regions. Based on<br />
our recent achievements (4) that include CTCF<br />
in the interplay between PARylation and DNA<br />
methylation, we suggest an alternative model of<br />
epigenetic control of CTCF boundary function<br />
based on PARylation. In this model, CTCF stimulates<br />
PARP activity at its DNA target sites and<br />
prevents de novo methylation of CpG dinucleotides<br />
by inhibiting Dnmt1 activity.<br />
40<br />
Data, obtained in L929 mouse fibroblasts, showing<br />
that CTCF forms a complex both in vitro (Pulldown)<br />
and in vivo (Co-IP e ChIP e RE-ChIP) with<br />
PARylated PARP-1 and Dnmt1 at the ICR, confirm<br />
our expectations. Moreover, the depletion of PARs,<br />
obtained by PARG overexpression, causes loss of<br />
imprinted expression of Igf2, hypermethylation of<br />
CTCF-associated CpGs and removal of both CTCF<br />
and PARP-1 from the ICR. All this suggests that<br />
PARs are required to preserve the DNA methylation<br />
profile of CTCF binding targets (within the<br />
maternal ICR), manuscript in preparation.<br />
References<br />
1) Caiafa et al. (2008) Faseb J 23, 672-8.<br />
2) Caiafa and Zampieri (2005) J Cell Biochem 94,<br />
257-65.<br />
3) Reale et al. (2005) Oncogene 24, 13-9.<br />
4) Guastafierro et al. (2008) J Biol Chem 283, 21873-<br />
80.<br />
5) Zampieri et al. (2009) PLos One 4:e4717.<br />
6) Witcher et al. (2009) Mol Cell 34, 271-84.<br />
7) Wallace and Felsenfeld (2007) Curr Opin Genet<br />
Dev 17, 400-7.<br />
8) Bell and Felsenfeld (2000) Nature 405, 482-85.<br />
9) Schoenherr et al. (2003) Nature Gen 33, 66-69.<br />
10) Yu et al. (2004) Nature Gen 36, 1105-10.<br />
Publications<br />
Caiafa P, Guastafierro T, Zampieri M. Epigenetics:<br />
poly(ADP-ribosyl)ation of PARP-1 regulates<br />
genomic methylation patterns. FASEB J. 2009, 23:<br />
672-8. doi: 10.1096/fj.08-123265.<br />
Caiafa P, Zlatanova J. CCCTC-binding factor meets<br />
poly(ADP-ribose) polymerase-1. J Cell Physiol. 2009,<br />
219: 265-70. doi: 10.1002/jcp.21691.<br />
Zampieri M, Passananti C, Calabrese R, Perilli M,<br />
Corbi N, De Cave F, Guastafierro T, Bacalini MG,<br />
Reale A, Amicosante G, Calabrese L, Zlatanova J,<br />
Caiafa P. Parp1 localizes within the Dnmt1 promoter<br />
and protects its unmethylated state by its enzymatic<br />
activity. PLoS One. 2009, 4:e4717. doi: 10.1371/journal.pone.0004717.<br />
Zlatanova J, Caiafa P. CCCTC-binding factor: to<br />
loop or to bridge. Cell Mol Life Sci. 2009, 66: 1647-60.<br />
doi: 10.1007/s00018-009-8647-z.<br />
Zlatanova J, Caiafa P. CTCF and its protein partners:<br />
divide and rule? J Cell Sci. 2009, 122: 1275-84. doi:<br />
10.1242/jcs.039990.
Chevanne M, Zampieri M, Caldini R, Rizzo A,<br />
Ciccarone F, Catizone A, D’Angelo C, Guastafierro<br />
T, Biroccio A, Reale A, Zupi G, Caiafa P. Inhibition<br />
of PARP activity by PJ-34 leads to growth impair-<br />
41<br />
AREA 3<br />
ment and cell death associated with aberrant mitotic<br />
pattern and nucleolar actin accumulation in M14<br />
melanoma cell line. J Cell Physiol. 2010, 222: 401-10.<br />
doi:10.1002/jcp.21964.
Principal investigator: Antonella De Jaco<br />
Researcher in Cell Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912310; Fax: (+39) 06 49912351<br />
antonella.dejaco@uniroma1.it<br />
43<br />
AREA 3<br />
Processing of the Neuroligins proteins and autism-related mutations<br />
Participants:<br />
Lisa Ulbrich, post-doc fellow; Flores Lietta Favaloro,<br />
undergraduate student.<br />
Collaborations:<br />
University of California, San Diego, USA (Prof. Palmer Taylor,<br />
Prof. Davide Comoletti); Stanford University, CA, USA (Prof.<br />
Michael Lin); University of Cambridge, UK (Prof. Stefan<br />
Marciniak).<br />
Report of activity<br />
Background<br />
Neuroligins (NLGNs) are post-synaptic cell adhesion<br />
proteins that form eterophilic trans-synaptic bridges<br />
with the presynaptic Neurexins (NRXNs). Their<br />
interaction plays a role in organizing both inhibitory<br />
and excitatory synapses, and is essential for selectivity<br />
in synaptic function (Sudhof, Nature, 2008).<br />
Support in favor of the role played by these proteins<br />
at the synapse comes from human genetic studies in<br />
patients affected by autism spectrum disorders (ASD),<br />
a group of neurodevelopmental disorders characterized<br />
by impairments in social reciprocal interactions<br />
and language. Several rare missense mutations have<br />
been <strong>report</strong>ed in the genes encoding for NLGN 3<br />
and 4 and for NRNX 1, highlighting that the cross<br />
talk between both partner proteins is essential for<br />
correct synaptic function (Persico and Bourgeron,<br />
Trends Neurosci, 2006). NLGNs are single pass multi<br />
domain proteins and belong to the α/β-hydrolase<br />
fold family because of the extracellular domain structure<br />
(Ichtchenko et al., J Biol Chem, 1996). The R451C<br />
substitution in the α/β-hydrolase fold domain of<br />
NLGN3 was found in two brothers affected by<br />
ASD (Jamain et al., Nat Genet, 2003). Several groups<br />
have shown that R451C NLGN3 is predominantly<br />
retained in the endoplasmic reticulum (ER) when<br />
transfected into HEK293, COS and hippocampal<br />
neurons (Comoletti, J Neurosci, 2004; De Jaco, J Biol<br />
Chem, 2006). Moreover construction of knock-in mice<br />
confirmed in vivo the effect of this amino acid change<br />
(Tabuchi et al., Science, 2007). Crystal structure of<br />
NLGN1/NRXN1-β complex reveals that R451C<br />
maps to a position remote from the binding site for<br />
NRXN1-β (Fabrichny et al., Neuron, 2007).<br />
Research objective<br />
The objective of this study is to analyze folding,<br />
disulfide bonds formation, post-translational glycosylation<br />
processing and trafficking of NLGN3 protein by<br />
biochemical and cell biological approaches. The final<br />
goal is to understand how the autism-related mutations<br />
are interfering with the normal assembling and<br />
exposure of the protein to the cell surface. Although the<br />
ER retention of NLGN3-R451C mutant protein has<br />
been well documented in different cell model systems,<br />
the molecular basis underlying the block in trafficking<br />
haven’t been clarified yet. A direct comparison of the<br />
processing of NLGN3 wild type and R451C mutant<br />
proteins in HEK-293 and trafficking in neurons should<br />
show where alterations are arising in the making of the<br />
NLGN3 protein. Especially since most of the NLGNs<br />
mutations map in the α/β-hydrolase fold domain<br />
that represents the protein extracellular domain, the<br />
identifications of ER molecular chaperones taking part<br />
in building its tridimensional structure and the ones<br />
involved in the retention mechanism are valuable information<br />
for future designing of rescue strategies.<br />
Results<br />
The following data have been included in the publications<br />
that acknowledge the support of the <strong>Istituto</strong><br />
<strong>Pasteur</strong> Fondazione Cenci-Bolognetti: De Jaco et al.,<br />
Chem Biol Interact, 2010; De Jaco et al., J Biol Chem, 2010.<br />
Protein misfolding of R451C NLGN3<br />
The R451C mutation affects folding of the α/βhydrolase<br />
fold domain of NLGN3. Protein folding
Antonella De Jaco<br />
was studied by trypsin digestion experiments after<br />
expression of NLGN3 wild type and R451C c-DNA<br />
in HEK-293 cells. R451C mutant protein results<br />
more sensitive to the trypsin digestion compared<br />
to the wild type protein suggesting that NLGN3<br />
R451C is more loosely folded around the mutation<br />
(De Jaco et al., 2010a). Nonetheless it appears that<br />
the extra cysteine is not interfering with the formation<br />
of pre-existing disulfide bonds as shown by<br />
reaction with PEG5000-maleimide and estimation<br />
of the molecular mass (De Jaco et al., 2010a). G221R,<br />
positioned deep in the core of NLGN3 α/β-hydrolase<br />
domain, was chosen as a control mutation to<br />
compare the extent of the misfolding induced by the<br />
R451C. Introduction of G221R makes NLGN3 to be<br />
retained in the ER in a more severe way than what<br />
observed for R451C mutant protein. Moreover, by<br />
comparing the sensitivity to protease digest, G221R<br />
is more sensitive to the digestion with trypsin<br />
than R451C, suggesting that this mutation affects<br />
the overall protein folding while R451C mutation<br />
results causing a locally expanded conformation (De<br />
Jaco et al., 2010b).<br />
Association with ER molecular chaperones<br />
By co-immunoprecipitation, we have identified the<br />
differential association of wild type, R451C and<br />
G221R mutant proteins with ER molecular chaperones.<br />
GRP78/BiP and GRP94 associate more avidly<br />
with R451C and G221R mutant proteins than the<br />
wild type protein, probably because these chaperones<br />
recognize them as misfolded. Calnexin and calreticulin<br />
don’t show a preferential association for either<br />
wild type or mutant proteins. Because NLGN3 presents<br />
both disulfide bonds and free cysteines association<br />
with PDI family members such as PDIr, Erp57<br />
and Erp72 was tested. These chaperones, associate<br />
with the mutant proteins but not with the wild type,<br />
suggesting involvement of a thiol-retention mechanism<br />
(De Jaco et al., 2010b).<br />
Processing of NLGN3 in HEK-293<br />
Metabolic labeling experiments in HEK-293 cells stably<br />
expressing either wild type or R451C or G221R<br />
mutant proteins show that the introduction of the<br />
R451C mutation slows down processing of NLGN3.<br />
At 3 hours of chase, processing of wild type protein<br />
is complete while R451C remains mainly unprocessed<br />
with only a small fraction of fully glycosylated protein<br />
that increases at 6 hours and disappears faster<br />
44<br />
compared to the wild type protein. As expected by the<br />
greatest severity of the misfolding, NLGN3 G221R<br />
remains unprocessed and degrades faster than either<br />
wild type or R451C (De Jaco et al., 2010b).<br />
Trafficking of NLGN3 in neurons<br />
The influence of R451C and G221R mutations on<br />
the trafficking of NLGN3 is analyzed in hippocampal<br />
neurons by using NLGN3 proteins fused to a novel<br />
fluorescent <strong>report</strong>er system that allows visualization<br />
of newly produced proteins from the soma to<br />
the dendrites. R451C reduces the export of NLGN3<br />
from the soma to a dendritic location in comparison<br />
to the wild type protein whereas G221R completely<br />
abolishes NLGN3 translocation to the dendritic<br />
synapses. These results in neurons are in accord<br />
with the data in HEK293 cells where the R451C and<br />
G221R mutations hinder protein processing, with<br />
the R451C causing a local misfolding and G221R the<br />
most severe defect (De Jaco et al., 2010b).<br />
Future directions<br />
Characterization of the rare R451C mutation is providing<br />
information about residues crucial for folding<br />
of the α/β-hydrolase fold domain of NLGN3.<br />
Acquiring information about the molecular basis of<br />
ER retention mechanism for autism related mutant<br />
proteins, such as the NLGN3 R451C, offers a solid<br />
base for designing strategies for rescue protein<br />
expression on the cell surface. The following up of<br />
the project will be focused on individuating other<br />
mutations that affect processing of NLGN3 and further<br />
characterization of the NLGN3 R451C mutant<br />
protein in regard to ER stress signalling.<br />
Publications<br />
De Jaco A, Dubi N, Comoletti D, Taylor P. Folding<br />
anomalies of neuroligin3 caused by a mutation in the<br />
α/β-hydrolase fold domain. Chem Biol Interact. 2010,<br />
187: 56-8. doi: 10.1074/jbc.M110.139519.<br />
De Jaco A, Lin MZ, Dubi N, Comoletti D, Miller<br />
MT, Camp S, Ellisman M, Butko MT, Tsien RY,<br />
Taylor P. Neuroligin trafficking arising from mutations<br />
in the α/β-hydrolase fold protein family. J<br />
Biol Chem. 2010, 285: 28674-82. doi: 10.1016/j.<br />
cbi.2010.03.012.
Epigenetic modifications in neurodegenerative diseases<br />
Principal investigator: Maria D’Erme<br />
Professor of Biochemistry<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 49910923; Fax: (+39) 06 4440062<br />
maria.derme@uniroma1.it<br />
Participants:<br />
Luciana Mosca, researcher; Alessandra Masci, Italo<br />
Tempera, post-doc fellows.<br />
Collaborations:<br />
Dipartimento di Farmacologia, Sapienza-Università di Roma (Dr.<br />
Luciano Saso), Università Paris 6 Pierre et Marie Curie, Paris,<br />
France (Prof. Francesco Visioli), <strong>Istituto</strong> di Biologia e Patologia<br />
Molecolari, CNR, Roma (Dr. Gianni Colotti).<br />
Report of activity<br />
In neurodegenerative diseases such as Alzheimer’s<br />
and Parkinson’s disease, injury might be associated<br />
with altered function of misfolded proteins leading<br />
to the formation of aggregated proteins, the Amyloid<br />
beta and the α-synuclein, respectively. It is known<br />
that the process of protein aggregation gives rise<br />
to soluble oligomers which are redox active and can<br />
generate reactive oxygen species (Smith et al., 2007;<br />
Uversky, 2008). Oxidative stress is responsible for<br />
DNA damage, which in turn is the major activator<br />
of poly (ADP-ribose) polymerase (PARP-1).<br />
The PARP-1 uses NAD + as a substrate to transfer<br />
ADP-ribose units to nuclear acceptor proteins forming<br />
polymers of varying lengths. The PARP-1 is<br />
involved in several physiological processes such as<br />
gene expression, genome integrity and apoptosis.<br />
PARP-1 inhibitors have been developed not only to<br />
study the role of this enzyme in cell biology, but also<br />
to improve the effectiveness of chemotherapy. Over<br />
the last decade many studies have shown that the<br />
activation of PARP-1 represents a key mediator of<br />
neuronal death during excitotoxicity, ischemia and<br />
oxidative stress, and how pharmacological inhibition<br />
of the enzyme can significantly enhance neuronal<br />
survival. Recently, several authors have noticed a<br />
correlation between PARP-1 and neurodegenerative<br />
processes. Indeed, nuclear poly(ADP-ribosylated)<br />
proteins were found in autoptic specimens of<br />
45<br />
AREA 3<br />
Alzheimer’s patients (Love et al., 1999). Outeiro et<br />
al. (2007) have shown that inhibitors of PARP-1 significantly<br />
reduced the neurotoxicity in experimental<br />
models of Parkinson’s diseases.<br />
In order to gain more insight into the molecular<br />
mechanisms underlying neurodegenerative diseases,<br />
this project was focused on the study of the relationship<br />
between poly(ADP-ribosylation) and neurodegeneration.<br />
As Alzheimer’s disease model, human SH-SY5Y<br />
dopaminergic neuroblastoma cell line were treated<br />
with the Aβ25-35 fragment, which retains all<br />
the neurotoxic properties of the full length Aβ<br />
1-42. Parkinsonian neurotoxicity, was induced by<br />
5-S-cysteinyldopamine (CysDA) or 6-hydroxydopamine<br />
(6OH-DA) treatment of mice or SH-SY5Y<br />
cells. The CysDA was recently indicated as a possible<br />
parkinsonian neurotoxin, but its effect in vivo<br />
has not yet been elucidated (Mosca et al., 2008). All<br />
the experiments were performed in the presence or<br />
in the absence of the novel water soluble PARP-1<br />
inhibitor, the quinazolinone derivative MC2050 provided<br />
by Prof. A. Mai, Sapienza University.<br />
In the first part of the project, the new molecule<br />
MC2050 was characterized and the obtained results<br />
led to the formulation of a paper in ChemMedChem.<br />
Since the activation of PARP-1 has been identified<br />
in Alzheimer’s disease, but its role has not yet<br />
been clarified, the second part of this research was<br />
to examine the influence of amyloid peptide on the<br />
activity of PARP-1 in the presence or absence of the<br />
inhibitor of MC2050. The Aβ was able to induce a<br />
marked increase in ROS within the first two hours<br />
of treatment. Because ROS are major agents of DNA<br />
damage resulting in activation of PARP-1, the activity<br />
of PARP-1 in the presence or absence of MC2050<br />
was assessed. The results showed that in SH-SY5Y<br />
cells treated with Aβ25-35 the activity of PARP-1<br />
increases about 70% in 24 hours. The pre-treatment<br />
with the inhibitor is able to counteract this increase.
Maria D’Erme<br />
To demonstrate whether the activation of PARP-1 is<br />
related to the generation of ROS due to fibrillation.<br />
The treatment of SH-SY5Y cells with Aβ25-35 with<br />
or without quercetin, which is a known antioxidant<br />
also endowed with the ability to prevent Aβ aggregation,<br />
led to prevent the PARP-1 activity caused by<br />
amyloid peptide by counteracting oxidative stress.<br />
Several studies have shown that Aβ can activate<br />
NF-kB in neuronal cells (Kuner et al., 1998) and<br />
Hassa and Hottinger (2002) have shown that also<br />
PARP-1 regulates the function of NF-kB. Our data<br />
demostrate that Aβ activates NF-kB, and this activation<br />
was significantly reduced in cells pretreated<br />
with the inhibitor MC2050, supporting the hypothesis<br />
that Aβ may modulate the activity of NF-kB<br />
through a PARP-1-dependent mechanism..<br />
The third part of this project was devoted to further<br />
study on the effects of 5-S-cysteinyldopamine<br />
(CysDA) as a parkinsonian neurotoxin. Experiments<br />
were carried on in vivo in C57BL/6 mice and in<br />
vitro on SH-SY5Y cells. Briefly, DA-denervating<br />
lesions were induced in male CD1 mice by bilateral<br />
inoculation of either CysDA or 6-OHDA into the<br />
dorsal striatum of mice. Sham operated mice were<br />
injected with the same volume of saline solution.<br />
21 days after treatment, mice were sacrificed and to<br />
evaluate the extent of neurodegeneration the striata<br />
were analyzed for monoamine neurotransmitters<br />
46<br />
level. Intrastriatal injection of CysDA in mice causes<br />
a long-lasting depletion of dopamine without affecting<br />
serotonergic or noradrenergic systems. CysDA<br />
exerts oxidative stress as demonstrated both in<br />
cells and in mice by a significative increase in protein<br />
carbonylation and oxidized glutathione. Brain<br />
regions of mice treated with both neurotoxins show<br />
an appreciable increase of stress-proteins ERp57 and<br />
α-synuclein levels in the striatum, compared to hippocampus<br />
and pre-frontal cortex regions.<br />
The neuroblastoma cells treatment induces an increase<br />
in cytosolic ERp57 levels, within 8 hours, whereas in<br />
the nuclei it is not significantly altered. α-Syn protein<br />
levels are affected within 1 hour of treatment both<br />
in the cytosol and nuclei. This increase seems to be<br />
transient, rapidly returning to basal level. Preliminary<br />
RealTime-PCR analyses confirm that CysDA modulates<br />
the expression of both ERp57 and α-Syn. The<br />
treatment with CysDA also induces an increase of<br />
PARylated proteins both in cells and mice.<br />
Publications<br />
Tempera I, Deng Z, Atanasiu C, Chen CJ, D’Erme<br />
M, Lieberman PM. Regulation of Epstein-Barr virus<br />
OriP replication by poly(ADP-ribose) polymerase 1. J<br />
Virol. 2010, 84: 4988-97. doi: 10.1128/JVI.02333-09.
DNA replication mechanisms and genome stability in<br />
Saccharomyces cerevisiae<br />
Principal investigator: Lucia Fabiani<br />
Researcher in Molecular Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: 06 49912243; Fax: (+39) 06 49912351<br />
lucia.fabiani@uniroma1.it<br />
Participants:<br />
Federica Lo Sardo, graduate student.<br />
Collaborations:<br />
Department of Microbiology and Molecular Genetics, UMDNJ,<br />
New Jersey Medical School, Newark, USA (Prof. Carol S.<br />
Newlon); Dipartimento di Scienze Biomolecolari e Biotecnologie,<br />
Università degli Studi di Milano and <strong>Istituto</strong> FIRC di oncologia<br />
Molecolare, Milano (Prof. Marco Foiani); <strong>Istituto</strong> FIRC di<br />
Oncologia Molecolare, Milano (Dr. Giordano Liberi).<br />
Report of activity<br />
Different biological processes are involved in the<br />
maintenance of genome stability as DNA replication,<br />
repair and checkpoint mechanisms that coordinate<br />
DNA metabolism with the progression of cell<br />
cycle. These mechanisms have been well studied in<br />
Saccharomyces cerevisiae and many of them are common<br />
to higher eukaryotic organisms. Accurate and<br />
complete DNA replication is essential for the maintenance<br />
of the genetic integrity. Chromosome duplication<br />
is controlled at the level of replication initiation,<br />
which occurs at cis-acting replicator sequences<br />
spaced at intervals of approximately 40kb along the<br />
chromosomes of S. cerevisiae. Surprisingly, it has been<br />
recently shown that derivatives of chromosome III<br />
that lack known replicators segregated properly in at<br />
least 96% of cell divisions. To understand the mechanisms<br />
that maintain these “originless” fragments,<br />
mutants defective in the maintenance of an “originless”<br />
chromosome fragment were isolated (originless<br />
fragment maintenance mutants). The study of ofm<br />
mutants can help to define the mechanisms responsible<br />
of the replication of the “originless” fragment and<br />
a more detailed analysis of one of the mutants, ofm5,<br />
will allow us also to understand the mechanisms and<br />
the genes involved in chromosome stability because<br />
this mutant is characterized by a high loss rate of the<br />
wildtype derivative of chromosome III.<br />
47<br />
AREA 3<br />
We identified the genomic clone by the capability to<br />
complement HU sensitivity of ofm5. Further analysis<br />
revealed that ofm5 is an allele of CHL1 (Chromosome<br />
Loss 1). Chl1p (861 aa) is a member of DEAH subfamily<br />
of Superfamily 2 (SF2) helicases. Its helicase<br />
activity is important for the prevention of chromosome<br />
loss or missegregation. It is required for the<br />
establishment of proper sister chromatid cohesion<br />
(SCC) during S phase but its role is poorly understood.<br />
Sister chromatid cohesion is essential for<br />
chromosome segregation and DNA double-strand<br />
break repair by homologous recombination. The<br />
establishment of SSC requires the coordinated activites<br />
of cohesins, and proteins required for cohesin<br />
deposition and cohesion establishment. Mutations in<br />
any of these factors result in precocious sister separation,<br />
aneuploidy and cell death. Mutations in human<br />
cohesion factors are known to contribute to cancer<br />
progression and premature aging.<br />
Correlations between DNA replication and sister<br />
chromatid cohesion have been shown, SCC can<br />
be established only during S phase, and proteins<br />
important for DNA replication interact directly with<br />
proteins involved in sister chromatid cohesion. The<br />
observation that CHL1 genetically and physically<br />
interacts with Eco1p (Establishment cohesion 1), a<br />
protein involved in the regulation of SCC establishment,<br />
supports the hypotesis of a role for Chl1p in<br />
preserving genome stability during S phase. Our<br />
interest is to understand the role of Chl1p during<br />
DNA replication in S. cerevisiae.<br />
Chl1p shares a high sequence similarity in the core<br />
helicase domain with a recently identified human<br />
Fanconi Anemia (FA) gene implicated in FA complementation<br />
group J, FANCJ. FANCJ was originally<br />
designed BACH1 (BRCA1 Associated C-terminal<br />
Helicase), a protein that binds to the tumor suppressor<br />
BRCA1. Mutations in FANCJ/BACH1 are<br />
associated with breast cancer and genetically linked<br />
to the bone marrow disease Fanconi Anemia (FA).<br />
We identified a point mutation in chl1 ofm5 , a transition
Lucia Fabiani<br />
of G to A at the level of nucleotide 275 of the coding<br />
sequence, converting a Trp codon (TGG) at the<br />
aminoacid 92 to a stop codon (TAG). This premature<br />
stop codon cause a null allele: all the important<br />
helicase domains are lost. chl1 deletion mutant is<br />
sensitive to hydroxyurea (HU) (depletion of dNTP<br />
pools), methylmethanesulfonate (MMS) (alkylation<br />
of bases) and UV radiation (pyrimidine dimer formation).<br />
We analyzed in wildtype and chl1 deletion mutant<br />
the correct activation of the S phase checkpoints<br />
(replication and intra-S checkpoints), by Western<br />
blot analysis following phosphorylation of Rad53p,<br />
and the replication intermediates, by two-dimensional<br />
gel electrophoresis, of ARS305 to understand the<br />
potential role of Chl1p at the replication fork. Both<br />
analysis were carried out after synchronization in G2<br />
and release in sublethal concentrations of HU and<br />
MMS. Both checkpoint mechanisms are efficiently<br />
activated and no difference was observed between<br />
wildtype and chl1 replication intermediates.<br />
Recently it has been proposed that different proteins<br />
are involved in binding and removal of cohesins<br />
in different chromosomal locations then these different<br />
locations can use different pathways for the<br />
establishment of SCC. Sister chromatid cohesion in<br />
ribosomal DNA (rDNA) is essential for the proper<br />
alignment of the chromatids to avoid unequal recombination<br />
between the repeats important to mantain<br />
their correct copy number. In S. cerevisiae rDNA is<br />
organized as 150-200 tandem repeats and is localized<br />
on chromosome XII. rDNA replication is preferen-<br />
48<br />
tially unidirectional for the presence of a replication<br />
fork barrier (RFB) to which the protein Fob1 (Fork<br />
blocking protein 1) is specifically associated, the<br />
replication is via the lagging strand apparatus and<br />
uses as template the G rich strand. G quadruplex<br />
DNA can be formed on this strand, this structure<br />
represents a problem to the fork progression. We<br />
analyzed the replication intermediates at the level of<br />
the region of chromosome XII containing the rDNA<br />
cluster and we finally found a difference between<br />
wildtype and chl1 mutant, we observed an increase<br />
of the RFB.<br />
FANCJ /BACH1, Chl1p human homolog, is able<br />
to unwind G4 DNA structure with 5′–3′ polarity,<br />
this recent result suggests a possible role of Chl1p<br />
in the maintenance of potential unstable genomic<br />
G/C tracts during replication. The 5′–3′ polarity<br />
is consistent with resolution of G4 structure on the<br />
lagging strand during replication. The loss of G4<br />
unwinding function in chl1 mutant during rDNA<br />
replication could correlate with the increase at the<br />
RFB of replication intermediates highly ricombinogenic<br />
and source of genomic instability.<br />
We verified the presence of Chl1p at the level of<br />
the replication fork by ChIP (Chromatin Immuno-<br />
Precitation) by adding a tag sequence at the<br />
C-terminus of the protein. Chl1p is able to bind to<br />
the rDNA origin of DNA replication. We are now<br />
analyzing the binding of Chl1p at the level of other<br />
origins of DNA replication and the presence of<br />
potential G4 DNA on the G rich strand of rDNA<br />
used as template.
New tools in deciphering aging and apoptotic pathways<br />
Principal investigator: Claudio Falcone<br />
Professor of Industrial Microbiology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912278; Fax: (+39) 06 49912256<br />
claudio.falcone@uniroma1.it<br />
Participants:<br />
Cristina Mazzoni, researcher; Vanessa Palermo, postdoc<br />
fellow; Mirko Torella, PhD student; Michele Saliola,<br />
technician.<br />
Collaborations:<br />
Institute of Molecular Biosciences, University of Graz, Austria<br />
(Prof. Frank Madeo); Dipartimento di Medicina e Patologia<br />
Sperimentale, Sapienza-Università di Roma (Prof. Patrizia<br />
Mancini); <strong>Istituto</strong> di Biologia e Patologia Molecolari, CNR, Roma<br />
(Dr. Enrico Cundari).<br />
Report of activity<br />
The aim of this project was the isolation of genes acting<br />
as suppressors of yeast apoptotic phenotypes and<br />
the study of their suppression mechanism.<br />
In recent years it has been demonstrated the existence<br />
of an apoptotic program in unicellular eukaryotes,<br />
and some crucial components of the apoptotic<br />
cascade such as AIF, caspase and Omi have been<br />
described in the yeast S. cerevisiae, indicating a<br />
high evolutionary conservation of this process. In<br />
addition, oxidative stress and the expression of proapoptotic<br />
genes involved in mammalian pathways,<br />
induce apoptosis also in yeast cells.<br />
The biological significance of apoptosis in unicellular<br />
organisms may be related to the possibility of eliminating<br />
older or damaged cells in favor of younger<br />
and healthy cells.<br />
Concerning aging, the relationships between this<br />
process and apoptosis in eukaryotes are controversial.<br />
Even though old cells are more resistant to apoptotic<br />
stimuli, they are naturally predisposed to cell death,<br />
favoring the long-term survival of the population.<br />
In our laboratory we have shown that mutants<br />
in genes involved in decapping (dcp1, dcp2, lsm1,<br />
Kllsm4D1, lsm6, lsm7), one of the first steps for the<br />
degradation of mRNAs, show an increased loss of viability<br />
during the stationary phase, compared to wild<br />
49<br />
AREA 3<br />
cells, indicating the occurring of premature aging.<br />
Moreover, this kind of cell death is linked to apoptotic<br />
events because cells show the accumulation of<br />
reactive oxygen species (ROS), DNA fragmentation<br />
and chromatin condensation.<br />
We also demonstrated that the stabilization of mRNA<br />
triggers apoptosis via a caspase-dependent pathway.<br />
Decapping mutants, affected at very early stages<br />
of the apoptotic cascade, show increased sensitivity<br />
inducers of ROS production and apoptosis, representing<br />
a good tool for the isolation of genes that<br />
counteract the aging process and cell death.<br />
To this purpose, the mutant strain Kllsm4Δ1 was<br />
transformed with a yeast genomic library and clones<br />
resistant to acetic acid, a known inducer of oxidative<br />
stress and apoptosis in yeast, were selected for<br />
further studies.<br />
One of these clones carried a DNA fragment containing<br />
the HIR1 (Histone transcriptional repressor)<br />
gene, which encodes a transcriptional co-repressor<br />
of histone genes.<br />
The over-expression of HIR1 in the mutant Kllsm4Δ1<br />
prevented rapid cell death during chronological<br />
aging, reduced nuclei fragmentation and increased<br />
H 2 O 2 resistance, indicating a relationship between<br />
this gene and apoptosis.<br />
The absence of HIRA, the human gene corresponding<br />
to HIR1, has been associated with Di George<br />
syndrome in which a spectrum of malformations and<br />
diseases of development and a very large increase<br />
in spontaneous apoptosis in T lymphocytes are<br />
observed. Moreover, HIRA is involved, along with<br />
ASF1 in the formation of SAHF (senescence-associated<br />
heterochromatin foci).<br />
The existence of relations between HIR1 and apoptosis<br />
offers new opportunities to study this phenomenon<br />
in a simple model such as yeast.<br />
Transcriptional analysis revealed that the expression<br />
of histone genes was lowered in the Kllsm4Δ1mutant<br />
after HIR1 over-expression, suggesting that a reduction<br />
of transcription counteracts the increased stabil-
Claudio Falcone<br />
ity of mRNA observed in this mutant.<br />
To better understand this relationship, we checked<br />
the expression of the histone genes during the cell<br />
cycle, with cells in that their transcription is tightly<br />
regulated both transcriptionally and post-transcriptionally<br />
and coupled to rates of DNA synthesis.<br />
We demonstrated that in the Kllsm4Δ1 mutant,<br />
histone mRNAs are induced early in the S-phase<br />
and maintained at high level all along the entire cell<br />
cycle, showing a delay in the exit from S-phase and/<br />
or entry into M-phase.<br />
The overexpression of the HIR1 gene also restored<br />
the normal cycling of histone genes expression.<br />
We also found that low doses of hydroxyurea (HU),<br />
which inhibits the ribonucleotide<br />
reductase and the production of dNTPs required for<br />
DNA synthesis, neutralized the onset of the apoptotic<br />
phenotypes in Kllsm4Δ1, as well in another mRNA<br />
decapping mutants (lsm1). In these conditions, we<br />
observed an increase of the chronological lifespan in<br />
both strains, suggesting that a delayed entry into the<br />
S phase can recover some cellular defects in decapping<br />
mutants.<br />
Our work presents clear evidence for a link between<br />
regulation of S-phase progression, histone mRNA<br />
control and cell death. We speculated that the HU<br />
rescuing effects might result from adjusting a detrimental<br />
imbalance in the NTP/dNTP equilibrium<br />
that potentially arises upon increased mRNA stabilization<br />
(for instance through enhanced NTP incorporation<br />
in mRNA molecules). Another explanation for<br />
the phenotypes of lsm mutants is that, consequent<br />
to the high levels of histone mRNAs, these mutants<br />
may accumulate excess of histonic proteins that,<br />
similarly to dNTP depletion, induce DNA replication<br />
stress.<br />
In mammalian cells, this kind of stress activates inter-Sphase<br />
checkpoints and causes selective histone mRNA<br />
degradation. Importantly, this process requires both<br />
ATR, a key regulator of the intra S-phase checkpoint,<br />
as well as Upf1, a crucial factor of the nonsense-mediated<br />
decay pathway, which monitors mRNA quality and<br />
ensures degradation of defective mRNAs. In yeast, such<br />
interplay between the DNA intra S-phase checkpoint<br />
50<br />
and the nonsense-mediated mRNA decay pathway<br />
might assure the proper coordination of chromatin<br />
structure replication.<br />
Such coupling could occur at a bidirectional level, not<br />
only allowing a DNA-to-RNA but also a RNA-to-<br />
DNA signaling. In the latter case, aberrant mRNA<br />
levels could be signaled to the intra S-phase checkpoint<br />
to stall cell cycle progression. Such RNA-DNA<br />
regulation cycles are apparently closely connected<br />
to cell death control, though the underlying mechanisms<br />
remain to be explored.<br />
Western blot experiments revealed that the levels<br />
of Sir2p, the NAD-dependent histone deacetylase,<br />
are lower in the decapping mutants compared to the<br />
wild type.<br />
The reduced amount of Sir2 results in increased<br />
transcription of chromosomal sites, such as the<br />
rDNA and the subtelomeric regions, which are normally<br />
repressed by this deacetylase.<br />
ChIP experiments have indeed shown an increase in<br />
recruitment of RNA polymerase II (RNApolII) to these<br />
sites in the Kllsm4D1 mutant compared to the wild type.<br />
The connection between the lower amount of Sir2<br />
protein and the increased chronological aging of<br />
decapping mutants is still under investigation.<br />
Publications<br />
Mazzoni C, Torella M, Petrera A, Palermo V,<br />
Falcone C. PGK1, the gene encoding the glycolitic<br />
enzyme phosphoglycerate kinase, acts as a multicopy<br />
suppressor of apoptotic phenotypes in S. cerevisiae.<br />
Yeast. 2009, 26: 31-7. doi: 10.1002/yea.1647.<br />
Palermo V, Cundari E, Mangiapelo E, Falcone C,<br />
Mazzoni C. Yeast lsm pro-apoptotic mutants show<br />
defects in S-phase entry and progression. Cell Cycle.<br />
2010, 9: 3991-96. doi: 10.4161/cc.9.19.13210.<br />
Palermo V, Falcone C, Calvani M, Mazzoni C.<br />
Acetyl-L-carnitine protects yeast cells from apoptosis<br />
and aging and inhibits mitochondrial fission.<br />
Aging Cell. 2010, 9: 570-9. doi: 10.1111/j.1474-<br />
9726.2010.00587.
Principal investigator: Laura Frontali<br />
Professor of Microbial Chemistry<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 4453950, Fax: (+39) 06 4461980<br />
laura.frontali@uniroma1.it<br />
51<br />
AREA 3<br />
Mitochondrial studies and the use of simple eukaryotic models for<br />
the analysis of molecular and cellular aspects of human pathologies<br />
Participants:<br />
Claudio Palleschi, Michele M. Bianchi, professors; Silvia<br />
Francisci, Teresa Rinaldi, Daniela Uccelletti, researchers;<br />
Cristina De Luca, post-doc fellow; Michela Esposito, Chiara<br />
Micolonghi, PhD students.<br />
Collaborations:<br />
Laboratoire de Génétique Moléculaire, Université de Paris Sud,<br />
Orsay, France (Prof. Monique Bolotin-Fukuhara, Dr. Agnès<br />
Delahodde); Department of Biology, The Technion, University of<br />
Haifa, Israel (Dr. Michael Glickman); Laboratoire de Génétique<br />
des Levures, Université Lyon 1, France (Prof. Micheline<br />
Wésolowski-Louvel).<br />
Report of activity<br />
Many human pathologies are related to mutations in<br />
very conserved genes. The present project aims to<br />
study the molecular and cellular aspects of some of<br />
these pathologies by the use of models derived from<br />
simple eukaryotes such as yeasts and the nematode<br />
Caenorhabditis elegans, with the purpose of verifying<br />
the possibility that the coordinate use of these models<br />
might increase our understanding of the above mentioned<br />
dysfunctions.<br />
The pathologies we are planning to investigate<br />
are mainly connected with mitochondrial functions<br />
and respiratory regulation, which are specially well<br />
known in yeasts, and could be more deeply investigated<br />
in the worm.<br />
Pathologies due to mutations in mit tRNA<br />
genes<br />
Base substitutions in mit tRNA genes cause in human<br />
very severe diseases mainly neurodegenerative. No<br />
therapies exist at present for these chronic devastating<br />
pathologies.<br />
We have set up a yeast model, by exploiting the special<br />
characteristics of yeast, which is the only eukaryotic<br />
cell amenable, by biolistic procedures, to mitochondrial<br />
transformation and which can survive on glucose by<br />
fermentation even when respiration is deeply affected.<br />
We have produced, by biolistic procedures a series of<br />
human equivalent mt yeast mutants, which proved to<br />
exhibit impaired respiration. The molecular mechanism<br />
of the defects was also shown to be similar in<br />
human and yeast cells (Montanari et al., 2008).<br />
The most important result we have obtained in yeast<br />
mutants was the observation that respiratory defects<br />
were rescued (suppressed) by the overexpression of<br />
some nuclearly encoded tRNA interactors such as<br />
the mit protein synthesis elongation factor Ef-Tu and<br />
the aminoacyl-tRNA synhetases (aa-RS) ( De Luca et<br />
al., 2009).<br />
Several laboratories recently confirmed that the<br />
same suppression could be obtained by overexpression<br />
of the horthologous human genes in patient cell<br />
lines and cybrids, thus reinforcing the possibility of<br />
a therapeutic perspective.<br />
The mechanism of suppression was studied in detail, in<br />
particular for the aminoacyl-tRNA synthetases (aa-RS)<br />
and was found to be independent from catalytic activity.<br />
This suggested the hypothesis that the suppressive<br />
effect may be due to the chaperon-like activity of a polypeptide<br />
restoring the altered structure of the mutated<br />
tRNA. We also found that both yeast and human<br />
mit leucyl-RS can suppress the respiratory defects of<br />
human equivalent yeast mutants in tRNALeu, tRNAIle<br />
and tRNAVal (Montanari et al., 2010).<br />
We plan now to localize the suppressor activity in<br />
the different domains of leu-RS. Our recent results<br />
on this aspect may have a clinical importance and<br />
Rome University is filing a patent on this subject.<br />
Mitochondrial pathologies caused by<br />
defective nuclear genes<br />
We have set up a yeast model for SPG13 spastic paraplegia,<br />
associated with mutations in the HSPD1 locus.<br />
We have indeed shown that the human HSPD1 gene is<br />
a functional homologue of the yeast HSP60. In particular,<br />
we verified that the cDNA for HSPD1 suppresses
Laura Frontali<br />
the lethality of the ΔHSP60 S. cerevisiae strain. We<br />
constructed two expression plasmids for yeast, bearing<br />
the mutated cDNA HSPD1. They are pV98I, producing<br />
the replacement of valine with isoleucine and<br />
pQ461E, producing the replacement of glutamine with<br />
glutamic acid. The transformation of the heterozygous<br />
diploid strain of S. cerevisiae HSP60/hsp60::kan<br />
with pV98I and subsequent sporulation showed that<br />
the mutant human gene is not able to replace functionally<br />
the HSP60 gene. When the heterozygous<br />
diploid strain of S. cerevisiae HSP60/hsp60::kan was<br />
transformed with the plasmid pQ461E, viable haploid<br />
strains expressing hsp60::kan and the mutated form<br />
of hspd1 were obtained after sporulation. However,<br />
the resulting strains are defective in mitochondrial<br />
function, as shown by reduced oxygen consumption<br />
capabilities, formation of petites, altered mitochondrial<br />
morphology (manuscript in preparation).<br />
In addition, we found that the HSPD1 cDNA suppresses<br />
oxidative defects in the yeast Kluyveromyces<br />
lactis due to inactivation of the Ca 2+ -ATPase, KlPMR1<br />
gene. This suppression was not observed when the<br />
K. lactis mutant was instead transformed with the<br />
plasmid pV98I. We found that this mutation is not<br />
dominant in the yeast model, as in the case of patients.<br />
In parallel, studies were also conducted revealing a<br />
cross-talk between two important cellular processes<br />
such as glycosylation and mitochondrial biogenesis<br />
and in which calcium homeostasis plays an important<br />
role (Zanni et al., 2009).<br />
In order to set up the nematode C. elegans as a model<br />
of SPG13, we got some transgenic strains in which<br />
HSPD1 was overexpressed, however, this construct<br />
was toxic to the nematode, with consequent loss of the<br />
construct in the progeny. We were not able to isolate<br />
other transgenic strains with an integrated HSPD1,<br />
in order to minimize the effects of toxicity. At the<br />
moment, we are trying to transform the nematodes by<br />
biolistic methods, with a plasmid in which the HSPD1<br />
expression is induced only under specific conditions,<br />
such as high temperatures, in order to isolate transgenic<br />
strains that may be stable and useful for our model.<br />
Proteasomal defects<br />
The proteasomal system and in particular the deubiquitinating<br />
enzyme Rpn11 is involved in mitochondrial<br />
dynamics (Rinaldi et al., 2008; Hofmann et<br />
al., 2009) and in several related diseases.<br />
Concerning the human spastic paraplegia, one of<br />
the candidate human genes mutated in the disease<br />
is the Heat Shock Protein 60 (HSPD1). We have<br />
observed in our yeast mutant rpn11-m1 the accumulation<br />
inside mitochondria of proteolysis products of<br />
52<br />
Hsp60. However, we did not succeed in identifying<br />
the mit protease involved, and we only could rule out<br />
Yme1 and Pim1 proteases.<br />
On the other side, we demonstrated a genetic interaction<br />
between the proteasomal mutant rpn11-m1<br />
(exhibiting cell cycle defects) and the yeast strain<br />
deleted for the calcium pump Pmr1(Δpmr1). We<br />
studied the PMR1 gene because the phenotypes<br />
of the deletant of this gene are suppressed by an<br />
overexpression of the HSP60 gene. The absence of<br />
the calcium pump allows a complete suppression of<br />
the cell cycle defects of rpn11-m1. The mechanism<br />
of suppression involves Swe1 activating pathway<br />
(Esposito et al., 2010). This result is supported by the<br />
observation that in the absence of Calcineurin CNB1<br />
gene (an activator of Swe1), the bud elongation phenotype<br />
is still present (Rinaldi et al., 2010).<br />
Since mitochondrial morphology is severely affected<br />
in the Δpmr1, strain and the presence of Calcineurin<br />
is essential for the control of mitochondrial fission in<br />
human, the yeast system could be useful to investigate<br />
the involvement of mitochondrial morphology<br />
in the human spastic paraplegia.<br />
Publications<br />
De Luca C, Zhou Y, Montanari A, Morea V, Oliva<br />
R, Besagni C, Bolotin-Fukuhara M, Frontali L,<br />
Francisci S. Can yeast be used to study mitochondrial<br />
diseases? Biolistic tRNA mutants for the analysis<br />
of mechanisms and suppressors. Mitochondrion. 2009,<br />
9: 408-17. doi: 10.1016/j.mito.2009.07.004.<br />
Hofmann L, Saunier R, Cossard R, Esposito M, Rinaldi<br />
T, Delahodde A. A non-proteolytic activity of the proteasome<br />
controls fission of organelles in yeast. J of Cell<br />
Science. 2009, 122: 3673-83. doi: 10.1242/jcs.050229.<br />
Zanni E, Farina F, Ricci A, Frank C, Mancini P,<br />
Palleschi C, Uccelletti D. The Golgi alpha 1,6 mannosyltransferase<br />
KlOch1p of Kluyveromyces lactis is<br />
required for Ca2+/calmodulin-based signaling and<br />
for proper mitochondrial functionality. BMC Cell<br />
Biology. 2009, 10: 86. doi: 10.1186/1471-2121-10-86.<br />
Montanari A, De Luca C, Frontali L, Francisci S.<br />
Aminoacyl-tRNA synthetases are multivalent suppressors<br />
of defects due to human equivalent mutations in<br />
yeast mt tRNA genes. Biochimica et Biophysica Acta. 2010,<br />
1803: 1050-57. doi:10.1016/j.bbamcr.2010.05.003.<br />
Rinaldi T, Dallabona C, Ferrero I, Frontali L,<br />
Bolotin-Fukuhara M. Mitochondrial diseases and the<br />
role of the yeast models. FEMS Yeast Res. 2010, 10:<br />
1006-22. doi: 10.1111/j.1567-1364.2010.00685.
The role of membrane trafficking in Drosophila cytokinesis<br />
Principal investigator: Maurizio Gatti<br />
Professor of Genetics<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912842; Fax: (+39) 06 4456866<br />
maurizio.gatti@uniroma1.it<br />
Participants:<br />
Silvia Bonaccorsi, Maria Grazia Giansanti, Patrizia Somma,<br />
Fiammetta Vernì, researchers; Elisabetta Bucciarelli,<br />
Violaine Mottier, Valeria Naim, Alan Wainman, post-doc<br />
fellows; Claudia Pellacani, PhD student.<br />
Collaborations:<br />
Stanford University, USA (Prof. Margareth Fuller); Cornell<br />
University, USA (Prof. Michel L. Goldberg); Exeter University,<br />
UK (Prof. James Wakefield).<br />
Report of activity<br />
During animal cell cytokinesis, constriction of the<br />
acto-myosin ring leads to the formation of a furrow<br />
in the plasma membrane, which invaginates until the<br />
two daughter cells remain connected by a thin cytoplasmic<br />
bridge, called the midbody. This bridge is<br />
ultimately cleaved during the final step of cytokinesis,<br />
named abscission, which results in the complete<br />
separation of daughter cells. Both cleavage furrow<br />
ingression and abscission require substantial membrane<br />
remodelling. Membrane addition to the invaginating<br />
furrow involves vesicle delivery through<br />
both the secretory and the endocytic pathways.<br />
In the secretory pathway, vesicles are transported<br />
from the endoplasmic reticulum (ER) to the Golgi<br />
and then to the plasma membrane. In the endocytic<br />
pathway, plasma membrane-derived vesicles proceed<br />
to the recycling endosome (RE), which directs them<br />
back to the plasma membrane.<br />
Our goal is elucidation of the molecular mechanisms<br />
underlying membrane addition to the advancing<br />
cleavage furrow in Drosophila spermatocytes.<br />
We have recently identified mutations in several<br />
genes required for furrow ingression during meiotic<br />
cytokinesis of Drosophila males. Most of these genes<br />
encode products involved in membrane structure<br />
and/or trafficking such as an Elovl protein, the<br />
Trs120p subunit of the TRAPP II complex, Rab1,<br />
53<br />
AREA 3<br />
the Exocist complex components Exo84 and Sec8,<br />
the ortholog of PACS-1 and Zw10. We are defining<br />
the roles of these proteins in furrow ingression,<br />
and investigating their functional relationships with<br />
other proteins involved in membrane traffic during<br />
cytokinesis.<br />
In the past three years we have carried out three<br />
main research projects.<br />
The role of bond in furrow ingression during<br />
cytokinesis in Drosophila spermatocytes<br />
Recent work indicates that plasma membrane lipids<br />
influence membrane biophysical properties such<br />
as membrane curvature and elasticity and play an<br />
active role in cell function. We found that mutations<br />
in the gene bond, which encodes a Drosophila<br />
member of the family of Elovl proteins that mediate<br />
elongation of very-long-chain fatty acids, block or<br />
dramatically slow cleavage-furrow ingression during<br />
early telophase in dividing spermatocytes. Bond<br />
shares sequence motifs with human and yeast Elovl<br />
family members, including five to seven predicted<br />
transmembrane domains, and can substitute for elovl<br />
genes in S. cerevisiae. In bond mutant cells at late<br />
stages of meiotic division, the contractile ring frequently<br />
detaches from the cortex and constricts or<br />
collapses to one side of the cell, and the cleavage furrow<br />
regresses. These findings implicate very-longchain<br />
fatty acids or their derivative complex lipids<br />
in allowing supple membrane deformation and the<br />
stable connection of cortical contractile components<br />
to the plasma membrane during cell division.<br />
brunelleschi (bru) interacts with Rab11 and is<br />
required for meiotic cytokinesis in Drosophila<br />
males<br />
We have found that successful furrow ingression<br />
during meiotic cytokinesis in Drosophila males<br />
requires function of the brunelleschi (bru) gene, which<br />
encodes the ortholog of the yeast transport protein
Maurizio Gatti<br />
particle (TRAPP) II complex subunit, Trs120p.<br />
Dividing spermatocytes from bru mutants assemble<br />
a normal contractile ring, which initiates but fails to<br />
complete constriction. The Bru protein fused to GFP<br />
is dispersed throughout the cytoplasm and enriched<br />
at Golgi organelles, similarly to another Drosophila<br />
TRAPPII subunit, dBet3. bru genetically interacts<br />
with Rab11, which encodes a small GTPase, and with<br />
the phosphatidylinositol 4-kinase (PI4K)-encoding<br />
gene four wheel drive (fwd). Consistent with these<br />
findings, localization of the Rab11 protein to the<br />
cleavage furrow requires wild-type function of bru.<br />
These results suggest that Bru cooperates with<br />
Rab11 and PI4K to regulate the efficiency of membrane<br />
addition to the cleavage furrow. The genetic<br />
interactions between bru, fwd (PI4K) and Rab11 are<br />
consistent with the known relationships between<br />
their orthologs in budding yeast, suggesting that<br />
membrane trafficking processes that are essential to<br />
growth and viability in yeast support cleavage furrow<br />
ingression in Drosophila.<br />
Zw10 is required for cytokinesis with a role in<br />
membrane addition during cleavage furrow<br />
ingression<br />
Zw10, along with the other members of the conserved<br />
Rod-Zwilch-Zw10 (RZZ) complex, is required for<br />
proper functioning of the mitotic spindle assembly<br />
checkpoint (SAC). Recent studies in mammalian cells<br />
have shown that Zw10 forms a complex with syntaxin-18<br />
and is involved in membrane traffic between<br />
the Golgi and the endoplasmic reticulum (ER). We<br />
found that male meiotic divisions in zw10 mutants<br />
display defects in cytokinesis; the acto-myosin ring<br />
forms and begins to ingress but then regresses.<br />
Surprisingly, this cytokinesis failure is observed<br />
in zw10 mutants but not in rod or zwilch mutants,<br />
even though all three members of the RZZ complex<br />
localize across the mid-zone of the spindle envelope<br />
54<br />
during telophase. In zwilch and rod mutants Zw10 no<br />
longer localizes to the kinetochores and the spindle<br />
envelope mid-zone but concentrates in the ER and<br />
the Golgi-derived vesicles. These results indicate<br />
that Zw10 association to the spindle envelope is not<br />
essential for cytokinesis and suggest that Zw10 regulates<br />
the ER-Golgi membrane traffic required for<br />
proper membrane addition to the cytokinetic furrow.<br />
Perspectives<br />
A paper on Zw10 is currently in preparation, and our<br />
work on Rab1 and the Exocist complex components<br />
Exo84 and Sec8 is almost complete. We will continue<br />
the study of the Drosophila ortholog of PACS-1 encoded<br />
by smeagol (sgo). We believe that the analysis of the<br />
role of these proteins in spermatocyte cytokinesis will<br />
help unravel the mechanism of membrane addition to<br />
the cleavage furrow, and the relationships between<br />
membrane traffic and acto-myosin ring constriction<br />
Publications<br />
Bucciarelli E, Pellacani C, Naim V, Palena A, Gatti<br />
M, Somma MP. Drosophila Dgt6 protein interacts<br />
with Ndc80, Msps/XMAP215 and gamma-tubulin to<br />
promote kinetochore-driven MT formation. Curr Biol.<br />
2009, 19: 1839-45. doi:10.1016/j.cub.2009.09.043.<br />
Robinett CC, Giansanti MG, Gatti M, Fuller MT.<br />
TRAPPII function is required for cleavage furrow<br />
ingression and localization of Rab11 in dividing<br />
male meiotic cells of Drosophila. J Cell Sci. 2009, 122:<br />
4526-34. doi: 10.1242/jcs.054536.<br />
Wainman A, Creque J, Williams B, Williams EV,<br />
Bonaccorsi S, Gatti M, and Goldberg ML. Roles of<br />
the Drosophila NudE protein in kinetochore function<br />
and centrosome migration. J Cell Sci. 2009, 122:<br />
1747-58. doi:10.1242/jcs.041798.
Principal investigator: Marco Lucarelli<br />
Professor of Clinical Biochemistry and Clinical Molecular Biology<br />
Dipartimento di Biotecnologie Cellulari ed Ematologia<br />
Tel: (+39) 06 4451784<br />
marco.lucarelli@uniroma1.it<br />
55<br />
AREA 3<br />
The interplay between epigenetics, cell cycle and homologous<br />
recombination in the gene therapy by Small Fragment Homologous<br />
Replacement (SFHR)<br />
Participants:<br />
Roberto Strom, professor; Fabrizio Ceci, Giampiero<br />
Ferraguri, researchers; Silvia Pierandrei, PhD student.<br />
Collaborations:<br />
Dipartimento di Biopatologia e Diagnostica per Immagini,<br />
Università di Roma Tor Vergata (Prof. Federica Sangiuolo, Prof.<br />
Giuseppe Novelli, Dr. Paola Borgiani, Dr. Annalisa Botta,<br />
Dr. Andrea Luchetti, Dr. Arianna Malgieri).<br />
Report of activity<br />
The aim of this project is to clarify the molecular<br />
mechanisms underlying a gene therapy approach,<br />
called Small Fragment Homologous Replacement<br />
(SFHR), focusing on the scarcely studied relationships<br />
between SFHR, DNA methylation and repair,<br />
chromatin structure and cell cycle. Homologous<br />
recombination (HR) is the DNA recombination during<br />
meiosis. In mitotic cells, however, HR is a basic<br />
mechanism to repair DNA. SFHR is an in situ gene<br />
targeting approach able to stably modify a genomic<br />
sequence by small DNA fragment (SDF), probably<br />
based on HR and DNA repair pathways, allowing the<br />
recovery of a normal gene function. The molecular<br />
mechanism of SFHR is poorly understood, with its<br />
potential currently limited by a low and variable frequency<br />
of correction, with several factors influencing<br />
its efficiency.<br />
Before the start of this project two cell lines have<br />
been established from a mouse model of Spinal<br />
Muscular Atrophy (SMA; OMIM #253300) type I:<br />
immortalized murine embryonic fibroblasts (MEF)<br />
and murine embryonic stem (mES) cells. They are<br />
both deleted for the unique murine SMN1 gene, but<br />
contain human SMN2 gene. The wild type human<br />
SMN1 gene produces the functional full length transcript<br />
(FL-SMN); the human non-functional SMN2<br />
gene has a single C>T nucleotide transition that<br />
results in exon 7 skipping (Δ7-SMN). The SMN2 is<br />
unable to complement the deletion of SMN1 gene<br />
that originates SMA; however, as the SMN2 gene<br />
can express SMN protein, correction of SMN2 is an<br />
attractive therapeutic option. The MEF have been<br />
stably modified integrating within their genome<br />
the wild type enhanced green fluorescent protein<br />
wtEGFP sequence, in which a non sense mutation<br />
was also created (mEGFP). In MEF-mEGFP, a SDF<br />
homologous to the wtEGFP sequence can integrate<br />
and restore the fluorescence of the cells. The efficiency<br />
of SFHR may be evaluated at DNA and RNA<br />
levels, as well as functionally quantified by cytofluorimetric<br />
analysis. In mES-SMN2 cells, a SDF homologous<br />
to the SMN1 gene can replace the point mutation<br />
of SMN2 to restore the wild type phenotype. In<br />
this case, the efficiency of SFHR may be evaluated<br />
at DNA and RNA levels, as well as by induction of<br />
differentiation to functional motor neurons, evaluated<br />
at morphological and biochemical levels. The<br />
MEF-mEGFP and the mES-SMN2 are two <strong>report</strong>er<br />
systems that provide a platform for developing and<br />
testing new strategies for gene repair.<br />
In this project, the MEF-mEGFP was firstly used<br />
with the aim of later transfer the main results<br />
obtained to the mES-SMN2 system. The best experimental<br />
conditions in term of transfection, correction<br />
efficiency and cell viability were selected in unsynchronized<br />
MEF-mEGFP: the best electroporation<br />
program on the Amaxa nucleofection system, the<br />
optimal SDF length (from 850 to 900 bp) and the<br />
optimal SDF concentration (12 x 10 6 SDF molecules/cell).<br />
Double strand, blunt end or PCR amplified<br />
(SDF-PCR) SDFs showed higher correction efficiency<br />
than, respectively, single strand, sticky ends<br />
or plasmid-digested (SDF-DIG) SDFs. To determine<br />
the cell cycle influence, we evaluated gene targeting<br />
in G0/G1 (by mimosine), G1/S (by thymidine) and<br />
G2/M (by vinblastine) phases. In G2/M the SFHR<br />
resulted favored, reaching 0.5% of correction, probably<br />
because of the absence of nuclear envelope or to<br />
the tetraploid situation present before cell mitosis.
