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Istituto Pasteur
Fondazione Cenci Bolognetti
Report of activity
2009-2010

© 2011 - Sapienza-Università di Roma
P.le Aldo Moro, 5 - 00185 Roma
Edited by Lucia Ugo
Photos by Giovanni Canitano
www.istitutopasteur.it

Contents
• Forward ..........................................................................................................................................................................
by Paolo Amati, President, and Angela Santoni, Scientific Director
• Boards and Staff .......................................................................................................................................................
• Fellowships awarded in 2009 and 2010 ........................................................................................................
Fellowships awarded for two years for training in foreign laboratories
Fellowships awarded to students who had a two-year experience abroad
Fellowships awarded for one year to students who have completed their PhD and are seeking a position
Fellows working on the research programs of the Foundation
• Seminars ........................................................................................................................................................................
• Meetings and Conferences ..................................................................................................................................
• Scientific Reports
Research area 1: Molecular biology of microorganisms and viruses
Alberto BOFFI, Bacterial globins as regulators of thiol redox equilibrium in bacteria ....................................
Daniela DE BIASE, The acid resistance genes of Escherichia coli: does their complex transcriptional control
hide novel biological roles? ........................................................................................................................................
Alberto FAGGIONI, Epstein-Barr virus interactions with cellular microRNAs .................................................
Paola LONDEI, Translational regulation: from the archaea to the eukarya ........................................................
Research area 2: Pathogenic mechanisms of microbially associated diseases
Guido ANTONELLI, Molecular characterization of viruses causing bronchiolitis and study of viral and
host factors affecting Type I IFN antiviral response induced by respiratory viruses ...............................
Andrea BELLELLI, A structural biology study of schistosomiasis .........................................................................
Maria Lina BERNARDINI, Lipopolysaccharide and peptidoglycan adaptation to host as an immune evasion
strategy of gram-negative pathogens ....................................................................................................................
Bianca COLONNA, Involvement of sRNA molecules in the complex regulatory circuits of virulence gene
expression in Shigella flexneri and in enteroinvasive E. coli ..............................................................................
Anna Teresa PALAMARA, Redox mediated mechanisms involved in the influenza virus replication and in
the pathogenesis of influenza associated diseases ...............................................................................................
Paolo SARTI, Defense mechanisms against oxidative and nitrosative stress in pathogenic protozoa
Maria Rosaria TORRISI, Role of the keratinocyte growth factor receptor on the molecular and cellular
alterations induced by the expression of HPV16 E5 oncoprotein .................................................................
Research area 3: Molecular genetics of eukaryotes
Fiorentina ASCENZIONI, CFTR gene engineering by means of prokaryotic and eukaryotic artificial
chromosome ..................................................................................................................................................................
Irene BOZZONI, RNA-RNA and RNA-protein interactions: role of small non-coding RNAs in gene
expression control .......................................................................................................................................................
Emanuele CACCI, Role of metalloproteinases and their tissue inhibitors in the regulation of neurogenesis
and gliogenesis from neural stem /progenitor cells ...........................................................................................
Paola CAIAFA, Does PARylated PARP-1 introduce an epigenetic mark on chromatin? .................................
Antonella DE JACO, Processing of the Neuroligins proteins and autism-related mutations ........................
Maria D’ERME, Epigenetic modifications in neurodegenerative diseases ............................................................
Lucia FABIANI, DNA replication mechanisms and genome stability in Saccharomyces cerevisiae ....................
Claudio FALCONE, New tools in deciphering aging and apoptotic pathways .....................................................
Laura FRONTALI, Mitochondrial studies and the use of simple eukaryotic models for the analysis of
molecular and cellular aspects of human pathologies .......................................................................................
Maurizio GATTI, The role of membrane trafficking during Drosophila cytokinesis .........................................
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Marco LUCARELLI, The interplay between epigenetics, cell cycle and homologous recombination in gene
therapy by Small Fragment Homologous Replacement (SFHR) ....................................................................
Rossella MAIONE, Interplay between myogenic factors and cell cycle control: regulation and role of the
cdk inhibitor p57kip2 ..................................................................................................................................................
Franco MANGIA, Molecular regulation of cell proliferation in early embryo blastomeres and neuronal
precursors of the mouse ............................................................................................................................................
Antonio MUSARO’, Study of the molecular and cellular mechanisms of sarcopenia: role of mIGF-1 and
oxidative stress .............................................................................................................................................................
Rodolfo NEGRI, Role of the COP9 signalosome in transcription modulation and chromatin organization
in yeast and plants .......................................................................................................................................................
Sergio PIMPINELLI, The biogenesis of piRNAs and their involvement in transposon silencing and
heterochromatin formation in Drosophila ..............................................................................................................
Sabrina SABATINI, To the root of organ growth: the control of root meristem activity in Arabidopsis ....
Mario STEFANINI, Biological characterization and in vitro culture of spermatogonial stem cells ................
Marco TRIPODI, Molecular mechanisms driving liver stem cell fate ....................................................................
Giuseppe ZARDO, Identification of novel genetic and epigenetic targets in Leukemia by genome wide
approaches .....................................................................................................................................................................
Research area 4: Molecular recognition in biomolecules
Fabio ALTIERI, Cellular response to oxidative stress: involvement of protein disulfide isomerases ............
Maurizio BRUNORI, How proteins recognize their biochemical partners: ligand binding and folding
pathways of PDZ domains .......................................................................................................................................
Felice CERVONE, Plant innate immunity: cell wall-mediated signalling and recognition in plant defense
Ernesto DI MAURO, Spontaneous formation and evolution of informational nucleic polymers ...................
Martino Luigi DI SALVO, Synthesis of pyridoxal phosphate in the vitamin B6 salvage pathway and
targeting of the cofactor to apo-enzymes .............................................................................................................
Francesco GASPARRINI, Integrating advanced functional materials and chromatography-mass
spectrometry to examine protein-ligand interactions ........................................................................................
Cristina LIMATOLA, Molecular and functional approaches to investigate the physiopathological role of the
chemokines and their receptors in the central nervous system .......................................................................
Paola PAGGI, Neurone response to experimental injury and lack of dystrophin: a molecular, functional
and structural study in autonomic ganglia in vivo and in vitro ........................................................................
Maria SAVINO, Structural and superstructural features of human telomeric chromatin ...............................
Research area 5: Cellular and molecular immunology
Vincenzo BARNABA, Interplay amongst chronic immune activation, apoptosis, cross-presentation,
immune-regulation, and autoimmunity .................................................................................................................
Roberto FOA’, Potential role of miRNAS in IgM-mediated signal transduction in normal and neoplastic
B cells ..............................................................................................................................................................................
Rossella PAOLINI, Signalling events negatively regulating FcepsilonRI expression and mast cell
functional responses ....................................................................................................................................................
Enza PICCOLELLA, Analysis of the molecular mechanisms regulating FOXP3 gene and protein expression
in TCR- and CD28-activated CD4+CD25-T cells and their influence on regulatory functions ............
Angela SANTONI, Anti-tumor effector functios of NK cells in tumor microenviroment ...............................
Isabella SCREPANTI, Dissection of Notch signaling-dependent pathways involved in the progression of
T cell leukemia .............................................................................................................................................................
Rosa SORRENTINO, The role of HLA-B27 in autoimmunity: from the genetics to the function .................
Loretta TUOSTO, CD28 co-stimulatory molecule as a key regulator of T lymphocyte differentiation and
survival: characterisation of the biochemical pathways and molecules coupling CD28 to NF-kB activation
Elio ZIPARO, The role of Toll Like Receptors in immune responses to infections and in inflammationassociated
pathologies of the male reproductive system ..................................................................................
Research area 6: New antimicrobial and antiviral agents
Donatella BARRA, Peptide effectors of innate immunity ........................................................................................
Mariangela BIAVA, New pyrrole derivatives of BM 212: a new class of antimycobacterial agents. Design,
synthesis, biological evaluation and study of their mode of action ...............................................................
Roberto DI SANTO, Pyrrolyl diketo hexenoic acid derivatives as novel anti-HIV agents targeted to the
ribonuclease H function of the HIV-1 reverse transcriptase enzyme ............................................................
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Antonello MAI, Design, synthesis and biological evaluation of small molecule epigenetic modulators: a
novel approach for anticancer, antifungal, and antiviral chemotherapy ........................................................
Elena MATTIA, Effects of resveratrol on Epstein Barr Virus latent and lytic phases of infection ..............
Romano SILVESTRI, Drug design and synthesis of non-nucleoside inhibitors of both HIV-1 wild type and
resistant mutant strains reverse transcriptase, and Coxsackie b4 virus .......................................................
Research area 7: Biology of Malaria and other vector-borne diseases
Alessandra DELLA TORRE e Vincenzo PETRARCA, Genetic and phenotypic characterization of species and
“molecular forms” of the Anopheles gambiae complex (Diptera: Culicidae), afrotropical malaria vectors
David MODIANO, Immune responses to malaria and autoimmune disorders: investigating common generegulatory
networks ...................................................................................................................................................
Anna TRAMONTANO, Computational Analysis of the gene products of the Plasmodium falciparum
genome and their interaction with human proteins ...........................................................................................
Start-up program
Giuseppe LUPO, Specification and maintenance of retinal stem cells ..................................................................
• Bibliography ...............................................................................................................................................................
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Forward
The Istituto Pasteur-Fondazione Cenci Bolognetti is a private non-profit foundation which was
established according to the terms of the bequest of Princess Beatrice Fiorenza Cenci Bolognetti to
Sapienza University of Rome for the general purpose of encouraging scientific research in the area of
pasteurian sciences and specifically for founding a Pasteur Institute in Rome. Since 1970 the Institute
has been part of a global network of Pasteur Institutes known as Réseau International des Instituts
Pasteur et Instituts Associés. The funding of its activity derives from the rent of three historical palaces
in Rome and during the period of the present report of activity the Istituto Pasteur-Fondazione
Cenci has been supported by the Fondazione Roma and the BNP Paribas BNL that are gratefully
acknowledged.
The research activity of the Istituto Pasteur-Fondazione Cenci Bolognetti is committed to
studying the mechanisms that regulate the basic processes of life. This mission is pursued through
the funding of research projects, the award of fellowships and the organization of scientific meetings
and seminars.
At a time when attention of granting agencies and science policy makers in Italy and Europe is largely
focused towards application, it should not be forgotten that important breakthroughs in biology and
medicine often originated from curiosity driven research. Starting from this premise, the Istituto
Pasteur-Fondazione Cenci Bolognetti has concentrated on the selection of applications exclusively
on scientific merit, without bias for application. Our scientific programs reflect this philosophy. The
research projects illustrated in this Report of activity, were selected by a peer review system based
on highly qualified international referees, to whom we are very grateful. The general themes witness
a modern interpretation of the sciences pasteuriennes, with adherence to the mission of the Foundation
Training young researchers has always been a top priority of the Foundation, which is achieved by:
i) providing fellowships to work abroad as well as at the Sapienza-University of Rome; ii) co-funding
the international Ph.D. program in Pasteurian Science at the Sapienza; and iii) financing fellowships for
the first year after the PhD; iv) beginning in 2009, the funding of start-up grants for young brilliant
researchers.
The institute’s development plan has scheduled the construction of its own laboratories for the near
future. These facilities will host leading researchers of all nationalities operating in the field of
pasteurian sciences, with special reference to the molecular bases of human pathologies.
The results obtained during the years 2009 and 2010, documented in this volume, witness the
enthusiasm and productivity of the scientists involved.
Paolo Amati
President
VII
Angela Santoni
Scientific Director

Boards and Staff
Administrative Board
The Board of Administration is presided over
by a President appointed by the Rector of
Sapienza-University of Rome chosen from a list
of three names selected during a joint session of
the board and the Scientific Council. Members
of the board include a Scientific Director, four
members of the Faculties of Pharmacy and
Medicine, Natural Sciences, and Medicine and
Dentistry. Other members are a legal expert
designated by the University of Rome Board
of Administration, three auditors nominated by
the University, the Ministry of the Treasury,
and the Ministry of Universities and Scientific
Research.
President
Paolo Amati (Pharmacy and Medicine)
Members
Paolo Costantino (Natural Sciences), Alberto
Faggioni (Medicine and Dentistry), David
Modiano (Pharmacy and Medicine), Angela
Santoni (Pharmacy and Medicine), Romano
Silvestri (Pharmacy and Medicine)
Secretary
Emanuela Gloriani
Administrative Expert
Giuseppina Capaldo
Auditors
Anna Carmela Ferrante, Simona Ranalli,
Carla Vassallo
VIII
Scientific Council
The Scientific Council is a board of seven
scholars in the field of the pasteurian sciences.
Members are elected to a four-year term by the
Faculties Natural Sciences, and Pharmacy and
Medicine. The scientific director, appointed by
the Scientific Council, is ex-officio a member
of the Board of Administration. The Scientific
Council attends to examine and co-ordinate the
research programs as well as the several scientific
activities.
Scientific Director
Angela Santoni (Pharmacy and Medicine)
Members
Ernesto Di Mauro (Natural Sciences), Laura
Frontali (Natural Sciences), Anna Teresa
Palamara (Pharmacy and Medicine), Anna
Tramontano (Pharmacy and Medicine), Marco
Tripodi (Pharmacy and Medicine), Carlo
Turano (Pharmacy and Medicine)
Secretariat
Sarah Gainsforth, Maria Pia Lorenzoni,
Lynda Romani, Nicoletta Silvestri, Lucia Ugo
Consultants
Tommaso De Dominicis (legal affairs), Anna
Maria Pivetti (architectural supervision),
Barbara Hell (financial affairs)

Fellowships awarded in 2009 and 2010
Fellowships awarded for two years for
training in foreign laboratories
Fabio DI DOMENICO, at University of Kentucky,
from Department of Biochemical Sciences
Daniela DIMASTROGIOVANNI, at Department of
Biochemistry, University of Cambridge, UK, from
Department of Biochemical Sciences
Michela ESPOSITO, at Institut de Génétique
et Microbiologie, Université Paris XI, from
Department of Biology and Biotechnologies «Charles
Darwin»
Roberto GAETANI, at Department of Cardiology,
University Medical Center, Utrecht, NL, from
Department of Molecular Medicine
Isabelle JOURDAN, at Inserm, Université Pierre et
Marie Curie, Paris, France, from Department of
Anatomical, Histological, Forensic & Orthopaedic
Sciences
Marta MORETTI, at Division of Immunology and
Inflammation, Imperia College, London, UK, from
Department of Molecular Medicine
Tommaso MOSCHETTI, at Department of
Biochemistry, University of Cambridge, UK, from
Department of Biochemical Sciences
Giuseppe SCIUMÈ, at NIH/NIAMS, Bethesda, MD,
USA, from Department of Molecular Medicine
Piera SMERIGLIO, at ISERM, Université Pierre
et Marie Curie, from Department of Anatomical,
Histological, Forensic & Orthopaedic Sciences
Fellowships awarded to students who had a
two-year experience abroad
Daniela CECCARELLI, at Department of Biology and
Biotechnologies “Charles Darwin”, from Université
de Sherbrooke, Québec, Canada
Antonio COLUCCIA, at Department of Medicinal
Chemistry and Technologies, from School of
Pharmacy, University of Cardiff, UK
Valentina MANGANO, at Department of Public
Health and Infectious Diseases, from MRC Centre
for Genomics and Global Health, Oxford, UK
Bich Lich NGUYEN, at Department of Cardiovalular
Pathophysiology, Anesthesiology and General
Surgery, from Cardiology Department of the Cedars-
Sinai Medical Center, California, USA
Fellowships awarded to students who have
completed their PhD and are seeking a position
Eleni ANASTASIADOU, at Department of
Experimental Medicine
IX
Tonino ANELLI, at Department of Biology and
Biotechnologies “Charles Darwin”
Cristina CAPUANO, at Department of Molecular
Medicine
Isotta CHIMENTI, at Department of Molecular
Medicine
Alessandra GALATI, at Department of Biology and
Biotechnologies “Charles Darwin”
Francesca GASPARRINI, at Department of
Molecular Medicine
Luigi LEMBO FAZIO, at Department of Biology and
Biotechnologies “Charles Darwin”
Adriana MAGNACCA, at Department of Biology
and Biotechnologies “Charles Darwin”
Marianna PELLONI, at Department of Molecular
Medicine
Marianna Nicoletta ROSSI, at Department of Cell
Biotechnology and Hematology
Fellows working on the research programs
of the Foundation
Salvatore ALFONSO, at Department of Medicinal
Chemistry and Technologies
Francesco ANGELUCCI, at Department of
Biochemical Sciences
Valentina ANNESE, at Department of Biology and
Biotechnologies “Charles Darwin”
Chiara ARDICCIONI, at Department of Biochemical
Sciences
Pamela AVELLINO, at Department of Public Health
and Infectious Diseases
Enrico M. BATASSA, at Department of Cell
Biotechnology and Hematology
Nicoletta CALOSCI, at Department of Biochemical
Sciences
Cristina CAMPERIO, at Department of Biology and
Biotechnologies “Charles Darwin”
Cristina CAPUANO, at Department of Molecular
Medicine
Maria Rosaria CARBONE, at Department of Cell
Biotechnology and Hematology
Nicoletta CASTIGLIONE, at Department of
Biochemical Sciences
Ignacio Celestino, at Department of Public Health
and Infectious Diseases
Marcella CESANA, at Department of Biology and
Biotechnologies “Charles Darwin”
Anna CHAILYAN, at Department of Biochemical
Sciences
Isotta CHIMENTI, at Department of Molecular

Medicine
Alberto CIOLFI, at Department of Cell
Biotechnology and Hematology
Ivan COLADARCI, at Department of Biology and
Biotechnologies “Charles Darwin”
Antonio COLUCCIA, at Department of Medicinal
Chemistry and Technologies
Domenico COZZETTO, at Department of
Biochemical Sciences
Simona CUGUSI, at Department of Biology and
Biotechnologies “Charles Darwin”
Cristina DE LUCA, at Department of Biology and
Biotechnologies “Charles Darwin”
Francesca DE VITO, at Department of Biology and
Biotechnologies “Charles Darwin”
Donatella D’ELISEO, at Department of Molecular
Medicine
Enea Gino DI DOMENICO, at Department of
Biology and Biotechnologies “Charles Darwin”
Maria Letizia DI MARTINO, at Department of
Biology and Biotechnologies “Charles Darwin”
Matteo DI PENTIMA, at Department of Biology and
Biotechnologies “Charles Darwin”
Michela ESPOSITO, at Department of Biology and
Biotechnologies “Charles Darwin”
Antonella FARINA, at Department of Experimental
Medicine
Fedra FRANCOCCI, at Department of Biology and
Biotechnologies “Charles Darwin”
Alessandra GALATI, at Department of Biology and
Biotechnologies “Charles Darwin”
Francesca GARIBALDI, at Department of Cell
Biotechnology and Hematology
Francesca GASPARRINI, at Department of
Molecular Medicine
Roberta GONNELLA, at Department of
Experimental Medicine
Caterina GRILLO, at Department of Biochemical
Sciences
Vittoria IONTA, at Department of Molecular
Medicine
Ilias KOUTRIS, at Department of Biochemical
Sciences
Loredana LE PERA, at Department of Biochemical
Sciences
Lanfranco LEO, at Department of Biology and
Biotechnologies “Charles Darwin”
Rosalba LEPORE, at Department of Biochemical
Sciences
Fabrizio LOMBARDO, at Department of Public
Health and Infectious Diseases
Vincenzo LUCA, at Department of Biochemical
Sciences
Emiliano MANCINI, at Department of Public Health
and Infectious Diseases
X
Paolo MARCATILI, at Department of Biochemical
Sciences
Roberto MATTIOLI, at Department of Biology and
Biotechnologies “Charles Darwin”
Emanuela MICHELI, at Department of Biology and
Biotechnologies “Charles Darwin”
Chiara MICOLONGHI, at Department of Biology
and Biotechnologies “Charles Darwin”
Vanessa MODESTI, at Department of Biology and
Biotechnologies “Charles Darwin”
Rosa MOLFETTA, at Department of Molecular
Medicine
Arianna MONTANARI, at Department of Biology
and Biotechnologies “Charles Darwin”
Linda Celeste MONTEMIGLIO, at Department of
Biochemical Sciences
Gaëlle NOEL, at Department of Biology and
Biotechnologies “Charles Darwin”
Vanessa PALERMO, at Department of Biology and
Biotechnologies “Charles Darwin”
Valeria Luciana PALUMBO, at Department of
Biology and Biotechnologies “Charles Darwin”
Claudia PELLACANI, at Department of Biology and
Biotechnologies “Charles Darwin”
Serena PERILLI, at Department of Biology and
Biotechnologies “Charles Darwin”
Francesco PISCITELLI, at Department of Medicinal
Chemistry and Technologies
Alessandra PORZIA, at Department of Molecular
Medicine
Pasqualina PUNZI, at Department of Biology and
Biotechnologies “Charles Darwin”
Wilhelmina RENSEN, at Department of Biology and
Biotechnologies “Charles Darwin”
Cinzia RIZZO, at Department of Public Health and
Infectious Diseases
Dimitri RYKUNOV, at Department of Biochemical
Sciences
Maria Isabel SANTORI, at Department of Biology
and Biotechnologies “Charles Darwin”
Daniel SAVATIN, at Department of Biology and
Biotechnologies “Charles Darwin”
Stefano SECHI, at Department of Biology and
Biotechnologies “Charles Darwin”
Riccardo SILIGATO, at Department of Biology and
Biotechnologies “Charles Darwin”
Aise Ozlem TASTAN BISHOP, at Department of
Biochemical Sciences
Simona TROMBETTI, at Department of Molecular
Medicine
Lucia TUFANO, at Department of Biology and
Biotechnologies “Charles Darwin”
Elena ZANNI, at Department of Biology and
Biotechnologies “Charles Darwin”

Seminars
2009
February 2 • Protein folding, misfolding and neurodegenerative
diseases
Prof. Mikael Oliveberg, University of Stockholm,
Sweden
March 16 • Final touches to nucleosome positioning
sequence pattern
March 23 • The earliest stages of (bio) molecular life
Prof. Edward N. Trifonov, University of Haifa,
Israel, and Masaryk University, Brno, Czech
Republic
April 22 • Inflammatory cytokine gene regulation
through intrinsic RNA elements that activate the cell’s
RNA-dependent protein kinase
May 11 • How Ebola virus blocks the host antiviral
response
Prof. Raymond Kaempfer, Faculty of Medicine,
The Hebrew University, Jerusalem, Israel
May 4 • Challenging the delusion: Histone H2A.Bbd
structure and function
Prof. Juan Ausio, University of Victoria, Canada
May 5 • Estimating an upper bound for transition path
times in protein folding from single molecule photon
trajectories
Prof. William A. Eaton, Laboratory of Chemical
Pysics, NIH, MD, USA
June 19 • Understanding the regulation and specific
inhibition of protein kinases using large scale structural
comparison
Prof. Stefan Knapp, Structural Genomics
Consortium, Oxford University, UK
June 19 • Vieving Neuronal Signaling by NMR
Spectroscopy and X-ray Crystallography
Prof. Mingjie Zhang, University of Hong Kong
June 30 • Darwin e il Vaticano: Storia di una evoluzione
Prof. Antonio Lazcano, Universidad Nacional
Autónoma de México
October 16 • Structural studies of antiviral lectins,
potential ingredients of anti-HIV microbicides
Prof. Alex Wlodawer, National Cancer Institute
at Frederick, MD, USA
XI
November 19 • Molecular mechanisms of daily biological
clock, the tale from a simple eukaryotic organism
Prof. Yi Liu, University of Texas Southwestern
Medical Center, Dallas, USA
December 18 • Darwin’s Revolution: Natural
Theology to Natural Selection
Prof. Francisco J. Ayala, University of California,
Irvine, USA
2010
March 30 • Folding-induced ion transfer in the ALSassociated
protein SOD1
Prof. Mikael Oliveberg, University of Stockholm,
Sweden
April 30 • Sex and Death in Bdelloid Rotifers
Prof. Mathew Meselson, Harvard University,
Cambridge, MA, USA
May 20 • Control of protective immunity through
dynamic refolding of mRNA
May 27 • Making hemoglobin: why stress is good for you,
a story of mRNA splicing
Prof. Raymond Kaempfer, Faculty of Medicine,
The Hebrew University, Jerusalem, Israel
October 18 • Leucemia acuta mieloide con mutazioni
di nucleofosmina (NPM1): dalla lesione genetica alla
clinica
Prof. Brunangelo Falini, Università degli Studi di
Perugia, Italy
October 25 • Regolazione della latenza nel virus di
Epstein Barr. L’interessante caso di CTCF, una proteina
multi-funzione
Dr. Italo Tempera, Wistar Institute, University
of Philadelphia, USA
December 6 • Identification of new drugs targeting
redox biochemistry for the control of Schistosomiasis
Prof. David Williams, Rush University Medical
Center, Chicago, USA

Meetings and Conferences organized or supported by the Foundation
Istituto Pasteur Science Days
Liver development and disease
Rome, October 9-10, 2009
• Organogenesis
• Immunity and Immunopathology
• Fibrosis and tumorigenesis
Joint Workshop
New frontiers in the pathogenesis and
treatment of infectious diseases
Rome, March 25, 2010
In collaboration with the Stephan Angeloff Institute
of Microbiology of Sofia
European Day of Immunology
Un amico sorprendente: il tuo sistema
immunitario
Rome, April 29, 2010
Interactive study experience on the basic mechanisms
of the immune response
22° Pasteur-Weizmann Symposium – 35° Anniversary
of Pasteur-Weizmann
Stem cells: from basic biology to translational
medicine
Rome, June 7-9, 2010
In collaboration with the Foundation
• Muscle stem cells
• Epithelial stem cells and tissue
reprogramming
• Embryo and postnatal stem cells
• Embryonic stemm cells and induced
pluripotency
• Neuronal stem cells
• Stem cell niches
XII
Mini-Workshop in
Epigenetics
Rome, September 10, 2010
Associazione Genetica Italiana - Genetics School
Lessons
Le malattie genetiche: genetica, epigenetica
e altro…
Cortona, November 25-26, 2010
with the contribution of the Foundation
Lincei International Symposium on
Protein structure and dynamics
Rome, December 1-3, 2010
with the contribution of the Foundation
• Folding and binding
• Dynamics and complexity of the
conformational landscape
• Single molecule biophysics

AREA 1
Molecular
biology of
microorganisms
and
viruses

AREA 1
Bacterial globins as regulators of thiol redox equilibrium in bacteria
Principal investigator: Alberto Boffi
Professor of Molecular Biology
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910990; Fax: (+39) 06 4440062
alberto.boffi@uniroma1.it
Participants:
Alessandra Bonamore, Alberto Macone, research fellows;
Andrea Ilari, CNR researcher; Paola Baiocco, post-doc fellow.
Collaborations:
Dipartimento di Chimica, Università di Firenze (Prof. Giulietta
Smulevich), CPC Biotech srl, Napoli (Dr. Fabio Arenghi),
Departamento de Química Inorgánica, Analítica y Química Física,
Universidad de Buenos Aires (Dr. Leonardo Boechi).
Report of activity
The specific targets of the project concern the elucidation
of the mechanism of heme based thiol oxidation
in bacterial truncated globins (trHbs) and the
discovery of novel pathways in thiol redox homeostasis
in bacteria. The first point entails a complete
biochemical characterization of the observed
catalytic activity towards sulfide reactive species and
model substrates such as cysteine and glutathione.
The understanding of the kinetic properties of thiol
oxidation is a prerequisite for the elucidation of
the reaction mechanism. Spectroscopic techniques
have been applied in order to characterize the reaction
profiles as a function of standard parameters
(pH, salt composition, reducing environment etc.,
see methods). In particular, a complete resonance
Raman spectroscopic characterization of the globins
from Thermobifida fusca and Bacillus subtilis has been
carried out both on native proteins and in selected
active-site mutants (Droghetti et al., 2010; Nicoletti
et al., 2010). The Raman studies brought out that
these globins display the spectroscopic signature
characteristics of heme peroxidases, with a strong
electron donation from the heme iron to the distal
ligand both in their ferrous and ferric adducts.
X-ray crystallography, coupled to X-Ray absorption
(EXAFS) and fluorescence (XANES) studies have
ascribed the peroxidase character to an unusually
3
short bond distance between the iron atom and the
proximal histidine residue (Arcovito et al., 2010). On
these basis, a genuine peroxidase activity has also
been described in the interactions of Thermobifida
fusca truncated hemoglobin, and on a set of selected
active site mutants, with a battery of typical peroxidase
substrates (Torge et al., 2009). A very high specific
peroxidase activity has been observed towards
several substrates, different from those typical of
vegetal peroxidases. However, the most significant
observations in relation with bacterial biochemistry
and physiology are centered on the unusually high
affinity of both Thermobifida fusca and Bacillus subtilis
trHb towards thiol containing derivatives. In
particular, these proteins, when overexpressed in E.
coli, are capable of sequestering virtually all hydrogen
sulfide produced within the host metabolism
and of oxidizing cysteine and other thiol containing
compounds thus altering the thiol redox homeostasis
and sulphur related metabolic pathways (Nicoletti et
al., 2010). The nature of the heme-sulfide complexes
has been further explored in spectroscopic experiments
that allowed for the first time the assignment
of the specific iron-sulfur stretching frequencies in
a hemoprotein (Nicoletti et al., 2010). In parallel,
molecular dynamics of sulfide motion inside the
heme pocket has been investigated and highlighted
a novel mechanism of ligand stabilization within
the heme pocket, based on hydrogen bonding from
a conserved tryptophan residue to the iron bound
sulfur atom (Nicoletti et al., 2010). X-ray crystallographic
studies are in progress in order to determine
the geometry of thiol-globin complexes by means of
crystal soaking studies and/or co-cristallization with
non-reactive analogues (e.g. selenocysteine).
The second target of the research concerns the identification
of partner proteins involved in the complex
physiological network of thiol homeostasis. At least
in the case of Bacillus subtilis, the partner protein

Alberto Boffi
YjbH has been identified already (Larsson et al.,
2007). Bioinformatic methods have been applied in
order to single out possible paralogues in actinomyces,
including mycobacteria. The aim of the “in silico”
search is to establish potential redox partners belonging
to the thioredoxin superfamily and identify novel
pathways within the network of thiol-disulfide oxidoreductases.
On these basis, it is suggested that the
globin may be able to catalyze disulfide bonds formation
and thus act as a heme based peroxiredoxin. The
first step towards the identification of a novel thiol
redox pathway in Bacillus subtilis concerns the demonstration
of the postulated interaction between the
globin (yjbI) and the “disulfide isomerase-like” protein
(yjbH) located upstream within the same operon. In
this framework, the whole operon contaning yjbI and
yjbH has been cloned both as a copy of the original
operon from B. subtilis and as a synthetic gene, optimized
for expression in E. coli. Overexpression of
the whole operon allowed us to single out that trHb
is capable of direct oxidation of the -CxxC- motif
of YjbH thus regenerating active protein. YjbH has
been previously demonstrated to be necessary for the
proteolytic control of the global stress regulator Spx
under disulfide stress and directly interacts with the
global regulator Spx thus preventing its proteolitic
degradation. It is envisaged that only the oxidized
form of YjbH is able to perform such regulatory
interaction and thus, trHb (YjbI) should restore the
disulfide bond by directly interacting with YjbH. In
order to clarify the nature of trHb-YjbH interaction,
analytical ultracentifugation experiments are
in progress under different experimental conditions
(buffer composition, presence of reductants, absence
of oxygen). Thereafter, X-ray crystallographic studies
of the trHb-YjbH complex will be carried out. In
parallel, a metabolomic study has started in order to
verify whether trHb act by oxidizing the low molecular
weight thiol containing species within the cytosol
and thus modulates directly the thiol redox equilibrium.
In this case, low molecular weight thiols have
been monitored by LC/MS methods, using E. coli
overexpressing YjbIH operon as model organism. As
expected, a tenfold increase in cytosolic free cysteine
4
is observed with an unprecedented metabolic drift
towards the synthesis of sulfur aminoacids. However,
the final picture underlyning the role of the truncated
globins within the thiol redox homeostasis is still far
from complete. In our understanding, it is clear that
trHbs act as redox switches in that they are capable
of oxidizing disulfide bonds of the YjbH regulator
which, in turn, governs the activity of the global
stress regulator Spx by expiring its lifespan and
quenching the transcription of genes associated with
oxidative stress. In this framework, trHbs would be
terminators of the redox stress response. However,
trHbs are also capable of efficient sequestering of all
free hydrogen sulfide present in the cell thus shifting
the metabolism towards sulfur compound anabolism,
a role that point to trHbs as active effectors in counterbalancing
the oxidative stress through production
of more complex thiol species. The solution of this
apparently contrasting biological functions will be
addressed in the second year of the project, by examining
in detail the nature of the interactions of trHbs
with their partner proteins.
Publications
Arcovito A, Bonamore A, Hazemann JL, Boffi A,
D’Angelo P. Unusual proximal heme pocket geometry
in the deoxygenated Thermobifida fusca: a combined
spectroscopic investigation. Biophys Chem.
2010, 147: 1-7. doi:10.1016/j.bpc.2009.11.006.
Droghetti E, Nicoletti FP, Bonamore A, Boechi
L, Pau AM, Estrin D, Boffi A, Smulevich G, Feis
A. Heme pocket structural properties of a bacterial
truncated hemoglobin from Thermobifida fusca.
Biochemistry. 2010, 49: 10394-402. doi:10.1021/
bi101452k.
Nicoletti FP, Comandini A, Bonamore A, Boechi
L, Boubeta FM, Feis A, Smulevich G, Boffi A.
Sulfide binding properties of truncated hemoglobins.
Biochemistry. 2010, 49: 2269-78. doi: 10.1021/
bi901671d.