Marco Lucarelli<br />
To clarify the molecular mechanism for the lower<br />
correction efficiency of SDF-DIG than SDF-PCR,<br />
the methylation pattern of both SDFs was studied<br />
using methylation-sensitive restriction enzymes and<br />
subsequent PCR amplification. Both DCM and DAM<br />
bacterial methylation resulted present on the SDF-<br />
DIG fragment whereas, obviously, no methylation<br />
was present on the SDF-PCR. To directly check if<br />
the SDF methylation may be the molecular base for<br />
a diminished correction efficiency, SDF fragments<br />
with specific methylation patterns were produced by<br />
SssI, Dam, and both DNA-methyltransferases and<br />
transfected into G2/M synchronised cells. Results<br />
showed an SFHR efficiency 50% and 80% lower<br />
than that of, respectively, SDF-DIG and unmethylated<br />
SDF-PCR. This reduction in the SFHR efficiency<br />
may originate from the creation of complexes<br />
between methylated SDFs and nuclear proteins that<br />
inhibit the integration. The purity and stability of<br />
the correction were confirmed, after culture of sorted<br />
corrected (fluorescent) cells for several passages, by<br />
RFLP analysis, allelic discrimination real time PCR<br />
and Southern blot analysis; this last technique also<br />
excluded any random integration of SDF within<br />
genomic DNA.<br />
With increasing cell passages, corrected cells gradually<br />
lost EGFP fluorescence. As retromutation was<br />
excluded by RFLP analysis and sequencing, the<br />
hypothesis was that DNA methylation may cause<br />
transcriptional inactivation of the corrected EGFP<br />
gene. In effect, the corrected cells that progressively<br />
lost their fluorescence resulted, at a HpaII/<br />
PCR assay, more methylated than the corrected still<br />
fluorescent cells, with a specific methylation pattern<br />
involving only some CpG sites. In addition, the<br />
methylation pattern appeared to be influenced by the<br />
SFHR process as it appeared different between corrected<br />
MEF-mEGFP and MEF-wtEGFP, which did<br />
not undergo correction, both showing progressively<br />
inactivating EGFP. A cause/effect relationship was<br />
evidenced by the fact the DNA methylation and<br />
EGFP expression were reverted by the treatment<br />
56<br />
with the demethylating agent 5-aza-2’-deoxycytidine.<br />
To test their influence on the SFHR mechanism,<br />
3 main biological pathways involved in the<br />
repair of the DNA damage were investigated: a) the<br />
DNA double-strand break repair based on the ATM<br />
kinase pathway, using its inhibitor KU-55933; b)<br />
the transcription-coupled nucleotide excision repair<br />
based on the RNA polymerase II activity, using its<br />
inhibitor α-amanitine; c) the non homologous recombination<br />
based on the poly-(ADP-ribose) polymerase<br />
pathway, using its inhibitor 1,5-isoquinolinediol. A<br />
modulation of SFHR efficiency was evidenced by<br />
each of these inhibitors, highlighting the interconnection<br />
of SFHR mechanism with all these biological<br />
pathways.<br />
Using the best experimental conditions optimized<br />
by the MEF–mEGFP assay system, we also treated<br />
the mES-SMN2 system. A SDF-PCR homologous to<br />
human SMN1 sequence was used to correct the nonfunctional<br />
SMN2 gene, in undifferentiated mES cells<br />
synchronized in G2/M phase. After transfection the<br />
levels of FL-SMN and Δ7-SMN were quantitatively<br />
evaluated at RNA level, by real time PCR, and at protein<br />
level, by western blot. The increase of FL-SMN<br />
transcript, due to SMN2 to SMN1 sequence conversion,<br />
resulted to be about 70%, moreover a 14%<br />
decrease in Δ7-SMN transcript was detected. Both<br />
the transcript FL-SMN/Δ7-SMN ratio and the<br />
protein FL-SMN/tubulin ratio increased 1.8 fold in<br />
respect to controls non-transfected or transfected<br />
with an heterologous SDF.<br />
These results contribute to the comprehension of<br />
the molecular mechanism of the SFHR, in both stem<br />
and differentiated embryonic cells, also providing<br />
fundamental suggestions for increasing gene repair<br />
efficiency. This can open new perspectives for a<br />
SFHR gene targeting strategy applicable to ex vivo<br />
protocols.<br />
A manuscript concerning these first results is in<br />
preparation for Molecular Therapy.
Interplay between myogenic factors and cell cycle control:<br />
regulation and role of the cdk inhibitor p57kip2<br />
Principal investigator: Rossella Maione<br />
Professor of Applied Biology<br />
Dipartimento di Biotecnologie Cellulari ed Ematologia<br />
Tel: (+39) 06 4457737; Fax: (+39) 06 4462891<br />
maione@bce.uniroma1.it<br />
Participants:<br />
Anna Busanello, Rosaria Carbone, Marianna Rossi, postdoc<br />
fellows; Cecilia Battistelli, Cassandra Mostocotto, PhD<br />
students.<br />
Collaborations:<br />
<strong>Istituto</strong> di Neurobiologia e Medicina Molecolare, CNR, Roma (Dr.<br />
Maurizia Caruso).<br />
Report of activity<br />
Objectives<br />
The general aim of this research is to gain insight<br />
into the mechanisms by which myogenic factors<br />
activate growth arrest pathways and into the roles<br />
played by cell cycle regulators during the process of<br />
skeletal myogenesis.<br />
Several cdk inhibitors (CKIs) are up-regulated during<br />
myogenesis. The function and the regulation of p57kip2<br />
are particularly interesting. p57, the most structurally<br />
complex member of the Cip/Kip family, is the only CKI<br />
which individual ablation causes developmental defects<br />
in mice and shows a restricted expression pattern during<br />
development and in some adult tissues including<br />
muscle. Moreover the gene coding for p57 is located<br />
within an imprinted cluster, involved in growth regulation<br />
and subject to a complex epigenetic control.<br />
The present project has two main objectives.<br />
The first is to identify the epigenetic mechanisms<br />
involved in the transcriptional control of p57 during<br />
myogenesis. The second is to investigate the role of<br />
cell cycle regulators in the control of cell cycle exit<br />
and maintenance of quiescence during skeletal muscle<br />
differentiation and regeneration.<br />
Results<br />
We had previously demonstrated that p57 transcription<br />
is induced by the myogenic factor MyoD<br />
through a pathway requiring intermediate factors<br />
(3,8), a molecular mechanism distinct from that<br />
57<br />
AREA 3<br />
employed for induction of other genes involved in<br />
cell cycle arrest. Furthermore, compared to p21,<br />
p57 showed a more restricted expression pattern<br />
in differentiating muscle cells. More recent results<br />
indicated that the accessibility of p57 promoter to<br />
MyoD-dependent regulation is prevented, in undifferentiated<br />
myoblasts and in muscle cell types unable<br />
to induce p57, by epigenetic constraints involving<br />
DNA methylation (4). We hypothesized that<br />
KvDMR1, a distant DNA region known to regulate<br />
in cis the hypermethylation and silencing of the<br />
non-expressed p57 allele during imprinting (5), is<br />
also involved in preventing p57 expression in undifferentiated<br />
and un-responsive cells. This hypothesis<br />
has been explored through the following approaches:<br />
Since KvDMR1 is a regulatory element shared by<br />
several genes of the p57 imprinting domain, we compared<br />
the kinetics of p57 induction (and its restriction<br />
in unresponsive cells), with that of other genes of the<br />
domain. The expression analysis showed that Kvlqt1,<br />
a gene co-imprinted with p57, was also co-induced<br />
with p57, while Lit1, a gene oppositely imprinted, was<br />
unaffected or rather down-regulated. Importantly,<br />
Kvlqt1, just like p57, was not induced in un-responsive<br />
cells. These results encouraged us to investigate<br />
the functional interaction of MyoD with KvDMR1.<br />
KvDMR1 is a multifunctional regulatory region<br />
consisting of multiple, independent cis-acting modules<br />
acting as enhancers, promoters, silencers or<br />
enhancer-blocking elements (5). The finding of several<br />
putative MyoD-binding sites within this region<br />
suggested us that the myogenic factor could regulate<br />
the expression of p57 and, possibly, of other genes of<br />
the cluster, by directly binding to KvDMR1. By performing<br />
ChIP assays, specific for different fragments<br />
of the region, we could identify a KvDMR1 subregion<br />
to which MyoD binds in vivo in a differentiation-<br />
and responsiveness-dependent manner. This<br />
finding strongly supports the conclusion that both<br />
the induction and the restriction of p57 expression<br />
involve a long distance regulatory element overlap-
Rossella Maione<br />
ping with the imprinting control region.<br />
The availability of allele-specific polymorphisms in<br />
the imprinting domain allowed us to determine that,<br />
although MyoD binds to both maternal and paternal<br />
KvDMR1 alleles, the induction of p57 involves<br />
the up-regulation of the maternal and not the derepression<br />
of the paternal imprinted p57 allele. The<br />
suggestion emerging is that in undifferentiated and<br />
unresponsive cells p57 expression is prevented by<br />
biallelic silencing and that differentiation signals<br />
are capable to relieve this constraint only from the<br />
maternal allele, and only in responsive cells.<br />
These results are the subject of a manuscript that we<br />
are going to submit.<br />
Concerning the second task, our plan was to identify<br />
specific roles of CKIs in the establishment of the differentiated<br />
state and in the control of satellite cell<br />
function. To this end, we have set up a system for the<br />
conditional silencing of p57 and of its related CKI<br />
p21, based on doxycyclin-regulatable microRNAbased<br />
retroviral vectors. The specific effects of the<br />
knock-down of each CKI are under investigation.<br />
A related research, not initially scheduled in the program<br />
but showing encouraging results, deals with<br />
the role of epigenetic modifications in controlling<br />
the two distinct states of cell cycle arrest occurring<br />
in muscle cells: terminal differentiation of myofibers<br />
and reversible quiescence of satellite reserve cells (2).<br />
Our interest is focused on poly(ADP-ribosyl)ation, a<br />
post-translational modification of chromatin proteins<br />
involved in a variety of physiological processes (6).<br />
We have previously demonstrated that the activity<br />
PARP-1, the main enzyme responsible for poly(ADPribosylation,<br />
is involved in the exit of fibroblasts from<br />
a quiescent state (G0) induced by serum deprivation<br />
(1). This modification is required for the upregulation<br />
of immediate early response genes (IEGs), such<br />
as c-Fos and c-Myc, after mitogen stimulation. We<br />
have recently set up an in vitro skeletal muscle cell<br />
system mimicking the function of satellite cells. C2<br />
myoblasts can be arrested in G0 through suspension<br />
culture in methyl-cellulose. This condition leads to a<br />
quiescent, undifferentiated state, which is characterized<br />
by the expression of satellite cell markers and<br />
which can be reverted by restoring cell adhesion<br />
in the presence of growth factors (7). Western blot<br />
analysis of cell extracts from quiescent myoblasts<br />
showed that poly(ADP-ribose), indicative of PARPactivity,<br />
rapidly accumulates within 2 hours upon cell<br />
cycle re-entry. Moreover, we demonstrated that the<br />
inhibition of PARP activity not only interferes with<br />
the up-regulation of immediate-early genes and with<br />
58<br />
cell cycle re-entry, but also delays the induction of<br />
the myogenic factor MyoD, a hallmark of satellite cell<br />
activation. The involvement of PARP-1 was further<br />
demonstrated by specific silencing through shRNAs.<br />
Perspectives<br />
Through the study of p57 regulation we have highlighted<br />
the coordinate control of a group of imprinted<br />
genes during myogenesis. Moreover, we have<br />
revealed that the myogenic factor MyoD directly<br />
communicates with a long-distance element overlapped<br />
with an imprinting control region. We plan to<br />
continue this research with the aim of characterizing<br />
the molecular mechanisms by which MyoD affects the<br />
activity of KvDMR1 and the functional interaction of<br />
this regulatory region with the p57 encoding gene.<br />
These studies promise the identification of a new epigenetic<br />
pathway by which MyoD orchestrates the coordinate<br />
expression of genes involved in growth control.<br />
Moreover, the results obtained will allow us to gain<br />
insights into the epigenetic constraints underlying the<br />
tissue and cell type restriction of p57 induction during<br />
physiological and pathological differentiation processes.<br />
The results concerning the functional interplay<br />
of PARP-1 with cell cycle arrest suggests that<br />
poly(ADP-ribosyl)ation may regulate the quiescence<br />
of satellite cells, the muscle stem cells. Although the in<br />
vitro model described above faithfully reproduce many<br />
characteristics of muscle stem cells, we have also<br />
planned preliminary approaches to explore a possible<br />
role of PARP-1 in the function of in vivo satellite cells.<br />
These will be propedeutical to a functional analysis of<br />
the role of PARP activity in muscle regeneration.<br />
References<br />
1) Carbone M, Rossi MN, Cavaldesi M, Notari A,<br />
Amati P, Maione R. 2008. Oncogene. 27:6083-92.<br />
2) Dhawan J, Rando TA. 2005. Trends Cell Biol.<br />
15:666-73.<br />
3) Figliola R, Maione R. 2004. J Cell Physiol. 200:<br />
468-75.<br />
4) Figliola R, Busanello A, Vaccarello G, Maione R.<br />
2008. J Mol Biol. 380:265-77.<br />
5) Fitzpatrick GV, Pugacheva EM, Shin JY,<br />
Abdullaev Z, Yang Y, Khatod K, Lobanenkov VV,<br />
Higgins MJ. 2007. Mol Cell Biol. 27:2636-47.<br />
6) Kim MY, Zhang T, Kraus WL. 2005. Genes Dev.<br />
19:1951-67.<br />
7) Sambasivan R, Pavlath GK, Dhawan J. 2008. J<br />
Biosci. 33:27-44.<br />
8) Vaccarello G, Figliola R, Cramerotti S, Novelli F,<br />
Maione R. 2006. J Mol Biol. 356:578-88.
Principal investigator: Franco Mangia<br />
Professor of Neurobiology<br />
Dipartimento di Psicologia, Sezione di Neuroscienze<br />
Tel: (+39) 06 49917784; Fax: (+39) 06 49917873<br />
franco.mangia@uniroma1.it<br />
59<br />
AREA 3<br />
Molecular regulation of cell proliferation and apoptosis in early<br />
embryo blastomeres and granule neuron precursors of the mouse<br />
Participants:<br />
Maria Teresa Fiorenza, Arturo Bevilacqua, professors; Sonia<br />
Canterini, researcher; Adriana Bosco, Andrea Fuso, post-doc<br />
fellows, Valentina Carletti, Stefania Nusca, PhD students.<br />
Collaborations:<br />
<strong>Istituto</strong> Dermopatico dell’Immacolata, Rome (Dr. Giandomenico<br />
Russo, Dr. Maria Grazia Narducci); Dipartimento di Scienze<br />
Anatomiche, Istologiche, Medico-Legali e dell’Apparato<br />
Locomotore, Sapienza-Università di Roma (Prof. Carla Boitani,<br />
Dr. Rossella Puglisi); Dipartimento di Psicologia, Sapienza-<br />
Università di Roma (Prof. Simona Cabib); Fondazione San<br />
Raffaele, Milano (Prof. Francesco Blasi); Università di Torino<br />
(Dr. Annalisa Buffo); University of Antwerp, Belgium (Dr.<br />
Michele Giugliano).<br />
Report of activity<br />
We have exploited two different model systems, the<br />
preimplantation mouse embryo development and<br />
the cerebellum granule neuron differentiation and<br />
apoptosis, to investigate the balance between cell<br />
proliferation-survival and apoptosis, by specifically<br />
studying: (i) the function of the oncogenic factor<br />
T-cell Leukemia Factor 1 (TCL1) and its interactions<br />
with AKT/PKB; and (ii) the role of the putative<br />
tumor suppressor THG-1pit/Tsc22d4 in cerebellum<br />
granule neuron differentiation and apoptosis.<br />
Besides T-cell leukemias, TCL1 is physiologically<br />
expressed in both embryonic stem cells, where<br />
it is expressed downstream from the Oct4 gene,<br />
and the early preimplantation embryos, in which<br />
it promotes early blastomere proliferation. TCL1<br />
binds and transphosphorylates AKT/PKB (AKT)<br />
at the level of plasma membrane and then mediates<br />
phosphorylated AKT transfer to nucleus. In spite<br />
of recent advances in the field (rev. by Gonzalez<br />
and McGraw, Cell Cycle 8:2505-8, 2009; Dillon and<br />
Muller, Cancer Res. 70:4260-4, 2010), however, the<br />
specific AKT isoform(s) TCL1 interacts with in<br />
early embryos is(are) still largely unknown. We<br />
have directly addressed this questions by depleting<br />
one-cell embryos of a specific AKT isoform with<br />
microinjection of anti-AKT1, anti-AKT2 or anti-<br />
AKT3 antibodies and subsequent analysis of embryo<br />
development in vitro. Depletion of AKT2 delayed/<br />
blocked embryo development to blastocyst, with features<br />
similar to those of Tcl1 KO embryos (Narducci<br />
et al., PNAS 99, 11712-7, 2002). In contrast, depletion<br />
of AKT1/AKT3 had no apparent effect on<br />
embryos, pinpointing the AKT2 isoform as putative<br />
TCL1 interactor in embryos. Moreover, immunofluorescence<br />
experiments showed that AKT2, but<br />
not AKT1 nor AKT3, migrates to nucleus in concomitance<br />
with TCL1 nuclear localization. Results<br />
obtained with anti-AKT antibodies have recently<br />
been confirmed by subcellular TCL1 localization<br />
in embryos genetically depleted of a single AKT<br />
isoform. AKT1-, AKT2- andAKT3-KO mice were<br />
kindly provided by Dr. B.A. Hemmings and used to<br />
generate -/- embryos. In agreement with results of<br />
antibody microinjections, TCL1 normally localized<br />
into blastomere nuclei in AKT1 -/- and AKT3 -/- twocell<br />
embryos, but was fully restricted to cytoplasm<br />
in AKT2 -/- embryos, conclusively showing that<br />
TCL1 specifically interacts with AKT2, but not<br />
AKT1 nor AKT3, in this system. The possibility<br />
that AKT Ser473/Thr308 phosphorylation depended<br />
on upstream factors/kinases, including PI3K,<br />
PDK1, and HSP90 protein has also been probed by<br />
treating embryos with specific inhibitors, showing<br />
that Ser473/Thr308-phosphorylated AKT2 is fully<br />
inherited from oogenesis and that, following fertilization,<br />
the ratio between AKT phosphorylation-<br />
dephosphorytlation does not significantly change.<br />
This indirectly indicates that TCL1 is not required<br />
for AKT phosphorylation, whereas it represents<br />
an absolute requirement for phosphorylated AKT<br />
transfer to nucleus.<br />
Cerebellum granule neurons (CGNs) proliferation, differentiation<br />
and apoptosis has also been investigated
Franco Mangia<br />
by studying Tsc22d4, a gene belonging to the TGFb1-stimulated<br />
TSC22D gene family and encoding a 42<br />
kDa protein that is post-translationally modified by<br />
O-glycosylation and phosphorylation, giving rise to<br />
55, 67 and 72 KDa forms. TSC22D4 is normally localized<br />
in CGN somata and neurites, but is rapidly and<br />
transiently translocated to nucleus following commitment<br />
to apoptosis. This is easily achieved by exposing<br />
in vitro cultured CGNs to a decrease in external KCl<br />
from 25 mM (a depolarizing condition mimicking synaptic<br />
inputs from mossy fibers), to 5 mM (a hyperpolarizing<br />
condition triggering CGNs to apoptosis). We<br />
have recently addressed the possibility that TSC22D4<br />
is relevant to CGN balance between survival, differentiation<br />
and apoptosis. Investigating TSC22D4 intracellular<br />
localization during early postnatal cerebellum<br />
development (PN6-40; a period of dramatic CGN/<br />
Purkinje cell change in morphology and membrane<br />
excitability), it appeared that TSC22D4 is localized<br />
in both nuclear and somatodendritic compartments<br />
before PN12-18 (the differentiation time at which<br />
CGN viability becomes dependent on membrane<br />
electrical activity), and that it apparently leaves the<br />
nucleus and accumulates at the level of cerebellum<br />
glomeruli (synaptic contacts with mossy fibers) in<br />
mature CGNs. Similar features were also observed in<br />
cerebellum slices cultured in vitro under conditions<br />
favoring/inhibiting CGN differentiation. We thus<br />
concluded that TSC22D4 functions take place: (i) in<br />
the nucleus while CGNs differentiate; (ii) in synaptic<br />
terminals when CGNs mature; and (iii) again in the<br />
nucleus, but with subnuclear targets different than<br />
those of immature cells, when CGNs are committed to<br />
apoptosis (Canterini et al., 2009; Canterini et al., 2010),<br />
suggesting multiple TSC44D4 roles. The intracellular<br />
localization of different TSC22D4 form(s) has<br />
recently been studied on subcellular fractions from<br />
differentiating, differentiated and apoptosis-committed<br />
CGNs. Mature TSC22D4 forms, including the<br />
phosphorylated 67 kDa and the phosphorylated and<br />
O-glycosylated 72 kDa forms that are normally present<br />
in the cytoplasm apparently decrease in relative<br />
abundance following cell commitment to apoptosis.<br />
60<br />
It also appeared that TSC22D4 is localized in neurite<br />
and at synaptic termini mithocondria (Canterini et<br />
al., in preparation). Moreover, the nuclear chromatin<br />
fraction selectively contains the mature 72 kDa form,<br />
irrespectively of normal or apoptotic condition, but<br />
not other TSC22D4 forms. In contrast, the nuclear<br />
matrix fraction contains 42 kDa form under normal<br />
conditions and is strongly enriched with the 42 kDa<br />
and 67 kDa forms following CGC commitment to<br />
apoptosis. TSC22D4 role in CGN differentiation has<br />
been investigated by exploiting siRNAs in in vitro cultured<br />
CGNs, in which a block of differentiation results<br />
in cell death, and in N1E-115 cells, a neuroblastomaderived<br />
cell line expressing TSC22D4 at a high level,<br />
in which the block of differentiation does not interfere<br />
with cell viability. As expected, siRNA silencing<br />
resulted in extensive death of CGNs and inhibition<br />
of neurite elongation, but did cell death, in N1E-115<br />
cells, showing that TSC22D4 is essential for CGN<br />
differentiation.<br />
Pubblications<br />
Canterini S, Bosco A, De Matteis V, Mangia F,<br />
Fiorenza MT. THG-1pit moves to nucleus at the<br />
onset of cerebellar granule neurons apoptosis. Mol<br />
and Cell Neurosci. 2009, 40: 249-57. doi: 10.1016/j.<br />
mcn.2008.10.013.<br />
Colelli V, Fiorenza MT., Conversi D, Orsini C, Cabib<br />
S. Strain-specific proportion of the two isoforms<br />
of the dopamine D2 receptor in the mouse striatum:<br />
associated neural and behavioral phenotypes.<br />
Genes, Brain and Behavior. 2010, 9: 703-711. doi:<br />
10.1111/j.1601-183X.2010.00604.x.<br />
Fernandez-Diaz LC, Laurent A, Girasoli S, Turco M,<br />
Longobardi E, Iotti G, Jenkins NA, Fiorenza MT,<br />
Copeland NG, Blasi F. The absence of Prep1 causes<br />
p53-dependent apoptosis of mouse pluripotent epiblast<br />
cells. Development. 2010, 137: 3393-3403. doi:<br />
10.1242/dev.050567.
Study of the molecular and cellular mechanisms of sarcopenia:<br />
role of mIGF-1 and oxidative stress<br />
Principal investigator: Antonio Musarò<br />
Professor of Biotechnology<br />
Dip. di Scienze Anatomiche, Istologiche, Medico-Legali e dell’Apparato Locomotore<br />
Tel: (+39) 06 49766956; Fax: (+39) 06 4462854<br />
antonio.musaro@uniroma1.it<br />
Participants:<br />
Gabriella Dobrowolny, Emanuele Rizzuto, Michela<br />
Aucello, post-doc fellows; Carmine Nicoletti, technician.<br />
Collaborations:<br />
Ce.S.I. Centro Scienze dell’Invecchiamento, IIM, Università degli<br />
Studi G. d’Annunzio, Chieti (Prof. Giorgio Fanò, Prof. Feliciano<br />
Protasi), Dipartimento di Scienze Biomediche, Università di<br />
Padova (Dr. Marco Sandri); EMBL Mouse Biology Program,<br />
Monterotondo, Rome (Dr. Nadia Rosenthal); Dipartimento di<br />
Neuroscienze, IIT, Genova (Dr. Maria Pennuto).<br />
Report of activity<br />
The aim of the project was to define the molecular<br />
mechanisms that modulate muscle homeostasis. In<br />
particular, we analysed the role of oxidative stress<br />
and the growth factor IGF-1 on muscle muscle<br />
atrophy and wasting. Generation of ROS represents<br />
one of the most prominent events during contractile<br />
activity, suggesting that it could influence muscle<br />
function and health (Fig. 1). How do ROS act as<br />
physiological signaling molecules? When does the<br />
oxidative stress become a damaging factor? It is well<br />
recognized that moderate, non-exhaustive physical<br />
exercise has beneficial effects and might prevents<br />
several chronic diseases. However, a crucial system<br />
severely affected in different pathological conditions<br />
is the antioxidative defense, leading to a pathological<br />
accumulation of ROS (Fig. 1).<br />
The combined facts that mice lacking the major<br />
antioxidant enzyme superoxide dismutase 1 (SOD1)<br />
display a dramatic acceleration in age-related loss of<br />
skeletal muscle mass, that elevated levels of reactive<br />
oxygen species (ROS) may contribute to chronic diseases,<br />
and that mutation in SOD1 is associated with<br />
one fifth of familial ALS, have implicated oxidative<br />
stress as a key mechanism underlying the pathogenesis<br />
of aging and neuromuscular diseases. However,<br />
how such an oxidative insult plays a role in the<br />
61<br />
AREA 3<br />
diseases-related decrease of muscle performance and<br />
mass and whether skeletal muscle is a direct target of<br />
SOD1 mutation remained largely unknown.<br />
In this project we undertook a transgenic approach<br />
to define the direct role of oxidative stress on muscle<br />
homeostasis and function. To this purpose and to<br />
verify whether skeletal muscle is a direct target of<br />
SOD1 mutation we generated transgenic mice with<br />
the mutated isoform of human superoxide dismutase<br />
1 (SOD1 G93A ) cDNA driven by skeletal muscle specific<br />
regulatory elements from the rat myosin light chain<br />
(MLC)-1/3 locus. Expression of the MLC/SOD1 G93A<br />
transgene in adult mice was restricted to skeletal muscle,<br />
predominated in muscles enriched in fast fibers and<br />
reduced in slow muscles such as the soleus, where the<br />
MLC regulatory cassette is characteristically expressed<br />
at very low levels. Notably, no expression of human<br />
SOD1 protein and transcript were found in heart, brain,<br />
liver, spleen, or spinal cord of transgenic mice.<br />
Muscle-restricted expression of mutant SOD1 G93A was<br />
sufficient to induces severe muscle atrophy associated<br />
with significant reduction in muscle strength, sarcomere<br />
disorganization, significant changes of mitochondria<br />
morphology and of their sarcomeric disposition,<br />
and disorganization of the sarcotubular system.<br />
In this study, we also disclosed the potential molecular<br />
mechanisms associated with muscle atrophy induced<br />
by selective accumulation of oxidative stress.<br />
We <strong>report</strong> that accumulation of ROS serves as signalling<br />
to initiate autophagy, one of the major intracellular<br />
degradation mechanisms that we demonstrated<br />
to be a key determinant for the induction of muscle<br />
atrophy associated with oxidative stress (Fig. 2).<br />
In particular, the critical role of autophagy in the promotion<br />
of muscle atrophy was disclosed by genetic<br />
manipulation of LC3 expression, a molecular marker<br />
of autophagy. In vivo electroporation of siRNA<br />
against LC3 gene rescued the atrophic phenotype<br />
in MLC/SOD1 G93A mice, suggesting that autophagy<br />
is the dominant pathway that mediates the atrophic<br />
stimulus of oxidative stress and that the modula-
Antonio Musarò<br />
tion of the autophagy pathway can be a potential<br />
therapeutic mechanism to counteract muscle atrophy<br />
associated with oxidative stress. Moreover, we<br />
addressed the involvement of caspases in the induction<br />
of muscle atrophy in experimental models of<br />
ALS, expressing the mutant SOD1 G93A transgene<br />
either locally or ubiquitously. We demonstrated that<br />
SOD1 G93A -mediated muscle atrophy is independent<br />
from caspases activity. In particular the expression<br />
of SOD1 G93A promotes a reduction of the PI3K/AKT<br />
pathway associated with activation of FOXO3 (Fig.<br />
2). In contrast, the activation of caspases is a later<br />
event causally linked to motor neuron degeneration,<br />
associated with an exacerbation of the atrophic phenotype<br />
and to a shift in fiber type composition.<br />
Thus, our study is the first to (1) establish skeletal<br />
muscle as a primary target for the dominant action<br />
of inherited SOD1 mutations, (2) implicate oxidative<br />
stress as the primary trigger of muscle atrophy associated<br />
with SOD1 mutation, (3) disjoin muscle atrophy<br />
and function from motor neuron degeneration.<br />
More recently, we analyzed the role of the growth<br />
factor mIGF-1 in counteracting muscle wasting in<br />
different animal models of neuromuscular diseases and<br />
wasting, including aging, Spinal and Bulbar Muscular<br />
Atrophy (SBMA), and muscular dystrophy. We demonstrated<br />
that augmentation of IGF-1/Akt signaling<br />
rescues behavioural and histopathological abnormalities,<br />
extends the life span, and reduces both muscle<br />
and spinal cord pathology of SBMA mice. This study<br />
demonstrates that skeletal muscle is a viable target<br />
tissue for therapeutic intervention in SBMA.<br />
It is also known that aging and neuromuscular<br />
diseases, including ALS and muscular dystrophy,<br />
compromise the viscoelastic properties of tendon.<br />
Confocal microscopy and mechanical properties<br />
measurements revealed that MLC/SOD1 G93A and<br />
dystrophic mdx tendons, compared to WT ones,<br />
have an increase in the number of dead cells and a<br />
significant reduction in tissue elasticity for all the<br />
frequencies that were tested. Notably, local expression<br />
of IGF-1 was able to preserve the viscoelastic<br />
properties of tendons in the different pathological<br />
animal models. Our results demonstrate that neuromuscular<br />
diseases involves not only muscle wasting,<br />
but also alteration in the viscoelastic properties of<br />
tendons, suggesting a paracrine effect of altered skeletal<br />
muscle on tendinous tissue.<br />
The results of this project contribute to clarify the<br />
molecular mechanisms underlying muscle atrophy<br />
62<br />
and wasting and will allow us to develop specific<br />
therapeutic strategies in order to prevent or attenuate<br />
the muscle atrophy and frailty associated with<br />
aging and neuro-muscular diseases.<br />
Publications<br />
Aucello M, Dobrowolny G, Musarò A. Localized<br />
accumulation of oxidative stress causes muscle atrophy<br />
through activation of an autophagic pathway.<br />
Autophagy. 2009, 5: 527-9. doi: 10.4161/auto.5.4.7962.<br />
Barberi L, Dobrowolny G, Pelosi L, Giacinti C,<br />
Musarò A. Muscle involvement and IGF-1 signaling<br />
in genetic disorders: new therapeutic approaches.<br />
Endocr Dev. 2009, 14: 29-37. doi: 10.1159/000207474.<br />
Messina S, Bitto A, Aguennouz M, Mazzeo A,<br />
Migliorato A, Polito F, Irrera N, Altavilla D, Vita<br />
GL, Russo M, Naro A, De Pasquale MG, Rizzuto<br />
E, Musarò A, Squadrito F, Vita G. Flavocoxid counteracts<br />
muscle necrosis and improves functional<br />
properties in mdx mice: a comparison study with<br />
methylprednisolone. Exp Neurol. 2009, 220: 349-58.<br />
doi: 10.1016/j.expneurol.2009.09.015.<br />
Palazzolo I, Stack C, Kong L, Musarò A, Adachi<br />
H, Katsuno M, Sobue G, Taylor JP, Sumner CJ,<br />
Fischbeck KH, Pennuto M. Overexpression of IGF-1<br />
in muscle attenuates disease in a mouse model of spinal<br />
and bulbar muscular atrophy. Neuron. 2009, 13:<br />
316-28. doi: 10.1016/j.neuron.2009.07.019.<br />
Rizzuto E, Musarò A, Catizone A, Del Prete Z.<br />
Measuring tendon properties in mdx mice: cell viability<br />
and viscoelastic characteristics. J Biomech. 2009,<br />
42: 2243-8. doi: 10.1016/j.jbiomech.2009.06.041.<br />
Scicchitano BM, Rizzuto E, Musarò A. Counteracting<br />
muscle wasting in aging and neuromuscular diseases:<br />
the critical role of IGF-1. Aging. 2009, 11: 451-7.<br />
Musarò A, Fulle S, Fanò G. Oxidative stress and muscle<br />
homeostasis. Curr Opin Clin Nutr Metab Care. 2010,<br />
13: 236-42. doi:10.1097/MCO.0b013e3283368188.<br />
Musarò A, Barberi L. Isolation and culture of mouse<br />
satellite cells. Methods Mol Biol. 2010, 633: 101-11.<br />
doi: 10.1007/978-1-59745-019-5_8.
Fig. 1 - A summary of the role of oxidative stress in the physiopathology of skeletal muscle.<br />
Fig. 2 - A summary of the signal pathways activated in SOD1 G93A transgenic mice.<br />
63<br />
AREA 3
Role of the COP9 signalosome in transcription modulation and<br />
chromatin organization in yeast and plants<br />
Principal investigator: Rodolfo Negri<br />
Professor of Molecular Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 44917790; Fax: (+39) 06 44917594<br />
rodolfo.negri@uniroma1.it<br />
Partecipants:<br />
Giovanna Serino, professor; Teresa Rinaldi, researcher;<br />
Angela Alagia, Micaela Esposito, PhD students.<br />
Collaborations:<br />
Università degli Studi di Torino (Prof. Gianni Balliano); University<br />
of Haifa, Israel (Prof. Elah Pick).<br />
Report of activity<br />
Accurate regulation of transcription in time and<br />
space is critical for the establishment and maintenance<br />
of gene expression patterns in eukaryotes. In<br />
the past years, several components of the ubiquitin/<br />
proteasome system (UPS) have been shown to be<br />
necessary for the tight regulation of gene expression<br />
with possible important implications for cellular<br />
homeostasis. Recent evidence shows that part of<br />
this regulatory action is at transcriptional level and<br />
mediated by direct action of UPS components on<br />
chromatin at defined genomic loci.<br />
A key component of the UPS is the COP9 signalosome<br />
(CSN), a protein complex conserved in<br />
all eukaryotes. At the biochemical level, the CSN<br />
removes the small peptide NEDD8 (an ubiquitin like<br />
modifier) from the cullin-RING family of E3 ubiquitin<br />
ligases. E3 ligases are the enzymes responsible<br />
for poly-ubiquitination of specific substrates, which<br />
can be subsequently recognized and degraded by the<br />
proteasome. The reaction catalyzed by CSN is necessary<br />
for the tight regulation of the assembly/disassembly<br />
cycles of these ligases and is essential for<br />
all higher eukaryotes, since null CSN mutants from<br />
different organisms ranging from plants to mammals<br />
are lethal at very early developmental stages.<br />
At the cellular level, CSN mutants from different<br />
organisms display de-repression and, more in general,<br />
miss-regulation of several sets of genes. This<br />
could be due simply to the fact that CSN is necessary<br />
for the proper degradation of several transcription<br />
65<br />
AREA 3<br />
factors. However, recent evidence suggests that<br />
CSN might perform specific tasks directly on the<br />
genomic chromatin.<br />
The CSN from budding yeast S. cerevisiae has been<br />
recently characterized and, like in higher eukaryotes,<br />
is responsible for the de-neddylation of cullin-RING<br />
ubiquitin ligases. However, while the CSN in most<br />
higher eukaryotes is composed of 8 subunits and is<br />
a paralog of the lid of the proteasome, the CSN from<br />
budding yeast consists of six subunits, four with a<br />
PCI domain (Csn9, Csn10, Csn11/Pci8 and Csn12),<br />
one with a JAMM/MPN+ domain (Csn5/Rri1), and<br />
a sixth protein called Csi1. In addition, in contrast to<br />
other eukaryotes, all the CSN subunits from S. cerevisiae<br />
are non-essential. The non essentiality of CSN<br />
components and the availability of powerful genetic<br />
tools make S. cerevisiae a very promising model system<br />
to elucidate some aspects of the nuclear role of<br />
this complex.<br />
Interestingly, the CSN localization in S. cerevisiae is<br />
prevalently nuclear and deletion of Csn12 leads to<br />
Csn5 delocalisation to the cytoplasm. This observation<br />
opens up the perspective of a nuclear function<br />
of the CSN in S. cerevisiae, which could be related to<br />
chromatin organization and/or direct transcriptional<br />
control, as it is the case for the proteosomal lid, and<br />
as it has been suggested for CSN in other organisms.<br />
This project aims at testing the hypothesis of a direct<br />
role of the CSN in the regulation of transcription and<br />
chromatin structure. The core experiments are carried<br />
out in the budding yeast S. cerevisiae and the results<br />
will be further validated in the plant Arabidopsis thaliana,<br />
where the CSN was initially identified.<br />
Trascriptomic analysis of a delta-csn5 strain<br />
We performed a transcriptomic analysis of a S. cerevisiae<br />
strain deleted in the Ylr100w ORF, coding<br />
for the JAMM/MPN+ domain containing the Csn5<br />
protein component (for the de-neddylating activity<br />
of the yeast Cop9 complex), as compared with its<br />
isogenic wild type strain. The transcriptome screen-
Rodolfo Negri<br />
ing was performed on two independent cultures<br />
of the wt and csn5-deleted strains by two-colour<br />
competitive hybridization of microarray slides containing<br />
the whole repertoire of S. cerevisiae ORFs<br />
(UHN Microarray Centre (http://data.microarrays.<br />
ca/arrays/index.htm). Data analysis showed that<br />
183 genes were significantly (p
The biogenesis of piRNAs and their involvement in transposon<br />
silencing and heterochromatin formation in Drosophila<br />
Principal investigator: Sergio Pimpinelli<br />
Professor of Genetics<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912876; Fax: (+39) 06 4456866<br />
sergio.pimpinelli@uniroma1.it<br />
Participants:<br />
Laura Fanti, professor; Lucia Piacentini, researcher; Marcella<br />
Marchetti, post-doc fellow; Enzo Marchetti, technician.<br />
Collaborations:<br />
Università di Lecce (Prof. Maria Pia Bozzetti, Dr. Maria<br />
Berloco).<br />
Report of activity<br />
Heterochromatin is a nearly ubiquitous component<br />
of the eukaryotic chromosome that is usually located<br />
at the pericentromeric regions and telomeres. Such<br />
material constitutes a good model for studying<br />
epigenetic gene silencing. Many significant results<br />
have already been obtained and much information is<br />
already available about the molecular determinants<br />
of heterochromatin and their involvement in gene<br />
silencing. However, many problems still remain to<br />
be solved such as the identification of all of the components<br />
of heterochromatic multiprotein complexes<br />
and the mechanisms of their targeting. Growing<br />
evidence strongly suggests that the RNA interference<br />
phenomenon is involved in heterochromatin<br />
formation and perhaps in other chromatin modifications<br />
responsible for more general transcriptional<br />
gene silencing.<br />
The aim of this research is to systematically search,<br />
by a combined genetic, cytological and molecular<br />
approach, genes involved in piRNAs biogenesis,<br />
heterochromatin formation and transposons silencing.<br />
To this end we are collecting and studying<br />
mutants that induce the expression of the repetitive<br />
Stellate sequences of Drosophila melanogaster normally<br />
repressed by piRNAs interference mechanism.<br />
The crystal-Stellate system is a clear example of<br />
“natural” piRNAs mediated silencing. In wild type<br />
males a Stellate mRNA is degraded due to a homologous<br />
piRNAs coded by the crystal region on the Y<br />
chromosome. This phenomenon is mediated by the<br />
RNA interference machinery. A mature 750 nucleo-<br />
67<br />
AREA 3<br />
tide Stellate mRNA is instead produced when the<br />
Y chromosome is deleted in the crystal region. The<br />
protein obtained after translation forms crystalline<br />
aggregates in spermatocytes and spermatids. The<br />
existence of modifiers of the crystal-Stellate interaction<br />
and that show the same phenotype as the crystal<br />
deletion has strongly suggested to us that mutations<br />
that abolish Stellate silencing are good candidates for<br />
genes involved in piRNAs biogenesis, heterochromatin<br />
formation and gene silencing.<br />
We have already performed a sistematic search for<br />
such mutations and we have collected and identified<br />
about 15 genes that derepress Stellate sequences and<br />
we developed meaningful experimental criteria to<br />
identify those genes involved in each, or more, of the<br />
phenomena considered above.<br />
The research activity during the last two years has<br />
been mainly focused on analysis of different chromatin<br />
proteins that are involved in heterochromatin<br />
formation and/or piRNA biogenesis in Drosophila<br />
melanogaster.<br />
HP1 and gene expression<br />
Recently, by a wide expression analysis, using a RIPchip<br />
assays (RNA-immunoprecipitation on microarrays),<br />
we have shown that HP1 associates with the<br />
transcripts of more than one hundred euchromatic<br />
genes and interacts with the heterogeneous nuclear<br />
ribonucleoproteins (hnRNPs) that are known to be<br />
involved in RNA processing. So that, HP1 seems to<br />
be part of a complex that stabilizes RNA transcripts.<br />
Though previously unsuspected, it was also found<br />
that HP1-interacting hnRNPs have a functional role<br />
in heterochromatin formation. These proteins bind<br />
heterochromatin and are dominant suppressors of<br />
position effect variegation. Taken together, the results<br />
open a window on a possible new conceptual landscape<br />
in which similar epigenetic mechanisms could<br />
have a significant role, both in the metabolism of RNA<br />
transcripts and in heterochromatin formation, produc-
Sergio Pimpinelli<br />
ing opposite functional effects. These data seem to<br />
establish a functional link between euchromatin and<br />
heterochromatin. Interestingly, we found that some of<br />
the hnRNP induce the expression of the Ste sequences<br />
thus suggesting a lpossible involvement of piRNAs in<br />
heterochromatin formation.<br />
Hsp90, piRNAs biogenesis and transposon<br />
silencing<br />
We have recently shown that, in Drosophila, functional<br />
alterations of Hsp90 affect the piRNA silencing mechanism<br />
leading to transposon activation and the induction<br />
of morphological mutants. The clear involvement<br />
of Hsp90 in piRNA biogenesis and transposon silencing<br />
has also suggested an alternative explanation to the<br />
Waddington’s “Canalization” concept. Hsp90, instead<br />
to be a “capacitor” that buffers phenotypic variance, is a<br />
68<br />
“mutator” that can actually generate new variation by<br />
trasposon-mediated canonical mutagenesis.<br />
Publications<br />
Piacentini L, Fanti L, Negri R, Del Vescovo V, Fatica<br />
A, Altieri F, Pimpinelli S. Heterochromatin protein 1<br />
(HP1a) positively regulates euchromatic gene expression<br />
through RNA transcript association and interaction<br />
with hnRNPs in Drosophila. PLoS Genet. 2009, 5:<br />
e1000670. doi: 10.1371/journal.pgen.1000670.<br />
Specchia V, Piacentini L, Tritto P, Fanti L,<br />
D’Alessandro R, Palumbo G, Pimpinelli S, Bozzetti<br />
MP. HSP90 prevents phenotypic variation by suppressing<br />
the mutagenic activity of transposons.<br />
Nature. 2010, 463: 662-665. doi:10.1038/nature08739.
To the root of organ growth: the control of root meristem<br />
activity in Arabidopsis<br />
Principal investigator: Sabrina Sabatini<br />
Researcher in Molecular Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49917916; Fax: (+39) 06 49917594<br />
sabrina.sabatini@uniroma1.it<br />
Participants:<br />
Laila Moubayidin, Serena Perilli, Riccardi Di Mambro, PhD<br />
students.<br />
Report of activity<br />
Introduction<br />
Plant post-embryonic development takes place in the<br />
meristems. In the root of the model plant Arabidopsis<br />
thaliana, stem cells organized in a stem cell niche<br />
in the apex of the root meristem generate transitamplifying<br />
cells, which undergo additional division<br />
in the proximal meristem, and differentiate in the<br />
distal meristem transition zone that encompasses the<br />
boundary between dividing and expanding (differentiating)<br />
cells in the different cell files (Dello Ioio et<br />
al., 2007). For meristem maintenance, and therefore<br />
continuous root growth, the rate of cell differentiation<br />
must equal the rate of generation of new cells:<br />
how this balance is achieved is a central question in<br />
plant development. By means of a genetic and molecular<br />
analysis, we have shown that in the Arabidopsis<br />
root meristem the balance between cell differentiation<br />
and cell division necessary for controlling root<br />
meristem size and root growth is the result of the<br />
interaction between two plant hormones, cytokinin<br />
and auxin, through a simple regulatory circuit converging<br />
on a single gene, the SHY2 gene (Dello Ioio<br />
et al., 2008). In particular, in the vascular tissue of the<br />
transition zone, a primary cytokinin-response transcription<br />
factor, ARR1 (Sakai et al., 2001), activates<br />
the gene SHY2 (Kepinsky et al., 2006), a repressor<br />
of auxin signaling that negatively regulates the PIN<br />
genes that encode auxin transport facilitators (Blilou<br />
et al., 2005). Thus, cytokinin causes redistribution of<br />
auxin, prompting cell differentiation (Dello Ioio et al.,<br />
2008). Conversely, auxin mediates degradation of the<br />
SHY2 protein (Kepinsky et al., 2006), sustaining the<br />
activity of the PIN genes and prompting cell division<br />
(Dello Ioio et al., 2008).<br />
69<br />
AREA 3<br />
Thus, our work has shown that a specific developmental<br />
domain of the Arabidopsis root - the vascular<br />
tissue of the transition zone - is where the interaction<br />
between cytokinin and auxin takes place, and<br />
has provided a genetic and molecular framework for<br />
this interaction, clarifying how the size of the root<br />
meristem is maintained. However, two important<br />
questions concerning the ARR1/SHY2/PIN regulatory<br />
network remain to be answered:<br />
- First, since cytokinin affects PIN activity in the<br />
vascular tissue of the transition zone, how is this<br />
change perceived by the neighbouring cell files in<br />
the transition zone in order for them to shift from<br />
the meristem into the differentiation zone (radial cell<br />
non-autonomous effect)<br />
- Second, in maintaining a balance between cell differentiation<br />
and cell division, how does the mechanism<br />
that controls the former (which takes place in the distal<br />
meristem transition zone) influence the division of stem<br />
cells (which takes place in the apical stem cell niche)<br />
Objectives<br />
The work proposed addresses the two above-mentioned<br />
questions, as part of a larger effort of our<br />
laboratory - which includes elucidating how the basic<br />
ARR1/SHY2/PIN regulatory circuit interacts with<br />
other (hormonal) signals and regulatory networks<br />
that affect root growth - aiming to clarify the molecular<br />
mechanisms through which cytokinin and auxin<br />
control Arabidopsis root growth, and ensure the<br />
robustness of such a complex developmental process.<br />
As of this proposal, the work will consist of two main<br />
lines of research aimed to:<br />
1) Identify and characterize the molecular components<br />
that ensure perception by all cell files in the<br />
transition zone of the cytokinin-dependent redistribution<br />
of auxin that occurs in the vascular tissue<br />
(radial cell non-autonomous effect).<br />
2) Identify and characterize the molecular components<br />
that allow the cytokinin/auxin interaction to<br />
influence stem cell activities.
Sabrina Sabatini<br />
Results<br />
While trying to identify the nature of the radial cell<br />
non-autonomous signals (point 1 of the Objectives)<br />
we notice that the plant hormones gibberellins, in<br />
analogy to auxin, positively control root growth and<br />
meristem size, by sustaining cell division. Based on<br />
this observation we were able to show that in allowing<br />
growth of the root meristem after seed germination<br />
and for the meristem to reach its final size, the<br />
ARR1/SHY2/PIN circuit necessary to maintain the<br />
meristem final size is integrated by two additional<br />
components: the cytokinin-responsive ARR12 transcription<br />
factor, and the plant hormones gibberellins.<br />
ARR12, in analogy to ARR1, drives a low level of<br />
expression of SHY2 during the meristem growth<br />
phase for ensuring a prevalence of cell division over<br />
cell differentiation. ARR1 eventually joins ARR12 in<br />
increasing SHY2 expression, leading to an increase<br />
of cell differentiation that thus balances cell division<br />
and stops meristem growth. Gibberellins, necessary<br />
for seed germination and radicle protrusion, repress<br />
expression of the ARR1 gene during post-germination<br />
meristem growth, and a subsequent decrease<br />
in their activity, allows ARR1 expression and the<br />
consequent up-regulation of SHY2 via the stabilization<br />
of RGA, a gibberellin inhibitor DELLA protein.<br />
These results, although do not reveal the nature of<br />
the radial cell non-autonomous signals that coordinate<br />
cell differentiation at the transition zone, take<br />
into account another important aspect of meristem<br />
development: how a defined final meristem size is set,<br />
i.e. how a change in the relative rates of cell division<br />
and cell differentiation is brought about for meristem<br />
growth to stop. We showed, for the first time, that<br />
70<br />
the balance between cell division and differentiation<br />
that ensures meristem maintenance is achieved by<br />
controlling the rate of cell differentiation rather than<br />
the rate of cell division, which remains constant.<br />
Progress in point 2 of the Objectives shows that<br />
ARR1 expression in the stem cell niche is suppressed<br />
by the transcription factor SCARECROW (SCR,<br />
Sabatini et al., 2003) corroborating the idea that SCR<br />
maintains stem cell division by preventing cytokinin<br />
action in the stem cell niche.<br />
References<br />
- Blilou I, Xu J, Wildwater M, Willemsen V,<br />
Paponov I, Friml J, Heidstra R, Aida M, Palme K,<br />
Scheres B. (2005) Nature 433, 39-44.<br />
- Dello Ioio R, Linhares FS, Scacchi E, Casamitjana-<br />
Martinez E, Heidstra R, Costantino P, Sabatini S.<br />
(2007) Curr Biol. 17, 678-682.<br />
- Dello Ioio R, Nakamura K, Moubayidin L, Perilli S,<br />
Taniguchi M, Morita MT, Aoyama T, Costantino<br />
P, Sabatini S. (2008) Science 322, 1380-1384.<br />
- Kepinski S. (2006) Curr Opin Plant Biol. 9, 28-34<br />
- Sabatini S, Heidstra R, Scheres B. (2003). Genes &<br />
Development 17, 354-358.<br />
- Sakai H, Honma T, Aoyama T, Sato S, Kato T,<br />
Tabata S, Oka A. (2001) Science 294, 1519-1521.<br />
Publications<br />
Moubayidin L, Perilli S, Dello Ioio R, Di Mambro R,<br />
Costantino P, Sabatini S. The rate of cell differentiation<br />
controls the Arabidopsis root meristem growth<br />
phase. Curr Biol. 2010, 20: 1138-1143. doi:10.1016/j.<br />
cub.2010.05.035.
71<br />
AREA 3<br />
Biological characterization and in vitro culture of spermatogonial<br />
stem cells<br />
Principal investigator: Mario Stefanini<br />
Professor of Histology and Embryology<br />
Dip. di Scienze Anatomiche, Istologiche, Medico-Legali e dell’Apparato Locomotore<br />
Tel: (+39) 06 49766570; Fax:(+39) 06 4462854<br />
mario.stefanini@uniroma1.it<br />
Participants:<br />
Carla Boitani, professor; Elena Vicini, researcher; Barbara<br />
Muciaccia, Rossella Pugliesi, post-doc fellows; Margherita<br />
Grasso, Lisa Dovere, PhD students; Stefania Fera, Tiziana<br />
Menna, technicians.<br />
Collaborations:<br />
Dipartimento di Sanità Pubblica e Biologia Cellulare, Università di<br />
Roma Tor Vergata (Prof. Massimo De Felici, Dr. Maria Lucia<br />
Scaldaferri); Dipartimento di Biologia Cellulare e Neuroscienza,<br />
<strong>Istituto</strong> Superiore di Sanità, Rome (Dr. Massimo Sanchez).<br />
Report of activity<br />
In the last years the activity of our group has focused<br />
on the isolation and characterization of spermatogonial<br />
stem cells (SSCs), the stem cells that sustain<br />
spermatogenesis in mammals.<br />
In the accepted model of SSCs self-renewal, Asingle<br />
spermatogonia (As) are the stem cells of spermatogenesis<br />
whereas chained undifferentiated spermatogonia<br />
are irreversibly committed to differentiation. This<br />
long-lasting model has been recently challenged by<br />
evidence from our and other laboratories indicating<br />
that As and chained early A spermatogonia are heterogeneous<br />
in terms of gene expression and function<br />
and different subsets of stem cells may be generated by<br />
an asymmetric mitotic division (Grisanti et al., 2009).<br />
The purpose of our project is to provide insight into<br />
the biological function of the different subsets of SSCs.<br />
This line of research may shed light on the molecular<br />
mechanisms regulating early step of spermatogenesis,<br />
for future applications on male infertility, and to unveil<br />
molecular mechanisms underlying pluripotency.<br />
Development of animal models to obtain<br />
isolation and enrichment of spermatogonial<br />
stem cells<br />
We have developed an animal model expressing<br />
endogenous GFRA1 tagged with the enhanced green<br />
fluorescent protein (EGFP-GFRA1). GFRA1 and its<br />
co-receptor Ret are the receptors for the niche-derived<br />
factor GDNF. GDNF is essential for the correct balance<br />
of self-renewal and differentiation of SSCs and<br />
its specific co-receptor GFRA1 is expressed by a subsets<br />
of As. GFRA1 is a glycosylphosphatidylinositol<br />
(GPI)-anchored plasma membrane protein which is<br />
synthesized as precursor with a cleavable signal peptide<br />
and a cleavable carboxy-terminal signal sequence<br />
that directs GPI anchoring. In the targeting vector<br />
the EGFP was cloned at the N-terminal of GFRA1<br />
because it has been demonstrated that this region is<br />
not involved in interactions with other components<br />
of the receptor-ligand complex. To avoid processing<br />
and excision of the EGFP tag with the signal peptide<br />
after protein synthesis the signal peptide has been<br />
placed N-terminal to EGFP. We first assessed that<br />
GFRA1-EGFP fusion protein retains its biological<br />
activity. We found that the fusion protein: i) retains<br />
its subcellular localization by confocal microscopy; ii)<br />
upon GDNF treatment interact with Ret as shown by<br />
co-immunoprecipitation; iii) upon GDNF treatment<br />
retains its ability to allow Ret auto-phosphorylation.<br />
These data indicated that the EGFP- GFRA1 fusion<br />
protein retains its biological activity and validate<br />
the strategy to create the “knock-in” modified mice.<br />
Therefore targeting vector was linearized, transfected<br />
into ES cells and positive clones were injected<br />
into C57BL/6 blastocyst. We have now obtained<br />
germline transmission. This new animal model will<br />
allow: i) isolation and enrichment of spermatogonial<br />
stem cells by FACS e prerequisite to biochemical<br />
analysis; ii) video-time laps analysis performed on isolated<br />
seminiferous tubules or on spermatogonial stem<br />
cells in vitro. This animal model will be used to gain<br />
more insight into the SSCs phenotypic heterogeneity.<br />
Ontogeny of side-population (SP) phenotype<br />
in the germ stem cell lineage<br />
The SP phenotype, defined as the ability of cells to<br />
efflux fluorescent dyes such as Hoechst, is common<br />
to several stem/progenitor cell types. We have pre-
Mario Stefanini<br />
viously shown that a subset of normal spermatogonial<br />
stem cells display an SP phenotype (Falciatori et<br />
al., 2004). SSCs displaying the SP phenotype are clonogenic<br />
in vivo and do not express GFRA1 (Grisanti<br />
et al., 2009). We tested the hypothesis that SP phenotype<br />
arise early and is maintained in the stem<br />
germ cell lineage throughout testis development.<br />
We analyzed and characterized the Hoechst SP via<br />
cytofluorimetric analysis of disaggregated gonads<br />
at different time points during embryonic development<br />
in mice. To directly test the hypothesis that<br />
the SP phenotype is a feature of germ cell lineage,<br />
experiments were performed on transgenic animals<br />
expressing EGFP under the control of the Oct4 promoter,<br />
to identify early germ cells up to primordial<br />
gem cells (PGCs) the embryonic precursors of SSCs.<br />
We found that prenatal gonads contain a fraction of<br />
SP cells at each stage analyzed, and the percentage<br />
of cells in the SP fraction decreases as development<br />
proceeds. Surprisingly, more than 50% of the PGCs<br />
displayed the SP phenotype at 11.5 dpc (days post<br />
coitum). The percentage of germ cells with the SP<br />
phenotype decreased steadily with development, to<br />
less than 1% at 18.5 dpc approaching the percentage<br />
of SP cells in postnatal testis (Falciatori et al., 2004).<br />
Cytofluorimetric analysis along with immunocyto-<br />
72<br />
chemistry performed on sorted cells indicate that the<br />
SP fraction of prenatal gonads, as in the adult testis,<br />
is heterogeneous, being composed of both somatic<br />
and germ cells. Both cell types express the ABC<br />
transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These<br />
findings provide evidence that SP phenotype is a<br />
common feature of germline development and identifies<br />
a subpopulation of the fetal testis cells including<br />
prospermatogonia whose differentiation fate remains<br />
to be investigated (Scaldaferri et al., in press).<br />
Publications<br />
Grisanti L, Corallini S, Fera S, Muciaccia B, Witke<br />
W, Stefanini M, Vicini E. Inactivation of numb and<br />
numblike in spermatogonial stem cells by cell-permeant<br />
Cre recombinase. Differentiation. 2009, 78:131-<br />
136. doi:10.1016/j.diff.2009.05.005.<br />
Grisanti L, Falciatori I, Grasso M, Dovere L, Fera S,<br />
Muciaccia B, Fuso A, Berno V, Boitani C, Stefanini<br />
M, Vicini E. Identification of spermatogonial stem<br />
cell subsets by morphological analysis and prospective<br />
isolation. Stem Cell. 2009, 27: 3043-52. doi:<br />
10.1002/stem.206.
Molecular mechanisms of the epithelial to mesenchymal<br />
transition in hepatocyte<br />
Principal investigator: Marco tripodi<br />
Professor of Genetics<br />
Dipartimento di Biotecnologie Cellulari ed Ematologia<br />
Tel: (+39) 06 4461387; Fax: (+39) 06 4462891<br />
tripodi@bce.uniroma1.it<br />
Participants:<br />
Laura Amicone, Carla Cicchini, Alessandra Marchetti,<br />
researchers; Alice Conigliaro, Carmine Mancone, post-doc<br />
fellows; Laura Santangelo, Angela Cozzolino, AIRC fellows;<br />
Francesca Garibaldi, PhD student; Claudio Cavallari, technician.<br />
Report of activity<br />
Aims of the proposal<br />
1) Study of the molecular mechanisms controlling liver<br />
stem cell EMT/MET oscillations and the influence of<br />
micro-environmental factors on the stem cell fate.<br />
In particular we have proposed to study the crosstalk<br />
between the niche factors Wnt and TGFβ in<br />
controlling liver stemness starting from the hypothesis,<br />
supported by our previous observations, that the<br />
transcriptional inhibitor Snail (respectively induced<br />
and stabilized by TGFb and Wnt) is required for<br />
stem cell maintenance.<br />
Moreover, we have planned to investigate on the<br />
cooperation between Wnt and FGF signaling in<br />
controlling hepatic differentiation, starting from the<br />
hypothesis that FGF pathway, crosstalking with Wnt,<br />
could drive Resident Liver Stem Cells (RLSCs) differentiation<br />
into hepatocyte promoting HNF4a-induced<br />
MET. The hypothesis was supported by our previous<br />
results (HNF4a dominancy on Snail in regulation of<br />
EMT) and from several literature data indicating common<br />
mediators and targets for FGFs and Wnts.<br />
2) Study of the stem cell contribution to the onset<br />
and progression of hepatic fibrosis. We have hypothesized<br />
that chronic liver injury could alter the niche<br />
signals triggering an abnormal activation of the stem<br />
cell compartment. In particular, we hypothesized<br />
that liver stem cells could respond to chronic injury<br />
proliferating and activating differentiative program<br />
towards fibrogenic cells. Our previous results, in<br />
fact, suggested the capability of our RLSCs to differentiate<br />
into mesenchymal cells quite similar to<br />
activated Hepatic Stellate Cells or myofibroblasts.<br />
73<br />
AREA 3<br />
Results<br />
We recently <strong>report</strong>ed the isolation, characterization<br />
and reproducible establishment in line of resident<br />
liver stem cells (RLSCs) with immunophenotype<br />
(Sca1+, CD34-, CD45-, Alpha-fetoprotein+,<br />
Albumin) that locate them in a pre-hepatoblast/<br />
liver precursor cells hierarchical position and differentiative<br />
potentiality spanning from endodermal<br />
to mesenchymal and ectodermal derivatives.<br />
(Conigliaro et al., 2008). RLSCs plasticity is coherent<br />
with their metastable molecular phenotype typical of<br />
embryonic and adult stem cells and characterized by<br />
co-expression of epithelial and mesenchymal markers<br />
as well as of a variety of chromatin remodeling<br />
genes. This highly dynamic cell state, thought to be<br />
the major determinant of stem cell fate, may be considered<br />
as the result of continue oscillations between<br />
mesenchymal-epithelial and epithelial-mesenchymal<br />
transitions (EMT/METs).<br />
Aim 1: We investigated on the molecular mechanisms<br />
controlling RLSC stemness, in particular on the role<br />
of the transcriptional regulator Snail, a TGFβinduced<br />
EMT master gene. We previously observed<br />
that TGFβ, as well as exogenous Snail, induces<br />
EMT in hepatocyte while impedes the differentiation<br />
into hepatocytes (Spagnoli et al., 2000) of liver bipotential<br />
precursor cells. RLSCs constitutively express<br />
Snail and their spontaneous differentiation into<br />
hepatocytes is underlined by negative regulation of<br />
this transcription factor. We gathered evidences that<br />
have led us to conclude that Snail is a key element<br />
in stemness maintenance. Moreover we unveiled the<br />
stemness inhibitinging role of a number of microR-<br />
NAs that are in turn repressed by Snail; Chromatin<br />
Immunoprecipitation assays and qRT-PCR analysis<br />
showed as the Snail recruitment (dynamically regulated<br />
by TGFβ) on specific consensus of miRNAs<br />
promoters correlates with down regulation of their<br />
expression (manuscript in preparation).<br />
In frame of the study of micro-environmental influ-
Marco Tripodi<br />
ence on liver stem cell fate, we published a work<br />
describing the effects of Wnt pathway on post-differentiation<br />
patterning of hepatocyte. Our data showed<br />
a direct and hitherto unknown convergence of the<br />
canonical Wnt signaling (contributing also the to<br />
liver stem cell maintenance in the stem niche) on the<br />
HNF4-driven transcription providing evidences of<br />
a mechanism controlling liver zonated gene expression<br />
(Colletti et al., 2009).<br />
We also attempt to further characterize the molecular<br />
mechanisms controlling epithelial identity of<br />
heptocytes. In brief, we unveiled a new mechanism<br />
for HNF4α (already characterized as master gene of<br />
the hepatocyte differentiation) in the induction and<br />
maintenance of hepatocyte differentiation ascribed<br />
to the capacity of this factor to continuously repress<br />
the mesenchymal program. Overall, both HNF4α<br />
activator and repressor functions appear necessary<br />
for the identity of hepatocytes (Santangelo et al.,<br />
submitted)<br />
Finally, we collected evidences of the major role played<br />
by the contact inhibition in the execution of hepatocyte<br />
terminal differentiation (Mancone et al., 2010).<br />
Aim 2: We studied the involvement of RLSC compartment<br />
in the onset and progression of hepatic<br />
fibrosis starting from the hypothesis that the<br />
Hepatic Stellate Cells (HSCs), the main players in<br />
fibrosis pathogenesis which origin is yet unknown,<br />
may derive from liver stem cells. This hypothesis<br />
started from the observation that RLSCs, when<br />
grown without instructive dominant signals, are<br />
able to express in culture a Mixed Epithelial and<br />
Mesenchymal Phenotype (MEMP) constituted by<br />
two different cell populations; while the characterization<br />
of the epithelial component confirmed<br />
the RLSCs ability to spontaneously differentiate<br />
toward hepatocytes, the mesenchymal one sug-<br />
74<br />
gested their possible differentiation toward HSCs.<br />
qRT-PCR analysis and Immunofluorescence analysis,<br />
in fact, showed the expression of markers typical<br />
of quiescent stellate cells that switch into activated<br />
miofibroblasts after treatment with the niche factor<br />
TGFβ (the most important cytokine involved in<br />
vivo in the activation of HSCs). Notably, RLSCs,<br />
injected intra-hepatically in new born mice, colonize<br />
recipient livers contributing to both hepatocyte and<br />
mesenchymal populations. The involvement of this<br />
last cell component in liver fibrosis will be verified<br />
in murine model of chemically induced hepatic<br />
fibro/cirrhosis.<br />
Publications<br />
Colletti M, Cicchini C, Conigliaro A, Santangelo<br />
L, Alonzi T, Pasquini E, Tripodi M, Amicone L.<br />
Convergence of Wnt signaling on the HNF4alphadriven<br />
transcription in controlling liver zonation.<br />
Gastroenterology. 2009, 137: 660-72. doi:10.1053/j.<br />
gastro.2009.05.038.<br />
Wegert M, La Monica N, Tripodi M, Adler G,<br />
Dikopoulos N. Impaired interferon type I signalling<br />
in the liver modulates the hepatic acute phase response<br />
in hepatitis C virus transgenic mice. J Hepatol. 2009,<br />
51: 271-8. doi: 10.1016/j.jhep.2009.03.014.<br />
Mancone C, Conti B, Amicone L, Bordoni V, Cicchini<br />
C, Calvo L, Basulto Perdomo A, Fimia G M, Tripodi<br />
M, Alonzi T. Proteomic analysis reveals a major role<br />
of contact inhibition in the execution of hepatocyte<br />
terminal differentiation. J Hepatol. 2010, 52: 234-43.<br />
doi:10.1016/j.jhep.2009.11.013.
Principal investigator: Giuseppe Zardo<br />
Researcher in Clinical Chemistry<br />
Dipartimento di Biotecnologie Cellulari ed Ematologia<br />
Tel: (+39) 06 80319026; Fax: (+39) 06 80319054<br />
zardo@bce.uniroma1.it<br />
75<br />
AREA 3<br />
Identification of novel genetic and epigenetic targets in Leukemia<br />
by genome wide approaches<br />
Participants:<br />
Alberto Ciolfi, PhD student; Carmen Maresca, technician.<br />
Collaborations:<br />
Dipartimento di Biotecnologie Cellulari ed Ematologia (Prof.<br />
Robin Foà, Dr. S. Chiaretti); Dipartimento di Scienze e<br />
Biotecnologie Medico-Chirurgiche (Prof. Clara Nervi); UCSF<br />
Cancer Center, University of California, San Francisco, USA (Prof.<br />
Joseph F. Costello); Roswell Park Cancer Institute, Buffalo, NY,<br />
USA (Prof. Dominic J. Smiraglia).<br />
Report of activity<br />
The dysregulation of sequential, coordinated, and<br />
combinatorial expression of lineage-affiliated transcription<br />
factors, modulating specific gene expression<br />
programs and lineage commitment, results in the<br />
onset and progression of hematopoietic malignancies<br />
as acute myeloid leukemia (AML). The pathogenesis<br />
of AML is correlated to recurrent genetic alterations<br />
such as chromosomal translocations that can<br />
allow the expression of oncogenic fusion proteins.<br />
However, in the inbred FVB/N mouse model the<br />
presence of an oncogenic fusion protein determines<br />
only a pre-leukemic status implying the necessity of<br />
additional hits for the establishment of a proper leukemic<br />
status. Additional genomic alterations might<br />
include aberrant epigenetic mechanisms of gene<br />
silencing, namely DNA methylation, modifications<br />
of histone tails, and mechanisms of epigenetic targeting<br />
guided by non-coding RNAs. Therefore, by<br />
Restriction Landmark Genome Scanning (RLGS), a<br />
genome wide approach to study DNA methylation,<br />
we aimed to identify specific DNA methylation patterns<br />
in AMLs and their association with Polycomb/<br />
Trythorax chromatin modifications. We analyzed<br />
the aberrant DNA methylation patterns in human<br />
leukemia cell lines and in human AML patients’ samples.<br />
Either AML human blats or cell lines differed<br />
for their FAB classification, block of differentiation,<br />
and presence of oncogenic fusion proteins. In order<br />
to identify the targets of aberrant DNA methylation<br />
in AML cell lines and human blasts we compared<br />
their DNA methylation RLGS profiles to a “master<br />
DNA methylation profile”, obtained by the integration<br />
of the CD34+ HSCs/HPCs and differentiated<br />
CD34- blood cells RLGS profiles. We observed that:<br />
i) the global levels of DNA methylation in AML<br />
cell lines were higher than in AML human blasts<br />
(25,9%±6,1% vs 6,44±2,23); ii) despite this difference,<br />
the cross-comparisons between the different AML<br />
cell lines and human samples evidenced common<br />
patterns of DNA methylation. For instance, either in<br />
AML cell lines or human samples chromosomes 5, 8,<br />
15 and 20 resulted as less hit by aberrant DNA methylation<br />
than chromosomes 4, 6, 10, 12, 13 and 18,<br />
which were the most affected; iii) we also observed<br />
specific and FAB grouping dependent DNA methylation<br />
signatures. Recent data identify, among the<br />
epigenetic mechanisms of gene regulation, the polycomb<br />
(PcG)/trythorax (TrxG) proteins and complexes<br />
as main regulators of stem cell identity and<br />
subsequent lineage choices and suggest their pivotal<br />
role in normal and leukemic hematopoiesis. It was<br />
shown that polycomb protein Ezh2 controls DNA<br />
methylation on target genes. However, a recent<br />
<strong>report</strong> investigating the extent to which distinct<br />
epigenetic mechanisms participate in carcinogenesis,<br />
shows that repressive mechanisms of tumor suppressor<br />
genes as PcG and DNA hypermethylation act<br />
independently, affecting different genes. Thus, we<br />
integrated our RLGS DNA methylation data with<br />
the PcG/TrxG signatures of human ES. Our analysis<br />
showed that DNA promoter regions of genes with<br />
the highest recurrence of aberrant DNA methylation<br />
in AMLs present a bivalent domain in ES and have<br />
a higher sensitivity to become aberrantly methylated<br />
than those presenting only the activating mark<br />
(H3K4me3) or the repressive mark (H3K27me3) in<br />
leukemia. We concluded that AMLs have shared<br />
but also specific and FAB grouping dependent DNA
Giuseppe Zardo<br />
methylation patterns, which are related to the PcG/<br />
TrxG gene specific chromatin signature in human<br />
ES. These observations have the potential to make<br />
significant contributions for a rational use of epigenetic<br />
drugs in AML, and for monitoring the minimal<br />
residual disease in AML especially in absence of specific<br />
molecular markers.<br />
Among the targets of aberrant DNA methylation<br />
identified in AML samples we focused our attention<br />
on a new hematopoietic transcription factor, namely<br />
NFI-A. NFI-A down-regulation is instrumental for<br />
granulopoiesis whereas its expression boosts erythropoiesis.<br />
In AML cell lines, tested for NFI-A and<br />
miR-223 mRNA expression, we observed an inverse<br />
correlation between their mRNA expression levels:<br />
at high miR-223 levels corresponded low NFI-A<br />
mRNA expression and viceversa. In particular, in<br />
HL60 cells induced to granulocytic differentiation<br />
by retinoic acid treatment (RA) and in CD34- blood<br />
cells vs CD34+ HCSs/HPCs, at high levels of<br />
mir-223 corresponded low levels of NFI-A mRNA<br />
expression. Thus, we investigated the epigenetic<br />
mechanisms of NFI-A gene regulation in normal<br />
and leukemic hematopoiesis. Our results showed that<br />
NFI-A down-regulation, which is required during<br />
normal granulocytopoiesis, is driven by the resolution<br />
of PcG/TrxG bimodal chromatin domains<br />
targeting the regulatory region of NFI-A gene.<br />
By ChIPs, we found that, as the HL60 RA induced<br />
granulocytic differentiation preceded, NFI-A regulatory<br />
region enriched for the chromatin repressive<br />
mark H3K27me3 and for the polycomb proteins YY1<br />
and Suz12. These features were also observed on<br />
76<br />
NFI-A regulatory region of CD34- cells and in AML<br />
human blasts showing low NFI-A mRNA levels and<br />
high miR-223 amounts. In accordance, HL60 cells<br />
knocked down for Suz12 failed to resolve the bivalent<br />
domains on NFI-A regulatory region, and rendered<br />
HL60 cells less responding to the differentiating<br />
stimulus of RA. As expected, in K562 cells induced to<br />
erytroid differentiation by ARAc treatment, which<br />
required high NFI-A expression, we found lower<br />
levels of the repressive mark H3K27me3 and of the<br />
polycomb protein YY1. This finding is relevant as<br />
we demonstrate that bivalent domains predispose<br />
not only for gene activation but also for inactivation<br />
during cell lineage specification. Previous <strong>report</strong>s<br />
associated transcriptional gene silencing (TGS) with<br />
non-coding RNAs and elements of RNAi machinery,<br />
our ChIP experiments supported these findings as<br />
NFI-A regulatory region enriched for Dicer1 during<br />
RA induced HL60 granulocytic differentiation,<br />
while Ago1 increased and then gradually diminished.<br />
In addition, the diminished recruitment of<br />
YY1 and Dicer1 on NFI-A regulatory region after<br />
the treatment of native chromatins with RNAseA<br />
and H implied the involvement of RNA molecules in<br />
NFI-A transcriptional gene silencing, and the ability<br />
of Dicer1 to immunoprecipitate YY1 indicated the<br />
close connection existing between PcG and RNAi<br />
in TGS. In this contest, the inverse correlation<br />
observed between NFI-A and miR-223 mRNAs levels<br />
could not be considered as a simple association<br />
of biological events but rather as two pathways intimately<br />
connected and cooperating in the regulation<br />
of NFI-A gene expression.
AREA 4<br />
Molecular<br />
recognition in<br />
biomolecules
Cellular response to oxidative stress: involvement of protein<br />
disulfide isomerases<br />
Principal investigator: Fabio Altieri<br />
Professor of Chemistry and Biochemistry<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 49910880/7; Fax: (+39) 06 4440062<br />
fabio.altieri@uniroma1.it<br />
Participants:<br />
Margherita Eufemi, professor; Caterina Grillo, post-doc<br />
fellow; Rossana Cocchiola, PhD student.<br />
Report of activity<br />
The aim of this research project is to elucidate the<br />
involvement of PDI in the cellular response to oxidative<br />
stress focusing our attention on ERp57. To<br />
address this question we designed several cellular<br />
models in which a study of the cell response to oxidative<br />
stress can be carried out. Preliminary results<br />
indicated that HeLa cells stably transfected with a<br />
DNA sequence coding for GFP-tagged ERp57 show<br />
a certain degree of protection against oxidative<br />
stress induced by H 2 O 2 , as result of an increase of<br />
vitality after 24h of treatment. At the same time, we<br />
could observe a nuclear translocation of the tagged<br />
protein, following the GFP fluorescence by microscopy.<br />
Similar experiments have done on SHSY-5Y<br />
neuronal cells transfected with DNA sequences coding<br />
for GFP-tagged ERp57 and for GFP alone as<br />
a control. MTT assay performed on wild type and<br />
transfected cells showed that ERp57 overexpression<br />
doesn’t influence cells vitality after 24 hours of<br />
stress treatment with H 2 O 2 at different concentrations<br />
(0-1.5mM). Cellular distribution of ERp57, in<br />
either wild type or transfected cells, was investigated<br />
by confocal microscope. Cells subjected to oxidative<br />
stress using 100µM H 2 O 2 for six hours were analyzed<br />
by immunofluorescence using antibodies to detect<br />
ERp57 and Ref1, a previously identified ERp57<br />
interactor. We observed a co-localization and nuclear<br />
translocation of both proteins after stress. We<br />
also tested 6-hydroxydopamine, who causes selective<br />
damage in dopaminergic neurons and induce<br />
ROS production. The results obtained, again, did not<br />
show a correlation between cell vitality and ERp57<br />
overexpression.<br />
With the aim to investigate the role of ERp57 in<br />
79<br />
AREA 4<br />
thyroid cells we analyzed the effects of oxidative<br />
stress on ARO, WRO and NPA cell lines, which correspond<br />
to anaplastic, follicular and papillary thyroid<br />
carcinomas respectively. Cells were stressed with<br />
0.3mM H 2 O 2 and analyzed during the following 24<br />
hours. We monitored survival and, by western blot<br />
analysis, the cellular distribution of selected proteins<br />
including ERp57, Ref1, STAT3 and Ku70/86.<br />
The latter were selected as proteins involved in<br />
stress response (redox regulation of gene expression<br />
and DNA repair) and previously identified as<br />
ERp57 interactors. We also evaluated the intracellular<br />
levels of reduced and oxidized glutathione. In<br />
a comparison analysis, ARO cells, which correspond<br />
to the most aggressive carcinoma and better survive<br />
to oxidative stress, showed the highest amount of<br />
ERp57 and the lowest concentration glutathione. In<br />
ARO cells can be also observed a nuclear translocation<br />
of ERp57, Ref1 and Ku70/86 as a consequence<br />
of the stress treatment. Probably, the presence of<br />
these proteins in the nucleus could play a protective<br />
role against oxidative stress. WRO cells, which are<br />
characterized by low cytosolic levels of ERp57 and<br />
Ref1, were more sensitive to stress treatment despite<br />
of the highest glutathione concentration. STAT3<br />
and pSTAT3 levels were very low in WRO cells and<br />
their amount was further reduced when cells were<br />
subjected to H 2 O 2 treatment. pSTAT3 levels were<br />
quite high in NPA cells but H 2 O 2 treatment induces<br />
pSTAT3 de-phosphorylation and this could explain<br />
the growth arrest observed.<br />
What we may conclude from these data is that<br />
ERp57 overexpression will not turnout always in<br />
an increase in cell viability. However, we generally<br />
observe a cellular redistribution of ERp57 and<br />
its interactors, thus confirming the involvement of<br />
ERp57 in the oxidative stress response. For this reason,<br />
we are going to apply in vivo crosslinking with<br />
formaldehyde and immunoprecipitation steps on<br />
wild type (HeLa, SHSY and thyroid cell lines) and<br />
transfected cells to detect ERp57 interactors before
Fabio Altieri<br />
and during oxidative stress. To avoid any effect due<br />
to the large tag used (GFP) we planned to switch to<br />
a different tag, like the flag peptide.<br />
To isolate and indentify the proteins substrate of<br />
ERp57 redox activity we designed a functional<br />
mutant protein where the second cysteine residue in<br />
each redox active site is replaced with a serine one.<br />
Mutant can react with target proteins but undergoes<br />
an incomplete redox reaction since the mixed<br />
disulfide ERp57-substrate can’t be resolved by the<br />
serine residue. Mutations have been generated by<br />
PCR overlap-extension and confirmed by DNA<br />
sequencing. The mutated ERp57 was expressed as<br />
GST-tagged protein in E. coli and used in an interaction<br />
assay with cellular extracts of HeLa cells. The<br />
mixed disulfide complexes containing the mutated<br />
ERp57 were purified on GSH-Sepharose, which<br />
traps the GST tag ERp57 interactors were specifically<br />
eluted and subjected to mass spectrometry<br />
analysis. Preliminary results indicate actin as a redox<br />
substrate of ERp57. To get better information we<br />
planned to transfect cell lines with the mutated<br />
ERp57 and analyze in vivo the redox interactors.<br />
Our results demonstrate that ERp57, beside its<br />
known functions in the ER, is involved in regulatory<br />
processes at the nuclear level. We showed<br />
that in M14 cells, where STAT3 is constitutively<br />
activated, ERp57 binds with STAT3 to a subset of<br />
well-characterized STAT3-associated promoters/<br />
enhancers. The presence of ERp57 in these sites<br />
appears to be essential for the regulatory activity of<br />
STAT3 on transcription, since inhibition of ERp57<br />
expression by RNA interference is accompanied by<br />
the decrease of expression of several STAT3 dependent<br />
genes. Moreover, the structure and/or composition<br />
of DNA-bound STAT3 multiprotein complexes<br />
may not be the same in the various promoters/<br />
80<br />
enhancers, even in the same cell type. ERp57 is a<br />
redox-sensitive and stress-responsive protein, whose<br />
expression is regulated by a variety of stimuli. One<br />
of these is the oxidative stress, which is known to<br />
lead also to the activation of STAT3 by an increased<br />
phosphorylation. It is conceivable that the functional<br />
interaction of ERp57 with STAT3 could reinforce<br />
the response of STAT3 to the oxidative stress conditions.<br />
It is interesting to note that another member<br />
of the protein disulfide isomerases, the PDI, is<br />
involved at the nuclear level in the transcriptional<br />
regulation, in this case by influencing the conformation<br />
of the estrogen receptor and by modulating its<br />
affinity for the estrogen-responsive element.<br />
The interaction between ERp57 and Ref1, both<br />
expressed in E. coli and purified, was further analyzed<br />
in vitro. Using a thiol probe able to modify<br />
the electrophoretic mobility of proteins we confirmed<br />
the reducing activity of ERp57 on oxidized<br />
Ref1. Reduced form of ERp57 was generated by the<br />
NADPH/NADPH-dependent thioredoxin reductase<br />
system, as previously demonstrated. In addition,<br />
ERp57 is able to remove the glutathione moiety on<br />
Ref1 oxidized with a biotin-labeled glutathione. By<br />
means of the ERp57 mutant above described, we<br />
also observed the transferring of glutathione from<br />
Ref1 to ERp57, thus confirming the redox activity of<br />
ERp57 towards Ref1.<br />
Publications<br />
Chichiarelli S, Gaucci E, Ferraro A, Grillo C, Altieri<br />
F, Cocchiola R, Arcangeli V, Turano C, Eufemi M.<br />
Role of ERp57 in the signaling and transcriptional<br />
activity of STAT3 in a melanoma cell line. Arch<br />
Biochem Biophys. 2010, 494: 178-83. doi: 10.1016/j.<br />
abb.2009.12.004.
Principal investigator: Maurizio Brunori<br />
Professor of Chemistry and Biochemistry<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 49910544; Fax: (+39) 06 4440062<br />
maurizio.brunori@uniroma1.it<br />
81<br />
AREA 4<br />
How proteins recognize their biochemical partners: ligand binding<br />
and folding pathways of PDZ domains<br />
Participants:<br />
Carlo Travaglini-Allocatelli, professor; Stefano Gianni, CNR<br />
researcher; Ylva Ivarsson, post-doc fellow; Rajanish Giri,<br />
Angela Morrone, PhD students.<br />
Collaborations:<br />
University of Uppsala, Sweden (Prof. Per Jemth); University of<br />
Cambridge, UK (Prof. Michele Vendruscolo).<br />
Report of activity<br />
The intricate nature of chemical reactions taking<br />
place in living systems demands these reactions to be<br />
highly specific. In general, such specificity is achieved<br />
by displaying a geometrical precision in molecular<br />
recognition with two interacting molecules displaying<br />
complementary features; for example, a substrate<br />
and an enzyme fit together like a key fits a lock. It is<br />
therefore clear that the process of protein folding is<br />
vital for any living organism.<br />
Recent studies have shown that changes in the cellular<br />
milieu or mutations may affect the stability of<br />
proteins, resulting in a totally or partially denatured<br />
state under physiological conditions; this event may<br />
be involved in many life threatening diseases ranging<br />
from Cystic Fibrosis to Alzheimer’s syndrome.<br />
Moreover, residual structure in the denatured state,<br />
even if minimal, can dictate nucleation and propagation<br />
that is carried through the folding pathway.<br />
This is exactly what we observed in the case<br />
of nucleophosmin, a protein crucially involved in<br />
human hematological malignancies: the characterization<br />
of its folding pathway allowed us to show that<br />
the protein folds through a compact denatured state<br />
(Scaloni et al., 2009, 2010), a finding which is pertinent<br />
to the properties of the mutated and thereby<br />
pathological variants.<br />
In the research project we employed extensive protein<br />
engineering and exploited circularly permuted<br />
proteins to probe the role of sequence connectivity,<br />
rather than composition, in dictating folding pathways.<br />
In particular:<br />
(i) we have characterized the folding pathway of<br />
an engineered variant based on the sequence of<br />
a canonical PDZ domain, cpPDZ (Ivarsson et al.,<br />
2009). To understand the role of sequence connectivity,<br />
we carried out a complete Φ-value analysis<br />
for an engineered circularly permuted PDZ domain,<br />
which displays a peculiar symmetrical distribution<br />
of secondary structure elements (i.e. ββαββαββ)<br />
while maintaining the canonical tertiary interactions.<br />
Reliable Φ values were obtained for both a<br />
low-energy intermediate and the late rate-limiting<br />
transition state, allowing a description of both early<br />
and late events in folding. A comparison with the<br />
wild-type PDZ2 reveals that while the structure<br />
of the late transition state is robust and unaffected<br />
by circular permutation, the folding intermediate is<br />
stabilized by a different nucleus involving residues<br />
located at the new N- and C-termini. This novel<br />
result suggested that folding is driven by competing<br />
nuclei whose stabilities may be selectively tuned by<br />
circular permutation.<br />
(ii) we have obtained for the first time the 3D<br />
structure of a misfolded intermediate populated<br />
in the folding pathway of a naturally occurring<br />
PDZ domain from the D1p protease of the green<br />
alga Scenedesemus obliquus (Gianni et al., 2010).<br />
Incorrectly folded states transiently populated during<br />
the protein folding process are potentially prone<br />
to aggregation and have been implicated in a range<br />
of devastating misfolding disorders that include<br />
Alzheimer’s and Parkinson’s diseases. Despite their<br />
medical importance, however, the structures of these<br />
states and the mechanism of formation have largely<br />
escaped detailed characterization because of their<br />
short-lived nature. We have been able to obtain the<br />
structures of all the major states involved in the<br />
folding process of this PDZ domain, including an offpathway<br />
“abnormal” intermediate. By using a com-
Maurizio Brunori<br />
bination of transient kinetics, protein engineering<br />
and computational techniques, we have shown that<br />
although the productive folding of D1pPDZ is similar<br />
to that observed in a canonical PDZ domain, the<br />
off-pathway intermediate is less compact and characterized<br />
by an “abnormal” docking of the N-terminal<br />
β-hairpin, though its overall topology largely resembles<br />
that of the native state. These results, that we<br />
published in Nature SMB (Gianni et al., 2010), suggest<br />
that misfolded intermediates may often display<br />
an unexpected high degree of native-like structure,<br />
mis-folding being dictated by the local mis-assembly<br />
of a few structural elements (a swapped β-hairpin in<br />
the case of D1pPDZ).<br />
Publications<br />
Gianni S, Brunori M, Jemth P, Oliveberg M, Zhang<br />
M. Distinguishing between smooth and rough free<br />
energy barriers in protein folding. Biochemistry. 2009,<br />
48: 11825-30. doi: 10.1021/bi901585q.<br />
Ivarsson Y, Travaglini-Allocatelli C, Brunori M,<br />
Gianni S. Engineered symmetric connectivity of secondary<br />
structure elements highlights malleability of<br />
82<br />
protein folding pathways. J Am Chem Soc. 2009, 131:<br />
11727-33. doi: 10.1021/ja900438b.<br />
Scaloni F, Gianni S, Federici L, Falini B, Brunori<br />
M. Folding mechanism of the C-terminal domain of<br />
nucleophosmin: residual structure in the denatured<br />
state and its pathophysiological significance. FASEB<br />
J. 2009, 8: 2360-5. doi: 10.1096/fj.08-128306.<br />
Travaglini-Allocatelli C, Ivarsson Y, Jemth, P, Gianni<br />
S. Folding and stability of globular proteins and implications<br />
for function. Curr Opin Struct Biol. 2009,<br />
19: 3-7. doi: 10.1016/j.sbi.2008.12.001.<br />
Gianni S, Ivarsson Y, De Simone A, Travaglini-<br />
Allocatelli C, Brunori M, Vendruscolo M. Structural<br />
characterization of a misfolded intermediate populated<br />
during the folding process of a PDZ domain. Nat Struct<br />
Mol Biol. 2010, 12: 1431-7. doi:10.1038/nsmb.1956.<br />
Scaloni F, Federici L, Brunori M, Gianni S.<br />
Deciphering the folding transition state structure<br />
and denatured state properties of nucleophosmin<br />
C-terminal domain. Proc Natl Acad Sci USA. 2010,<br />
107: 5447-52. doi: 10.1073/pnas.0910516107.
Plant innate immunity: cell wall-mediated signalling and<br />
recognition in plant defense<br />
Principal investigator: Felice Cervone<br />
Professor of Plant Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912517; Fax: (+39) 06 49912446<br />
felice.cervone@uniroma1.it<br />
Participants:<br />
Giulia De Lorenzo, Daniela Bellincampi, professors; Simone<br />
Ferrari, Benedetta Mattei, researchers; Alexander Brutus,<br />
Manuela Casasoli, Roberta Galletti, Vincenzo Lionetti,<br />
Francesca Sicilia, Francesco Spinelli, post-doc fellows; Fedra<br />
Francocci, Lorenzo Mariotti, Manuel Benedetti, Daniel<br />
Savatin, PhD students; Daniela Pontiggia,Giovanni Salvi,<br />
Isabel Santori, Lucia Tufano, technicians.<br />
Report of activity<br />
Plants are continually exposed to pathogens and,<br />
in most cases, successfully defend themselves. They<br />
are capable of detecting the presence of pathogens<br />
and activating the signalling pathways that lead to<br />
defence responses. This is part of the “plant innate<br />
immunity” mechanism that is based on recognition<br />
of the so-called pathogen-associated molecular patterns<br />
(PAMPs) present in different microbes. The<br />
cell wall plays an important role in plant innate<br />
immunity since cell wall-derived oligolacturonides<br />
(OGs) are formed during attempted attacks of pathogens<br />
and trigger the typical responses elicited by<br />
PAMPs. OGs may be regarded as DAMPs (Damage-<br />
Associated Molecular Patterns).<br />
The molecular basis of the plant innate immunity<br />
elicited by DAMPs is the focus of this project. In<br />
Arabidopsis the accumulation of OGs takes place<br />
during the degradation of pectin by microbial polygalacturonases<br />
(PGs) and is favoured when PGs interact<br />
with specific plant inhibitors (polygalacturonaseinhibiting<br />
proteins or PGIPs). OGs activate the<br />
expression of defense genes through a signal transduction<br />
pathway that has been partially elucidated<br />
during this project. We have achieved the important<br />
result of identifying the receptor of OGs.<br />
Members of the Wall-Associated Kinase (WAK)<br />
family have been thought for a long time to be candidate<br />
receptors of OGs, due to their ability to bind<br />
in vitro these oligosaccharides. Because lethality and<br />
83<br />
AREA 4<br />
redundancy have hampered the study of WAKs by<br />
reverse genetics, we have adopted a chimeric receptor<br />
approach to elucidate the role of Arabidopsis<br />
WAK1. In a test-of-concept study, we first defined<br />
the appropriate chimera design and demonstrated<br />
that the Arabidopsis pattern recognition receptor<br />
(PRR) EFR is amenable to the construction of functional<br />
and resistance conferring chimeric receptors<br />
carrying the ectodomain of another Arabidopsis<br />
PRR, FLS2. After, we analyzed chimeras derived<br />
from EFR and WAK1 and demonstrated that, upon<br />
stimulation with OGs, the WAK1 ectodomain is<br />
capable of activating the EFR kinase domain. On the<br />
other hand, upon stimulation with the cognate ligand<br />
elf18, the EFR ectodomain activates the WAK1<br />
kinase, triggering defense responses that mirror<br />
those normally activated by OGs and are effective<br />
against fungal and bacterial pathogens. Finally, we<br />
demonstrated that transgenic plants overexpressing<br />
WAK1 are more resistant to the fungal pathogen<br />
Botrytis cinerea.<br />
Mitogen-activated protein kinases (MAPKs) are<br />
important components of the plant innate immune<br />
system. In Arabidopsis, the single kinases AtMPK3,<br />
AtMPK6 and AtMPK4 are activated by pathogens<br />
and elicitors; however, their specific contribution to<br />
the regulation of defense responses is not completely<br />
defined. We have shown that overexpression of the<br />
protein phosphatase AP2C1 impedes the phosphorylation<br />
of AtMPK3, AtMPK4 and AtMPK6 triggered<br />
by OGs and by flg22, a well-known microbeassociated<br />
molecular pattern (MAMP). The simultaneous<br />
dephosphorylation of the three MAPKs<br />
impairs the activation of responses to these elicitors<br />
such as the expression of a subset of marker genes<br />
and induction of resistance against Botrytis cinerea. In<br />
contrast, production of reactive oxygen species and<br />
the expression of a MAPK-independent gene are not<br />
affected by AP2C1. Single mutant analysis showed<br />
that only atmpk6, but not atmpk3 or mekk1, a triple<br />
kinase upstream of AtMPK4 activation, has reduced
Felice Cervone<br />
OG- and flg22-triggered gene activation. Our results<br />
indicate that AtMPK3 acts as a negative regulator of<br />
specific responses to DAMPs and MAMPs important<br />
for plant immunity.<br />
A plant protein (PGIP) localized in the cell wall<br />
modulates the accumulation of OGs by interacting<br />
with microbial PGs. PGIPs are leucine-rich repeat<br />
(LRR) proteins and represent a useful model system<br />
to study ligand-receptor coevolution dynamics at<br />
the protein level. We have applied codon substitution<br />
models to PGIP sequences of different eudicotyledonous<br />
families to identify putative positively<br />
selected sites and then compared these sites with<br />
the propensity of protein surface residues to interact<br />
with protein partners, based on desolvation energy<br />
calculations. The two approaches remarkably correlated<br />
in pinpointing several residues in the concave<br />
face of the LRR domain. These residues were mutated<br />
into alanine and their effect on the recognition of<br />
several PGs was tested, leading to the identification<br />
of unique hotspots for the PGIP-PG interaction.<br />
We have obtained a low resolution structure of the<br />
complex formed by PG from Fusarium phyllophylum<br />
and PGIP from Phaseolus vulgaris as determined<br />
by small-angle x-ray scattering (SAXS) analysis.<br />
The inhibitor interacts with the enzyme through<br />
its concave surface of the LRR domain. Both sides<br />
of the enzyme active site cleft are recognized by the<br />
84<br />
inhibitor, accounting for the competitive mechanism<br />
of inhibition observed. The structure is in agreement<br />
with site-directed mutagenesis data and subsequent<br />
assay of the inhibitory activity.<br />
Publications<br />
Casasoli M, Federici L, Spinelli F, Di Matteo A,<br />
Vella N, Scaloni F, Fernandez-Recio J, Cervone F,<br />
De Lorenzo G. Integration of evolutionary and desolvation<br />
energy analysis identifies functional sites in<br />
a plant immunity protein. Proc Natl Acad Sci USA.<br />
2009, 106: 7666-71. doi: 10.1073/pnas.0812625106.<br />
Lionetti V, Francocci F, Ferrari S, Volpi C,<br />
Bellincampi D, Galletti R, D’Ovidio R, De Lorenzo<br />
G, Cervone F. Engineering the cell wall by reducing<br />
de-methyl-esterified homogalacturonan improves<br />
saccharification of plant tissues for bioconversion.<br />
Proc Natl Acad Sci USA. 2010, 107: 616-21. doi:<br />
10.1073/pnas.0907549107.<br />
Brutus A, Sicilia F, Macone A, Cervone F, De<br />
Lorenzo G. A domain swap approach reveals a role of<br />
the plant wall-associated kinase 1 (WAK1) as a receptor<br />
of oligogalacturonides. Proc Natl Acad Sci USA.<br />
2010, 107: 9453-7. doi: 10.1073/pnas.1000675107.
Spontaneous formation and evolution of informational nucleic<br />
polymers<br />
Principal investigator: Ernesto Di Mauro<br />
Professor of Molecular Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912880; Fax: (+39) 06 4440812<br />
ernesto.dimauro@uniroma1.it<br />
Participants:<br />
Giovanna Costanzo, researcher; Fabiana Ciciriello, post-doc<br />
fellow; Samanta Pino, PhD student; Silvia Lopizzo, collaborator.<br />
Collaborations:<br />
Dipartimento di Agrobiologia e Agrochimica, Università della<br />
Tuscia, Viterbo (Prof. Raffaele Saladino); Università Tor<br />
Vergata, Rome (Dr. Claudia Crestini); INAF, Osservatorio<br />
Astrofisico di Arcetri, Firenze (Prof. John R. Brucato); University<br />
of Haifa, Israel (Prof. Edward N. Trifonov).<br />
Report of activity<br />
The general goal of our research has been the determination<br />
of a plausible and experimentally verifiable<br />
ensemble of chemical processes allowing the spontaneous<br />
organization of informational polymers in abiotic<br />
conditions. Once identified, this system could provide<br />
invaluable information both on the origin of informational<br />
polymers and on the persistence capacity of the<br />
extant genetic systems in terrean and non-terrean environments.<br />
In this frame of reference we have analyzed:<br />
Synthesis of nucleic precursors<br />
The reaction yielding nucleic bases from formamide<br />
in the presence of catalysts but in the absence of<br />
any biotic (cellular or enzymatic) effector has been<br />
carefully analyzed. The analyses that have been performed<br />
pertain to a series of sulphur and iron containing<br />
minerals, of zirconium-based minerals and<br />
of a large panel of boron-containing compounds. The<br />
rationale for the first type of analyses is that iron and<br />
sulphur minerals are involved in the hypothesized<br />
initial chemical energy-transfer mechanisms and<br />
that zirconia formed only in the presence of water.<br />
The protective function of borates of various kinds<br />
towards nucleosides has been recently identified.<br />
Hence the interest of the exploration of their synthetic<br />
capacity. The overall analysis has provided a<br />
complete picture for the syntheses of nucleic bases<br />
85<br />
AREA 4<br />
in the presence of the mineral compounds which are<br />
major constituents of the solar system.<br />
Quite interestingly the products obtained are precursors<br />
of nucleic acids and related compounds, namely<br />
purine, (1H)-pyrimidinone, isocytosine, adenine,<br />
2-aminopurine, carbodiimide, urea, oxalic acid. The<br />
stability properties of nucleic acids in the presence of<br />
these minerals were analyzed. The results clearly show<br />
that iron-based catalysts have strongly degradative<br />
capacity and that the origin of informational polymers<br />
with these catalysts only can be based upon the evolution<br />
of specific protective mechanisms of the polymers<br />
(“encapsulation” in lipids, interaction in the clays, etc.).<br />
The results obtained with zirconium-based catalysts<br />
have provided a similar set of results: components of<br />
the nucleic acid bases are synthesized from formamide<br />
in the presence of minerals which were present in the<br />
early formation of planet. Earth Synthesis of nucleic<br />
acid precursors has been obtained also in the presence<br />
of a large panel of boron-containing minerals<br />
(unpublished) and of powdered minerals of the meteorite<br />
Murchison (unpublished). Also in these cases a<br />
cornucopia of nucleic acid precursors was obtained,<br />
along with a large number of carboxylic acids and of<br />
formylated glycine and formylated alanine.<br />
Taken together, these results show that:<br />
- Formamide is the compound that may explain the<br />
abiotic origin of nucleic acids, both for the production<br />
in moderate-to-high yield of the necessary<br />
building blocks and for the relative independence<br />
upon a specific catalyst.<br />
- Formamide may have created the conditions for the<br />
activation (namely: phosphorylation) of abiotically<br />
synthesized nucleosides and, as described in the following<br />
section, of their polymerization.<br />
- Stability assays have been developed and carried<br />
out with all catalysts studied. The stability of RNA<br />
in their presence has been measured, allowing to<br />
define the conditions and the environments in which<br />
the abiotically generated RNA polymers could have<br />
been able to survive.
Ernesto Di Mauro<br />
Polymerizations and stability<br />
After the synthesis of nucleic bases and their possible<br />
transformation in nucleosides, next step towards<br />
complexity is the activation by phosphorylation of<br />
abiotically synthesized nucleosides. This problem<br />
has been solved by our group, as <strong>report</strong>ed (1).<br />
This observation has allowed us to answer the question:<br />
may an ensemble of monomers give rise to<br />
informational molecules endowed with pre-genetic<br />
possibilities? We have previously determined the<br />
physical-chemical conditions in which this may occur<br />
(2,3). An extended set of analyses has determined<br />
the properties of RNA molecules allowing the possibility<br />
of evolution in abiotic environments and<br />
the conditions in which this may occur. The results<br />
are <strong>report</strong>ed in (4). Starting from these findings, we<br />
have focused on the determination of the resilience<br />
properties of nucleic polymers in various prebiotic<br />
scenarios, in both terrestrial and space-wise conditions.<br />
Namely, experiments have been conducted to<br />
answer the questions:<br />
- How does the structure of nucleic acids affect their<br />
resistance to the hydrolytic process?<br />
- Do different environmental conditions affect<br />
sequence evolution? Conditions were determined for<br />
the polymerization of prebiotically formed monomers<br />
into oligomers and increasingly complex informational<br />
structures. Three levels of abiotic polymerization<br />
were attained:<br />
1) from 3’,5’ cyclic nucleotides: 3’,5’ cAMP and 3’,5’<br />
cGMP. In the first case, short chains of RNA long<br />
up to 8 nucleotides were obtained, which undergo<br />
successive ligations, finally resulting in the production<br />
of RNA chains up to 200 nucleotides long. In the<br />
second case, serial growth of oligomers resulted in<br />
the production of oligoG fragments up to 30 nucleotides<br />
long.<br />
2) Terminal ligation was observed for both polyA<br />
oligonucleotides and sequence-complementary<br />
polyC and polyG oligonucleotides. The mechanism<br />
of reaction for these non-enzymatic ligations was<br />
determined. These ligation reactions provide an<br />
explanation to the production of RNA chains whose<br />
size exceeds that expected from a synthesis/degradation<br />
quickly reacing steady-state that would limit the<br />
size of the observed polymers to molecules shorter<br />
than a few tens of nucleotides.<br />
3) Terminal growth of preformed oligonucleotides.<br />
We have <strong>report</strong>ed that 3’.5’ cyclic GMP can polymerize<br />
on the 3’ tail of preformed oligoC polymers.<br />
The net result is in the non-enzymatic formation of<br />
mixed sequences molecules, pointing to the possible<br />
86<br />
mechanism for sequence-complexity evolution.<br />
These results provide the proof-of-principle that<br />
abiotic polymerizations are obtained spontaneously.<br />
In addition, a set of conditions are established which<br />
may provide the test systems for the analysis of the<br />
evolution of genetic information in acellular, non.<br />
biotic conditions. The results obtained are instrumental<br />
to the formulation of a chemical theory of the<br />
origin of informational polymers on this planet and<br />
for the search of life in non-terrean environments.<br />
References<br />
1) G. Costanzo et al. Nucleoside phosphorylation<br />
by phosphate minerals. J Biol Chem (2007) 282,<br />
16729-35.<br />
2) R. Saladino et al. Origin of informational polymers:<br />
differential stability of 3’- and 5’-phosphoester<br />
bonds in deoxy monomers and oligomers. J<br />
Biol Chem (2005) 280, 35658-69.<br />
3) R. Saladino et al. Origin of Informational<br />
Polymers: Differential Stability of Phosphoester<br />
Bonds in Ribo Monomers and Oligomers. J Biol<br />
Chem (2006) 281, 5790-96.<br />
4) F. Ciciriello, G. Costanzo, S. Pino, C. Crestini,<br />
R. Saladino, E. Di Mauro. Molecular complexity<br />
favors the evolution of ribopolymers. Biochemistry<br />
(2008) 47, 2732-42.<br />
Publications<br />
Costanzo G, Pino S, Ciciriello F, Di Mauro E.<br />
Generation of long RNA chains in water. J Biol Chem.<br />
2009, 284: 33206-16. doi: 10.1074/jbc.M109.041905.<br />
Saladino R, Crestini C, Ciciriello F, Pino S, Costanzo<br />
G, Di Mauro E. From formamide to RNA: the roles<br />
of formamide and water in the evolution of chemical<br />
information. Res Microbiol. 2009, 160: 441-8. doi:<br />
10.1016/j.resmic.2009.06.001.<br />
Barks HL, Buckley R, Grieves GA, Di Mauro E, Hud<br />
N, Orlando T. Guanine, Adenine, Hypoxanthine<br />
production in UV-irradiated Formamide solutions:<br />
relaxation of the requirements for prebiotic purine<br />
nucleobase formation. Chembiochem. 2010, 11: 1240-<br />
43. doi: 10.1002/cbic.201090037.<br />
Saladino R, Neri V, Crestini C, Costanzo G, Graciotti<br />
M, Di Mauro E. The role of the formamide/zirconia<br />
system in the synthesis of nucleobases and biogenic<br />
carboxylic acid derivatives. J Mol Evol. 2010, 71:<br />
100-10. doi: 10.1007/s00239-010-9366-7.
Synthesis of pyridoxal phosphate in the vitamin B6 salvage<br />
pathway and targeting of the cofactor to apo-enzymes<br />
Principal investigator: Martino Luigi di Salvo<br />
Researcher in Biochemistry<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 49917684; Fax: (+39) 06 49917566<br />
martino.disalvo@uniroma1.it<br />
Participants:<br />
Francesco Bossa, professor; Roberto Contestabile,<br />
Sebastiana Angelaccio, researchers; Rita Florio, post-doc<br />
fellow; Mirella Vivoli, PhD student.<br />
Collaborations:<br />
Department of Medicinal Chemistry, Virginia Commonwealth<br />
University, USA (Prof. Martin Safo); Department of Medical<br />
Laboratory Science and Biotechnology, National Cheng Kung<br />
University, Taiwan (Prof. Tzu-Fan Fu).<br />
Report of activity<br />
In the 2009-2010 period, our research activity has<br />
focused on the following topics:<br />
The mechanism of PLP addition to apo-SHMT<br />
during protein folding<br />
Several mutant forms of E. coli SHMT, localized in<br />
the so-called third hydrophobic cluster, have been<br />
characterized for their enzymatic activity, spectroscopic<br />
features and PLP binding capability. The<br />
results show that the hydrophobic cluster is essential<br />
for enzyme assembly and formation of the correct<br />
quaternary structure, even though it is not localized<br />
in the subunit interface. The cluster actually stabilizes<br />
a bridging structural element, which is essential<br />
for the overall enzyme structure; this element connects<br />
the two protein domains, is involved in the<br />
modeling of the active site and participates in the<br />
quaternary assembly. Moreover, we have shown that<br />
the final slow step of cofactor addition to SHMT<br />
requires either free PLP or PLP-substrate (glycine or<br />
L-serine) complex; other PLP-amino acid complexes<br />
do not trigger the needed conformational change.<br />
We also carried out a structural and functional study<br />
on a conserved cation-π interaction (Arg-Tyr) in the<br />
active site of SHMT. Both residues involved in this<br />
cation-π interaction seem to have a catalytic role in<br />
the transaldimination reaction, a common step in all<br />
PLP-dependent catalyzed reactions.<br />
87<br />
AREA 4<br />
Studies on the putative PLP channelling<br />
between PNPOx, PLK and SHMT<br />
The channelling mechanism requires a direct interaction<br />
between the donor enzyme and the acceptor<br />
enzyme, through the formation of a protein complex.<br />
In order to detect such interaction by means of<br />
fluorescence anisotropy, we produced PNPOx and<br />
PLK forms labelled with maleimide fluorophore.<br />
All apo-B6 enzymes used in the experiments were<br />
members of the Fold-Type I group. The experiments<br />
demonstrated that PNPOx and PLK interact with<br />
all apo-B6 enzymes tested. The calculated dissociation<br />
constants ranged from 60 nM to 400 nM in the<br />
case both donor and acceptor enzymes were from<br />
E. coli. Dissociation constants with either human<br />
PLK or PNPOx and E. coli apo-B6 enzymes ranged<br />
from 1 to 20 µM. These results showed that the<br />
transfer of PLP takes place more efficiently when<br />
donor and acceptor enzymes from the same source<br />
are employed, in agreement with the hypothesis of<br />
the channeling mechanism. We have also developed<br />
a new methodology, based on the employment of<br />
a chemical quenched-flow instrument, that allows<br />
rapid kinetic measurements of the PLP transfer from<br />
either PNPOx or PLK to the apo-B6 enzymes.<br />
Studies on PNPOx<br />
We have produced and characterized the R229W<br />
mutant forms of human PNPOx, known to cause<br />
neonatal epileptic encephalopathy. This variant is<br />
850-fold less efficient than the wild-type enzyme;<br />
there is also a 50-fold reduction in the affinity for the<br />
FMN cofactor. The crystal structure of the R229W<br />
shows that the substitution of Arg229 has led to a<br />
loss of hydrogen-bond interactions between FMN<br />
and active site residues. Additionally, the mutation has<br />
led to an alteration of the configuration of a β-strandloop-β-strand<br />
structure at the active site, which is<br />
important for substrate binding and orientation for<br />
catalysis. These results provided a molecular basis for<br />
the phenotype associated with the R229W mutation,
Martino Luigi di Salvo<br />
allowing for a better understanding of the pathophysiological<br />
consequences of PNPOx mutations.<br />
Studies on PLK<br />
For what PLK is concerned, we first focused on the<br />
mechanism of action of the enzyme, and particularly<br />
on the role of the active site residue Asp235, which<br />
we showed to be involved in the deprotonation of the<br />
substrate, an essential step for catalysis. During the<br />
crystallographic studies on Asp235, we realized the<br />
presence of a PLP molecule tightly bound to the active<br />
site of the enzyme, with the formation of a high affinity<br />
ternary complex between PLK, PLP and the substrate<br />
MgATP. Then, PLP might work as a slow tight binding<br />
inhibitor of PLK. Understanding the mechanism of<br />
regulation of PLK is particularly important considering<br />
the negative impact of several commonly used natural<br />
products and drugs on the activity of the PLK (and,<br />
consequently on the homeostasis of PLP in the cell).<br />
For this reason, we have determined the inhibition<br />
constants and mechanism of some of these compounds,<br />
such as theophylline, gingkotoxin, lamotrigine, roscovitine.<br />
The three-dimensional structure determination<br />
of PLK in complex with the above inhibitors has been<br />
determined and a manuscript is under submission.<br />
88<br />
Publications<br />
Gandhi AK, Ghatge M, Musayev FN, Sease A,<br />
Aboagye SO, di Salvo ML, Schirch V, Safo MK.<br />
Kinetic and structural studies of the role of the active<br />
site residue Asp235 of human pyridoxal kinase.<br />
Biochem Biophys Res Commun. 2009, 381: 12-15. doi:<br />
10.1016/j.bbrc.2009.01.170.<br />
Musayev FN, Di Salvo ML, Saavedra MA,<br />
Contestabile R, Ghatge MS, Haynes A, Schirch V,<br />
Safo MK. Molecular basis of reduced pyridoxine<br />
5’-phosphate oxidase catalytic activity in neonatal<br />
epileptic encephalopathy disorder. J Biol Chem. 2009,<br />
284: 30949-56. doi: 10.1074/jbc.M109.038372.<br />
Vivoli M, Angelucci F, Ilari A, Morea V, Angelaccio<br />
S, di Salvo ML, Contestabile R. Role of a conserved<br />
active site cation-π interaction in Escherichia coli<br />
serine hydroxymethyltransferase. Biochemistry. 2009,<br />
48: 12034-46. doi: 10.1021/bi901568b.<br />
Wilkins BJ, Marionni S, Young DD, Liu J, Wang Y,<br />
Di Salvo ML, Deiters A, Cropp TA. Site-specific incorporation<br />
of fluorotyrosines into proteins in Escherichia<br />
coli by photochemical disguise. Biochemistry. 2010; 49:<br />
1557-9. doi: 10.1021/bi100013s.
Integrating advanced functional materials and chromatographymass<br />
spectrometry to examine protein-ligand interactions<br />
Principal investigator: Francesco Gasparrini<br />
Professor of Organic Chemistry<br />
Dipartimento di Chimica e Tecnologie del Farmaco<br />
Tel: (+39) 06 49912776; Fax: (+39) 06 49912780<br />
francesco.gasparrini@uniroma1.it<br />
Participants:<br />
Bruno Botta, Claudio Villani, professors; Ilaria D’Acquarica,<br />
Marco Pierini, researchers; Alessia Ciogli, Deborah<br />
Subissati, Laura Nevola, post-doc fellows; Giovanna<br />
Cancelliere, technician.<br />
Report of activity<br />
The covalent immobilization of carbohydrates<br />
on solid supports has recently been developed by<br />
employing copper-catalyzed azide-alkyne cycloaddition<br />
(CuAAC), which is in agreement with the<br />
demonstrated potential of this reaction in surface<br />
modification. In “click saccharides”, prepared by<br />
Liang using the click chemistry approach, two-step<br />
azido-modified silica gels were coupled to sugar<br />
alkynes in the presence of a Cu catalyst to yield the<br />
above packing materials, in which the linked carbohydrates<br />
retained their inherent configuration and<br />
properties. In this context, we prepared novel sugarbased<br />
silica gels by exploiting the CuAAC of two<br />
sugar alkynes with new single-step azido-activated<br />
silica gels. The fully characterised stationary phases<br />
were generally used for hydrophilic interaction chromatography<br />
(HILIC), with particular application in<br />
the stereoselective separation of monosaccharides.<br />
Dynamic HILIC (DHILIC) experiments were performed<br />
to evaluate the influence of anomerization on<br />
the chromatographic peak shapes. The potential of<br />
such materials was shown in the separation of other<br />
highly polar compounds, including amino acids and<br />
flavonoids.<br />
We recently <strong>report</strong>ed a new hybrid organic/inorganic<br />
chiral stationary phase (CSP) for HPLC synthesized<br />
by the grafting-from (g-from) radical polymerization<br />
of an enantiopure diacryloyl derivative of trans-<br />
1,2-diaminocyclohexane (DACH) in the presence of<br />
mesoporous, azo-activated silica micro-particles. This<br />
was the first example of application of the g-from<br />
approach to the synthesis of a CSP for HPLC applica-<br />
89<br />
AREA 4<br />
tions. However, the thermal grafting process could<br />
not be surface confined only, since concomitant solution<br />
polymerization may lead to ungrafted polymer<br />
chains. Thus, we moved to study another way<br />
to induce polymerization of the above monomer,<br />
and focused our attention on photo-induced polymerization,<br />
which has been obtaining much attention<br />
in recent years because of its numerous industrial<br />
applications. Two hybrid polyacrylamide CSPs<br />
were thus prepared by the surface-initiated photoinduced<br />
radical polymerization of enantiopure N,N’diacryloyl<br />
derivatives of (1R,2R)-DACH and (1R,2R)diphenylethylenediamine<br />
(DPEDA), initiated by the<br />
system consisting of trichloroacetyl groups on mesoporous<br />
silica particles and dimanganese decacarbonyl<br />
Mn 2 (CO) 10 under UV irradiation. Physico-chemical<br />
properties and chromatographic performances of the<br />
two phases were evaluated in comparison with those<br />
exhibited by the homologue CSPs obtained by the<br />
g-from thermal-induced process. The new photopolymerization<br />
approach yielded higher grafting density<br />
than the thermal-induced one, especially in the case of<br />
the less reactive monomer (the diacryloyl derivative<br />
of (1R,2R)-DPEDA), good chromatographic efficiency<br />
and a broad application field under normal phase and<br />
polar organic mode conditions.<br />
With regard to our previous studies of biorecognition<br />
events monitored by Fourier-Transform Ion<br />
Cyclotron Resonance (FT-ICR) mass spectrometry,<br />
we investigated the nature of the non-covalent interactions<br />
between ethanolamine neurotransmitters<br />
and some chiral resorc[4]arene receptors in the<br />
gas phase, under conditions mimicking the extensive<br />
desolvation complementary to the uptake of<br />
neurotransmitters inside their biological receptors.<br />
The kinetic experiments, supported by molecular<br />
mechanics calculations and molecular dynamics<br />
simulations, indicate that the relative stability of<br />
the diastereomeric proton-bound adducts between<br />
ethanolamine neurotransmitters and the chiral hosts
Francesco Gasparrini<br />
depends on (i) the relative position and orientation<br />
of the hydroxyl- and amino-functionalities on the<br />
neurotransmitter side chain, (ii) the presence of the<br />
methyl substituent on its nitrogen atom, and (iii) the<br />
specific group of the receptor proton-bonded to the<br />
amino group of the ethanolamine.<br />
Publications<br />
Moni L, Ciogli A, D’Acquarica I, Dondoni A,<br />
Gasparrini F, Marra A. Synthesis of sugar-based<br />
90<br />
silica gels by copper-catalysed azide-alkyne cycloaddition<br />
via a single-step azido-activated silica intermediate<br />
and the use of the gels in hydrophilic<br />
interaction chromatography. Chem Eur J. 2010, 16:<br />
5712-22. doi: 10.1002/chem.201000106.<br />
Ciogli A, D’Acquarica I, Gasparrini F, Molinaro C,<br />
Rompietti R, Simone P, Villani C, Zappia G. Hybrid<br />
polyacrylamide chiral stationary phases for HPLC prepared<br />
by surface-initiated photopolymerization. J Sep<br />
Sci. 2010, 33: 3022-32. doi: 10.1002/jssc.201000355.