The acid resistance genes of Escherichia coli: does their complex
transcriptional control hide novel biological roles?
Principal investigator: Daniela De Biase
Professor of Biochemistry
Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche
Sede di Latina
Tel: (+39) 0773 1757212; Fax: (+39) 0773 1757254
daniela.debiase@uniroma1.it
Participants:
Francesco Bossa, professor; Angela Tramonti, CNR
researcher; Eugenia Pennacchietti, post-doc fellow; Daniela
Bastianelli, PhD student; Denise Galzerano, Gaia Grassini,
undergraduate students.
Collaborations:
Paul Scherrer Institute, Villigen PSI, Switzerland (Dr. Guido
Capitani); Wageningen University and Research Centre,
Wageningen, The Netherlands (Prof. Maurice C.R. Franssen);
School of Biosciences University of Wales, Cardiff, UK (Prof.
Robert A. John); CNRS-ENS de Cachan, Cachan, France (Dr.
Sylvie Rimsky); Institute of Molecular Cell Biology, VU University
Amsterdam, The Netherlands (Prof. Joen Luirink).
Report of activity
Our research project focusses on the analysis of the
molecular bases that in Escherichia coli lead to acid
resistance (AR), a necessary pre-requisite to breach
the gastric barrier and gain access into the host gut.
In particular we are investigating the regulation and
the mechanism of action of the most efficient acid
resistance system in E. coli, the glutamate-dependent
acid resistance (GDAR) system, which is functional
not only in commensal and pathogenic E. coli, but
also in Shigella flexneri and Listeria monocytogenes. We
aim to unveil the specific role played by the transcriptional
regulators GadX and GadE, also with respect
to the genome silencer H-NS, as well as the structurefunction
relationships in the structural components of
the GDAR system. Recent reports in the fields of
quorum sensing and biofilm formation showed that
some GDAR genes are switched on or off upon specific
stimuli or growth conditions. Therefore we are
performing experiments aimed at uncovering novel
biological roles of the GDAR genes.
The GDAR system requires the intracellular protonconsuming
activity of two glutamate decarboxylase
paralogs, GadA and GadB, as well as the antiport
activity of an integral membrane protein, GadC, which
5
AREA 1
exchanges glutamate for the decarboxylation product
γ-aminobutyrate (GABA). The gadB and gadC structural
genes are co-transcribed, while the gadA structural
gene, which is 2.1 Mb from gadBC, can be either
independently transcribed or co-transcribed with the
downstream gadX gene. The gadA gene belongs to
the so-called acid fitness island (AFI), a 15-kb genome
region repressed by H-NS (the histone-like nucleoidstructuring
protein) and controlled by RpoS, (the stationary
phase σ factor of the RNA polymerase). The
AFI contains 13 genes that encode 12 proteins and a
small RNA (GadY); the genes are mainly organised
into operons and the protein products of the AFI contribute
to E. coli AR in different ways.
The gadA and gadBC promoter regions are under
the control of a very complex regulatory network
that integrates multiple environmental signals and
physiological adaptations. The essential regulator
GadE is under the control of three different activation
circuits. The GadX/GadW-dependent circuit,
the main object of our investigation, involves the
specific transcriptional regulators GadX, GadW and
GadY, encoded by the corresponding genes in the
AFI, and also the global regulators H-NS, CRP and
RpoS. GadX and GadW are AraC-like transcriptional
regulators, capable of up-regulating the gadA and
gadBC structural genes by direct binding to a 42-bp
sequence in their 5’ regulatory regions (Tramonti et
al., J. Bacteriol. 2006, 188:8118-8127). Interestingly,
also the 5’ regulatory regions of the AFI operons
slp-yhiF, hdeAB-yhiD, hdeD, gadE-mtdEF and gadY
are direct targets of these activators (Tramonti et
al., Mol. Microbiol. 2008, 70:965-982). We generated
a 42-bp GadX/GadW consensus sequence by aligning
the six 42-bp long GadX/GadW-binding sites
identified by DNAse I footprinting analyses. This
sequence arises from the tandem arrangement of two
21-bp sequences, in which nine positions (43%) are
highly-to-strictly conserved.
In the period 2009-2010 we carried out experiments
aimed at fully characterizing the GadX regulon

Daniela De Biase
(manuscript in preparation) and at analyzing GadB
site-specific mutants to establish the sequence of
steps that allow this enzyme to control its activity
in the cell. We also set up the basis to investigate
the GadX-H-NS interplay at different targets and
produced GDAR genes deletion mutants to be used
in motility and biofilm formation assays.
Characterization of the GadX regulon
To fully characterize the GadX regulon in the E. coli
K12 strain MG1655, we used chromatin immunoprecipitation
coupled with microarrays (ChIP on chip).
Cultures expressing the 3xFlag-tagged GadX were
grown in LB-MES, pH 5.5, to the stationary phase
and then treated with formaldehyde to crosslink the
DNA-protein complexes. Immunoprecipitation using
anti-Flag antibodies yielded DNA-GadX complexes.
The corresponding DNA fragments were analysed
on microarrays specifically designed for Cy5 (or
Cy3)-labelled DNA fragments obtained from ChIP
experiments and consisting of 21321 60-mer oligonucleotide
probes that match MG1655 sequences at
intervals of 160 bp (OGT, UK). For each probe, the
Cy5/Cy3 (or Cy3/Cy5) ratio was measured and this
was plotted against the corresponding position on the
E. coli MG1655 genome. More than 60 peaks in the
fluorescence ratio are easily distinguishable from the
background signal and therefore identify in vivo GadX
binding sites. Some of the highest peaks are located in
the genomic positions corresponding to known GadX
targets, i.e. gadBC and AFI regulatory regions. More
than 30 DNA fragments corresponding to the genomic
regions encompassing the major peaks were analysed
by EMSA in the presence of GadX. In most of
the cases, we were able to reproduce in vitro (EMSA)
the in vivo data (ChIP-chip). Some of the GadX targets
were also selected for DNaseI footprinting analysis to
improve the GadX consensus sequence.
6
Structure-function relationships in GadB
GadB exhibits an acidic pH optimum and undergoes
a spectroscopically detectable and strongly cooperative
pH-dependent conformational change involving
at least six protons. Crystallographic studies showed
that at mildly alkaline pH GadB is inactive because all
active sites are locked by the C-termini and that the
340 nm absorbance is an aldamine formed by the pyridoxal
5’-phosphate-Lys276 Schiff base with the distal
nitrogen of His465, the penultimate residue in the
GadB sequence (Gut et al., EMBO J. 2006, 25:2643-
2651). We have shown that His465 has a massive
influence on the equilibrium between active and
inactive forms, the former being favoured when this
residue is absent. His465 contributes with n ≈ 2.5 to
the overall cooperativity of the system. The residual
cooperativity (n ≈ 3) is associated with the conformational
changes still occurring at the N-terminal
ends, regardless of the mutation. His465, dispensable
for the cooperativity that affects enzyme activity, is
essential to include the conformational change of the
N-termini into the cooperativity of the whole system.
In the absence of His465, a 330-nm absorbing species
appears, with fluorescence emission spectra more
complex than model compounds and consisting of two
maxima at 390 and 510 nm. Because His465 mutants
are active at pH well above 5.7, we suggest that they
might be employed for biotechnological applications.
Publications
Pennacchietti E, Lammens TM, Capitani G, Franssen
MC, John RA, Bossa F, De Biase D. Mutation of
His465 alters the pH-dependent spectroscopic properties
of Escherichia coli glutamate decarboxylase
and broadens the range of its activity toward more
alkaline pH. J Biol Chem. 2009, 284: 31587-96. doi:
10.1074/jbc.M109.049577.

Epstein-Barr virus interactions with cellular microRNAs
Principal investigator: Alberto Faggioni
Professor of General Pathology
Dipartimento di Medicina Sperimentale
Tel: (+39) 06 4461500; Fax: (+39) 06 4454820
alberto.faggioni@uniroma1.it
Participants:
Antonio Angeloni, Pankaj Trivedi, professors; Roberta
Santarelli, Mara Cirone, researchers; Eleni Anastasiadou,
Antonella Farina, Roberta Gonnella, Marisa Granato,
post-doc fellows; Francesco Boccellato, Paola Rosato, PhD
students; Claudia Zompetta, technician.
Report of activity
Epstein-Barr virus (EBV) has the unique distinction of
being the first human oncogenic virus. Its discovery
caused a renewal of interest in the field of viral oncology
which laid dormant for more than half a century after
the initial discovery of avian Rous sarcoma virus (RSV)
which Peyton Rous discovered in 1911. Initially found
in Burkitt lymphoma (BL) biopsies by Epstein and Barr
in 1964, EBV did not disappoint the expectation that it
might have an important role in cancer development.
Besides being tightly associated with several other
human cancers like nasopharyngeal carcinoma (NPC),
Hodgkin’s disease (HD), it is the only virus which
transforms normal B lymphocytes into continuously
growing lymphoblastoid cell lines (LCLs). The virus
achieves this by virtue of its nine latent proteins out of
which six are located in the nucleus of the infected cell
and the other three are membrane proteins.
The discovery of microRNA (miR) represents a novel
paradigm in RNA based regulation of gene expression
and their dysregulation has become a hallmark
of many a tumor. The miRs participate in gene regulation
of a broad spectrum of biological pathways
through fine-tuning of protein expression levels.
Their aberrant expression is a hallmark of a variety
of diseases. The initial evidence of their involvement
in cancer came in light by studies in chronic lymphocytic
leukemia (CLL). It is estimated that more than
50% human miRs are located to genomic regions
like common breakpoints and fragile sites, involved
in cancers. Some miRs are overexpressed in cancers
in comparison to normal tissues, while others are
7
AREA 1
downregulated. The former thus may be considered
oncomiRs and the latter tumor supressor miRs.
In virally associated cancers, the host-pathogen interaction
could involve alteration in miR expression.
Interestingly, EBV besides encoding its own miRs and
can also influence cellular miRs. The initial evidence
that this is so came from studies showing that EBV
negative and EBV positive BLs differ in their miR
expression signature (Kluiver et al., 2007). Moreover,
among EBV positive BLs, it was shown that the miR
profile of restricted latency expressing BLs (latency I)
and broader latency expressors (latency III) is diverse.
Given the importance on the one hand, of LMP1
and EBNA2 in EBV driven transformation and on
the other, the dysregulation of miRs in cancer, we
asked how these viral proteins influence cellular miR
expression. To this end, the following tasks were put
forward in our original proposal: 1: miR profiling
of tumor cell lines and tissues derived from different
stages of B cell differentiation. 2: Expression of
cellular miRs in LMP1 and EBNA2 transfected B
lymphoma cells. 3: Differential regulation of miR-
146a by EBNA2 and LMP1. 4: Regulation of TCL1
by LMP1 through miR-29b. 5: miR-21 regulation
by EBNA2. 6: Effect of miR-29b expression on EBV
latency shift and 7: EBV regulated cellular miRs and
their influence on Hedgehog signalling pathway.
Microarray miR profile of EBV infected cells and
compared it to the same cells transfected with either
LMP1 or EBNA2 only was established (Anastasiadou
et al., 2009, 2010; Imig et al., 2010). The progress on
the first five tasks is the following: EBV encoded
LMP1 is noted for its transforming potential. Yet, it
also acts as a cytostatic and growth-relenting factor
in Burkitt’s lymphoma (BL) cells. The underlying
molecular mechanisms of the growth inhibitory
property of LMP1 have remained largely unknown.
In the 1st 2nd, 3rd and 4th task, we investigated
how LMP1 negatively regulates a major oncogene,
TCL1, in diffuse large B-cell lymphoma (DLBCL)
and BL cells. MicroRNA (miR) profiling of LMP1

Alberto Faggioni
transfectants showed that among others, miR-29b,
is upregulated. LMP1 diminished TCL1 by inducing
miR-29b through C-terminus activation region 1
(CTAR1) and CTAR2. miR-29b locked nucleic acid
(LNA) antisense oligonucleotide transfection into
LMP1-expressing cells reduced miR-29b expression
and consequently reconstituted TCL1, suggesting
that LMP1 negatively regulates TCL1 through miR-
29b upregulation (Anastasiadou et al., 2010). The
miR-29b increase by LMP1 was due to an increase
in the cluster pri-miR-29b1-a transcription, derived
from human chromosome 7. Using pharmacological
inhibitors, we found that p38 mitogen-activated protein
kinase-activating function of LMP1 is important
for this effect. The ability of LMP1 to negatively
regulate TCL1 through miR-29b might underlie
its B-cell lymphoma growth antagonistic property.
As LMP1 is also important for B-cell transformation,
we suggest that the functional dichotomy of
this viral protein may depend on a combination of
levels of its expression, lineage and differentiation of
the target cells and regulation of miRs, which then
directs the outcome of the cellular response.
EBNA2 is indispensable for the capacity of the
virus to transform B cells in vitro. In the 2nd, 3rd
and 5 th task, we studied how it affects cellular miRs.
Extensive miR profiling of the virus infected and
EBNA2 transfected B lymphoma cells revealed
that oncomiR miR-21 is positively regulated by
this viral protein. Conversely, BL cell lines infected
with EBNA2 lacking P3HR1 strain did not show
any increase in miR-21. EBNA2 increased pAKT
expression and this was directly correlated with
increased miR-21. The primary miR-21 transcripts
were unaltered by EBNA2 and this suggests that
the increase in mature miR-21 is due to higher
post-transcriptional processing. Interestingly,
miR-146a, whose expression is known to be induced
by LMP1 was severely downregulated by EBNA2
in U2932 DLBCL cells. The primary miR-146a
transcription and the promoter activity was hampered
by EBNA2 whereas LMP1 had no such effect.
Low miR-146a expression generally correlates with
high IRAK1 and consequently higher type I interferon
level. Indeed an elevated level of IRAK1 and
type I interferon was found in EBNA2 transfectants.
Further studies will be required to investigate
precise molecular mechanisms how EBNA2 affects
miR expression and in particular miR-21 and miR-
146a. However the data suggest that EBNA2 might
contribute to EBV induced B cell transformation
by a positive regulation of oncomiR miR-21 and
by interfering with the innate immunity through
8
regulating a key regulator of it, namely miR-146a
(Rosato et al., submitted). Additionally, this miR
has been attributed with oncosuppressive functions.
Our data thus suggest that EBNA2 may promote
B cell transformation by simultaneously increasing
an oncomiR and reducing a miR with noted tumor
suppressing functions.
Publications
Anastasiadou E, Boccellato F, Rosato P, Junker S,
Winberg G, Frati L, Wade P, Faggioni A, Trivedi
P. Epstein-Barr virus infection leads to phenotypic
reversion of terminally differentiated B cells
of malignant origin. Cancer Lett. 2009, 284: 165-174.
doi: 10.1016/j.canlet.2009.04.025.
Anastasiadou E, Boccellato F, Vincenti S, Rosato P,
Bozzoni I, Frati L, Faggioni A, Presutti C, Trivedi
P. Epstein-Barr virus encoded LMP1 downregulates
TCL1 oncogene through miR-29b. Oncogene. 2010,
29: 1316-28. doi: 10.1038/onc.2009.439.
Imig J, Motsch N, Zhu J, Barth S, Okonievski
M, Tinguely M, Kurrer M, Schraml P, Moch H,
Faggioni A, Trivedi P, Meister G, Renner C, Grasser
MG. microRNA profiling in EBV-associated B-cell
lymphomas. Nucleic Acids Res. 2010. doi: 10.1093/
nar/gkg1043.
Heat map of global microarray profile of EBV infected
and EBNA2 transfected U2932 DLBCL
A: Two EBV infected clones of U2932 cells, U2932 EBVGFP cl-A
(EBNA2 negative) and U2932 EBVGFP cl-B (EBNA2 positive) were
compared for their miR profile. B: Overview of miR profiling of
EBNA2 transfected U2932 cells. C: a comparison of principally
altered miRs in U2932 EBVGFP cl-B and U2932 EBNA2 cl-2

Translational regulation: from the archaea to the eukarya
Principal investigator: Paola Londei
Professor of Applied Biology
Dipartimento Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 4940463; Fax: (+39) 06 4462891
londei@bce.uniroma1.it
Participants:
Dario Benelli, research fellow; Antimo Naspi, Michela
Pinzaglia, PhD students; Dorina Polinari, undergraduate
student.
Collaborations:
University of Wien, Austria (Prof. Udo Blaesi); University J.W.
Goethe Frankfurt, Germany (Prof. Joerg Soppa and Prof.
Robert Tampè); CNRS Strasbourg, France (Dr. Bruno Klaholtz);
Università Politecnica delle Marche, Ancona (Prof. Anna La
Teana); Università di Camerino (Prof. Claudio Gualerzi).
Report of activity
Summary
It is well known that the process of gene expression
in Archaea has many features in common with that of
Eukarya. This is especially true for the initiation step
of translation, which in Archaea and in Eukarya has
a similar high complexity. The project is specifically
centred on investigating the function and mechanism
of action in Archaea of two translation initiation factors,
a/eIF2 and aIF6, shared by the Archaea and the
Eukarya but absent in Bacteria. The eukaryal factors
(eIF2 and eIF6) have important roles in regulating
protein synthesis in both physiological and pathological
situations, but several aspects of their function remain
to be clarified. The function of a/eIF2 and aIF6 in
Archaea is currently poorly understood; its elucidation
will help to reconstruct the early evolution of these
interesting proteins, thus gaining valuable insight into
the onset of eukaryotic-type translational regulation.
An important regulator of translation: a/e IF2
In both Archaea and Eukarya, a/eIF2 is trimeric
protein consisting of the α, β and γ subunits. It interacts
with initiator tRNA (met-tRNAi) and delivers it
to the ribosome. A mechanism of translational control
essential and widespread in eukaryotic cells, but
lacking in Bacteria, targets specifically eIF2, inac-
9
AREA 1
tivating it by the phosphorylation of its α-subunit.
Why the Archaea, as the Eukarya, should use a
trimeric protein as the met-tRNAi binding factor is
as yet unclear. To investigate this problem, we initially
analyzed the modification pattern of the a/eIF2
α-subunit in the archaeon Sulfolobus solfataricus. In
vitro and in vivo studies with wild-type and mutated
a/eIF2-α led to the conclusion that this protein is
most probably not phosphorylated in archaea, suggesting
a mechanism of translational control different
from the eukaryal one.
However, our studies have revealed an interesting and
unexpected function of a/eIF2. Both trimeric a/eIF2
and its isolated γ-subunit appear to interact with the 5’
end of mRNAs, protecting them against exonuclease
degradation. Strikingly, only mRNA 5’ ends carrying
three phosphate groups are recognized by the factor,
while mono-phosphorylated and dephosphorylated
mRNAs are not. In living cells, the physiological
ligand of mRNA 5’-3P end is probably the isolated a/
eIF2 γ-subunit, which is 2-3 fold more abundant than
the α and β ones in normal conditions and is further
over-expressed under stress, e.g. heat- or cold-shock.
This situation is reminiscent of eukaryotic capping,
and may well be an evolutionary precursor of this
critical feature of eukaryotic translation. Work is in
progress to ascertain whether the IF2/mRNA interaction
has also a role in promoting translation.
In the last stage of the project we have extended
our studies to the analysis of eIF2 modifications in
certain human tumours. Specifically, we have found
that in metastatic melanomas, but not primary ones,
eIF2-α is hyperphosphorylated. Unexpectedly, we
have also found that phosphor-IF2-α is relocated to
the cell nucleus during the transition from primary to
metastatic tumours, hinting to some unprecedented
function of the factor in modulating gene expression
of the metastatic cells. Work is in progress to identify
the nuclear interactors of eIF2-α and to obtain
insight into the nuclear function of the protein.

Paola Londei
A still mysterious protein: IF6
The factor known as aIF6 (archaea) and eIF6
(eukarya) is a monomeric protein of about 25 kDa.
Eukaryotic IF6 functions as a ribosome anti-association
factors and probably has also a role in the
biogenesis of 60S ribosomal subunits. To enter the
translation cycle, 60S subunits must release eIF6; in
mammals, this is triggered by the phosphorylation of
the factor carried out by certain regulatory kinases.
Recently, it has also been reported that eIF6 depletion
inhibits miRNA-induced gene silencing in both
human cells and in nematodes. However, the mechanism
of action of eIF6 remains elusive.
Archaeal IF6 is highly homologous with eIF6
in both sequence and structure. We have found
that S. solfataricus aIF6, similar to its eukaryal
counterpart, inhibits association of the ribosomal
subunits. For the first time, we have mapped the
ribosomal binding site of the factor, locating it in
the middle of the 30S-interacting face of the large
ribosomal subunit. Our model of the aIF6/50S
complex reveals that the factor masks certain
RNA and protein domains critical for bridging
the two subunits. Work is in progress to unravel
the mechanism whereby aIF6 dissociates from
the 50S subunit. Unlike the eukaryal homologue,
aIF6 is not phosphorylated, suggesting that the
mechanism of release from ribosomes is not conserved.
Our recent data indicate that the release
of aIF6 from the ribosomes may entail a novel
type of protein modification, possibly in associa-
10
tion with a ribosome-dependent GTPase activity,
yet to be identified.
Lastly, we have begun to study the involvement of
eukaryal IF6 in cellular transformation, suggested by
the fact that eIF6 is over-expressed in many spontaneous
tumours. Our recent results have revealed that
Notch-1, a receptor protein implicated in cell proliferation
and survival, directly controls the expression
of eIF6. We found that the eIF6 promoter contains
two binding sites for RBP-jk, a downstream target
of Notch signalling. Indeed, the transcription of a
reporter construct containing the eIF6 promoter was
down-regulated when human leukaemia cell lines,
having constitutively activated Notch signalling,
were treated with Notch-1 inhibitors. Conversely,
the transcription of the eIF6 reporter construct was
up-regulated upon over-expression of Notch-1 in
NIH3T3 cells. Work is in progress to determine the
physiological significance of these findings.
Publications
Benelli D, Londei P. Begin at the beginning: evolution
of translational initiation. Res Microbiol. 2009,
160: 493-501. doi:10.1016/j.resmic.2009.06.003.
Benelli D, Marzi S, Mancone C, Alonzi T, la Teana
A, Londei P. Function and ribosomal localization
of aIF6, a translational regulator shared by archaea
and eukarya. Nucleic Acids Res. 2009, 37: 256-67. doi:
10.1093/nar/gkn959.

AREA 2
Pathogenetic
mechanisms
of microbially
associated
diseases

Molecular characterization of viruses causing bronchiolitis and
study of viral and host factors affecting Type I IFN antiviral
response induced by respiratory viruses
Principal investigator: Guido Antonelli
Professor of Virology
Dipartimento di Medicina Molecolare
Tel: (+39) 06 4474122; Fax: (+39) 06 44741236
guido.antonelli@uniroma1.it
Participants:
Ombretta Turriziani, Professor; Laura Antonelli, Fabio
Midulla, Paola Papoff, Alessandra Pierangeli, Carolina
Scagnolari, researchers; Carla Selvaggi, research fellow;
Katia Monteleone, PhD student.
Report of activity
Bronchiolitis severity and subsequent episodes of
wheezing in children are likely to be due to either
viral factors (e.g. genotype and strain, viral load) or
to many other, yet under recognized, host factors
determining effectiveness of innate and acquired
immune response. Most previous studies had been
focused on respiratory syncytial virus (RSV), either
in vitro or in patient groups; correlation of RSV
genotypes or strains, and viral load with disease
severity and recurrent wheezing is still controversial.
Recently, increasing evidence suggested that
rhinoviruses (RV) were important agents of bronchiolitis
and represented the major risk factor in
recurrent wheezing episodes in children. However,
it is not yet clear whether viral bronchiolitis directly
contributes to recurrent wheezing and asthma causation
or infants are at risk for subsequent wheezing as
a result of genetic conditions. Hence, we proposed
to study viral species and strains involved in bronchiolitis,
and the role of type I IFNs in antiviral
response, either in patient samples or in cell lines,
properly infected with respiratory viruses.
In the first year of the project, we characterized
viral infections, detecting 14 different viruses with
a comprehensive panel of PCR-based reactions, in
respiratory samples (pharyngeal swabs and nasal
washings) from patients seeking medical care in
Emergency Departments (ED). Patient signs and
symptoms, laboratory results and clinical outcome
were compared with virus results: no respiratory
signs/symptoms could differentiate virus infected
individuals (either infants, children or adult patients)
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from other cause of acute respiratory syndrome and
clinical presentations were not useful to discriminate
viral pathogens (Pierangeli et al., CMI 2010).
Bronchiolitis (defined as a history of upper respiratory
tract infection followed by acute onset of respiratory
distress with cough, tachypnea, retraction
and diffuse crackles on auscultation in infants within
one year) was the more frequent clinical diagnosis
overall in children attending ED; RSV single infection
represented about 50% of bronchiolitis cases,
in nearly 30% of bronchiolitis cases no virus was
found, 10-15% of bronchiolitis were due to RV single
infections, whereas other viruses or coinfections
were detected in the remaining cases. Clinical data
of enrolled patients confirmed that RSV is the pathogen
with the highest severity of infection in children,
with highest PICU admissions, even though total
days of hospitalization were not significantly different
from the other virus groups (“Virological and
clinical characterizations of respiratory infections in
children attending an emergency department” manuscript
in preparation).
To perform viral strain characterizations in respiratory
samples from children with bronchiolitis, either
using primers from published papers or designed for
the project, we sequenced genomic region longer and
less conserved than those used for virus detection in
PCR tests: RSV G glycoprotein and RV 5’ UTR and
VP4/VP2 coding region. Preliminary results from
RV sequencing identified 16 RV cases encountered in
the epidemic season 2009/10 as being RV-A N = 9,
RV-B N = 1, RV-C N = 6, reporting for the first time,
RV-C (the novel RV species identified in 2007) circulation
in central Italy (manuscript in preparation).
Moreover, viral genome determination by real-time
PCR of RSV, influenza A and RV were performed
in respiratory samples taken at admittance and at
follow-up visits; preliminary results showed no
clear association of viral loads with illness severity
scores, but we are currently performing a thoughtful
statistical analysis of the data, to evaluate different

Guido Antonelli
contribution of viral genotypes and strains (e.g. RSV
A or B, RV A, B and C).
In the framework of the project, we studied cases
of infants younger than one year hospitalised during
several epidemic seasons for their first episode
of acute bronchiolitis, to determine the incidence of
severe bronchiolitis and factors predicting disease
severity in previously healthy term infants. Groups
of infants were assessed on a severity score: (0)
required only conservative treatment; (1) needed
intravenous fluids or oxygen treatment or both for
less than 12 hours; (2) needed oxygen for more than
12 hours without ventilatory support and intravenous
fluids; (3) needed either mechanical ventilation
or noninvasive respiratory support. Data analyzed
included: specific viral infection, patient demographics
and clinical data, chest radiographs. Younger
age on admission (

A structural biology study of schistosomiasis
Principal investigator: Andrea Bellelli
Professor of Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910955; Fax: (+39) 06 4440062
andrea.bellelli@uniroma1.it
Participants:
Maurizio Brunori, Adriana Erica Miele, professors;
Francesco Angelucci, Ilyas Koutris, post-doc fellows;
Daniela Dimastrogiovanni, Fulvio Saccoccia, PhD students;
Giovanna Boumis, technician.
Collaborations:
Rush University, Chicago, IL, USA (Prof. David L. Williams,
Dr. Ahmed A. Sayed); Sapienza Università di Roma (Prof.
Alfredo di Nola, Dr. Massimiliano Anselmi); University of
Lund, Sweden (Dr. Linn Petterson, Dr. Frida Pauly); Istituto
di Biologia Cellulare, CNR, Monterotondo, Roma (Prof. Donato
Cioli, Prof. Livia Pica Mattoccia, Dr. Cristiana Valle).
Report of activity
Schistosomiasis is a parasitic disease affecting over
200 million people in tropical and subtropical countries.
The disease is currently treated with only one
drug, Praziquantel; since the appearance of strains of
Schistosoma mansoni displaying reduced sensitivity
to Praziquantel has been reported, the study of alternative
drugs is important (Cioli et al., 2008).
A possible drug target is the seleno-protein thioredoxin-glutathione
reductase (TGR), a key flavoenzyme
in the pathway of the parasite for detoxification
of reactive oxygen species. The enzyme is a unique
fusion of a glutaredoxin domain with a thioredoxin
reductase domain, which contains a selenocysteine
(Sec) as the penultimate amino acid. TGR bridges
two detoxification pathways crucial for the parasite
survival in the host’s organism: those based on
glutathione and thioredoxin respectively. The parasite’s
reduction system is very different from that
of the host, which is based on two separated (even
though evolutionarily related) enzymes, Thioredoxin
Reductase and Glutathione Reductase respectively.
SmTGR is a validated drug target since (i) selective
inhibition of its biosynthesis by RNA interference kills
the parasite in vitro; and (ii) inhibition of its function
15
AREA 2
by Auranofin and other drugs kills the parasite both in
vitro and in vivo (Kuntz et al., 2007).
In the project “A structural biology study of schistosomiasis”
we solved the crystal structure (at 2.2 Å resolution)
of TGR from Schistosoma mansoni (SmTGR),
deleted in the last two residues; our structure has been
the first for a Thioredoxin Glutathione Reductase
(see Angelucci et al., 2008). The structure reveals the
peculiar architecture of this chimeric enzyme: the small
Glutaredoxin (Grx) domain at the N-terminus is joined
to the large thioredoxin reductase (TrxR) one via an
extended complementary surface, involving residues
not conserved in the Grx superfamily; the TR domain
interacts with an identical partner via its C-terminal
domain, forming a dimer with a twisted “W” shape.
Although lacking the penultimate Selenocysteine residue
(Sec), the enzyme is still able to reduce oxidized
glutathione. These data update the interpretation of the
interdomain communication in TGR enzymes.
Auranofin (AF), a gold-containing compound already
in clinical use as an anti-arthritic drug, has been
shown to inhibit TGR and to substantially reduce
worm burden in mice. Using X-ray crystallography
we solved (at 2.5 Å resolution) the structure of wild
type TGR incubated with AF (see Angelucci et al.,
2009). The electron density maps show that the
actual inhibitor is gold, released from AF. Gold is
bound at three different sites not directly involving
the C-terminal Sec residue; however, because the C
terminus in the electron density maps is disordered,
we cannot exclude the possibility that gold may also
bind to Sec. To investigate the possible role of Sec in
the inactivation kinetics, we tested the effect of AF
on a model enzyme of the same superfamily, i.e. the
naturally Sec-lacking glutathione reductase, and on
truncated TGR. We demonstrated that the role of
selenium in the onset of inhibition by AF is catalytic
and can be mimicked by the external source of selenium
benzeneselenol. Therefore, we proposed that
Sec mediates the transfer of gold from its ligands in
AF to the redox-active Cys couples of TGR.