91<br />
AREA 4<br />
Molecular and functional approaches to investigate the<br />
physiopathological role of the chemokines and their receptors in<br />
the central nervous system<br />
Principal investigator: Cristina Limatola<br />
Professor of Physiology<br />
Dipartimento di Fisiologia e Farmacologia<br />
Tel: (+39) 06 49690243; Fax: (+39) 06 49910851<br />
cristina.limatola@uniroma1.it<br />
Participants:<br />
Sergio Fucile, Francesca Grassi, Eleonora Palma, Davide<br />
Ragozzino, professors; Flavia Trettel, Laura Maggi,<br />
researchers; Myriam Catalano, Raffaela Cipriani, Clotilde<br />
Lauro, post-doc fellows; Giuseppina d’Alessandro, Maria<br />
Rosito, Mariella Scianni, PhD students; Giuseppina Chece,<br />
technician.<br />
Collaborations:<br />
<strong>Istituto</strong> Mario Negri, Milano (Dr. Maria Grazia De Simone, Dr.<br />
Pia Villa); Dipartimento di Fisiologia e Farmacologia, Sapienza-<br />
Università di Roma (Dr. Letizia Antonilli, Dr. Valentina<br />
Brusadin); Karolinska Institute, Stockholm, Sweden (Dr. Bertil<br />
Fredholm); Università di Camerino (Prof. Gloria Cristalli);<br />
Università di Perugia (Prof. Fabio Franciolini).<br />
Report of activity<br />
Chemokines are proteins endogenously expressed<br />
in the central nervous system, where they play different<br />
roles during development modulating the<br />
timing of neuronal migration in different brain area<br />
and in the adulthood, regulating glia-neuron communication<br />
and leukocyte entry across the blood<br />
brain barrier under physiological and pathological<br />
conditions. The present research project aims to<br />
investigate the many functions of chemokines and<br />
their receptors in the central nervous system, with<br />
particular interest on the cross talk between glial<br />
cells and neurons and the molecular mechanisms<br />
adopted by these molecules to mediate neuroprotection,<br />
neuromodulation and cell migration. In<br />
particular, in the last year, we focused our attention<br />
on 1) the neuroprotective activity of the chemokine<br />
CX3CL1 in in vitro models of excitotoxicity and<br />
in in vivo model of cerebral ischemia; 2) the neuromodulatory<br />
activity of CX3CL1 on glutamatergic<br />
neurotransmission; 3) the chemotactic activity<br />
induced by CXCL12 on glioblastoma cells and the<br />
involvement of K+ channels.<br />
Neuroprotective and neuromodulatory<br />
activities of CX3CL1: involvement of<br />
adenosine receptors<br />
CX3CL1 is constitutively expressed in the nervous<br />
system, but levels in the brain can be modulated under<br />
diverse pathological conditions. The presence and the<br />
stimulation of the CX3CL1 receptor CX3CR1 has<br />
been correlated with a reduced release of interleukin-<br />
1-b (IL-1-b) and tumor necrosis factor-a (TNF-a) from<br />
microglial cells and a lower rate of neuronal degeneration<br />
in different experimental models of neuropathologies<br />
such as experimental autoimmune encephalomyelitis,<br />
1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine<br />
hydrochloride striatal injection, lipopolysaccharide<br />
administration, and superoxide dismutase (SOD1)<br />
mutation. These data attest to a role of the pair<br />
CX3CL1/CX3CR1 in reducing neuronal degeneration<br />
on several types of brain injury. The neurotrophic<br />
activity of exogenously administered CX3CL1 has<br />
been related to the simultaneous production of protective<br />
factors from microglial cells and in particular with<br />
the activation of adenosine receptors (ARs, Lauro et<br />
al., 2008). We showed earlier that A1R are probably<br />
involved in the neurotrophic activity of CX3CL1 as it<br />
was blocked by a relatively selective antagonist (Lauro<br />
et al., 2008). In the last year we strongly supported<br />
this conclusion in experiments where the protective<br />
effect of CX3CL1 is absent in A1R-/- mice. In addition<br />
we performed evidence that extracellularly produced<br />
adenosine mainly derived from the activity of ectonucleotidases,<br />
suggesting a predominant involvement of<br />
glial cell-dependent nucleotide release in this process.<br />
Furthermore, since it is proposed that microglia has<br />
a prominent role in mediating the neuroprotective<br />
effects of CX3CL1 and we have recently shown that<br />
CX3CL1-stimulated microglia releases neuroprotective<br />
substances that reduce Glu-induced cell death<br />
(Lauro et al., 2008) we further investigated this aspect<br />
showing that the selective ablation of microglia from<br />
hippocampal cultures, using clodronate-encapsulating
Cristina Limatola<br />
liposomes, has the effect to fully abolish the neuroprotective<br />
activity of CX3CL1 toward excitotoxic death<br />
of hippocampal neurons, confirming that these cells<br />
represent the first target, which primes the functional<br />
effects of CX3CL1 (Lauro et al., 2010).<br />
CX3CL1 is known to modulate glutamatergic neurotransmission<br />
with a mechanism that requires GluR1<br />
AMPA-type Glu receptors in hippocampal neurons<br />
(Ragozzino et al., 2006). We previously demonstrated<br />
that adenosine receptor type 1 (A1R) are involved in<br />
CX3CL1-induced depression of glutamatergic currents<br />
in hippocampal neurons and transfected HEK cells<br />
(Lauro et al., 2008). In the last year we characterized the<br />
role of different ARs in CX3CL1-induced modulation of<br />
hippocampal glutamatergic transmission, using cultured<br />
neurons and brain slices, demonstrating that CX3CL1<br />
action requires the activation of A1A, A2AR and A3R,<br />
by locally released or tonic adenosine. Specifically,<br />
CX3CL1 causes glutamatergic depression through A1R<br />
and A3R and potentiation through A2AR stimulation.<br />
Thus, CX3CL1 might affect glutamatergic synapses in<br />
opposite way, depending on the prevailing functional<br />
expression of AR subtypes (Piccinin et al., 2010).<br />
Modulation of glioblastoma chemotaxis by<br />
CXCL12: requirement of IK channel activity<br />
Growing evidence shows a role for the chemokine<br />
CXCL12 and its receptor CXCR4 in the migratory<br />
behavior of glioblastoma cells: CXCL12 expression in<br />
low-grade glioma has been associated with a shorter<br />
time to tumor progression, whereas CXCR4 is widely<br />
expressed in glioblastoma tissues, overexpressed in<br />
angiogenic areas and in infiltrating glioblastoma<br />
cells in vivo, and a phosphorylated/activated form<br />
of CXCR4 is largely found in astrocytomas. It has<br />
been further shown that the expression of CXCR4<br />
in highly malignant gliomas is associated with poor<br />
prognosis and that CXCL12 stimulates the proliferation<br />
of glioma progenitor cells. A chemotactic role of<br />
CXCL12/CXCR4 axis has been demonstrated in several<br />
glioblastoma cell lines in vitro, whereas a role in<br />
tumor progression has been proved in animal models,<br />
thus confirming its potentially active role in glioblastoma<br />
invasiveness in vivo. Despite the large body of<br />
information available on CXCL12/CXCR4 expression<br />
and promigratory activity in glioblastoma, the<br />
underlying mechanisms are still poorly understood.<br />
In the last year we investigate the signaling mechanisms<br />
involved in the chemotactic action of CXCL12<br />
on highly invasive human glioblastoma cells and<br />
compare them with those induced by EGF, a growth<br />
factor deeply involved in glioblastoma progression.<br />
We found that both CXCL12 and EGF induce IKCa<br />
92<br />
channel activation but, only in case of CXCL12, IKCa<br />
channels undergo a long-term functional upregulation<br />
and their activity is required for chemotaxis. These<br />
findings suggest the possibility that IKCa channel<br />
mediates the in vivo promigratory capability of<br />
CXCL12 in invasive subpopulations of glioblastoma<br />
cells overexpressing CXCR4 (Sciaccaluga et al., 2010).<br />
Publications<br />
Maggi L, Trettel F, Scianni M, Bertollini C, Eusebi<br />
F, Fredholm BB, Limatola C. LTP impairment by<br />
fractalkine/CX3CL1 in mouse hippocampus is mediated<br />
through the activity of adenosine receptor type<br />
3 (A3R). J Neuroimmunol. 2009, 215: 36-42. doi:<br />
10.1016/j.jneuroim.2009.07.016.<br />
Roseti C, Palma E, Martinello K, Fucile S, Morace<br />
R, Esposito V, Cantore G, Arcella A, Giangaspero<br />
F, Aronica E, Mascia A, Di Gennaro G, Quarato PP,<br />
Manfredi M, Cristalli G, Lambertucci C, Marucci G,<br />
Volpini R, Limatola C, Eusebi F. Blockage of A2A<br />
and A3 adenosine receptors decreases the desensitization<br />
of human GABA(A) receptors microtransplanted<br />
to Xenopus oocytes. Proc Natl Acad Sci USA.<br />
2009, 106: 15927-31. doi: 10.1073/pnas.0907324106.<br />
Lauro C, Cipriani R, Catalano M, Trettel F, Chece<br />
G, Brusadin V, Antonilli L, Van Rooijen N, Eusebi<br />
F, Fredholm BB, Limatola C. Adenosine A recep-<br />
1<br />
tors and microglial cells mediate CX3CL1-induced<br />
protection of hippocampal neurons against Gluinduced<br />
death. Neuropsychopharmacol. 2010, 35: 1550-<br />
59. doi:10.1038/npp.2010.26.<br />
Sciaccaluga M, Fioretti B, Catacuzzeno L, Pagani<br />
F, Bertollini C, Rosito C, Catalano M, D’Alessandro<br />
G, Santoro A, Cantore G, Ragozzino D, Castigli E,<br />
Franciolini F, Limatola C. CXCL12-induced glioblastoma<br />
cell migration requires intermediate-conductance<br />
Ca2+ -activated K + channel activity. Am J<br />
Physiol Cell Physiol. 2010, 299: C175-84 doi: 10.1152/<br />
ajpcell.00344.2009.<br />
Trettel F, Di Angelantonio S, Limatola C, Ransohoff<br />
RR. Conference <strong>report</strong>: Chemokines and chemokine<br />
receptors in the nervous system, Rome, 24/25<br />
October, 2009; 2nd workshop. J Neuroimmunol. 2010,<br />
224: 1-7. doi:10.1016/j.jneuroim.2010.05.001.<br />
Piccinin S, Di Angelantonio S, Piccioni A, Volpini<br />
R, Cristalli G, Fredholm BB, Limatola C, Eusebi F,<br />
Ragozzino D. CX 3 CL1-induced modulation at CA1<br />
synapses reveals multiple mechanisms of EPSC modulation<br />
involving AR subtypes. J Neuroimmunol. 2010,<br />
224: 85-92. doi:10.1016/j.jneuroim.2010.05.012.
Principal investigator: Paola Paggi<br />
Professor of Physiology<br />
Dipartimento di Biologia e biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912323; Fax: (+39) 06 49912351<br />
paola.paggi@uniroma1.it<br />
93<br />
AREA 4<br />
Neurone response to experimental injury and lack of dystrophin:<br />
a molecular, functional and structural study in autonomic ganglia<br />
in vivo and in vitro<br />
Participants:<br />
Maria Egle De Stefano, professor; Silvia Di Angelantonio,<br />
researcher; Loredana Lombardi, post-doc fellow; Valentina<br />
Annese, Valerio Licursi, PhD students.<br />
Collaborations:<br />
<strong>Istituto</strong> Superiore di Sanità, Laboratorio di Biologia Cellulare,<br />
Roma (Dr. Tamara Petrucci); Centro di Farmacologia Cellulare<br />
e Molecolare, CNR, Dipartimento di Farmacologia Medica,<br />
Università di Milano (Dr. Cecilia Gotti); Dipartimento di<br />
Biologia e Biotecnologie “Charles Darwin”, Sapienza-Università<br />
di Roma (Prof. Irene Bozzoni, Prof. Ernesto Di Mauro, Prof.<br />
Andrea Mele, Prof. Rodolfo Negri, Prof. Alberto Oliverio);<br />
Dipartimento di Scienze Biologiche, Università di Napoli Federico<br />
II (Prof. Carla Perrone Capano); Erasmus Medical Center,<br />
Department of Neuroscience, Rotterdam, The Netherlands (Dr.<br />
Casper Hoogenraad); Paracelsus University of Salzsburg,<br />
Austria (Dr. Falk Schroedl).<br />
Report of activity<br />
Aim of this project is the characterisation, in rodent<br />
superior cervical ganglion (SCG), of the molecular<br />
mechanisms and structural changes involved in the<br />
establishment, maintenance and plasticity of the reciprocal<br />
interactions between pre- and post-ganglionic<br />
neurones and between post-ganglionic neurones and<br />
their target organs. Damage of the ganglionic connectivities,<br />
consequent to pre- and post-ganglionic nerve<br />
crush, or to the lack of dystrophin, are used as a tool<br />
to perform our investigation. Moreover, on the basis<br />
of our previous data (De Stefano et al., 2007, Mol Cell<br />
Neurosci 36: 174-184), showing the involvement of the<br />
plasminogen activators/plasmin enzymatic system<br />
in the synaptic plasticity induced by postganglionic<br />
nerve crush in the rat SCG, we collaborated to a work<br />
by Prof. A. Oliverio and Prof. A. Mele, investigating<br />
whether this enzymatic complex is also involved in<br />
the ventral striatal plasticity associated to the spatial<br />
memory in mice.<br />
Specifically, we focused on: (i) the modulation of<br />
voltage-gated K+ channel expression after pre- and<br />
post-ganglionic neuron axotomy in the rat SCG; (ii)<br />
the role of dystrophin on nicotine and acetylcholineevoked<br />
currents through the nicotinic acetylcholine<br />
receptors (nAChRs) present in mouse SCG neurons<br />
in vitro; (iii) levels of tissue plasminogen activator<br />
(tPA) in the ventral striatum during consolidation of<br />
spatial memory.<br />
(i) Voltage-gated K + channels (Kv) are constituted<br />
by four alfa subunits and a cytoplasmic regulatory<br />
subunit, important for channel dynamics. As Kv are<br />
responsible for K + inward current (I A ), a reduction of<br />
which has been demonstrated after axotomy of rat<br />
SCG neurons (Sacchi et al., 2005, Neuroscience 135:<br />
31-45; Sacchi et al., 2006, J Neurophysiol 95: 2832-44)<br />
we investigated whether I A reduction was associated<br />
to changes in Kv expression. Therefore, we analyzed<br />
mRNA and protein levels of members of the Kv4<br />
and Kv1 families and of their respective regulatory<br />
proteins, β2 and KChIP, known to be expressed in<br />
rat SCG. Ganglion neurons of adult male rats<br />
were either axotomized or denervated by crushing<br />
the post- and pre-ganglionic nerves, respectively.<br />
Animals were killed 8h (hour), 1d (day), 3d and 6d<br />
after surgery for mRNA level evaluation and up to<br />
20 d for protein level evaluation. After axotomy, all<br />
mRNA examined decrease significantly, compared to<br />
control, by 1d and, except for KChIP, remain lower<br />
in the following dates. Differently from its mRNA,<br />
Kv1.4 protein levels increase significantly by 1d and<br />
remain higher than control up to 6d, while those<br />
of its β2 subunit decrease. The axotomy-induced<br />
decrease in mRNA levels is in accord with the reduction<br />
in gene expression of proteins not essential for<br />
axonal regeneration. Since Kv1 channels are mainly<br />
presynaptic and axonal, the increase in Kv1.4 protein<br />
levels suggests its accumulation in the perikaryon<br />
induced by reduced export of this protein consequent<br />
to distal axon degeneration. As the b2 subunit is<br />
required for channel insertion into membrane, its
Paola Paggi<br />
reduction is also consistent with the degeneration<br />
of axonal plasma membrane. Denervation does not<br />
affect Kv1.4 mRNA levels, while those of Kv4.2,<br />
which is mainly somatodendritic, are reduced, as<br />
expected after functionally silencing the ganglionic<br />
neurons induced by the removal of pre-synaptic<br />
boutons (De Stefano et al., 2010, 7th FENS forum of<br />
European Neuroscience. Abstract 013.10; manuscript<br />
in preparation).<br />
(ii) In the sympathetic superior cervical ganglion<br />
(SCG), nicotinic acetylcholine receptors (nAChRs)<br />
mediate fast synaptic transmission. We previously<br />
demonstrated that in SCG neurons of mdx mice, an<br />
animal model for Duchenne muscular dystrophy,<br />
lack of dystrophin causes a decrease, compared to<br />
the wild-type, in post-synaptic nAChRs containing<br />
the α3 subunit associated with β2 and/or β4 (α3β2/<br />
β4-nAChRs), but not of those containing the α7<br />
subunit (α7-nAChRs). We showed, by whole cell<br />
patch-clamp recordings from cultured SCG neurons,<br />
that both nicotine and acetylcholine-evoked currents<br />
through α3β2/β4-nAChRs are significantly reduced<br />
in mdx mice compared to wild-type, while those<br />
through α7-nAChR are unaffected. This reduction<br />
associates with that of the protein levels of α3, β2<br />
and β4 subunits. We suggest that, in mdx mouse SCG<br />
neurons, lack of dystrophin, by specifically affecting<br />
membrane stabilization of α3β2/β4-nAChRs, could<br />
determine an increase in receptor internalization and<br />
degradation, with consequent reduction in the fast<br />
intraganglionic cholinergic transmission.<br />
(iii) To determine whether tissue plasminogen<br />
activator (tPA) activity in the ventral striatum is<br />
required for spatial memory consolidation, we examined<br />
the long-term object place association memory<br />
in mice treated in the ventral striatum, immediately<br />
after training, with the tPA inhibitor PAI-1.<br />
94<br />
Administration of PAI-1 to the ventral striatum<br />
significantly and dose-dependently decreased spatial<br />
memory recall 24 h (hour) after training, compared<br />
with vehicle treated mice, as measured by relative<br />
exploration of the displaced object. In a further<br />
experiment, we assessed the effects of immediate<br />
posttraining injections of PAI-1 into the ventral striatum<br />
on spatial memory performance in the Morris<br />
water maze task. Mice were trained in the water<br />
maze, administered immediately after the final training<br />
session with 0.5 µg PAI-1 per dose, and tested in<br />
a probe trial 24 h later. Control mice spent significantly<br />
more time in the correct quadrant compared<br />
with the other three quadrants, thus demonstrating<br />
correct memory for the platform location. PAI-1treated<br />
mice failed to show a preference for any<br />
quadrant during the probe trial. The specificity of<br />
the effect observed in the spatial version of the water<br />
maze after intra- ventral striatum administrations of<br />
PAI-1 was evaluated in mice trained in the cue version<br />
of the task and treated immediately posttraining<br />
with 0.5 µg PAI-1 per side. In this case, PAI-1 did<br />
not affect performance of the mice, with both groups<br />
spending more time in the correct quadrant than in<br />
the other three. These data demonstrate that tPA<br />
activity is required immediately after spatial learning<br />
for long-term, but not short-term, memory.<br />
Publications<br />
Ferretti V, Roullet P, Sargolini F, Rinaldi A, Perri<br />
V, Del Fabbro M, Costantini VJA, Annese V, Scesa<br />
G, De Stefano ME, Oliverio A, Mele A. Ventral<br />
striatal plasticity and spatial memory. Proc Natl<br />
Acad Sci USA. 2010, 107: 7945-50. doi: 10.1073/<br />
pnas.0911757107.
Principal investigator: Maria Savino<br />
Professor of Biophysical Chemistry<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912238; Fax: (+39) 06 4440812<br />
maria.savino@uniroma1.it<br />
95<br />
AREA 4<br />
Structural and superstructural features of human telomeric chromatin<br />
Participants:<br />
Stefano Cacchione, researcher; Sabrina Pisano, Alessandra<br />
Galati, post-doc fellows; Emanuela Micheli, PhD student.<br />
Collaborations:<br />
Dipartimento di Chimica, Sapienza-Università di Roma (Prof.<br />
Armandodoriano Bianco, Prof. Pasquale De Santis, Prof.<br />
Giancarlo Ortaggi, Dr. Marco Franceschin, Dr. Anita Scipioni);<br />
Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli<br />
Federico II (Prof. Luciano Mayol, Dr. Michela Varra); Medical<br />
Research Council, Laboratory of Molecular Biology, Cambridge,<br />
UK (Dr. Daniela Rhodes, Dr. Linda Chapman); Laboratory of<br />
Molecular and Cellular Biology, ENS, Lyon (Prof. Eric Gilson).<br />
Report of Activity<br />
The recent few years in Molecular Biology have been<br />
characterized by the development of very powerful<br />
tools, which makes possible the study of whole<br />
genomes at molecular level. Nucleosome positioning,<br />
along genomes, can be considered as the first necessary<br />
step to clarify chromosomes organization. Recently,<br />
a strongly increasing interest is developing in the<br />
building of models capable of predicting nucleosomes<br />
positioning along different genomes in vitro and then<br />
to explore their correlations with the corresponding in<br />
vivo systems (Segal, Trends in Genetics 2009).<br />
These models are, generally, based on the identification<br />
of some recurrent nucleotide sequence features,<br />
obtained from the statistical analysis of very large<br />
pools of nucleosomal DNA sequences, experimentally<br />
obtained. The sequence dependent statistical<br />
model of nucleosome positioning appears rather<br />
efficient in predicting in vitro nucleosome positioning<br />
and more importantly, also the correlation with<br />
corresponding in vivo systems seems possible.<br />
Very recently, a different outcome was <strong>report</strong>ed<br />
showing that the correlation between in vitro and in<br />
vivo nucleosome postioning could be flawed and that<br />
in vivo nucleosomes positioning in coding regions<br />
arises primarily from statistical positioning from a<br />
barrier, a poorly defined parameter at molecular level,<br />
that should depend on chromatin assembly factors as<br />
well as by RNA polymerase II (Zhang, Nature 2009).<br />
This unsatisfactory situation enhances the necessity<br />
to build satisfactory physical models of nucleosome<br />
positioning, on the basis of sequence dependent physical<br />
features of DNA such as curvature, flexibility and<br />
local hydration. It is reasonable to consider that chromatin<br />
remodeling complexes or polymerase could<br />
only modulate nucleosome basic positioning, due to<br />
DNA wrapping around histone octamer according to<br />
sequence dependent DNA physical features. In fact<br />
histone octamer concentration is dramatically higher<br />
with respect to that of other chromatin proteins.<br />
We have developed an original physical model to connect<br />
sequence dependent DNA physical features to<br />
nucleosome positioning. Based on statistical mechanical<br />
approach, the model allows the calculation of the<br />
sequence dependent canonical ensemble free energy<br />
involved in nucleosome formation. The theoretical free<br />
energy was evaluated for one hundred single nucleosome<br />
tracts and successfully compared with those<br />
obtained, experimentally, with nucleosome competitive<br />
reconstitution. These results, obtained for single<br />
nucleosomes, could in principle allow the calculation<br />
of the intrinsic affinity of nucleosomes along DNA<br />
sequences virtually opening the possibility of predicting<br />
nucleosome positioning along genomes on physical<br />
basis. The theoretical nucleosome distribution was<br />
compared and validated with that of yeast and human<br />
genomes experimentally determined (Fig. 1).<br />
Theoretical nucleosome positioning on physical basis<br />
represents the key to clarify, at molecular level, the<br />
structural and superstructural features of chromatin<br />
organization at telomeres which is important to clarify<br />
the basic mechanisms of replication, transcription<br />
and DNA repair.<br />
Our research is aimed to explore the relationship<br />
between telomeric nucleosomes and telomeric proteins.<br />
In most eukaryotic genomes, telomeric nucleosomes<br />
are characterized by the lowest thermodynamic sta-
Maria Savino<br />
bility and by the highest mobility, as we, first, have<br />
shown (Filesi, FEBS 1997). Recently, using an original<br />
approach based on single molecule visualization, by<br />
AFM, we found that the telomeric protein TRF1 is able<br />
to significantly increase the mobility of telomeric nucleosomes,<br />
connecting telomeric proteins interactions to<br />
the dynamic features of telomeric chromatin.<br />
An unresolved problem in telomeric chromatin organization<br />
is the folding of the nucleosome array into the<br />
fiber. The fiber modulates DNA accessibility and thus<br />
is important both in interacting with telomeric proteins<br />
and telomeric RNA, very recently discovered.<br />
We approached the problem by integrating the experimental<br />
findings by electron microscopy visualization<br />
of reconstituted nucleosome arrays with geometrical,<br />
conformational and topological restraints, under the<br />
hypothesis of minimum distortion of the nucleosome<br />
and linker DNA structures. The torsional energy of<br />
the chromatin fiber appears crucial in determining<br />
the kind of nucleosome packing for short nucleosome<br />
repeat length as in telomeres. The presence of<br />
high torsional energy and topological extra-energy<br />
could represent an important basic mechanism for<br />
the formation of telomeric G-quadruplex structure.<br />
Telomeric DNA G-quadruplex represents one of the<br />
more exciting findings in telomeres research in the<br />
last few years. In fact, the possibility to induce and<br />
stabilize this structure, by small organic molecules in<br />
mammalian cells, could represent a powerful tool to<br />
control pharmacologically cellular processes, ruled by<br />
telomerase, such as cancer and aging.<br />
We have developed two different approaches aimed to<br />
inhibit telomerase in mammalian cells, based on the<br />
stabilization of G-quadruplex structures either at 3’<br />
telomeric DNA overhang or at the proximal promoter<br />
of gene coding for the enzymatic moiety of telomerase,<br />
hTERT. A new class of hydrosoluble perylene<br />
derivatives have been developed in collaboration with<br />
a group of organic chemists. We found that these<br />
molecules are able to selectively induce G-quadruplex<br />
structure and inhibit telomerase in cell free systems,<br />
both in the case of telomeric G-quadruplex than in the<br />
96<br />
case of G-quadruplex in hTERT core promoter (Fig.<br />
2). Their activity in vivo systems are actually under<br />
investigation in our research group.<br />
Publications<br />
Micheli E, D’Ambrosio D, Franceschin M, Savino M.<br />
Water soluble cationic perylene derivatives as possible<br />
telomerase inhibitors: the search for selective<br />
G-quadruplex targeting. Mini Rev Med Chem. 2009,<br />
9: 1622-32. doi:10.2174/138955709791012274.<br />
Micheli E, Lombardo CM, D’Ambrosio D, Franceschin<br />
M, Neidle S, Savino M. Selective G-quadruplex ligands:<br />
the significant role of side chain charge density in a<br />
series of perylene derivatives. Bioorg Med Chem Lett.<br />
2009, 19: 3903-8. doi: 10.1016/j.bmcl.2009.03.106.<br />
Scipioni A, Morosetti S, De Santis P. A statistical<br />
thermodynamic approach for predicting the sequencedependent<br />
nucleosome positioning along genomes.<br />
Biopolymers. 2009, 91: 1143-53. doi: 10.1002/bip.21276.<br />
De Santis P, Morosetti S, Scipioni A. Prediction of<br />
nucleosome positioning in genomes: limits and perspectives<br />
of physical and bioinformatic approaches. J<br />
Biomol Struct Dyn. 2010, 27: 747-64.doi:<br />
Pisano S, Leoni D, Galati A, Rhodes D, Savino M,<br />
Cacchione S. The human telomeric protein hTRF1<br />
induces telomere-specific nucleosome mobility. Nucleic<br />
Acids Res. 2010, 38: 2247-55. doi: 10.1093/nar/gkp1228.<br />
Scipioni A, Turchetti G, Morosetti S, De Santis P.<br />
Geometrical, conformational and topological restraints<br />
in regular nucleosome compaction in chromatin. Biophys<br />
Chem. 2010, 148: 56-67. doi: 10.1016/j.bpc.2010.02.010.<br />
Micheli E, Martufi M, Cacchione S, De Santis P,<br />
Savino M. Self-organization of G-quadruplex structures<br />
in the hTERT core promoter stabilized by polyaminic<br />
side chain perylene derivatives. Biophys Chem.<br />
2010, 153: 43-53. doi: 10.1016/j.bpc.2010.10.003.
97<br />
AREA 4<br />
Fig. 1 - Comparison between the profiles of the experimental nucleosome distribution (dashed line) and the theoretical free energy difference<br />
(full line) for a 6000 bp region of chromosome III of S. cerevisiae genome.<br />
Fig. 2 - Representative model of POL-3 (a hydrosoluble perylene derivative)<br />
with human telomeric G-quadruplex.
AREA 5<br />
Cellular and<br />
molecular<br />
immunology
Interplay amongst chronic immune activation, apoptosis,<br />
cross-presentation, immune-regulation, and autoimmunity<br />
Principal investigator: Vincenzo Barnaba<br />
Professor of Internal Medicine<br />
Dipartimento di Medicina Interna e Specialità Mediche<br />
Tel: (+39) 06 4453994; Fax: (+39) 06 49383333<br />
vincenzo.barnaba@uniroma1.it<br />
Participants:<br />
Marino Paroli, professor; Daniele Accapezzato, researcher;<br />
Vittorio Francavilla, Pisana Moroni, Antonella Propato,<br />
post-doc fellows; Melissa Videtta, Tiziana Donato, Debora<br />
Franceschini, Francesca Meloni, Laura Altieri, PhD students.<br />
Report of activity<br />
In the first year of our project, we addressed the role of<br />
regulatory T (Treg) cells in the establishment of a state<br />
of chronic low-level inflammation that is instrumental<br />
to limit immunopathology, to limit the pathogen spread,<br />
and ultimately to ensure a long-lasting survival of the<br />
host. Treg cells expressing the transcription factor<br />
forkhead box P (Foxp)3 develop either in the thymus<br />
(natural) or in the periphery from conventional CD4 +<br />
T cells (induced). As a result of the expression of the<br />
Il-2 gene-inhibitory Foxp3 transcription factor, Treg<br />
cells do not produce IL-2 and are unable to respond to<br />
antigens (anergy). However, Foxp3 activity maintains<br />
high levels of IL-2 receptors (CD25 hi ) on Treg cells,<br />
hence compensating the incapacity of producing IL-2.<br />
Indisputably, CD25 hi Treg cells promptly proliferate<br />
both in vitro and in vivo in response to relevant antigens<br />
in the presence of paracrine IL-2, which is mainly<br />
produced by responder (effector) T lymphocytes, but it<br />
is dominantly absorbed by Treg cells expressing higher<br />
CD25 levels than responder T cells. This appears to<br />
represent a key suppression mechanism.<br />
Treg cells are induced and proliferate in response<br />
to hepatitis C virus (HCV) and seem to modulate<br />
liver inflammation in the course of chronic infection.<br />
Therefore, the model of HCV infection supports the<br />
idea that Treg cells participate in the establishment<br />
of a fine equilibrium between immunopathology and<br />
immune protection, ultimately resulting in the longlasting<br />
survival of the host during chronic infections.<br />
It is unclear how Treg cells control unwarranted<br />
inflammation without completely suppressing the<br />
protective immune responses.<br />
101<br />
AREA 5<br />
We demonstrated that PD-1 is over-expressed on<br />
Foxp3 + Treg cells and limits Treg cell proliferation<br />
and function during chronic HCV infection. We first<br />
observed substantial Treg cell accumulation in the<br />
inflamed liver of HCV patients, and that some of<br />
this accumulation was caused by increased proliferation<br />
(as calculated by Ki67 expression) occurring<br />
selectively at the site of infection in vivo. Moreover,<br />
accumulation and proliferation of Treg cells were<br />
synchronized with those of T effector (Teff) cells<br />
at the site of infection, but at a level significantly<br />
and constantly lower than the parallel expansion of<br />
intra-hepatic (IH)-Teff lymphocytes. Nonetheless,<br />
IH-Treg cells were intrinsically “professional”,<br />
because they exerted efficient suppression function<br />
that was directly related with the level of Foxp3<br />
expression, the decline of IH-Teff cells (as well as of<br />
Ki67 + cells), and the clinical consequences of downregulation<br />
of anti-viral (i.e., increased viral load), and<br />
immunopathological responses (i.e., decreased liver<br />
inflammation) in vivo. Because we were able to demonstrate<br />
a notable production of IL-2 by Teff cells<br />
derived from inflamed liver (known to be the most<br />
important source of HCV antigens), the capacity of<br />
Treg cells to proliferate in vivo is likely due to the<br />
sustained amounts of viral antigens and paracrine<br />
IL-2 at the site of infection. Our antigen-specific<br />
Treg cell proliferation assay in vitro supported this<br />
view because the combination of viral antigens and<br />
IL-2 efficiently rescued Treg cells from anergy in the<br />
majority of HCV patients tested, indicating a key role<br />
of HCV in the generation of functional Treg cells.<br />
To understand why the expansion and proliferation<br />
of IH-Treg cells were (although parallel) constantly<br />
overwhelmed by those of IH-Teff cells in all HCV<br />
patients tested, we investigated the possibility that<br />
Treg cells were more susceptible than the effectors to<br />
the programmed death (PD-1) dependent exhaustion<br />
mechanism in the inflamed tissue. The lower Treg<br />
cell expansion correlated with a significantly higher<br />
expression of PD-1 on Treg than on Teff cells infil-
Vincenzo Barnaba<br />
trating the inflamed liver. Further in vivo observations<br />
revealed that the PD-1/Foxp3 cell ratio correlated<br />
inversely with the viral load, and directly with the<br />
histological activity index (HAI) score, suggesting<br />
that PD-1 expression on Treg cells contraregulates<br />
the suppression of both the anti-viral and the<br />
immunopathological responses. Data showed that the<br />
PD-1/PD-L1 blockade significantly increased both<br />
the expansion and the relative suppression function of<br />
Treg cells when the latter were stimulated with either<br />
specific or non-specific TCR ligands plus paracrine<br />
IL-2 in the presence of a blocking mAb to PD-ligand1<br />
(PD-L1) in vitro, thus providing evidence for the<br />
hypothesis stating that the inhibition of suppression<br />
is PD-1-dependent. The potential role of PD-1dependent<br />
inhibitory signals in controlling Treg cell<br />
proliferation is emphasized by the observation that<br />
PD-1/PD-L1 blockade increased the expansion of<br />
Ki67 + cells in the Treg cells upon TCR-dependent<br />
stimulation plus IL-2 in the presence of anti-PD-L1<br />
in vitro, a finding consistent with the inverse correlation<br />
observed between Foxp3 + and Ki67 + Treg cells in<br />
vivo. Notably, the PD-1-mediated inhibition of Treg<br />
cell function is dependent on its capacity to decrease<br />
expansion and, ultimately, the number of Treg cells<br />
rather than the intrinsic Treg cell suppression capacity.<br />
This finding is consistent with both the evidence<br />
that Foxp3 expression dictates and fixes the suppression<br />
function of the single Treg cells, irrespective of<br />
the presence of inhibitory signals, and the hypothesis<br />
that the decreased expansion of Treg cells by PD-1<br />
can, however, result in a decreased suppression function<br />
in a given inflammatory context. By contrast, the<br />
observation that no correlation was evident when we<br />
considered the peripheral Treg cells further demonstrates<br />
that peripheral immune settings do not necessarily<br />
reflect the local immune responses present at a<br />
given inflammatory site.<br />
High CD25 expression by Treg cells drives a positive<br />
feedback loop, as the dominant IL-2 capture<br />
increases STAT-5 phosphorylation (pSTAT-5) that<br />
in turn drives Treg cell proliferation and function.<br />
We demonstrated that pSTAT-5 was spontaneously<br />
overexpressed in IH-Treg cells (significantly more<br />
than in IH-Teff cells), and paralleled the expression<br />
of both CD25 hi and PD-1 in vivo. In addition, kinetic<br />
studies in patients with severe hepatitis reactivation,<br />
showed that pSTAT-5 and PD-1 expression in Treg<br />
cells were up-regulated in an alternate fashion in<br />
vivo: pSTAT-5 upregulation followed the peak and<br />
anticipated the dramatic suppression of disease activity,<br />
whereas PD-1 upregulation preceded the return<br />
of both pSTAT-5 and disease activity to steady lev-<br />
102<br />
els. Finally, PD-1 blockade increased both proliferation<br />
and pSTAT-5 expression by fresh IH-Treg cells<br />
significantly more than by IH-Teff cells ex vivo.<br />
Taken together, these data provide evidence for<br />
the hypothesis that PD-1, upon the contact with its<br />
own ligands, inhibits pSTAT-5 via the activation of<br />
Src homology 2–containing tyrosine phosphatases<br />
(SHPs) (Fig. 1). As a consequence, responder T cells<br />
can escape from excessive expansion of Treg cells<br />
and render them available for responding to possible<br />
novel waves of infection (likely leading to recovery).<br />
This negative feedback loop assumes a different<br />
significance during chronic infections, such as HCV.<br />
The incapacity to clear HCV by the immune system<br />
maintains a vicious spiral, whereby responder T cells<br />
are chronically stimulated to produce IL-2 that will<br />
be dominantly adsorbed by CD25 hi Treg cells that in<br />
turn will continuously suppress the effector responses.<br />
The PD-1 upregulation limits the excessive<br />
expansion of Treg cells by controlling pSTAT-5 and<br />
fine-tunes the Treg function in order to minimize<br />
the immunopathology without completely switch off<br />
those intended to limit excessive viral spread (Fig.<br />
1). This may represent a critical contra-suppression<br />
mechanism that has evolved to control that Treg<br />
cells have a limited suppression. Homeostatic balance<br />
participates in establishing a status of chronic<br />
low-level liver inflammation that is in turn instrumental<br />
to ensure a long-lasting survival of the host.<br />
Publications<br />
Franceschini D, Paroli M, Francavilla V, Videtta M,<br />
Morrone S, Labbadia G, Cerino A, Mondelli MU, Barnaba<br />
V. PD-L1 negatively regulates CD4+CD25+Foxp3+<br />
Tregs by limiting STAT-5 phosphorylation in patients<br />
chronically infected with HCV. J Clin Invest. 2009, 119:<br />
551-64. doi: 10.1172/JCI36604.<br />
Curtale G, Citarella F, Carissimi C, Goldoni M, Carucci<br />
N, Fulci V, Franceschini D, Meloni F, Barnaba V,<br />
Macino G. An emerging player in the adaptive immune<br />
response: microRNA-146a is a modulator of IL-2<br />
expression and AICD in T lymphocytes. Blood. 2010,<br />
115: 265-73. doi: 10.1182/blood-2009-06-225987.<br />
Fulci V, Scappucci G, Sebastiani GD, Giannitti C,<br />
Franceschini D, Meloni F, Colombo T, Citarella F,<br />
Barnaba V, Galeazzi M, Macino G. miR-223 is overexpressed<br />
in Tlymphocytes of patients affected by<br />
rheumatoid arthritis. Hum Immunol. 2010; 71: 206-<br />
11. doi: 10.1016/j.humimm.2009.11.008.
Beauvillain C, Meloni F, Sirard JC, Blanchard S,<br />
Jarry U, Scotet M, Magistrelli G, Delneste Y,<br />
Barnaba V, Jeannin P. The scavenger receptors<br />
SRA-1 and SREC-I cooperate with TLR2 in the<br />
recognition of the hepatitis C virus non-structural<br />
protein 3 by dendritic cells. J Hepatol. 2010, 52: 644-<br />
51 doi: 10.1016/j.jhep.2009.11.031.<br />
103<br />
AREA 5<br />
Barnaba V. Hepatitis C virus infection: a “liaison a<br />
trois” amongst the virus, the host, and chronic lowlevel<br />
inflammation for human survival. J Hepatol.<br />
2010, 53: 752-61. doi:10.1016/j.jhep.2010.06.003.<br />
Fig. 1 - PD-1 controls Treg cells in HCV infection. (a) Responder (CD25 low Foxp3 – ) T effector memory (T EM ) cells proliferate in<br />
response to HCV antigens, and produce IL-2, which through the IL-2R (CD25) signaling, induces pSTAT-5. This leads to the development<br />
of the genetic program dictating their effector phenotype. In parallel, the same phenomena occur for the (CD25 hi Foxp3 + ) Treg cells that do<br />
not proliferate to viral antigens alone because of the expression of the Il-2 gene-inhibitory Foxp3. Their proliferation is dependent on the<br />
dominant capture of paracrine IL-2 that is initially produced by responder T cells. Followed by, CD25 hi Foxp3 + Treg cells can proliferate by the<br />
engagement of the Jak3/STAT-5 pathway, and extrinsically down-regulate the T EM cell responses. (b) In the late phases of T cell activation,<br />
the death receptors intrinsically deliver negative signals to activated T cells (including Fas, CTLA-4, and PD-1) in order to terminate the T<br />
cell responses. PD-1 is upregulated on both responder and Treg cells and upon contact with PD-L1/2 inhibits pSTAT-5 possibly via SHP2.<br />
This mechanism results in limiting both T EM cell responses and excessive Treg cell function. Under conditions resulting in the HCV resolution,<br />
this loop is self-limited because of the disappearance of the viral antigenic stimuli. During a chronic HCV infection, in which responder T<br />
cells have been unable to clear HCV, the negative loop is maintained by the persisting HCV antigens that chronically stimulate IL-2 producing<br />
responder T cells. The chronic PD-1 expression on both T EM and Treg cells modulate the potential excessive pSTAT-5-dependent cell<br />
proliferation. The resulting contra-regulation of Treg cells will have an important role to limit excessive suppression of immune responses<br />
allowing the control of the viral spread, at the cost of the maintenance of the chronic low-level liver immunopathology. This mechanism<br />
establishes a long-lasting survival of the host.
Potential role of miRNAS in IgM-mediated signal transduction in<br />
normal and neoplastic B cells<br />
Principal investigator: Roberto Foà<br />
Professor of Hematology<br />
Dipartimento di Biotecnologie Cellulari ed Ematologia<br />
Tel: (+39) 06 85795753; Fax: (+39) 06 85795792<br />
rfoa@bce.uniroma1.it<br />
Participants:<br />
Anna Guarini, Franca Citarella, Sabina Chiaretti,<br />
researchers; Simona Tavolaro, post-doc fellow; Marilisa<br />
Marinelli, Monica Messina, Nadia Peragine, PhD students.<br />
Report of activity<br />
Background<br />
Chronic lymphocytic leukemia (CLL) is the most<br />
common leukemia in the Western countries. It is<br />
characterized by a clonal accumulation of small,<br />
mature-appearing lymphocytes in the blood, marrow<br />
and secondary lymphoid tissue. The disease presents<br />
a highly variable clinical course, with some patients<br />
surviving for many years without requiring treatment<br />
and others who witness a rapidly progressing<br />
disease, associated with a short life expectancy,<br />
despite aggressive treatment. Several biological and<br />
genetic properties of the leukemic cells – e.g. the<br />
mutational status of the Ig heavy (H) chain variable<br />
(V) genes, cytogenetic alterations, CD38 and ZAP-70<br />
antigen expression, and p53 dysfunctions – that bear<br />
an important prognostic value have been identified<br />
and have enabled to stratify patients into risk categories.<br />
These aberrations are important independent<br />
predictors of disease progression and survival.<br />
MicroRNAs (miRNAs) are endogenous, non-coding<br />
small RNAs that negatively regulate gene expression<br />
in a sequence specific manner via translational<br />
repression and/or mRNA degradation. The important<br />
role of miRNAs has been demonstrated in<br />
normal and neoplastic hemopoietic cells. Our group<br />
has shown the different expression of some miRNA<br />
in CLL cells. We have identified 4 miRNAs (miR-<br />
21, miR-150, miR-155 and miR-92) as differentially<br />
expressed between CLL samples and healthy donors<br />
(Fulci et al., Blood 2007). Furthermore, we and others<br />
observed an overexpression of miR-150, miR-223,<br />
and miR-29b and miR-29c in the IGHV mutated<br />
CLL cases compared to the IGHV unmutated cases.<br />
105<br />
AREA 5<br />
Primary aim of the project<br />
The primary aim of this project was an extension of<br />
the study previously carried out by our group following<br />
IgM stimulation of CLL cases. It was focused in<br />
particular on the characterization of the gene expression<br />
profile of leukemic cells after B-cell receptor<br />
(BCR) stimulation and the importance of the role of<br />
miRNA in the behavior of CLL cells. The support of<br />
the <strong>Istituto</strong> <strong>Pasteur</strong> - Fondazione Cenci Bolognetti also<br />
contributed to complete other related studies.<br />
Results<br />
In CLL, gene expression profiling analysis upon IgM<br />
ligation has shown an upregulation of genes involved<br />
in signal transduction, cell cycle and cytoskeleton<br />
organization exclusively in IGHV unmutated<br />
patients (poor prognosis patients), and functional<br />
studies confirmed these findings, showing a significant<br />
increase of cells in G1-phase only in IGHV<br />
unmutated patients.<br />
With regard to IgD stimulation, cells have been<br />
evaluated at two different time-points (24 and 48<br />
hours). At 24 hours, gene expression profiling highlighted<br />
a downmodulation of a set of genes, most<br />
involved in signal transduction; in contrast, at 48<br />
hours, an activation of genes involved mainly in<br />
gene expression regulation and transcription was<br />
observed. Furthermore, genes encoding for BCR<br />
signaling molecules were modulated. Finally, genes<br />
involved in differentiation and apoptosis were overrepresented<br />
at both time points.<br />
These findings were corroborated at the functional<br />
level in 17 patients (12 IGHV mutated and 5 IGHV<br />
mutated), by evaluating cell cycle changes and apoptotic<br />
rate upon IgD stimulus. At both 24 and 48<br />
hours and in all cases, we did not record any proliferation,<br />
coupled to an increased apoptotic rate. There<br />
were no differences between IGHV mutated and<br />
unmutated patients.
Roberto Foà<br />
By flow cytometry, in agreement with the gene<br />
expression profiling results, there was a downmodulation<br />
of the CD79a, CD79b, CD52, CD27<br />
and CD62L molecules. The downmodulation was<br />
observed in all cases, regardless of the IGHV mutational<br />
status and the clinical behavior of the disease,<br />
at both 24 and 48 hours.<br />
Overall, gene expression, functional and flow cytometry<br />
data have shown substantial differences between<br />
IgM and IgD stimulation: in fact, while CLL cell activation<br />
upon IgM cross-linking is strictly associated<br />
to the IGHV mutational status, IgD cross-linking<br />
results in similar changes in all the CLL examined,<br />
independently of the IGHV mutational status, thus<br />
indicating that IgM and IgD stimuli induce different<br />
patterns of response, which might play a variable role<br />
in disease progression (manuscript in preparation).<br />
Finally, extensive miRNA expression profiling is<br />
currently ongoing using the Affymetrix platform at<br />
our center: so far, 4 patients and 2 pools from healthy<br />
donors have been evaluated upon IgM and IgD<br />
stimulation. The data are currently been analyzed.<br />
Further studies completed with the support<br />
of the <strong>Istituto</strong> <strong>Pasteur</strong> - Fondazione Cenci<br />
Bolognetti<br />
The support of the <strong>Istituto</strong> <strong>Pasteur</strong> - Fondazione Cenci<br />
Bolognetti has helped to complete other related studies.<br />
The profile of miRNAs expression represents a key<br />
of interpretation to some peculiar characteristics of<br />
gene profile of leukemic cells. We have demonstrated<br />
in a subgroup of adult T-lineage acute lymphocytic<br />
leukemia (ALL) that the overexpression of myeloid<br />
genes (MPO, CEBPA, CEBPB, GRN and IL8) was<br />
correlated to the expression levels of miRNA-223,<br />
a miRNA involved in myeloid differentiation. These<br />
cases had significantly higher levels of miR-223 than<br />
the other cases of T-ALL, with values comparable to<br />
those observed in acute myeloid leukemia (Chiaretti<br />
et al., Haematologica 2010).<br />
We have completed the analysis of the gene profile<br />
of 100 CLL with particular attention to the tyrosine<br />
kinase (TK) pathway expression. We performed gene<br />
expression profile on 505 PK genes. The analysis<br />
highlighted an homogeneous upmodulation of several<br />
PKs, 16 also overexpressed in two additional CLL<br />
cohorts. Q-PCR analysis confirmed these findings.<br />
No differences were observed in the main prognostic<br />
subclasses, indicating that PK overexpression in<br />
CLL appears to be disease-associated. Tests in vitro<br />
have shown that the multi-TK inhibitor Dasatinib<br />
partially reduces CLL cell viability, mostly in IGHV<br />
germline patients (Tavolaro et al., Leukemia Research<br />
106<br />
2010). These findings suggest that treatment with<br />
second generation TK inhibitors may represent an<br />
attractive therapeutic strategy for CLL patients.<br />
In view of the clinical results obtained in BCR/<br />
ABL+ ALL patients treated with TK inhibitors, the<br />
above gene expression analysis has been investigated<br />
in adult ALL, subdivided on the basis of the genetic<br />
features. The results obtained have documented an<br />
altered expression profile of various protein kinases<br />
in the different ALL subgroups, indicating that TK<br />
inhibitors may be considered for the management<br />
of ALL, beyond BCR/ABL+ cases (Messina et al.,<br />
Cancer 2010).<br />
We have also investigated the p53 gene status in<br />
CLL because alterations of this gene represent<br />
the worse prognostic parameter in CLL. We utilized<br />
a novel non time-consuming microarray-based<br />
resequencing assay and investigated exons 2-11.<br />
Furthermore, we performed microarrays analysis on<br />
TP53 mutated and/or deleted cases. This study has<br />
allowed to confirm a high percentage of TP53 mutations<br />
in CLL with unfavorable outcome and a significant<br />
association between TP53 aberrations and<br />
del(17p). Finally, specific gene expression profiles<br />
are recognized for TP53 alterations (Chiaretti et al.,<br />
Genes Chromosomes and Cancer, in press).<br />
Publications<br />
Tavolaro S, Chiaretti S, Messina M, Peragine N, Del<br />
Giudice I, Marinelli M, Santangelo S, Mauro FR,<br />
Guarini A, Foà R. Gene expression profile of protein<br />
kinases reveals a distinctive signature in chronic<br />
lymphocytic leukemia and in vitro experiments support<br />
a role of second generation protein kinase inhibitors.<br />
Leukemia Res. 2010, 34: 733-41. doi:10.1016/j.<br />
leukres.2009.11.005.<br />
Chiaretti S, Messina M, Tavolaro S, Zardo G, Elia L,<br />
Vitale A, Fatica A, Gorello P, Piciocchi A, Scappucci G,<br />
Bozzoni I, Fozza C, Candoni A, Guarini A, Foà R. Gene<br />
expression profiling identifies a subset of adult T-cell<br />
acute lymphoblastic leukemia with myeloid-like gene<br />
features and over-expression of miR-223. Haematologica.<br />
2010, 95: 1114-21. doi: 10.1002/cncr.25113.<br />
Messina M, Chiaretti S, Tavolaro S, Peragine N,<br />
Vitale A, Elia L, Sica S, Levis A, Guarini A, Foà<br />
R. Protein kinase gene expression profiling and in<br />
vitro functional experiments identify novel potential<br />
therapeutic targets in adult acute lymphoblastic<br />
leukemia. Cancer. 2010, 116: 3426-37. doi: 10.1002/<br />
cncr.25113.
Signalling events negatively regulating FcepsilonRI expression<br />
and mast cell functional responses<br />
Principal investigator: Rossella Paolini<br />
Professor of Immunology<br />
Dipartimento di Medicina Molecolare<br />
Tel/Fax: (+39) 06 4468448<br />
rossella.paolini@uniroma1.it<br />
Participants:<br />
Stefania Morrone, professor, Rosa Molfetta, researcher;<br />
Francesca Gasparrini, Alessandra Porzia, post-doc fellows.<br />
Report of activity<br />
Mast cells are key effectors in allergic diseases and<br />
play essential roles in host defense against certain<br />
types of parasitic infections. They release a wide<br />
array of pro-inflammatory mediators after activation<br />
by the ligation of several receptors, being the high<br />
affinity receptor for IgE (FceRI) the most efficient<br />
and best characterized.<br />
FceRI is a multisubunit complex consisting of an IgEbinding<br />
α-chain, a four transmembrane-spanning<br />
β-chain with cytoplasmic N-terminal and C-terminal<br />
edges, and a disulphide linked γ-γ homodimer. The<br />
β and γ subunits have no role in ligand binding, but<br />
they share a conserved immunoreceptor tyrosinebased<br />
activation motif (ITAM) within their long<br />
cytoplasmic tails that, upon FceRI aggregation, is<br />
rapidly phosphorylated on tyrosines by the Src family<br />
kinase Lyn, thus allowing the recruitment and<br />
activation of the cytoplasmic kinase Syk, responsible<br />
for signal propagation (1).<br />
The full activation of FceRI requires the recruitment<br />
of aggregated receptors into lipid rafts, membrane<br />
domains rich in cholesterol and sphingolipids that<br />
form ordered but dynamic structures floating in the<br />
less ordered surrounding membrane.<br />
Upon FceRI engagement, membrane rafts coalesce<br />
into larger and more stable structures where<br />
engaged receptors are concentrated (2) and can<br />
more easily interact with signaling molecules, such<br />
as active Lyn (3), favouring phosphorylation events.<br />
The outcome of this activated state is the release of<br />
preformed mediators and the de novo synthesis of<br />
eicosanoids, cytokines and chemokines that result<br />
in both immediate and late-phase immune reactions.<br />
Besides these positive signals, FceRI engagement<br />
107<br />
AREA 5<br />
has recently been understood to generate negative<br />
intracellular signals capable of limiting the rate<br />
and the extent of mast cell functional responses (4).<br />
Relevant to this, others and we have demonstrated<br />
that the Cbl family proteins control the amplitude<br />
of FceRI-generated signals by specific ubiquitin<br />
modification of activated receptor subunits and associated<br />
protein tyrosine kinases (5,6). Concurrently,<br />
engaged receptors trigger their own endocytosis to<br />
extinguish signaling through removal of activated<br />
FceRI complexes from the cell surface and delivery<br />
to lysosomes for degradation (7).<br />
The cytosolic effectors of positive signalling have<br />
been analyzed extensively, but very little is known<br />
about the pathways and effectors involved in negative<br />
signalling.<br />
The present study is aimed at identifying the molecular<br />
mechanisms ensuring the clearance of antigenstimulated<br />
FceRI complexes from the cell surface,<br />
thus contributing to the termination of mast cell<br />
functional program.<br />
Our first objective was to investigate the relationship<br />
occurring between receptor ubiquitination, lipid<br />
rafts, and receptor endocytosis.<br />
We initially demonstrated that FceRI β and γ<br />
subunits are mainly monoubiquitinated by c-Cbl at<br />
multiple sites upon antigen stimulation, and provided<br />
evidence that this modification controls receptor<br />
internalization: under conditions of impaired<br />
receptor ubiquitination we reproducibly observed a<br />
decrease of FceRI down-regulation (8).<br />
We have then shown that the recruitment of engaged<br />
FceRI subunits into lipid rafts precedes their ubiquitination,<br />
and that the integrity of these membrane<br />
microdomains is required to allow both receptor<br />
ubiquitination and endocytosis (8).<br />
Thus, a cohesive model is one in which lipid rafts,<br />
in addition to assemble the machinery needed for<br />
the downstream propagation of the signal from the<br />
engaged FceRI complexes, also serve as platforms to<br />
recruit the necessary machinery for their endocytosis.