Andrea Bellelli
Moreover, with the aim of achieving a better understanding
of the electron flow inside the enzyme
macromolecule, we dissected the catalytic cycle of
Schistosoma mansoni TGR by structural and functional
analysis of the U597C mutant. Crystallographic
data were obtained for (i) the oxidized form (at 1.9 Å
resolution); (ii) the NADPH- and GSH-bound forms
(2.3 and 1.9 Å, respectively); and (iii) a different
crystal form of the (partially) reduced enzyme (3.1
Å), showing the physiological dimer and the entire
C terminus of one subunit. Whenever possible, we
determined the rate constants for the interconversion
between the different oxidation states of TGR
by kinetic methods. By combining the crystallographic
analysis with computer modeling, we were
able to throw further light on the mechanism of
action of S. mansoni TGR. In particular, we were
able to model the putative functionally relevant
conformational change of the C terminus after the
transfer of reducing equivalents from NADPH to the
redox sites of the enzyme (see Angelucci et al., 2010).
Another enzyme belonging to the same metabolic
pathway that has been indicated (but not yet validated)
as a possible drug target is Glutathione peroxidase
(Gpx). Glutathione peroxidases are a family
of sulfur or selenium-dependent isozymes sharing
the ability to reduce peroxides using the reducing
equivalents provided by glutathione or possibly
small proteins such as thioredoxin. The genome of
Schistosoma mansoni encodes for a Gpx that, as
for other frontline antioxidant enzymatic systems,
is localized in the worm’s tegument, the outermost
defense layer. We solved the first crystal structure at
1.0 and 1.7 Å resolution of two recombinant SmGpx
16
variants, carrying the active site mutations Sec43Cys
and Sec43Ser, respectively (see Dimastrogiovanni et
al., 2010). The structures confirm that this enzyme
belongs to the monomeric class 4 (phospholipid
hydroperoxide) Gpx. In the case of the Sec to Cys
mutant, the catalytic Cys residue is oxidized to sulfonic
acid. By combining static crystallography with
molecular dynamics simulations, we obtained insight
into the substrate binding sites and the conformational
changes relevant to catalysis, proposing a role
for the unusual reactivity of the catalytic residue.
Publications
Angelucci F, Sayed AA, Williams DL, Boumis G,
Brunori M, Dimastrogiovanni D, Miele AE, Pauly
F, Bellelli A.Inhibition of Schistosoma mansoni thioredoxin-glutathione
reductase by auranofin: structural
and kinetic aspects. J Biol Chem. 2009, 284: 28977-85.
doi: 10.1074/jbc.M109.020701.
Angelucci F, Dimastrogiovanni D, Boumis G,
Brunori M, Miele AE, Saccoccia F, Bellelli A.
Mapping the catalytic cycle of Schistosoma mansoni
thioredoxin glutathione reductase by X-ray crystallography.
J Biol Chem. 2010, 285: 32557-67. doi:
10.1074/jbc.M110.141960.
Dimastrogiovanni D, Anselmi M, Miele AE, Boumis
G, Petersson L, Angelucci F, Di Nola A, Brunori M,
Bellelli A. Combining crystallography and molecular
dynamics: the case of Schistosoma mansoni phospholipid
glutathione peroxidase. Proteins. 2010, 78: 259-
70. doi: 10.1002/prot.22536.

Lipopolysaccharide and peptidoglycan adaptation to host as an
immune evasion strategy of gram-negative pathogens
Principal investigator: Maria Lina Bernardini
Researcher in Cellular Microbiology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49917850; Fax: (+39) 06 49917994
marialina.bernardini@uniroma1.it
Participants:
Luigi Lembo-Fazio, post-doc fellow; Laura Curcurù, Gaëlle
Noël, PhD students.
Collaborations:
Dipartimento di Immunologia, Trapianti e Malattie infettive,
Fondazione S. Raffaele del Monte Tabor (Prof. Alessandra
Bragonzi); Dipartimento di Chimica Organica e Biochimica,
Università di Napoli “Federico II” (Prof. Antonio Molinaro);
Dipartimento di Oncologia Sperimentale, Istituto Europeo di
Oncologia, Milano (Prof. Maria Rescigno).
Report of activity
This study focuses on the molecular mechanisms
underlining the adaptation to host of two pathogens,
Pseudomonas aeruginosa (PA) and Shigella flexneri. P.
aeruginosa (PA) is an opportunistic pathogen, which
causes severe infections in immune-compromised
individuals, as in the lung of patients suffering from
cystic fibrosis (CF) (Lyczak et al., 2000). During
the colonization of the host airways, PA undergoes
genetic changes, which allow it to evade detection
and clearance by the immune system and to establish
a chronic infection (Gibson et al., 2003). S. flexneri is a
Gram-negative enteroinvasive bacterium that causes
shigellosis, a severe inflammatory dysentery, characterized
by strong abdominal cramps, fever and stools
containing blood and mucus (Schroeder & Hilbi,
2008). During dissemination through the intestinal
epithelium, bacterial components are released, which
activate immune responses of the host, inducing
proinflammatory cytokines and chemokines production
(Phalipon & Sansonetti, 2007). The mammalian
innate immune system detects the presence of microbial
infection through germ-line encoded pattern
recognition receptors (PRRs) (Kawai & Akira, 2009).
These proteins are implicated in the recognition of a
limited, but highly conserved, set of molecular structures
that are produced by micro-organisms and are
17
AREA 2
absent from the host cell, called pathogen associated
molecular patterns (PAMPs). The activation of a
PRR by its cognate PAMP induces pro-inflammatory
signaling pathways such as that governed by
NF-κB and MAPKs, leading to the clearance of the
intruder. However, pathogens have evolved immune
evasion strategies, including the alteration of their
structures, in order to escape or to modulate the
immune system recognition.
The lypopolisaccharide (LPS) and peptidoglycan
(PGN) of three sequential, clonal, PA clinical isolates
from a CF patient were analyzed (Bragonzi et
al., 2009). The AA2 strain was isolated at the initial
stage of lung infection, while the strains AA43
and AA44 during the chronic infection. Chemical
analysis of AA2 and AA43 P. aeruginosa strain LPS
demonstrated that these structures were composed
essentially by tetra-, penta- and hexa-acylated species,
but present at different percent concentrations.
In contrast, the AA44 LPS of the other strain, AA44,
isolated at the chronic stage of the disease, was constituted
by hexa-acylated and hepta-acylated moieties.
These findings are in accordance with previous
studies, describing that P. aeruginosa synthesizes
more highly acylated (hexa- and hepta-acylated) lipid
A during the passage from acute to chronic infection
(Ernst et al., 2007). The presence of the hepta-acylated
lipid A in PA AA44 was supported by genetic
data, indicating a mutation in pagL gene, encoding
for a lipid A deacylase (Rutten et al., 2006). This
observation is in accordance with results obtained
with other pathogens, such as Salmonella spp., showing
that microorganisms modify their LPS structure
through deacylase activity in order to modulate lipid
A recognition by innate immune receptor of the host
(Kawasaki et al., 2004; Kawasaki & Manabe, 2010).
On this basis, the biological activity of LPS was
evaluated in different cell lines in vitro and in a mouse
model in vivo. The LPS of the “chronic” strains AA43
and AA44 displayed a reduced immunostimulatory
activity compared to the LPS of AA2 in different

Maria Lina Bernardini
cell models, including HEK293 cells expressing the
LPS receptor TLR4, together with MD2 and CD14,
the macrophage-like THP-1 cells and a CF airway
epithelial IB3-1 cell line and in its isogenic non-CF
C38 cells. In accordance with in vitro results, in vivo
in a murine model of intranasal injection of LPS,
neutrophil recruitment and cytokines (MIP-2, KC
and IL-1β) release was major after stimulation with
PA AA2 LPS than AA43 and AA44 strain LPS.
As for LPS, the PGN structure of the three CF strains
was elucidated, through extraction and muramidase
digestion. HPLC analysis of PA PGN showed the
same muramyl peptide species in the three strains
albeit in different relative amount. HEK293 cells
expressing Nod1 and CF IB3 cell line were stimulated
with the PGN of the three clinical strains, and
the inflammatory responses, i.e. NF-κB activity and
IL-8 production, were evaluated. The results are in
accordance with those obtained with LPS: in fact,
PGN of the “chronic” strains displayed a reduced
immunopotential, compared to the PGN of the strain
from initial colonization. A recent study reported
that in the airways of cystic fibrosis patients the high
levels of the inflammatory cytokine IL-17 prime CF
epithelial cells to increase PRR receptors, and in
particular Nod1, stressing the involvement of Nod1
in PA infection (Roussel & Rousseau, 2009). These
issues reinforce the interest to study the relationship
between PGN of PA and Nod1 activity.
Finally, the results about PA adaptation during the
passage from acute to chronic infection, suggest that
the pathogen promotes its survival into the host
airways through PAMP modification. This strategy
allows the bacterium to evade innate immune system
recognition and to impair the homeostasis of the
infected tissues.
To identify parallel mechanisms in the pathogenesis
of the highly inflammatory S. flexneri the
relevant bacterial structures were analyzed during
infection. It was demonstrated that approximately
one-quarter of S. flexneri genes altered their
expression profile, during infection of epithelial
and macrophagic cell lines (Lucchini et al., 2005).
On this basis, the LPS structure of intracellular
bacteria was evaluated and compared to that of the
bacteria grown in vitro. Chemical analysis showed
the same O-chain structure of the LPS of the intracellular
and the in vitro grown shigellae (Clément
et al., 2003). The only difference resides on the
core region of LPS, where a phosphate replaced
a phosphoethanolamine. Surprisingly, the lipid
A region of intracellular S. flexneri was underacylated,
compared to the hexa-acylated lipid A of
18
the same in vitro grown strain. According to this
structure, the LPS of the intracellular bacterium
showed a lower capability to induce innate immune
system activation in HEK293/TLR4-MD2-CD14
cells, with respect to the in vitro maintained strain.
This immune evasion strategy of the intracellular
S. flexneri strain is in agreement with previous
findings reporting that TLR4 is internalized from
plasma membrane into early endosomes, after
ligand binding (Kagan et al., 2008).
In conclusion, these data showed parallel mechanisms
of adaptation of two bacteria with different behavior
into the host. The highly inflammatory pathogen S.
flexneri and P. aeruginosa from lung chronic infection
adapt their LPS in order to evade immune recognition
by TLR4 receptor. However, while P. aeruginosa modifies
its PAMP in a permanent manner, as indicated by
pagL mutation, it is presumable that Shigella alters its
LPS transiently, through expression profile adaptation.
References
- Bragonzi et al., 2009, Am J Respir Crit Care Med
180:138-45.
- Clément et al., 2003, J Biol Chem 278:47928-36.
- Ernst et al., 2007, J Infect Dis 196:1088-92.
- Gibson et al., 2003, Am J Respir Crit Care Med
168:918-51.
- Kagan et al. 2008, Nat Immunol 9:361-368.
- Kawai T, Akira S. 2009, Int Immunol 21:317-337.
- Kawasaki et al., 2004, J Biol Chem 279:20044-
20048.
- Kawasaki et al., 2010, J Bacteriol 192:5837-5840.
- Lucchini et al., 2005, Infect Immun 73:88-102.
- Lyczak et al., 2000, Microbes Infect 2:1051-1060.
- Phalipon A, Sansonetti JP. 2007, Immunol Cell Biol
85:119-129.
- Roussel L, Rousseau S. 2010, Biochem Biophys Res
Commun 391:505-509.
- Rutten et al., 2006, Proc Natl Acad Sci USA
103:7071-7076.
- Schroeder GN, Hilbi H. 2008, Clin Microbiol Rev
21:134-156.
Publications
Cigana C, Curcurù L, Leone MR, Ieranò T, Lorè
NI, Bianconi I, Silipo A, Cozzolino F, Lanzetta R,
Molinaro A, Bernardini ML, Bragonzi A. Pseudomonas
aeruginosa exploits lipid A and muropeptides modification
as a strategy to lower innate immunity during
cystic fibrosis lung infection. PLoS One. 2009,
4:e8439. doi: 10.1371/journal.pone.0008439.

Involvement of sRNA molecules in the complex regulatory
circuits of virulence gene expression in Shigella flexneri and in
enteroinvasive E. coli
Principal investigator: Bianca Colonna
Professor of General Microbiology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49917580, 06 49917582; Fax: (+39) 06 49917594
bianca.colonna@uniroma.it
Participants:
Gianni Prosseda, researcher; Marialuisa Barbagallo, Rosaria
Campilongo, Maria Letizia Di Martino, PhD students.
Collaborations:
Dipartimento di Biologia, Università di Roma-Tre (Prof.
Mariassunta Casalino); Dipartimento di Biologia Molecolare,
Cellulare e Animale, Università di Camerino (Dr. Maurizio
Falconi); Istituto di Biologia e Patologia Molecolari, CNR, Roma
(Dr. Gioacchino Micheli).
Report of activity
Shigella is a highly adapted human pathogen that
causes bacillary dysentery, a severe enteric syndrome,
mainly in the developing world. The pathogenicity
mechanism of Shigella strains and enteroinvasive
E. coli (EIEC) is based on their capacity
to reach and invade colonic epithelial cells, leading
to intracellular bacterial multiplication and spread
to adjacent cells with consequent cell death and
destruction of the colonic mucosa. The cellular
pathogenesis and clinical presentation of shigellosis
result from the complex action of a large number
of bacterial virulence factors mainly located on a
large virulence plasmid (pINV). The expression of
pINV genes is controlled by multiple environmental
stimuli through a regulatory cascade involving proteins
encoded by both, pINV and the chromosome.
Transcriptional activation of the invasivity operons
in response to temperature, pH, and osmolarity
conditions found in the human intestine requires
two positively acting plasmid-encoded regulators,
VirF and VirB. Among environmental stimuli, temperature
is a crucial factor since transcription of the
virulence genes is strongly repressed at 30°C by the
chromosomal-encoded protein H-NS. Within the
virF promoter, the nucleoid protein H-NS interacts
- only at low temperature - with two sites spaced by
an intrinsically curved DNA region. With increasing
19
AREA 2
temperature a consequent decrease in DNA curvature
and in H-NS binding occur, and at a temperature
higher than 30°C the virF promoter becomes
insensitive to H-NS repression, strongly suggesting
that the environmentally induced structural collapse
of the promoter’s intrinsic bend is a key event in the
regulation of virulence gene expression.
Recent observations from our group confirm that
among the molecular strategies bacteria have set up
to quickly match their transcriptional program to
new environments, changes in sequence mediated
DNA curvature play a crucial role. By analyzing the
temperature dependent expression of a reporter gene
in a construct carrying a progressively decreasing
bent sequence upstream from the promoter, we have
shown that with decreasing temperature a narrow
curvature range accounts for a significant enhancement
of promoter activity. This strengthen the view
that DNA-curvature-mediated regulation of gene
expression is likely a strategy offering fine-tuning
control possibilities and that, considering the widespread
presence of curved sequences upstream from
bacterial promoters, it may represent one of the most
primitive forms of gene regulation.
Recently small non-coding RNAs (sRNA or ncRNA)
have emerged as major elements in bacterial cell regulation.
Moreover, in several human bacterial pathogens
sRNAs appear to be crucial for integrating
environmental stimuli into outputs that are relevant
for pathogenicity. Therefore, we decided to investigate
the potential involvement of sRNA transcripts
in the modulation of virulence gene expression in
Shigella. By analysing the large intergenic region
separating the virA and icsA genes, located on the
virulence plasmid of S. flexneri, we identified a novel
gene coding for an antisense RNA (RnaG). Mapping
the 5’- and 3’-ends of RnaG reveals that this sRNA
molecule is quite long (450 bases) and is transcribed
from a promoter located within the sequence corresponding
to the 5’-untranslated region of the icsA
mRNA. The icsA gene encodes a structural protein

Bianca Colonna
involved in the reorganization of the cytoskeleton
upon penetration of bacteria into the host cells and
its activity is crucial for the intra- and inter-spreading
of the bacterial pathogen. In order to understand
if and how RnaG is able to interfere with the expression
of icsA, the promoters of icsA and RnaG were
alternatively inactivated by introducing mutations in
the corresponding -10 elements. As opposed to other
sRNAs which affect the ability of the target mRNAs
to be translated, we found that RnaG is capable to
downregulate icsA transcription by means of two,
not mutually esclusive mechanisms.
First we showed that icsA and RnaG are subject
to Transcriptional Interference (TI) regulation: the
activity of the icsA promoter (P icsA ) is significantly
reduced when a transcription process starts from the
convergent RnaG promoter (P RnaG ). In this case TI is
an asymmetric phenomenon as P RnaG acts as aggressive
promoter reducing the expression of the weak
one, P icsA . The extent of TI is higher in vitro than in
vivo, suggesting that other factors besides promoter
strength could contribute to the regulation of this
genetic system. At the moment our results do not
allow to figure out if the TI mechanism depends on
the collision between the two RNA polymerases moving
in opposite directions or on the removal of the
promoter-bound complexes by the passage of RNA
polymerases from the opposite promoter. We hope to
obtain more detailed information about the TI mechanism
by altering the arrangement of the P icsA and P RnaG
sequences, as well as by introducing a transcriptional
terminator between the two face-to-face promoters.
In addition to TI, RnaG can also cause a direct
repression of icsA transcription by acting primarily
as antisense RNA targeting the icsA messenger.
By combining computational predictions and data
from chemical and enzymatic RNA probing we
demonstrated that binding of RnaG to the target
icsA mRNA alters the secondary structure of the
untranslated region of the sense RNA. This alternate
structure, which originates in the course of the transcriptional
process but only in the presence of RnaG,
gives rise to an intrinsic terminator in the nascent
icsA messenger that likely leads to premature termination.
The proposed model is further strengthened
by the fact that mutations which destabilize the
conserved stem structure of this intrinsic terminator,
significantly affect the ability of RnaG to inhibit
icsA transcription. This RnaG-mediated attenuation
20
process constitutes a very powerful strategy adopted
by a sRNA molecule to modulate the expression of
a crucial virulence gene at the transcriptional level.
The expression of icsA is subject to a complex regulatory
network that, besides being affected by RnaG,
also depends on the coordinated action of two major
players of the Shigella regulon, VirF and H-NS. We
are currently investigating how the action of these
regulatory factors is coordinated on the icsA promoter
in order to obtain the full expression of the
icsA only when the bacterium needs to move inside
the host cell and towards the adjacent cells.
Since current evidence indicates that regulation
mediated by sRNA molecules appears to be more
common than previously thought, we plan to perform
genome-wide analyses to deeper understand
the transcriptome of Shigella. This will allow us to
identify new sRNAs molecules, with emphasis on
those transcribed during peculiar environmental
conditions, like high osmolarity or low oxygen availability,
that more closely mimic the intracellular
habitat of Shigella.
Publications
Rocco F, De Gregorio E, Colonna B, Di Nocera PP.
Stenotrophomonas maltophilia genomes: a start-up
comparison. Int J Med Microbiol. 2009, 299: 535-46.
doi: 10.1016/j.ijmm.2009.05.004.
Casalino M, Prosseda G, Barbagallo M, Iacobino A,
Ceccarini P, Latella M C, Nicoletti M, Colonna B.
Interference of the CadC regulator in the argininedependent
acid resistance system of Shigella and
enteroinvasive E. coli. Int J Med Microbiol. 2010, 300:
289-95. doi:10.1016/j.ijmm.2009.10.008.
Giangrossi M, Prosseda G, Tran CN, Brandi A,
Colonna B, Falconi M. A novel antisense RNA regulates
at transcriptional level the virulence gene icsA
of Shigella flexneri. Nucleic Acids Res. 2010, 38: 3 362-
75. doi: 10.1093/nar/gkq025.
Prosseda G, Mazzola A, Di Martino M L, Tielker D,
Micheli G, Colonna B. A temperature-induced narrow
DNA curvature range sustains the maximum
activity of a bacterial promoter in vitro. Biochemistry.
2010, 49: 2778-85. doi: 10.1021/bi902003g.

Principal investigator: Anna teresa Palamara
Professor of Microbiology
Dipartimento di Sanità Pubblica e Malattie Infettive
Tel: (+39) 06 4468622; Fax: (+39) 06 4468625
annateresa.palamara@uniroma1.it
21
AREA 2
Redox mediated mechanisms involved in the influenza virus
replication and in the pathogenesis of influenza associated diseases
Participants:
Lucia Nencioni, Claudio Passariello, researchers; Rossella
Sgarbanti, Donatella Amatore, post-doc fellows; Ignacio
Celestino, PhD student.
Collaborations:
Dipartimento di Biologia, Università di Roma Tor Vergata (Prof.
M. R. Ciriolo); Istituto di Biochimica “Giorgio Fornaini”,
Università di Urbino (Prof. M. Magnani); Institute of Molecular
Virology, University of Monaco (Prof. S. Ludwig).
Report of activity
Every year, influenza epidemics cause numerous
deaths and millions of hospitalizations, but the
most frightening effects are seen when new strains
of the virus emerge from different species, causing
worldwide outbreaks of infection. All the antiviral
compounds developed against influenza virus target
viral structures but unfortunately, the efficacy
of these drugs is often limited by toxicity and the
almost inevitable selection of drug-resistant viral
mutants. Thus, the discovery of novel anti-influenza
drugs targeting specific cell pathways essential for
viral replication, could reduce the emergence of drug
resistance and increase the effectiveness towards different
viral strains.
Influenza viruses use several strategies to manipulate
host cell machinery to their advantage. Among
these, the imbalance of intracellular redox state can
play an important role in modulating the activity of
several signal pathways (MAP kinases, ER oxidoreductases)
involved in viral replication.
The scientific activities of our group in 2008-2010
were aimed at defining the mechanisms through
which influenza virus activates redox regulated
intracellular pathways and their potential role in
regulating viral life cycle. Since secondary bacterial
pneumonia are one of the primary cause of
excess mortality associated with influenza A virus
(IAV) infection we also investigated the potential
involvement of redox-mediated mechanisms in the
increased internalization of Staphylococcus aureus in
influenza virus-infected cells. The obtained results
are described in the following sections:
Role of p38MAPK in influenza virus
replication and viral induced apoptosis
In cells, which are fully permissive to viral infection,
virus-induced p38MAPK activation contributed to
the phosphorylation of viral nucleoprotein (NP) that
is an essential step for its export from the nucleus
and virus assembly. Interestingly, p38MAPK-mediated
NP phosphorylation was impaired in cells that
are characterized by high levels of glutathione (GSH)
and the expression of the antiapoptotic protein Bcl-
2. In these cells Bcl-2 did not prevent infected cells
from undergoing virally triggered apoptosis despite
its negative impact on viral replication. The protein’s
reduced antiapoptotic capacity was related to
its phosphorylation by virally activated p38MAPK.
Our data suggest that p38MAPK’s impact on the
IAV life cycle and the apoptotic response of host
cells to infection depend on whether or not the cells
express Bcl-2, highlighting the possibility that the
virus’ pathological effects are partly determined by
the type of cell it targets. These results are reported
in the paper Nencioni et al., Journal of Biological
Chemistry 2009.
Role of virus-induced oxidative stress in the
activation of intracellular signaling pathways
On the basis of results described above, we wondered
if the oxidative stress induced by IAV could play a
role in the activation of p38MAPK. We found that
few minutes after viral challenge, i.e., during the
period of viral adsorption to the cells, IAV caused
a significant but transient increase in the intracellular
levels of reactive oxygen species (ROS).
The ROS over-production was efficiently inhibited

Anna Teresa Palamara
by NADPH-oxidase inhibitor diphenyl iodonium
(DPI), thus suggesting a significant contribution
of this enzyme to virus-induced oxidative stress.
ROS increase paralleled with the phosphorylation
of p38MAPK, a kinase involved in the regulation
of viral replication, as well as in the induction of
virus-induced inflammatory responses. p38MAPK
activation was also inhibited by treatment with DPI,
suggesting that virus-induced oxidative stress is
involved in its activation.
In the late steps of influenza virus life-cycle, we
found a further increase in ROS production that was
associated to a decrease in the content of GSH, the
main intracellular antioxidant. This second round
of oxidative stress is still related to an activation of
p38MAPK. ROS over-production, p38MAPK activation
and viral replication were efficiently inhibited
by DPI, suggesting that the virus-induced oxidative
stress plays a key role in regulating influenza viral
replication. These results suggest that redox-regulated
intracellular pathways might represent useful
targets for new anti-influenza approaches. A paper
reporting these results is in preparation (Amatore
et al.).
Definition of redox mediated pathways
involved in hemagglutinin (HA) maturation
Folding of influenza HA, a disulfide-rich viral glycoprotein,
is an event strictly dependent on intracellular
redox state. Recently our attention has been
focused on the effect of GSH-C4, a GSH derivative
with hydrophobic properties, on HA maturation
process. In MDCK cells, the treatment with GSH-
C4 decreased by 90-95% the replication of different
human and avian strains of influenza viruses,
without producing toxic effects on uninfected cells.
This effect was associated to a specific block of HA
folding: the viral protein remains in the endoplasmic
reticulum as a reduced monomer instead of undergoing
oligomerization and plasma-membrane insertion.
HA maturation depends on the host-cell oxidoreductase,
PDI, whose activity is probably facilitated
by virus-induced glutathione depletion. GSH-C4induced
correction of this depletion increases the
presence of reduced PDI, thereby impairing its
oxidizing ability. Host-cell glycoprotein expression
and secretion in uninfected cells was unaffected,
22
suggesting that GSH-C4 acts only on glutathionedepleted
cells. Antinfluenza activity of GSH-C4 was
confirmed also in a murine model of lethal infection.
Collectively, these results suggest that redox-sensitive
host-cell pathways exploited specifically by
the virus are promising targets for effective antiinfluenza
strategies. A paper reporting these results
has been under revision (Sgarbanti et al.).
Mechanisms involved in the internalization
of Staphylococcus aureus in influenza virusinfected
cells
We have investigated in vitro the cellular and molecular
mechanisms underlying the synergism between
S. aureus and IAV. Experimental data show a significant
increase in the efficiency of internalisation
of S. aureus into cultured pneumocytes. In particular
we demonstrated that S. aureus bind the viral HA
exposed at the surface of infected cells and that the
affinity of this binding is enhanced by altered redox
environment that the virus generate extracellularly.
A paper reporting these results has been accepted for
pubblication (Passariello et al., 2011)
Selected publications
Nencioni L, De Chiara G, Sgarbanti R, Amatore D,
Aquilano K, Marcocci ME, Serafino A, Torcia M,
Cozzolino F, Ciriolo MR, Garaci E, and Palamara
AT. Bcl-2 expression and p38MAPK activity in cells
infected with influenza A virus: impact on virally
induced apoptosis and viral replication. J Biol Chem.
2009, 284: 16004-15. doi: 10.1074/jbc.M900146200.
De Chiara G, Marcocci ME, Civitelli L, Argnani
R, Piacentini R, Ripoli C, Manservigi R, Grassi C,
Garaci E, Palamara AT. APP processing induced by
herpes simplex virus type 1 (HSV-1) yelds several
APP fragments in human and rat neuronal cells.
PLoS One. 2010, 5: e13989. doi:10.1371/journal.
pone.0013989.
Saladino R, Barontini M, Crucianelli M, Nencioni
L, Sgarbanti R, Palamara AT. Current advances in
Anti-influenza Therapy. Curr Med Chem. 2010, 17:
2101-40.

Defense mechanisms against oxidative and nitrosative stress in
pathogenic protozoa
Principal investigator: Paolo Sarti
Professor of Chemistry and Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910944; Fax: (+39) 06 4440062
paolo.sarti@uniroma1.it
Participants:
Alessandro Giuffrè, CNR researcher; Elena Forte, researcher;
Daniela Mastronicola, post-doc fellow; Fabrizio Testa, PhD
student.
Collaborations:
Instituto de Tecnologia Química e Biológica, Universidade Nova
de Lisboa, Portugal (Prof. Miguel Teixeira); Dipartimento di
Scienze Biomediche, Università di Sassari (Prof. Pier Luigi Fiori);
Istituto Nazionale per le Malattie Infettive “L. Spallanzani”, Roma
(Dr. Leopoldo Paolo Pucillo).
Report of Activity
Introduction
Our research has been mainly focused on the human
protozoan parasite Giardia intestinalis, the causative
agent of giardiasis, a very common human intestinal
disease with an estimated incidence of 280 million
cases per year worldwide. This air-sensitive anaerobic
parasite, though lacking some of the conventional
ROS-scavenging systems (such as catalase
and superoxide dismutase), preferentially colonizes
a fairly aerobic tract of the gut, i.e., the proximal
small intestine. Parasite survival in the host is thus
expected to rely on antioxidant defense systems,
largely unknown as yet. Elucidating these defense
systems at a molecular level, one of the goals of the
current project, may thus provide the basis for pharmacological
applications. In the recently sequenced
genome of Giardia, a gene coding for a flavodiiron
protein (FDP) was identified. FDPs are typically
expressed in facultative anaerobic prokaryotes where
they afford from oxidative and/or nitrosative stress.
Based on an extensive structural and functional
characterization of the recombinant Giardia FDP, we
previously reported that such a protein is an efficient
H O-forming O -reductase, whose physiological role
2 2
may be to protect the parasite from O toxicity.
2
23
AREA 2
State of Art
In the current project, the studies on Giardia FDP
have been extended. Despite their remarkable
structural similarity, unlike some of the homologous
prokoryatic FDPs able to use also nitric oxide (NO)
as the substrate, Giardia FDP is exclusively reactive
towards O . The equilibrium redox properties of
2
the Giardia FDP cofactors (FMN and Fe-Fe) were
investigated by potentiometry combined with EPR
or UV/visible absorption spectroscopy. We reported
that the Giardia enzyme has virtually identical
FMN
reduction potentials for the FMN cofactor (E1 FMN = -66 mV and E = -83 mV) and considerably
2
up-shifted reduction potentials for the diiron center
Fe-Fe Fe-Fe (E = +163 ± 20 mV and E2 = +2 ± 20 mV),
1
if compared with those measured for NO-consuming
FDPs and similarly with those reported for the FDP
from the pathogenic protozoan Trichomonas vaginalis.
These different redox properties are suggestive
of subtle, yet unidentified, structural differences in
protozoan FDPs that may account for the substrate
selectivity.
By immunoblotting analysis, the Giardia FDP was
shown to be expressed at detected levels in the
disease-causing cells of the parasite (the trophozoites),
grown under standard anaerobic reducing
conditions. Evidence was provided that, whereas
nitrosative stress does not change the intracellular
levels of FDP, after shortly (10 minutes) exposing
the parasitic cells to ≥ 100 µM H O , the protein is
2 2
degraded and cellular O -consumption is severely
2
impaired, eventually causing cell death. The FDP
degradation was shown to involve a cellular proteolytic
pathway, sensitive to the proteasome-inhibitor
MG132. Based on this notable susceptibility of
Giardia to H O , we raised the hypothesis that the
2 2
peculiar localization of the parasite in the proximal
small intestine (where microbial flora is much less
abundant than in the large intestine) is a strategy to
escape oxidative stress: the small intestine is indeed

Paolo Sarti
endowed with a significantly higher redox buffering
capacity, compared to the large intestine, thereby
providing a more favorable microenvironment for
Giardia proliferation.
Nitric oxide (NO) is an antimicrobial agent produced
together with ROS by the host immune
system to control pathogens infections. In vitro,
NO-stress has been reported to have cytostatic,
rather than cytotoxic effects on Giardia. This effect
leads to the suggestion that Giardia is endowed with
defense mechanisms against NO, though, as yet,
none NO-detoxifying enzyme has been described for
the parasite.
In the genome of Giardia, a prokaryotic gene coding
for a flavohemoglobin (FlavoHb) was recently
identified. As far as our knowledge, this is the first
flavohemoglobin identified from parasitic protozoa.
We have purified and characterized the recombinant
FlavoHb from Giardia. Under aerobic conditions and
in the presence of NADH, it was shown that Giardia
FlavoHb metabolizes NO with high efficacy (V max =
116 ± 10 s -1 at [O 2 ] = 200 µM). The enzyme shows
an apparent O 2 affinity (K m = 22 ± 7 µM) compatible
with the physiological O 2 levels in the small intestine.
In response to nitrosative stress, the expression
of Giardia FlavoHb markedly increases (~ 8 fold) in
the trophozoites, which thereby become able to efficiently
metabolize NO under aerobic conditions. In
24
line with previous studies on bacterial FlavoHbs, by
using NO-releasers, NO has been shown to enhance
FlavoHb expression in Giardia. Based on these
results, we propose that physiologically FlavoHb
protects Giardia from NO toxicity.
Publications
Borisov VB, Forte E, Giuffrè A, Konstantinov A,
Sarti P. Reaction of nitric oxide with the oxidized diheme
and heme-copper oxygen-reducing centers of
terminal oxidases: Different reaction pathways and
end-products. J Inorg Biochem. 2009, 103: 1185-7.
doi:10.1016/j.jinorgbio.2009.06.002.
Vicente JB, Testa F, Mastronicola D, Forte E,
Sarti P, Teixeira M, Giuffrè A. Redox properties
of the oxygen-detoxifying flavodiiron protein
from the human parasite Giardia intestinalis. Arch
Biochem Biophys. 2009, 488: 9-13. doi: 10.1016/j.
abb.2009.06.011.
Mastronicola D, Testa F, Forte E, Bordi E, Pucillo
L P, Sarti P, Giuffrè A. Flavohemoglobin and
Nitric Oxide Detoxification in the Human Protozoan
Parasite Giardia intestinalis. Biochem Biophys Res
Commun. 2010, 399: 654-8. doi: 10.1016/j.
bbrc.2010.07.137.