Rossella Paolini<br />
Our second objective was to identify the main endocytic<br />
adapters involved in receptor endocytosis.<br />
Likely candidates for coupling ubiquitinated FceRI<br />
to the endocytic machinery are the ubiquitous adapter<br />
proteins harboring Ub interacting motifs (UIMs),<br />
namely Eps15, Eps15R, epsin and Hrs. We found<br />
that all of them co-immunoprecipitate with endogenous<br />
FceRI in an antigen-dependent fashion and<br />
within a time-course compatible with function(s) at<br />
different steps of the endocytic route (8).<br />
Eps15, Eps15R and Epsin display similar functions:<br />
they control the early steps of the endocytic route<br />
coupling ubiquitinated receptors with components<br />
of the budding vesicles (9,10). The key role of Hrs<br />
is, instead, the delivery of ubiquitinated proteins to<br />
the outer membrane of the late endosomes and the<br />
sorting of the cargo into internal vesicles of multivesicular<br />
bodies for lysosomal degradation (11).<br />
Although we failed to observe a significant decrease<br />
of FceRI entry in early endosomes upon individual<br />
depletion of Eps15, Eps15R or Epsin, the simultaneous<br />
depletion of all of them impaired ligand-induced<br />
receptor endocytosis (8), suggesting a partial overlapping<br />
function of these adapters in ubiquitinated<br />
FceRI uptake.<br />
Notably, we have also demonstrated a critical role<br />
for Hrs in controlling the fate of internalized receptor<br />
complexes: Hrs depletion retains ubiquitinated<br />
receptors into early endosomes and partially prevents<br />
their sorting into lysosomes (8).<br />
In summary, our data support a key role for the ubiquitin<br />
pathway to ensure proper endocytic trafficking<br />
of an immune receptor to the lysosomal compartment<br />
where degradation of the complexes can take<br />
place (8,12).<br />
The outcome of our proposal would be a better<br />
comprehension of the molecular mechanisms that<br />
functionally regulate the endocytic adaptor Hrs, thus<br />
ensuring a proper delivery of internalized FceRI<br />
complexes to lysosomes for degradation.<br />
Elucidating the molecular mechanisms able to downregulate<br />
the main activating receptor of mast cell<br />
may be critical for the future development of effective<br />
therapeutic strategies in the treatment of disorders<br />
that involve mast cell activation.<br />
108<br />
References<br />
1) Nadler MJ, Matthews SA, Turner H, Kinet JP.<br />
Adv Immunol. 76: 325-355, 2000.<br />
2) Field KA, Holowka D, Baird B. J Biol Chem. 272:<br />
4276-4280, 1997.<br />
3) Sheets ED, Holowka D, Baird B. J Cell Biol. 145:<br />
877-887,1999.<br />
4) Molfetta R, Peruzzi G, Santoni A, Paolini R. Arch<br />
Immunol Ther Exp. 55: 219-229, 2007.<br />
5) Paolini R, Molfetta R, Beitz LO, Zhang J,<br />
Scharenberg AM, Piccoli M, Frati L, Siraganian R,<br />
Santoni A. J Biol Chem. 277: 36940-36947, 2002.<br />
6) Kyo S, Sada K, Qu X, Maeno K, Miah SM,<br />
Kawauchi-Kamata K, Yamamura H. Genes Cells.<br />
8: 825-836, 2003.<br />
7) Molfetta R, Belleudi F, Peruzzi G, Morrone S,<br />
Leone L, Dikic I, Piccoli M, Frati L, Torrisi MR,<br />
Santoni A, Paolini R. J Immunol. 175: 4208-4216,<br />
2005.<br />
8) Molfetta R, Gasparrini F, Peruzzi G, Vian L,<br />
Piccoli M, Frati L, Santoni A, Paolini R. PLoS<br />
One. 4:e5604, 2009.<br />
9) Carbone R, Fré S, Cannolo G, Belleudi F, Mancini<br />
P, Pelicci PG, Torrisi MR, Di Fiore PP. Cancer<br />
Res. 57: 5498-5504, 1997.<br />
10) Chen H, Fre S, Slepnev VI, Capua MR, Takei K,<br />
Butler MH, Di Fiore PP, De Camilli P. Nature.<br />
394: 793-797, 1998.<br />
11) Bache K., Brech A, Mehlum A, Stenmark H. J Cell<br />
Biol. 162: 435-442, 2003.<br />
12) Molfetta R, Gasparrini F, Santoni A, Paolini R.<br />
Mol Immunol. 47: 2427-2434, 2010.<br />
Publications<br />
Molfetta R, Gasparrini F, Peruzzi G, Vian L, Piccoli<br />
M, Frati L, Santoni A, Paolini R. Lipid raft-dependent<br />
FcepsilonRI ubiquitination regulates receptor<br />
endocytosis through the action of ubiquitin binding<br />
adaptors.PLoS One. 2009, 4:e5604. doi: 10.1371/<br />
journal.pone.0005604.<br />
Molfetta R, Gasparrini F, Santoni A, Paolini R.<br />
Ubiquitination and endocytosis of the high affinity<br />
receptor for IgE. Mol Immunol. 2010, 47: 2427-34.<br />
doi: 10.1016/j.molimm.2010.06.003.
Principal investigator: Enza Piccolella<br />
Professor of Immunology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49917584; Fax: (+39) 06 49917584<br />
enza.piccolella@uniroma1.it<br />
109<br />
AREA 5<br />
Analysis of the molecular mechanisms regulating FOXP3 gene and<br />
protein expression in TCR- and CD28-activated CD4+CD25-T cells<br />
and their influence on regulatory functions<br />
Participants:<br />
Cristina Camperio, Marzia Soligo, Cristiano Scottà, postdoc<br />
fellows.<br />
Collaborators:<br />
Dipartimento di Dermatologia, Università di Tor Vergata, Roma<br />
(Prof. Antonio Costanzo); Center of Allergy and Environment<br />
(ZAUM), Technical University and Helmholtz Center Munich,<br />
Germany (Prof. Carsten B Schimdt-Weber).<br />
Report of activity<br />
The CD4 + CD25 + FOXP3 + regulatory T cells (Treg)<br />
are a specialized subset of CD4 + T cells that can<br />
suppress the responses of effector T cells. Treg<br />
maintain peripheral immune tolerance and prevent<br />
chronic inflammation as well as autoimmune diseases.<br />
Treg are characterized by the expression of<br />
the a-chain of the interleukin-2 receptor (CD25)<br />
and by the transcription factor forkhead box P3<br />
(FOXP3). Peripheral CD4 + CD25 - T cell could be<br />
converted into FOXP3-expressing Treg (induced<br />
Treg) by signaling through T-cell antigen receptor<br />
(TCR) and accessory molecules such as TGFb1 and<br />
IL-2. Moreover, co-stimulation through the interaction<br />
of CD28 with its ligand B7 is strictly required<br />
for the activation of FOXP3 and for the maturation<br />
and differentiation of Treg either in vivo or in vitro.<br />
However, the molecular mechanisms whereby CD28<br />
alone or associated with TCR can regulate FOXP3<br />
expression remain unknown.<br />
We have explored this function by using our previously<br />
described CD28/B7 selective stimulation<br />
system. Our results have shown that CD28 signals<br />
independent from TCR and dependent on NF-kB<br />
pathways are sufficient to induce the transcription<br />
of FOXP3. Starting from this data, in this project<br />
we have clarified the following unresolved issues:<br />
1) the characterization of the NF-kB/Rel members<br />
recruited on FOXP3 promoter after CD28 activation<br />
and their interaction with NFAT to trans activate<br />
FOXP3 promoter; 2) the determination whether, in<br />
CD28-stimulated T cell, the region of FOXP3 promoter<br />
is in an active chromatin conformation, and<br />
whether epigenetic changes occur in specific binding<br />
sites; 3) the ascertainment whether CD28- and TCRmediated<br />
biochemical pathways function in independent<br />
or dependent manner in regulating FOXP3<br />
protein expression in activated T cells.<br />
1. First, we have explored whether FOXP3<br />
-1657/+176 promoter construct presented NF-kB<br />
binding motifs. To this aim, HEK 293 cells were<br />
co-transfected with FOXP3 -1657/+176 construct<br />
and empty vector or with expression vector encoding<br />
RelA or cRel. The results have shown that both<br />
RelA and cRel homodimers interacted with DNA.<br />
Furthermore, we have shown that DNA region<br />
between -936 and -646 nucleotides of the human<br />
FOXP3 promoter is characterized by at least three<br />
functional and not redundant kB sites for the NF-kB<br />
subunit RelA. However, these sites could not be<br />
required for c-Rel-mediated FOXP3 activation, since<br />
their deletion did not modify the effect of c-Rel on<br />
FOXP3 promoter. We have also investigated the<br />
effect of the co-expression of NFAT and RelA on<br />
the activation of human FOXP3 promoter. In the<br />
absence of the binding of RelA to FOXP3 promoter,<br />
NFAT failed to mediate trans activating function,<br />
suggesting that RelA and not NFAT is primarily<br />
required to trans activate FOXP3 gene, and that the<br />
occupancy of kB sites favours the recruitment of<br />
NFAT on the specific binding sites.<br />
2. FOXP3 promoter shows a stronger association<br />
with acetylated histones in Treg than in conventional<br />
T cells, suggesting that in order to induce<br />
the activation of the FOXP3 gene, the histones that<br />
cover FOXP3 DNA have to be acetylated. Therefore<br />
CD28 could mediate the recruitment of RelA on the<br />
specific binding sites and the induction of epigenetic<br />
modification of the FOXP3 gene at the level<br />
of DNA region distinct. Therefore, to verify this, we
Enza Piccolella<br />
performed chromatin immunoprecipitation (ChIP)<br />
assays with anti-RelA and anti-acetyl histone H4 in<br />
CD4 + CD25 - T cells cultured with Dap3 or Dap3/<br />
B7. The presence of RelA on the identified kB sites<br />
and the histone acetylation of this region allowed<br />
us to conclude to have identified a region on human<br />
FOXP3 promoter where RelA can be recruited following<br />
CD28 signaling and can contribute to the<br />
transcription of FOXP3 gene by inducing histone<br />
H4 acetylation and subsequent loading of RNA<br />
polymerase II. This region is distinct from that<br />
where AP-1 and Rel-NFAT binding sites have been<br />
characterized.<br />
3. To investigate the influence of TCR and CD28<br />
signals on the activation of FOXP3 promoter, we<br />
analysed the transcriptional activation of FOXP3<br />
promoter construct expressed in CD4 + CD25 - T<br />
cells activated by anti-CD28 and anti-CD3 mAbs.<br />
Interestingly, the activation of TCR and CD28<br />
signaling pathways increased the trans activation of<br />
FOXP3 promoter, impling that TCR and CD28 may<br />
induce the sequential and independent activation of<br />
both RelA and c-Rel by using different pathways that<br />
110<br />
synergize in the activation of FOXP3. Therefore,<br />
CD28 unique signals could mediate a fast engagement<br />
of kB sites by RelA for initiating FOXP3 transcription,<br />
but maximal transcription will occur only<br />
upon c-Rel recruitment. Moreover, since TCR signaling<br />
in the absence of CD28 costimulation did not<br />
result in any epigenetic change, RelA could favor the<br />
promoter occupancy by other transcription factors.<br />
The evidence that siRNA-mediated knockdown of<br />
RelA inhibited trans activation of FOXP3 promoter<br />
in CD4 + CD25 - T cells full activated by TCR and<br />
CD28 sustains this hypothesis.<br />
In conclusion, the current study reveals a new<br />
mechanism of FOXP3 gene regulation, and supports<br />
a scenario where CD28-mediated recruitment of RelA<br />
on FOXP3 gene is necessary for the induction of<br />
FOXP3 transcription in CD4 + CD25 - T cells, while<br />
supplementary signals such as those mediated by<br />
TCR are required to allow sustained and maximal<br />
FOXP3 synthesis. On the basis of our data it will be<br />
possible to explore receptors and signaling partners<br />
that may give access to specific interventions aiming<br />
at regulatory T cells.
Anti-tumor effector functions of NK cells in tumor<br />
microenvironment<br />
Principal investigator: Angela Santoni<br />
Professor of Immunology<br />
Dipartimento di Medicina Molecolare<br />
Tel/Fax: (+39) 06 44340632<br />
angela.santoni@uniroma1.it<br />
Participants:<br />
Angela Gismondi, Marco Cippitelli, professors; Giovanni<br />
Bernardini, Alessandra Zingoni, Cristina Cerboni,<br />
Alessandra Soriani, researchers; Francesca Di Rosa,<br />
CNR researcher; Cinzia Fionda, Helena Stabile, Michele<br />
Ardolino, Giuseppe Sciumè, post-doc fellows; Maria Luisa<br />
Iannitto, PhD student.<br />
Collaborators:<br />
Dipartimento di Biotecnologie ed Ematologia, Sapienza-Università<br />
di Roma (Prof. Maria Teresa Petrucci, Prof. Maria Rosaria<br />
Ricciardi, Prof. Anna Guarini, Prof. Robin Foà).<br />
Report of activity<br />
Natural Killer (NK) cells represent one of the main<br />
effectors of the immunosurveillance against tumors<br />
by exhibiting cytotoxic functions and secreting<br />
a number of cytokines and chemokines. They are<br />
extremely heterogeneous both in BM and periphery,<br />
as acquisition of NK cell receptor repertoire, signalling<br />
ability, and functional competence are developmentally<br />
regulated. The specificity of NK cells relies<br />
on the interplay between inhibitory receptors for<br />
MHC class I molecules, and a wide array of activating<br />
receptors that act in concert to induce efficient<br />
elimination of tumor cells. Among the non MHC<br />
I-recognizing activating receptors whose ligands<br />
have been defined so far, particularly relevant for<br />
tumor cell recognition are NKG2D, the receptor for<br />
the MHC I-related molecules MICA/B and ULBPs,<br />
and DNAM-1, the receptor for the nectin/nectinlike<br />
family. NKG2D and DNAM-1 ligands are by no<br />
means tumor-specific molecules, and the mechanisms<br />
responsible for their expression regulation are still<br />
rather obscure.<br />
This research proposal is aimed at identifying the<br />
molecular mechanisms underlying the NK cell recognition<br />
of tumor cells, and the defects of NK cell<br />
maturation and functional competence promoted<br />
111<br />
AREA 5<br />
by tumor microenvironment. To this end, a tumor<br />
type particularly suitable is the multiple myeloma<br />
(MM), a clonal B cell malignancy characterized by<br />
an excess of mature plasma cells mainly in the BM<br />
where NK cell maturation occurs. We have previously<br />
shown that treatment of MM cells with low<br />
doses of therapeutic agents, such as doxorubicin,<br />
melphalan and bortezomib, up-regulate DNAM-1<br />
and NKG2D ligands. Accordingly, drug treatment<br />
of MM cells increases NK cell degranulation, being<br />
the NKG2D and DNAM-1 receptors the major<br />
triggering molecules. Drug-induced DNAM-1 and<br />
NKG2D ligand expression was abolished following<br />
treatment with the pharmacological inhibitors (caffeine<br />
and KU-55933) of the DNA Damage Response<br />
(DDR)-initiating kinases ATM and ATR, and<br />
was preferentially associated with senescent cells<br />
arrested in the G2 phase of the cell cycle (Soriani et<br />
al., Blood 2009).<br />
More recently, we have investigated the signaling<br />
pathways underlying NK cell recognition of druginduced<br />
senescent MM cells with particular attention<br />
to the role played by the generation of reactive<br />
oxygen species (ROS) in the control of senescenceassociated<br />
NKG2D and DNAM-1 ligand expression.<br />
We have found that up-regulation of MICA and PVR<br />
expression on MM cells by low doses of doxorubicin<br />
and melphalan shown to induce ROS generation,<br />
is inhibited by the potent scavenger of ROS NAC<br />
(N-acetyl-L-cysteine), both at protein and mRNA<br />
levels. Moreover, NAC completely abrogated drugtriggered<br />
activation of ATM and of some signaling<br />
components of DDR including the phosphorylation<br />
of the ATM substrates H2AX and p53. Finally,<br />
we found that ROS scavenging resulted in impairment<br />
of cell cycle arrest in G2-M phase and of the<br />
p14Arf senescent marker expression. Based on the<br />
results obtained in vitro with MM cell lines, we<br />
also assessed whether drug-induced increased ROS<br />
production was responsible for the up-regulated<br />
expression of NKG2D and DNAM-1 ligands on
Angela Santoni<br />
patient-derived malignant plasma cells treated with<br />
melphalan, doxorubicin or bortezomib, a proteasome<br />
inhibitor largely used in the therapy of MM.<br />
We observed that NAC pretreatment was able to<br />
completely block the increased surface expression of<br />
NKG2D and DNAM-1 ligands.<br />
Overall our findings demonstrate that low doses<br />
drug-induced NKG2D and DNAM-1 ligand upregulation<br />
on Multiple Myeloma cells is triggered<br />
by ROS-dependent activation of DNA Damage<br />
Response and that the redox-dependent MICA<br />
and PVR expression is preferentially enhanced on<br />
p14ARF senescent cells.<br />
Our study suggests a model in which the senescence<br />
program promotes tumor cell recognition and elimination<br />
by NK cells (Fig. 1).<br />
Our attention was also focused on the analysis of in<br />
vivo NK cell-mediated tumor cell recognition and<br />
elimination upon treatment with chemotherapeutic<br />
drugs. To this end we have established a model<br />
of syngeneic orthotopic MM in C57BL/KaLwRij<br />
mice using the 5TGM1 cell line a subclone of the<br />
5T33 tumor cell line. NK cells from this mouse<br />
strain express NKR-P1C recognized by anti-NK1.1<br />
mAb and display similar maturation markers (our<br />
unpublished observations). Disease progression was<br />
followed by measurement of IgG2b serum titers<br />
and analysis of paraplegia, and in live mice by using<br />
5TGM1 cells constitutively expressing firefly luciferase<br />
and the Xenogen IVIS Optical In Vivo Imaging<br />
System. Preliminary data show that NK cell depletion<br />
by administration of anti-NK1.1 mAb results in<br />
enhanced tumor growth in the BM already at twothree<br />
weeks after tumor challenge. The results were<br />
also confirmed by evaluation of tumor burden by<br />
intracellular IgG2b staining and FACS analysis on<br />
cell samples from spleen and BM at three-four weeks.<br />
In addition, we found that ip injection of suboptimal<br />
doses of melphalan enhanced NKG2DL expression<br />
on MM cells, suggesting that drug-induced enhancement<br />
of NKG2D-dependent NK cell recognition of<br />
tumor cells can also occur in vivo.<br />
Delineating the receptors and functional competence<br />
of discrete NK cell populations on one hand and<br />
the phenotypic analysis of ligand expression on the<br />
other, would allow prediction of tumor cell sensitivity<br />
to NK cell-mediated killing and thus the efficacy<br />
of NK cell-mediated immunotherapy.<br />
112<br />
Publications<br />
Cerboni C, Ardolino M, Santoni A, Zingoni A.<br />
Detuning CD8+ T lymphocytes by down-regulation<br />
of the activating receptor NKG2D: role of NKG2D<br />
ligands released by activated T cells. Blood. 2009,<br />
113: 2955-64. doi: 10.1182/blood-2008-06-165944.<br />
Fionda C, Soriani A, Malgarini G, Iannitto ML,<br />
Santoni A, Cippitelli M. Heat shock protein-90<br />
inhibitors increase MHC class I-related chain A<br />
and B ligand expression on multiple myeloma cells<br />
and their ability to trigger NK cell degranulation.<br />
J Immunol. 2009, 183: 4385-94. doi: 10.4049/jimmunol.0901797.<br />
Soriani A, Zingoni A, Cerboni C, Iannitto ML,<br />
Ricciardi MR, Di Gialleonardo V, Cippitelli M,<br />
Fionda C, Petrucci MT, Guarini A, Foà R, Santoni<br />
A. ATM-ATR-dependent up-regulation of DNAM-<br />
1 and NKG2D ligands on multiple myeloma cells<br />
by therapeutic agents results in enhanced NK-cell<br />
susceptibility and is associated with a senescent<br />
phenotype. Blood. 2009,113: 3503-11. doi: 10.1182/<br />
blood-2008-08-173914.<br />
Kettner A, Di Matteo M, Santoni A. Insulin potentiates<br />
FcepsilonRI-mediated signaling in mouse bone<br />
marrow-derived mast cells. Mol Immunol. 2010, 47:<br />
1039-46. doi:10.1016/j.molimm.2009.11.013.<br />
Porzia A, Lanzardo S, Citti A, Cavallo F, Forni G,<br />
Santoni A, Galandrini R, Paolini R. Attenuation of<br />
PI3K/Akt-mediated tumorigenic signals through<br />
PTEN activation by DNA vaccine-induced anti-<br />
ErbB2 antibodies. J Immunol. 2010, 184: 4170-7. doi:<br />
10.4049/jimmunol.0903375.<br />
Sciumè G, Santoni A, Bernardini G. Chemokines and<br />
glioma: invasion and more. J Neuroimmunol. 2010,<br />
224(1-2): 8-12. doi:10.1016/j.jneuroim.2010.05.019.<br />
Stabile H, Carlino C, Mazza C, Giliani S, Morrone<br />
S, Notarangelo LD, Notarangelo LD, Santoni A,<br />
Gismondi A.Impaired NK-cell migration in WAS/<br />
XLT patients: role of Cdc42/WASp pathway in<br />
the control of chemokine-induced beta2 integrin<br />
high-affinity state. Blood. 2010, 115: 2818-26. doi:<br />
10.1182/blood-2009-07-235804.
Fig.1 - NK cell-mediated recognition of tumor cells undergoing genotoxic stress.<br />
113<br />
AREA 5
Dissection of Notch signaling-dependent pathways involved in<br />
the progression of T cell leukemia<br />
Principal investigator: Isabella Screpanti<br />
Professor of General Pathology<br />
Dipartimento di Medicina Sperimentale;<br />
Tel: (+39) 06 44700816; Fax: (+39) 06 4464129<br />
isabella.screpanti@uniroma1.it<br />
Participants:<br />
Maria Pia Felli, professor; Diana Bellavia, Antonio F.<br />
Campese, researchers; Paola Grazioli, Gaia Scafetta, postdoc<br />
fellows; Maria Pelullo, Roberta Quaranta, PhD students.<br />
Collaborations:<br />
Department of Immunology, Imperial College of London, UK<br />
(Prof. Guido Franzoso); Molecular Oncology Research Program,<br />
Division of Surgical Oncology, University of Miami, FL, USA (Prof.<br />
Anthony J. Capobianco).<br />
Report of activity<br />
The role of Notch signaling in the development of<br />
T cell leukemia is sufficiently established, whereas<br />
its role in promoting and sustaining its progression<br />
remains undefined. The triggering of the canonical<br />
pathway of the transcription factor NF-kB,<br />
sustained by the constitutive surface expression of<br />
an active preTCR, that induces in turn, the activation<br />
of several anti-apoptotic and pro-proliferative<br />
signals (Bcl2-A1, IL2, Ciclina D1) appears to exert<br />
an essential role during the expansion phase of the<br />
disease. In this context, it has been recently suggested<br />
that the combined use of pharmacological<br />
compounds that inhibit Notch signaling and NF-kB<br />
activation, is able to improve the therapy response<br />
in mouse models of Notch-induced T-ALL. We previously<br />
demonstrated that the deletion of PKCtheta<br />
kinase, that plays an important role in signal<br />
transduction ending in the triggering of NF-kB<br />
canonical pathway, significantly reduces the disease<br />
aggressiveness in the Notch3-induced T-ALL<br />
mouse model, and induces an important delay in the<br />
progression of the disease.<br />
To clarify the Notch/NF-kB relationships in the<br />
onset and development of T cell leukemia, we decided<br />
to inhibit NF-kB canonical pathway in N3-tg mice.<br />
We generated double mutant mice, deleted of<br />
the NF-kB/p50 subunit in a Notch3 transgenic<br />
115<br />
AREA 5<br />
background (N3-tg/p50 -/- ). The follow-up of<br />
N3-tg/p50 -/- versus N3-tg mice was carried out to<br />
analyze the potential effects of NF-kB inhibition on<br />
the aggressiveness and progression of T-ALL.<br />
The immunophenotypic analysis of hematopoietic<br />
cell populations was performed at different age<br />
and in multiple tissues from the indicated animals<br />
(thymus, spleen, bone-marrow and blood), by flowcytometry<br />
tecniques.<br />
As expected, p50 deletion virtually abolished the<br />
onset of T-ALL in N3-tg/p50 -/- mice, as defined<br />
primarily by the development of splenomegaly and<br />
the peripheral expansion of immature CD4 + CD8 +<br />
T cells. However, the mortality curves evidenced<br />
that double mutant mice succumb earlier than<br />
N3-tg counterparts. Surprisingly, moribund N3-tg/<br />
p50 -/- mice display the trait of a myeloproliferative<br />
disease, with the dramatic expansion of Mac1 + Gr1 +<br />
myeloid cells in both spleen, peripheral blood and<br />
Bone Marrow. Preliminary data indicate that these<br />
cells do not express Notch3, suggesting that in the<br />
absence of p50 expression, Notch3 is able to mainly<br />
influence the equilibrium of the myeloid compartment<br />
in trans.<br />
Our results suggest that the ablation of NF-kB<br />
canonical pathway may strongly impact on the outcomes<br />
of a T cell specific deregulation of Notch signaling.<br />
Thus, providing a useful experimental model<br />
to extend our understanding of Notch/NF-kB interplay<br />
and to unravel novel strategies for the therapy<br />
of different hematological malignancies.<br />
Publications<br />
Canettieri G, Coni S, Della Guardia M, Nocerino<br />
V, Antonucci L, Di Magno L, Screaton R, Screpanti<br />
I, Giannini G, Gulino A. The coactivator CRTC1<br />
promotes cell proliferation and transformation via<br />
AP-1. Proc Natl Acad Sci USA. 2009, 106: 1445-50.<br />
doi: 10.1073/pnas.0808749106.
Isabella Screpanti<br />
Ferretti E, De Smaele E, Po A, Di Marcotullio<br />
L, Tosi E, Espinola MS, Di Rocco C, Riccardi R,<br />
Giangaspero F, Farcomeni A, Nofroni I, Laneve P,<br />
Gioia U, Caffarelli E, Bozzoni I, Screpanti I, Gulino A.<br />
MicroRNA profiling in human medulloblastoma. Int<br />
J Cancer. 2009, 124: 568-77. doi: 10.1002/ijc.23948.<br />
116<br />
Checquolo S, Palermo R, Cialfi S, Ferrara G, Oliviero<br />
C, Talora C, Bellavia D, Giovenco A, Grazioli P,<br />
Frati L, Gulino A, Screpanti I. Differential subcellular<br />
localization regulates c-Cbl E3 ligase activity<br />
upon Notch3 protein in T-cell leukemia. Oncogene.<br />
2010, 1129: 1463-74. doi: 10.1038/onc.2009.446.
The role of HLA-B27 in autoimmunity: from the genetics to the<br />
function<br />
Principal investigator: Rosa Sorrentino<br />
Professor of Pathology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49917706; Fax: (+39) 06 49917594<br />
rosa.sorrentino@uniroma1.it<br />
Participants:<br />
Maria Teresa Fiorillo, researcher; Fabiana Paladini, Adriana<br />
Magnacca, post-doc fellows; Elisa Cocco, Elisa Nurzia, PhD<br />
students.<br />
Collaborations:<br />
<strong>Istituto</strong> di Biologia Cellulare, CNR, Roma (Dr. Isabella<br />
Cascino); Department for Crystallography, FU Berlin, Germany<br />
(Prof. Wolfang Saenger); Institut für Immungenetik,<br />
Universitätsklinikum Charité Humboldt-Universität zu Berlin,<br />
Germany (Prof. Andreas Ziegler, Dr. Barbara Uchanska-<br />
Ziegler); Dipartimento di Scienze Mediche, Università di Cagliari<br />
(Prof. Alessandro Mathieu).<br />
Report of activity<br />
The association between HLA-B27 gene and<br />
Spondyloarthropathies has been known since many<br />
decades. However the role of these molecules is still<br />
controversial. The main contribution of our research<br />
group has been the identification of a novel allele<br />
in Sardinia, HLA-B*2709, which does not confer<br />
susceptibility to Ankylosing Spondylitis (AS). This<br />
allele differs for a single amino acid change, H116D<br />
from the B*2705 the most common and AS associated<br />
allele, and the functional consequences have<br />
been analysed in detail by us. Crystallographic studies<br />
have shown how the two molecules, the ancestral<br />
B*2705 and the B*2709, can differ in the way they<br />
bind and present to T lymphocytes some peptides<br />
rich in Arginines. We have identified two peptides<br />
from self-proteins, pVIPR1 and pGlucR, that can<br />
be bound in a dual mode: pVIPR1 when bound to<br />
B*2705 and pGlucR when bound to either subtype.<br />
Interestingly, this observation correlates with the<br />
presence of autoreactive T cells: pVIPR-specific<br />
present in B*2705 positive patients only whereas<br />
pGlucR-specific in both patients and B*2709 positive<br />
healthy controls. This suggests that the presence<br />
of more than one conformation, possibly with<br />
117<br />
AREA 5<br />
a dynamic behaviour, can hamper the correct T cell<br />
selection in the thymus interfering with the recognition<br />
by TCR. The crystallographic structure of the<br />
three party, TCR, HLA-B27 molecules and peptide<br />
could give insights on how the recognition is influenced<br />
by the double structure. A second hypothesis<br />
that could explain the differential association is that<br />
different haplotypes can harbour the two HLA-B27<br />
alleles and therefore additional HLA genes can<br />
co-operate to confer susceptibility to AS. In this<br />
context, our observation that patients with AS in<br />
Sardinia show a higher frequency of homozygous<br />
markers in the region between HLA-A and HLA-B<br />
compared to HLA-B27 controls, seems to support<br />
this second, not mutually exclusive, hypothesis. A<br />
possible candidate gene is the HLA-E mapping in<br />
between HLA-A and HLA-B. The HLA-E gene<br />
encodes for the ligand of NKG2A receptor and is stabilized<br />
by peptides derived from the leader sequence<br />
of HLA-class I genes. This represents an inhibitory<br />
signal for the Natural Killer cells and, interestingly,<br />
the signal sequence of the HLA-B27 allele shows a<br />
polymorphism that interferes with the binding of the<br />
HLA-E molecules. A peak of disease association has<br />
been <strong>report</strong>ed by us with a functional polymorphism<br />
of the HLA-E gene. Experiments are in progress to<br />
verify this hypothesis.<br />
We have also characterized an interesting alternative<br />
pathway for antigen cross-presentation which is<br />
exploited by the HLA-B27 and not by the HLA-A2<br />
molecules and takes place in the TGN, a cellular<br />
compartment in which the molecules go through<br />
their way to the cell surface. In this compartment<br />
the HLA-B27 molecules can load peptides embedded<br />
in chimeric proteins even in not professional antigen<br />
presenting cells. This appears as particularly interesting<br />
given the specificity for the HLA-B27 molecules<br />
and might have important implications in cells<br />
that are not equipped to perform cross-presentation.<br />
Other investigated genes are in the frame of a metaanalysis<br />
of the IL-1 gene and the regulation of the
Rosa Sorrentino<br />
gene encoding for VIP receptor 1 (VPAC1) whose<br />
interest for us was twofold: this is the gene from<br />
which the pVIPR peptide derives and the VIP-<br />
VPAC1 system plays a relevant regulatory role during<br />
immune-mediated inflammation. A correlation<br />
between some polymorphisms in the 3’-UTR regulatory<br />
region of VPAC1 and its down-modulation in<br />
the presence of an inflammatory stimulus such as the<br />
bacterial LPS was observed. We therefore postulated<br />
that a possible regulatory mechanism could be due<br />
to the action of a microRNA whose complementary<br />
sequence maps in the 3’-UTR region of the VPAC1<br />
gene. The hypothesis was found to be correct and<br />
the microRNA, MiR 525-5p, was shown to be upregulated<br />
by LPS and to target the region in close<br />
proximity of a SNP. The experimental protocol did<br />
not allow such a fine modulation to verify subtle<br />
quantitative variations eventually due to the polymorphism.<br />
This is something that will be verified in<br />
the next future.<br />
A second line of research has been more theoretical<br />
and it is based on the observation that the distribution<br />
of the HLA-B27 molecules in general and B*2705<br />
in particular follows a clear north to south gradient<br />
along the world and is opposite to that of malaria. On<br />
the contrary, the establishment of the HLA-B27 subtypes<br />
derived by point mutations from the ancestral<br />
B*2705 type and associated or not with AS, shows<br />
a gradient coincident with that of malaria. This has<br />
allowed us to hypothesize, based on several observations<br />
<strong>report</strong>ed by others, that HLA-B27, although<br />
being an efficient antigen presenting molecule that<br />
protects from infections such as those due to HIV or<br />
Hepatitis C, has indeed being negatively selected by<br />
malaria and the enrichment of some HLA-B27 sub-<br />
118<br />
types in particular areas where the malaria is endemic,<br />
could be due to a selective pressure driven by the parasite.<br />
The association or not with AS, is likely to be a<br />
secondary effect of this variation which might be due<br />
to either the functional consequences of these mutations<br />
or to other genes belonging to the haplotypes<br />
harbouring the different B27 alleles.<br />
Publications<br />
Mathieu A, Paladini F, Vacca A, Cauli A, Fiorillo<br />
MT, and Sorrentino R. The interplay between the<br />
geographic distribution of HLA-B27 alleles and their<br />
role in disease: an unifying hypothesis. Autoimmun Rev.<br />
2009, 8: 420-25. doi: 10.1016/j.autrev.2009.01.003.<br />
Paladini F, Belfiore F, Cocco E, Carcassi C, Cauli<br />
A, Vacca A, Fiorillo MT, Mathieu A, Cascino I,<br />
Sorrentino R. HLA-E gene polymorphism associates<br />
with Ankylosing Spondylitis in Sardinia. Arthritis Res<br />
Ther. 2009, 11: R171. doi: 10.1186/ar2860.<br />
Nurzia E, Panimolle F, Cauli A, Mathieu A,<br />
Magnacca A, Paladini F, Sorrentino R, Fiorillo M T.<br />
CD8+ T-cell mediated self-reactivity in HLA-B27<br />
context as a consequence of dual peptide conformation.<br />
Clin Immunol. 2010, 135: 476-82. doi: 10.1016/j.<br />
clim.2010.01.009.<br />
Cocco E, Paladini F, Macino G, Fulci V, Fiorillo<br />
MT, Sorrentino R. The expression of vasoactive<br />
intestinal peptide receptor 1 is negatively modulated<br />
by microRNA 525-5p. PLoS One. 2010, 105:e12067.<br />
doi:10.1371/journal.pone.0012067.
Principal investigator: Loretta tuosto<br />
Researcher in Molecular Immunology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49917595, Fax: (+39) 06 49917594<br />
loretta.tuosto@uniroma1.it<br />
119<br />
AREA 5<br />
CD28 co-stimulatory molecule as a key regulator of T lymphocyte<br />
differentiation and survival: characterisation of the biochemical<br />
pathways and molecules coupling CD28 to NF-κB activation<br />
Participants:<br />
Michela Muscolini, PhD student; Elisa Montagni, student,<br />
Silvana Caristi, technician.<br />
Collaborations:<br />
Centro de Biología Molecular Severo Ochoa, Universidad<br />
Autónoma de Madrid, Cantoblanco, Madrid, Spain (Prof. Balbino<br />
Alarcón); Dipartimento di Biologia Cellulare, Università di Tor<br />
Vergata, Roma (Prof. Mauro Piacentini); Dipartimento di<br />
Dermatologia, Università di Tor Vergata, Roma (Dr. Antonio<br />
Costanzo); Laboratorio di Oncogenomica Traslazionale, <strong>Istituto</strong><br />
Regina Elena, Roma (Prof. Giovanni Blandino), Hoddaido<br />
University, Sapporo, Japan (Prof. Kazuyasu Sakaguchi).<br />
Report of activity<br />
The signalling pathway that leads from antigenreceptor<br />
and/or co-receptor triggering to the activation<br />
of transcription factors of the NF-κB family has<br />
a crucial role in the regulation of cell survival, and<br />
is controlled by highly similar molecular events in<br />
B and T cells. The understanding of the molecular<br />
mechanisms that control NF-κB activation in lymphocytes<br />
has important implications for the design<br />
of specific therapies for immune diseases.<br />
CD28 is one of the most important co-stimulatory<br />
receptors necessary for full T lymphocyte activation.<br />
By binding its cognate ligands, B7.1/CD80 or B7.2/<br />
CD86 on the surface of professional antigen presenting<br />
cells (APC), CD28 plays a critical role in the<br />
recruitment of key molecules to the T cell receptor<br />
(TCR), thus lowering the activation threshold and<br />
enhancing TCR-mediated signalling pathways.<br />
The existence of autonomous CD28 signalling<br />
has been proposed by Schwartz in 1990, when he<br />
observed that CD28 was able to act as a TCRindependent<br />
signalling unit and to deliver in trans<br />
co-stimulation (Schwartz, 1992). The efforts made<br />
in an attempt to characterize the signalling pathways<br />
activated by CD28/B7 interaction in a TCR-<br />
independent manner, led to the identification of<br />
NF-kB as the most relevant CD28 biochemical<br />
target. In this context, our research group evidenced<br />
that CD28 engagement by B7.1/CD80 expressed on<br />
the surface of APCs is, indeed, sufficient to induce<br />
NF-kB activation in the absence of TCR. In particular,<br />
CD28/B7 interaction alone leads to the activation<br />
of a non-canonical IKKα/NF-kB2-like cascade<br />
and to the nuclear translocation of p52/RelA dimers<br />
(Marinari et al., 2004). IKKα is mainly involved in the<br />
activation of the alternative NF-kB pathway, which<br />
is characterized by IKKα-mediated phosphorylation<br />
of p100, its degradation by proteasome and the<br />
nuclear translocation of active of RelB/p52 or RelA/<br />
p52 dimers. We found that RelA/p52 dimers control<br />
the trans-activation and expression of IL-8, BAFF<br />
and Bcl-xL genes in CD28-stimulated memory T<br />
cells (Marinari et al., 2004). All these data evidence<br />
the existence of an autonomous CD28 signalling<br />
that, through the activation of an alternative-like<br />
IKKα-dependent NF-kB pathway, selectively regulates<br />
the expression of pro-inflammatory cytokines/<br />
chemokines as well as of pro-survival proteins.<br />
CD28 co-stimulation regulates T cell survival by upregulating<br />
the anti-apoptotic Bcl-2 family member,<br />
Bcl-xL through NF-kB (Boise et al., 1995; Kerstan<br />
& Hunig, 2004; Khoshnan et al., 2000). However, the<br />
up-regulation of Bcl-2 family anti-apoptotic genes, in<br />
particular A1/Bfl-1 and Bcl-xL, may account only in<br />
part for the protective effects exerted by NF-kB in T<br />
cells. Recently, co-stimulation dependent activation<br />
of NF-kB has been described to counteract TCRdriven<br />
apoptosis by inhibiting p73 expression (Wan &<br />
DeGregori, 2003). p73 is a member of the p53 family<br />
that integrates stimuli from both DNA damage and<br />
receptor-mediated apoptosis in T cells (Lissy et al.,<br />
2000). Similarly to p53, p73 regulates apoptosis by<br />
inducing the expression of several Bcl-2 family proapoptotic<br />
proteins, including Bax (Melino et al., 2002).<br />
We found that CD28 stimulation by B7 and in<br />
the absence of TCR protects primary T cells from
Loretta Tuosto<br />
IR-mediated apoptosis through two mechanisms. In<br />
one hand, CD28 induces the nuclear translocation<br />
of active RelA/p52 dimers, which together with the<br />
histone acetylase p300/CBP co-activator trans-activate<br />
the bcl-xL promoter. In the other hand, CD28<br />
engagement also stimulates the formation of histone<br />
deacetylase HDAC1/RelA co-repressor complexes,<br />
which inhibit p73 transcriptional activation of bax.<br />
The key clue associated to the nature of CD28 signalling<br />
derives from the composition of its small cytoplasmic<br />
domain (41 aa) and its interaction with intracellular<br />
proteins. It contains a N-terminal YMNM<br />
motif that following phosphorylation binds the p85<br />
subunit of phosphatidylinositol 3-kinase (PI3K) and<br />
Grb2 (Cai et al., 1995; Okkenhaug et al., 2001).<br />
Downstream of the YMNM motif, CD28 has two<br />
proline-rich regions, the N-terminal P 196 RRP that<br />
binds the SH3 domain of the IL-2 inducible kinase<br />
(Itk), and a C-terminal motif Y 206 QP 208 YAPP containing<br />
two proline (P) residues, that binds the SH3<br />
domain of Lck (Holdford et al., 1999; Tavano et al.,<br />
2004), Grb2 (Kim et al., 1998) and FLNa (Tavano et<br />
al., 2006), a large cytoplasmic protein that crosslinks<br />
cortical actin.<br />
By using the CD28-negative Jurkat T cells<br />
(CH7C17) stably transfected with human wild-type<br />
CD28 (CD28 WT), or with CD28 mutants in the<br />
Y 191 MNM 194 motif (CD28Y 191 F or CD28M 194 Q), or in<br />
the N-terminal P 196 RRP 199 motif (CD28AA 196-199 ) or<br />
in the C-terminal proline-rich Y 206 QP 208 YAPP motif<br />
(CD28-3A, or CD28FF 206/209 ), we found that mutation<br />
of either proline (CD28-3A) or tyrosine residues<br />
(CD28FF 206/209 ) in the C-terminal proline-rich motif<br />
strongly reduced both CD28-mediated IKKα and<br />
NF-kB activation.<br />
The C-terminal Y 206 QP 208 YAPP motif of CD28<br />
regulates the recruitment of FLNa to the membrane.<br />
FLNa is a scaffold protein that binds several proteins<br />
involved in cell signalling (Zhou et al., 2010) and<br />
participates in T cell activation (Tavano et al., 2006).<br />
In searching for specific proteins associating FLNa<br />
and coupling CD28 to IKKα and a non-canonical<br />
NF-kB2-like pathway, we looked at the IKKα activator<br />
NIK (Senftleben et al., 2001). Our data evidence<br />
that NIK constitutively associated with FLNa in<br />
both Jurkat and primary T cells. Confocal analyses<br />
revealed that in CD28WT cells NIK was efficiently<br />
recruited to the membrane and co-localized with<br />
FLNa in the T:APC contact zone, while CD28-3A<br />
and CD28FF 206/209 mutants failed to do that. CD28<br />
engagement by B7 significantly induced the kinase<br />
activity of NIK-associated IKKα, without affecting<br />
the amount of co-precipitated IKKα. Both CD28-3A<br />
120<br />
and FF 206/209 mutants failed to induce NIK-associated<br />
IKKα kinase activity. Similar analyses performed<br />
in primary T lymphocytes, revealed that CD28<br />
stimulation induced the stabilization of NIK, its association<br />
with IKKα and NIK-associated IKKα kinase<br />
activity. Thus, the C-terminal proline-rich motif of<br />
CD28 regulates the recruitment of both FLNa/NIK<br />
complexes, thus leading to NIK/IKKα activation<br />
and to the induction of the non-canonical NF-kB2<br />
pathway.<br />
Selected publications<br />
Muscolini M, Montagni E, Caristi S, Nomura T,<br />
Kamada R, Di Agostino S, Corazzari M, Piacentini<br />
M, Blandino G, Costanzo A, Sakaguchi K, Tuosto L.<br />
Characterization of a new cancer-associated mutant<br />
of p53 with a missense mutation (K351N) in the<br />
tetramerization domain. Cell Cycle. 2009, 8: 3396-405.<br />
doi: 10.4161/cc.8.20.9910.
Principal investigator: Elio Ziparo<br />
Professor of Embryology<br />
Dip. di Scienze Anatomiche, Istologiche, Medico-Legali e dell’Apparato Locomotore<br />
Tel: (+39) 06 49766586; Fax: (+39) 0649766340<br />
elio.ziparo@uniroma1.it<br />
121<br />
AREA 5<br />
The role of Toll Like Receptors in immune responses to infections<br />
and in inflammation associated pathologies of the male<br />
reproductive system<br />
Participants:<br />
Antonio Filippini, professor; Anna Riccioli, researcher;<br />
Claudia Giampietri, post-doc fellow; Roberta Galli,<br />
Alessio Paone, PhD students; Fabrizio Padula, Simonetta<br />
Petrungaro, Donatella Starace, technicians.<br />
Collaborations:<br />
Dipartimento di Medicina Sperimentale, Università de L’Aquila<br />
(Prof. Paola De Cesaris).<br />
Report of activity<br />
Hypoxia inducible factor 1 (HIF-1) is a transcription<br />
factor, which regulates a number of genes required<br />
for hypoxic response via binding specific regions of<br />
their promoters, named hypoxia responsive elements<br />
(HREs). HIF-1 is active only as a heterodimer of<br />
HIF-1α and HIF-1β subunits. HIF-1β is constitutively<br />
expressed in all cell types, whereas HIF-1α<br />
levels are tightly controlled.<br />
It has been clearly demonstrated that HIF-1 regulates<br />
genes relevant to cancer progression, especially<br />
as predictor of clinical outcome in patients<br />
with adenocarcinomas (Hoffmann et al., 2008). In<br />
particular, HIF-1α has emerged as a potential prognostic<br />
biomarker in proteomic assessment of prostate<br />
cancer (Kimbro and Simons, 2006) since clinical<br />
observation of high-grade prostate intraepithelial<br />
neoplasia (PIN) lesion (precursor of most prostate<br />
adenocarcinoma (PCa), showed increased HIF-1α<br />
expression (Zhong et al., 2004) and HIF-1α upregulation<br />
in PCa as well as in prostate cancer bone<br />
metastases has been observed (Zhong et al., 1999).<br />
PCa is a prevalent tumour among elderly men and<br />
survival benefit with current PCa therapies is often<br />
limited (Gomella et al., 2009). Indeed standard pharmacological<br />
therapy, consisting of withdrawal of<br />
androgens, leads to only transient regression of the<br />
disease and there is no cure for prostate cancer once<br />
become androgen refractory. Although HIF-1α pro-<br />
tein is mainly induced by hypoxic conditions, other<br />
stimuli can strongly increase the HIF-1 complex in<br />
normoxic conditions and modulate the transcription<br />
of hypoxic genes. Interestingly, also a variety<br />
of molecular components derived from bacteria<br />
or viruses has been described to activate HIF-1α<br />
in normoxia through specific Toll Like Receptors<br />
(TLRs) (Blouin et al., 2004; Nicholas and Sumbayev,<br />
2009; Ramanathan et al., 2009). TLRs are a group<br />
of transmembrane proteins (11 in humans) that<br />
recognize pathogen-associated molecular patterns<br />
(PAMPs). Recent studies demonstrate that tumour<br />
cells express functional TLRs and that TLR signaling<br />
can promote opposite outcomes: tumour growth<br />
and immune evasion, or apoptosis and cell cycle<br />
arrest (Huang et al., 2005; Kelly et al., 2006; Paone et<br />
al., 2008b; Salaun et al., 2006).<br />
The TLR3-ligand poly(I:C) mimics the action of<br />
double stranded RNA (dsRNA), the genetic material<br />
of many viruses, and TLR3 engagement, directly<br />
inhibits cell proliferation and induces tumour cell<br />
death (Salaun et al., 2006; Salaun et al., 2007). We<br />
have previously demonstrated that LNCaP cells, an<br />
androgen-dependent human prostate cancer cell line,<br />
are sensitive to poly(I:C)-induced apoptosis, whereas<br />
PC3 cells, a more aggressive androgen-independent<br />
prostate cancer cell line, show a weak sensitivity to<br />
the same stimulus (Paone et al., 2008b). Recently, a<br />
number of articles <strong>report</strong>ed the ability of HIF-1 complex<br />
to mediate the resistance to several apoptotic<br />
stimuli (Chen et al., 2009; Peng et al., 2006). Based<br />
on these data, we have hypothesized that the limited<br />
response of PC3 cells to poly(I:C) could be due to the<br />
induction of a parallel pro-tumoral signal involving<br />
HIF-1 complex activation.<br />
We described a novel pro-tumour machinery triggered<br />
by TLR3 activation in PC3 cells consisting<br />
in increased expression of the specific I.3 isoform of<br />
HIF-1α and nuclear accumulation of HIF-1 complex<br />
in normoxia, resulting in reduced apoptosis and in<br />
secretion of functional VEGF. Moreover, we <strong>report</strong>
Elio Ziparo<br />
that in the less aggressive LNCaP cells, TLR3 activation<br />
fails to induce nuclear accumulation of HIF-<br />
1α. However, the transfection of I.3 isoform of hif-1α<br />
in LNCaP cells allows poly(I:C)-induced HIF-1 activation,<br />
resulting in apoptosis protection and VEGF<br />
secretion. Altogether, our findings demonstrate that<br />
differences in basal level of HIF-1α expression in<br />
different prostate cancer cell lines underlie their differential<br />
response to TLR3 activation, suggesting a<br />
correlation between different stages of malignancy,<br />
hypoxic gene expression and beneficial responsiveness<br />
to TLR agonists (Paone et al., 2010).<br />
Mounting evidence shows that the enhancement of<br />
innate and adaptive immunity represents the principal<br />
mechanism by which TLR stimulation produces<br />
anti-tumour activity. Apart from a direct effect of<br />
TLR3 on prostate cells apoptosis, we studied also the<br />
ability of TLR ligands to affect the recruitment of<br />
different immune cell populations by human prostate<br />
cancer cell lines and the underlying mechanisms. We<br />
have investigated the expression of TLRs in different<br />
human prostate cancer cell lines, LNCaP, DU-145<br />
and PC3 and shown that only TLR3 and TLR5 are<br />
shared by the three cell lines. Treatment with their<br />
respective agonists poly(I:C) and flagellin rapidly<br />
triggers NF-κB-dependent up-regulation of different<br />
inflammatory molecules, as assayed by microarray<br />
and ELISA. Furthermore, we demonstrate that conditioned<br />
media from poly(I:C)- and flagellin-treated<br />
LNCaP and DU-145 cells induce the recruitment of<br />
different leukocyte subpopulations, suggesting that<br />
TLR stimulation is able to activate the earliest step<br />
of immune response mediated by soluble factors.<br />
Interestingly, the more aggressive cancer cell line<br />
PC3 was found to express TLR3 and TLR5, but<br />
failed to respond to TLR agonists in terms of NF-κB<br />
activation and ability to attract immune effectors<br />
(Galli et al., 2010). Altogether, our data indicate that<br />
both TLR3 and TLR5 are expressed and functionally<br />
active in LNCaP and DU-145 prostate cancer<br />
cells, whereas in PC3 cells TLR3/5 stimulation does<br />
not affect NF-kB activation, cytokine/chemokine<br />
expression and immune cell recruitment, suggesting<br />
that PC3 cells, representing the most aggressive<br />
castration-resistant stage of prostate malignancy, are<br />
not able to activate an anti-tumor immune response.<br />
In conclusion, the ability to mount an immune<br />
response following TLR stimulation seems to be in<br />
reverse correlation with neoplastic progression.<br />
122<br />
Overall, our data show that poly(I:C), by exerting<br />
both a direct pro-apoptotic effect on LNCaP cells<br />
(Paone et al., 2008a), and an immune-mediated effect<br />
due to the recruitment of NK and cytotoxic CD8 cells<br />
(Galli et al., 2010), is a potentially valid therapeutic<br />
agent in prostate cancer. In addition to the known<br />
anti-tumour effect for HIF-1 knock-down, our data<br />
indicate that targeting HIF-1 in the most aggressive<br />
PC3 cells has also the potential to improve the<br />
efficacy of treatment with TLR3 agonists as direct<br />
effectors of prostate cancer cell apoptosis (Paone et<br />
al., 2010).<br />
Selected publications<br />
Antonangeli F, Giampietri C, Petrungaro S, Filippini<br />
A, Ziparo E. Expression profile of a 400-bp Stra8<br />
promoter region during spermatogenesis. Microsc<br />
Res Tech. 2009, 72: 816-22. doi: 10.1002/jemt.20724.<br />
Antonangeli F, Petrungaro S, Coluccia P, Filippini<br />
A, Ziparo E, Giampietri C. Testis atrophy and<br />
reduced sperm motility in transgenic mice overexpressing<br />
c-FLIP(L). Fertil Steril. 2010, 93: 1407-14.<br />
doi: 10.1016/j.fertnstert.2009.01.122.<br />
Starace D, Muciaccia B, Morgante E, Russo MA,<br />
Pensini S, D‘agostino A, De Cesaris P, Filippini A,<br />
Ziparo E, Riccioli A. Peculiar subcellular localization<br />
of Fas antigen in human and mouse spermatozoa.<br />
Microsc Res Tech. 2009, 72: 573-9. doi: 10.1002/<br />
jemt.20700.<br />
Galli R, Starace D, Busà R, Angelini DF, Paone<br />
A, De Cesaris P, Filippini A, Sette C, Battistini L,<br />
Ziparo E, Riccioli A. TLR stimulation of prostate<br />
tumor cells induces chemokine-mediated recruitment<br />
of specific immune cell types. J Immunol. 2010,<br />
15184: 6658-69. doi:10.4049/jimmunol.0902401.<br />
Paone A, Galli R, Gabellini C, Lukashev D, Starace<br />
D, Gorlach A, De Cesaris P, Ziparo E, Del Bufalo<br />
D, Sitkovsky MV, Filippini A, Riccioli A. Toll-like<br />
receptor 3 regulates angiogenesis and apoptosis in<br />
prostate cancer cell lines through hypoxia-inducible<br />
factor 1 alpha. Neoplasia. 2010, 7: 539-49. doi:<br />
10.1593/neo.92106.