Principal investigator: Maria Rosaria torrisi
Professor of General Pathology
Dipartimento di Medicina Clinica e Molecolare
Tel/Fax: (+39) 06 49979797
Azienda Ospedaliera Sant’Andrea
Tel: (+39) 06 33775502; Fax: (+39) 06 33775257
mara.torrisi@uniroma1.it
25
AREA 2
Role of the keratinocyte growth factor receptor on the molecular and
cellular alterations induced by the expression of HPV16 E5 oncoprotein
Participants:
Maurizio Alimandi, Deborah French, Patrizia Mancini,
Vincenzo Visco, professors; Francesca Belleudi, Salvatore
Raffa, researchers; Laura Leone, post-doc fellow; Danilo
Ranieri, Valeria Purpura, PhD students; Antonio Sabatucci,
technician.
Collaboration:
Laboratorio di Virologia, Istituto Regina Elena IFO-IRE, Roma (Dr.
Aldo Venuti).
Report of activity
The high-risk human papillomaviruses (HPVs) play
key roles in the pathogenesis of cervical cancers. The
E5 protein encoded by HPV type 16 is an oncoprotein
which contributes to epithelial transformation
and cooperates in tumor progression by deregulating
cell growth, survival and differentiation through
the modulation of growth factor receptors, such as
the epidermal growth factor receptor (EGFR). The
expression of the16E5 protein at the skin level in a
transgenic mouse model induces epidermal hyperplasia,
aberrant differentiation and skin tumors. Among
the epithelial growth factors, the keratinocyte growth
factor (KGF/FGF7) and the fibroblast growth factor
10 (FGF10/KGF2) are major paracrine mediators of
proliferation, differentiation, survival and migration
of epithelial cells. Both KGF and FGF10 bind to and
activate exclusively the keratinocyte growth factor
receptor (KGFR/FGFR2b). The KGFR, in contrast
to most of the growth factor receptors, appears to
play an unique and unusual role in epithelial tissues,
exerting a tumor suppressive function in vitro and in
vivo. Interestingly, the KGFR/FGFR2b null-mice
phenotype closely reminds that shown by the 16E5
transgenic mice, characterized by a similar behaviour
in skin carcinogenic model. Thus, KGFR and 16E5
might be inversely correlated in their expression
and might exert opposite and interplaying roles in
skin homeostasis and tumorigenesis. With the aim
to better elucidate the molecular events involved in
the pathological effects induced by HPV infection,
our research project attempted: a) to establish in
vitro the correlation between the expression levels
of 16E5 and those of KGFR and other epithelial
RTKs and the keratinocyte growth, differentiation,
survival, and transformation; b) to identify the
mechanisms and signaling pathways controlling the
possible effects of 16E5 expression in modulating the
ligand-dependent KGFR activation and endocytosis.
During the first year of the project, to investigate if
16E5 expression would be able to modulate KGFR in
vitro, we had used human cultured HaCaT keratinocytes
expressing 16E5 under the control of dexamethasone-inducible
promoter, as a model to study the 16E5
effects on the receptor activation and modulation and on
the related epithelial proliferation/differentiation. The
results of that preliminary part of the study have shown
KGFR transcriptional down-modulation induced by
E5 expression. In the second and third year of the project
this receptor down-modulation was confirmed in
HaCaT cells singly or doubly transfected with pCIneo
E5 (kindly provided by Dr. Venuti) or pCIneo KGFR;
the E5-induced down-regulation was demonstrated also
at the protein level by Western blot analysis. According
to this KGFR down-modulation, we found that 16E5
expression in human keratinocytes decreased the KGFinduced
proliferation and differentation.
Because the altered expression of transforming growth
factor β (TGFβ) and of its receptor serine/threosine
kinase TGF-βRII are also involved in the pathogenesis
of cervical carcinomas, during this project we have analyzed
also the possible relationship between HPV-16
integration, E5 transcription and TGF-βRII modulation
in the progression of cervical neoplasia. To this
aim quantitative Real Time PCR was performed for
the detection of HPV-16 E5 and TGF-βRII mRNA in
Low-Grade Squamous Intraepithelial Lesions (L-SIL)
and High-Grade Squamous Intraepithelial Lesions
(H-SIL), all E6/E7 positive for HPV-16. We found that
the transcript levels of TGF-βRII, compared to control
samples, were down-regulated in all HSILs, while in

Maria Rosaria Torrisi
LSILs we observed a clear decrease of the receptor
expression only in relation to high levels of E5 mRNA,
suggesting that E5 expression could play a transforming
role in cervical lesions through down-modulation
of TGF-βRII expression. These results have been
confirmed by a series of in vitro experiments showing
receptor down-regulation and deregulation of TGFβ
signaling (Belleudi, French et al., in preparation).
Since 16E5 is known to inhibit the EGFR endocytic
degradative pathway and to enhance receptor recycling,
with the aim to better elucidate the molecular
events involved in the pathological effects induced by
HPV infection we investigated if HPV16 E5 protein
would be able: a) to interfere with the two alternative
KGFR endocytic pathways and b) to affect the proliferative
and differentiative response to the ligands. In
fact, we have previously demonstrated that FGF10/
KGF2 induces receptor recycling, whereas KGF/
FGF7 stimulates receptor degradation (Belleudi et al.,
Traffic 2007): since the recycling endocytic pathway
followed by KGFR upon FGF10 stimulation correlates
with the higher mitogenic activity exerted by this
ligand on epithelial cells compared to KGF, suggesting
that the two ligands may play different functional
roles through the regulation of the receptor endocytic
transport, our hypothesis was that 16E5 could play a
similar role in targeting KGFR to the recycling route.
To this aim we studied in detail the intracellular endocytic
traffic followed by KGFR in HaCaT E5/KGFR
cotrasfected cells. In particular, we focused our attention
on the late steps of KGFR internalization to search
for a possible differential sorting of receptors destined
to recycling or degradation in the presence of E5. The
KGFR endocytic intracellular transport was analyzed
at immunofluorescence and confocal microscopy: we
found that the expression of E5 inhibits the KGFinduced
KGFR transport to the degradative pathway
but does not affect the FGF10-induced receptor traffic
through the recycling compartment. In addition, we
wondered if, in the presence of 16E5, KGFR would
recycle back to the plasma membrane upon KGF
treatment: the results showed receptor reappearance at
the plasma membrane upon KGF stimulation only in
cells expressing the 16E5 protein, indicating that the
receptor could be sorted to the slow indirect recycling
pathway and implying intracellular traffic through the
juxtanuclear compartment. Consistent with the 16E5
effect on the KGFR traffic, parallel biochemical western
blot analysis revealed that E5 protein expression
was able to induce a block of KGF-dependent degradation
(Belleudi et al., submitted).
Our results lead to the interesting hypothesis that
KGFR direct recycling is a default pathway, being
activated only when the degradative ruote is blocked,
while the indirect perinuclear recycling would be
26
inducible and regulated by a specific sorting machinery
(Belleudi et al., 2009): thus we cannot exclude that
E5 protein might be directly involved in the shifting
of KGFR trafficking from degradation to recycling.
Therefore, to investigate if 16E5 expression could
redirect the KGF-induced endocytic trafficking from
the degradative to the slow recycling pathway inducing
an inefficient recruitment of the Grb2/c-Cbl complex
and consequent ligand-dependent ubiquitination
of the receptor, as occurring upon FGF10 treatment
(Belleudi et al., 2007), we analyzed the phosphorylation
level of tyrosine 196 in the KGFR substrate FRS2,
which represent the principal docking site for Grb2,
and we found that E5 expression correlates with a
reduced phosphorylation of the FRS2 tyrosine 196.
Thus, the KGFR alternative endocytic traffic triggered
by the two ligands may provide a useful physiological
model for further investigations directed to
elucidate the distinct functions of the molecular components
of the endosomal sorting complexes as well as
the mechanisms controlling the endocytic traffic and
intracellular receptor signaling. In addition, we propose
that HPV16 E5 expression can be used as a model
to analyze the endocytic indirect recycling pathway.
Publications
Belleudi F, Leone L, Maggio M, Torrisi MR. Hrs
regulates the endocytic sorting of the fibroblast
growth factor receptor 2b. Exp Cell Res. 2009, 315:
2181-91. doi:10.1016/j.yexcr.2009.03.022.
Visco V, Bava FA, d’Alessandro F, Cavallini M,
Ziparo V, Torrisi MR. Human colon fibroblasts
induce differentiation and proliferation of intestinal
epithelial cells through the direct paracrine action of
keratinocyte growth factor. J Cell Physiol. 2009, 220:
204-13. doi: 10.1002/jcp.21752.
Belleudi F, Cardinali G, Kovacs D, Picardo M, Torrisi
MR. KGF promotes paracrine activation of the SCF/c-
KIT axis from human keratinocytes to melanoma cells.
Transl Oncol. 2010, 3: 80-90. doi: 10.1593/tlo.0919.
Kovacs D, Cardinali G, Aspite N, Cota C, Luzi F,
Bellei B, Briganti S, Amantea A, Torrisi MR, Picardo
M. Role of fibroblast-derived growth factors in
regulating hyperpigmentation of solar lentigo. Br J
Dermatol. 2010, 163: 1020-27. doi: 10.1111/j.1365-
2133.2010.09946.x.
Santonico E, Belleudi F, Panni S, Torrisi MR,
Cesareni G, Castagnoli L. Multiple modification
and protein interaction signals drive the Ring finger
protein 11 (RNF11) E3 ligase to the endosomal compartment.
Oncogene. 2010, 29: 5604-18. doi:10.1038/
onc.2010.294.

AREA 3
Molecular
genetics
of
eukaryotes

CFTR gene engineering by means of prokaryotic and eukaryotic
artificial chromosome
Principal investigator: Fiorentina Ascenzioni
Professor of Microbiology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49917577/7614; Fax: (+39) 06 49917594
fiorentina.ascenzioni@uniroma1.it
Participants:
Cristina Auriche, Enea Gino Di Domenico, Francesca
Tilesi, post-doc fellows; Ivan Coladarci, graduate student.
Collaborations:
Dipartimento di Scienze Biomediche, Università di Foggia (Prof.
Massimo Conese); Dipartimento di Biotecnologie Cellulari
ed Ematologia, Sapienza-Università di Roma (Prof. Marco
Lucarelli); Laboratorio di Genetica Molecolare, Istituto Giannina
Gaslini, Genova (Prof. Olga Zegarra-Moran); Istituto di
Neurobiologia e Medicina Molecolare, CNR, Roma (Prof. Ettore
D’Ambrosio).
Report of activity
Cystic fibrosis (CF) is a recessive congenital disease
that shows high incidence in Caucasian populations
(ca. 1 in 2 000). CF is caused by mutations in the gene
coding for CF transmembrane conductance regulator
(CFTR; OMIM 602421), a Cl - channel expressed
in the apical membrane of several epithelia. CFTR is
the main pathway for Cl - secretion; therefore, normal
CFTR activity is necessary for the maintenance of
the level of the airway surface liquid. Indeed, CFTR
loss-of-function leads not only to reduced Cl - secretion,
but also to an increase in Na + and water absorption,
probably because of a predominant activity of
the epithelial Na + channel (ENaC). In vivo as well as
in vitro models of well-differentiated CF airway epithelia
have shown isotonic reduction of the surface
liquid, and the consequent impaired movement of
the overlying mucus; leading to increased adherence
of micro-organisms such as Pseudomonas aeruginosa.
Despite recent developments in the treatment of CF,
there is no definitive cure for this disease, and the
life expectancy for the patient with CF is around
30-40 years. Since localization of the CFTR gene to
the long arm of chromosome 7 in 1989, a huge effort
has been put into the development of gene therapies
for this life-threatening disease. To date, more than
29
AREA 3
30 clinical gene therapy trials have been performed
worldwide, although with only very limited success.
As effective therapy by such in-vivo delivery of therapeutic
DNA requires physiological levels of expression
and long-term maintenance, it has been hypothesized
that this can be achieved by using genomic
fragments that contain all of the long-range controlling
elements that will allow tissue-specific gene
expression to a physiological level. Although CFTR
expression was initially thought to be dictated by a
“housekeeping” promoter, it appears that regulation
of CFTR expression involves interactions between a
number of different regulatory factors and elements.
Indeed, cell-specific expression is determined by regulatory
elements that are upstream and downstream
of the coding region, and in various introns.
The human CFTR locus was isolated in a YAC vector
of about 320 kb in size (YAC 37AB12) by Anand
and collaborators in 1991, and subsequently Manson
and collaborators showed that this vector complemented
CF defects in cftr-/- mice.
From the YAC- to the BAC-CFTR
The YAC37AB12 containing the intact CFTR
locus, was converted into a circular BAC by using
the retrofitting plasmid pNK-G418. Subsequently, to
insert a selectable marker for mammalian cells into
the circularized YAC, we performed a second DNA
modification by replacing the region between -50 kb
to -25 kb from the CFTR start codon, with a fragment
containing markers for selection of mammalian
cells (Zeocin) and for yeast cells (Phleomicin). Such
substitution caused a size reduction of the circular
YAC of about 20 kb, leading to a circular molecule
smaller than 300 kb. Next, both circular YAC/BACs,
cCFTR5A and cCFTRD12, were recovered into E.
coli EC100 cell and analyzed by sequencing.
Sequence analysis demonstrated that the cCFTR5A
contained an insert running from -49,923 bp upstream
the CFTR start codon (position 42245379 of the reference
contig NT_00793.14) to +58,214 bp downstream

Fiorentina Ascenzioni
the stop codon (position 42542218, NT_00793.14)
(Fig. 1). The two vectors cCFTR5A and cCFTRD12
showed the same 5’ and 3’ ends confirming that the
pruning procedure occurred as expected, additionally
we found that the 5’ upstream DNA removed in
the cCFTRD12 vector corresponded to the region
spanning from -25,696 bp to -49,788 bp (position
42277897 to 42253805 of the reference contig
NT_00793.14). Sequence analysis of all the introns,
intron/exon junctions and the proximal 5’-flanking
region of the CFTR locus resulted wild type and the
studied polymorphisms were as follows: the repeats in
intron 6a were (GATT) 7 ; the repeats in intron 8 were
(TG) 11 T 7 ; the polymorphic site at the aminoacid 470
was V470. Overall the BAC cCFTR5A, and its derivative
cCFTRD12, contain the most common wild type
CFTR haplotype in the Caucasian population.
Next cCFTRD12 was transferred into Fisher Rat
Thyroid (FRT) cells for functional analysis. The
resulting clones were analyzed for the CFTR mRNA
and the transepithelial Cl - current. The results
obtained clearly showed CFTR mRNA in half of
clones analysed (FRTcl2-7), although at different
level, and CFTR Cl- currents in 5 out of the 6 CFTR
mRNA positive clone. One of these (FRTcl7) was the
best clone responding to the cAMP agonist with a
short-circuit current (Isc) increase of about 18 µA/
cm 2 (Fig. 2). Finally, by confocal microscopy analysis
revealed that the CFTR protein was localized both
to the apical membrane and to the cytoplasm.
CFTR-bacteria interaction
It was shown that CFTR functions as an internalizing
receptor for P. aeruginosa by binding of amino
acids 108-117 to the outer core LPS of P. aeruginosa.
According to this finding, we hypothesized that
CFTR expression and proper trafficking to the apical
membrane of FRT cells should increase the rate
of bacteria internalization. Infection assays with P.
aeruginosa (ATCC 27853) clearly demonstrated
that FRT cells expressing CFTR internalized much
30
more bacteria than FRT cells, additionally treatment
of the cells with the CFTR inhibitor (CFTR Ihn172 )
reduced the percentage of internalized bacteria in
CFTR-expressing cells but not in parental FRT
cells. These results not only confirm the role of
CFTR as P. aeruginosa receptor but also suggest a
possible role of CFTR activity in the survival of bacteria
within the cells, soon after infection.
Publications
Di Domenico EG, Auriche C, Viscardi V, Longhese
MP, Gilson E, Ascenzioni F. The Mec1p and Tel1p
checkpoint kinases allow humanized yeast to tolerate
chronic telomere dysfunctions by suppressing
telomere fusions. DNA Rep. 2009, 8: 209-18. doi:
10.1016/j.dnarep.2008.10.005.
Tilesi F, Fradiani P, Socci V, Willems D, Ascenzioni
F. Design and validation of siRNAs and shRNAs.
Curr Opin Mol Ther. 2009, 11: 156-64.
Auriche C, Di Domenico EG, Pierandrei S, Lucarelli
M, Castellani S, Conese M, Melani R, Zegarra-Moran
O, Ascenzioni F. CFTR expression and activity from
the human CFTR locus in BAC vectors, with regulatory
regions, isolated by a single-step procedure. Gene
Ther. 2010, 17: 1341-54. doi:10.1038/gt.2010.89.
Fradiani PA, Petrucca A, Ascenzioni F, Di Nucci
G, Teggi A, Bilancini S, Cipriani P. Endocarditis
caused by Lactobacillus jensenii in an immunocompetent
patient. J Med Microbiol. 2010, 59: 607-9. doi:
10.1099/jmm.0.017764-0.
Rocchi L, Braz C, Cattani S, Ramalho A, Christan
S, Edlinger M, Ascenzioni F, Laner A, Kraner S,
Amaral M, Schindelhauer D. Escherichia coli-cloned
CFTR loci relevant for human artificial chromosome
therapy. Hum Gene Ther. 2010, 21: 1077-92.
doi:10.1089/hum.2009.225.

31
AREA 3
Fig. 1 - Schematic representation of the contig NT_00793.14 (in the middle); the CFTR locus is flanked upstream by ASZ1 (ankyrin repeat,
SAM and basic leucin zipper) and downstream by CTTNBP2 (cortactin binding protein 2). The arrows point to the positions 42245379 and
42542218 delimitating the fragment cloned into the BACs; bent arrow represents the initiation site of translation. cCFTR5A (below the
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
as cCFTR5A but the upstream region between position 42253805-42277897 (dashed line) was deleted.
Fig. 2 - Short-circuit current recordings. Summary of CFTR-dependent short-circuit currents obtained with each clone. Data are means ±SE
(n = 3). The negative control was parental FRT cells.

RNA-RNA and RNA-protein interactions: role of small non-coding
RNAs in gene expression control
Principal investigator: Irene Bozzoni
Professor of Molecular Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912202; Fax: (+39) 06 49912500
irene.bozzoni@uniroma1.it
Participants:
Carlo Presutti, professor; Elisa Caffarelli, Alessandro
Fatica, researchers; Monica Ballarino, Davide Cacchiarelli,
Erika Girardi, Tania Incitti, Pietro Laneve, Julie Martone,
Mariangela Morlando, Tiziana Santini, post-doc fellows;
Marcella Cesana, PhD student.
Collaborations:
Dipartimento di Medicina Molecolare, Sapienza-Università di
Roma (Prof. Alberto Gulino); Scienze Anatomiche, Istologiche,
Medico-Legali e dell’Apparato Locomotorio; Sapienza-Università
di Roma (Prof. Antonio Musarò); TIGEM, Napoli (Prof. Alberto
Auricchio); City of Hope, Duarte, USA (Prof. John Rossi).
Report of activity
The aim of this project was to exploit and further
develop the high potential of RNA-based methodologies
for the comprehension of the molecular basis of
several processes of gene regulation and to apply this
knowledge to the study and cure of several genetic
disorders, such as cancer or inherited diseases.
The project covers topics ranging from the biosynthesis
of small-non coding RNAs to their function in
normal and pathological conditions. In particular, we
have focused on the study of the role that miRNAs
play in biological processes as important as developmental
control, cell proliferation and apoptosis.
Accordingly, altered miRNA expression can have
profound effects on human pathology, including
cancer, making it crucial to understand how miRNA
genes are regulated.
In a parallel line of research, we have exploited the
vast potential of different RNA activities (antisense
and interference) for human therapy, based on an
advanced understanding of the underlying mechanisms.
To achieve these goals, integrated and multidisciplinary
action was required including basic research
and development, genetic approaches, as well as cell
biology assessment.
33
AREA 3
miRNA function
miRNA in neuronal differentiation
We previously showed that miR-9, which is almost
exclusively expressed in the brain, is upregulated
during in vitro neuronal differentiation, whereas it
is downregulated in 50% of primary neuroblastoma
(NB) tumours, suggesting its potential function as an
oncosuppressor.
In the last years we have characterized the transcriptional
regulation of the miR-9-2 gene and found that,
despite its localization inside an exon of a host-gene,
it is expressed as an independent unit with the promoter
located in the upstream intron. We also show
that the repressor REST inhibits expression of the
miR-9-2 promoter in undifferentiated neuroblastoma
cells, whereas REST dismissal and simultaneous
binding of the phosphorylated active form of CREB
triggers transcription in differentiating cells. Finally,
we have been able to highlight a regulatory feed-back
mechanism in which the reciprocal action of miR-9
and the antineurogenic transcription factor REST
may be relevant for the maintenance of the neuronal
differentiation program (Laneve et al., 2010).
A high throughput microRNA expression profile
was also performed in human primary medulloblastoma
specimens. We identified specific micro-
RNA expression patterns which distinguish medulloblastoma
differing in histotypes (anaplastic, classic
and desmoplastic), in molecular features (ErbB2 or
c-Myc overexpressing tumors) and in disease-risk
stratification (Ferretti et al., 2009). We found a
limited number of miRNAs displaying differential
expression between tumours and controls, indicating
that specific miRNAs may represent biological markers
that could be useful in predicting MB prognosis
and further subdivision of patients.
miRNA in muscle differentiation
miRNA deregulation linked to pathology finds an
interesting example in the Duchenne Muscular
Dystrophy (DMD), characterized by the absence of

Irene Bozzoni
dystrophin. We found that this deficiency at the sarcolemma
delocalizes and downregulates Nitric Oxide
Synthase (NOS) and that this alters S-nitrosylation
of HDAC2 and its chromatin association. During
this study we showed that the differential HDAC2
nitrosylation state in Duchenne versus wild-type conditions
deregulates the expression of a specific subset
of microRNA genes (Cacchiarelli et al., 2010a).
Several circuitries controlled by the identified micro-
RNAs, such as the one linking miR-1 to the G6PD
enzyme and the redox state of cell, or miR-29
to extracellular proteins and the fibrotic process,
explain some of the DMD pathogenetic traits. We
also showed that, at variance with other myomiRs,
miR-206 escapes from the dystrophin-nNOS control
being produced in activated satellite cells before
dystrophin expression; in these cells, it contributes
to muscle regeneration through repression of the
satellite specific factor, Pax7.
We concluded that the pathway activated by dystrophin/nNOS
controls several important circuitries
increasing the robustness of the muscle differentiation
programme.
miRNAs in hematopoietic differentiation
In the last years we have identified several miRNAs
playing a crucial role in myeloid differentiation
(Fazi et al., 2005; Rosa et al., 2007). Recently, the
list has included the newly identified miR-342. We
demonstrated that miR-342 and its host transcript
are under the control of the same promoter: the
transcriptional factor IRF-1 (interferon regulatory
factor-1) is required for maintaining basal levels of
miR-342, while IRF-9 and PU.1 are responsible for
its responsivenes to RA. Notably, PU.1 and IRF-1
play a crucial role in myeloid differentiation and
PU1 is very often mutated in myeloid leukemia (De
Marchis et al., 2009).
With the aim of identifying the molecular circuitries
controlled by miR-342 we have pursued the SILAC
methodology on NB4 cells infected with a lentiviral
vector expressing miR-342. 100 proteins have been
identified whose expression appears to be controlled
by miR-342 and among them approximately 15% are
predicted to be its direct targets. Validation of these
targets and the building of integrated circuitries connecting
these different factors is currently undergoing.
Use of RNA for the therapy of Duchenne
Muscular Dystrophy
The use of the mdx mouse as the animal model for
the Duchenne Muscular Dystrophy has allowed us
to demonstrate the effectiveness of the antisense
34
strategy in restoring the synthesis of dystrophin in
vivo for the entire life of the animal. Therefore, this
approach provided solid basis for a systemic use of
AAV-mediated antisense-U1 snRNA expression for
the therapeutic treatment of DMD.
However, when thinking of possible intervention on
human, it is very crucial to identify the most appropriate
antisense sequences able to provide the highest
possible skipping efficiency.
In the course of the last year, we compared the
exon 51 skipping activity of ten different antisense
molecules, raised against splice junctions and/or
exonic splicing enhancers, expressed as part of the
U1 snRNA. The effectiveness of each construct
was tested in human DMD myoblasts carrying
the deletion of exons 48-50 which can be cured by
skipping of exon 51. Our results showed that the
highest skipping activity and dystrophin rescue is
achieved upon expression of a U1 snRNA-derived
antisense molecule targeting exon 51 splice sites
in combination with an internal exon sequence.
The efficacy of this molecule was further proven
on an exon 45-50 deletion background, utilizing
patient’s fibroblasts trans-differentiated into myoblasts.
In this system, we showed that the selected
antisense was able to produce 50% skipping of exon
51. These data allowed us to select the most active
molecule for the skipping of exon 51 that has been
selected for entering human clinical trials (Incitti
et al., 2010).
Publications
Ballarino M, Pagano F, Girardi E, Morlando M,
Cacchiarelli D, Marchioni M, Proudfoot NJ, Bozzoni
I. Coupled RNA processing and transcription of
intergenic primary microRNAs. Mol Cell Biol. 2009,
29: 5632-8. doi:10.1128/MCB.00664-09.
De Marchis ML, Ballarino M, Salvatori B, Puzzolo
MC, Bozzoni I, Fatica A. A new molecular network
comprising PU.1, interferon regulatory factor proteins
and miR-342 stimulates ATRA-mediated granulocytic
differentiation of acute promyelocytic leukemia cells.
Leukemia. 2009, 23: 856-62. doi:10.1038/leu.2008.372.
Fatica A, Bozzoni I. Role of microRNAs in hematological
malignancies. Expert Review Hematol. 2009, 2:
415-23. doi: 10.1586/ehm.09.32.
Ferretti E, De Smaele E, Po A, Di Marcotullio
L, Tosi E, Espinola MS, Di Rocco C, Riccardi R,
Giangaspero F, Farcomeni A, Nofroni I, Laneve P,

Gioia U, Caffarelli E, Bozzoni I, Screpanti I, Gulino
A. MicroRNA profiling in human medulloblastoma.
Int J Cancer. 2009, 124: 568-77. doi: 10.1002/ijc.23948.
Puttini S, Lekka M, Dorchies OM, Saugy D, Incitti
T, Ruegg UT, Bozzoni I, Kulik AJ, Mermod N.
Gene-mediated restoration of normal myofiber elasticity
in dystrophic muscles. Mol Ther. 2009, 17:
19-25. doi: 10.1038/mt.2008.239.
Anastasiadou E, Boccellato F, Vincenti S, Rosato P,
Bozzoni I, Frati L, Faggioni A, Presutti C, Trivedi
P. Epstein-Barr virus encoded LMP1 downregulates
TCL1 oncogene through miR-29b. Oncogene. 2010,
29: 1316-28. doi:10.1038/onc.2009.439.
Cacchiarelli D, Martone J, Girardi E, Cesana M,
Incitti T, Nicoletti C, Santini T, Sthandier O,
Auricchio A, Musarò A, Bozzoni I. MicroRNAs
involved in molecular circuitries relevant for the
Duchenne muscular dystrophy pathogenesis are controlled
by the dystrophin/nNOS pathway. Cell Metab.
2010, 12: 341-51. doi: 10.1016/j.cmet.2010.07.008.
Chiaretti S, Messina M, Tavolaro S, Zardo G, Elia L,
Vitale A, Fatica A, Gorello P, Piciocchi A, Scappucci
35
AREA 3
G, Bozzoni I, Fozza C, Candoni A, Guarini A, Foà
R. Gene expression profiling identifies a subset of
adult T-cell acute lymphoblastic leukemia (T-ALL)
with myeloid-like gene features and overexpression
of miR-223. Haematologica. 2010, 95: 1114-21. doi:
10.3324/haematol.2009.015099.
Incitti T, De Angelis FG, Cazzella V, Sthandier O,
Pinnarò C, Legnini I, Bozzoni I. Exon skipping and
Duchenne muscular dystrophy therapy: selection of
the most active U1 snRNA-antisense able to induce
dystrophin exon 51. Mol Ther. 2010, 18: 1675-82.
doi:10.1038/mt.2010.123.
Laneve P, Gioia U, Andriotto A, Moretti F, Bozzoni
I, Caffarelli E. A minicircuitry involving REST and
CREB controls miR-9-2 expression during human
neuronal differentiation. Nucleic Acids Res. 2010, 38:
6895-905. doi: 10.1093/nar/gkq604.
Rinaldi A, Vincenti S, De Vito F, Bozzoni I, Oliverio
A, Presutti C, Fragapane P, Mele A. Stress induces
region specific alterations in microRNAs expression
in mice. Behav Brain Res. 2010, 208: 265-9.
doi:10.1016/j.bbr.2009.11.012.

Role of metalloproteinases and their tissue inhibitors in the
regulation of neurogenesis and gliogenesis from neural stem/
progenitor cells
Principal investigator: Emanuele Cacci
Researcher in Clinical Biochemistry and clinical Molecular Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912206; Fax: (+39) 06 49912351
emanuele.cacci@uniroma1.it
Participants:
Stefano Biagioni, professor, Tonino Anelli, post-doc fellow;
Pasquale Caramanica, PhD student.
Collaborations:
Università Carlo Bo, Urbino (Prof. Ferdinando Mannello, Dr.
Gaetana A. Tonti); Istituto Superiore di Sanità, Roma (Dr.
Luisa Minghetti, Dr. Maria Antonietta Ajmone-Cat); King’s
College, London, UK (Prof. Noel Bukley).
Report of activity
Neural stem cells (NSC), which generate new neurons
and glia under physiological and pathological
conditions, persist in the adult brain. Regulation
of adult neurogenesis involves a wide spectrum of
signals from NSC microenvironment, the so-called
neurogenic niche. As demonstrated by our and other
groups, microglial cells are crucial components of the
niche and they exert either beneficial or detrimental
effects on neurogenesis, depending on their activation
state and ranges of mediators produced.
The identification of intrinsic and extrinsic factors
implicated in the modulation of neurogenesis is crucial
to define strategies to successfully manipulate
the neurogenic and gliogenic potential of NSC.
Among extrinsic factors, matrix metalloproteinases
(MMPs) and their natural tissue inhibitors (TIMPs)
are emerging as contributors to neurogenesis modulation.
In addition, MMPs are multifaceted contributors
to neuroinflammation. Particularly MMP-2 and
MMP-9, have been extensively studied in the brain,
where they are found expressed at low levels under
physiological conditions and up-regulated in various
pathological conditions. MMPs and TIMPs are
expressed in NSCs and other brain cell types, including
microglia. Noteworthy, MMPs and TIMPs regulate
cytokine production in microglia in autocrine/
paracrine manner and, vice versa, cytokines appear to
37
AREA 3
be potent regulators of microglial MMP production.
In light of these considerations, we started the present
project with the following major aims:
- Analysis of the possible roles of MMPs and
TIMPs in the regulation of NSC properties.
- Characterization of MMP and TIMP profile in
NSCs.
- Analysis of the molecular mechanisms regulating
MMP and TIMP expression during NSC differentiation.
- Analysis of inflammatory mediators (cytokines,
MMPs, and TIMPs) produced in resting and activated
microglia and their possible implications for
neurogenesis and gliogenesis modulation.
Results
a) We first generated cell lines, which display NSC
properties, from embryonic and adult mouse brain.
We also isolated and extensively characterized several
clonal lines from adult mouse brain. These lines
express typical markers of NSCs (e.g. Pax6, Sox2,
Nestin, Blbp, RC2) and retain self-renewal and multipotency
(ability to differentiate into neurons, astrocytes
and oligodendrocytes), two functional criteria
to define stem cells. Once established suitable cellular
models, we investigated the role of MMPs in the
regulation of NSC properties. We used a broad spectrum
MMP pharmacological inhibitor. The inhibitor
increased NSC differentiation into neurons (almost
doubling the percentage of neurons compared with
control cultures) and reduced the generation of glial
cells in a dose-dependent manner. The inhibitor did
not affect NSC differentiation if added once the differentiation
process was ongoing. From these and other
data we conclude that: 1) MMPs modulate NSC commitment;
2) The inhibitor treatment is an efficient
and simple protocol for the in vitro generation of neurons
from NSC. The generation of highly enriched
neuronal (or glial) cultures is highly demanded, as
it enables to dissect differentiation and maturation
processes at both biochemical and molecular levels.

Emanuele Cacci
b) Since MMP-2 and MMP-9 are the main MMPs
in the brain, we speculated that the MMP inhibitor
effects on NSCs were mediated through MMP-2
and/or MMP-9 inhibition. To clarify this point we
first investigated the expression profile of MMP-2
and MMP-9 in NSCs. RT-PCR analysis and gel
zymography showed that MMP-2 is expressed in
NSCs and increases during differentiation, whereas
MMP-9 is undetectable under our culture conditions.
To directly assess if MMP-2 is important in
the modulation of NSC differentiation we silenced
these proteins by means of specific siRNAs. Our
results indicate that MMP-2 is not the major controller
of NSC differentiation into neurons and that
other MMPs could be involved in the modulation of
this process (manuscript in preparation).
c) In this first year project we also addressed the
molecular mechanisms involved in the regulation
of NSC properties. We focused our attention on the
RE1-silencing transcription factor (REST), a master
regulator of neuronal genes that can induce neuronal
differentiation in NSC. Our computational analysis
and experimental data suggest that REST can modulate,
among others, several genes of the MMP and
TIMP families.
To clarify which genes are transcriptionally repressed
by REST we have used an adenovirus carrying the
dominant negative of REST (DN:REST), or shRNA
against REST. DN:REST comprises the REST DNA
binding domain, but lacks the N and C termini; it
thus derepresses transcription of REST target genes.
NSCs were infected with DN:REST vector and with
the corresponding control vector and after 48 h the
cells were lysated and RNA extracted. Analysis of
global gene expression changes by DNA microarray
is currently ongoing (in collaboration with Prof.
Bukley). In parallel, we immunoprecipitated chromatin
extracted from DN:REST infected and control
cells by using an anti-REST antibody. Preliminary
data suggest REST enrichment at the RE1 elements
of well known neuronal genes; Real time PCR analysis
will be performed to demonstrate specific target
gene occupancy by REST. These studies will help
38
to identify novel mechanisms for the regulation of
genes known, or yet unexplored, as possible modulators
of NSC properties.
d) To identify released factors produced by microglia
under different activation conditions (i.e. acute or
chronic stimulation) and their consequences on neurogenesis,
we characterized the expression profile of
several pro- and anti-inflammatory cytokines. We
focused our attention on IL1a and IL1b, abundantly
produced under acute but not chronic activation, and
found that IL1a and IL1b are important modulators
of NSC differentiation (Ajmone-Cat et al., 2010).
We aim to further characterize the expression profile
of inflammatory mediators, including MMPs
and TIMPs, under different activation conditions.
We will test the effects of MMP inhibition on the
functional phenotypes of activated microglia and in
particular we will investigate if MMPs inhibition
shapes activated microglia towards a pro- or antineurogenic
phenotype.
Perspective
We aim to pursue the following goals: 1) To complete
transcriptome and ChIp analysis to deepen
our knowledge on the role of REST in NSC differentiation
and in the regulation of specific genes,
such as MMPs and TIMPs; 2) To understand the
contribution of MMPs and TIMPs to the modulation
of microglial activation and the consequences on
the neurogenic and gliogenic processes. Altogether
these experiments will contribute to a better comprehension
of the events and cues that regulate brain
repair mechanisms and will offer new opportunities
to identify possible targets for the successful therapeutic
manipulation of endogenous neurogenesis.
Publications
Ajmone-Cat MA, Cacci E, Ragazzoni Y, Minghetti
L, Biagioni S. Pro-gliogenic effect of IL-1alpha in
the differentiation of embryonic neural precursor
cells in vitro. J Neurochem. 2010, 113: 1060-72. doi:
10.1111/j.1471-4159.2010.06670.x.

Crosstalk between poly(ADP-ribosyl)ation and DNA methylation
in the regulation of gene expression
Principal investigator: Paola Caiafa
Professor of Clinical Biochemistry and Molecular Biology
Dipartimento di Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 49976530; Fax: (+39) 06 44231961
caiafa@bce.uniroma1.it
Participants:
Anna Reale, professor; Michele Zampieri, Tiziana
Guastafierro, post-doc fellows; Maria Giulia Bacalini, PhD
student.
Collaborations:
Istituto di Biologia e Patologia Molecolari, CNR, Roma (Dr.
Claudio Passananti).
Report of activity
Our findings showed (1-2) that along the chain of
events that induce DNA methylation-dependent
chromatin condensation/decondensation, a postsynthetic
modification, namely poly(ADP-ribosyl)
ation (PARylation), participates in the establishment
and maintenance of genome methylation pattern.
To explain how PARP activity controls DNA
methylation pattern we suggested a model (3) in
which ADP-ribose polymers (PARs), present on
PARylated PARP-1, interact noncovalently with
Dnmt1, preventing its enzymatic activity. We found
that Dnmt1 is able to bind long and branched PARs
in a noncovalent way and PARs, either free or
PARP-1 bound, inhibit human recombinant Dnmt1
activity both in vitro and in vivo (3). In absence of
PARylated PARP-1, Dnmt1 is free to methylate
DNA; if, on the other hand, high levels of PARylated
PARP-1 persist, Dnmt1 will be stably inhibited,
preventing DNA methylation. Our new data supported
this model and have now included CTCF as
an important player in the PARP-1/Dnmt1 interplay
(4). CTCF over-expression caused genomic
hypomethylation. In vitro data confirmed that the
inhibitory role on Dnmt1 was not due to CTCF,
but to PARylated PARP-1. In fact, CTCF was per
se capable of activating PARP-1 automodification
and PARylated PARP-1 was, in turn, able to inhibit
Dnmt1 activity, according to the above mentioned
model. Considering all the above mentioned data, it
39
AREA 3
was suggested that CTCF marks DNA regions for
PARP-1 binding, and thus for maintaining CTCF
target sequences in an unmethylated state. The
maintenance of proper DNA methylation pattern on
these sites determines the correct expression of the
correlated genes, e.g. imprinted genes.
By using PARG overexpression to deplete cells of
PARs, we showed that the Dnmt1 gene promoter is
protected from methylation by PARylated PARP-1,
adding another dimension to the regulatory functions
of PARylated PARP-1, through its control
of DNA methylation pattern of housekeeping gene
promoters. We demonstrated that the CpG island
located in the promoter of Dnmt1 becomes methylated
in absence of PARs (5). ChIP analyses, performed
to verify if PARP-1 and/or PARs are involved in the
control of DNA methylation pattern of Dnmt1 promoter,
showed that only the fragment that we found
methylated following PARG overexpression, was
present in ChIP with both antibodies anti PARP-1
and anti PARs, Figure 1. Therefore the PARylated
form of PARP-1 sits on Dnmt1 promoter maintaining
this region non methylated and this introduces
PARylation as an epigenetic mark capable of preventing
the methylation of some regions that need
to maintain their unmethylated state in normal cells,
e.g. some CpG islands.
A model can be put forward: when the cells are depleted
of PARylated PARP-1 and PARs, Dnmt1, not inhibited,
is free to introduce new aberrant methyl groups
onto DNA. Otherwise, PARP-1, already PARylated
or when PARylated because of binding to CTCF, is
capable of attracting Dnmt1, through the polymers
present on its PARylated form and polymers, in turn,
inhibit the Dnmt1 methyltransferase activity. These
data raise the possibility that PARylated PARP-
1 marks those DNA sequences that must remain
unmethylated, thus playing a role in the epigenetic
regulation of gene expression. The eventuality that
PARP-1 activity could confer in trans the protection
against aberrant DNA methylation to CpG islands of

Paola Caiafa
housekeeping gene promoters is of great interest and
it is also strongly supported by data on p16 gene (6).
It could provide important information on one possible
mechanism by which proper DNA methylation
patterns, which determine the correct expression of
correlated genes, is protected under physiological
conditions.
Our aim is to verify if PARylated PARP-1 introduces
an epigenetic code on chromatin. The aim has been
to verify if the interplay between PARylation and
DNA methylation could be responsible for the maintenance
of the methylation state of specific CTCF
target sequences. To this purpose, we have been
using as model some well characterised CTCF binding
regions, i.e. the differentially methylated region
(ICR) in the Igf2/H19 imprinted locus.
Here the two genes are adjacent and share the same
enhancer region but, while H19 is expressed only
by the maternal allele, Igf2 is expressed only by
the paternal one (7). The unmethylated ICR DNA
region, localized between the two genes, exerts
the function of insulator by acting as a boundary
to limit the function of the enhancer region at the
Igf2 promoter in the maternal allele. The insulator
function of the ICR requires the binding of the
CTCF protein that occurs only when the ICR is
unmethylated. It has recently been demonstrated
that also PARylation plays a role in imprinting
control (10). In fact, a covalently PARylated form
of CTCF, whose MW shifts from 130 to 180 kDa
as a consequence of the modification, has been
found to be bound to the ICR. Moreover, the
evidence that the treatment of cells with competitive
PARP inhibitors causes loss of imprinting at
the Igf2/H19 locus together with almost 140
CTCF target sites in the genome (10), strongly
suggested that PARylation of CTCF is required
in the control of imprinted gene expression. The
notion of a functional link between PARylation
and DNA methylation (1) prompted us to verify
the hypothesis that these two epigenetic events do
not act separately but work together in the control
of imprinting and in particular that PARylation
is required to maintain proper DNA methylation
patterns at imprinted DNA regions. Based on
our recent achievements (4) that include CTCF
in the interplay between PARylation and DNA
methylation, we suggest an alternative model of
epigenetic control of CTCF boundary function
based on PARylation. In this model, CTCF stimulates
PARP activity at its DNA target sites and
prevents de novo methylation of CpG dinucleotides
by inhibiting Dnmt1 activity.
40
Data, obtained in L929 mouse fibroblasts, showing
that CTCF forms a complex both in vitro (Pulldown)
and in vivo (Co-IP e ChIP e RE-ChIP) with
PARylated PARP-1 and Dnmt1 at the ICR, confirm
our expectations. Moreover, the depletion of PARs,
obtained by PARG overexpression, causes loss of
imprinted expression of Igf2, hypermethylation of
CTCF-associated CpGs and removal of both CTCF
and PARP-1 from the ICR. All this suggests that
PARs are required to preserve the DNA methylation
profile of CTCF binding targets (within the
maternal ICR), manuscript in preparation.
References
1) Caiafa et al. (2008) Faseb J 23, 672-8.
2) Caiafa and Zampieri (2005) J Cell Biochem 94,
257-65.
3) Reale et al. (2005) Oncogene 24, 13-9.
4) Guastafierro et al. (2008) J Biol Chem 283, 21873-
80.
5) Zampieri et al. (2009) PLos One 4:e4717.
6) Witcher et al. (2009) Mol Cell 34, 271-84.
7) Wallace and Felsenfeld (2007) Curr Opin Genet
Dev 17, 400-7.
8) Bell and Felsenfeld (2000) Nature 405, 482-85.
9) Schoenherr et al. (2003) Nature Gen 33, 66-69.
10) Yu et al. (2004) Nature Gen 36, 1105-10.
Publications
Caiafa P, Guastafierro T, Zampieri M. Epigenetics:
poly(ADP-ribosyl)ation of PARP-1 regulates
genomic methylation patterns. FASEB J. 2009, 23:
672-8. doi: 10.1096/fj.08-123265.
Caiafa P, Zlatanova J. CCCTC-binding factor meets
poly(ADP-ribose) polymerase-1. J Cell Physiol. 2009,
219: 265-70. doi: 10.1002/jcp.21691.
Zampieri M, Passananti C, Calabrese R, Perilli M,
Corbi N, De Cave F, Guastafierro T, Bacalini MG,
Reale A, Amicosante G, Calabrese L, Zlatanova J,
Caiafa P. Parp1 localizes within the Dnmt1 promoter
and protects its unmethylated state by its enzymatic
activity. PLoS One. 2009, 4:e4717. doi: 10.1371/journal.pone.0004717.
Zlatanova J, Caiafa P. CCCTC-binding factor: to
loop or to bridge. Cell Mol Life Sci. 2009, 66: 1647-60.
doi: 10.1007/s00018-009-8647-z.
Zlatanova J, Caiafa P. CTCF and its protein partners:
divide and rule? J Cell Sci. 2009, 122: 1275-84. doi:
10.1242/jcs.039990.

Chevanne M, Zampieri M, Caldini R, Rizzo A,
Ciccarone F, Catizone A, D’Angelo C, Guastafierro
T, Biroccio A, Reale A, Zupi G, Caiafa P. Inhibition
of PARP activity by PJ-34 leads to growth impair-
41
AREA 3
ment and cell death associated with aberrant mitotic
pattern and nucleolar actin accumulation in M14
melanoma cell line. J Cell Physiol. 2010, 222: 401-10.
doi:10.1002/jcp.21964.

Principal investigator: Antonella De Jaco
Researcher in Cell Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912310; Fax: (+39) 06 49912351
antonella.dejaco@uniroma1.it
43
AREA 3
Processing of the Neuroligins proteins and autism-related mutations
Participants:
Lisa Ulbrich, post-doc fellow; Flores Lietta Favaloro,
undergraduate student.
Collaborations:
University of California, San Diego, USA (Prof. Palmer Taylor,
Prof. Davide Comoletti); Stanford University, CA, USA (Prof.
Michael Lin); University of Cambridge, UK (Prof. Stefan
Marciniak).
Report of activity
Background
Neuroligins (NLGNs) are post-synaptic cell adhesion
proteins that form eterophilic trans-synaptic bridges
with the presynaptic Neurexins (NRXNs). Their
interaction plays a role in organizing both inhibitory
and excitatory synapses, and is essential for selectivity
in synaptic function (Sudhof, Nature, 2008).
Support in favor of the role played by these proteins
at the synapse comes from human genetic studies in
patients affected by autism spectrum disorders (ASD),
a group of neurodevelopmental disorders characterized
by impairments in social reciprocal interactions
and language. Several rare missense mutations have
been reported in the genes encoding for NLGN 3
and 4 and for NRNX 1, highlighting that the cross
talk between both partner proteins is essential for
correct synaptic function (Persico and Bourgeron,
Trends Neurosci, 2006). NLGNs are single pass multi
domain proteins and belong to the α/β-hydrolase
fold family because of the extracellular domain structure
(Ichtchenko et al., J Biol Chem, 1996). The R451C
substitution in the α/β-hydrolase fold domain of
NLGN3 was found in two brothers affected by
ASD (Jamain et al., Nat Genet, 2003). Several groups
have shown that R451C NLGN3 is predominantly
retained in the endoplasmic reticulum (ER) when
transfected into HEK293, COS and hippocampal
neurons (Comoletti, J Neurosci, 2004; De Jaco, J Biol
Chem, 2006). Moreover construction of knock-in mice
confirmed in vivo the effect of this amino acid change
(Tabuchi et al., Science, 2007). Crystal structure of
NLGN1/NRXN1-β complex reveals that R451C
maps to a position remote from the binding site for
NRXN1-β (Fabrichny et al., Neuron, 2007).
Research objective
The objective of this study is to analyze folding,
disulfide bonds formation, post-translational glycosylation
processing and trafficking of NLGN3 protein by
biochemical and cell biological approaches. The final
goal is to understand how the autism-related mutations
are interfering with the normal assembling and
exposure of the protein to the cell surface. Although the
ER retention of NLGN3-R451C mutant protein has
been well documented in different cell model systems,
the molecular basis underlying the block in trafficking
haven’t been clarified yet. A direct comparison of the
processing of NLGN3 wild type and R451C mutant
proteins in HEK-293 and trafficking in neurons should
show where alterations are arising in the making of the
NLGN3 protein. Especially since most of the NLGNs
mutations map in the α/β-hydrolase fold domain
that represents the protein extracellular domain, the
identifications of ER molecular chaperones taking part
in building its tridimensional structure and the ones
involved in the retention mechanism are valuable information
for future designing of rescue strategies.
Results
The following data have been included in the publications
that acknowledge the support of the Istituto
Pasteur Fondazione Cenci-Bolognetti: De Jaco et al.,
Chem Biol Interact, 2010; De Jaco et al., J Biol Chem, 2010.
Protein misfolding of R451C NLGN3
The R451C mutation affects folding of the α/βhydrolase
fold domain of NLGN3. Protein folding

Antonella De Jaco
was studied by trypsin digestion experiments after
expression of NLGN3 wild type and R451C c-DNA
in HEK-293 cells. R451C mutant protein results
more sensitive to the trypsin digestion compared
to the wild type protein suggesting that NLGN3
R451C is more loosely folded around the mutation
(De Jaco et al., 2010a). Nonetheless it appears that
the extra cysteine is not interfering with the formation
of pre-existing disulfide bonds as shown by
reaction with PEG5000-maleimide and estimation
of the molecular mass (De Jaco et al., 2010a). G221R,
positioned deep in the core of NLGN3 α/β-hydrolase
domain, was chosen as a control mutation to
compare the extent of the misfolding induced by the
R451C. Introduction of G221R makes NLGN3 to be
retained in the ER in a more severe way than what
observed for R451C mutant protein. Moreover, by
comparing the sensitivity to protease digest, G221R
is more sensitive to the digestion with trypsin
than R451C, suggesting that this mutation affects
the overall protein folding while R451C mutation
results causing a locally expanded conformation (De
Jaco et al., 2010b).
Association with ER molecular chaperones
By co-immunoprecipitation, we have identified the
differential association of wild type, R451C and
G221R mutant proteins with ER molecular chaperones.
GRP78/BiP and GRP94 associate more avidly
with R451C and G221R mutant proteins than the
wild type protein, probably because these chaperones
recognize them as misfolded. Calnexin and calreticulin
don’t show a preferential association for either
wild type or mutant proteins. Because NLGN3 presents
both disulfide bonds and free cysteines association
with PDI family members such as PDIr, Erp57
and Erp72 was tested. These chaperones, associate
with the mutant proteins but not with the wild type,
suggesting involvement of a thiol-retention mechanism
(De Jaco et al., 2010b).
Processing of NLGN3 in HEK-293
Metabolic labeling experiments in HEK-293 cells stably
expressing either wild type or R451C or G221R
mutant proteins show that the introduction of the
R451C mutation slows down processing of NLGN3.
At 3 hours of chase, processing of wild type protein
is complete while R451C remains mainly unprocessed
with only a small fraction of fully glycosylated protein
that increases at 6 hours and disappears faster
44
compared to the wild type protein. As expected by the
greatest severity of the misfolding, NLGN3 G221R
remains unprocessed and degrades faster than either
wild type or R451C (De Jaco et al., 2010b).
Trafficking of NLGN3 in neurons
The influence of R451C and G221R mutations on
the trafficking of NLGN3 is analyzed in hippocampal
neurons by using NLGN3 proteins fused to a novel
fluorescent reporter system that allows visualization
of newly produced proteins from the soma to
the dendrites. R451C reduces the export of NLGN3
from the soma to a dendritic location in comparison
to the wild type protein whereas G221R completely
abolishes NLGN3 translocation to the dendritic
synapses. These results in neurons are in accord
with the data in HEK293 cells where the R451C and
G221R mutations hinder protein processing, with
the R451C causing a local misfolding and G221R the
most severe defect (De Jaco et al., 2010b).
Future directions
Characterization of the rare R451C mutation is providing
information about residues crucial for folding
of the α/β-hydrolase fold domain of NLGN3.
Acquiring information about the molecular basis of
ER retention mechanism for autism related mutant
proteins, such as the NLGN3 R451C, offers a solid
base for designing strategies for rescue protein
expression on the cell surface. The following up of
the project will be focused on individuating other
mutations that affect processing of NLGN3 and further
characterization of the NLGN3 R451C mutant
protein in regard to ER stress signalling.
Publications
De Jaco A, Dubi N, Comoletti D, Taylor P. Folding
anomalies of neuroligin3 caused by a mutation in the
α/β-hydrolase fold domain. Chem Biol Interact. 2010,
187: 56-8. doi: 10.1074/jbc.M110.139519.
De Jaco A, Lin MZ, Dubi N, Comoletti D, Miller
MT, Camp S, Ellisman M, Butko MT, Tsien RY,
Taylor P. Neuroligin trafficking arising from mutations
in the α/β-hydrolase fold protein family. J
Biol Chem. 2010, 285: 28674-82. doi: 10.1016/j.
cbi.2010.03.012.

Epigenetic modifications in neurodegenerative diseases
Principal investigator: Maria D’Erme
Professor of Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910923; Fax: (+39) 06 4440062
maria.derme@uniroma1.it
Participants:
Luciana Mosca, researcher; Alessandra Masci, Italo
Tempera, post-doc fellows.
Collaborations:
Dipartimento di Farmacologia, Sapienza-Università di Roma (Dr.
Luciano Saso), Università Paris 6 Pierre et Marie Curie, Paris,
France (Prof. Francesco Visioli), Istituto di Biologia e Patologia
Molecolari, CNR, Roma (Dr. Gianni Colotti).
Report of activity
In neurodegenerative diseases such as Alzheimer’s
and Parkinson’s disease, injury might be associated
with altered function of misfolded proteins leading
to the formation of aggregated proteins, the Amyloid
beta and the α-synuclein, respectively. It is known
that the process of protein aggregation gives rise
to soluble oligomers which are redox active and can
generate reactive oxygen species (Smith et al., 2007;
Uversky, 2008). Oxidative stress is responsible for
DNA damage, which in turn is the major activator
of poly (ADP-ribose) polymerase (PARP-1).
The PARP-1 uses NAD + as a substrate to transfer
ADP-ribose units to nuclear acceptor proteins forming
polymers of varying lengths. The PARP-1 is
involved in several physiological processes such as
gene expression, genome integrity and apoptosis.
PARP-1 inhibitors have been developed not only to
study the role of this enzyme in cell biology, but also
to improve the effectiveness of chemotherapy. Over
the last decade many studies have shown that the
activation of PARP-1 represents a key mediator of
neuronal death during excitotoxicity, ischemia and
oxidative stress, and how pharmacological inhibition
of the enzyme can significantly enhance neuronal
survival. Recently, several authors have noticed a
correlation between PARP-1 and neurodegenerative
processes. Indeed, nuclear poly(ADP-ribosylated)
proteins were found in autoptic specimens of
45
AREA 3
Alzheimer’s patients (Love et al., 1999). Outeiro et
al. (2007) have shown that inhibitors of PARP-1 significantly
reduced the neurotoxicity in experimental
models of Parkinson’s diseases.
In order to gain more insight into the molecular
mechanisms underlying neurodegenerative diseases,
this project was focused on the study of the relationship
between poly(ADP-ribosylation) and neurodegeneration.
As Alzheimer’s disease model, human SH-SY5Y
dopaminergic neuroblastoma cell line were treated
with the Aβ25-35 fragment, which retains all
the neurotoxic properties of the full length Aβ
1-42. Parkinsonian neurotoxicity, was induced by
5-S-cysteinyldopamine (CysDA) or 6-hydroxydopamine
(6OH-DA) treatment of mice or SH-SY5Y
cells. The CysDA was recently indicated as a possible
parkinsonian neurotoxin, but its effect in vivo
has not yet been elucidated (Mosca et al., 2008). All
the experiments were performed in the presence or
in the absence of the novel water soluble PARP-1
inhibitor, the quinazolinone derivative MC2050 provided
by Prof. A. Mai, Sapienza University.
In the first part of the project, the new molecule
MC2050 was characterized and the obtained results
led to the formulation of a paper in ChemMedChem.
Since the activation of PARP-1 has been identified
in Alzheimer’s disease, but its role has not yet
been clarified, the second part of this research was
to examine the influence of amyloid peptide on the
activity of PARP-1 in the presence or absence of the
inhibitor of MC2050. The Aβ was able to induce a
marked increase in ROS within the first two hours
of treatment. Because ROS are major agents of DNA
damage resulting in activation of PARP-1, the activity
of PARP-1 in the presence or absence of MC2050
was assessed. The results showed that in SH-SY5Y
cells treated with Aβ25-35 the activity of PARP-1
increases about 70% in 24 hours. The pre-treatment
with the inhibitor is able to counteract this increase.

Maria D’Erme
To demonstrate whether the activation of PARP-1 is
related to the generation of ROS due to fibrillation.
The treatment of SH-SY5Y cells with Aβ25-35 with
or without quercetin, which is a known antioxidant
also endowed with the ability to prevent Aβ aggregation,
led to prevent the PARP-1 activity caused by
amyloid peptide by counteracting oxidative stress.
Several studies have shown that Aβ can activate
NF-kB in neuronal cells (Kuner et al., 1998) and
Hassa and Hottinger (2002) have shown that also
PARP-1 regulates the function of NF-kB. Our data
demostrate that Aβ activates NF-kB, and this activation
was significantly reduced in cells pretreated
with the inhibitor MC2050, supporting the hypothesis
that Aβ may modulate the activity of NF-kB
through a PARP-1-dependent mechanism..
The third part of this project was devoted to further
study on the effects of 5-S-cysteinyldopamine
(CysDA) as a parkinsonian neurotoxin. Experiments
were carried on in vivo in C57BL/6 mice and in
vitro on SH-SY5Y cells. Briefly, DA-denervating
lesions were induced in male CD1 mice by bilateral
inoculation of either CysDA or 6-OHDA into the
dorsal striatum of mice. Sham operated mice were
injected with the same volume of saline solution.
21 days after treatment, mice were sacrificed and to
evaluate the extent of neurodegeneration the striata
were analyzed for monoamine neurotransmitters
46
level. Intrastriatal injection of CysDA in mice causes
a long-lasting depletion of dopamine without affecting
serotonergic or noradrenergic systems. CysDA
exerts oxidative stress as demonstrated both in
cells and in mice by a significative increase in protein
carbonylation and oxidized glutathione. Brain
regions of mice treated with both neurotoxins show
an appreciable increase of stress-proteins ERp57 and
α-synuclein levels in the striatum, compared to hippocampus
and pre-frontal cortex regions.
The neuroblastoma cells treatment induces an increase
in cytosolic ERp57 levels, within 8 hours, whereas in
the nuclei it is not significantly altered. α-Syn protein
levels are affected within 1 hour of treatment both
in the cytosol and nuclei. This increase seems to be
transient, rapidly returning to basal level. Preliminary
RealTime-PCR analyses confirm that CysDA modulates
the expression of both ERp57 and α-Syn. The
treatment with CysDA also induces an increase of
PARylated proteins both in cells and mice.
Publications
Tempera I, Deng Z, Atanasiu C, Chen CJ, D’Erme
M, Lieberman PM. Regulation of Epstein-Barr virus
OriP replication by poly(ADP-ribose) polymerase 1. J
Virol. 2010, 84: 4988-97. doi: 10.1128/JVI.02333-09.

DNA replication mechanisms and genome stability in
Saccharomyces cerevisiae
Principal investigator: Lucia Fabiani
Researcher in Molecular Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: 06 49912243; Fax: (+39) 06 49912351
lucia.fabiani@uniroma1.it
Participants:
Federica Lo Sardo, graduate student.
Collaborations:
Department of Microbiology and Molecular Genetics, UMDNJ,
New Jersey Medical School, Newark, USA (Prof. Carol S.
Newlon); Dipartimento di Scienze Biomolecolari e Biotecnologie,
Università degli Studi di Milano and Istituto FIRC di oncologia
Molecolare, Milano (Prof. Marco Foiani); Istituto FIRC di
Oncologia Molecolare, Milano (Dr. Giordano Liberi).
Report of activity
Different biological processes are involved in the
maintenance of genome stability as DNA replication,
repair and checkpoint mechanisms that coordinate
DNA metabolism with the progression of cell
cycle. These mechanisms have been well studied in
Saccharomyces cerevisiae and many of them are common
to higher eukaryotic organisms. Accurate and
complete DNA replication is essential for the maintenance
of the genetic integrity. Chromosome duplication
is controlled at the level of replication initiation,
which occurs at cis-acting replicator sequences
spaced at intervals of approximately 40kb along the
chromosomes of S. cerevisiae. Surprisingly, it has been
recently shown that derivatives of chromosome III
that lack known replicators segregated properly in at
least 96% of cell divisions. To understand the mechanisms
that maintain these “originless” fragments,
mutants defective in the maintenance of an “originless”
chromosome fragment were isolated (originless
fragment maintenance mutants). The study of ofm
mutants can help to define the mechanisms responsible
of the replication of the “originless” fragment and
a more detailed analysis of one of the mutants, ofm5,
will allow us also to understand the mechanisms and
the genes involved in chromosome stability because
this mutant is characterized by a high loss rate of the
wildtype derivative of chromosome III.
47
AREA 3
We identified the genomic clone by the capability to
complement HU sensitivity of ofm5. Further analysis
revealed that ofm5 is an allele of CHL1 (Chromosome
Loss 1). Chl1p (861 aa) is a member of DEAH subfamily
of Superfamily 2 (SF2) helicases. Its helicase
activity is important for the prevention of chromosome
loss or missegregation. It is required for the
establishment of proper sister chromatid cohesion
(SCC) during S phase but its role is poorly understood.
Sister chromatid cohesion is essential for
chromosome segregation and DNA double-strand
break repair by homologous recombination. The
establishment of SSC requires the coordinated activites
of cohesins, and proteins required for cohesin
deposition and cohesion establishment. Mutations in
any of these factors result in precocious sister separation,
aneuploidy and cell death. Mutations in human
cohesion factors are known to contribute to cancer
progression and premature aging.
Correlations between DNA replication and sister
chromatid cohesion have been shown, SCC can
be established only during S phase, and proteins
important for DNA replication interact directly with
proteins involved in sister chromatid cohesion. The
observation that CHL1 genetically and physically
interacts with Eco1p (Establishment cohesion 1), a
protein involved in the regulation of SCC establishment,
supports the hypotesis of a role for Chl1p in
preserving genome stability during S phase. Our
interest is to understand the role of Chl1p during
DNA replication in S. cerevisiae.
Chl1p shares a high sequence similarity in the core
helicase domain with a recently identified human
Fanconi Anemia (FA) gene implicated in FA complementation
group J, FANCJ. FANCJ was originally
designed BACH1 (BRCA1 Associated C-terminal
Helicase), a protein that binds to the tumor suppressor
BRCA1. Mutations in FANCJ/BACH1 are
associated with breast cancer and genetically linked
to the bone marrow disease Fanconi Anemia (FA).
We identified a point mutation in chl1 ofm5 , a transition

Lucia Fabiani
of G to A at the level of nucleotide 275 of the coding
sequence, converting a Trp codon (TGG) at the
aminoacid 92 to a stop codon (TAG). This premature
stop codon cause a null allele: all the important
helicase domains are lost. chl1 deletion mutant is
sensitive to hydroxyurea (HU) (depletion of dNTP
pools), methylmethanesulfonate (MMS) (alkylation
of bases) and UV radiation (pyrimidine dimer formation).
We analyzed in wildtype and chl1 deletion mutant
the correct activation of the S phase checkpoints
(replication and intra-S checkpoints), by Western
blot analysis following phosphorylation of Rad53p,
and the replication intermediates, by two-dimensional
gel electrophoresis, of ARS305 to understand the
potential role of Chl1p at the replication fork. Both
analysis were carried out after synchronization in G2
and release in sublethal concentrations of HU and
MMS. Both checkpoint mechanisms are efficiently
activated and no difference was observed between
wildtype and chl1 replication intermediates.
Recently it has been proposed that different proteins
are involved in binding and removal of cohesins
in different chromosomal locations then these different
locations can use different pathways for the
establishment of SCC. Sister chromatid cohesion in
ribosomal DNA (rDNA) is essential for the proper
alignment of the chromatids to avoid unequal recombination
between the repeats important to mantain
their correct copy number. In S. cerevisiae rDNA is
organized as 150-200 tandem repeats and is localized
on chromosome XII. rDNA replication is preferen-
48
tially unidirectional for the presence of a replication
fork barrier (RFB) to which the protein Fob1 (Fork
blocking protein 1) is specifically associated, the
replication is via the lagging strand apparatus and
uses as template the G rich strand. G quadruplex
DNA can be formed on this strand, this structure
represents a problem to the fork progression. We
analyzed the replication intermediates at the level of
the region of chromosome XII containing the rDNA
cluster and we finally found a difference between
wildtype and chl1 mutant, we observed an increase
of the RFB.
FANCJ /BACH1, Chl1p human homolog, is able
to unwind G4 DNA structure with 5′–3′ polarity,
this recent result suggests a possible role of Chl1p
in the maintenance of potential unstable genomic
G/C tracts during replication. The 5′–3′ polarity
is consistent with resolution of G4 structure on the
lagging strand during replication. The loss of G4
unwinding function in chl1 mutant during rDNA
replication could correlate with the increase at the
RFB of replication intermediates highly ricombinogenic
and source of genomic instability.
We verified the presence of Chl1p at the level of
the replication fork by ChIP (Chromatin Immuno-
Precitation) by adding a tag sequence at the
C-terminus of the protein. Chl1p is able to bind to
the rDNA origin of DNA replication. We are now
analyzing the binding of Chl1p at the level of other
origins of DNA replication and the presence of
potential G4 DNA on the G rich strand of rDNA
used as template.