AREA 6<br />
New<br />
antimicrobial<br />
and antiviral<br />
agents
Peptide effectors of innate immunity<br />
Principal investigator: Donatella Barra<br />
Professor of Biochemistry<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 4456663; Fax: (+39) 06 4440062<br />
donatella.barra@uniroma1.it<br />
Participants:<br />
Maurizio Simmaco, Giuseppina Mignogna, professors; M.<br />
Luisa Mangoni, Rossella Miele, Alessandro Paiardini,<br />
researchers; Marina Borro, Giovanna Gentile, post-doc<br />
fellows; Ludovica Marcellini Hercolani Gaddi, Sara<br />
Marsango, Vincenzo Luca, PhD students; Alessandra<br />
Franco, Alessandra Giorgi, technicians.<br />
Collaborations:<br />
Institute of Molecular Biology, Austrian Academy od Sciences,<br />
Salzburg, Austria (Prof. Günther Kreil); Centro de Investigaciones<br />
Biológicas (CSIC) Madrid, Spain (Prof. Luis Rivas); Dipartimento<br />
di Scienze e Tecnologie Chimiche, Università di Roma di Tor<br />
Vergata (Prof. Lorenzo Sella); Dipartimento di Fisiologia e<br />
Farmacologia “V. Erspamer”, Sapienza-Università di Roma (Prof.<br />
Lucia Negri).<br />
Report of activity<br />
Because of the increasing emergence of microbes<br />
which are resistant to conventional antibiotics, the<br />
discovery of new antimicrobial agents with a new<br />
mode of action is urgently needed. Naturally occurring<br />
antimicrobial peptides (AMPs), which are produced<br />
by almost all forms of life, represent promising<br />
candidates for the development of new anti-infective<br />
drugs. Amphibian skin is one of the richest sources<br />
for such molecules. In contrast with conventional<br />
antibiotics, most AMPs interact and increase the<br />
permeability of the bacterial membrane as part of<br />
their killing mechanism. However, before reaching<br />
it, they need to cross the cell wall that, in Gramnegative<br />
bacteria, is surrounded by the lipopolysaccharide<br />
(LPS)-outer membrane, which forms a very<br />
efficient barrier against a variety of hydrophilic and<br />
hydrophobic molecules.<br />
We had previously found that some isoforms of<br />
temporins, produced within the same frog specimen<br />
(temporins A and B), can synergize when combined<br />
each with temporin L, to overcome the bacterial<br />
125<br />
AREA 6<br />
resistance imposed by the LPS protective layer. In<br />
addition, we discovered that these peptides can synergize<br />
in the detoxification of LPS molecules, by suppressing<br />
the release of TNF-α from LPS-activated<br />
macrophages. Note that both synergisms inversely<br />
depend on the length of the LPS-polysaccharide<br />
chain. Studies on the underlying molecular mechanisms<br />
are in progress.<br />
In parallel, we carried out an in-depth study on<br />
the in vitro and in vivo antimicrobial properties of<br />
esculentin fragments. With regards to the in vitro<br />
experiments, the mode(s) of action of Esc(1-18)<br />
(GIFSKLAGKKLKNLLISG-NH 2 ) on Escherichia coli<br />
was investigated. We found that the peptide rapidly<br />
kills E. coli at the minimal inhibitory concentration<br />
(MIC) and that the lethal event is concomitant with<br />
the membrane perturbation. Note that many hostdefense<br />
peptides do not destabilize membranes at<br />
their MICs. Importantly, proteomic analysis revealed<br />
that Esc(1-18) has a limited ability to modify the bacterium’s<br />
protein expression profile, at both sub-lethal<br />
and lethal concentrations. This suggests the bacterial<br />
membrane as the major target for the killing<br />
mechanism of this peptide, rather than intracellular<br />
processes. In addition, Esc(1-18) displays a strong<br />
synergism with conventional antibiotics, presumably<br />
by increasing the intracellular influx of these drugs,<br />
as a result of an increased membrane permeability.<br />
With regards to the in vivo studies, we analyzed<br />
the antimicrobial activity of Esc(1-18) and temporins<br />
on the minihost model of Caenorhabditis elegans<br />
infected by a multidrug resistant (MDR) strain of<br />
Pseudomonas aeruginosa. The data showed that both<br />
temporin B and Esc(1-18) can cause a drastic reduction<br />
in the number of viable bacteria within the<br />
worm’s gut, promoting survival of the infected nematodes.<br />
The peptides’ effect on the bacterial colonization<br />
was also visualized by fluorescence microscopy,<br />
using a GFP-expressing strain of P. aeruginosa. After<br />
90 min of exposure to a sub-lethal concentration of<br />
Esc(1-18), the fluorescence intensity throughout the
Donatella Barra<br />
gut of the infected worms decreased considerably.<br />
Remarkably, this peptide is able to alter the permeability<br />
of the membrane of Pseudomonas cells within<br />
the animal. To the best of our knowledge, this is the<br />
first case showing the ability of an AMP to permeate<br />
microbial membranes within a living organism.<br />
Concerning in vivo studies with more complex<br />
animals, such as mammals, we tested the activity<br />
of Esc(1-21) (GIFSKLAGKKIKNLLISGKG-NH 2 )<br />
on cows affected by mastitis, the most common<br />
and expensive illness of dairy cows throughout the<br />
world. Although stress and physical injuries may<br />
give rise to infiammation of the udders, infections<br />
by bacteria or other microorganisms remain the<br />
principal cause: infusion of antibiotics is the major<br />
treatment approach. However, the increased emergence<br />
of MDR pathogens and the production of<br />
milk contaminated with antibiotics has become a<br />
serious threat in the livestock. Our data indicated<br />
that Esc(1-21) is highly potent against the most<br />
common mastitis-causing microbes (e.g. Streptococcus<br />
agalactiae). Most importantly, the peptide is active in<br />
vivo, causing a visible regression of the clinical stage<br />
of mastitis in dairy cows after 1 week of treatment.<br />
Altogether, these results encourage further study of<br />
esculentin peptides to clarify their in vivo antimicrobial<br />
activity, pharmacokinetics and half-life.<br />
Another interesting family of frog skin AMPs is<br />
given by bombinins H, isolated from amphibia of<br />
Bombina genus. They include isomers with a single<br />
D-amino acid resulting from a post-translational<br />
epimerization of the corresponding L-residue.<br />
Specifically, we evaluated the antimicrobial activity<br />
of the pair H2 (IIGPVLGLVGSALGGLLKKI-NH 2 )<br />
and H4, differing by only the configuration of the<br />
second amino acid (an L-isoleucine in H2 and a<br />
D-alloisoleucine in H4), against a large panel of<br />
reference and clinical isolates of Gram-negative and<br />
-positive bacteria; we have performed membrane<br />
permeation assays on both intact cells and model<br />
membranes; used biochemical tools, such as trypsinencapsulated<br />
liposomes and capillary electrophoresis,<br />
to monitor the peptides’ ability to translocate<br />
through the membrane of liposomes mimicking the<br />
E. coli inner membrane. Our current results have<br />
pointed out interesting relationships between the<br />
presence of a single D-amino acid in the sequence<br />
of an AMP and its target microbial cell selectivity/<br />
membrane perturbing activity. Such findings will<br />
contribute to the development of new peptide-based<br />
therapeutic drugs.<br />
Bv8/prokineticins are small proteins that regulate<br />
diverse biological processes including gastro-<br />
126<br />
intestinal motility, angiogenesis, circadian rhythm<br />
and innate immune response. The Bv8 gene from<br />
Bombina orientalis has been cloned, sequenced and<br />
its promoter has been functionally characterized.<br />
Recombinant expression of Bv8 has been obtained in<br />
Pichia pastoris.<br />
Prokineticins bind two closed related G-protein coupled<br />
receptors (GPCRs), PKR1 and PKR2. In general,<br />
these receptors act as molecular switches to relay<br />
activation to heterotrimeric G proteins and a growing<br />
body of evidence points to the fact that GPCRs<br />
exist as homo- or heterodimers. We have shown<br />
by Western blot analysis that PKR2 has a dimeric<br />
structure in neutrophils. By heterologous expression<br />
of PKR2 in Saccharomyces cerevisiae, we examined the<br />
mechanisms of intermolecular interaction of PKR2<br />
dimerization. The potential involvement of three types<br />
of mechanisms was investigated: coiled-coil, disulfide<br />
bridges and hydrophobic interactions between transmembrane<br />
domains. Characterization of differently<br />
deleted or site-directed PKR2 mutants suggest that<br />
dimerization proceeds through interactions between<br />
transmembrane domains. We demonstrated that coexpressing<br />
binding-deficient and signalling-deficient<br />
forms of PKR2 can re-establish receptor functionality,<br />
possibly through a domain-swapping mechanism.<br />
Publications<br />
Islas-Rodríguez AE, Marcellini L, Orioni B, Barra<br />
D, Stella L, Mangoni ML. Esculentin 1-21: a linear<br />
antimicrobial peptide from frog skin with inhibitory<br />
effect on bovine mastitis-causing bacteria. J Pept Sci.<br />
2009, 15: 607-14. doi: 10.1002/psc.1148.<br />
Maisetta G, Mangoni ML, Esin S, Pichierri G, Capria<br />
AL, Brancatisano FL, Di Luca M, Barnini S, Barra D,<br />
Campa M, Batoni G. In vitro bactericidal activity of the<br />
N-terminal fragment of the frog peptide esculentin-1b<br />
(Esc 1-18) in combination with conventional antibiotics<br />
against Stenotrophomonas maltophilia. Peptides.<br />
2009, 30: 1622-6. doi: 10.1016/j.peptides.2009.06.004.<br />
Mangoni ML, Shai Y. Temporins and their synergism<br />
against Gram-negative bacteria and in lipopolysaccharide<br />
detoxification. Biochim Biophys Acta. 2009,<br />
1788: 1610-19. doi: 10.1016/j.bbamem.2009.04.021.<br />
Marcellini L, Borro M, Gentile G, Rinaldi AC, Stella<br />
L, Aimola P, Barra D, Mangoni ML. Esculentin-<br />
1b(1-18)--a membrane-active antimicrobial peptide<br />
that synergizes with antibiotics and modifies the<br />
expression level of a limited number of proteins in
Escherichia coli. FEBS J. 2009, 276: 5647-64. doi:<br />
10.1111/j.1742-4658.2009.07257.x.<br />
Marsango S, Bonaccorsi di Patti M C, Barra D, Miele<br />
R. The Bv8 gene from Bombina orientalis: molecular<br />
cloning, genomic organization and functional<br />
characterization of the promoter. Peptides. 2009, 30:<br />
2182-90. doi: 10.1007/s00018-010-0601-6.<br />
Simmaco M, Kreil G, Barra D. Bombinins, antimicrobial<br />
peptides from Bombina species. Biochim<br />
Biophys Acta. 2009, 1788: 1551-5. doi: 10.1016/j.<br />
bbamem.2009.01.004.<br />
Miele R, Lattanzi R, Bonaccorsi di Patti M C,<br />
Paiardini A, Negri L, Barra D. Expression of Bv8<br />
127<br />
AREA 6<br />
in Pichia pastoris to identify structural features for<br />
receptor binding. Protein Expr Purif. 2010, 73: 10-4.<br />
doi: 10.1016/j.pep.2010.04.012.<br />
Marcellini L, Giammatteo M, Aimola P, Mangoni<br />
ML. Fluorescence and electron microscopy methods<br />
for exploring antimicrobial peptides mode(s)<br />
of action. Methods Mol Biol. 2010, 618:249-66. doi:<br />
10.1007/978-1-60761-594-1_16.<br />
Uccelletti D, Zanni E, Marcellini L, Palleschi C,<br />
Barra D, Mangoni ML. Anti-Pseudomonas activity<br />
of frog skin antimicrobial peptides in a Caenorhabditis<br />
elegans infection model: a plausible mode of action in<br />
vitro and in vivo. Antimicrob Agents Chemother. 2010,<br />
54: 3853-60. doi: 10.1128/AAC.00154-10.
New pyrrole derivatives of BM 212: a new class of<br />
antimycobacterial agents. Design, synthesis, biological evaluation<br />
and study of their mode of action<br />
Principal investigator: Mariangela Biava<br />
Professor of Medicinal Chemistry<br />
Dipartimento di Chimica e Tecnologie del Farmaco<br />
Tel: (+39) 06 49913812; Fax: (+39) 06 49913133<br />
mariangela.biava@uniroma1.it<br />
Participants:<br />
Maurizio Botta, Alessandro De Logu, Edda De Rossi,<br />
Raffaello Pompei, Giulio Cesare Porretta, professors;<br />
Fabrizio Manetti, researcher; Giovanna Poce, post-doc fellow;<br />
Claudio Battilocchio, PhD student; Roberto Torri, technician.<br />
Collaborations:<br />
Department of Medicinal Chemistry and Pharmacognosy,<br />
University of Illinois at Chicago College of Pharmacy, Chicago,<br />
IL, USA (Prof. Scott G. Franzblau); Department of Immunology<br />
and Infectious Diseases, Harvard School of Public Health, Boston,<br />
USA (Prof. Eric Rubin); CNRS, Immunologie et Embryologie<br />
Moléculaires, Université Orléans, France (Prof. Bernhard Ryffel).<br />
Report of activity<br />
The recrudescence of Tuberculosis (TB) has led to a<br />
situation that the World Health Organization describes<br />
as having the potential to cause a “global catastrophe”. 1<br />
The major factors explaining the explosion in numbers<br />
of infections with Mycobacterium tuberculosis (MTB) are:<br />
i) the deadly synergy with HIV, 2-4 ii) the resistance to<br />
the majority of the common antitubercular drugs as<br />
impermeable to these agents, 5 iii) the ability of mycobacteria<br />
to establish a persistent phase, living in a slow<br />
growing or not-growing state, 6,7 recalcitrant to treatment<br />
by conventional anti TB drugs. 8<br />
For all this reason, the aim of this project is to synthesize:<br />
1) new antimycobacterial compounds endowed<br />
with a mechanism of action different from that of<br />
drugs employed in the current therapy, towards<br />
which mycobacteria proved to be resistant; 2) bactericidal<br />
molecules more active, an less cytotoxic than<br />
the previously synthesized ones; 3) low-cost drugs,<br />
that can be used not only in industrialized countries<br />
but also in developing ones in which tuberculosis, in<br />
the last years, is rapidly growing.<br />
The work of this first year has been devoted to the synthesis<br />
of new derivatives of the lead compounds previously<br />
identified, BM212 and BM521 (see Figure), on<br />
129<br />
AREA 6<br />
the basis of both structure-activity relationship (SAR)<br />
and molecular modelling studies previously performed.<br />
The rational approach started with the generation<br />
of a first subclass 9 of compounds in which the parafluoro-phenyl<br />
moiety was placed in turn at N1 or C5<br />
of the central nucleus, while the remaining position<br />
was arranged (ortho or para positions) with different<br />
methyl-derived moieties (CF 3 , SCH 3 or OCH 3 ) to evaluate<br />
the activity, when replacing the C5 methyl group<br />
present in BM521, with different groups endowed with<br />
different electronic properties, but bioisosteres, and the<br />
following superposition on the pharmacophoric model.<br />
Then, to verify the hypothesis that the presence of a<br />
fluorine atom, an alkyl moiety or an electron-releasing<br />
moieties at the para position of the phenyl rings were<br />
crucial parameters for activity we develop a second<br />
subclass 9 of compounds characterized by an electrondonating<br />
group (methoxyl or methylsulphanyl) and<br />
an alkyl one (ethyl, propyl and i-propyl) alternatively<br />
placed at N1 and C5 of the pyrrole core, by maintaining<br />
the thiomorpholino-methyl group positioned at C3.<br />
Finally, in the attempt to conjugate lipophilic and<br />
structural parameters, a further enlargement of SAR<br />
was carried out developing a third subclass 10 of compounds<br />
in which the methyl chain at C2 of the central<br />
nucleus was replaced with an ethyl one. This was<br />
accomplished to find out if the introduction of a more<br />
lipophilic moiety at C2 of the pyrrole, instead of at the<br />
phenyl rings in N1 or C5, was able to affect the activity,<br />
being the C2 methyl group devoid of interactions<br />
with the pharmacophore features, but involved in the<br />
conformational modulation of the other substituents.<br />
New derivatives were synthesized following the<br />
scheme previously <strong>report</strong>ed, characterized by only<br />
three steps, very versatile and cheep, that allowed us<br />
to assure their potential low-cost, satisfying the goal 3.<br />
All the products have been evaluated for their cytotoxicity<br />
and tested for their in vitro activity against<br />
i) intra and extra-cellular MTB; ii) atypical mycobacteria,<br />
such as M. avium, particularly incident in HIVimmunocompromised<br />
patients; iii)clinical strains
Mariangela Biava<br />
resistant to the conventional drugs. The study of the<br />
possible target is in progress, and it is performed by<br />
external research groups.<br />
Microbiology<br />
All compounds were assayed to assess their in vitro<br />
activity towards Mycobacterium tuberculosis 10347. The<br />
activities of compounds were evaluated as minimal<br />
concentration that completely inhibits the bacteria<br />
growth (MIC). Cytotoxicity was evaluated in a cellbased<br />
assay employing Vero Cells and expressed as<br />
50 % maximum non-toxic dose, MNTD or as 50%<br />
50<br />
cytotoxic concentration, CC . The protection index<br />
50<br />
(PI) was calculated as MNTD (or CC ) vs MIC ratio.<br />
50 50<br />
Compounds belonging to the first subclass, shared<br />
good MICs. N1-halo-phenyl derivatives are endowed<br />
with lower inhibitory concentrations with respect to<br />
corresponding C5 analogs. One compound of this series<br />
showed an outstanding activity in terms of cytotoxicity,<br />
with a PI (>1000) higher than that shown by rifampin.<br />
In general, chlorinated derivatives seem to generate<br />
less cytotoxic effect and better biological profiles with<br />
respect to the corresponding fluorine derivatives.<br />
Furthermore, the para substitution leads to derivatives<br />
more potent than corresponding ortho.<br />
All derivatives belonging to the second subclass showed<br />
very low inhibitory concentration and the results highlighted<br />
that an increase in electronic density of the phenyl<br />
rings at N1 and C5 translates into activity enhancement.<br />
It can be clearly seen that N1/C5 alkyl/methoxy<br />
or alkyl/methylsulphanyl subclasses of compounds are<br />
endowed with very good potential and their activities<br />
follow an almost resembling trend, with inhibitory<br />
concentrations ranging from 0.125 to 2 µg/mL. On the<br />
other hand, this subclass of molecules were characterized<br />
by moderate cytotoxicity and only one compound<br />
in the series proved to perform cytotoxic effects comparable<br />
to that shown by reference compounds (PI >512).<br />
In this sense, the substitution of the fluorine atom with<br />
a para-methoxyl group and the methyl with a propyl<br />
one does not seem to change the biological interactions<br />
(in terms of MICs and cytotoxicity). It is also worthy<br />
to note that an increase of lipophylicity in the alkyl/<br />
methylsulphanyl subseries reflects in an improvement<br />
130<br />
of activity, both in terms of MICs and PIs.<br />
Compounds bearing an ethyl chain at C2 of the<br />
pyrrole core, belonging to the third subclass, are<br />
characterized by MIC values two- to four fold higher<br />
than those <strong>report</strong>ed for methyl analogs. This result<br />
shows that correlation between higher lipophilicity<br />
and antimycobacterial activity may depend on<br />
structural modifications mainly involving the phenyl<br />
rings at N1 and C5, that are able to match the hydrophobic<br />
features of the pharmacophoric model.<br />
Perspective<br />
The future work will be devoted to perform the<br />
microsomial stability for the most active compounds,<br />
and their pharmacokinetic study as preliminary evaluation<br />
for the following in vivo test. Further modifications<br />
to the structure will be performed as well, in<br />
the attempt to improve the biological profile of these<br />
molecules.<br />
References<br />
1) WHO Report 2009. Geneva, Switzerland.<br />
2) Corbett EL et al., Arch Intern Med 2003, 163,1009.<br />
3) Breen RA et al., Drugs 2006, 66, 2299.<br />
4) Phillips KD, J Assoc Nurses AIDS Care 2007, 18, 75.<br />
5) Inderlied CB et al., Clin Microbiol Rev, 1993, 6, 266.<br />
6) Stewart GR et al., Nat Rev Microbiol 2003, 1, 97.<br />
7) Dooley KE et al., Clin Chest Med 2006, 26, 313.<br />
8) Wallis RS et al., Antimicrob Agents Chemother<br />
1999, 43, 2600.<br />
9) Biava M et al., Bioorganic & Medicinal Chemistry, 2010,<br />
18, 8076.<br />
10) Biava M et al., ChemMedChem, submitted.<br />
Publications<br />
Biava M, Porretta GC, Poce G, Battilocchio C,<br />
Alfonso S, De Logu A, Serra N, Manetti F, Botta<br />
M. Identification of a novel pyrrole derivative<br />
endowed with antimycobacterial activity and protection<br />
index comparable to that of the current<br />
antitubercular drugs streptomycin and rifampin,<br />
Bioorg Med Chem. 2010, 18: 8076–84. doi: 10.1016/j.<br />
bmc.2010.09.006.
Principal investigator: Roberto Di Santo<br />
Professor of Farmaceutical Chemistry and Toxicology<br />
Dipartimento di Chimica e Tecnologie del Farmaco<br />
Tel: (+39) 06 49913150; Fax: (+39) 06 491491<br />
roberto.disanto@uniroma1.it<br />
131<br />
AREA 6<br />
Pyrrolyl diketo hexenoic acid derivatives as novel anti-HIV agents<br />
targeted to the ribonuclease H function of the HIV-1 reverse<br />
transcriptase enzyme<br />
Participants:<br />
Roberta Costi, professor; Gaetano Miele, post-doc fellow;<br />
Giuliana Cuzzucoli Crucitti, PhD student; Federica Rosi,<br />
Alberto Iacovo, graduate students; Giovanni Santilli, technician.<br />
Collaborations:<br />
Università di Napoli “Federico II” (Prof. Ettore Novellino,<br />
Dr. Luciana Marinelli); Università di Cagliari (Prof. Enzo<br />
Tramontano); NCI at Bethesda, NIH, Bethesda, USA (Prof. Yves<br />
Pommier); ChiefKatholieke Universiteit Leuven, Belgium (Prof.<br />
Christophe Pannecouque); Politechnika Lodzka, Poland (Prof.<br />
Grzegorz Bujacz).<br />
Report of activity<br />
The HIV-1 RT-associated ribonuclease H (RNase H)<br />
function is a validated and very attractive new target<br />
for HIV/AIDS drug development (1-3); up to today<br />
no drug against the HIV-1 RT-associated RNase<br />
H is i) approved for therapy, ii) under evaluation in<br />
clinical trial, iii) under later stages of pre-clinical<br />
evaluation. The current available information on the<br />
3D structure of the HIV-1 RT (comprising its RNase<br />
H domain) give a solid support for drug development<br />
by both in silico screening and lead compound<br />
optimization through docking studies and the first<br />
crystal structure of the HIV-1 RT with an inhibitor<br />
bound to the RNase H domain has been very recently<br />
<strong>report</strong>ed by an american academic team (3).<br />
Recently, two interesting DKA derivatives have been<br />
<strong>report</strong>ed. The first derivative, 4-[5-(benzoylamino)<br />
thien-2-yl]-2,4-dioxobutanoic acid (BTDBA), originally<br />
synthesized for HIV-1 IN inhibition, has been<br />
shown to inhibit the HIV-1 RT RNase H function<br />
without affecting its polymerase activity (4). BTDBA<br />
provided the proof of concept for direct inhibition of<br />
the HIV-1 RT RNase H associated activity by DKAs,<br />
even though it was not highly selective for RNase H<br />
since i) it inhibited in the same concentration range<br />
also the HIV-1 IN in enzyme assays and ii) it did not<br />
block the viral replication in cell-based assays.<br />
The second DKA derivative, 6-[1-(4-fluorophenyl)<br />
methyl-1Hpyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid<br />
ethyl ester (RDS 1643) was recently <strong>report</strong>ed by<br />
our research group, has been <strong>report</strong>ed to have anti-<br />
RNase H activity, as well BTDB (5). RDS 1643,<br />
inhibited the HIV-1 RNase H activity in enzyme<br />
assays with an IC 50 value of 13 µM, showing lower<br />
potency if compared to BTDBA. It did not affect neither<br />
the HIV-1 RDDP function nor the AMV and E.<br />
coli RNase H activity, while it inhibited the HIV-1 IN<br />
reaction (IC 50 = 90 µM). Differently from BTDBA,<br />
RDS 1643 inhibited the replication of wild type<br />
HIV-1 in cell-based assays showing an EC 50 value of<br />
13 µM and a CC 50 value of 63 µM. Furthermore, RDS<br />
1643 blocked the replication of three HIV-1 NNRTI<br />
resistant viral mutants (RT mutations were Y181C;<br />
K103N/Y181C; K103R/V179D/P225H) with EC 50<br />
values ranging from 7 to 19 µM. Mode of action<br />
studies demonstrated that the RDS 1643 maximum<br />
adsorbance shifted in the presence of the Mg 2+ ions,<br />
suggesting a mechanism of action similar to that<br />
shown by BTDBA. Recent molecular modeling studies<br />
supported the hypothesis that RDS1643 may bind<br />
to the HIV-1 RNase H domain similarly to BTDBA<br />
(7). However, RDS 1643 would not reach towards<br />
the p51 subunit as far as BTDBA, showing therefore<br />
a less favorable interaction with RT, consistently<br />
with the lower potency of RDS 1643 inhibition as<br />
compared to BTDBA inhibition. Therefore, even<br />
though no straight demonstration that RDS 1643<br />
inhibits the RNase H activity inside the cells has<br />
been published yet, these docking results support the<br />
claim that RDS 1643 inhibits the HIV-1 replication<br />
through the selective inhibition of the RT-associated<br />
RNase H function.<br />
Since RDS 1643 was originally designed as HIV-1<br />
IN inhibitor (8), a few RDS 1643 derivatives were<br />
synthesized by our research group and assayed on<br />
both HIV-1 RT-associated enzyme activities. Firstly,<br />
20 derivatives (PDKAs) with different substituents<br />
on the benzyl portion linked to the 1-position of
Roberto Di Santo<br />
the pyrrole ring were synthesized and tested on i)<br />
RT enzyme to determine their ability to inhibit its<br />
RNase H function, and ii) infected cells with HIV-1<br />
and HIV-2 (Table 1).<br />
All tested compounds within this series were found<br />
active against RNase H. Moreover, among the tested<br />
DKA derivatives, both ester and acid derivatives<br />
showed comparable anti-RNase H activities. The<br />
compounds <strong>report</strong>ed in Table 1 were all mono-substituted<br />
on the benzyl ring. It is worthy to note that<br />
the activities seem to be not highly dependent by<br />
the nature or position of the substituent. In fact, the<br />
IC 50 s are still contained within a fairly narrow range<br />
(2.5-26 µM). The above compounds were tested also<br />
in cell-based assays regarding their antiretroviral<br />
activities against both HIV-1 and HIV-2 viruses,<br />
to assess whether the enzyme inhibition also corresponded<br />
to an ex vivo activity. It has been noted<br />
that, in general, the anti-HIV activity is higher than<br />
the inhibition of RNase H. So we hypothesize that<br />
inhibition of further targets could be involved in the<br />
antiretroviral activity, and decided to test these compounds<br />
also against IN in enzyme assays. This was<br />
conceived as a logical approach since many RNase H<br />
inhibitors, including aryl diketo acids, showed activity<br />
also against IN (Test by Pommier, NIH). The data<br />
that we obtained, confirmed that these compounds<br />
can be considered as dual inhibitors (Table 1).<br />
Therefore, we designed and synthesized new PDKAs<br />
to better understand SAR within this class of derivatives,<br />
to obtain both dual and selective inhibitors.<br />
The RNase H selective inhibitors will be particularly<br />
useful to understand the mechanism of action<br />
of this function of RNase H that is far to be totally<br />
elucidated. Three of the most potent compound are<br />
under investigation about their mechanism of action<br />
and under studies to obtain crystal structure of the<br />
complex RNase H-PDKHA inhibitor. This information<br />
could be useful to define the structural elements<br />
in this series that can move their activity to one or<br />
other of the two targets. Thus, further 30 derivatives<br />
mono- and di-substituted on the benzene ring have<br />
been obtained by means of parallel synthesis (structure<br />
A). After this step about 100 inhibitors have<br />
been designed characterized by more pronounced<br />
structural modifications, whose structures are shown<br />
in Table 2 (selected derivatives).<br />
These compounds have been designed also based<br />
on information obtained by RX and MM studies<br />
<strong>report</strong>ed in literature, which described the interac-<br />
132<br />
tions between various RNase H inhibitors, including<br />
RDS 1643, and the catalytic site of RNase H.<br />
We obtained excellent dual inhibitors, in particular<br />
derivative RDS 1610 active on both targets and<br />
also in cell-based assays against HIV-1 and HIV-2<br />
viruses. Additional studies are currently ongoing<br />
that will certainly allow to design the next generation<br />
of RNase H inhibitors to be considered as new<br />
preclinical trial drug candidates.<br />
Finally, due to the discovery of dual inhibitors<br />
activity, we decide to test also more than 100 quinolonyl<br />
diketo acid derivatives that were previously<br />
described by us as IN inhibitors, and found a number<br />
of hit with good anti-RNase H activities. These<br />
compounds will be developed soon as novel RNase<br />
H inhibitors. During this screening also an activator<br />
of RNase H was identified, which is endowed of<br />
antiretroviral activity.<br />
References<br />
1) De Clercq E. J Med Chem. 48: 1297-1313 (2005).<br />
2) Tramontano E. Mini Rev Med Chem. 6: 727-737<br />
(2006).<br />
3) Himmel DM, Serafianos SG, Dharmasena S,<br />
Hossain MM, McCoy-Simandle K, Ilina T, Clark<br />
Jr AD, Knight JL, Julias JG, Clark PK, Krogh-<br />
Jespersen K, Levy RM, Hughes SH, Parniak MA,<br />
Arnold E. ACS Chem Biol. 1: 702-712 (2006).<br />
4) Beutler J, Legrice SFJ, Budihas SR, Wamiru A,<br />
Gardella R, Wilson J, Goncharova K. PCT Int<br />
Appl. 2006026619 A2 (2006).<br />
5) Tramontano E, Esposito F, Badas R, Di Santo R,<br />
Costi R, La Colla P. Antiv Res. 65: 117-124 (2005).<br />
6) Mizrahi V, Brooksbank R, Nkabinde N. J Biol<br />
Chem. 269: 19245-49 (1994).<br />
7) Klumpp K, Mirzadegan T. Curr Pharm Des. 12:<br />
1909-22 (2006).<br />
8) Di Santo R, Costi R, Artico M, Ragno R, Greco G,<br />
Novellino E, Marchand C, Pommier Y. Farmaco.<br />
60: 409–417 (2005).<br />
Pubblications<br />
Di Santo R. Natural products as antifungal agents<br />
against clinically relevant pathogens. Nat Prod Rep.<br />
2010, 27: 1084-1098. doi: 10.1039/b914961a.<br />
Tramontano E, Di Santo R. HIV-1 RT-associated<br />
RNase H function inhibitors: recent advances in drug<br />
development. Curr Med Chem. 2010, 17: 2837-2851.
Cpd<br />
RDS<br />
R 1<br />
R<br />
IC 50 RH<br />
(µM)<br />
IC 50 IN<br />
(ST)(µM)<br />
133<br />
IC 50 HIV-1<br />
(µM)<br />
IC 50 HIV-2<br />
(µM)<br />
CC 50<br />
(µM)<br />
1698 H Et - 15 1.46±0.03 2.32±0.53 24.00±7.58<br />
1699 H H 15 0.09 0.83±0.49 2.11±0.33 66.55±7.45<br />
1683 2-F Et 6.3 0.98 1.20±0.21 2.70±0.32 16.90±3.28<br />
1684 2-F H 6.4 0.98<br />
1743 3-F Et 9.0 11 5.39±4.75 ≥6.86 43.95±15.89<br />
1744 3-F H - 0.92 2.61±1.77 9.52±1.81 64.30±4.85<br />
1643 4-F Et 8.0 2.3 1.70±0.06 Borderline 16.78±5.64<br />
1644 4-F H 2.5 0.026 1.71±0.42 3.09±0.66 60.33±9.13<br />
1759 2-Cl Et 9.8 8.0 ≥2.86 ≥13.73 13.73±1.33<br />
1760 2-Cl H 5.0 4.0 4.19±3.63 7.73±0.86 52.83±11.40<br />
1713 4-Cl Et 8.0 42 ≥14.78 ≥14.78 14.78±1.37<br />
1714 4-Cl H 5.0 ≥4.1 ≥59.28 ≥59.28 59.28±8.74<br />
1687 2-Me Et 3.0 32 ≥2.23 ≥13.48 13.48±1.43<br />
1688 2-Me H 26 0.17 1.56±0.31 3.19±1.00 14.43±2.52<br />
1736 3-Me Et 9.6 8.0 2.17±0.36 ≥25.63 25.63±19.6<br />
1737 3-Me H 4.6 1.3 4.66±2.84 8.35±1.08 72.53±2.96<br />
1692 4-Me Et 7.3 ≥111 ≥12.30 ≥12.30 12.30±1.54<br />
1693 4-Me H 17 1.2 ≥13.33 ≥13.33 13.33±1.52<br />
1703 4-NO 2 Et 12 - ≥59.98 ≥59.98 59.98±5.57<br />
1715 4-OMe Et 6.7 110 ≥13.28 ≥13.28 13.28±1.09<br />
1716 4-OMe H 3.0 4.1<br />
Table 1 - Anti-RNase H and antiretroviral activities of mono-substituted PDKHAs derivatives.<br />
AREA 6<br />
Cpd Str R1 R IC (µM)IN 50 IC (µM)RH 50 IC (µM)HIV1 50 IC (µM)HIV2 50 CC (µM) 50<br />
1610 B =O Et 0.31 10 1.53 2.32 30<br />
1711 C - Et ≥100 3.0 ≥13.80 ≥13.80 13.80<br />
1712 C - H 0.73 7.0 7.30 11.00 43.05<br />
1790 D Me H - 28 ≥13.23 ≥13.23 13.23<br />
1791 D Me Et - 20 ≥13.45 ≥13.45 13.45<br />
2400 E 4-F Et 90.00 55 >50<br />
2401 E 4-F H 26.00 69 >50<br />
2200 F 4-F Et 1.6 > 100 >50<br />
2201 F 4-F H 0.024 41 >50<br />
2600 G 4-F H >111 64 >50<br />
2231 H 4-F Et 0.79 6 3.9<br />
2232 H 4-F H 0.019 14 >50<br />
Table 2 - Activities of selected new PDKA derivatives.
Design, synthesis and biological evaluation of small molecule<br />
epigenetic modulators: a novel approach for anticancer,<br />
antifungal, and antiviral chemotherapy<br />
Principal investigator: Antonello Mai<br />
Professor on Medicinal Chemistry<br />
Dipartimento di Chimica e Tecnologie del Farmaco<br />
Tel: (+39) 06 49913392; Fax: (+39) 06 491491<br />
antonello.mai@uniroma1.it<br />
Participants:<br />
Giovanna Simonetti, professor; Rino Ragno, researcher;<br />
Dante Rotili, Sergio Valente, Silvia Simeoni, post-doc<br />
students; Antonia Caroli, Giorgia Botta, Domenico<br />
Tarantino, PhD students.<br />
Collaborations:<br />
Università di Siena (Prof. Silvio Massa), University of Innsbruck,<br />
Austria (Prof . Gerald Brosch), II Università di Napoli (Prof.<br />
Lucia Altucci), Università di Milano & <strong>Istituto</strong> Europeo di<br />
Oncologia, Milano (Prof. Saverio Minucci), University of Texas,<br />
USA (Prof. M. T. Bedford, Dr. Donghang Cheng).<br />
Report of Activity<br />
HDAC inhibitors. In cancer cells, HDAC inhibition<br />
causes histone hyperacetylation and leads to<br />
transcriptional activation of genes associated with<br />
growth arrest, terminal differentiation, and/or apoptosis,<br />
both in vitro and in vivo. A number of HDAC<br />
inhibitors is actually in >100 clinical trials, alone or<br />
in combination, for the treatment of several types<br />
of cancer, and two of them, vorinostat (SAHA) and<br />
romidepsin (FK-228) have been approved by FDA<br />
for the treatment of cutaneous T cell lymphoma<br />
(CTCL). Since 2001 we <strong>report</strong>ed various series of<br />
hydroxamate compounds as HDACi, from pyrrole<br />
derivatives to uracil-based and cinnamyl hydroxamates.<br />
In this Project, we continued our searches focusing<br />
on class I- and class II-selective HDAC inhibitors<br />
(HDACi). A) Class I-selective HDACi. MS-275 is<br />
a class I-selective HDACi from the Bayer Schering<br />
AG Pharma group, that inhibits tumor growth in<br />
vivo and promotes differentiation or apoptosis. Since<br />
the main chemical difference between MS-275 and<br />
vorinostat (a pan-HDACi) is the zinc-binding-group<br />
(the 2-aminoanilide in MS and the hydroxamate<br />
in vorinostat), we prepared a number of 2-aminoanilide<br />
analogues in addition to new hydroxamates.<br />
Among the newly synthesized aroylamino-cinnamyl<br />
135<br />
AREA 6<br />
hydroxamates, the majority of them behaved as class<br />
I-selective HDACi. Moreover, they showed high<br />
apoptosis induction (higher than vorinostat) and low<br />
differentiating activity (U937 cells). Differently, the<br />
cinnamic 2-aminoanilides showed class I HDACselectivity<br />
joined to low apoptosis and high cytodifferentiation<br />
effects, similar to that of MS-275.<br />
Among the uracil-based 2-aminoanilides, the most<br />
potent derivatives inhibited class I and IIb HDACs<br />
but not class IIa, displaying high antiproliferative<br />
and dose-dependent granulocytic differentiation on<br />
U937 cells. B) Class II-selective HDACi. In cancer<br />
therapy, inhibition of class I HDACs is clinically<br />
relevant, nevertheless a role of class II HDACs in<br />
cancer cannot be ruled out. Our class II-selective<br />
HDACi (MC1568 and MC1575) have been extensively<br />
used to dissect and to elucidate the biological<br />
role of class II HDACs in some conditions. In human<br />
breast cancer, MC1568 and MC1575 displayed antiproliferative<br />
activity without induction of apoptosis<br />
or cytodifferentiation. In thyroid carcinoma,<br />
MC1568 was ineffective whereas class I-selective<br />
HDACi (MS-275) reduced TRAIL degradation by<br />
interfering with the proteasomal pathway. In muscle<br />
cells, MC1568 arrest myogenesis through reduction<br />
of MEF2D expression and stabilisation of the<br />
HDAC3-HDAC4-MEF2D complex. Other applications<br />
involving MC1568 have been described by<br />
us in HIV-1 induction from latency, inflammation<br />
(TNF-α expression during LPS stimulation), RAR-<br />
and PPARγ-induced adipocyte differentiation, chromatin<br />
folding in HUVEC through the NO-PP2Aclass<br />
II HDACs pathway, and muscular (DMD) and<br />
neurodegenerative (Huntington) diseases.<br />
SIRT modulators. In a search for potent sirtuin inhibitors<br />
we prepared a series of sirtinol analogues by<br />
studying the replacement of the benzamide linkage<br />
of the prototype with other bioisoster such as anilide,<br />
sulfonamide, sulfide, 1-oxopropyl groups. These new<br />
compounds were evaluated against human SIRT1
Antonello Mai<br />
and SIRT2, and their effects in nematodes expressing<br />
a muscular dystrophy protein have been described.<br />
Moreover, the new compounds were tested in U937<br />
cells showing high apoptosis induction and/or cytodifferentiation.<br />
One of them, salermide, was well tolerated<br />
by mice at concentrations up to 100 µM, and prompted<br />
tumor-specific apoptosis in a wide range of human<br />
cancer cell lines. Genetic p53 knockdowns showed that<br />
the SIRT1-dependent pro-apoptotic effect of salermide<br />
is p53-independent, but it is due to the reactivation of<br />
pro-apoptotic genes epigenetically repressed exclusively<br />
in cancer cells by SIRT1. In addition, we designed<br />
some barbituric and thiobarbituric acid analogues to<br />
evaluate as SIRTi and anticancer agents. When tested<br />
as SIRT1 and SIRT2 inhibitors, only the tetracyclic<br />
compound MC2141 showed high and selective SIRT1<br />
inhibiting activity joined to high apoptosis induction<br />
on U937 cells. Searching new putative SIRTi based<br />
on the nicotinamide scaffold, we identified some SIRT<br />
activators, able to increase SIRT1 as well as SIRT2<br />
activity. Cellular screening (on U937 at 50 µM) did<br />
not show any significant apoptosis- or differentiationinducing<br />
properties, but such new SIRT activators<br />
showed anti-senescence effect as well as activation of<br />
PGC1α and increase of mithocondrial effects similar<br />
as resveratrol.<br />
HMT and HD inhibitors. In addition to histone and<br />
non-histone protein acetylation, histone methylation<br />
has also been shown to be important in epigenetic<br />
regulation of gene expression. Histone methylation<br />
can occur on both Lys (K, by HKMTs) or Arg (R,<br />
by PRMTs) residues. Several Lys residues have been<br />
found to undergo mono-, di-, or tri-methylation, the<br />
result of the chemical modification being transcriptional<br />
activation as well as silencing. In 2008 we<br />
prepared and tested some bis(3-bromo-4-hydroxy-<br />
and 3,5-dibromo-4-hydroxyphenyl) compounds and<br />
their analogues against PRMT1, CARM1/PRMT4,<br />
SET7 (a HKMT), p300/CBP (an HAT enzyme),<br />
SIRT1, and SIRT2. Depending on the extension of<br />
bromination of the molecule and on the nature of the<br />
linker connecting the two dibromophenol moieties<br />
some of such compounds behaved as epigenetic multiple<br />
ligands (epi-MLs), they being active against all<br />
the tested enzymes. The H3K4 demethylase LSD1<br />
has been shown to be up-regulated in high-risk<br />
tumors. Very few molecules have been <strong>report</strong>ed<br />
as able to inhibit the LSD1 demethylating action.<br />
Starting from the <strong>report</strong> of the LSD1 inhibiting<br />
activity of the tranylcypromine (TCPA), we prepared<br />
two series of TCPA derivatives by inserting<br />
at its benzene ring an amide or amino acid function.<br />
136<br />
Among such compounds, MC2580 showed K i = 1.3<br />
µM against LSD1 (~ 200-fold more potent than<br />
TCPA) and was inactive against MAO-B. MC2580<br />
also dramatically enhanced the efficacy of RA on<br />
growth and differentiation of acute promyelocytic<br />
leukemia cells, including primary murine APL blasts.<br />
Publications<br />
Cirilli R, Ferretti R, La Torre F, Borioni A, Fares<br />
V, Camalli M, Faggi C, Rotili D, Mai A. Chiral<br />
HPLC separation and absolute configuration of<br />
novel S-DABO derivatives. Chirality. 2009, 21: 604-<br />
12. doi: 10.1002/chir.20654.<br />
Lara E, Mai A, Calvanese V, Altucci L, López-Nieva<br />
P, Martínez-Chantar ML, Varela-Rey M, Rotili<br />
D, Nebbioso A, Ropero S, Montoya G, Oyarzabal<br />
J, Velasco S, Serrano M, Witt M, Villar-Garea<br />
A, Imhof A, Mato JM, Esteller M, Fraga MF.<br />
Salermide, a Sirtuin inhibitor with a strong cancerspecific<br />
proapoptotic effect. Oncogene. 2009, 28: 781-<br />
91. doi:10.1038/onc.2008.436.<br />
Mai A, Altucci L. Epi-drugs to fight cancer: From<br />
chemistry to cancer treatment, the road ahead. Int<br />
J Biochem Cell Biol. 2009, 41: 199-213. 10.1016/j.<br />
biocel.2008.08.020.<br />
Mai A, Rotili D, Tarantino D, Nebbioso A, Castellano<br />
S, Sbardella G, Tini M, Altucci L. Identification of<br />
4-hydroxyquinolines inhibitors of p300/CBP histone<br />
acetyltransferases. Bioorg Med Chem Lett. 2009, 19:<br />
1132-5. doi: 10.1016/j.bmcl.2008.12.097.<br />
Mai A, Rotili D, Valente S, Kazantsev AG. Histone<br />
deacetylase inhibitors and neurodegenerative disorders:<br />
holding the promise. Curr Pharm Des. 2009, 15:<br />
3940-57.<br />
Mai A, Valente S, Meade S, Carafa V, Tardugno M,<br />
Nebbioso A, Galmozzi A, Mitro N, De Fabiani E,<br />
Altucci L, Kazantsev A. Study of 1,4-dihydropyridine<br />
structural scaffold: discovery of novel sirtuin activators<br />
and inhibitors. J Med Chem. 2009, 52: 5496-<br />
5504. doi: 10.1021/jm9008289.<br />
Mai A, Valente S, Nebbioso A, Simeoni S, Ragno R,<br />
Massa S, Brosch G, De Bellis F, Manzo F, Altucci<br />
L. New pyrrole-based histone deacetylase inhibitors:<br />
Binding mode, enzyme- and cell-based investigations.<br />
Int J Biochem Cell Biol. 2009, 41: 235-47.<br />
doi:10.1016/j.biocel.2008.09.002.
Naldi M, Calonghi N, Masotti L, Parolin C, Valente<br />
S, Mai A, Andrisano V. Histone post-translational<br />
modifications by HPLC-ESI-MS after HT29 cell<br />
treatment with HDAC inhibitors. Proteomics. 2009,<br />
9: 5437-45. doi: 10.1002/pmic.200800866.<br />
Nebbioso A, Manzo F, Miceli M, Conte M, Manente<br />
L, Baldi A, De Luca A, Rotili D, Valente S, Mai A,<br />
Usiello A, Gronemeyer H, Altucci L. Selective class<br />
II HDAC inhibitors impair myogenesis by modulating<br />
the stability and activity of HDAC-MEF2 complexes.<br />
EMBO Rep. 2009, 10: 776-82. doi: 10.1038/<br />
embor.2009.88.<br />
Rotili D, Simonetti G, Savarino A, Palamara AT,<br />
Migliaccio AR, Mai A. Non-cancer uses of histone<br />
deacetylase inhibitors: effects on infectious diseases<br />
and beta-hemoglobinopathies. Curr Top Med Chem.<br />
2009, 9: 272-91.<br />
Binda C, Valente S, Romanenghi M, Pilotto S, Cirilli<br />
R, Karytinos A, Ciossani G, Botrugno OA, Forneris<br />
F, Tardugno M, Edmondson DE, Minucci S, Mattevi<br />
A, Mai A. Biochemical, structural, and biological<br />
evaluation of tranylcypromine derivatives as inhibitors<br />
of histone demethylases LSD1 and LSD2. J Am<br />
Chem Soc. 2010, 132: 6827-6833.<br />
Borbone E, Berlingieri MT, De Bellis F, Nebbioso<br />
A, Chiappetta G, Mai A, Altucci L, Fusco A. Histone<br />
deacetylase inhibitors induce thyroid cancer-specific<br />
apoptosis through proteasome-dependent inhibition<br />
of TRAIL degradation. Oncogene. 2010, 29: 105-116.<br />
doi:10.1038/onc.2009.306.<br />
Castellano S, Milite C, Ragno R, Simeoni S, Mai<br />
A, Limongelli V, Novellino E, Bauer I, Brosch G,<br />
Spannhoff A, Cheng D, Bedford M T, Sbardella G.<br />
Design, synthesis and biological evaluation of carboxy<br />
analogues of arginine methyltransferase inhibitor<br />
1 (AMI-1). ChemMedChem. 2010, 5: 398-414. doi:<br />
10.1002/cmdc.200900459.<br />
Colussi C, Banfi C, Brioschi M, Tremoli E, Straino<br />
S, Spallotta F, Mai A, Rotili D, Capogrossi MC,<br />
Gaetano C. Proteomic profile of differentially<br />
expressed plasma proteins from dystrophic mice and<br />
following suberoylanilide hydroxamic acid treatment.<br />
Proteomics Clin Appl. 2010, 4: 71-83. doi:<br />
10.1002/prca.200900116.<br />
Colussi C, Berni R, Rosati J, Straino S, Vitale S,<br />
Spallotta F, Baruffi S, Bocchi L, Delucchi F, Rossi S,<br />
137<br />
AREA 6<br />
Savi M, Rotili D, Quaini F, Macchi E, Stilli D, Musso<br />
E, Mai A, Gaetano C, Capogrossi MC. The histone<br />
deacetylase inhibitor suberoylanilide hydroxamic<br />
acid reduces cardiac arrhythmias in dystrophic mice.<br />
Cardiovasc Res. 2010, 87: 73-82. doi: 10.1093/cvr/<br />
cvq035.<br />
Colussi C, Illi B, Rosati J, Spallotta F, Farsetti A,<br />
Grasselli A, Mai A, Capogrossi M C, Gaetano C.<br />
Histone deacetylase inhibitors: keeping momentum<br />
for neuromuscular and cardiovascular diseases treatment.<br />
Pharmacol Res. 2010, 62: 3-10. doi: 10.1016/j.<br />
phrs.2010.02.014.<br />
Musmuca I, Caroli A, Mai A, Kaushik-Basu N, Arora<br />
P, Ragno R. Combining 3-D quantitative structureactivity<br />
relationship with ligand based and structure<br />
based alignment procedures for in silico screening of<br />
new hepatitis C virus NS5B polymerase inhibitors.<br />
J Chem Inf Model. 2010, 50: 662-76. doi:10.1021/<br />
ci9004749.<br />
Nebbioso A, Dell’Aversana C, Bugge A, Sarno R,<br />
Valente S, Rotili D, Manzo F, Teti D, Mandrup S,<br />
Ciana P, Maggi A, Mai A, Gronemeyer H, Altucci<br />
L. HDACs class II-selective inhibition alters nuclear<br />
receptor-dependent differentiation. J Mol Endocrinol.<br />
2010, 45: 219-28. doi: 10.1677/JME-10-0043.<br />
Palacios D, Mozzetta C, Consalvi S, Caretti G,<br />
Saccone V, Proserpio V, Marquez V E, Valente S,<br />
Mai A, Forcales S V, Sartorelli V, Puri P L. TNF/<br />
p38α/polycomb signaling to Pax7 locus in satellite<br />
cells links inflammation to the epigenetic control of<br />
muscle regeneration. Cell Stem Cell. 2010, 7: 455-69.<br />
doi: 10.1016/j.stem.2010.08.013.<br />
Pasco M Y, Rotili D, Altucci L, Farina F, Rouleau G<br />
A, Mai A, Néri C. Characterization of sirtuin inhibitors<br />
in nematodes expressing a muscular dystrophy<br />
protein reveals muscle cell and behavioral protection<br />
by specific sirtinol analogues. J Med Chem. 2010, 53:<br />
1407-11. doi: 10.1021/jm9013345.<br />
Quinti L, Chopra V, Rotili D, Valente S, Amore A,<br />
Franci G, Meade S, Valenza M, Altucci L, Maxwell<br />
MM, Cattaneo E, Hersch S, Mai A, Kazantsev A.<br />
Evaluation of histone deacetylases as drug targets<br />
in Huntington’s disease models. Study of HDACs<br />
in brain tissues from R6/2 and CAG140 knock-in<br />
HD mouse models and human patients and in a neuronal<br />
HD cell model. PLoS Curr. 2010, Sep 22. pii:<br />
RRN1172. doi: 10.1371/currents.RRN1172.
Antonello Mai<br />
Rotili D, Tarantino D, Carafa V, Lara E, Meade S,<br />
Botta G, Nebbioso A, Schemies J, Jung M, Kazantsev<br />
AG, Esteller M, Fraga MF, Altucci L, Mai A.<br />
Identification of tri- and tetracyclic pyrimidinediones<br />
as sirtuin inhibitors. ChemMedChem. 2010, 5: 674-77.<br />
doi: 10.1002/cmdc.201000030.<br />
Ruotolo R, Tosi F, Vernarecci S, Ballario P, Mai<br />
A, Filetici P, Ottonello S. Chemogenomic profiling<br />
of the cellular effects associated with histone H3<br />
acetylation impairment by a quinoline-derived compound.<br />
Genomics. 2010, 96: 272-80. doi:10.1016/j.<br />
ygeno.2010.08.005.<br />
Spallotta F, Rosati J, Straino S, Nanni S, Grasselli A,<br />
Ambrosino V, Rotili D, Valente S, Farsetti A, Mai<br />
A, Capogrossi MC, Gaetano C, Illi B. Nitric oxide<br />
determines mesodermic differentiation of mouse<br />
138<br />
embryonic stem cells by activating class IIa histone<br />
deacetylases: potential therapeutic implications in a<br />
mouse model of hindlimb ischemia. Stem Cells. 2010,<br />
28: 431-42 doi: 10.1002/stem.300.<br />
Thaler F, Varasi M, Colombo A, Boggio R, Munari<br />
D, Regalia N, Rozio MG, Reali V, Resconi AE, Mai<br />
A, Gagliardi S, Dondio G, Minucci S, Mercurio C.<br />
Synthesis and biological characterization of amidopropenyl<br />
hydroxamates as HDAC inhibitors<br />
ChemMedChem. 2010, 5: 1359-1372. doi: 10.1002/<br />
cmdc.201000166.<br />
Zhu H, Shan L, Schiller PW, Mai A, Peng T. Histone<br />
deacetylase-3 activation promotes TNF-{alpha}<br />
expression in cardiomyocytes during lipopolysaccharide<br />
stimulation. J Biol Chem. 2010, 285: 9429-9436.<br />
doi: 10.1074/jbc.M109.071274.
Principal investigator: Elena Mattia<br />
Professor in Microbiology<br />
Dipartimento di Scienze di Sanità Pubblica<br />
Tel: (+39) 06 49914608; Fax: (+39) 06 49914626<br />
elena.mattia@uniroma1.it<br />
139<br />
AREA 6<br />
Effects of resveratrol on Epstein-Barr Virus latent and lytic phases<br />
of infection<br />
Participants:<br />
Livia Di Renzo, researcher; Alessandra De Leo, Giulia<br />
Matusali, PhD students; Giuseppe Arena, graduate student;<br />
Egidio Lacanna, Claudia Stecca, Giorgio Oliviero,<br />
undergraduate students.<br />
Collaborations:<br />
Dipartimento di Biologia e Biotecnologie “C. Darwin”, Sapienza-<br />
Università di Roma (Prof. Enzo Marchetti); The Wistar Institute,<br />
Philadelphia, USA (Prof. Paul M. Lieberman).<br />
Report of activity<br />
EBV, the causative agent of infectious mononucleosis,<br />
is associated with a variety of different human<br />
tumors including Burkitt’s lymphoma (BL), nasopharyngeal<br />
carcinoma (NPC), Hodgkin’s desease<br />
(HD) and post transplant lymphoproliferative disorders<br />
(PTLDs) occurring in immunocompromized<br />
patients. In all these malignancies the virus establishes<br />
a latent infection characterized by different<br />
EBV gene expression profiles. Three latency programs<br />
have been described depending on the selective<br />
expression of six nuclear (EBNAs) and three<br />
membrane antigens (LMPs). EBV lytic cycle activation<br />
which results in the production of infectious<br />
particles, is initiated by the two immediate early proteins<br />
BZLF1 (ZEBRA or Zta) and BRLF1 (Rta) that<br />
function as transcriptional activators of EBV early<br />
genes. In vitro, latency can be disrupted by a variety<br />
of different agents such as phorbol esters, sodium<br />
butyrate, TGFβ, anti-immunoglobulins (anti-IgG)<br />
and calcium ionophores.<br />
Resveratrol (3,4’,5-trihidroxystilbene; RV), a polyphenolic<br />
phytoalexin has been <strong>report</strong>ed to have cardioprotective,<br />
anti-oxidant, anti inflammatory, antibacterial<br />
and anti-viral activity. In addition, RV was<br />
found to inhibit proliferation and induce apoptosis in<br />
several human tumor cells.<br />
Because EBV infection might confer additional sur-<br />
vival potential to malignant B cells, in this study, we<br />
have examined the antiproliferative activity of RV on<br />
BLs with different forms of restricted virus latency.<br />
Thus, in vitro systems enforcing the full Latency III,<br />
Latency II-like and Latency I program in a BL cell<br />
background, we have investigated the contribution<br />
that different patterns of EBV gene expression give<br />
to RV-induced susceptibility to apoptosis and studied<br />
the molecular mechanisms underneath. In addition,<br />
the antiviral effects of resveratrol on viral replication<br />
have been studied. EBV lytic cascate was activated<br />
in Raji cells by introduction of the BZLF1 expression<br />
vector while in Akata cells EBV lytic cycle was<br />
induced by cross-linking surface immunoglobulin G<br />
(IgG) with anti-Ig , a method which mimics in vivo<br />
activation of the B-cell receptor.<br />
Resveratrol and EBV latent infection<br />
As we aimed to determine whether EBV gene expression<br />
played a role on the susceptibility of BL cells to<br />
RV, we tested two EBV (-) (2A8 and Ramos) and<br />
three EBV(+) BL cell lines, the latter expressing different<br />
patterns of EBV latency genes. In particular,<br />
Raji cells express all EBV latency genes (latency III),<br />
Jijoye M13, a subline of Jijoye cells which is classified<br />
as group II-like do not express LMP1, and Akata<br />
cells show a Latency I phenotype. All cell lines were<br />
exposed to concentrations of the polyphenol varying<br />
from 20 to 300 µM for different periods of time.<br />
We observed that that RV inhibited proliferation of<br />
each cell line in a dose and time-dependent manner.<br />
To investigate the antiproliferative effect of RV on<br />
BL cells, we analyzed the cell cycle distribution after<br />
incubation with the polyphenol and found that a 12<br />
hours treatment with RV increased significantly the<br />
percentage of cells in the G1 phase with a reduction<br />
of those in S and G2/M phases. Cell cycle arrest<br />
by RV treatment was associated with apoptosis as<br />
revealed by Annexin V binding and PARP cleavage<br />
tests. However, our data indicated that EBV(-)<br />
BL cells and latency I Akata were more prone to
Elena Mattia<br />
RV-induced apoptosis than Latency II and Latency<br />
III cell lines. We then examined the expression of<br />
several cell cycle and apoptosis-related proteins by<br />
Western blot analysis. Exposure to RV increased<br />
the expression of the cyclin dependent kinase (cdk)<br />
inhibitor p27 and that of cyclin E in all cell lines<br />
while the analysis of the levels of antiapoptotic proteins<br />
revealed a strong down-regulation of the c-myc<br />
oncogene but also that of IAP1, FLIP and XIAP.<br />
Furthermore, RV treatment of BL cell lines strongly<br />
activated p38 and dramatically inhibited ERK1/2,<br />
two MAPKs pathways important in determining<br />
cell proliferation or apoptosis; in addition, it caused<br />
a marked inhibition of NFkB activity in Latency I<br />
and Latency II cell lines but not in Latency III cells.<br />
Therefore, we looked for EBV genes expressed in<br />
the Latency III program that would confer additional<br />
survival advantage against the effects of the<br />
polyphenol. LMP1 is the principal EBV oncogene<br />
with the potential to antagonize apoptosis as well as<br />
to promote cellular survival and proliferation. We<br />
demonstrated that silencing LMP1gene by siRNA<br />
dramatically increased the sensitivity of Latency III<br />
Raji cells to RV.<br />
Resveratrol and EBV lytic infection<br />
Raji cells were electroporated with BZLF1 plasmid<br />
to induce EBV lytic cycle and exposed to RV at concentrations<br />
of 130 or 300 mM for 24 and 48 hours<br />
to evaluate the expression of the immediate early<br />
antigen (IEA) BZLF1 as well as that of different<br />
EBV early genes expressed in the lytic cycle. We<br />
found that at the lower concentration of RV, BZLF1<br />
expression was stimulated while it was inhibited at<br />
the higher one. Nevertheless, both RV concentrations<br />
were effective in suppressing the expression<br />
of several EBV early antigen (EA) examined, such<br />
as BFRF1, BFLF2 and BALF5. Interestingly, RV<br />
inhibited EBV EA expression also when added 4<br />
hours after EBV activation, confirming that the<br />
polyphenol is able to block lytic gene cascade also<br />
downstream of IEA expression.<br />
The inhibition of EBV lytic cycle in Akata cells<br />
was concentration dependent and RV, at 130 mM,<br />
completely suppressed EBV activation as judged<br />
by immunological detection of IEA and EA expression.<br />
Analysis of the corresponding transcripts by<br />
RT-PCR revealed that RV down-regulated EBV<br />
lytic gene expression at the transcriptional level.<br />
Our study also demonstrated that RV inhibition of<br />
EBV lytic cycle, actually decreased the production<br />
140<br />
of mature viral particles. We examined the activity<br />
of transcription factors involved in regulating EBV<br />
gene expression. The results obtained by EMSA<br />
carried out on Akata cells treated with IgG, revealed<br />
that RV negatively affects the binding activity of<br />
NFkB, SP1 and AP1. Moreover, evaluation of the<br />
levels of reactive oxygen species (ROS), <strong>report</strong>ed to<br />
increase during EBV lytic cycle, revealed a drastic<br />
decrement indicating a rapid intracellular uptake of<br />
the compound and a strong reducing activity. The<br />
decrement of ROS appears to be largely responsible<br />
for RV-mediated inhibition of EBV lytic cycle.<br />
Conclusions<br />
The results of this research project show that RV<br />
induces cell cycle arrest and apoptosis of EBVinfected<br />
B cells; however, each form of EBV infection<br />
is associated with a specific degree of protection from<br />
apoptosis, more extended as a larger set of viral genes<br />
is expressed. Moreover, the finding that silencing the<br />
viral oncogene LMP1 decreased the protection of<br />
Latency III cells to RV-induced apoptosis suggests<br />
that the combined use of RV and siRNA directed to<br />
viral genes may configure a novel therapy for the<br />
treatment of EBV-positive B cell malignancies with<br />
a known pattern of viral gene expression.<br />
We found that RV inhibits the transcription of EBV<br />
immediate early and early genes, the expression of<br />
the viral lytic proteins and decreases the production<br />
of viral particles.<br />
The molecular mechanisms underlying RV inhibition<br />
of EBV lytic cycle include a significant reduction<br />
in the activity of transcription factors as well as<br />
redox-dependent epigenetic events involved in the<br />
activation of viral lytic genes expression.<br />
Publications<br />
Matusali G, Arena G, De Leo A, Di Renzo L, Mattia<br />
E. Inhibition of p38 MAP kinase pathway induces<br />
apoptosis and prevents Epstein Barr virus reactivation<br />
in Raji cells exposed to lytic cycle inducing compounds.<br />
Mol Cancer. 2009, 8: 18. doi: 10.1186/1476-<br />
4598-8-18.<br />
De Leo A, Matusali G, Arena G, Di Renzo L,<br />
Mattia E. Epstein-Barr virus lytic cycle activation<br />
alters proteasome subunit expression in Burkitt’s<br />
lymphoma cells. Biol Chem. 2010, 391: 1041-46. doi:<br />
10.1515/BC.2010.107.