New tools in deciphering aging and apoptotic pathways
Principal investigator: Claudio Falcone
Professor of Industrial Microbiology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912278; Fax: (+39) 06 49912256
claudio.falcone@uniroma1.it
Participants:
Cristina Mazzoni, researcher; Vanessa Palermo, postdoc
fellow; Mirko Torella, PhD student; Michele Saliola,
technician.
Collaborations:
Institute of Molecular Biosciences, University of Graz, Austria
(Prof. Frank Madeo); Dipartimento di Medicina e Patologia
Sperimentale, Sapienza-Università di Roma (Prof. Patrizia
Mancini); Istituto di Biologia e Patologia Molecolari, CNR, Roma
(Dr. Enrico Cundari).
Report of activity
The aim of this project was the isolation of genes acting
as suppressors of yeast apoptotic phenotypes and
the study of their suppression mechanism.
In recent years it has been demonstrated the existence
of an apoptotic program in unicellular eukaryotes,
and some crucial components of the apoptotic
cascade such as AIF, caspase and Omi have been
described in the yeast S. cerevisiae, indicating a
high evolutionary conservation of this process. In
addition, oxidative stress and the expression of proapoptotic
genes involved in mammalian pathways,
induce apoptosis also in yeast cells.
The biological significance of apoptosis in unicellular
organisms may be related to the possibility of eliminating
older or damaged cells in favor of younger
and healthy cells.
Concerning aging, the relationships between this
process and apoptosis in eukaryotes are controversial.
Even though old cells are more resistant to apoptotic
stimuli, they are naturally predisposed to cell death,
favoring the long-term survival of the population.
In our laboratory we have shown that mutants
in genes involved in decapping (dcp1, dcp2, lsm1,
Kllsm4D1, lsm6, lsm7), one of the first steps for the
degradation of mRNAs, show an increased loss of viability
during the stationary phase, compared to wild
49
AREA 3
cells, indicating the occurring of premature aging.
Moreover, this kind of cell death is linked to apoptotic
events because cells show the accumulation of
reactive oxygen species (ROS), DNA fragmentation
and chromatin condensation.
We also demonstrated that the stabilization of mRNA
triggers apoptosis via a caspase-dependent pathway.
Decapping mutants, affected at very early stages
of the apoptotic cascade, show increased sensitivity
inducers of ROS production and apoptosis, representing
a good tool for the isolation of genes that
counteract the aging process and cell death.
To this purpose, the mutant strain Kllsm4Δ1 was
transformed with a yeast genomic library and clones
resistant to acetic acid, a known inducer of oxidative
stress and apoptosis in yeast, were selected for
further studies.
One of these clones carried a DNA fragment containing
the HIR1 (Histone transcriptional repressor)
gene, which encodes a transcriptional co-repressor
of histone genes.
The over-expression of HIR1 in the mutant Kllsm4Δ1
prevented rapid cell death during chronological
aging, reduced nuclei fragmentation and increased
H 2 O 2 resistance, indicating a relationship between
this gene and apoptosis.
The absence of HIRA, the human gene corresponding
to HIR1, has been associated with Di George
syndrome in which a spectrum of malformations and
diseases of development and a very large increase
in spontaneous apoptosis in T lymphocytes are
observed. Moreover, HIRA is involved, along with
ASF1 in the formation of SAHF (senescence-associated
heterochromatin foci).
The existence of relations between HIR1 and apoptosis
offers new opportunities to study this phenomenon
in a simple model such as yeast.
Transcriptional analysis revealed that the expression
of histone genes was lowered in the Kllsm4Δ1mutant
after HIR1 over-expression, suggesting that a reduction
of transcription counteracts the increased stabil-

Claudio Falcone
ity of mRNA observed in this mutant.
To better understand this relationship, we checked
the expression of the histone genes during the cell
cycle, with cells in that their transcription is tightly
regulated both transcriptionally and post-transcriptionally
and coupled to rates of DNA synthesis.
We demonstrated that in the Kllsm4Δ1 mutant,
histone mRNAs are induced early in the S-phase
and maintained at high level all along the entire cell
cycle, showing a delay in the exit from S-phase and/
or entry into M-phase.
The overexpression of the HIR1 gene also restored
the normal cycling of histone genes expression.
We also found that low doses of hydroxyurea (HU),
which inhibits the ribonucleotide
reductase and the production of dNTPs required for
DNA synthesis, neutralized the onset of the apoptotic
phenotypes in Kllsm4Δ1, as well in another mRNA
decapping mutants (lsm1). In these conditions, we
observed an increase of the chronological lifespan in
both strains, suggesting that a delayed entry into the
S phase can recover some cellular defects in decapping
mutants.
Our work presents clear evidence for a link between
regulation of S-phase progression, histone mRNA
control and cell death. We speculated that the HU
rescuing effects might result from adjusting a detrimental
imbalance in the NTP/dNTP equilibrium
that potentially arises upon increased mRNA stabilization
(for instance through enhanced NTP incorporation
in mRNA molecules). Another explanation for
the phenotypes of lsm mutants is that, consequent
to the high levels of histone mRNAs, these mutants
may accumulate excess of histonic proteins that,
similarly to dNTP depletion, induce DNA replication
stress.
In mammalian cells, this kind of stress activates inter-Sphase
checkpoints and causes selective histone mRNA
degradation. Importantly, this process requires both
ATR, a key regulator of the intra S-phase checkpoint,
as well as Upf1, a crucial factor of the nonsense-mediated
decay pathway, which monitors mRNA quality and
ensures degradation of defective mRNAs. In yeast, such
interplay between the DNA intra S-phase checkpoint
50
and the nonsense-mediated mRNA decay pathway
might assure the proper coordination of chromatin
structure replication.
Such coupling could occur at a bidirectional level, not
only allowing a DNA-to-RNA but also a RNA-to-
DNA signaling. In the latter case, aberrant mRNA
levels could be signaled to the intra S-phase checkpoint
to stall cell cycle progression. Such RNA-DNA
regulation cycles are apparently closely connected
to cell death control, though the underlying mechanisms
remain to be explored.
Western blot experiments revealed that the levels
of Sir2p, the NAD-dependent histone deacetylase,
are lower in the decapping mutants compared to the
wild type.
The reduced amount of Sir2 results in increased
transcription of chromosomal sites, such as the
rDNA and the subtelomeric regions, which are normally
repressed by this deacetylase.
ChIP experiments have indeed shown an increase in
recruitment of RNA polymerase II (RNApolII) to these
sites in the Kllsm4D1 mutant compared to the wild type.
The connection between the lower amount of Sir2
protein and the increased chronological aging of
decapping mutants is still under investigation.
Publications
Mazzoni C, Torella M, Petrera A, Palermo V,
Falcone C. PGK1, the gene encoding the glycolitic
enzyme phosphoglycerate kinase, acts as a multicopy
suppressor of apoptotic phenotypes in S. cerevisiae.
Yeast. 2009, 26: 31-7. doi: 10.1002/yea.1647.
Palermo V, Cundari E, Mangiapelo E, Falcone C,
Mazzoni C. Yeast lsm pro-apoptotic mutants show
defects in S-phase entry and progression. Cell Cycle.
2010, 9: 3991-96. doi: 10.4161/cc.9.19.13210.
Palermo V, Falcone C, Calvani M, Mazzoni C.
Acetyl-L-carnitine protects yeast cells from apoptosis
and aging and inhibits mitochondrial fission.
Aging Cell. 2010, 9: 570-9. doi: 10.1111/j.1474-
9726.2010.00587.

Principal investigator: Laura Frontali
Professor of Microbial Chemistry
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 4453950, Fax: (+39) 06 4461980
laura.frontali@uniroma1.it
51
AREA 3
Mitochondrial studies and the use of simple eukaryotic models for
the analysis of molecular and cellular aspects of human pathologies
Participants:
Claudio Palleschi, Michele M. Bianchi, professors; Silvia
Francisci, Teresa Rinaldi, Daniela Uccelletti, researchers;
Cristina De Luca, post-doc fellow; Michela Esposito, Chiara
Micolonghi, PhD students.
Collaborations:
Laboratoire de Génétique Moléculaire, Université de Paris Sud,
Orsay, France (Prof. Monique Bolotin-Fukuhara, Dr. Agnès
Delahodde); Department of Biology, The Technion, University of
Haifa, Israel (Dr. Michael Glickman); Laboratoire de Génétique
des Levures, Université Lyon 1, France (Prof. Micheline
Wésolowski-Louvel).
Report of activity
Many human pathologies are related to mutations in
very conserved genes. The present project aims to
study the molecular and cellular aspects of some of
these pathologies by the use of models derived from
simple eukaryotes such as yeasts and the nematode
Caenorhabditis elegans, with the purpose of verifying
the possibility that the coordinate use of these models
might increase our understanding of the above mentioned
dysfunctions.
The pathologies we are planning to investigate
are mainly connected with mitochondrial functions
and respiratory regulation, which are specially well
known in yeasts, and could be more deeply investigated
in the worm.
Pathologies due to mutations in mit tRNA
genes
Base substitutions in mit tRNA genes cause in human
very severe diseases mainly neurodegenerative. No
therapies exist at present for these chronic devastating
pathologies.
We have set up a yeast model, by exploiting the special
characteristics of yeast, which is the only eukaryotic
cell amenable, by biolistic procedures, to mitochondrial
transformation and which can survive on glucose by
fermentation even when respiration is deeply affected.
We have produced, by biolistic procedures a series of
human equivalent mt yeast mutants, which proved to
exhibit impaired respiration. The molecular mechanism
of the defects was also shown to be similar in
human and yeast cells (Montanari et al., 2008).
The most important result we have obtained in yeast
mutants was the observation that respiratory defects
were rescued (suppressed) by the overexpression of
some nuclearly encoded tRNA interactors such as
the mit protein synthesis elongation factor Ef-Tu and
the aminoacyl-tRNA synhetases (aa-RS) ( De Luca et
al., 2009).
Several laboratories recently confirmed that the
same suppression could be obtained by overexpression
of the horthologous human genes in patient cell
lines and cybrids, thus reinforcing the possibility of
a therapeutic perspective.
The mechanism of suppression was studied in detail, in
particular for the aminoacyl-tRNA synthetases (aa-RS)
and was found to be independent from catalytic activity.
This suggested the hypothesis that the suppressive
effect may be due to the chaperon-like activity of a polypeptide
restoring the altered structure of the mutated
tRNA. We also found that both yeast and human
mit leucyl-RS can suppress the respiratory defects of
human equivalent yeast mutants in tRNALeu, tRNAIle
and tRNAVal (Montanari et al., 2010).
We plan now to localize the suppressor activity in
the different domains of leu-RS. Our recent results
on this aspect may have a clinical importance and
Rome University is filing a patent on this subject.
Mitochondrial pathologies caused by
defective nuclear genes
We have set up a yeast model for SPG13 spastic paraplegia,
associated with mutations in the HSPD1 locus.
We have indeed shown that the human HSPD1 gene is
a functional homologue of the yeast HSP60. In particular,
we verified that the cDNA for HSPD1 suppresses

Laura Frontali
the lethality of the ΔHSP60 S. cerevisiae strain. We
constructed two expression plasmids for yeast, bearing
the mutated cDNA HSPD1. They are pV98I, producing
the replacement of valine with isoleucine and
pQ461E, producing the replacement of glutamine with
glutamic acid. The transformation of the heterozygous
diploid strain of S. cerevisiae HSP60/hsp60::kan
with pV98I and subsequent sporulation showed that
the mutant human gene is not able to replace functionally
the HSP60 gene. When the heterozygous
diploid strain of S. cerevisiae HSP60/hsp60::kan was
transformed with the plasmid pQ461E, viable haploid
strains expressing hsp60::kan and the mutated form
of hspd1 were obtained after sporulation. However,
the resulting strains are defective in mitochondrial
function, as shown by reduced oxygen consumption
capabilities, formation of petites, altered mitochondrial
morphology (manuscript in preparation).
In addition, we found that the HSPD1 cDNA suppresses
oxidative defects in the yeast Kluyveromyces
lactis due to inactivation of the Ca 2+ -ATPase, KlPMR1
gene. This suppression was not observed when the
K. lactis mutant was instead transformed with the
plasmid pV98I. We found that this mutation is not
dominant in the yeast model, as in the case of patients.
In parallel, studies were also conducted revealing a
cross-talk between two important cellular processes
such as glycosylation and mitochondrial biogenesis
and in which calcium homeostasis plays an important
role (Zanni et al., 2009).
In order to set up the nematode C. elegans as a model
of SPG13, we got some transgenic strains in which
HSPD1 was overexpressed, however, this construct
was toxic to the nematode, with consequent loss of the
construct in the progeny. We were not able to isolate
other transgenic strains with an integrated HSPD1,
in order to minimize the effects of toxicity. At the
moment, we are trying to transform the nematodes by
biolistic methods, with a plasmid in which the HSPD1
expression is induced only under specific conditions,
such as high temperatures, in order to isolate transgenic
strains that may be stable and useful for our model.
Proteasomal defects
The proteasomal system and in particular the deubiquitinating
enzyme Rpn11 is involved in mitochondrial
dynamics (Rinaldi et al., 2008; Hofmann et
al., 2009) and in several related diseases.
Concerning the human spastic paraplegia, one of
the candidate human genes mutated in the disease
is the Heat Shock Protein 60 (HSPD1). We have
observed in our yeast mutant rpn11-m1 the accumulation
inside mitochondria of proteolysis products of
52
Hsp60. However, we did not succeed in identifying
the mit protease involved, and we only could rule out
Yme1 and Pim1 proteases.
On the other side, we demonstrated a genetic interaction
between the proteasomal mutant rpn11-m1
(exhibiting cell cycle defects) and the yeast strain
deleted for the calcium pump Pmr1(Δpmr1). We
studied the PMR1 gene because the phenotypes
of the deletant of this gene are suppressed by an
overexpression of the HSP60 gene. The absence of
the calcium pump allows a complete suppression of
the cell cycle defects of rpn11-m1. The mechanism
of suppression involves Swe1 activating pathway
(Esposito et al., 2010). This result is supported by the
observation that in the absence of Calcineurin CNB1
gene (an activator of Swe1), the bud elongation phenotype
is still present (Rinaldi et al., 2010).
Since mitochondrial morphology is severely affected
in the Δpmr1, strain and the presence of Calcineurin
is essential for the control of mitochondrial fission in
human, the yeast system could be useful to investigate
the involvement of mitochondrial morphology
in the human spastic paraplegia.
Publications
De Luca C, Zhou Y, Montanari A, Morea V, Oliva
R, Besagni C, Bolotin-Fukuhara M, Frontali L,
Francisci S. Can yeast be used to study mitochondrial
diseases? Biolistic tRNA mutants for the analysis
of mechanisms and suppressors. Mitochondrion. 2009,
9: 408-17. doi: 10.1016/j.mito.2009.07.004.
Hofmann L, Saunier R, Cossard R, Esposito M, Rinaldi
T, Delahodde A. A non-proteolytic activity of the proteasome
controls fission of organelles in yeast. J of Cell
Science. 2009, 122: 3673-83. doi: 10.1242/jcs.050229.
Zanni E, Farina F, Ricci A, Frank C, Mancini P,
Palleschi C, Uccelletti D. The Golgi alpha 1,6 mannosyltransferase
KlOch1p of Kluyveromyces lactis is
required for Ca2+/calmodulin-based signaling and
for proper mitochondrial functionality. BMC Cell
Biology. 2009, 10: 86. doi: 10.1186/1471-2121-10-86.
Montanari A, De Luca C, Frontali L, Francisci S.
Aminoacyl-tRNA synthetases are multivalent suppressors
of defects due to human equivalent mutations in
yeast mt tRNA genes. Biochimica et Biophysica Acta. 2010,
1803: 1050-57. doi:10.1016/j.bbamcr.2010.05.003.
Rinaldi T, Dallabona C, Ferrero I, Frontali L,
Bolotin-Fukuhara M. Mitochondrial diseases and the
role of the yeast models. FEMS Yeast Res. 2010, 10:
1006-22. doi: 10.1111/j.1567-1364.2010.00685.

The role of membrane trafficking in Drosophila cytokinesis
Principal investigator: Maurizio Gatti
Professor of Genetics
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912842; Fax: (+39) 06 4456866
maurizio.gatti@uniroma1.it
Participants:
Silvia Bonaccorsi, Maria Grazia Giansanti, Patrizia Somma,
Fiammetta Vernì, researchers; Elisabetta Bucciarelli,
Violaine Mottier, Valeria Naim, Alan Wainman, post-doc
fellows; Claudia Pellacani, PhD student.
Collaborations:
Stanford University, USA (Prof. Margareth Fuller); Cornell
University, USA (Prof. Michel L. Goldberg); Exeter University,
UK (Prof. James Wakefield).
Report of activity
During animal cell cytokinesis, constriction of the
acto-myosin ring leads to the formation of a furrow
in the plasma membrane, which invaginates until the
two daughter cells remain connected by a thin cytoplasmic
bridge, called the midbody. This bridge is
ultimately cleaved during the final step of cytokinesis,
named abscission, which results in the complete
separation of daughter cells. Both cleavage furrow
ingression and abscission require substantial membrane
remodelling. Membrane addition to the invaginating
furrow involves vesicle delivery through
both the secretory and the endocytic pathways.
In the secretory pathway, vesicles are transported
from the endoplasmic reticulum (ER) to the Golgi
and then to the plasma membrane. In the endocytic
pathway, plasma membrane-derived vesicles proceed
to the recycling endosome (RE), which directs them
back to the plasma membrane.
Our goal is elucidation of the molecular mechanisms
underlying membrane addition to the advancing
cleavage furrow in Drosophila spermatocytes.
We have recently identified mutations in several
genes required for furrow ingression during meiotic
cytokinesis of Drosophila males. Most of these genes
encode products involved in membrane structure
and/or trafficking such as an Elovl protein, the
Trs120p subunit of the TRAPP II complex, Rab1,
53
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the Exocist complex components Exo84 and Sec8,
the ortholog of PACS-1 and Zw10. We are defining
the roles of these proteins in furrow ingression,
and investigating their functional relationships with
other proteins involved in membrane traffic during
cytokinesis.
In the past three years we have carried out three
main research projects.
The role of bond in furrow ingression during
cytokinesis in Drosophila spermatocytes
Recent work indicates that plasma membrane lipids
influence membrane biophysical properties such
as membrane curvature and elasticity and play an
active role in cell function. We found that mutations
in the gene bond, which encodes a Drosophila
member of the family of Elovl proteins that mediate
elongation of very-long-chain fatty acids, block or
dramatically slow cleavage-furrow ingression during
early telophase in dividing spermatocytes. Bond
shares sequence motifs with human and yeast Elovl
family members, including five to seven predicted
transmembrane domains, and can substitute for elovl
genes in S. cerevisiae. In bond mutant cells at late
stages of meiotic division, the contractile ring frequently
detaches from the cortex and constricts or
collapses to one side of the cell, and the cleavage furrow
regresses. These findings implicate very-longchain
fatty acids or their derivative complex lipids
in allowing supple membrane deformation and the
stable connection of cortical contractile components
to the plasma membrane during cell division.
brunelleschi (bru) interacts with Rab11 and is
required for meiotic cytokinesis in Drosophila
males
We have found that successful furrow ingression
during meiotic cytokinesis in Drosophila males
requires function of the brunelleschi (bru) gene, which
encodes the ortholog of the yeast transport protein

Maurizio Gatti
particle (TRAPP) II complex subunit, Trs120p.
Dividing spermatocytes from bru mutants assemble
a normal contractile ring, which initiates but fails to
complete constriction. The Bru protein fused to GFP
is dispersed throughout the cytoplasm and enriched
at Golgi organelles, similarly to another Drosophila
TRAPPII subunit, dBet3. bru genetically interacts
with Rab11, which encodes a small GTPase, and with
the phosphatidylinositol 4-kinase (PI4K)-encoding
gene four wheel drive (fwd). Consistent with these
findings, localization of the Rab11 protein to the
cleavage furrow requires wild-type function of bru.
These results suggest that Bru cooperates with
Rab11 and PI4K to regulate the efficiency of membrane
addition to the cleavage furrow. The genetic
interactions between bru, fwd (PI4K) and Rab11 are
consistent with the known relationships between
their orthologs in budding yeast, suggesting that
membrane trafficking processes that are essential to
growth and viability in yeast support cleavage furrow
ingression in Drosophila.
Zw10 is required for cytokinesis with a role in
membrane addition during cleavage furrow
ingression
Zw10, along with the other members of the conserved
Rod-Zwilch-Zw10 (RZZ) complex, is required for
proper functioning of the mitotic spindle assembly
checkpoint (SAC). Recent studies in mammalian cells
have shown that Zw10 forms a complex with syntaxin-18
and is involved in membrane traffic between
the Golgi and the endoplasmic reticulum (ER). We
found that male meiotic divisions in zw10 mutants
display defects in cytokinesis; the acto-myosin ring
forms and begins to ingress but then regresses.
Surprisingly, this cytokinesis failure is observed
in zw10 mutants but not in rod or zwilch mutants,
even though all three members of the RZZ complex
localize across the mid-zone of the spindle envelope
54
during telophase. In zwilch and rod mutants Zw10 no
longer localizes to the kinetochores and the spindle
envelope mid-zone but concentrates in the ER and
the Golgi-derived vesicles. These results indicate
that Zw10 association to the spindle envelope is not
essential for cytokinesis and suggest that Zw10 regulates
the ER-Golgi membrane traffic required for
proper membrane addition to the cytokinetic furrow.
Perspectives
A paper on Zw10 is currently in preparation, and our
work on Rab1 and the Exocist complex components
Exo84 and Sec8 is almost complete. We will continue
the study of the Drosophila ortholog of PACS-1 encoded
by smeagol (sgo). We believe that the analysis of the
role of these proteins in spermatocyte cytokinesis will
help unravel the mechanism of membrane addition to
the cleavage furrow, and the relationships between
membrane traffic and acto-myosin ring constriction
Publications
Bucciarelli E, Pellacani C, Naim V, Palena A, Gatti
M, Somma MP. Drosophila Dgt6 protein interacts
with Ndc80, Msps/XMAP215 and gamma-tubulin to
promote kinetochore-driven MT formation. Curr Biol.
2009, 19: 1839-45. doi:10.1016/j.cub.2009.09.043.
Robinett CC, Giansanti MG, Gatti M, Fuller MT.
TRAPPII function is required for cleavage furrow
ingression and localization of Rab11 in dividing
male meiotic cells of Drosophila. J Cell Sci. 2009, 122:
4526-34. doi: 10.1242/jcs.054536.
Wainman A, Creque J, Williams B, Williams EV,
Bonaccorsi S, Gatti M, and Goldberg ML. Roles of
the Drosophila NudE protein in kinetochore function
and centrosome migration. J Cell Sci. 2009, 122:
1747-58. doi:10.1242/jcs.041798.

Principal investigator: Marco Lucarelli
Professor of Clinical Biochemistry and Clinical Molecular Biology
Dipartimento di Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 4451784
marco.lucarelli@uniroma1.it
55
AREA 3
The interplay between epigenetics, cell cycle and homologous
recombination in the gene therapy by Small Fragment Homologous
Replacement (SFHR)
Participants:
Roberto Strom, professor; Fabrizio Ceci, Giampiero
Ferraguri, researchers; Silvia Pierandrei, PhD student.
Collaborations:
Dipartimento di Biopatologia e Diagnostica per Immagini,
Università di Roma Tor Vergata (Prof. Federica Sangiuolo, Prof.
Giuseppe Novelli, Dr. Paola Borgiani, Dr. Annalisa Botta,
Dr. Andrea Luchetti, Dr. Arianna Malgieri).
Report of activity
The aim of this project is to clarify the molecular
mechanisms underlying a gene therapy approach,
called Small Fragment Homologous Replacement
(SFHR), focusing on the scarcely studied relationships
between SFHR, DNA methylation and repair,
chromatin structure and cell cycle. Homologous
recombination (HR) is the DNA recombination during
meiosis. In mitotic cells, however, HR is a basic
mechanism to repair DNA. SFHR is an in situ gene
targeting approach able to stably modify a genomic
sequence by small DNA fragment (SDF), probably
based on HR and DNA repair pathways, allowing the
recovery of a normal gene function. The molecular
mechanism of SFHR is poorly understood, with its
potential currently limited by a low and variable frequency
of correction, with several factors influencing
its efficiency.
Before the start of this project two cell lines have
been established from a mouse model of Spinal
Muscular Atrophy (SMA; OMIM #253300) type I:
immortalized murine embryonic fibroblasts (MEF)
and murine embryonic stem (mES) cells. They are
both deleted for the unique murine SMN1 gene, but
contain human SMN2 gene. The wild type human
SMN1 gene produces the functional full length transcript
(FL-SMN); the human non-functional SMN2
gene has a single C>T nucleotide transition that
results in exon 7 skipping (Δ7-SMN). The SMN2 is
unable to complement the deletion of SMN1 gene
that originates SMA; however, as the SMN2 gene
can express SMN protein, correction of SMN2 is an
attractive therapeutic option. The MEF have been
stably modified integrating within their genome
the wild type enhanced green fluorescent protein
wtEGFP sequence, in which a non sense mutation
was also created (mEGFP). In MEF-mEGFP, a SDF
homologous to the wtEGFP sequence can integrate
and restore the fluorescence of the cells. The efficiency
of SFHR may be evaluated at DNA and RNA
levels, as well as functionally quantified by cytofluorimetric
analysis. In mES-SMN2 cells, a SDF homologous
to the SMN1 gene can replace the point mutation
of SMN2 to restore the wild type phenotype. In
this case, the efficiency of SFHR may be evaluated
at DNA and RNA levels, as well as by induction of
differentiation to functional motor neurons, evaluated
at morphological and biochemical levels. The
MEF-mEGFP and the mES-SMN2 are two reporter
systems that provide a platform for developing and
testing new strategies for gene repair.
In this project, the MEF-mEGFP was firstly used
with the aim of later transfer the main results
obtained to the mES-SMN2 system. The best experimental
conditions in term of transfection, correction
efficiency and cell viability were selected in unsynchronized
MEF-mEGFP: the best electroporation
program on the Amaxa nucleofection system, the
optimal SDF length (from 850 to 900 bp) and the
optimal SDF concentration (12 x 10 6 SDF molecules/cell).
Double strand, blunt end or PCR amplified
(SDF-PCR) SDFs showed higher correction efficiency
than, respectively, single strand, sticky ends
or plasmid-digested (SDF-DIG) SDFs. To determine
the cell cycle influence, we evaluated gene targeting
in G0/G1 (by mimosine), G1/S (by thymidine) and
G2/M (by vinblastine) phases. In G2/M the SFHR
resulted favored, reaching 0.5% of correction, probably
because of the absence of nuclear envelope or to
the tetraploid situation present before cell mitosis.

Marco Lucarelli
To clarify the molecular mechanism for the lower
correction efficiency of SDF-DIG than SDF-PCR,
the methylation pattern of both SDFs was studied
using methylation-sensitive restriction enzymes and
subsequent PCR amplification. Both DCM and DAM
bacterial methylation resulted present on the SDF-
DIG fragment whereas, obviously, no methylation
was present on the SDF-PCR. To directly check if
the SDF methylation may be the molecular base for
a diminished correction efficiency, SDF fragments
with specific methylation patterns were produced by
SssI, Dam, and both DNA-methyltransferases and
transfected into G2/M synchronised cells. Results
showed an SFHR efficiency 50% and 80% lower
than that of, respectively, SDF-DIG and unmethylated
SDF-PCR. This reduction in the SFHR efficiency
may originate from the creation of complexes
between methylated SDFs and nuclear proteins that
inhibit the integration. The purity and stability of
the correction were confirmed, after culture of sorted
corrected (fluorescent) cells for several passages, by
RFLP analysis, allelic discrimination real time PCR
and Southern blot analysis; this last technique also
excluded any random integration of SDF within
genomic DNA.
With increasing cell passages, corrected cells gradually
lost EGFP fluorescence. As retromutation was
excluded by RFLP analysis and sequencing, the
hypothesis was that DNA methylation may cause
transcriptional inactivation of the corrected EGFP
gene. In effect, the corrected cells that progressively
lost their fluorescence resulted, at a HpaII/
PCR assay, more methylated than the corrected still
fluorescent cells, with a specific methylation pattern
involving only some CpG sites. In addition, the
methylation pattern appeared to be influenced by the
SFHR process as it appeared different between corrected
MEF-mEGFP and MEF-wtEGFP, which did
not undergo correction, both showing progressively
inactivating EGFP. A cause/effect relationship was
evidenced by the fact the DNA methylation and
EGFP expression were reverted by the treatment
56
with the demethylating agent 5-aza-2’-deoxycytidine.
To test their influence on the SFHR mechanism,
3 main biological pathways involved in the
repair of the DNA damage were investigated: a) the
DNA double-strand break repair based on the ATM
kinase pathway, using its inhibitor KU-55933; b)
the transcription-coupled nucleotide excision repair
based on the RNA polymerase II activity, using its
inhibitor α-amanitine; c) the non homologous recombination
based on the poly-(ADP-ribose) polymerase
pathway, using its inhibitor 1,5-isoquinolinediol. A
modulation of SFHR efficiency was evidenced by
each of these inhibitors, highlighting the interconnection
of SFHR mechanism with all these biological
pathways.
Using the best experimental conditions optimized
by the MEF–mEGFP assay system, we also treated
the mES-SMN2 system. A SDF-PCR homologous to
human SMN1 sequence was used to correct the nonfunctional
SMN2 gene, in undifferentiated mES cells
synchronized in G2/M phase. After transfection the
levels of FL-SMN and Δ7-SMN were quantitatively
evaluated at RNA level, by real time PCR, and at protein
level, by western blot. The increase of FL-SMN
transcript, due to SMN2 to SMN1 sequence conversion,
resulted to be about 70%, moreover a 14%
decrease in Δ7-SMN transcript was detected. Both
the transcript FL-SMN/Δ7-SMN ratio and the
protein FL-SMN/tubulin ratio increased 1.8 fold in
respect to controls non-transfected or transfected
with an heterologous SDF.
These results contribute to the comprehension of
the molecular mechanism of the SFHR, in both stem
and differentiated embryonic cells, also providing
fundamental suggestions for increasing gene repair
efficiency. This can open new perspectives for a
SFHR gene targeting strategy applicable to ex vivo
protocols.
A manuscript concerning these first results is in
preparation for Molecular Therapy.

Interplay between myogenic factors and cell cycle control:
regulation and role of the cdk inhibitor p57kip2
Principal investigator: Rossella Maione
Professor of Applied Biology
Dipartimento di Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 4457737; Fax: (+39) 06 4462891
maione@bce.uniroma1.it
Participants:
Anna Busanello, Rosaria Carbone, Marianna Rossi, postdoc
fellows; Cecilia Battistelli, Cassandra Mostocotto, PhD
students.
Collaborations:
Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma (Dr.
Maurizia Caruso).
Report of activity
Objectives
The general aim of this research is to gain insight
into the mechanisms by which myogenic factors
activate growth arrest pathways and into the roles
played by cell cycle regulators during the process of
skeletal myogenesis.
Several cdk inhibitors (CKIs) are up-regulated during
myogenesis. The function and the regulation of p57kip2
are particularly interesting. p57, the most structurally
complex member of the Cip/Kip family, is the only CKI
which individual ablation causes developmental defects
in mice and shows a restricted expression pattern during
development and in some adult tissues including
muscle. Moreover the gene coding for p57 is located
within an imprinted cluster, involved in growth regulation
and subject to a complex epigenetic control.
The present project has two main objectives.
The first is to identify the epigenetic mechanisms
involved in the transcriptional control of p57 during
myogenesis. The second is to investigate the role of
cell cycle regulators in the control of cell cycle exit
and maintenance of quiescence during skeletal muscle
differentiation and regeneration.
Results
We had previously demonstrated that p57 transcription
is induced by the myogenic factor MyoD
through a pathway requiring intermediate factors
(3,8), a molecular mechanism distinct from that
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employed for induction of other genes involved in
cell cycle arrest. Furthermore, compared to p21,
p57 showed a more restricted expression pattern
in differentiating muscle cells. More recent results
indicated that the accessibility of p57 promoter to
MyoD-dependent regulation is prevented, in undifferentiated
myoblasts and in muscle cell types unable
to induce p57, by epigenetic constraints involving
DNA methylation (4). We hypothesized that
KvDMR1, a distant DNA region known to regulate
in cis the hypermethylation and silencing of the
non-expressed p57 allele during imprinting (5), is
also involved in preventing p57 expression in undifferentiated
and un-responsive cells. This hypothesis
has been explored through the following approaches:
Since KvDMR1 is a regulatory element shared by
several genes of the p57 imprinting domain, we compared
the kinetics of p57 induction (and its restriction
in unresponsive cells), with that of other genes of the
domain. The expression analysis showed that Kvlqt1,
a gene co-imprinted with p57, was also co-induced
with p57, while Lit1, a gene oppositely imprinted, was
unaffected or rather down-regulated. Importantly,
Kvlqt1, just like p57, was not induced in un-responsive
cells. These results encouraged us to investigate
the functional interaction of MyoD with KvDMR1.
KvDMR1 is a multifunctional regulatory region
consisting of multiple, independent cis-acting modules
acting as enhancers, promoters, silencers or
enhancer-blocking elements (5). The finding of several
putative MyoD-binding sites within this region
suggested us that the myogenic factor could regulate
the expression of p57 and, possibly, of other genes of
the cluster, by directly binding to KvDMR1. By performing
ChIP assays, specific for different fragments
of the region, we could identify a KvDMR1 subregion
to which MyoD binds in vivo in a differentiation-
and responsiveness-dependent manner. This
finding strongly supports the conclusion that both
the induction and the restriction of p57 expression
involve a long distance regulatory element overlap-

Rossella Maione
ping with the imprinting control region.
The availability of allele-specific polymorphisms in
the imprinting domain allowed us to determine that,
although MyoD binds to both maternal and paternal
KvDMR1 alleles, the induction of p57 involves
the up-regulation of the maternal and not the derepression
of the paternal imprinted p57 allele. The
suggestion emerging is that in undifferentiated and
unresponsive cells p57 expression is prevented by
biallelic silencing and that differentiation signals
are capable to relieve this constraint only from the
maternal allele, and only in responsive cells.
These results are the subject of a manuscript that we
are going to submit.
Concerning the second task, our plan was to identify
specific roles of CKIs in the establishment of the differentiated
state and in the control of satellite cell
function. To this end, we have set up a system for the
conditional silencing of p57 and of its related CKI
p21, based on doxycyclin-regulatable microRNAbased
retroviral vectors. The specific effects of the
knock-down of each CKI are under investigation.
A related research, not initially scheduled in the program
but showing encouraging results, deals with
the role of epigenetic modifications in controlling
the two distinct states of cell cycle arrest occurring
in muscle cells: terminal differentiation of myofibers
and reversible quiescence of satellite reserve cells (2).
Our interest is focused on poly(ADP-ribosyl)ation, a
post-translational modification of chromatin proteins
involved in a variety of physiological processes (6).
We have previously demonstrated that the activity
PARP-1, the main enzyme responsible for poly(ADPribosylation,
is involved in the exit of fibroblasts from
a quiescent state (G0) induced by serum deprivation
(1). This modification is required for the upregulation
of immediate early response genes (IEGs), such
as c-Fos and c-Myc, after mitogen stimulation. We
have recently set up an in vitro skeletal muscle cell
system mimicking the function of satellite cells. C2
myoblasts can be arrested in G0 through suspension
culture in methyl-cellulose. This condition leads to a
quiescent, undifferentiated state, which is characterized
by the expression of satellite cell markers and
which can be reverted by restoring cell adhesion
in the presence of growth factors (7). Western blot
analysis of cell extracts from quiescent myoblasts
showed that poly(ADP-ribose), indicative of PARPactivity,
rapidly accumulates within 2 hours upon cell
cycle re-entry. Moreover, we demonstrated that the
inhibition of PARP activity not only interferes with
the up-regulation of immediate-early genes and with
58
cell cycle re-entry, but also delays the induction of
the myogenic factor MyoD, a hallmark of satellite cell
activation. The involvement of PARP-1 was further
demonstrated by specific silencing through shRNAs.
Perspectives
Through the study of p57 regulation we have highlighted
the coordinate control of a group of imprinted
genes during myogenesis. Moreover, we have
revealed that the myogenic factor MyoD directly
communicates with a long-distance element overlapped
with an imprinting control region. We plan to
continue this research with the aim of characterizing
the molecular mechanisms by which MyoD affects the
activity of KvDMR1 and the functional interaction of
this regulatory region with the p57 encoding gene.
These studies promise the identification of a new epigenetic
pathway by which MyoD orchestrates the coordinate
expression of genes involved in growth control.
Moreover, the results obtained will allow us to gain
insights into the epigenetic constraints underlying the
tissue and cell type restriction of p57 induction during
physiological and pathological differentiation processes.
The results concerning the functional interplay
of PARP-1 with cell cycle arrest suggests that
poly(ADP-ribosyl)ation may regulate the quiescence
of satellite cells, the muscle stem cells. Although the in
vitro model described above faithfully reproduce many
characteristics of muscle stem cells, we have also
planned preliminary approaches to explore a possible
role of PARP-1 in the function of in vivo satellite cells.
These will be propedeutical to a functional analysis of
the role of PARP activity in muscle regeneration.
References
1) Carbone M, Rossi MN, Cavaldesi M, Notari A,
Amati P, Maione R. 2008. Oncogene. 27:6083-92.
2) Dhawan J, Rando TA. 2005. Trends Cell Biol.
15:666-73.
3) Figliola R, Maione R. 2004. J Cell Physiol. 200:
468-75.
4) Figliola R, Busanello A, Vaccarello G, Maione R.
2008. J Mol Biol. 380:265-77.
5) Fitzpatrick GV, Pugacheva EM, Shin JY,
Abdullaev Z, Yang Y, Khatod K, Lobanenkov VV,
Higgins MJ. 2007. Mol Cell Biol. 27:2636-47.
6) Kim MY, Zhang T, Kraus WL. 2005. Genes Dev.
19:1951-67.
7) Sambasivan R, Pavlath GK, Dhawan J. 2008. J
Biosci. 33:27-44.
8) Vaccarello G, Figliola R, Cramerotti S, Novelli F,
Maione R. 2006. J Mol Biol. 356:578-88.

Principal investigator: Franco Mangia
Professor of Neurobiology
Dipartimento di Psicologia, Sezione di Neuroscienze
Tel: (+39) 06 49917784; Fax: (+39) 06 49917873
franco.mangia@uniroma1.it
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AREA 3
Molecular regulation of cell proliferation and apoptosis in early
embryo blastomeres and granule neuron precursors of the mouse
Participants:
Maria Teresa Fiorenza, Arturo Bevilacqua, professors; Sonia
Canterini, researcher; Adriana Bosco, Andrea Fuso, post-doc
fellows, Valentina Carletti, Stefania Nusca, PhD students.
Collaborations:
Istituto Dermopatico dell’Immacolata, Rome (Dr. Giandomenico
Russo, Dr. Maria Grazia Narducci); Dipartimento di Scienze
Anatomiche, Istologiche, Medico-Legali e dell’Apparato
Locomotore, Sapienza-Università di Roma (Prof. Carla Boitani,
Dr. Rossella Puglisi); Dipartimento di Psicologia, Sapienza-
Università di Roma (Prof. Simona Cabib); Fondazione San
Raffaele, Milano (Prof. Francesco Blasi); Università di Torino
(Dr. Annalisa Buffo); University of Antwerp, Belgium (Dr.
Michele Giugliano).
Report of activity
We have exploited two different model systems, the
preimplantation mouse embryo development and
the cerebellum granule neuron differentiation and
apoptosis, to investigate the balance between cell
proliferation-survival and apoptosis, by specifically
studying: (i) the function of the oncogenic factor
T-cell Leukemia Factor 1 (TCL1) and its interactions
with AKT/PKB; and (ii) the role of the putative
tumor suppressor THG-1pit/Tsc22d4 in cerebellum
granule neuron differentiation and apoptosis.
Besides T-cell leukemias, TCL1 is physiologically
expressed in both embryonic stem cells, where
it is expressed downstream from the Oct4 gene,
and the early preimplantation embryos, in which
it promotes early blastomere proliferation. TCL1
binds and transphosphorylates AKT/PKB (AKT)
at the level of plasma membrane and then mediates
phosphorylated AKT transfer to nucleus. In spite
of recent advances in the field (rev. by Gonzalez
and McGraw, Cell Cycle 8:2505-8, 2009; Dillon and
Muller, Cancer Res. 70:4260-4, 2010), however, the
specific AKT isoform(s) TCL1 interacts with in
early embryos is(are) still largely unknown. We
have directly addressed this questions by depleting
one-cell embryos of a specific AKT isoform with
microinjection of anti-AKT1, anti-AKT2 or anti-
AKT3 antibodies and subsequent analysis of embryo
development in vitro. Depletion of AKT2 delayed/
blocked embryo development to blastocyst, with features
similar to those of Tcl1 KO embryos (Narducci
et al., PNAS 99, 11712-7, 2002). In contrast, depletion
of AKT1/AKT3 had no apparent effect on
embryos, pinpointing the AKT2 isoform as putative
TCL1 interactor in embryos. Moreover, immunofluorescence
experiments showed that AKT2, but
not AKT1 nor AKT3, migrates to nucleus in concomitance
with TCL1 nuclear localization. Results
obtained with anti-AKT antibodies have recently
been confirmed by subcellular TCL1 localization
in embryos genetically depleted of a single AKT
isoform. AKT1-, AKT2- andAKT3-KO mice were
kindly provided by Dr. B.A. Hemmings and used to
generate -/- embryos. In agreement with results of
antibody microinjections, TCL1 normally localized
into blastomere nuclei in AKT1 -/- and AKT3 -/- twocell
embryos, but was fully restricted to cytoplasm
in AKT2 -/- embryos, conclusively showing that
TCL1 specifically interacts with AKT2, but not
AKT1 nor AKT3, in this system. The possibility
that AKT Ser473/Thr308 phosphorylation depended
on upstream factors/kinases, including PI3K,
PDK1, and HSP90 protein has also been probed by
treating embryos with specific inhibitors, showing
that Ser473/Thr308-phosphorylated AKT2 is fully
inherited from oogenesis and that, following fertilization,
the ratio between AKT phosphorylation-
dephosphorytlation does not significantly change.
This indirectly indicates that TCL1 is not required
for AKT phosphorylation, whereas it represents
an absolute requirement for phosphorylated AKT
transfer to nucleus.
Cerebellum granule neurons (CGNs) proliferation, differentiation
and apoptosis has also been investigated

Franco Mangia
by studying Tsc22d4, a gene belonging to the TGFb1-stimulated
TSC22D gene family and encoding a 42
kDa protein that is post-translationally modified by
O-glycosylation and phosphorylation, giving rise to
55, 67 and 72 KDa forms. TSC22D4 is normally localized
in CGN somata and neurites, but is rapidly and
transiently translocated to nucleus following commitment
to apoptosis. This is easily achieved by exposing
in vitro cultured CGNs to a decrease in external KCl
from 25 mM (a depolarizing condition mimicking synaptic
inputs from mossy fibers), to 5 mM (a hyperpolarizing
condition triggering CGNs to apoptosis). We
have recently addressed the possibility that TSC22D4
is relevant to CGN balance between survival, differentiation
and apoptosis. Investigating TSC22D4 intracellular
localization during early postnatal cerebellum
development (PN6-40; a period of dramatic CGN/
Purkinje cell change in morphology and membrane
excitability), it appeared that TSC22D4 is localized
in both nuclear and somatodendritic compartments
before PN12-18 (the differentiation time at which
CGN viability becomes dependent on membrane
electrical activity), and that it apparently leaves the
nucleus and accumulates at the level of cerebellum
glomeruli (synaptic contacts with mossy fibers) in
mature CGNs. Similar features were also observed in
cerebellum slices cultured in vitro under conditions
favoring/inhibiting CGN differentiation. We thus
concluded that TSC22D4 functions take place: (i) in
the nucleus while CGNs differentiate; (ii) in synaptic
terminals when CGNs mature; and (iii) again in the
nucleus, but with subnuclear targets different than
those of immature cells, when CGNs are committed to
apoptosis (Canterini et al., 2009; Canterini et al., 2010),
suggesting multiple TSC44D4 roles. The intracellular
localization of different TSC22D4 form(s) has
recently been studied on subcellular fractions from
differentiating, differentiated and apoptosis-committed
CGNs. Mature TSC22D4 forms, including the
phosphorylated 67 kDa and the phosphorylated and
O-glycosylated 72 kDa forms that are normally present
in the cytoplasm apparently decrease in relative
abundance following cell commitment to apoptosis.
60
It also appeared that TSC22D4 is localized in neurite
and at synaptic termini mithocondria (Canterini et
al., in preparation). Moreover, the nuclear chromatin
fraction selectively contains the mature 72 kDa form,
irrespectively of normal or apoptotic condition, but
not other TSC22D4 forms. In contrast, the nuclear
matrix fraction contains 42 kDa form under normal
conditions and is strongly enriched with the 42 kDa
and 67 kDa forms following CGC commitment to
apoptosis. TSC22D4 role in CGN differentiation has
been investigated by exploiting siRNAs in in vitro cultured
CGNs, in which a block of differentiation results
in cell death, and in N1E-115 cells, a neuroblastomaderived
cell line expressing TSC22D4 at a high level,
in which the block of differentiation does not interfere
with cell viability. As expected, siRNA silencing
resulted in extensive death of CGNs and inhibition
of neurite elongation, but did cell death, in N1E-115
cells, showing that TSC22D4 is essential for CGN
differentiation.
Pubblications
Canterini S, Bosco A, De Matteis V, Mangia F,
Fiorenza MT. THG-1pit moves to nucleus at the
onset of cerebellar granule neurons apoptosis. Mol
and Cell Neurosci. 2009, 40: 249-57. doi: 10.1016/j.
mcn.2008.10.013.
Colelli V, Fiorenza MT., Conversi D, Orsini C, Cabib
S. Strain-specific proportion of the two isoforms
of the dopamine D2 receptor in the mouse striatum:
associated neural and behavioral phenotypes.
Genes, Brain and Behavior. 2010, 9: 703-711. doi:
10.1111/j.1601-183X.2010.00604.x.
Fernandez-Diaz LC, Laurent A, Girasoli S, Turco M,
Longobardi E, Iotti G, Jenkins NA, Fiorenza MT,
Copeland NG, Blasi F. The absence of Prep1 causes
p53-dependent apoptosis of mouse pluripotent epiblast
cells. Development. 2010, 137: 3393-3403. doi:
10.1242/dev.050567.

Study of the molecular and cellular mechanisms of sarcopenia:
role of mIGF-1 and oxidative stress
Principal investigator: Antonio Musarò
Professor of Biotechnology
Dip. di Scienze Anatomiche, Istologiche, Medico-Legali e dell’Apparato Locomotore
Tel: (+39) 06 49766956; Fax: (+39) 06 4462854
antonio.musaro@uniroma1.it
Participants:
Gabriella Dobrowolny, Emanuele Rizzuto, Michela
Aucello, post-doc fellows; Carmine Nicoletti, technician.
Collaborations:
Ce.S.I. Centro Scienze dell’Invecchiamento, IIM, Università degli
Studi G. d’Annunzio, Chieti (Prof. Giorgio Fanò, Prof. Feliciano
Protasi), Dipartimento di Scienze Biomediche, Università di
Padova (Dr. Marco Sandri); EMBL Mouse Biology Program,
Monterotondo, Rome (Dr. Nadia Rosenthal); Dipartimento di
Neuroscienze, IIT, Genova (Dr. Maria Pennuto).
Report of activity
The aim of the project was to define the molecular
mechanisms that modulate muscle homeostasis. In
particular, we analysed the role of oxidative stress
and the growth factor IGF-1 on muscle muscle
atrophy and wasting. Generation of ROS represents
one of the most prominent events during contractile
activity, suggesting that it could influence muscle
function and health (Fig. 1). How do ROS act as
physiological signaling molecules? When does the
oxidative stress become a damaging factor? It is well
recognized that moderate, non-exhaustive physical
exercise has beneficial effects and might prevents
several chronic diseases. However, a crucial system
severely affected in different pathological conditions
is the antioxidative defense, leading to a pathological
accumulation of ROS (Fig. 1).
The combined facts that mice lacking the major
antioxidant enzyme superoxide dismutase 1 (SOD1)
display a dramatic acceleration in age-related loss of
skeletal muscle mass, that elevated levels of reactive
oxygen species (ROS) may contribute to chronic diseases,
and that mutation in SOD1 is associated with
one fifth of familial ALS, have implicated oxidative
stress as a key mechanism underlying the pathogenesis
of aging and neuromuscular diseases. However,
how such an oxidative insult plays a role in the
61
AREA 3
diseases-related decrease of muscle performance and
mass and whether skeletal muscle is a direct target of
SOD1 mutation remained largely unknown.
In this project we undertook a transgenic approach
to define the direct role of oxidative stress on muscle
homeostasis and function. To this purpose and to
verify whether skeletal muscle is a direct target of
SOD1 mutation we generated transgenic mice with
the mutated isoform of human superoxide dismutase
1 (SOD1 G93A ) cDNA driven by skeletal muscle specific
regulatory elements from the rat myosin light chain
(MLC)-1/3 locus. Expression of the MLC/SOD1 G93A
transgene in adult mice was restricted to skeletal muscle,
predominated in muscles enriched in fast fibers and
reduced in slow muscles such as the soleus, where the
MLC regulatory cassette is characteristically expressed
at very low levels. Notably, no expression of human
SOD1 protein and transcript were found in heart, brain,
liver, spleen, or spinal cord of transgenic mice.
Muscle-restricted expression of mutant SOD1 G93A was
sufficient to induces severe muscle atrophy associated
with significant reduction in muscle strength, sarcomere
disorganization, significant changes of mitochondria
morphology and of their sarcomeric disposition,
and disorganization of the sarcotubular system.
In this study, we also disclosed the potential molecular
mechanisms associated with muscle atrophy induced
by selective accumulation of oxidative stress.
We report that accumulation of ROS serves as signalling
to initiate autophagy, one of the major intracellular
degradation mechanisms that we demonstrated
to be a key determinant for the induction of muscle
atrophy associated with oxidative stress (Fig. 2).
In particular, the critical role of autophagy in the promotion
of muscle atrophy was disclosed by genetic
manipulation of LC3 expression, a molecular marker
of autophagy. In vivo electroporation of siRNA
against LC3 gene rescued the atrophic phenotype
in MLC/SOD1 G93A mice, suggesting that autophagy
is the dominant pathway that mediates the atrophic
stimulus of oxidative stress and that the modula-

Antonio Musarò
tion of the autophagy pathway can be a potential
therapeutic mechanism to counteract muscle atrophy
associated with oxidative stress. Moreover, we
addressed the involvement of caspases in the induction
of muscle atrophy in experimental models of
ALS, expressing the mutant SOD1 G93A transgene
either locally or ubiquitously. We demonstrated that
SOD1 G93A -mediated muscle atrophy is independent
from caspases activity. In particular the expression
of SOD1 G93A promotes a reduction of the PI3K/AKT
pathway associated with activation of FOXO3 (Fig.
2). In contrast, the activation of caspases is a later
event causally linked to motor neuron degeneration,
associated with an exacerbation of the atrophic phenotype
and to a shift in fiber type composition.
Thus, our study is the first to (1) establish skeletal
muscle as a primary target for the dominant action
of inherited SOD1 mutations, (2) implicate oxidative
stress as the primary trigger of muscle atrophy associated
with SOD1 mutation, (3) disjoin muscle atrophy
and function from motor neuron degeneration.
More recently, we analyzed the role of the growth
factor mIGF-1 in counteracting muscle wasting in
different animal models of neuromuscular diseases and
wasting, including aging, Spinal and Bulbar Muscular
Atrophy (SBMA), and muscular dystrophy. We demonstrated
that augmentation of IGF-1/Akt signaling
rescues behavioural and histopathological abnormalities,
extends the life span, and reduces both muscle
and spinal cord pathology of SBMA mice. This study
demonstrates that skeletal muscle is a viable target
tissue for therapeutic intervention in SBMA.
It is also known that aging and neuromuscular
diseases, including ALS and muscular dystrophy,
compromise the viscoelastic properties of tendon.
Confocal microscopy and mechanical properties
measurements revealed that MLC/SOD1 G93A and
dystrophic mdx tendons, compared to WT ones,
have an increase in the number of dead cells and a
significant reduction in tissue elasticity for all the
frequencies that were tested. Notably, local expression
of IGF-1 was able to preserve the viscoelastic
properties of tendons in the different pathological
animal models. Our results demonstrate that neuromuscular
diseases involves not only muscle wasting,
but also alteration in the viscoelastic properties of
tendons, suggesting a paracrine effect of altered skeletal
muscle on tendinous tissue.
The results of this project contribute to clarify the
molecular mechanisms underlying muscle atrophy
62
and wasting and will allow us to develop specific
therapeutic strategies in order to prevent or attenuate
the muscle atrophy and frailty associated with
aging and neuro-muscular diseases.
Publications
Aucello M, Dobrowolny G, Musarò A. Localized
accumulation of oxidative stress causes muscle atrophy
through activation of an autophagic pathway.
Autophagy. 2009, 5: 527-9. doi: 10.4161/auto.5.4.7962.
Barberi L, Dobrowolny G, Pelosi L, Giacinti C,
Musarò A. Muscle involvement and IGF-1 signaling
in genetic disorders: new therapeutic approaches.
Endocr Dev. 2009, 14: 29-37. doi: 10.1159/000207474.
Messina S, Bitto A, Aguennouz M, Mazzeo A,
Migliorato A, Polito F, Irrera N, Altavilla D, Vita
GL, Russo M, Naro A, De Pasquale MG, Rizzuto
E, Musarò A, Squadrito F, Vita G. Flavocoxid counteracts
muscle necrosis and improves functional
properties in mdx mice: a comparison study with
methylprednisolone. Exp Neurol. 2009, 220: 349-58.
doi: 10.1016/j.expneurol.2009.09.015.
Palazzolo I, Stack C, Kong L, Musarò A, Adachi
H, Katsuno M, Sobue G, Taylor JP, Sumner CJ,
Fischbeck KH, Pennuto M. Overexpression of IGF-1
in muscle attenuates disease in a mouse model of spinal
and bulbar muscular atrophy. Neuron. 2009, 13:
316-28. doi: 10.1016/j.neuron.2009.07.019.
Rizzuto E, Musarò A, Catizone A, Del Prete Z.
Measuring tendon properties in mdx mice: cell viability
and viscoelastic characteristics. J Biomech. 2009,
42: 2243-8. doi: 10.1016/j.jbiomech.2009.06.041.
Scicchitano BM, Rizzuto E, Musarò A. Counteracting
muscle wasting in aging and neuromuscular diseases:
the critical role of IGF-1. Aging. 2009, 11: 451-7.
Musarò A, Fulle S, Fanò G. Oxidative stress and muscle
homeostasis. Curr Opin Clin Nutr Metab Care. 2010,
13: 236-42. doi:10.1097/MCO.0b013e3283368188.
Musarò A, Barberi L. Isolation and culture of mouse
satellite cells. Methods Mol Biol. 2010, 633: 101-11.
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.
Fig. 2 - A summary of the signal pathways activated in SOD1 G93A transgenic mice.
63
AREA 3

Role of the COP9 signalosome in transcription modulation and
chromatin organization in yeast and plants
Principal investigator: Rodolfo Negri
Professor of Molecular Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 44917790; Fax: (+39) 06 44917594
rodolfo.negri@uniroma1.it
Partecipants:
Giovanna Serino, professor; Teresa Rinaldi, researcher;
Angela Alagia, Micaela Esposito, PhD students.
Collaborations:
Università degli Studi di Torino (Prof. Gianni Balliano); University
of Haifa, Israel (Prof. Elah Pick).
Report of activity
Accurate regulation of transcription in time and
space is critical for the establishment and maintenance
of gene expression patterns in eukaryotes. In
the past years, several components of the ubiquitin/
proteasome system (UPS) have been shown to be
necessary for the tight regulation of gene expression
with possible important implications for cellular
homeostasis. Recent evidence shows that part of
this regulatory action is at transcriptional level and
mediated by direct action of UPS components on
chromatin at defined genomic loci.
A key component of the UPS is the COP9 signalosome
(CSN), a protein complex conserved in
all eukaryotes. At the biochemical level, the CSN
removes the small peptide NEDD8 (an ubiquitin like
modifier) from the cullin-RING family of E3 ubiquitin
ligases. E3 ligases are the enzymes responsible
for poly-ubiquitination of specific substrates, which
can be subsequently recognized and degraded by the
proteasome. The reaction catalyzed by CSN is necessary
for the tight regulation of the assembly/disassembly
cycles of these ligases and is essential for
all higher eukaryotes, since null CSN mutants from
different organisms ranging from plants to mammals
are lethal at very early developmental stages.
At the cellular level, CSN mutants from different
organisms display de-repression and, more in general,
miss-regulation of several sets of genes. This
could be due simply to the fact that CSN is necessary
for the proper degradation of several transcription
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factors. However, recent evidence suggests that
CSN might perform specific tasks directly on the
genomic chromatin.
The CSN from budding yeast S. cerevisiae has been
recently characterized and, like in higher eukaryotes,
is responsible for the de-neddylation of cullin-RING
ubiquitin ligases. However, while the CSN in most
higher eukaryotes is composed of 8 subunits and is
a paralog of the lid of the proteasome, the CSN from
budding yeast consists of six subunits, four with a
PCI domain (Csn9, Csn10, Csn11/Pci8 and Csn12),
one with a JAMM/MPN+ domain (Csn5/Rri1), and
a sixth protein called Csi1. In addition, in contrast to
other eukaryotes, all the CSN subunits from S. cerevisiae
are non-essential. The non essentiality of CSN
components and the availability of powerful genetic
tools make S. cerevisiae a very promising model system
to elucidate some aspects of the nuclear role of
this complex.
Interestingly, the CSN localization in S. cerevisiae is
prevalently nuclear and deletion of Csn12 leads to
Csn5 delocalisation to the cytoplasm. This observation
opens up the perspective of a nuclear function
of the CSN in S. cerevisiae, which could be related to
chromatin organization and/or direct transcriptional
control, as it is the case for the proteosomal lid, and
as it has been suggested for CSN in other organisms.
This project aims at testing the hypothesis of a direct
role of the CSN in the regulation of transcription and
chromatin structure. The core experiments are carried
out in the budding yeast S. cerevisiae and the results
will be further validated in the plant Arabidopsis thaliana,
where the CSN was initially identified.
Trascriptomic analysis of a delta-csn5 strain
We performed a transcriptomic analysis of a S. cerevisiae
strain deleted in the Ylr100w ORF, coding
for the JAMM/MPN+ domain containing the Csn5
protein component (for the de-neddylating activity
of the yeast Cop9 complex), as compared with its
isogenic wild type strain. The transcriptome screen-

Rodolfo Negri
ing was performed on two independent cultures
of the wt and csn5-deleted strains by two-colour
competitive hybridization of microarray slides containing
the whole repertoire of S. cerevisiae ORFs
(UHN Microarray Centre (http://data.microarrays.
ca/arrays/index.htm). Data analysis showed that
183 genes were significantly (p

The biogenesis of piRNAs and their involvement in transposon
silencing and heterochromatin formation in Drosophila
Principal investigator: Sergio Pimpinelli
Professor of Genetics
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912876; Fax: (+39) 06 4456866
sergio.pimpinelli@uniroma1.it
Participants:
Laura Fanti, professor; Lucia Piacentini, researcher; Marcella
Marchetti, post-doc fellow; Enzo Marchetti, technician.
Collaborations:
Università di Lecce (Prof. Maria Pia Bozzetti, Dr. Maria
Berloco).
Report of activity
Heterochromatin is a nearly ubiquitous component
of the eukaryotic chromosome that is usually located
at the pericentromeric regions and telomeres. Such
material constitutes a good model for studying
epigenetic gene silencing. Many significant results
have already been obtained and much information is
already available about the molecular determinants
of heterochromatin and their involvement in gene
silencing. However, many problems still remain to
be solved such as the identification of all of the components
of heterochromatic multiprotein complexes
and the mechanisms of their targeting. Growing
evidence strongly suggests that the RNA interference
phenomenon is involved in heterochromatin
formation and perhaps in other chromatin modifications
responsible for more general transcriptional
gene silencing.
The aim of this research is to systematically search,
by a combined genetic, cytological and molecular
approach, genes involved in piRNAs biogenesis,
heterochromatin formation and transposons silencing.
To this end we are collecting and studying
mutants that induce the expression of the repetitive
Stellate sequences of Drosophila melanogaster normally
repressed by piRNAs interference mechanism.
The crystal-Stellate system is a clear example of
“natural” piRNAs mediated silencing. In wild type
males a Stellate mRNA is degraded due to a homologous
piRNAs coded by the crystal region on the Y
chromosome. This phenomenon is mediated by the
RNA interference machinery. A mature 750 nucleo-
67
AREA 3
tide Stellate mRNA is instead produced when the
Y chromosome is deleted in the crystal region. The
protein obtained after translation forms crystalline
aggregates in spermatocytes and spermatids. The
existence of modifiers of the crystal-Stellate interaction
and that show the same phenotype as the crystal
deletion has strongly suggested to us that mutations
that abolish Stellate silencing are good candidates for
genes involved in piRNAs biogenesis, heterochromatin
formation and gene silencing.
We have already performed a sistematic search for
such mutations and we have collected and identified
about 15 genes that derepress Stellate sequences and
we developed meaningful experimental criteria to
identify those genes involved in each, or more, of the
phenomena considered above.
The research activity during the last two years has
been mainly focused on analysis of different chromatin
proteins that are involved in heterochromatin
formation and/or piRNA biogenesis in Drosophila
melanogaster.
HP1 and gene expression
Recently, by a wide expression analysis, using a RIPchip
assays (RNA-immunoprecipitation on microarrays),
we have shown that HP1 associates with the
transcripts of more than one hundred euchromatic
genes and interacts with the heterogeneous nuclear
ribonucleoproteins (hnRNPs) that are known to be
involved in RNA processing. So that, HP1 seems to
be part of a complex that stabilizes RNA transcripts.
Though previously unsuspected, it was also found
that HP1-interacting hnRNPs have a functional role
in heterochromatin formation. These proteins bind
heterochromatin and are dominant suppressors of
position effect variegation. Taken together, the results
open a window on a possible new conceptual landscape
in which similar epigenetic mechanisms could
have a significant role, both in the metabolism of RNA
transcripts and in heterochromatin formation, produc-

Sergio Pimpinelli
ing opposite functional effects. These data seem to
establish a functional link between euchromatin and
heterochromatin. Interestingly, we found that some of
the hnRNP induce the expression of the Ste sequences
thus suggesting a lpossible involvement of piRNAs in
heterochromatin formation.
Hsp90, piRNAs biogenesis and transposon
silencing
We have recently shown that, in Drosophila, functional
alterations of Hsp90 affect the piRNA silencing mechanism
leading to transposon activation and the induction
of morphological mutants. The clear involvement
of Hsp90 in piRNA biogenesis and transposon silencing
has also suggested an alternative explanation to the
Waddington’s “Canalization” concept. Hsp90, instead
to be a “capacitor” that buffers phenotypic variance, is a
68
“mutator” that can actually generate new variation by
trasposon-mediated canonical mutagenesis.
Publications
Piacentini L, Fanti L, Negri R, Del Vescovo V, Fatica
A, Altieri F, Pimpinelli S. Heterochromatin protein 1
(HP1a) positively regulates euchromatic gene expression
through RNA transcript association and interaction
with hnRNPs in Drosophila. PLoS Genet. 2009, 5:
e1000670. doi: 10.1371/journal.pgen.1000670.
Specchia V, Piacentini L, Tritto P, Fanti L,
D’Alessandro R, Palumbo G, Pimpinelli S, Bozzetti
MP. HSP90 prevents phenotypic variation by suppressing
the mutagenic activity of transposons.
Nature. 2010, 463: 662-665. doi:10.1038/nature08739.