Principal investigator: Romano Silvestri<br />
Professor of Medicinal Chemistry<br />
Dipartimento di Chimica e Tecnologie del Farmaco<br />
Tel: (+39) 06 49913800; Fax: (+39) 06 491491<br />
romano.silvestri@uniroma1.it<br />
141<br />
AREA 6<br />
Drug design and synthesis of non-nucleoside inhibitors of both<br />
HIV-1 wild type and resistant mutant strains reverse transcriptase,<br />
and Coxsackie B4 virus<br />
Participants:<br />
Giuseppe La Regina, researcher; Antonio Coluccia,<br />
Francesco Piscitelli, post-doc fellows; Valerio Gatti, PhD<br />
student.<br />
Collaborations:<br />
CNR, <strong>Istituto</strong> di Genetica Molecolare, Pavia (Dr. Giovanni<br />
Maga); Rega Institute for Medical Research, K.U., Leuven,<br />
Belgium (Dr. Jean Balzarini); Welsh School of Pharmacy,<br />
Cardiff University, UK (Dr Andrea Brancale); IrsiCaixa, Hospital<br />
Germans Trias i Pujol, Badalona, Spain (Prof. José A. Esté).<br />
Report of activity<br />
Human immunodeficiency virus (HIV) was identified<br />
as the causative agent of acquired immunodeficiency<br />
syndrome (AIDS) in 1983. Although significant progress<br />
has been made in the prevention and treatment<br />
of AIDS/HIV infection over the past twenty years,<br />
the number of people living with HIV is constantly<br />
growing. Worldwide, AIDS-related diseases are still<br />
one of the leading causes of death, and are predicted<br />
to cause significantly premature mortality in the<br />
coming decades. Highly active antiretroviral therapy<br />
(HAART) which combines three (recommended) or<br />
four different antiretroviral drugs, can slow down<br />
viral replication and reduce plasma viremia below<br />
the detection level, resulting in a substantial delay<br />
of disease progression. However, long-term HAART<br />
treatments lead to the emergence of complications<br />
due to drug resistance, toxicity and adverse effects.<br />
Therefore, new NNRTIs which display a broad spectrum<br />
of activity against clinically relevant HIV-1<br />
mutant strains are needed.<br />
Our research activity has been focused towards<br />
the development of indolylarylsulfone (IAS) HIV-1<br />
NNRTIs. Structure-activity relationship (SAR)<br />
studies led the identification of three regions of the<br />
IAS scaffold: (A) the spectrum of activity against<br />
mutant HIV-1 strains was significantly expanded by<br />
introduction of two methyl groups at positions 3’ and<br />
5’ of the 3-phenylsulfonyl moiety; (B) potent HIV-1<br />
inhibitors were obtained by coupling the indole-<br />
2-carboxamide with either natural or unnatural<br />
amino acids; (C) IASs bearing the 5-chloro-4-fluoro<br />
substitution pattern at the indole ring were potent<br />
inhibitors of RT WT and RTs carrying the K103N,<br />
Y181I, and L100I mutations.<br />
We synthesized new IASs characterized by (a) natural<br />
and unnatural amino acids at the 2-carboxamide<br />
and (b) different electron-withdrawing substituents<br />
at the positions 4 and 5 of the indole. These compounds<br />
proved to be highly potent against HIV-1<br />
replication in human T-lymphocyte (CEM) cells and<br />
showed inhibitory potencies in the low/subnanomolar<br />
range of concentrations that were comparable<br />
with those of the previously <strong>report</strong>ed lead compounds.<br />
Against HIV-1 WT, the inhibitory activity<br />
seemed only marginally affected by the substituent<br />
introduced on the indole nucleus. In general, none of<br />
the IASs proved to be markedly cytostatic. Against<br />
the L100I and K103N RT HIV-1 strains, five IASs<br />
were always superior to EFV and had similar activity<br />
to that <strong>report</strong>ed for MK-49652 and TMC-120. These<br />
IASs were also equipotent to EFV against the mutant<br />
Y181C RT HIV-1 strain. Several compounds were<br />
inhibitory to Coxsackie B4 virus at EC50 = 3-5 μM<br />
but were much less selective in their anti-Coxsackie<br />
virus activity than observed for HIV. In view that<br />
for picornaviruses, no single antiviral drug has ever<br />
been approved, these agents may serve as basis for<br />
the development of drugs endowed with both anti-<br />
HIV-1 RT and Coxsackie B4 virus inhibitory activities.<br />
In L100I mutated RT, the mutation of leucine<br />
to isoleucine does not affect the binding mode, and<br />
it was consistent with the interactions <strong>report</strong>ed for<br />
the WT strain. The lower antiviral activity against<br />
the mutant K103N and Y181I RT HIV-1 strains<br />
could be the consequence of the reduced interaction<br />
with the mutated residues. For the Y181I RT HIV-1<br />
strain, the limited anti-HIV efficiency could also be
Romano Silvestri<br />
caused by the loss of the favorable protein-ligand π-π<br />
interactions, while in the K103N mutant, the loss of<br />
the hydrophobic interaction between the ligand and<br />
the side chain of the lysine 103 might be responsible<br />
for the reduced activity.<br />
The di-halo-IAS 5-chloro-3-[(3,5-dimethylphenyl)<br />
sulfonyl]-4-fluoro-1H-indole-2-carboxamide showed<br />
improved activity towards the Y181I mutant enzyme<br />
and comparable activity towards the K103N mutant<br />
RT, in comparison with the previously characterized<br />
IAS derivatives, which preferentially associated with<br />
the free enzymatic form of HIV-1 RT. In addition,<br />
this compound showed much slower dissociation rates<br />
from both mutant enzymes with respect to HIV-1 RT<br />
WT and showed a superior activity profile towards<br />
the K103N, L100I and Y181I mutants with respect to<br />
the reference compounds NVP and EFV. The results<br />
obtained by this study, as well as the excellent activity<br />
demonstrated by these inhibitors in blocking the viral<br />
replication in infected cells, suggest a possible use of<br />
these compounds for the therapy of HIV.<br />
Second generation NNRTIs currently under development<br />
are potent inhibitors of HIV-1 WT and the<br />
most clinically relevant HIV-1 mutant strains, and<br />
show inhibitory activities in the nano- or subnanomolar<br />
range of concentration. Despite all compounds<br />
are endowed with potent and (more or less)<br />
similar antiretroviral potency, they do not share a<br />
common binding mode to the NNBS of the RT. In the<br />
past five years, NNRTIs based on the “butterfly-like”<br />
model seemed obsolete with respect to the newer<br />
“horseshoe-like” inhibitors. However, new drug candidates<br />
renewed the interest for the “butterfly-like”<br />
active conformation. In addition, a new concept of<br />
binding interaction with the HIV-1 drug-resistant<br />
mutant strains emerged: the “flexibility hypothesis”.<br />
According to this idea, a powerful inhibition of the<br />
142<br />
mutant variants of the HIV-1 should be correlated<br />
to the ability of the molecule to adopt variable binding<br />
conformations which would be not affected by<br />
the interchanges of the amino acids residues into the<br />
NNBS of the mutant RT. These findings support<br />
the opinion that a newer era for the HIV-1 NNRTIs<br />
is just begun. Future NNRTIs should be capable to<br />
adopt multi binding conformations for a powerful<br />
inhibition of HIV-1 WT and the most relevant drug<br />
resistant mutant strains.<br />
Publications<br />
Piscitelli F, Coluccia A, Brancale A, La Regina G,<br />
Sansone A, Giordano C, Balzarini J, Maga G, Zanoli<br />
Z, Samuele A, Cirilli R, La Torre F, Lavecchia A,<br />
Novellino E, Silvestri R. Indolyl aryl sulfones bearing<br />
natural and unnatural aminoacids. Discovery<br />
of potent inhibitors of both HIV-1 non-nucleoside<br />
wild type and resistant mutant strains reverse transcriptase,<br />
and coxsackie B4 virus. J Med Chem. 2009,<br />
52: 1922-34. doi: 10.1021/jm801470b.<br />
Samuele A, Kataropoulou A, Viola M, Zanolia S, La<br />
Regina G, Piscitelli F, Silvestri R, Maga G. Nonnucleoside<br />
HIV-1 reverse transcriptase inhibitors<br />
di-halo-indolyl aryl sulfones achieve tight binding<br />
to drug-resistant mutants by targeting the enzymesubstrate<br />
complex. Antivir Res. 2009, 81: 47-55. doi:<br />
10.1016/j.antiviral.2008.09.008.<br />
La Regina G, Coluccia A, Silvestri R. Looking for an<br />
active conformation of the future HIV-1 non-nucleoside<br />
reverse transcriptase inhibitors. Antiviral Chem<br />
Chemoth. 2010, 20: 231-7. doi: 10.3851/IMP1607.
AREA 7<br />
Biology of<br />
malaria<br />
and other<br />
vector-borne<br />
diseases
Genetic and phenotypic characterization of species and<br />
“molecular forms” of the Anopheles gambiae complex (Diptera:<br />
Culicidae), afrotropical malaria vectors<br />
Principal investigators: Alessandra della torre - Vincenzo Petrarca<br />
Professors of Parasitology<br />
Dipartimento di Sanità Pubblica e Malattie Infettive - Dipartimento di Biologia e<br />
Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49694268, 06 49914932; Fax: (+39 ) 06 49914653<br />
ale.dellatorre@uniroma1.it - vincenzo.petrarca@uniroma1.it<br />
Participants:<br />
Marco Pombi, researcher, Beniamino Caputo, Emiliano<br />
Mancini, Federica Santolamazza, post-doc fellows; Federica<br />
Tammaro, PhD student; Maria Calzetta, graduate student.<br />
Collaborations:<br />
Center for Global Health and Infectious Diseases, Department of<br />
Biological Sciences, University of Notre Dame, IN, USA (Prof. Nora<br />
J. Besansky); Centro de Malária e outras Doenças Tropicais,<br />
Instituto de Higiene e Medicina Tropical, Lisbon, Portugal (Dr.<br />
João Pinto); Dipartimento di Biologia Animale e Genetica,<br />
Università di Firenze (Dr. Francesca Dani, Prof. Stefano<br />
Turillazzi); Institut de Recherche pour le Développement, (France:<br />
Prof. Didier Fontenille; Burkina Faso: Dr. Frédéric Simard;<br />
Cameroun: Dr. Carlo Costantini); Medical Research Council<br />
Laboratories, Fajara, Banjul, The Gambia (Prof. David Conway,<br />
Dr. Davis Nwakanma); Division of Cell and Molecular Biology,<br />
Imperial College London, UK (Prof. F. Catteruccia); Department<br />
of Entomology, Virginia Tech, Blacksburg, VA, USA (Prof. I.<br />
Sharakhov); Lab. Réponse Immunitaire et Développement Chez<br />
les Insectes, CNRS, Strasburg, France (Prof. EA. Levashina);<br />
Vector Group, Liverpool School of Tropical Medicine, Liverpool,<br />
UK (Dr. H. Ranson, Dr. MJ. Donnelly); IRSS/Centre Muraz,<br />
Laboratoire de Parasitologie/Entomologie, Bobo-Dioulasso,<br />
Burkina Faso (Dr. Roch Dabiré); Dipartimento di Biologia<br />
delle Piante Agrarie, Università di Pisa, (Prof. P. Pelosi, Dr.<br />
A. Felicioli); Dipartimento di Medicina Sperimentale e Scienze<br />
Biochimiche, Università di Perugia, Terni (Prof. F. Catteruccia.<br />
Dr. F. Baldini); Dipartimento di Scienze Biochimiche, Sapienza-<br />
Università di Roma, Rome, (Prof. A. Tramontano, Dr. D.<br />
Raimondo, Dr. A. Via).<br />
Report of Activity<br />
Analysis of the genetic and ecological<br />
differentiation of Anopheles gambiae s.s. M<br />
and S molecular forms<br />
Molecular genotyping of M and S forms in area of high<br />
hybridization at the western extreme of their geographical<br />
145<br />
AREA 7<br />
range. The physically unlinked centromeric regions<br />
on all 3 chromosomes of M and S molecular forms<br />
contain fixed nucleotide differences which have been<br />
found in nearly complete linkage disequilibrium in<br />
geographic areas of no or low M-S hybridization.<br />
We applied recently developed diagnostic assays<br />
for detection of SNPs and structural differences<br />
between M and S forms in the 3 centromeric regions<br />
in samples from the western extreme of their range<br />
of sympatry, the only area where high frequencies<br />
of putative M/S hybrids have been <strong>report</strong>ed. The<br />
results revealed a level of admixture not observed in<br />
the rest of the range. In particular, we found: i) heterozygous<br />
genotypes at each marker at frequencies<br />
lower than expected under panmixia; ii) virtually all<br />
possible genotypic combinations between markers<br />
on different chromosomes, although genetic association<br />
was nevertheless detected; iii) discordant M and<br />
S genotypes at two X-linked markers near the centromere,<br />
suggestive of introgression and inter-locus<br />
recombination. These results suggest a secondary<br />
contact zone between M and S, although it is not<br />
possible to completely rule out the hypothesis of<br />
maintenance of ancestral polymorphisms. Overall,<br />
the above findings highlight the complexity and the<br />
variability of the biological and genetic differentiation<br />
of these incipient species. In order to get a more<br />
complete picture of this speciation model, we collected<br />
samples from a west to east transect in Guinea<br />
Bissau in October 2010 which are being currently<br />
identified and genotyped.<br />
Chromosomal characterization of M and S forms in area<br />
of high hybridization at the western extreme of their<br />
geographical range. Floating paracentric inversions<br />
on chromosome-2, probably involved in adaptation<br />
to sub-niches, are shared by the M and S forms,<br />
although with different frequencies of alternative<br />
inverted arrangements. Little is currently known<br />
about the distribution and chromosomal inversion<br />
patterns of molecular forms in the western limits of
Alessandra della Torre – Vincenzo Petrarca<br />
their geographical range. We completed the karyotyping<br />
of A. gambiae s.s. collected in The Gambia in<br />
2005-06, finding that the M form is mostly characterized<br />
by frequencies of 2Rb, 2Rd, 2La inversions<br />
increasing from 40% to 80% from the coast eastwards.<br />
The S form coastal populations are characterized<br />
by karyotypes similar to those of M, while the<br />
eastward populations by more complex arrangements<br />
also based on inversions 2Rj and 2Rbk. These results<br />
are currently being correlated with those from an<br />
extensive microsatellite analysis. Additionally, the M<br />
and S samples collected in October in Guinea Bissau<br />
are currently being identified and karyotyped.<br />
Analysis of genetic/phenotypic differentiation<br />
involved in pre/post-mating behaviour in the<br />
Anopheles gambiae complex<br />
Analysis of A. gambiae antennal proteins. Odourant<br />
Binding Proteins (OBPs) and Chemo-Sensory<br />
Proteins (CSPs) are proteins involved in the detection<br />
of odour molecules and in the activation of odorant<br />
receptors. Based on the published genome of A.<br />
gambiae, 57 genes encoding for putative OBPs and 7<br />
encoding for CSPs have been identified. So far, the<br />
expression pattern of these genes has been almost<br />
exclusively investigated by genomic approaches:<br />
some OBPs have been found ubiquitously, while<br />
others have been shown to be specific of the olfactory<br />
organs. The later genes are likely to encode<br />
for OBPs relevant to olfactory processes involved<br />
in behaviour, such as the mating partner choice or<br />
the search for sugar-sources and, for females, the<br />
search and selection of hosts for blood-meal or<br />
of appropriate oviposition sites. Traditional proteomic<br />
approaches have been proved difficult to<br />
be performed on mosquito antennae, due to low<br />
concentration of OBPs and CSPs, and difficulties in<br />
obtaining sufficient material for 2-dimensional gels.<br />
We have exploited an innovative shotgun proteomic<br />
approach to analyse OBPs and CSP expression in A.<br />
gambiae antennae. The sensitivity and efficiency of<br />
this approach is significantly higher than that based<br />
on the traditional 2-dimensional gel analysis: we<br />
identified 2.390 proteins in male and female antennal<br />
samples, 28 of which were olfactory soluble proteins,<br />
and showed that some OBPs are significantly more<br />
expressed in females than in males.<br />
Moreover, to clarify the role of some OBPs in A.<br />
gambiae, we have expressed six of these proteins<br />
146<br />
and investigated their ligand-binding properties.<br />
Ligand-binding experiments revealed marked differences<br />
between these OBPs. In situ hybridisation has<br />
revealed that some OBPs are co-expressed in antennal<br />
sensilla of A. gambiae, suggesting that OBPs also<br />
in mosquitoes might form complexes with novel<br />
ligand specificities, thus amplifying the repertoire of<br />
OBPs and the number of semiochemicals that could<br />
be discriminated.<br />
Analysis of differentiation of genes involved in postmating<br />
behaviour in the An. gambiae complex. In A.<br />
gambiae male accessory glands (MAG) products are<br />
transferred to females upon mating as a mating plug<br />
that induces a series of physiological post-mating<br />
responses in the female lower reproductive tract<br />
(LRT). We analysed the molecular evolution of<br />
two clusters of 3 LRT- and 3 MAG- specific genes<br />
potentially involved in post-mating mechanisms in 5<br />
species of the A. gambiae complex.<br />
MAG-specific paralog genes Agap009369 and<br />
Agap009370 were shown to be highly conserved<br />
among those 5 species. Moreover, we identified at<br />
3rd gene, clustering with the former two, which is<br />
not annotated in the A. gambiae genome: intriguingly,<br />
this paralog gene shows signs of positive<br />
selective pressure and purifying selection maintaining<br />
lineage-specific products.<br />
The 3 LRT-specific genes analyzed encode serineproteases<br />
that are down-regulated after mating, 2<br />
of which are expressed in the atrium and interact<br />
with the mating plug and 1 in the spermatheca. The<br />
analysis of polymorphisms revealed a high level of<br />
replacement polymorphisms consistent with relaxed<br />
evolutionary constraints of duplicated genes, allowing<br />
to rapidly fix novel replacements to perform new<br />
or more specific functions. Adaptive evolution was<br />
detected in several codons of the 3 genes and evidence<br />
of episodic selection were also found. In addition,<br />
the structural modeling of these proteases highlighted<br />
some important differences in their substrate<br />
specificity, and provided evidence that a number of<br />
sites evolving under selective pressures lie relatively<br />
close to the catalytic triad and/or on the edge of the<br />
specificity pocket, known to be involved in substrate<br />
recognition or binding. The observed patterns suggest<br />
that these proteases may interact with factors<br />
transferred by males during mating and that differently<br />
evolved in independent A. gambiae lineages.
Immune responses to malaria and autoimmune disorders:<br />
investigating common gene-regulatory networks<br />
Principal investigators: David Modiano<br />
Professor of Parasitology<br />
Dipartimento di Sanità Pubblica e Malattie Infettive<br />
Tel: (+39) 06 49914933, 06 33775629; Fax: (+39 ) 06 49914653<br />
david.modiano@uniroma1.it<br />
Participants:<br />
Valentina Mangano, Pamela Avellino, Cinzia Rizzo, postdoc<br />
fellows.<br />
Collaborations:<br />
Wellcome Trust Center for Human Genetics, Oxford, UK (Dr.<br />
Dominic Kwiatkowski); London School of Tropical Medicine<br />
and Hygiene, London, UK (Dr. Eleanor Riley); Centre National de<br />
Recherche et Formation sur le Paludisme, Ouagadougou, Burkina<br />
Faso (Dr. Bienvenu Sodiomon Sirima).<br />
Report of Activity<br />
Background<br />
The Fulani of West Africa have been shown to<br />
mount stronger immune responses to P. falciparum<br />
antigens and to be less susceptible to infection and<br />
mild disease than sympatric populations (Modiano<br />
et al., 1996). The Fulani also show a higher response<br />
to other pathogens, and both their Th1 and Th2<br />
responses are enhanced, suggesting that their resistance<br />
to malaria could result from a generally stronger<br />
immune activation. Indeed key genes related to T<br />
regulatory cell function are indeed down-regulated<br />
in the Fulani (Torcia et al., 2008). This disorder of<br />
immune homeostasis could be driven by genetic<br />
factors positively selected by P. falciparum and may<br />
underlie the higher susceptibility of the Fulani to<br />
diseases with autoimmune pathogenesis <strong>report</strong>ed in<br />
the literature. To investigate this hypothesis, we are<br />
conducting a large-scale epidemiological and immunogenetic<br />
study in Burkina Faso supported also by<br />
the Malaria Genomic Epidemiology Network (2008).<br />
Serological analyses<br />
Anti-Nuclear Antibodies (ANA) are markers of systemic<br />
autoimmune diseases. Their prevalence in the<br />
European population is about 5% while it ranges<br />
from 30 to 100% in European patients with systemic<br />
147<br />
AREA 7<br />
autoimmune diseases. Little is known on the prevalence<br />
of ANAs in African populations. We measured<br />
IgG against a combination of 11 self nuclear antigens<br />
(dsDNA, U1-snRNP, Sm, SS-A/Ro, SS-B/La, Scl-<br />
70, CENP, Jo-1, Histone, PM-Scl, Rib-P) in plasma<br />
samples including 474 Fulani and 900 Non Fulani<br />
using a commercial ELISA Kit (Varelisa, Phadia).<br />
We then validated the results by IFAT. We found<br />
a higher prevalence of ANA in Fulani (63%) than<br />
Non Fulani (54%) (P=0.002). These values are much<br />
higher than that of European controls (6%) and suggest<br />
that the response to self nuclear antigens could<br />
be highly enhanced in malaria endemic countries,<br />
for reasons yet to be investigated. Furthermore, this<br />
response is higher in the population with the lower<br />
susceptibility to malaria. The levels of ANA were<br />
also higher in Fulani than Non Fulani (P=0.001).<br />
In the same samples, in order to confirm previous<br />
observations, we measured IgG against four P. falciparum<br />
antigens (CSP, MSP1, MSP2, AMA1) by ELISA.<br />
As expected we observed higher prevalences and levels<br />
in Fulani than Non-Fulani (P
David Modiano<br />
ated with protection from infection and/or clinical<br />
malaria in both populations. We compared<br />
the Minor Allele Frequency (MAF) of 168 Single<br />
Nucleotide Polymorphisms (SNPs) - genotyped by<br />
the MalariaGEN Resource Centre in 2225 DNA<br />
samples - between Fulani and Non Fulani. For all<br />
SNPs showing a significant difference in allele frequency<br />
between the two populations (62 SNPs after<br />
Bonferroni correction) we performed the following<br />
allele-based (minor vs major allele) association analyses<br />
at the intra-population level: association with P.<br />
falciparum infection (yes/no) in 5 cross-sectional surveys<br />
in all age groups (Mantel-Haenszel Chi Square<br />
test stratified by survey); association with clinical<br />
malaria (T>37.5 and P. falciparum infection, yes/no)<br />
in 2 longitudinal surveys in the 0-5 years old age<br />
group (Mantel-Haenszel Chi Square test stratified<br />
by survey). We found no SNPs associated with either<br />
infection or clinical malaria in both populations.<br />
We then selected 60 loci with a <strong>report</strong>ed role in<br />
autoimmunity for genetic association analysis with<br />
humoral response and susceptibility to malaria, as<br />
well as with immunological markers of autoimmune<br />
diseases. The following selection criteria were used:<br />
immune system function; association with 1 or more<br />
autoimmune diseases in GWAS or in meta-analysis<br />
based on at least 5 papers (HuGE Navigator v3.1, Yu<br />
et al., 2008); differential expression in Treg cells of<br />
Fulani vs Mossi (Torcia et al., 2008). Polymorphisms<br />
for each gene (around 120 SNPs in total) have been<br />
selected based on their functional consequences<br />
(e.g. non-synonymous coding, transcription factor<br />
binding site, splicing site) and on their MAF in the<br />
Yoruba population from Nigeria (HapMap data).<br />
148<br />
These SNPs are currently being genotyped in collaboration<br />
with the WTCHG in Oxford.<br />
References<br />
- Modiano et al., Different response to Plasmodium<br />
falciparum malaria in West African sympatric ethnic<br />
groups. PNAS USA 1996, 93: 13206-13211.<br />
- Torcia et al., Functional deficit of T regulatory<br />
cells in Fulani, an ethnic group with low susceptibility<br />
to Plasmodium falciparum malaria. PNAS<br />
USA 2008, 105: 646-51.<br />
- Malaria Genomic Epidemiology Network,<br />
-<br />
Genome-wide and fine-resolution association analysis<br />
of malaria in West Africa. Nature Genetics 2009.<br />
Malaria Genomic Epidemiology Network. A global<br />
network for investigating the genomic epidemiology<br />
of malaria. Nature 2008; 456: 732-7.<br />
- Yu et al., A Navigator for Human Genome<br />
Epidemiology. Nature Genetics 2008, 40: 124-5.<br />
Publications<br />
Lulli P, Mangano VD, Onori A, Batini C, Luoni G,<br />
Sirima BS, Nebie I, Chessa L, Petrarca V, Modiano D.<br />
HLA-DRB1 and -DQB1 loci in three west African ethnic<br />
groups: genetic relationship with sub-Saharan African<br />
and European populations. Hum Immunol 2009, 42:<br />
903-9. ISSN: doi: 10.1016/j.humimm.2009.07.025.<br />
Gouagna LC, Bancone G, Yao F, Yameogo B,<br />
Dabiré KR, Costantini C, Simpore J, Ouedraogo JB,<br />
Modiano D. Genetic variation in human HBB is associated<br />
with Plasmodium falciparum transmission. Nat<br />
Genet. 2010, 42: 328-31. doi: 10.1038/ng.554.
Computational Analysis of the gene products of the Plasmodium<br />
falciparum genome and their interaction with human proteins<br />
Principal investigator: Anna tramontano<br />
Professor of Biochemistry<br />
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”<br />
Tel: (+39) 06 49910556; Fax: (+39) 06 49910717<br />
anna.tramontano@uniroma1.it<br />
Participants:<br />
Paolo Marcatili, Domenico Raimondo, Allegra Via, Mauro<br />
Chinappi, researchers; Alba Lepore, Le Pera Loredana, PhD<br />
students.<br />
Collaborations:<br />
<strong>Istituto</strong> Superiore di Sanità, Roma (Dr. Elisabetta Pizzi, Dr.<br />
Pietro Alano); Universita’ di Siena (Prof. Campiani), CRS4<br />
(Dr. Matteo Floris, Dr. Massimiliano Orsini), University<br />
of California Davis, USA (Dr. Krzysztof Fidelis, Dr. Andriy<br />
Kryshtafovych), CARB (Dr. John Moult), The Hebrew Univeristy<br />
of Jerusalem, Israel (Dr. Yaniv Lowenstein, Dr. Michal Linial),<br />
University College of London, UK (Dr. Christine Orengo), EBI<br />
(Dr. James Watson, Dr. Janet Thornton), Helmholtz Center,<br />
Munchen, Germany (Dr. Dmitrij Frishman).<br />
Report of activity<br />
This project aimed at exploiting the knowledge of<br />
the genomes and proteomes of Plasmodium, the etiological<br />
cause of the deadly parasitic malaria disease<br />
to understand the structure and function of their<br />
proteins, in particular those involved in host pathogen<br />
interaction and in the development of resistance.<br />
We focused on three parasite proteins, PfEMP1,<br />
EBA-175 and PfCRT. The first two interact with the<br />
human proteins ICAM-1 and Glycophorin A (GPA),<br />
respectively. The third is involved in resistance to<br />
chloroquine. They represent different challenges of<br />
increasing difficulty for computational methods as<br />
discussed below.<br />
The strategy we used in the first case (complex<br />
between PfEMP1 and ICAM-1) was to model the<br />
interacting plasmodium protein by comparative modelling<br />
techniques (i.e. using as first approximation the<br />
structure of an evolutionary related protein) and to<br />
use docking methods to predict the orientation of the<br />
protein in its complex with ICAM-1 (whose structure<br />
is experimentally known). The result was very convincing<br />
and could explain a number of experimental<br />
149<br />
AREA 7<br />
observations, which convinced us that we could use<br />
the model of the complex to identify regions of the<br />
human protein that could be mimicked by peptides in<br />
order to interfere with the interaction.<br />
The second case (complex between EBA-175 and<br />
GPA) was much more challenging for at least two<br />
reasons. We could not model the structure of GPA<br />
(and also of the related GPB protein known not to<br />
interact with EBA-175) by comparative modelling<br />
and had to recur to the more computer expensive<br />
and somewhat less reliable fragment based methods.<br />
Once again, however, the model was very convincing<br />
because it would fit with known experimental<br />
data. However, the protein is heavily glycosylated<br />
and sugar moieties are known to be involved in its<br />
interaction with the Plasmodium protein. Because of<br />
their high flexibility, modelling of the structure of<br />
sugars is an unsolved problem and we are developing<br />
methods to do so in order to be able to obtain the<br />
model of the complex.<br />
The third case is also very challenging since the<br />
involved protein (PfCRT) is a transmembrane protein<br />
and, because of the paucity of known structures<br />
of transmembrane proteins, their prediction is still<br />
very difficult. Our main question in the case of PfCRT<br />
regarded the details of the function of the protein.<br />
In its erythrocyte stage, P. falciparum invades the<br />
red blood cells where it forms a lysosomal isolated<br />
acidic compartment known as the digestive vacuole.<br />
Chloroquine is a diprotic weak base and, at physiological<br />
pH (~7.4), can be found in its un-protonated,<br />
mono-protonated and di-protonated forms.<br />
The uncharged chloroquine is the only membrane<br />
permeable form of the molecule and it freely diffuses<br />
into the erythrocyte up to the vacuole. In this compartment,<br />
chloroquine molecules become protonated<br />
and, since membranes are not permeable to charged<br />
species, the drug accumulates into the acidic digestive<br />
vacuole where it is believed to bind haematin, a<br />
toxic byproduct of the haemoglobin proteolysis, preventing<br />
its incorporation into the haemozoin crystal
Anna Tramontano<br />
and thereby causing damage to the Plasmodium<br />
membranes. Chloroquine sensitive parasites (CQS)<br />
accumulate much more chloroquine in the vacuole<br />
than chloroquine resistant strains (CQR) and this<br />
has been associated with point mutations in the<br />
gene encoding for the transmembrane P. falciparum<br />
chloroquine resistance transporter (PfCRT) protein.<br />
There are two alternative models for the function<br />
of PfCRT: 1) the channel model (i.e. a passive channel<br />
that enables charged chloroquine to leak out of<br />
the food vacuole down its electrochemical gradient)<br />
or 2) the carrier model (i.e. an active efflux carrier<br />
extruding chloroquine from the food vacuole).<br />
Understanding the molecular basis of the mutated<br />
PfCRT mechanism of action is obviously important<br />
and it would open the road to new strategies for<br />
circumventing, and perhaps suppressing its role in<br />
determining resistance.<br />
The question we addressed is whether available data,<br />
together with a combination of analytical and modeling<br />
approaches can provide insights into the mechanism<br />
of action of chloroquine and PfCRT.<br />
Our approach consisted in developing an analytical<br />
model and test the consistency of several plausible<br />
hypotheses about the binding mode of the drug to<br />
the heme related species inside the vacuole and the<br />
mechanism of action of PfCRT with experimental<br />
data. We could demonstrate that some of the hypotheses<br />
can be excluded on the basis of the available<br />
data, but also that the latter are compatible with<br />
both models of PfCRT being a passive channel and<br />
a transporter. Evolutionary analysis of the protein<br />
was therefore required to solve the issue. We demonstrated<br />
that sequence analysis, advanced database<br />
searching methods and structural prediction taken<br />
together strongly support the hypothesis that the<br />
protein acts as an active carrier and, based on the<br />
conclusions derived from the mathematical model,<br />
the carried molecule is either or both the mono and<br />
di-protonated forms of chloroquine.<br />
In the course of this project we used available computational<br />
tools, but we also needed to develop new<br />
ones. We set up a tool for mapping mutations from<br />
genomes to proteomes and vice versa, a method to<br />
analyse the three-dimensional context of post-translational<br />
modification, a system for identifying extracellular<br />
regions of proteins and, obviously, tools to build<br />
and assess the quality of three-dimensional models of<br />
150<br />
proteins. The scope of application of these methods<br />
is not limited to the specific problem of malaria and<br />
therefore they were also made available to the community<br />
in the form of publicly accessible web server.<br />
Publications<br />
Cozzetto D, Kryshtafovych A, Fidelis K, Moult J,<br />
Rost B, Tramontano A. Evaluation of templatebased<br />
models in CASP8 with standard measures.<br />
Proteins. 2009, 77: 18-28. doi: 10.1002/prot.22561.<br />
Cozzetto D, Kryshtafovych A, Tramontano A.<br />
Evaluation of CASP8 model quality predictions.<br />
Proteins. 2009, 77: 157-66. doi: 10.1002/prot.22561.<br />
Loewenstein Y, Raimondo D, Redfern OC, Watson<br />
J, Frishman D, Linial M, Orengo C, Thornton J,<br />
Tramontano A. Protein function annotation by<br />
homology-based inference. Genome Biol. 2009, 10:<br />
Article 207. doi: 10.1186/gb-2009-10-2-207.<br />
Maullu C, Raimondo D, Caboi F, Giorgetti A, Sergi<br />
M, Valentini M, Tonon G, Tramontano A. Sitedirected<br />
enzymatic PEGylation of the human granulocyte<br />
colony-stimulating factor FEBS J. 2009, 276:<br />
6741-50. doi: 10.1111/j.1742-4658.2009.07387.x.<br />
Moult J, Fidelis K, Kryshtafovych A, Rost B,<br />
Tramontano A. Critical assessment of methods of<br />
protein structure prediction - Round VIII. Proteins.<br />
2009, 77: 1-4. doi: 10.1002/prot.22589.<br />
Chinappi M, Via A, Marcatili P, Tramontano A.<br />
On the mechanism of chloroquine resistance in<br />
Plasmodium falciparum. PLoS One 2010, 5:e14064.<br />
doi: 10.1371/journal.pone.0014064.<br />
Giombini E, Orsini M, Carabino D, Tramontano A.<br />
An automatic method for identifying surface proteins<br />
in bacteria: SLEP. BMC Bioinformatics. 2010, 11: 39.<br />
doi: 10.1186/1471-2105-11-39.<br />
Le Pera L, Marcatili P, Tramontano A. Picmi: mapping<br />
point mutations on genomes. Bioinformatics.<br />
2010, 26: 2904-5. doi: 10.1093/bioinformatics/<br />
btq547.
Start-Up<br />
Specification<br />
and<br />
maintenance<br />
of retinal<br />
stem cells
Specification and maintenance of retinal stem cells<br />
Principal investigator: Giuseppe Lupo<br />
Researcher in Development Biology<br />
Dipartimento di Biologia e Biotecnologie “Charles Darwin”<br />
Tel: (+39) 06 49912206; Fax: (+39) 06 49912351<br />
giuseppe.lupo@uniroma1.it<br />
Participants:<br />
Nicoletta Carucci, post-doc fellow.<br />
Collaborations:<br />
Dipartimento di Biologia e Biotecnologie “C. Darwin” (Dr.<br />
Emanuele Cacci, Dr. Maria Elena Miranda Banos);<br />
Department of Physiology, Development and Neuroscience,<br />
University of Cambridge, UK (Prof. William Harris); Anne<br />
McLaren Laboratory for Regenerative Medicine, University<br />
of Cambridge, UK (Prof. Roger Pedersen); Department of<br />
Developmental Neurobiology, Kumamoto University, Japan (Prof.<br />
Kunimasa Ohta).<br />
Report of activity<br />
Background<br />
The retina is a favoured system to study formation of<br />
the central nervous system (CNS), due to its accessible<br />
position, and to its simple and well-characterized<br />
structure. The mature retina consists of only six cell<br />
classes, identifiable by their morphology and molecular<br />
markers. Several vision-impairing diseases, such<br />
as retinitis pigmentosa and macular degeneration,<br />
are due to the loss of retinal cells. Studies in animals<br />
suggest that they may be treatable by transplantation<br />
of healthy retinal cells, but obtaining a sufficient<br />
number of suitable donor cells remains a major problem.<br />
This has propelled stem cell based approaches<br />
to generate pure populations of retinal cells in vitro,<br />
to be used for cell-replacement therapy. Retinal stem<br />
cells (RSCs) are the embryonic progenitors that give<br />
rise to the retina during embryogenesis and early<br />
post-natal life. RCSs are specified early in development<br />
as a result of three consecutive steps. First, the<br />
neuroectoderm (NE) is induced in the dorsal part of<br />
the embryo. Subsequently, the NE is patterned along<br />
the embryonic antero-posterior (AP) axis, specifying<br />
forebrain (FB), midbrain, hindbrain and spinal cord<br />
at different AP levels. Finally, a subset of the FB is<br />
153<br />
START-UP<br />
specified as the eye field (EF), containing the RSCs.<br />
Although most of the RSCs eventually differentiate<br />
to form retinal neurons and glia, RSCs are retained<br />
at the periphery of the retina, at least during early<br />
post-natal life. The signals that promote self-renewal<br />
of RSCs and prevent them from differentiating prematurely<br />
are still largely unclear.<br />
Objectives<br />
1. The elucidation of the signalling systems that<br />
control specification of RSCs during early NE development,<br />
using pluripotent stem cell lines as a model<br />
system.<br />
2. The comparison of RSCs obtained from pluripotent<br />
cells in vitro, with RSCs in the embryonic retina<br />
or the post-natal eye.<br />
3. The comparison of RSCs with neural stem cells<br />
(NSCs) derived from other embryonic NE regions,<br />
in order to understand the mechanisms controlling<br />
regionalization of progenitor cells during CNS<br />
development.<br />
4. The identification of factors that can support proliferation<br />
and self-renewal of RSCs, while preventing<br />
loss of retinal identity and differentiation.<br />
Results<br />
In collaboration with Dr Cacci at the University<br />
of Rome “La Sapienza”, and confirming previous<br />
results by Dr Chiara Soldati, Prof. Biagoni and Prof.<br />
Augusti-Tocco at the same University, we have generated<br />
a repertoire of NSC lines, derived from various<br />
region of the E13.5 mouse CNS: the cerebral cortex,<br />
the lateral ganglionic eminence, the dorsal and<br />
ventral mesencephalon, the spinal cord. In all this<br />
cases, is has been possible to isolate and propagate<br />
in vitro NSCs that efficiently self-renew in the presence<br />
of FGF and Epidermal Growth Factor (EGF).<br />
Furthermore, these different NSC lines appear to<br />
retain in vitro a positional identity consistent with<br />
their region of derivation in vivo. For example, we
Giuseppe Lupo<br />
have confirmed that dopaminergic neuron differentiation<br />
can be activated in vitro in NSCs derived<br />
from the lateral ganglionic eminence or the ventral<br />
mesencephalon, in agreement with the presence of<br />
dopaminergic neurons in these areas in vivo.<br />
However, retinal progenitors isolated from the E13.5<br />
mouse retina stopped proliferating and completely<br />
differentiated in the same culture conditions and<br />
hence could not be propagated in vitro. These results<br />
are very interesting as they suggest that FGF and<br />
EGF are sufficient for self-renewal of NSCs located<br />
in the embryonic neural tube (brain and spinal cord),<br />
while RSC self-renewal requires alternative or additional<br />
factors.<br />
In collaboration with Prof. Ohta at Kumamoto<br />
University, Japan, we have also investigated the signals<br />
supporting proliferation of RSCs located at the<br />
periphery of the mouse retina. Our work has shown<br />
a crucial role for the secreted molecule Tsukuchi<br />
(TSK), belonging to the small leucine-rich proteoglycan<br />
(SLRP) family, in regulating RSC proliferation.<br />
TSK is expressed in the peripheral retina from<br />
embryonic stages to adulthood and TSK knock-out<br />
mice show increased proliferation in this region.<br />
Activation of the Wnt pathway also stimulates RSC<br />
proliferation and we found that TSK inhibits Wnt<br />
signalling by competing for Wnt ligands to receptor<br />
binding. These results suggest that RSC proliferation<br />
in the peripheral retina is modulated by the antagonistic<br />
interaction between TSK and Wnt ligands and<br />
they also raise the interesting possibility that TSK<br />
activity may be responsible for the limited or absent<br />
RSC self-renewal in the adult peripheral retina.<br />
Future plans<br />
Objective 1. We have obtained a Material Transfer<br />
Agreement with the University of Cambridge to<br />
import and use mouse pluripotent stem cell lines<br />
(mouse embryonic stem cells (ESCs) and epiblast<br />
stem cells (EpiSCs)) in our Department in Rome.<br />
We aim to apply to these cells the same protocol that<br />
154<br />
we previously devised in human ESCs in the labs of<br />
Prof. Harris and Prof. Pedersen at the University<br />
of Cambridge, UK, to obtain specification of FB<br />
and retinal progenitors. The use of mouse cells will<br />
offer the advantage of using cells lines derived from<br />
genetically modified mice. Of particular interest will<br />
be the use of cell lines bearing fluorescent <strong>report</strong>er<br />
proteins (eg GFP) inserted into the loci of RSC<br />
markers for flow cytometry analysis and sorting of<br />
RSCs. Furthermore, the use of mouse pluripotent<br />
stem cells for generation of RSCs in vitro will allow a<br />
direct comparison of these cells with RSCs and NSCs<br />
derived from the embryonic CNS within the same<br />
species. Our experimental priorities are: i) the identification<br />
of the molecular mechanisms influencing<br />
retinal progenitor specification in ESCs and EpiSCs;<br />
ii) the sorting of retinal progenitors produced in<br />
vitroto obtain pure populations of RSCs; iii) the<br />
comparison (eg by transcriptome analysis) of ESC-<br />
or EpiSC-derived RSCs with RSCs derived from the<br />
embryonic retina.<br />
Objectives 2-4. We will focus on the derivation of<br />
RSC lines from the mouse embryonic retina by<br />
employing a systematic screening approach where<br />
retinal progenitor cells will be cultured in the presence<br />
of various combinations of growth factors and<br />
cytokines. We will especially concentrate on molecules<br />
working in the Wnt signalling pathway, since<br />
our results on the study of the TSK molecule indicate<br />
a critical role for this pathway in RSC proliferation<br />
and self-renewal. Embryo-derived RSCs will be compared<br />
with ESCs- or EpiSC-derived RSCs in terms<br />
of both their gene expression profiles and growth<br />
factor requirement for self-renewal. Furthermore,<br />
we will also compare the differentiation potential of<br />
RSCs with NSCs derived from other CNS regions, in<br />
order to understand the developmental mechanisms<br />
controlling the specification of regional identities in<br />
CNS progenitors in vivo and the plasticity of NSCs<br />
of different origins cultured in vitro with regards to<br />
their regional identity.
2009-2010<br />
Bibliography
Year 2009<br />
Anastasiadou E, Boccellato F, Rosato P, Junker S,<br />
Winberg G, Frati L, Wade P, Faggioni A, Trivedi<br />
P. Epstein-Barr virus infection leads to phenotypic<br />
reversion of terminally differentiated B cells<br />
of malignant origin. Cancer Lett. 2009, 284: 165-174.<br />
doi: 10.1016/j.canlet.2009.04.025.<br />
Angelucci F, Sayed AA, Williams DL, Boumis G,<br />
Brunori M, Dimastrogiovanni D, Miele AE, Pauly<br />
F, Bellelli A. Inhibition of Schistosoma mansoni thioredoxin-glutathione<br />
reductase by auranofin: structural<br />
and kinetic aspects. J Biol Chem. 2009, 284: 28977-85.<br />
doi: 10.1074/jbc.M109.020701.<br />
Antonangeli F, Giampietri C, Petrungaro S, Filippini<br />
A, Ziparo E. Expression profile of a 400-bp Stra8 promoter<br />
region during spermatogenesis. Microsc Res<br />
Tech. 2009, 72: 816-22. doi: 10.1002/jemt.20724.<br />
Aucello M, Dobrowolny G, Musarò A. Localized<br />
accumulation of oxidative stress causes muscle atrophy<br />
through activation of an autophagic pathway.<br />
Autophagy. 2009, 5: 527-9. doi: 10.4161/auto.5.4.7962.<br />
Ballarino M, Pagano F, Girardi E, Morlando M,<br />
Cacchiarelli D, Marchioni M, Proudfoot NJ, Bozzoni<br />
I. Coupled RNA processing and transcription of<br />
intergenic primary microRNAs. Mol Cell Biol. 2009,<br />
29: 5632-8. doi:10.1128/MCB.00664-09.<br />
Barberi L, Dobrowolny G, Pelosi L, Giacinti C,<br />
Musarò A. Muscle involvement and IGF-1 signaling<br />
in genetic disorders: new therapeutic approaches.<br />
Endocr Dev. 2009, 14: 29-37. doi: 10.1159/000207474.<br />
Belleudi F, Leone L, Maggio M, Torrisi MR. Hrs<br />
regulates the endocytic sorting of the fibroblast<br />
growth factor receptor 2b. Exp Cell Res. 2009, 315:<br />
2181-91. doi:10.1016/j.yexcr.2009.03.022.<br />
Benelli D, Londei P. Begin at the beginning: evolution<br />
of translational initiation. Res Microbiol. 2009,<br />
160: 493-501. doi:10.1016/j.resmic.2009.06.003.<br />
Benelli D, Marzi S, Mancone C, Alonzi T, la Teana<br />
A, Londei P. Function and ribosomal localization<br />
of aIF6, a translational regulator shared by archaea<br />
and eukarya. Nucleic Acids Res. 2009, 37: 256-67. doi:<br />
10.1093/nar/gkn959.<br />
Borisov VB, Forte E, Giuffrè A, Konstantinov A,<br />
Sarti P. Reaction of nitric oxide with the oxidized diheme<br />
and heme-copper oxygen-reducing centers of<br />
terminal oxidases: Different reaction pathways and<br />
end-products. J Inorg Biochem. 2009, 103: 1185-7.<br />
doi:10.1016/j.jinorgbio.2009.06.002.<br />
157<br />
BIBLIOGRAPHY 2009<br />
Bucciarelli E, Pellacani C, Naim V, Palena A,<br />
Gatti M, Somma MP. Drosophila Dgt6 protein<br />
interacts with Ndc80, Msps/XMAP215 and gammatubulin<br />
to promote kinetochore-driven MT formation.<br />
Curr Biol. 2009, 19: 1839-45. doi:10.1016/j.<br />
cub.2009.09.043.<br />
Caiafa P, Guastafierro T, Zampieri M. Epigenetics:<br />
poly(ADP-ribosyl)ation of PARP-1 regulates<br />
genomic methylation patterns. FASEB J. 2009, 23:<br />
672-8. doi: 10.1096/fj.08-123265.<br />
Caiafa P, Zlatanova J. CCCTC-binding factor meets<br />
poly(ADP-ribose) polymerase-1. J Cell Physiol. 2009,<br />
219: 265-70. doi: 10.1002/jcp.21691.<br />
Canettieri G, Coni S, Della Guardia M, Nocerino<br />
V, Antonucci L, Di Magno L, Screaton R, Screpanti<br />
I, Giannini G, Gulino A. The coactivator CRTC1<br />
promotes cell proliferation and transformation via<br />
AP-1. Proc Natl Acad Sci USA. 2009, 106: 1445-50.<br />
doi: 10.1073/pnas.0808749106.<br />
Canterini S, Bosco A, De Matteis V, Mangia F,<br />
Fiorenza MT. THG-1pit moves to nucleus at the<br />
onset of cerebellar granule neurons apoptosis. Mol<br />
and Cell Neurosci. 2009, 40: 249-57. doi: 10.1016/j.<br />
mcn.2008.10.013.<br />
Casasoli M, Federici L, Spinelli F, Di Matteo A,<br />
Vella N, Scaloni F, Fernandez-Recio J, Cervone F,<br />
De Lorenzo G. Integration of evolutionary and desolvation<br />
energy analysis identifies functional sites in<br />
a plant immunity protein. Proc Natl Acad Sci USA.<br />
2009, 106: 7666-71. doi: 10.1073/pnas.0812625106.<br />
Cerboni C, Ardolino M, Santoni A, Zingoni A.<br />
Detuning CD8+ T lymphocytes by down-regulation<br />
of the activating receptor NKG2D: role of NKG2D<br />
ligands released by activated T cells. Blood. 2009,<br />
113: 2955-64. doi: 10.1182/blood-2008-06-165944.<br />
Cigana C, Curcurù L, Leone MR, Ieranò T, Lorè<br />
NI, Bianconi I, Silipo A, Cozzolino F, Lanzetta R,<br />
Molinaro A, Bernardini ML, Bragonzi A. Pseudomonas<br />
aeruginosa exploits lipid A and muropeptides modification<br />
as a strategy to lower innate immunity during<br />
cystic fibrosis lung infection. PLoS One. 2009,<br />
4:e8439. doi: 10.1371/journal.pone.0008439.<br />
Cirilli R, Ferretti R, La Torre F, Borioni A, Fares<br />
V, Camalli M, Faggi C, Rotili D, Mai A. Chiral<br />
HPLC separation and absolute configuration of<br />
novel S-DABO derivatives. Chirality. 2009, 21: 604-<br />
12. doi: 10.1002/chir.20654.
BIBLIOGRAPHY 2009<br />
Colletti M, Cicchini C, Conigliaro A, Santangelo<br />
L, Alonzi T, Pasquini E, Tripodi M, Amicone L.<br />
Convergence of Wnt signaling on the HNF4alphadriven<br />
transcription in controlling liver zonation.<br />
Gastroenterology. 2009, 137: 660-72. doi:10.1053/j.<br />
gastro.2009.05.038.<br />
Costanzo G, Pino S, Ciciriello F, Di Mauro E.<br />
Generation of long RNA chains in water. J Biol Chem.<br />
2009, 284: 33206-16. doi: 10.1074/jbc.M109.041905.<br />
Cozzetto D, Kryshtafovych A, Fidelis K, Moult<br />
J, Rost B, Tramontano A. Evaluation of templatebased<br />
models in CASP8 with standard measures.<br />
Proteins. 2009, 77: 18-28. doi: 10.1002/prot.22561.<br />
Cozzetto D, Kryshtafovych A, Tramontano A.<br />
Evaluation of CASP8 model quality predictions.<br />
Proteins. 2009, 77: 157-66. doi: 10.1002/prot.22561.<br />
De Luca C, Zhou Y, Montanari A, Morea V, Oliva<br />
R, Besagni C, Bolotin-Fukuhara M, Frontali L,<br />
Francisci S. Can yeast be used to study mitochondrial<br />
diseases? Biolistic tRNA mutants for the analysis<br />
of mechanisms and suppressors. Mitochondrion. 2009,<br />
9: 408-17. doi: 10.1016/j.mito.2009.07.004.<br />
De Marchis ML, Ballarino M, Salvatori B, Puzzolo<br />
MC, Bozzoni I, Fatica A. A new molecular network<br />
comprising PU.1, interferon regulatory factor proteins<br />
and miR-342 stimulates ATRA-mediated granulocytic<br />
differentiation of acute promyelocytic leukemia<br />
cells. Leukemia 2009, 23: 856-62. doi:10.1038/<br />
leu.2008.372.<br />
Di Domenico EG, Auriche C, Viscardi V, Longhese<br />
MP, Gilson E, Ascenzioni F. The Mec1p and Tel1p<br />
checkpoint kinases allow humanized yeast to tolerate<br />
chronic telomere dysfunctions by suppressing<br />
telomere fusions. DNA Rep. 2009, 8: 209-18. doi:<br />
10.1016/j.dnarep.2008.10.005.<br />
Fatica A., Bozzoni I. Role of microRNAs in hematological<br />
malignancies. Expert Review Hematol. 2009, 2:<br />
415-23. doi: 10.1586/ehm.09.32.<br />
Ferretti E, De Smaele E, Po A, Di Marcotullio<br />
L, Tosi E, Espinola MS, Di Rocco C, Riccardi R,<br />
Giangaspero F, Farcomeni A, Nofroni I, Laneve P,<br />
Gioia U, Caffarelli E, Bozzoni I, Screpanti I, Gulino<br />
A. MicroRNA profiling in human medulloblastoma.<br />
Int J Cancer. 2009, 124: 568-77. doi: 10.1002/ijc.23948.<br />
Fionda C, Soriani A, Malgarini G, Iannitto ML,<br />
Santoni A, Cippitelli M. Heat shock protein-90 inhibitors<br />
increase MHC class I-related chain A and B<br />
ligand expression on multiple myeloma cells and their<br />
ability to trigger NK cell degranulation. J Immunol.<br />
2009, 183: 4385-94. doi: 10.4049/jimmunol.0901797.<br />
158<br />
Franceschini D, Paroli M, Francavilla V, Videtta M,<br />
Morrone S, Labbadia G, Cerino A, Mondelli MU, Barnaba<br />
V. PD-L1 negatively regulates CD4+CD25+Foxp3+<br />
Tregs by limiting STAT-5 phosphorylation in patients<br />
chronically infected with HCV. J Clin Invest. 2009, 119:<br />
551-64. doi: 10.1172/JCI36604.<br />
Gandhi AK, Ghatge M, Musayev FN, Sease A,<br />
Aboagye SO, di Salvo ML, Schirch V, Safo MK.<br />
Kinetic and structural studies of the role of the active<br />
site residue Asp235 of human pyridoxal kinase.<br />
Biochem Biophys Res Commun 2009, 381: 12-15. doi:<br />
10.1016/j.bbrc.2009.01.170.<br />
Gianni S, Brunori M, Jemth P, Oliveberg M, Zhang<br />
M. Distinguishing between smooth and rough free<br />
energy barriers in protein folding. Biochemistry. 2009,<br />
48: 11825-30. doi: 10.1021/bi901585q.<br />
Grisanti L, Corallini S, Fera S, Muciaccia B, Witke<br />
W, Stefanini M, Vicini E. Inactivation of numb and<br />
numblike in spermatogonial stem cells by cell-permeant<br />
Cre recombinase. Differentiation. 2009, 78:131-<br />
136. doi:10.1016/j.diff.2009.05.005.<br />
Grisanti L, Falciatori I, Grasso M, Dovere L, Fera<br />
S, Muciaccia B, Fuso A, Berno V, Boitani C, Stefanini<br />
M, Vicini E. Identification of spermatogonial stem<br />
cell subsets by morphological analysis and prospective<br />
isolation. Stem Cell. 2009, 27: 3043-52. doi:<br />
10.1002/stem.206.<br />
Hofmann L, Saunier R, Cossard R, Esposito M,<br />
Rinaldi T, Delahodde A. A non-proteolytic activity<br />
of the proteasome controls fission of organelles<br />
in yeast. J of Cell Science. 2009, 122: 3673-83. doi:<br />
10.1242/jcs.050229.<br />
Islas-Rodríguez AE, Marcellini L, Orioni B, Barra<br />
D, Stella L, Mangoni ML. Esculentin 1-21: a linear<br />
antimicrobial peptide from frog skin with inhibitory<br />
effect on bovine mastitis-causing bacteria. J Pept Sci.<br />
2009, 15: 607-14. doi: 10.1002/psc.1148.<br />
Ivarsson Y, Travaglini-Allocatelli C, Brunori M,<br />
Gianni S. Engineered symmetric connectivity of secondary<br />
structure elements highlights malleability of<br />
protein folding pathways. J Am Chem Soc. 2009, 131:<br />
11727-33. doi: 10.1021/ja900438b.<br />
Lara E, Mai A, Calvanese V, Altucci L, López-Nieva<br />
P, Martínez-Chantar ML, Varela-Rey M, Rotili<br />
D, Nebbioso A, Ropero S, Montoya G, Oyarzabal<br />
J, Velasco S, Serrano M, Witt M, Villar-Garea<br />
A, Imhof A, Mato JM, Esteller M, Fraga MF.<br />
Salermide, a Sirtuin inhibitor with a strong cancerspecific<br />
proapoptotic effect. Oncogene. 2009, 28: 781-<br />
91. doi:10.1038/onc.2008.436.
Loewenstein Y, Raimondo D, Redfern OC, Watson<br />
J, Frishman D, Linial M, Orengo C, Thornton J,<br />
Tramontano A. Protein function annotation by<br />
homology-based inference. Genome Biol. 2009, 10:<br />
Article 207. doi: 10.1186/gb-2009-10-2-207.<br />
Lulli P, Mangano VD, Onori A, Batini C, Luoni G,<br />
Sirima BS, Nebie I, Chessa L, Petrarca V, Modiano<br />
D. HLA-DRB1 and -DQB1 loci in three west African<br />
ethnic groups: genetic relationship with sub-Saharan<br />
African and European populations. Hum Immunol.<br />
2009, 42: 903-9. doi: 10.1016/j.humimm.2009.07.025.<br />
Maggi L, Trettel F, Scianni M, Bertollini C, Eusebi<br />
F, Fredholm BB, Limatola C. LTP impairment by<br />
fractalkine/CX3CL1 in mouse hippocampus is mediated<br />
through the activity of adenosine receptor type<br />
3 (A3R). J Neuroimmunol. 2009, 215: 36-42. doi:<br />
10.1016/j.jneuroim.2009.07.016.<br />
Mai A, Altucci L. Epi-drugs to fight cancer: From<br />
chemistry to cancer treatment, the road ahead. Int<br />
J Biochem Cell Biol. 2009, 41: 199-213. 10.1016/j.<br />
biocel.2008.08.020.<br />
Mai A, Rotili D, Tarantino D, Nebbioso A, Castellano<br />
S, Sbardella G, Tini M, Altucci L. Identification of<br />
4-hydroxyquinolines inhibitors of p300/CBP histone<br />
acetyltransferases. Bioorg Med Chem Lett. 2009, 19:<br />
1132-5. doi: 10.1016/j.bmcl.2008.12.097.<br />
Mai A, Rotili D, Valente S, Kazantsev AG. Histone<br />
deacetylase inhibitors and neurodegenerative disorders:<br />
holding the promise. Curr Pharm Des. 2009, 15:<br />
3940-57.<br />
Mai A, Valente S, Meade S, Carafa V, Tardugno<br />
M, Nebbioso A, Galmozzi A, Mitro N, De Fabiani E,<br />
Altucci L, Kazantsev A. Study of 1,4-dihydropyridine<br />
structural scaffold: discovery of novel sirtuin activators<br />
and inhibitors. J Med Chem. 2009, 52: 5496-<br />
5504. doi: 10.1021/jm9008289.<br />
Mai A, Valente S, Nebbioso A, Simeoni S, Ragno<br />
R, Massa S, Brosch G, De Bellis F, Manzo F, Altucci<br />
L. New pyrrole-based histone deacetylase inhibitors:<br />
Binding mode, enzyme- and cell-based investigations.<br />
Int J Biochem Cell Biol. 2009, 41: 235-47.<br />
doi:10.1016/j.biocel.2008.09.002.<br />
Maisetta G, Mangoni ML, Esin S, Pichierri G,<br />
Capria AL, Brancatisano FL, Di Luca M, Barnini S,<br />
Barra D, Campa M, Batoni G. In vitro bactericidal<br />
activity of the N-terminal fragment of the frog peptide<br />
esculentin-1b (Esc 1-18) in combination with<br />
conventional antibiotics against Stenotrophomonas<br />
maltophilia. Peptides. 2009, 30: 1622-6. doi: 10.1016/j.<br />
peptides.2009.06.004.<br />
159<br />
BIBLIOGRAPHY 2009<br />
Mangoni ML, Shai Y. Temporins and their synergism<br />
against Gram-negative bacteria and in lipopolysaccharide<br />
detoxification. Biochim Biophys Acta. 2009,<br />
1788: 1610-19. doi: 10.1016/j.bbamem.2009.04.021.<br />
Marcellini L, Borro M, Gentile G, Rinaldi AC, Stella<br />
L, Aimola P, Barra D, Mangoni ML. Esculentin-<br />
1b(1-18)--a membrane-active antimicrobial peptide<br />
that synergizes with antibiotics and modifies the<br />
expression level of a limited number of proteins in<br />
Escherichia coli. FEBS J. 2009, 276: 5647-64. doi:<br />
10.1111/j.1742-4658.2009.07257.x.<br />
Marsango S, Bonaccorsi di Patti MC, Barra D,<br />
Miele R. The Bv8 gene from Bombina orientalis:<br />
molecular cloning, genomic organization and functional<br />
characterization of the promoter. Peptides.<br />
2009, 30: 2182-90. doi: 10.1007/s00018-010-0601-6.<br />
Mathieu A, Paladini F, Vacca A, Cauli A, Fiorillo<br />
MT, and Sorrentino R. The interplay between the<br />
geographic distribution of HLA-B27 alleles and their<br />
role in disease: an unifying hypothesis. Autoimmun Rev.<br />
2009, 8: 420-25. doi: 10.1016/j.autrev.2009.01.003.<br />
Matusali G, Arena G, De Leo A, Di Renzo L,<br />
Mattia E. Inhibition of p38 MAP kinase pathway<br />
induces apoptosis and prevents Epstein Barr virus<br />
reactivation in Raji cells exposed to lytic cycle<br />
inducing compounds. Mol Cancer. 2009, 8: 18. doi:<br />
10.1186/1476-4598-8-18.<br />
Maullu C, Raimondo D, Caboi F, Giorgetti A, Sergi<br />
M, Valentini M, Tonon G, Tramontano A. Sitedirected<br />
enzymatic PEGylation of the human granulocyte<br />
colony-stimulating factor FEBS J. 2009, 276:<br />
6741-50. doi: 10.1111/j.1742-4658.2009.07387.x.<br />
Mazzoni C, Torella M, Petrera A, Palermo V,<br />
Falcone C. PGK1, the gene encoding the glycolitic<br />
enzyme phosphoglycerate kinase, acts as a multicopy<br />
suppressor of apoptotic phenotypes in S. cerevisiae.<br />
Yeast. 2009, 26: 31-7. doi: 10.1002/yea.1647.<br />
Messina S, Bitto A, Aguennouz M, Mazzeo A,<br />
Migliorato A, Polito F, Irrera N, Altavilla D, Vita<br />
GL, Russo M, Naro A, De Pasquale MG, Rizzuto<br />
E, Musarò A, Squadrito F, Vita G. Flavocoxid counteracts<br />
muscle necrosis and improves functional<br />
properties in mdx mice: a comparison study with<br />
methylprednisolone. Exp Neurol. 2009, 220: 349-58.<br />
doi: 10.1016/j.expneurol.2009.09.015.<br />
Micheli E, D’Ambrosio D, Franceschin M, Savino<br />
M. Water soluble cationic perylene derivatives as<br />
possible telomerase inhibitors: the search for selective<br />
G-quadruplex targeting. Mini Rev Med Chem.<br />
2009, 9: 1622-32. doi:10.2174/138955709791012274.
BIBLIOGRAPHY 2009<br />
Micheli E, Lombardo CM, D’Ambrosio D,<br />
Franceschin M, Neidle S, Savino M. Selective<br />
G-quadruplex ligands: the significant role of side<br />
chain charge density in a series of perylene derivatives.<br />
Bioorg Med Chem Lett. 2009, 19: 3903-8. doi:<br />
10.1016/j.bmcl.2009.03.106.<br />
Molfetta R, Gasparrini F, Peruzzi G, Vian L,<br />
Piccoli M, Frati L, Santoni A, Paolini R. Lipid<br />
raft-dependent FcepsilonRI ubiquitination regulates<br />
receptor endocytosis through the action of ubiquitin<br />
binding adaptors. PLoS One. 2009, 4:e5604. doi:<br />
10.1371/journal.pone.0005604.<br />
Moult J, Fidelis K, Kryshtafovych A, Rost B,<br />
Tramontano A. Critical assessment of methods of<br />
protein structure prediction - Round VIII. Proteins.<br />
2009, 77: 1-4. doi: 10.1002/prot.22589.<br />
Musayev FN, Di Salvo ML, Saavedra MA,<br />
Contestabile R, Ghatge MS, Haynes A, Schirch V,<br />
Safo MK. Molecular basis of reduced pyridoxine<br />
5’-phosphate oxidase catalytic activity in neonatal<br />
epileptic encephalopathy disorder. J Biol Chem. 2009,<br />
284: 30949-56. doi: 10.1074/jbc.M109.038372.<br />
Muscolini M, Montagni E, Caristi S, Nomura T,<br />
Kamada R, Di Agostino S, Corazzari M, Piacentini<br />
M, Blandino G, Costanzo A, Sakaguchi K, Tuosto L.<br />
Characterization of a new cancer-associated mutant<br />
of p53 with a missense mutation (K351N) in the<br />
tetramerization domain. Cell Cycle. 2009, 8: 3396-405.<br />
doi: 10.4161/cc.8.20.9910.<br />
Naldi M, Calonghi N, Masotti L, Parolin C, Valente<br />
S, Mai A, Andrisano V. Histone post-translational<br />
modifications by HPLC-ESI-MS after HT29 cell<br />
treatment with HDAC inhibitors. Proteomics. 2009,<br />
9: 5437-45. doi: 10.1002/pmic.200800866.<br />
Nebbioso A, Manzo F, Miceli M, Conte M, Manente<br />
L, Baldi A, De Luca A, Rotili D, Valente S, Mai A,<br />
Usiello A, Gronemeyer H, Altucci L. Selective class<br />
II HDAC inhibitors impair myogenesis by modulating<br />
the stability and activity of HDAC-MEF2 complexes.<br />
EMBO Rep. 2009, 10: 776-82. doi: 10.1038/<br />
embor.2009.88.<br />
Nencioni L, De Chiara G, Sgarbanti R, Amatore<br />
D, Aquilano K, Marcocci ME, Serafino A, Torcia<br />
M, Cozzolino F, Ciriolo MR, Garaci E, Palamara<br />
AT. Bcl-2 expression and p38MAPK activity in cells<br />
infected with influenza A virus: impact on virally<br />
induced apoptosis and viral replication. J Biol Chem.<br />
2009, 284: 16004-15. doi: 10.1074/jbc.M900146200.<br />
Paladini F, Belfiore F, Cocco E, Carcassi C, Cauli<br />
A, Vacca A, Fiorillo MT, Mathieu A, Cascino I,<br />
Sorrentino R. HLA-E gene polymorphism associates<br />
160<br />
with Ankylosing Spondylitis in Sardinia. Arthritis Res<br />
Ther. 2009, 11: R171. doi: 10.1186/ar2860.<br />
Palazzolo I, Stack C, Kong L, Musarò A, Adachi<br />
H, Katsuno M, Sobue G, Taylor JP, Sumner CJ,<br />
Fischbeck KH, Pennuto M. Overexpression of IGF-1<br />
in muscle attenuates disease in a mouse model of spinal<br />
and bulbar muscular atrophy. Neuron. 2009, 13:<br />
316-28. doi: 10.1016/j.neuron.2009.07.019.<br />
Pennacchietti E, Lammens TM, Capitani G,<br />
Franssen MC, John RA, Bossa F, De Biase D.<br />
Mutation of His465 alters the pH-dependent spectroscopic<br />
properties of Escherichia coli glutamate<br />
decarboxylase and broadens the range of its activity<br />
toward more alkaline pH. J Biol Chem. 2009, 284:<br />
31587-96. doi: 10.1074/jbc.M109.049577.<br />
Piacentini L, Fanti L, Negri R, Del Vescovo V, Fatica<br />
A, Altieri F, Pimpinelli S. Heterochromatin protein 1<br />
(HP1a) positively regulates euchromatic gene expression<br />
through RNA transcript association and interaction<br />
with hnRNPs in Drosophila. PLoS Genet. 2009, 5:<br />
e1000670. doi: 10.1371/journal.pgen.1000670.<br />
Piscitelli F, Coluccia A, Brancale A, La Regina G,<br />
Sansone A, Giordano C, Balzarini J, Maga G, Zanoli<br />
Z, Samuele A, Cirilli R, La Torre F, Lavecchia A,<br />
Novellino E, Silvestri R. Indolyl aryl sulfones bearing<br />
natural and unnatural aminoacids. Discovery<br />
of potent inhibitors of both HIV-1 non-nucleoside<br />
wild type and resistant mutant strains reverse transcriptase,<br />
and coxsackie B4 virus. J Med Chem. 2009,<br />
52: 1922-34. doi: 10.1021/jm801470b.<br />
Puttini S, Lekka M, Dorchies OM, Saugy D,<br />
Incitti T, Ruegg UT, Bozzoni I, Kulik AJ, Mermod<br />
N. Gene-mediated restoration of normal myofiber<br />
elasticity in dystrophic muscles. Mol Ther. 2009, 17:<br />
19-25. doi: 10.1038/mt.2008.239.<br />
Rizzuto E, Musarò A, Catizone A, Del Prete Z.<br />
Measuring tendon properties in mdx mice: cell viability<br />
and viscoelastic characteristics. J Biomech. 2009,<br />
42: 2243-8. doi: 10.1016/j.jbiomech.2009.06.041.<br />
Robinett CC, Giansanti MG, Gatti M, Fuller MT.<br />
TRAPPII function is required for cleavage furrow<br />
ingression and localization of Rab11 in dividing<br />
male meiotic cells of Drosophila. J Cell Sci. 2009, 122:<br />
4526-34. doi: 10.1242/jcs.054536.<br />
Rocco F, De Gregorio E, Colonna B, Di Nocera<br />
PP. Stenotrophomonas maltophilia genomes: a start-up<br />
comparison. Int J Med Microbiol. 2009, 299: 535-46.<br />
doi: 10.1016/j.ijmm.2009.05.004.<br />
Roseti C, Palma E, Martinello K, Fucile S, Morace<br />
R, Esposito V, Cantore G, Arcella A, Giangaspero F,
Aronica E, Mascia A, Di Gennaro G, Quarato PP,<br />
Manfredi M, Cristalli G, Lambertucci C, Marucci G,<br />
Volpini R, Limatola C, Eusebi F. Blockage of A2A<br />
and A3 adenosine receptors decreases the desensitization<br />
of human GABA(A) receptors microtransplanted<br />
to Xenopus oocytes. Proc Natl Acad Sci USA.<br />
2009, 106: 15927-31. doi: 10.1073/pnas.0907324106.<br />
Rotili D, Simonetti G, Savarino A, Palamara AT,<br />
Migliaccio AR, Mai A. Non-cancer uses of histone<br />
deacetylase inhibitors: effects on infectious diseases<br />
and beta-hemoglobinopathies. Curr Top Med Chem.<br />
2009, 9: 272-91.<br />
Saladino R, Crestini C, Ciciriello F, Pino S, Costanzo<br />
G, Di Mauro E. From formamide to RNA: the roles<br />
of formamide and water in the evolution of chemical<br />
information. Res Microbiol. 2009, 160: 441-8. doi:<br />
10.1016/j.resmic.2009.06.001.<br />
Samuele A, Kataropoulou A, Viola M, Zanolia S,<br />
La Regina G, Piscitelli F, Silvestri R, Maga G. Nonnucleoside<br />
HIV-1 reverse transcriptase inhibitors<br />
di-halo-indolyl aryl sulfones achieve tight binding<br />
to drug-resistant mutants by targeting the enzymesubstrate<br />
complex. Antivir Res. 2009, 81: 47-55. doi:<br />
10.1016/j.antiviral.2008.09.008.<br />
Scaloni F, Gianni S, Federici L, Falini B, Brunori<br />
M. Folding mechanism of the C-terminal domain of<br />
nucleophosmin: residual structure in the denatured<br />
state and its pathophysiological significance. FASEB<br />
J. 2009, 8: 2360-5. doi: 10.1096/fj.08-128306.<br />
Scicchitano BM, Rizzuto E, Musarò A. Counteracting<br />
muscle wasting in aging and neuromuscular diseases:<br />
the critical role of IGF-1. Aging. 2009, 11: 451-7.<br />
Scipioni A, Morosetti S, De Santis P. A statistical<br />
thermodynamic approach for predicting the sequencedependent<br />
nucleosome positioning along genomes.<br />
Biopolymers. 2009, 91: 1143-53. doi: 10.1002/bip.21276.<br />
Simmaco M, Kreil G, Barra D. Bombinins, antimicrobial<br />
peptides from Bombina species. Biochim<br />
Biophys Acta. 2009, 1788: 1551-5. doi: 10.1016/j.<br />
bbamem.2009.01.004.<br />
Soriani A, Zingoni A, Cerboni C, Iannitto ML,<br />
Ricciardi MR, Di Gialleonardo V, Cippitelli M,<br />
Fionda C, Petrucci MT, Guarini A, Foà R, Santoni<br />
A. ATM-ATR-dependent up-regulation of DNAM-<br />
1 and NKG2D ligands on multiple myeloma cells<br />
by therapeutic agents results in enhanced NK-cell<br />
susceptibility and is associated with a senescent<br />
phenotype. Blood. 2009,113: 3503-11. doi: 10.1182/<br />
blood-2008-08-173914.<br />
Starace D, Muciaccia B, Morgante E, Russo MA,<br />
Pensini S, D’agostino A, De Cesaris P, Filippini A,<br />
161<br />
BIBLIOGRAPHY 2009<br />
Ziparo E, Riccioli A. Peculiar subcellular localization<br />
of Fas antigen in human and mouse spermatozoa.<br />
Microsc Res Tech. 2009, 72: 573-9. doi: 10.1002/<br />
jemt.20700.<br />
Tilesi F, Fradiani P, Socci V, Willems D, Ascenzioni<br />
F. Design and validation of siRNAs and shRNAs.<br />
Curr Opin Mol Ther. 2009, 11: 156-64. doi.<br />
Travaglini-Allocatelli C, Ivarsson Y, Jemth, P,<br />
Gianni S. Folding and stability of globular proteins<br />
and implications for function. Curr Opin Struct Biol.<br />
2009, 19: 3-7. doi: 10.1016/j.sbi.2008.12.001.<br />
Vicente JB, Testa F, Mastronicola D, Forte E, Sarti<br />
P, Teixeira M, Giuffrè A. Redox properties of the oxygen-detoxifying<br />
flavodiiron protein from the human<br />
parasite Giardia intestinalis. Arch Biochem Biophys.<br />
2009, 488: 9-13. doi: 10.1016/j.abb.2009.06.011.<br />
Visco V, Bava FA, d’Alessandro F, Cavallini M,<br />
Ziparo V, Torrisi MR. Human colon fibroblasts<br />
induce differentiation and proliferation of intestinal<br />
epithelial cells through the direct paracrine action of<br />
keratinocyte growth factor. J Cell Physiol. 2009, 220:<br />
204-13. doi: 10.1002/jcp.21752.<br />
Vivoli M, Angelucci F, Ilari A, Morea V, Angelaccio<br />
S, di Salvo ML, Contestabile R. Role of a conserved<br />
active site cation-π interaction in Escherichia coli<br />
serine hydroxymethyltransferase. Biochemistry. 2009,<br />
48: 12034-46. doi: 10.1021/bi901568b.<br />
Wainman A, Creque J, Williams B, Williams EV,<br />
Bonaccorsi S, Gatti M, and Goldberg ML. Roles of<br />
the Drosophila NudE protein in kinetochore function<br />
and centrosome migration. J Cell Sci. 2009, 122:<br />
1747-58. doi:10.1242/jcs.041798.<br />
Wegert M, La Monica N, Tripodi M, Adler G,<br />
Dikopoulos N. Impaired interferon type I signalling<br />
in the liver modulates the hepatic acute phase response<br />
in hepatitis C virus transgenic mice. J Hepatol. 2009,<br />
51: 271-8. doi: 10.1016/j.jhep.2009.03.014.<br />
Zampieri M, Passananti C, Calabrese R, Perilli M,<br />
Corbi N, De Cave F, Guastafierro T, Bacalini MG,<br />
Reale A, Amicosante G, Calabrese L, Zlatanova J,<br />
Caiafa P. Parp1 localizes within the Dnmt1 promoter<br />
and protects its unmethylated state by its enzymatic<br />
activity. PLoS One. 2009, 4: e4717. doi: 10.1371/journal.pone.0004717.<br />
Zanni E, Farina F, Ricci A, Frank C, Mancini P,<br />
Palleschi C, Uccelletti D. The Golgi alpha 1,6 mannosyltransferase<br />
KlOch1p of Kluyveromyces lactis is<br />
required for Ca2+/calmodulin-based signaling and<br />
for proper mitochondrial functionality. BMC Cell<br />
Biology. 2009, 10: 86. doi: 10.1186/1471-2121-10-86.
BIBLIOGRAPHY 2009<br />
Zlatanova J, Caiafa P. CCCTC-binding factor: to<br />
loop or to bridge. Cell Mol Life Sci. 2009, 66: 1647-60.<br />
doi: 10.1007/s00018-009-8647-z.<br />
162<br />
Zlatanova J, Caiafa P. CTCF and its protein partners:<br />
divide and rule? J Cell Sci. 2009, 122: 1275-84.<br />
doi: 10.1242/jcs.039990.
Year 2010<br />
Ajmone-Cat MA, Cacci E, Ragazzoni Y, Minghetti<br />
L, Biagioni S. Pro-gliogenic effect of IL-1alpha in<br />
the differentiation of embryonic neural precursor<br />
cells in vitro. J Neurochem. 2010, 113: 1060-72. doi:<br />
10.1111/j.1471-4159.2010.06670.x.<br />
Anastasiadou E, Boccellato F, Vincenti S, Rosato P,<br />
Bozzoni I, Frati L, Faggioni A, Presutti C, Trivedi<br />
P. Epstein-Barr virus encoded LMP1 downregulates<br />
TCL1 oncogene through miR-29b. Oncogene. 2010,<br />
29: 1316-28. doi: 10.1038/onc.2009.439.<br />
Angelucci F, Dimastrogiovanni D, Boumis G,<br />
Brunori M, Miele AE, Saccoccia F, Bellelli A.<br />
Mapping the catalytic cycle of Schistosoma mansoni<br />
thioredoxin glutathione reductase by X-ray crystallography.<br />
J Biol Chem. 2010, 285: 32557-67. doi:<br />
10.1074/jbc.M110.141960.<br />
Antonangeli F, Petrungaro S, Coluccia P, Filippini<br />
A, Ziparo E, Giampietri C. Testis atrophy and<br />
reduced sperm motility in transgenic mice overexpressing<br />
c-FLIP(L). Fertil Steril. 2010, 93: 1407-14.<br />
doi: 10.1016/j.fertnstert.2009.01.122.<br />
Arcovito A, Bonamore A, Hazemann JL, Boffi<br />
A, D‘Angelo P. Unusual proximal heme pocket<br />
geometry in the deoxygenated Thermobifida fusca: a<br />
combined spectroscopic investigation. Biophys Chem.<br />
2010:147: 1-7. doi:10.1016/j.bpc.2009.11.006.<br />
Auriche C, Di Domenico EG, Pierandrei S,<br />
Lucarelli M, Castellani S, Conese M, Melani R,<br />
Zegarra-Moran O, Ascenzioni F. CFTR expression<br />
and activity from the human CFTR locus in<br />
BAC vectors, with regulatory regions, isolated by a<br />
single-step procedure. Gene Ther. 2010, 17: 1341-54.<br />
doi:10.1038/gt.2010.89.<br />
Barks HL, Buckley R, Grieves GA, Di Mauro E,<br />
Hud N, Orlando T. Guanine, Adenine, Hypoxanthine<br />
production in UV-irradiated Formamide solutions:<br />
relaxation of the requirements for prebiotic purine<br />
nucleobase formation. Chembiochem. 2010, 11: 1240-<br />
43. doi: 10.1002/cbic.201090037.<br />
Barnaba V. Hepatitis C virus infection: a “liaison a<br />
trois” amongst the virus, the host, and chronic lowlevel<br />
inflammation for human survival. J Hepatol.<br />
2010, 53: 752-61. doi:10.1016/j.jhep.2010.06.003.<br />
Beauvillain C, Meloni F, Sirard JC, Blanchard<br />
S, Jarry U, Scotet M, Magistrelli G, Delneste Y,<br />
Barnaba V, Jeannin P. The scavenger receptors<br />
SRA-1 and SREC-I cooperate with TLR2 in the<br />
163<br />
BIBLIOGRAPHY 2010<br />
recognition of the hepatitis C virus non-structural<br />
protein 3 by dendritic cells. J Hepatol. 2010, 52: 644-<br />
51 doi: 10.1016/j.jhep.2009.11.031.<br />
Belleudi F, Cardinali G, Kovacs D, Picardo M,<br />
Torrisi MR. KGF promotes paracrine activation of<br />
the SCF/c-KIT axis from human keratinocytes to<br />
melanoma cells. Transl Oncol. 2010, 3: 80-90. doi:<br />
10.1593/tlo.0919.<br />
Biava M, Porretta GC, Poce G, Battilocchio<br />
C, Alfonso S, De Logu A, Serra N, Manetti F,<br />
Botta M. Identification of a novel pyrrole derivative<br />
endowed with antimycobacterial activity and<br />
protection index comparable to that of the current<br />
antitubercular drugs streptomycin and rifampin,<br />
Bioorg Med Chem. 2010, 18: 8076–84. doi: 10.1016/j.<br />
bmc.2010.09.006.<br />
Binda C, Valente S, Romanenghi M, Pilotto S,<br />
Cirilli R, Karytinos A, Ciossani G, Botrugno OA,<br />
Forneris F, Tardugno M, Edmondson DE, Minucci<br />
S, Mattevi A, Mai A. Biochemical, structural, and<br />
biological evaluation of tranylcypromine derivatives<br />
as inhibitors of histone demethylases LSD1 and<br />
LSD2. J Am Chem Soc. 2010, 132: 6827-6833.<br />
Borbone E, Berlingieri MT, De Bellis F, Nebbioso<br />
A, Chiappetta G, Mai A, Altucci L, Fusco A. Histone<br />
deacetylase inhibitors induce thyroid cancer-specific<br />
apoptosis through proteasome-dependent inhibition<br />
of TRAIL degradation. Oncogene. 2010, 29: 105-116.<br />
doi:10.1038/onc.2009.306.<br />
Brutus A, Sicilia F, Macone A, Cervone F, De<br />
Lorenzo G. A domain swap approach reveals a role of<br />
the plant wall-associated kinase 1 (WAK1) as a receptor<br />
of oligogalacturonides. Proc Natl Acad Sci USA.<br />
2010, 107: 9453-7. doi: 10.1073/pnas.1000675107.<br />
Cacchiarelli D, Martone J, Girardi E, Cesana<br />
M, Incitti T, Nicoletti C, Santini T, Sthandier O,<br />
Auricchio A, Musarò A, Bozzoni I. MicroRNAs<br />
involved in molecular circuitries relevant for the<br />
Duchenne muscular dystrophy pathogenesis are controlled<br />
by the dystrophin/nNOS pathway. Cell Metab.<br />
2010, 12: 341-51. doi: 10.1016/j.cmet.2010.07.008.<br />
Casalino M, Prosseda G, Barbagallo M, Iacobino<br />
A, Ceccarini P, Latella MC, Nicoletti M, Colonna B.<br />
Interference of the CadC regulator in the argininedependent<br />
acid resistance system of Shigella and<br />
enteroinvasive E. coli. Int J Med Microbiol. 2010, 300:<br />
289-95. doi:10.1016/j.ijmm.2009.10.008.
BIBLIOGRAPHY 2010<br />
Castellano S, Milite C, Ragno R, Simeoni S,<br />
Mai A, Limongelli V, Novellino E, Bauer I, Brosch<br />
G, Spannhoff A, Cheng D, Bedford M T, Sbardella<br />
G. Design, synthesis and biological evaluation of carboxy<br />
analogues of arginine methyltransferase inhibitor<br />
1 (AMI-1). ChemMedChem. 2010, 5: 398-414. doi:<br />
10.1002/cmdc.200900459.<br />
Checquolo S, Palermo R, Cialfi S, Ferrara G,<br />
Oliviero C, Talora C, Bellavia D, Giovenco A, Grazioli<br />
P, Frati L, Gulino A, Screpanti I. Differential subcellular<br />
localization regulates c-Cbl E3 ligase activity<br />
upon Notch3 protein in T-cell leukemia. Oncogene.<br />
2010, 1129: 1463-74. doi: 10.1038/onc.2009.446.<br />
Chevanne M, Zampieri M, Caldini R, Rizzo A,<br />
Ciccarone F, Catizone A, D’Angelo C, Guastafierro<br />
T, Biroccio A, Reale A, Zupi G, Caiafa P. Inhibition<br />
of PARP activity by PJ-34 leads to growth impairment<br />
and cell death associated with aberrant mitotic<br />
pattern and nucleolar actin accumulation in M14<br />
melanoma cell line. J Cell Physiol. 2010, 222: 401-10.<br />
doi:10.1002/jcp.21964.<br />
Chiaretti S, Messina M, Tavolaro S, Zardo G,<br />
Elia L, Vitale A, Fatica A, Gorello P, Piciocchi A,<br />
Scappucci G, Bozzoni I, Fozza C, Candoni A, Guarini<br />
A, Foà R. Gene expression profiling identifies a<br />
subset of adult T-cell acute lymphoblastic leukemia<br />
with myeloid-like gene features and over-expression<br />
of miR-223. Haematologica. 2010, 95: 1114-21. doi:<br />
10.1002/cncr.25113.<br />
Chichiarelli S, Gaucci E, Ferraro A, Grillo C, Altieri<br />
F, Cocchiola R, Arcangeli V, Turano C, Eufemi M.<br />
Role of ERp57 in the signaling and transcriptional<br />
activity of STAT3 in a melanoma cell line. Arch<br />
Biochem Biophys. 2010, 494: 178-83. doi: 10.1016/j.<br />
abb.2009.12.004.<br />
Chinappi M, Via A, Marcatili P, Tramontano A.<br />
On the mechanism of chloroquine resistance in<br />
Plasmodium falciparum. PLoS One. 2010, 5:e14064.<br />
doi: 10.1371/journal.pone.0014064.<br />
Ciogli A, D’Acquarica I, Gasparrini F, Molinaro C,<br />
Rompietti R, Simone P, Villani C, Zappia G. Hybrid<br />
polyacrylamide chiral stationary phases for HPLC prepared<br />
by surface-initiated photopolymerization. J Sep<br />
Sci. 2010, 33: 3022-32. doi: 10.1002/jssc.201000355.<br />
Cocco E, Paladini F, Macino G, Fulci V, Fiorillo<br />
MT, Sorrentino R. The expression of vasoactive<br />
intestinal peptide receptor 1 is negatively modulated<br />
by microRNA 525-5p. PLoS One. 2010, 105:e12067.<br />
doi:10.1371/journal.pone.0012067.<br />
Colelli V, Fiorenza MT, Conversi D, Orsini C,<br />
Cabib S. Strain-specific proportion of the two iso-<br />
164<br />
forms of the dopamine D2 receptor in the mouse<br />
striatum: associated neural and behavioral phenotypes.<br />
Genes, Brain and Behavior 2010, 9: 703-711.<br />
doi: 10.1111/j.1601-183X.2010.00604.x.<br />
Colussi C, Banfi C, Brioschi M, Tremoli E, Straino<br />
S, Spallotta F, Mai A, Rotili D, Capogrossi MC,<br />
Gaetano C. Proteomic profile of differentially<br />
expressed plasma proteins from dystrophic mice and<br />
following suberoylanilide hydroxamic acid treatment.<br />
Proteomics Clin Appl. 2010, 4: 71-83. doi:<br />
10.1002/prca.200900116.<br />
Colussi C, Berni R, Rosati J, Straino S, Vitale S,<br />
Spallotta F, Baruffi S, Bocchi L, Delucchi F, Rossi S,<br />
Savi M, Rotili D, Quaini F, Macchi E, Stilli D, Musso<br />
E, Mai A, Gaetano C, Capogrossi MC. The histone<br />
deacetylase inhibitor suberoylanilide hydroxamic<br />
acid reduces cardiac arrhythmias in dystrophic mice.<br />
Cardiovasc Res. 2010, 87: 73-82. doi: 10.1093/cvr/<br />
cvq035.<br />
Colussi C, Illi B, Rosati J, Spallotta F, Farsetti A,<br />
Grasselli A, Mai A, Capogrossi MC, Gaetano C.<br />
Histone deacetylase inhibitors: keeping momentum<br />
for neuromuscular and cardiovascular diseases treatment.<br />
Pharmacol Res. 2010, 62: 3-10. doi: 10.1016/j.<br />
phrs.2010.02.014.<br />
Curtale G, Citarella F, Carissimi C, Goldoni M,<br />
Carucci N, Fulci V, Franceschini D, Meloni F,<br />
Barnaba V, Macino G. An emerging player in the<br />
adaptive immune response: microRNA-146a is a<br />
modulator of IL-2 expression and AICD in T lymphocytes.<br />
Blood 2010, 115: 265-73. doi: 10.1182/<br />
blood-2009-06-225987.<br />
De Chiara G, Marcocci ME, Civitelli L, Argnani<br />
R, Piacentini R, Ripoli C, Manservigi R, Grassi C,<br />
Garaci E, Palamara AT. APP processing induced by<br />
herpes simplex virus type 1 (HSV-1) yelds several<br />
APP fragments in human and rat neuronal cells.<br />
PLoS One. 2010; 5: e13989. doi:10.1371/journal.<br />
pone.0013989.<br />
De Jaco A, Dubi N, Comoletti D, Taylor P. Folding<br />
anomalies of neuroligin3 caused by a mutation in the<br />
α/β-hydrolase fold domain. Chem Biol Interact. 2010,<br />
187: 56-8. DOI 10.1074/jbc.M110.139519.<br />
De Jaco A, Lin MZ, Dubi N, Comoletti D, Miller<br />
MT, Camp S, Ellisman M, Butko MT, Tsien RY,<br />
Taylor P. Neuroligin trafficking arising from mutations<br />
in the α/β-hydrolase fold protein family. J<br />
Biol Chem. 2010, 285: 28674-82. doi: 10.1016/j.<br />
cbi.2010.03.012.<br />
De Leo A, Matusali G, Arena G, Di Renzo L,<br />
Mattia E. Epstein-Barr virus lytic cycle activation
alters proteasome subunit expression in Burkitt’s<br />
lymphoma cells. Biol Chem. 2010, 391: 1041-46. doi:<br />
10.1515/BC.2010.107.<br />
De Santis P, Morosetti S, Scipioni A. Prediction of<br />
nucleosome positioning in genomes: limits and perspectives<br />
of physical and bioinformatic approaches. J<br />
Biomol Struct Dyn. 2010; 27: 747-64.<br />
Di Santo R. Natural products as antifungal agents<br />
against clinically relevant pathogens. Nat Prod Rep.<br />
2010, 27: 1084-1098. doi: 10.1039/b914961a.<br />
Dimastrogiovanni D, Anselmi M, Miele AE,<br />
Boumis G, Petersson L, Angelucci F, Di Nola A,<br />
Brunori M, Bellelli A. Combining crystallography<br />
and molecular dynamics: the case of Schistosoma mansoni<br />
phospholipid glutathione peroxidase. Proteins.<br />
2010, 78: 259-70. doi: 10.1002/prot.22536.<br />
Droghetti E, Nicoletti FP, Bonamore A, Boechi L,<br />
Pau AM, Estrin D, Boffi A, Smulevich G, Feis A. Heme<br />
pocket structural properties of a bacterial truncated<br />
hemoglobin from Thermobifida fusca. Biochemistry.<br />
2010, 49: 10394-402. doi:10.1021/bi101452k.<br />
Fernandez-Diaz LC, Laurent A, Girasoli S, Turco<br />
M, Longobardi E, Iotti G, Jenkins NA, Fiorenza<br />
MT, Copeland NG, Blasi F. The absence of Prep1<br />
causes p53-dependent apoptosis of mouse pluripotent<br />
epiblast cells. Development. 2010, 137: 3393-<br />
3403. doi: 10.1242/dev.050567.<br />
Ferretti V, Roullet P, Sargolini F, Rinaldi A, Perri<br />
V, Del Fabbro M, Costantini VJA, Annese V, Scesa<br />
G, De Stefano ME, Oliverio A, Mele A. Ventral<br />
striatal plasticity and spatial memory. Proc Natl<br />
Acad Sci USA. 2010, 107: 7945-50. doi: 10.1073/<br />
pnas.0911757107.<br />
Fradiani PA, Petrucca A, Ascenzioni F, Di Nucci<br />
G, Teggi A, Bilancini S, Cipriani P. Endocarditis<br />
caused by Lactobacillus jensenii in an immunocompetent<br />
patient. J Med Microbiol. 2010, 59: 607-9. doi:<br />
10.1099/jmm.0.017764-0.<br />
Fulci V, Scappucci G, Sebastiani GD, Giannitti C,<br />
Franceschini D, Meloni F, Colombo T, Citarella F,<br />
Barnaba V, Galeazzi M, Macino G. miR-223 is overexpressed<br />
in Tlymphocytes of patients affected by<br />
rheumatoid arthritis. Hum Immunol. 2010, 71: 206-<br />
11. doi: 10.1016/j.humimm.2009.11.008.<br />
Galli R, Starace D, Busà R, Angelini DF, Paone<br />
A, De Cesaris P, Filippini A, Sette C, Battistini L,<br />
Ziparo E, Riccioli A. TLR stimulation of prostate<br />
tumor cells induces chemokine-mediated recruitment<br />
of specific immune cell types. J Immunol. 2010,<br />
15184: 6658-69. doi:10.4049/jimmunol.0902401.<br />
165<br />
BIBLIOGRAPHY 2010<br />
Giangrossi M, Prosseda G, Tran CN, Brandi A,<br />
Colonna B, Falconi M. A novel antisense RNA regulates<br />
at transcriptional level the virulence gene icsA<br />
of Shigella flexneri. Nucleic Acids Res. 2010, 38: 3 362-<br />
75. doi: 10.1093/nar/gkq025.<br />
Gianni S, Ivarsson Y, De Simone A, Travaglini-<br />
Allocatelli C, Brunori M, Vendruscolo M. Structural<br />
characterization of a misfolded intermediate populated<br />
during the folding process of a PDZ domain. Nat Struct<br />
Mol Biol. 2010, 12: 1431-7. doi:10.1038/nsmb.1956.<br />
Giombini E, Orsini M, Carabino D, Tramontano A.<br />
An automatic method for identifying surface proteins<br />
in bacteria: SLEP. BMC Bioinformatics. 2010, 11: 39.<br />
doi: 10.1186/1471-2105-11-39.<br />
Gouagna LC, Bancone G, Yao F, Yameogo B, Dabiré<br />
KR, Costantini C, Simpore J, Ouedraogo JB, Modiano<br />
D. Genetic variation in human HBB is associated with<br />
Plasmodium falciparum transmission. Nat Genet. 2010,<br />
42: 328-31. doi: 10.1038/ng.554.<br />
Imig J, Motsch N, Zhu J, Barth S, Okonievski<br />
M, Tinguely M, Kurrer M, Schraml P, Moch H,<br />
Faggioni A, Trivedi P, Meister G, Renner C, Grasser<br />
MG. microRNA profiling in EBV-associated B-cell<br />
lymphomas. Nucleic Acids Res. 2010. doi: 10.1093/<br />
nar/gkg1043.<br />
Incitti T, De Angelis FG, Cazzella V, Sthandier O,<br />
Pinnarò C, Legnini I, Bozzoni I. Exon skipping and<br />
Duchenne muscular dystrophy therapy: selection of<br />
the most active U1 snRNA-antisense able to induce<br />
dystrophin exon 51. Mol Ther. 2010, 18: 1675-82.<br />
doi:10.1038/mt.2010.123.<br />
Kettner A, Di Matteo M, Santoni A. Insulin potentiates<br />
FcepsilonRI-mediated signaling in mouse bone<br />
marrow-derived mast cells. Mol Immunol. 2010, 47:<br />
1039-46. doi:10.1016/j.molimm.2009.11.013.<br />
Kovacs D, Cardinali G, Aspite N, Cota C, Luzi<br />
F, Bellei B, Briganti S, Amantea A, Torrisi MR,<br />
Picardo M. Role of fibroblast-derived growth factors<br />
in regulating hyperpigmentation of solar lentigo. Br<br />
J Dermatol. 2010, 163: 1020-27. doi: 10.1111/j.1365-<br />
2133.2010.09946.x.<br />
La Regina G, Coluccia A, Silvestri R. Looking for an<br />
active conformation of the future HIV-1 non-nucleoside<br />
reverse transcriptase inhibitors. Antiviral Chem<br />
Chemoth. 2010, 20: 231-7. doi: 10.3851/IMP1607.<br />
Laneve P, Gioia U, Andriotto A, Moretti F, Bozzoni<br />
I, Caffarelli E. A minicircuitry involving REST and<br />
CREB controls miR-9-2 expression during human<br />
neuronal differentiation. Nucleic Acids Res. 2010, 38:<br />
6895-905. doi: 10.1093/nar/gkq604.
BIBLIOGRAPHY 2010<br />
Lauro C, Cipriani R, Catalano M, Trettel F, Chece<br />
G, Brusadin V, Antonilli L, Van Rooijen N, Eusebi<br />
F, Fredholm BB, Limatola C. Adenosine A1 receptors<br />
and microglial cells mediate CX3CL1-induced<br />
protection of hippocampal neurons against Gluinduced<br />
death. Neuropsychopharmacol. 2010, 35: 1550-<br />
59. doi:10.1038/npp.2010.26.<br />
Le Pera L, Marcatili P, Tramontano A. Picmi: mapping<br />
point mutations on genomes. Bioinformatics.<br />
2010, 26: 2904-5. doi: 10.1093/bioinformatics/<br />
btq547.<br />
Lionetti V, Francocci F, Ferrari S, Volpi C,<br />
Bellincampi D, Galletti R, D’Ovidio R, De Lorenzo<br />
G, Cervone F. Engineering the cell wall by reducing<br />
de-methyl-esterified homogalacturonan improves<br />
saccharification of plant tissues for bioconversion.<br />
Proc Natl Acad Sci USA. 2010, 107: 616-21. doi:<br />
10.1073/pnas.0907549107.<br />
Mancone C, Conti B, Amicone L, Bordoni V,<br />
Cicchini C, Calvo L, Basulto Perdomo A, Fimia G<br />
M, Tripodi M, Alonzi T. Proteomic analysis reveals<br />
a major role of contact inhibition in the execution of<br />
hepatocyte terminal differentiation. J Hepatol. 2010,<br />
52: 234-43I. doi:10.1016/j.jhep.2009.11.013.<br />
Marcellini L, Giammatteo M, Aimola P, Mangoni<br />
ML. Fluorescence and electron microscopy methods<br />
for exploring antimicrobial peptides mode(s)<br />
of action. Methods Mol Biol. 2010, 618:249-66. doi:<br />
10.1007/978-1-60761-594-1_16.<br />
Mastronicola D, Testa F, Forte E, Bordi E, Pucillo<br />
LP, Sarti P, Giuffrè A. Flavohemoglobin and nitric<br />
oxide detoxification in the human protozoan parasite<br />
Giardia intestinalis. Biochem Biophys Res Commun.<br />
2010, 399: 654-8. doi: 10.1016/j.bbrc.2010.07.137.<br />
Messina M, Chiaretti S, Tavolaro S, Peragine N,<br />
Vitale A, Elia L, Sica S, Levis A, Guarini A, Foà R.<br />
Protein kinase gene expression profiling and in vitro<br />
functional experiments identify novel potential therapeutic<br />
targets in adult acute lymphoblastic leukemia.<br />
Cancer. 2010, 116: 3426-37. doi: 10.1002/cncr.25113.<br />
Micheli E, Martufi M, Cacchione S, De Santis<br />
P, Savino M. Self-organization of G-quadruplex<br />
structures in the hTERT core promoter stabilized<br />
by polyaminic side chain perylene derivatives.<br />
Biophys Chem. 2010, 153: 43-53. doi: 10.1016/j.<br />
bpc.2010.10.003.<br />
Miele R, Lattanzi R, Bonaccorsi di Patti MC,<br />
Paiardini A, Negri L, Barra D. Expression of Bv8<br />
in Pichia pastoris to identify structural features for<br />
receptor binding. Protein Expr Purif. 2010, 73: 10-4.<br />
doi: 10.1016/j.pep.2010.04.012.<br />
166<br />
Molfetta R, Gasparrini F, Santoni A, Paolini R.<br />
Ubiquitination and endocytosis of the high affinity<br />
receptor for IgE. Mol Immunol. 2010, 47: 2427-34.<br />
doi: 10.1016/j.molimm.2010.06.003.<br />
Moni L, Ciogli A, D’Acquarica I, Dondoni A,<br />
Gasparrini F, Marra A. Synthesis of sugar-based<br />
silica gels by copper-catalysed azide-alkyne cycloaddition<br />
via a single-step azido-activated silica intermediate<br />
and the use of the gels in hydrophilic<br />
interaction chromatography. Chem Eur J. 2010, 16:<br />
5712-22. doi: 10.1002/chem.201000106.<br />
Montanari A, De Luca C, Frontali L, Francisci<br />
S. Aminoacyl-tRNA synthetases are multivalent<br />
suppressors of defects due to human equivalent<br />
mutations in yeast mt tRNA genes. Biochimica et<br />
Biophysica Acta 2010, 1803: 1050-57. doi:10.1016/j.<br />
bbamcr.2010.05.003.<br />
Moubayidin L, Perilli S, Dello Ioio R, Di Mambro<br />
R, Costantino P, Sabatini S. The rate of cell differentiation<br />
controls the Arabidopsis root meristem<br />
growth phase. Curr Biol. 2010, 20: 1138-1143.<br />
doi:10.1016/j.cub.2010.05.035.<br />
Musarò A, Barberi L. Isolation and culture of<br />
mouse satellite cells. Methods Mol Biol. 2010, 633:<br />
101-11. doi: 10.1007/978-1-59745-019-5_8.<br />
Musarò A, Fulle S, Fanò G. Oxidative stress and muscle<br />
homeostasis. Curr Opin Clin Nutr Metab Care. 2010,<br />
13: 236-42. doi:10.1097/MCO.0b013e3283368188.<br />
Musmuca I, Caroli A, Mai A, Kaushik-Basu N,<br />
Arora P, Ragno R. Combining 3-D quantitative<br />
structure-activity relationship with ligand based and<br />
structure based alignment procedures for in silico<br />
screening of new hepatitis C virus NS5B polymerase<br />
inhibitors. J Chem Inf Model. 2010, 50: 662-76.<br />
doi:10.1021/ci9004749.<br />
Nebbioso A, Dell’Aversana C, Bugge A, Sarno R,<br />
Valente S, Rotili D, Manzo F, Teti D, Mandrup S,<br />
Ciana P, Maggi A, Mai A, Gronemeyer H, Altucci<br />
L. HDACs class II-selective inhibition alters nuclear<br />
receptor-dependent differentiation. J Mol Endocrinol.<br />
2010, 45: 219-28. doi: 10.1677/JME-10-0043.<br />
Nicoletti FP, Comandini A, Bonamore A, Boechi<br />
L, Boubeta FM, Feis A, Smulevich G, Boffi A.<br />
Sulfide binding properties of truncated hemoglobins.<br />
Biochemistry. 2010; 49: 2269-78. doi: 10.1021/<br />
bi901671d.<br />
Nurzia E, Panimolle F, Cauli A, Mathieu A,<br />
Magnacca A, Paladini F, Sorrentino R, Fiorillo MT.<br />
CD8+ T-cell mediated self-reactivity in HLA-B27<br />
context as a consequence of dual peptide conforma-
tion. Clin Immunol. 2010, 135: 476-82. doi: 10.1016/j.<br />
clim.2010.01.009.<br />
Palacios D, Mozzetta C, Consalvi S, Caretti G,<br />
Saccone V, Proserpio V, Marquez VE, Valente S,<br />
Mai A, Forcales SV, Sartorelli V, Puri PL. TNF/<br />
p38α/polycomb signaling to Pax7 locus in satellite<br />
cells links inflammation to the epigenetic control of<br />
muscle regeneration. Cell Stem Cell. 2010, 7: 455-69.<br />
doi: 10.1016/j.stem.2010.08.013.<br />
Palermo V, Cundari E, Mangiapelo E, Falcone C,<br />
Mazzoni C. Yeast lsm pro-apoptotic mutants show<br />
defects in S-phase entry and progression. Cell Cycle.<br />
2010, 9: 3991-96. doi: 10.4161/cc.9.19.13210.<br />
Palermo V, Falcone C, Calvani M, Mazzoni C.<br />
Acetyl-L-carnitine protects yeast cells from apoptosis<br />
and aging and inhibits mitochondrial fission.<br />
Aging Cell. 2010, 9: 570-9. doi: 10.1111/j.1474-<br />
9726.2010.00587.<br />
Paone A, Galli R, Gabellini C, Lukashev D, Starace<br />
D, Gorlach A, De Cesaris P, Ziparo E, Del Bufalo<br />
D, Sitkovsky MV, Filippini A, Riccioli A. Toll-like<br />
receptor 3 regulates angiogenesis and apoptosis in<br />
prostate cancer cell lines through hypoxia-inducible<br />
factor 1 alpha. Neoplasia. 2010; 7: 539-49. doi:<br />
10.1593/neo.92106.<br />
Pasco MY, Rotili D, Altucci L, Farina F, Rouleau<br />
GA, Mai A, Néri C. Characterization of sirtuin inhibitors<br />
in nematodes expressing a muscular dystrophy<br />
protein reveals muscle cell and behavioral protection<br />
by specific sirtinol analogues. J Med Chem. 2010, 53:<br />
1407-11. doi: 10.1021/jm9013345.<br />
Piccinin S, Di Angelantonio S, Piccioni A, Volpini<br />
R, Cristalli G, Fredholm BB, Limatola C, Eusebi F,<br />
Ragozzino D. CX3CL1-induced modulation at CA1<br />
synapses reveals multiple mechanisms of EPSC modulation<br />
involving AR subtypes. J Neuroimmunol. 2010,<br />
224: 85-92. doi:10.1016/j.jneuroim.2010.05.012.<br />
Pierangeli A, Scagnolari C, Gentile M, Spina MT,<br />
Iudicello A, Bertazzoni G, Antonelli G. Virological<br />
diagnosis of respiratory virus infection in patients<br />
attending an emergency department during the<br />
influenza season. Clin Microbiol Infect. 2010, 16: 391-<br />
3. doi: 10.1111/j.1469-0691.2009.03119.x.<br />
Pisano S, Leoni D, Galati A, Rhodes D, Savino M,<br />
Cacchione S. The human telomeric protein hTRF1<br />
induces telomere-specific nucleosome mobility. Nucleic<br />
Acids Res. 2010, 38: 2247-55. doi: 10.1093/nar/gkp1228.<br />
Porzia A, Lanzardo S, Citti A, Cavallo F, Forni G,<br />
Santoni A, Galandrini R, Paolini R. Attenuation of<br />
PI3K/Akt-mediated tumorigenic signals through<br />
167<br />
BIBLIOGRAPHY 2010<br />
PTEN activation by DNA vaccine-induced anti-<br />
ErbB2 antibodies. J Immunol. 2010, 184: 4170-7. doi:<br />
10.4049/jimmunol.0903375.<br />
Prosseda G, Mazzola A, Di Martino ML, Tielker<br />
D, Micheli G, Colonna B. A temperature-induced<br />
narrow DNA curvature range sustains the maximum<br />
activity of a bacterial promoter in vitro. Biochemistry<br />
2010, 49: 2778-85. doi: 10.1021/bi902003g.<br />
Quinti L, Chopra V, Rotili D, Valente S, Amore A,<br />
Franci G, Meade S, Valenza M, Altucci L, Maxwell<br />
MM, Cattaneo E, Hersch S, Mai A, Kazantsev A.<br />
Evaluation of histone deacetylases as drug targets<br />
in Huntington’s disease models. Study of HDACs<br />
in brain tissues from R6/2 and CAG140 knock-in<br />
HD mouse models and human patients and in a neuronal<br />
HD cell model. PLoS Curr. 2010, Sep 22. pii:<br />
RRN1172. doi: 10.1371/currents.RRN1172.<br />
Rinaldi A, Vincenti S, De Vito F, Bozzoni I,<br />
Oliverio A, Presutti C, Fragapane P, Mele A. Stress<br />
induces region specific alterations in microRNAs<br />
expression in mice. Behav Brain Res. 2010, 208: 265-<br />
9. doi:10.1016/j.bbr.2009.11.012.<br />
Rinaldi T, Dallabona C, Ferrero I, Frontali L,<br />
Bolotin-Fukuhara M. Mitochondrial diseases and the<br />
role of the yeast models. FEMS Yeast Res. 2010, 10:<br />
1006-22. doi: 10.1111/j.1567-1364.2010.00685.<br />
Rotili D, Tarantino D, Carafa V, Lara E, Meade S,<br />
Botta G, Nebbioso A, Schemies J, Jung M, Kazantsev<br />
AG, Esteller M, Fraga MF, Altucci L, Mai A.<br />
Identification of tri- and tetracyclic pyrimidinediones<br />
as sirtuin inhibitors. ChemMedChem. 2010, 5: 674-77.<br />
doi: 10.1002/cmdc.201000030.<br />
Ruotolo R, Tosi F, Vernarecci S, Ballario P, Mai<br />
A, Filetici P, Ottonello S. Chemogenomic profiling<br />
of the cellular effects associated with histone H3<br />
acetylation impairment by a quinoline-derived compound.<br />
Genomics. 2010, 96: 272-80. doi:10.1016/j.<br />
ygeno.2010.08.005.<br />
Saladino R, Barontini M, Crucianelli M, Nencioni<br />
L, Sgarbanti R, Palamara AT. Current advances in<br />
Anti-influenza Therapy. Curr Med Chem. 2010; 17:<br />
2101-40.<br />
Saladino R, Neri V, Crestini C, Costanzo G,<br />
Graciotti M, Di Mauro E. The role of the formamide/zirconia<br />
system in the synthesis of nucleobases<br />
and biogenic carboxylic acid derivatives. J Mol Evol.<br />
2010, 71: 100-10. doi: 10.1007/s00239-010-9366-7.<br />
Santonico E, Belleudi F, Panni S, Torrisi MR,<br />
Cesareni G, Castagnoli L. Multiple modification and<br />
protein interaction signals drive the Ring finger protein
BIBLIOGRAPHY 2010<br />
11 (RNF11) E3 ligase to the endosomal compartment.<br />
Oncogene. 2010, 29: 5604-18. doi:10.1038/onc.2010.294.<br />
Scaloni F, Federici L, Brunori M, Gianni S.<br />
Deciphering the folding transition state structure<br />
and denatured state properties of nucleophosmin<br />
C-terminal domain. Proc Natl Acad Sci USA. 2010,<br />
107: 5447-52. doi: 10.1073/pnas.0910516107.<br />
Sciaccaluga M, Fioretti B, Catacuzzeno L, Pagani<br />
F, Bertollini C, Rosito C, Catalano M, D’Alessandro<br />
G, Santoro A, Cantore G, Ragozzino D, Castigli E,<br />
Franciolini F, Limatola C. CXCL12-induced glioblastoma<br />
cell migration requires intermediate-conductance<br />
Ca2+-activated K+ channel activity. Am J<br />
Physiol Cell Physiol. 2010, 299: C175-84 doi: 10.1152/<br />
ajpcell.00344.2009.<br />
Scipioni A, Turchetti G, Morosetti S, De Santis<br />
P. Geometrical, conformational and topological<br />
restraints in regular nucleosome compaction in<br />
chromatin. Biophys Chem. 2010, 148: 56-67. doi:<br />
10.1016/j.bpc.2010.02.010.<br />
Sciumè G, Santoni A, Bernardini G. Chemokines<br />
and glioma: invasion and more. J Neuroimmunol. 2010,<br />
224(1-2): 8-12. doi:10.1016/j.jneuroim.2010.05.019.<br />
Spallotta F, Rosati J, Straino S, Nanni S, Grasselli<br />
A, Ambrosino V, Rotili D, Valente S, Farsetti A, Mai<br />
A, Capogrossi MC, Gaetano C, Illi B. Nitric oxide<br />
determines mesodermic differentiation of mouse<br />
embryonic stem cells by activating class IIa histone<br />
deacetylases: potential therapeutic implications in a<br />
mouse model of hindlimb ischemia. Stem Cells. 2010,<br />
28: 431-42 doi: 10.1002/stem.300.<br />
Specchia V, Piacentini L, Tritto P, Fanti L,<br />
D’Alessandro R, Palumbo G, Pimpinelli S and<br />
Bozzetti MP. HSP90 prevents phenotypic variation<br />
by suppressing the mutagenic activity of transposons.<br />
Nature. 2010, 463: 662-665. doi:10.1038/<br />
nature08739.<br />
Stabile H, Carlino C, Mazza C, Giliani S, Morrone<br />
S, Notarangelo LD, Notarangelo LD, Santoni A,<br />
Gismondi A. Impaired NK-cell migration in WAS/<br />
XLT patients: role of Cdc42/WASp pathway in<br />
the control of chemokine-induced beta2 integrin<br />
high-affinity state. Blood. 2010, 115: 2818-26. doi:<br />
10.1182/blood-2009-07-235804.<br />
168<br />
Tavolaro S, Chiaretti S, Messina M, Peragine N,<br />
Del Giudice I, Marinelli M, Santangelo S, Mauro<br />
FR, Guarini A, Foà R. Gene expression profile of<br />
protein kinases reveals a distinctive signature in<br />
chronic lymphocytic leukemia and in vitro experiments<br />
support a role of second generation protein<br />
kinase inhibitors. Leukemia Res. 2010, 34: 733-41.<br />
doi:10.1016/j.leukres.2009.11.005.<br />
Tempera I, Deng Z, Atanasiu C, Chen CJ, D’Erme<br />
M, Lieberman PM. Regulation of Epstein-Barr virus<br />
OriP replication by poly(ADP-ribose) polymerase 1. J<br />
Virol. 2010, 84: 4988-97. doi: 10.1128/JVI.02333-09.<br />
Thaler F, Varasi M, Colombo A, Boggio R, Munari<br />
D, Regalia N, Rozio MG, Reali V, Resconi AE, Mai<br />
A, Gagliardi S, Dondio G, Minucci S, Mercurio C.<br />
Synthesis and biological characterization of amidopropenyl<br />
hydroxamates as HDAC inhibitors<br />
ChemMedChem. 2010, 5: 1359-1372. doi: 10.1002/<br />
cmdc.201000166.<br />
Tramontano E, Di Santo R. HIV-1 RT-associated<br />
RNase H function inhibitors: recent advances in drug<br />
development. Curr Med Chem. 2010, 17: 2837-2851.<br />
Trettel F, Di Angelantonio S, Limatola C, Ransohoff<br />
RR. Conference <strong>report</strong>: Chemokines and chemokine<br />
receptors in the nervous system, Rome, 24/25<br />
October, 2009; 2nd workshop. J Neuroimmunol. 2010,<br />
224: 1-7. doi:10.1016/j.jneuroim.2010.05.001.<br />
Uccelletti D, Zanni E, Marcellini L, Palleschi C,<br />
Barra D, Mangoni ML. Anti-Pseudomonas activity<br />
of frog skin antimicrobial peptides in a Caenorhabditis<br />
elegans infection model: a plausible mode of action in<br />
vitro and in vivo. Antimicrob Agents Chemother. 2010,<br />
54: 3853-60. doi: 10.1128/AAC.00154-10.<br />
Wilkins BJ, Marionni S, Young DD, Liu J,<br />
Wang Y, Di Salvo ML, Deiters A, Cropp TA.<br />
Site-specific incorporation of fluorotyrosines into<br />
proteins in Escherichia coli by photochemical disguise.<br />
Biochemistry. 2010; 49: 1557-9. doi: 10.1021/<br />
bi100013s.<br />
Zhu H, Shan L, Schiller PW, Mai A, Peng T.<br />
Histone deacetylase-3 activation promotes TNF-<br />
{alpha} expression in cardiomyocytes during<br />
lipopolysaccharide stimulation. J Biol Chem. 2010,<br />
285: 9429-9436. doi: 10.1074/jbc.M109.071274.
Finito di stampare nel mese di maggio 2011<br />
presso il<br />
Centro Stampa Università<br />
Università degli Studi di Roma La Sapienza<br />
P.le Aldo Moro, 5 - 00185<br />
www.editriceateneo.it