To the root of organ growth: the control of root meristem
activity in Arabidopsis
Principal investigator: Sabrina Sabatini
Researcher in Molecular Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49917916; Fax: (+39) 06 49917594
sabrina.sabatini@uniroma1.it
Participants:
Laila Moubayidin, Serena Perilli, Riccardi Di Mambro, PhD
students.
Report of activity
Introduction
Plant post-embryonic development takes place in the
meristems. In the root of the model plant Arabidopsis
thaliana, stem cells organized in a stem cell niche
in the apex of the root meristem generate transitamplifying
cells, which undergo additional division
in the proximal meristem, and differentiate in the
distal meristem transition zone that encompasses the
boundary between dividing and expanding (differentiating)
cells in the different cell files (Dello Ioio et
al., 2007). For meristem maintenance, and therefore
continuous root growth, the rate of cell differentiation
must equal the rate of generation of new cells:
how this balance is achieved is a central question in
plant development. By means of a genetic and molecular
analysis, we have shown that in the Arabidopsis
root meristem the balance between cell differentiation
and cell division necessary for controlling root
meristem size and root growth is the result of the
interaction between two plant hormones, cytokinin
and auxin, through a simple regulatory circuit converging
on a single gene, the SHY2 gene (Dello Ioio
et al., 2008). In particular, in the vascular tissue of the
transition zone, a primary cytokinin-response transcription
factor, ARR1 (Sakai et al., 2001), activates
the gene SHY2 (Kepinsky et al., 2006), a repressor
of auxin signaling that negatively regulates the PIN
genes that encode auxin transport facilitators (Blilou
et al., 2005). Thus, cytokinin causes redistribution of
auxin, prompting cell differentiation (Dello Ioio et al.,
2008). Conversely, auxin mediates degradation of the
SHY2 protein (Kepinsky et al., 2006), sustaining the
activity of the PIN genes and prompting cell division
(Dello Ioio et al., 2008).
69
AREA 3
Thus, our work has shown that a specific developmental
domain of the Arabidopsis root - the vascular
tissue of the transition zone - is where the interaction
between cytokinin and auxin takes place, and
has provided a genetic and molecular framework for
this interaction, clarifying how the size of the root
meristem is maintained. However, two important
questions concerning the ARR1/SHY2/PIN regulatory
network remain to be answered:
- First, since cytokinin affects PIN activity in the
vascular tissue of the transition zone, how is this
change perceived by the neighbouring cell files in
the transition zone in order for them to shift from
the meristem into the differentiation zone (radial cell
non-autonomous effect)
- Second, in maintaining a balance between cell differentiation
and cell division, how does the mechanism
that controls the former (which takes place in the distal
meristem transition zone) influence the division of stem
cells (which takes place in the apical stem cell niche)
Objectives
The work proposed addresses the two above-mentioned
questions, as part of a larger effort of our
laboratory - which includes elucidating how the basic
ARR1/SHY2/PIN regulatory circuit interacts with
other (hormonal) signals and regulatory networks
that affect root growth - aiming to clarify the molecular
mechanisms through which cytokinin and auxin
control Arabidopsis root growth, and ensure the
robustness of such a complex developmental process.
As of this proposal, the work will consist of two main
lines of research aimed to:
1) Identify and characterize the molecular components
that ensure perception by all cell files in the
transition zone of the cytokinin-dependent redistribution
of auxin that occurs in the vascular tissue
(radial cell non-autonomous effect).
2) Identify and characterize the molecular components
that allow the cytokinin/auxin interaction to
influence stem cell activities.

Sabrina Sabatini
Results
While trying to identify the nature of the radial cell
non-autonomous signals (point 1 of the Objectives)
we notice that the plant hormones gibberellins, in
analogy to auxin, positively control root growth and
meristem size, by sustaining cell division. Based on
this observation we were able to show that in allowing
growth of the root meristem after seed germination
and for the meristem to reach its final size, the
ARR1/SHY2/PIN circuit necessary to maintain the
meristem final size is integrated by two additional
components: the cytokinin-responsive ARR12 transcription
factor, and the plant hormones gibberellins.
ARR12, in analogy to ARR1, drives a low level of
expression of SHY2 during the meristem growth
phase for ensuring a prevalence of cell division over
cell differentiation. ARR1 eventually joins ARR12 in
increasing SHY2 expression, leading to an increase
of cell differentiation that thus balances cell division
and stops meristem growth. Gibberellins, necessary
for seed germination and radicle protrusion, repress
expression of the ARR1 gene during post-germination
meristem growth, and a subsequent decrease
in their activity, allows ARR1 expression and the
consequent up-regulation of SHY2 via the stabilization
of RGA, a gibberellin inhibitor DELLA protein.
These results, although do not reveal the nature of
the radial cell non-autonomous signals that coordinate
cell differentiation at the transition zone, take
into account another important aspect of meristem
development: how a defined final meristem size is set,
i.e. how a change in the relative rates of cell division
and cell differentiation is brought about for meristem
growth to stop. We showed, for the first time, that
70
the balance between cell division and differentiation
that ensures meristem maintenance is achieved by
controlling the rate of cell differentiation rather than
the rate of cell division, which remains constant.
Progress in point 2 of the Objectives shows that
ARR1 expression in the stem cell niche is suppressed
by the transcription factor SCARECROW (SCR,
Sabatini et al., 2003) corroborating the idea that SCR
maintains stem cell division by preventing cytokinin
action in the stem cell niche.
References
- Blilou I, Xu J, Wildwater M, Willemsen V,
Paponov I, Friml J, Heidstra R, Aida M, Palme K,
Scheres B. (2005) Nature 433, 39-44.
- Dello Ioio R, Linhares FS, Scacchi E, Casamitjana-
Martinez E, Heidstra R, Costantino P, Sabatini S.
(2007) Curr Biol. 17, 678-682.
- Dello Ioio R, Nakamura K, Moubayidin L, Perilli S,
Taniguchi M, Morita MT, Aoyama T, Costantino
P, Sabatini S. (2008) Science 322, 1380-1384.
- Kepinski S. (2006) Curr Opin Plant Biol. 9, 28-34
- Sabatini S, Heidstra R, Scheres B. (2003). Genes &
Development 17, 354-358.
- Sakai H, Honma T, Aoyama T, Sato S, Kato T,
Tabata S, Oka A. (2001) Science 294, 1519-1521.
Publications
Moubayidin L, Perilli S, Dello Ioio R, Di Mambro R,
Costantino P, Sabatini S. The rate of cell differentiation
controls the Arabidopsis root meristem growth
phase. Curr Biol. 2010, 20: 1138-1143. doi:10.1016/j.
cub.2010.05.035.

71
AREA 3
Biological characterization and in vitro culture of spermatogonial
stem cells
Principal investigator: Mario Stefanini
Professor of Histology and Embryology
Dip. di Scienze Anatomiche, Istologiche, Medico-Legali e dell’Apparato Locomotore
Tel: (+39) 06 49766570; Fax:(+39) 06 4462854
mario.stefanini@uniroma1.it
Participants:
Carla Boitani, professor; Elena Vicini, researcher; Barbara
Muciaccia, Rossella Pugliesi, post-doc fellows; Margherita
Grasso, Lisa Dovere, PhD students; Stefania Fera, Tiziana
Menna, technicians.
Collaborations:
Dipartimento di Sanità Pubblica e Biologia Cellulare, Università di
Roma Tor Vergata (Prof. Massimo De Felici, Dr. Maria Lucia
Scaldaferri); Dipartimento di Biologia Cellulare e Neuroscienza,
Istituto Superiore di Sanità, Rome (Dr. Massimo Sanchez).
Report of activity
In the last years the activity of our group has focused
on the isolation and characterization of spermatogonial
stem cells (SSCs), the stem cells that sustain
spermatogenesis in mammals.
In the accepted model of SSCs self-renewal, Asingle
spermatogonia (As) are the stem cells of spermatogenesis
whereas chained undifferentiated spermatogonia
are irreversibly committed to differentiation. This
long-lasting model has been recently challenged by
evidence from our and other laboratories indicating
that As and chained early A spermatogonia are heterogeneous
in terms of gene expression and function
and different subsets of stem cells may be generated by
an asymmetric mitotic division (Grisanti et al., 2009).
The purpose of our project is to provide insight into
the biological function of the different subsets of SSCs.
This line of research may shed light on the molecular
mechanisms regulating early step of spermatogenesis,
for future applications on male infertility, and to unveil
molecular mechanisms underlying pluripotency.
Development of animal models to obtain
isolation and enrichment of spermatogonial
stem cells
We have developed an animal model expressing
endogenous GFRA1 tagged with the enhanced green
fluorescent protein (EGFP-GFRA1). GFRA1 and its
co-receptor Ret are the receptors for the niche-derived
factor GDNF. GDNF is essential for the correct balance
of self-renewal and differentiation of SSCs and
its specific co-receptor GFRA1 is expressed by a subsets
of As. GFRA1 is a glycosylphosphatidylinositol
(GPI)-anchored plasma membrane protein which is
synthesized as precursor with a cleavable signal peptide
and a cleavable carboxy-terminal signal sequence
that directs GPI anchoring. In the targeting vector
the EGFP was cloned at the N-terminal of GFRA1
because it has been demonstrated that this region is
not involved in interactions with other components
of the receptor-ligand complex. To avoid processing
and excision of the EGFP tag with the signal peptide
after protein synthesis the signal peptide has been
placed N-terminal to EGFP. We first assessed that
GFRA1-EGFP fusion protein retains its biological
activity. We found that the fusion protein: i) retains
its subcellular localization by confocal microscopy; ii)
upon GDNF treatment interact with Ret as shown by
co-immunoprecipitation; iii) upon GDNF treatment
retains its ability to allow Ret auto-phosphorylation.
These data indicated that the EGFP- GFRA1 fusion
protein retains its biological activity and validate
the strategy to create the “knock-in” modified mice.
Therefore targeting vector was linearized, transfected
into ES cells and positive clones were injected
into C57BL/6 blastocyst. We have now obtained
germline transmission. This new animal model will
allow: i) isolation and enrichment of spermatogonial
stem cells by FACS e prerequisite to biochemical
analysis; ii) video-time laps analysis performed on isolated
seminiferous tubules or on spermatogonial stem
cells in vitro. This animal model will be used to gain
more insight into the SSCs phenotypic heterogeneity.
Ontogeny of side-population (SP) phenotype
in the germ stem cell lineage
The SP phenotype, defined as the ability of cells to
efflux fluorescent dyes such as Hoechst, is common
to several stem/progenitor cell types. We have pre-

Mario Stefanini
viously shown that a subset of normal spermatogonial
stem cells display an SP phenotype (Falciatori et
al., 2004). SSCs displaying the SP phenotype are clonogenic
in vivo and do not express GFRA1 (Grisanti
et al., 2009). We tested the hypothesis that SP phenotype
arise early and is maintained in the stem
germ cell lineage throughout testis development.
We analyzed and characterized the Hoechst SP via
cytofluorimetric analysis of disaggregated gonads
at different time points during embryonic development
in mice. To directly test the hypothesis that
the SP phenotype is a feature of germ cell lineage,
experiments were performed on transgenic animals
expressing EGFP under the control of the Oct4 promoter,
to identify early germ cells up to primordial
gem cells (PGCs) the embryonic precursors of SSCs.
We found that prenatal gonads contain a fraction of
SP cells at each stage analyzed, and the percentage
of cells in the SP fraction decreases as development
proceeds. Surprisingly, more than 50% of the PGCs
displayed the SP phenotype at 11.5 dpc (days post
coitum). The percentage of germ cells with the SP
phenotype decreased steadily with development, to
less than 1% at 18.5 dpc approaching the percentage
of SP cells in postnatal testis (Falciatori et al., 2004).
Cytofluorimetric analysis along with immunocyto-
72
chemistry performed on sorted cells indicate that the
SP fraction of prenatal gonads, as in the adult testis,
is heterogeneous, being composed of both somatic
and germ cells. Both cell types express the ABC
transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These
findings provide evidence that SP phenotype is a
common feature of germline development and identifies
a subpopulation of the fetal testis cells including
prospermatogonia whose differentiation fate remains
to be investigated (Scaldaferri et al., in press).
Publications
Grisanti L, Corallini S, Fera S, Muciaccia B, Witke
W, Stefanini M, Vicini E. Inactivation of numb and
numblike in spermatogonial stem cells by cell-permeant
Cre recombinase. Differentiation. 2009, 78:131-
136. doi:10.1016/j.diff.2009.05.005.
Grisanti L, Falciatori I, Grasso M, Dovere L, Fera S,
Muciaccia B, Fuso A, Berno V, Boitani C, Stefanini
M, Vicini E. Identification of spermatogonial stem
cell subsets by morphological analysis and prospective
isolation. Stem Cell. 2009, 27: 3043-52. doi:
10.1002/stem.206.

Molecular mechanisms of the epithelial to mesenchymal
transition in hepatocyte
Principal investigator: Marco tripodi
Professor of Genetics
Dipartimento di Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 4461387; Fax: (+39) 06 4462891
tripodi@bce.uniroma1.it
Participants:
Laura Amicone, Carla Cicchini, Alessandra Marchetti,
researchers; Alice Conigliaro, Carmine Mancone, post-doc
fellows; Laura Santangelo, Angela Cozzolino, AIRC fellows;
Francesca Garibaldi, PhD student; Claudio Cavallari, technician.
Report of activity
Aims of the proposal
1) Study of the molecular mechanisms controlling liver
stem cell EMT/MET oscillations and the influence of
micro-environmental factors on the stem cell fate.
In particular we have proposed to study the crosstalk
between the niche factors Wnt and TGFβ in
controlling liver stemness starting from the hypothesis,
supported by our previous observations, that the
transcriptional inhibitor Snail (respectively induced
and stabilized by TGFb and Wnt) is required for
stem cell maintenance.
Moreover, we have planned to investigate on the
cooperation between Wnt and FGF signaling in
controlling hepatic differentiation, starting from the
hypothesis that FGF pathway, crosstalking with Wnt,
could drive Resident Liver Stem Cells (RLSCs) differentiation
into hepatocyte promoting HNF4a-induced
MET. The hypothesis was supported by our previous
results (HNF4a dominancy on Snail in regulation of
EMT) and from several literature data indicating common
mediators and targets for FGFs and Wnts.
2) Study of the stem cell contribution to the onset
and progression of hepatic fibrosis. We have hypothesized
that chronic liver injury could alter the niche
signals triggering an abnormal activation of the stem
cell compartment. In particular, we hypothesized
that liver stem cells could respond to chronic injury
proliferating and activating differentiative program
towards fibrogenic cells. Our previous results, in
fact, suggested the capability of our RLSCs to differentiate
into mesenchymal cells quite similar to
activated Hepatic Stellate Cells or myofibroblasts.
73
AREA 3
Results
We recently reported the isolation, characterization
and reproducible establishment in line of resident
liver stem cells (RLSCs) with immunophenotype
(Sca1+, CD34-, CD45-, Alpha-fetoprotein+,
Albumin) that locate them in a pre-hepatoblast/
liver precursor cells hierarchical position and differentiative
potentiality spanning from endodermal
to mesenchymal and ectodermal derivatives.
(Conigliaro et al., 2008). RLSCs plasticity is coherent
with their metastable molecular phenotype typical of
embryonic and adult stem cells and characterized by
co-expression of epithelial and mesenchymal markers
as well as of a variety of chromatin remodeling
genes. This highly dynamic cell state, thought to be
the major determinant of stem cell fate, may be considered
as the result of continue oscillations between
mesenchymal-epithelial and epithelial-mesenchymal
transitions (EMT/METs).
Aim 1: We investigated on the molecular mechanisms
controlling RLSC stemness, in particular on the role
of the transcriptional regulator Snail, a TGFβinduced
EMT master gene. We previously observed
that TGFβ, as well as exogenous Snail, induces
EMT in hepatocyte while impedes the differentiation
into hepatocytes (Spagnoli et al., 2000) of liver bipotential
precursor cells. RLSCs constitutively express
Snail and their spontaneous differentiation into
hepatocytes is underlined by negative regulation of
this transcription factor. We gathered evidences that
have led us to conclude that Snail is a key element
in stemness maintenance. Moreover we unveiled the
stemness inhibitinging role of a number of microR-
NAs that are in turn repressed by Snail; Chromatin
Immunoprecipitation assays and qRT-PCR analysis
showed as the Snail recruitment (dynamically regulated
by TGFβ) on specific consensus of miRNAs
promoters correlates with down regulation of their
expression (manuscript in preparation).
In frame of the study of micro-environmental influ-

Marco Tripodi
ence on liver stem cell fate, we published a work
describing the effects of Wnt pathway on post-differentiation
patterning of hepatocyte. Our data showed
a direct and hitherto unknown convergence of the
canonical Wnt signaling (contributing also the to
liver stem cell maintenance in the stem niche) on the
HNF4-driven transcription providing evidences of
a mechanism controlling liver zonated gene expression
(Colletti et al., 2009).
We also attempt to further characterize the molecular
mechanisms controlling epithelial identity of
heptocytes. In brief, we unveiled a new mechanism
for HNF4α (already characterized as master gene of
the hepatocyte differentiation) in the induction and
maintenance of hepatocyte differentiation ascribed
to the capacity of this factor to continuously repress
the mesenchymal program. Overall, both HNF4α
activator and repressor functions appear necessary
for the identity of hepatocytes (Santangelo et al.,
submitted)
Finally, we collected evidences of the major role played
by the contact inhibition in the execution of hepatocyte
terminal differentiation (Mancone et al., 2010).
Aim 2: We studied the involvement of RLSC compartment
in the onset and progression of hepatic
fibrosis starting from the hypothesis that the
Hepatic Stellate Cells (HSCs), the main players in
fibrosis pathogenesis which origin is yet unknown,
may derive from liver stem cells. This hypothesis
started from the observation that RLSCs, when
grown without instructive dominant signals, are
able to express in culture a Mixed Epithelial and
Mesenchymal Phenotype (MEMP) constituted by
two different cell populations; while the characterization
of the epithelial component confirmed
the RLSCs ability to spontaneously differentiate
toward hepatocytes, the mesenchymal one sug-
74
gested their possible differentiation toward HSCs.
qRT-PCR analysis and Immunofluorescence analysis,
in fact, showed the expression of markers typical
of quiescent stellate cells that switch into activated
miofibroblasts after treatment with the niche factor
TGFβ (the most important cytokine involved in
vivo in the activation of HSCs). Notably, RLSCs,
injected intra-hepatically in new born mice, colonize
recipient livers contributing to both hepatocyte and
mesenchymal populations. The involvement of this
last cell component in liver fibrosis will be verified
in murine model of chemically induced hepatic
fibro/cirrhosis.
Publications
Colletti M, Cicchini C, Conigliaro A, Santangelo
L, Alonzi T, Pasquini E, Tripodi M, Amicone L.
Convergence of Wnt signaling on the HNF4alphadriven
transcription in controlling liver zonation.
Gastroenterology. 2009, 137: 660-72. doi:10.1053/j.
gastro.2009.05.038.
Wegert M, La Monica N, Tripodi M, Adler G,
Dikopoulos N. Impaired interferon type I signalling
in the liver modulates the hepatic acute phase response
in hepatitis C virus transgenic mice. J Hepatol. 2009,
51: 271-8. doi: 10.1016/j.jhep.2009.03.014.
Mancone C, Conti B, Amicone L, Bordoni V, Cicchini
C, Calvo L, Basulto Perdomo A, Fimia G M, Tripodi
M, Alonzi T. Proteomic analysis reveals a major role
of contact inhibition in the execution of hepatocyte
terminal differentiation. J Hepatol. 2010, 52: 234-43.
doi:10.1016/j.jhep.2009.11.013.

Principal investigator: Giuseppe Zardo
Researcher in Clinical Chemistry
Dipartimento di Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 80319026; Fax: (+39) 06 80319054
zardo@bce.uniroma1.it
75
AREA 3
Identification of novel genetic and epigenetic targets in Leukemia
by genome wide approaches
Participants:
Alberto Ciolfi, PhD student; Carmen Maresca, technician.
Collaborations:
Dipartimento di Biotecnologie Cellulari ed Ematologia (Prof.
Robin Foà, Dr. S. Chiaretti); Dipartimento di Scienze e
Biotecnologie Medico-Chirurgiche (Prof. Clara Nervi); UCSF
Cancer Center, University of California, San Francisco, USA (Prof.
Joseph F. Costello); Roswell Park Cancer Institute, Buffalo, NY,
USA (Prof. Dominic J. Smiraglia).
Report of activity
The dysregulation of sequential, coordinated, and
combinatorial expression of lineage-affiliated transcription
factors, modulating specific gene expression
programs and lineage commitment, results in the
onset and progression of hematopoietic malignancies
as acute myeloid leukemia (AML). The pathogenesis
of AML is correlated to recurrent genetic alterations
such as chromosomal translocations that can
allow the expression of oncogenic fusion proteins.
However, in the inbred FVB/N mouse model the
presence of an oncogenic fusion protein determines
only a pre-leukemic status implying the necessity of
additional hits for the establishment of a proper leukemic
status. Additional genomic alterations might
include aberrant epigenetic mechanisms of gene
silencing, namely DNA methylation, modifications
of histone tails, and mechanisms of epigenetic targeting
guided by non-coding RNAs. Therefore, by
Restriction Landmark Genome Scanning (RLGS), a
genome wide approach to study DNA methylation,
we aimed to identify specific DNA methylation patterns
in AMLs and their association with Polycomb/
Trythorax chromatin modifications. We analyzed
the aberrant DNA methylation patterns in human
leukemia cell lines and in human AML patients’ samples.
Either AML human blats or cell lines differed
for their FAB classification, block of differentiation,
and presence of oncogenic fusion proteins. In order
to identify the targets of aberrant DNA methylation
in AML cell lines and human blasts we compared
their DNA methylation RLGS profiles to a “master
DNA methylation profile”, obtained by the integration
of the CD34+ HSCs/HPCs and differentiated
CD34- blood cells RLGS profiles. We observed that:
i) the global levels of DNA methylation in AML
cell lines were higher than in AML human blasts
(25,9%±6,1% vs 6,44±2,23); ii) despite this difference,
the cross-comparisons between the different AML
cell lines and human samples evidenced common
patterns of DNA methylation. For instance, either in
AML cell lines or human samples chromosomes 5, 8,
15 and 20 resulted as less hit by aberrant DNA methylation
than chromosomes 4, 6, 10, 12, 13 and 18,
which were the most affected; iii) we also observed
specific and FAB grouping dependent DNA methylation
signatures. Recent data identify, among the
epigenetic mechanisms of gene regulation, the polycomb
(PcG)/trythorax (TrxG) proteins and complexes
as main regulators of stem cell identity and
subsequent lineage choices and suggest their pivotal
role in normal and leukemic hematopoiesis. It was
shown that polycomb protein Ezh2 controls DNA
methylation on target genes. However, a recent
report investigating the extent to which distinct
epigenetic mechanisms participate in carcinogenesis,
shows that repressive mechanisms of tumor suppressor
genes as PcG and DNA hypermethylation act
independently, affecting different genes. Thus, we
integrated our RLGS DNA methylation data with
the PcG/TrxG signatures of human ES. Our analysis
showed that DNA promoter regions of genes with
the highest recurrence of aberrant DNA methylation
in AMLs present a bivalent domain in ES and have
a higher sensitivity to become aberrantly methylated
than those presenting only the activating mark
(H3K4me3) or the repressive mark (H3K27me3) in
leukemia. We concluded that AMLs have shared
but also specific and FAB grouping dependent DNA

Giuseppe Zardo
methylation patterns, which are related to the PcG/
TrxG gene specific chromatin signature in human
ES. These observations have the potential to make
significant contributions for a rational use of epigenetic
drugs in AML, and for monitoring the minimal
residual disease in AML especially in absence of specific
molecular markers.
Among the targets of aberrant DNA methylation
identified in AML samples we focused our attention
on a new hematopoietic transcription factor, namely
NFI-A. NFI-A down-regulation is instrumental for
granulopoiesis whereas its expression boosts erythropoiesis.
In AML cell lines, tested for NFI-A and
miR-223 mRNA expression, we observed an inverse
correlation between their mRNA expression levels:
at high miR-223 levels corresponded low NFI-A
mRNA expression and viceversa. In particular, in
HL60 cells induced to granulocytic differentiation
by retinoic acid treatment (RA) and in CD34- blood
cells vs CD34+ HCSs/HPCs, at high levels of
mir-223 corresponded low levels of NFI-A mRNA
expression. Thus, we investigated the epigenetic
mechanisms of NFI-A gene regulation in normal
and leukemic hematopoiesis. Our results showed that
NFI-A down-regulation, which is required during
normal granulocytopoiesis, is driven by the resolution
of PcG/TrxG bimodal chromatin domains
targeting the regulatory region of NFI-A gene.
By ChIPs, we found that, as the HL60 RA induced
granulocytic differentiation preceded, NFI-A regulatory
region enriched for the chromatin repressive
mark H3K27me3 and for the polycomb proteins YY1
and Suz12. These features were also observed on
76
NFI-A regulatory region of CD34- cells and in AML
human blasts showing low NFI-A mRNA levels and
high miR-223 amounts. In accordance, HL60 cells
knocked down for Suz12 failed to resolve the bivalent
domains on NFI-A regulatory region, and rendered
HL60 cells less responding to the differentiating
stimulus of RA. As expected, in K562 cells induced to
erytroid differentiation by ARAc treatment, which
required high NFI-A expression, we found lower
levels of the repressive mark H3K27me3 and of the
polycomb protein YY1. This finding is relevant as
we demonstrate that bivalent domains predispose
not only for gene activation but also for inactivation
during cell lineage specification. Previous reports
associated transcriptional gene silencing (TGS) with
non-coding RNAs and elements of RNAi machinery,
our ChIP experiments supported these findings as
NFI-A regulatory region enriched for Dicer1 during
RA induced HL60 granulocytic differentiation,
while Ago1 increased and then gradually diminished.
In addition, the diminished recruitment of
YY1 and Dicer1 on NFI-A regulatory region after
the treatment of native chromatins with RNAseA
and H implied the involvement of RNA molecules in
NFI-A transcriptional gene silencing, and the ability
of Dicer1 to immunoprecipitate YY1 indicated the
close connection existing between PcG and RNAi
in TGS. In this contest, the inverse correlation
observed between NFI-A and miR-223 mRNAs levels
could not be considered as a simple association
of biological events but rather as two pathways intimately
connected and cooperating in the regulation
of NFI-A gene expression.

AREA 4
Molecular
recognition in
biomolecules

Cellular response to oxidative stress: involvement of protein
disulfide isomerases
Principal investigator: Fabio Altieri
Professor of Chemistry and Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910880/7; Fax: (+39) 06 4440062
fabio.altieri@uniroma1.it
Participants:
Margherita Eufemi, professor; Caterina Grillo, post-doc
fellow; Rossana Cocchiola, PhD student.
Report of activity
The aim of this research project is to elucidate the
involvement of PDI in the cellular response to oxidative
stress focusing our attention on ERp57. To
address this question we designed several cellular
models in which a study of the cell response to oxidative
stress can be carried out. Preliminary results
indicated that HeLa cells stably transfected with a
DNA sequence coding for GFP-tagged ERp57 show
a certain degree of protection against oxidative
stress induced by H 2 O 2 , as result of an increase of
vitality after 24h of treatment. At the same time, we
could observe a nuclear translocation of the tagged
protein, following the GFP fluorescence by microscopy.
Similar experiments have done on SHSY-5Y
neuronal cells transfected with DNA sequences coding
for GFP-tagged ERp57 and for GFP alone as
a control. MTT assay performed on wild type and
transfected cells showed that ERp57 overexpression
doesn’t influence cells vitality after 24 hours of
stress treatment with H 2 O 2 at different concentrations
(0-1.5mM). Cellular distribution of ERp57, in
either wild type or transfected cells, was investigated
by confocal microscope. Cells subjected to oxidative
stress using 100µM H 2 O 2 for six hours were analyzed
by immunofluorescence using antibodies to detect
ERp57 and Ref1, a previously identified ERp57
interactor. We observed a co-localization and nuclear
translocation of both proteins after stress. We
also tested 6-hydroxydopamine, who causes selective
damage in dopaminergic neurons and induce
ROS production. The results obtained, again, did not
show a correlation between cell vitality and ERp57
overexpression.
With the aim to investigate the role of ERp57 in
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thyroid cells we analyzed the effects of oxidative
stress on ARO, WRO and NPA cell lines, which correspond
to anaplastic, follicular and papillary thyroid
carcinomas respectively. Cells were stressed with
0.3mM H 2 O 2 and analyzed during the following 24
hours. We monitored survival and, by western blot
analysis, the cellular distribution of selected proteins
including ERp57, Ref1, STAT3 and Ku70/86.
The latter were selected as proteins involved in
stress response (redox regulation of gene expression
and DNA repair) and previously identified as
ERp57 interactors. We also evaluated the intracellular
levels of reduced and oxidized glutathione. In
a comparison analysis, ARO cells, which correspond
to the most aggressive carcinoma and better survive
to oxidative stress, showed the highest amount of
ERp57 and the lowest concentration glutathione. In
ARO cells can be also observed a nuclear translocation
of ERp57, Ref1 and Ku70/86 as a consequence
of the stress treatment. Probably, the presence of
these proteins in the nucleus could play a protective
role against oxidative stress. WRO cells, which are
characterized by low cytosolic levels of ERp57 and
Ref1, were more sensitive to stress treatment despite
of the highest glutathione concentration. STAT3
and pSTAT3 levels were very low in WRO cells and
their amount was further reduced when cells were
subjected to H 2 O 2 treatment. pSTAT3 levels were
quite high in NPA cells but H 2 O 2 treatment induces
pSTAT3 de-phosphorylation and this could explain
the growth arrest observed.
What we may conclude from these data is that
ERp57 overexpression will not turnout always in
an increase in cell viability. However, we generally
observe a cellular redistribution of ERp57 and
its interactors, thus confirming the involvement of
ERp57 in the oxidative stress response. For this reason,
we are going to apply in vivo crosslinking with
formaldehyde and immunoprecipitation steps on
wild type (HeLa, SHSY and thyroid cell lines) and
transfected cells to detect ERp57 interactors before

Fabio Altieri
and during oxidative stress. To avoid any effect due
to the large tag used (GFP) we planned to switch to
a different tag, like the flag peptide.
To isolate and indentify the proteins substrate of
ERp57 redox activity we designed a functional
mutant protein where the second cysteine residue in
each redox active site is replaced with a serine one.
Mutant can react with target proteins but undergoes
an incomplete redox reaction since the mixed
disulfide ERp57-substrate can’t be resolved by the
serine residue. Mutations have been generated by
PCR overlap-extension and confirmed by DNA
sequencing. The mutated ERp57 was expressed as
GST-tagged protein in E. coli and used in an interaction
assay with cellular extracts of HeLa cells. The
mixed disulfide complexes containing the mutated
ERp57 were purified on GSH-Sepharose, which
traps the GST tag ERp57 interactors were specifically
eluted and subjected to mass spectrometry
analysis. Preliminary results indicate actin as a redox
substrate of ERp57. To get better information we
planned to transfect cell lines with the mutated
ERp57 and analyze in vivo the redox interactors.
Our results demonstrate that ERp57, beside its
known functions in the ER, is involved in regulatory
processes at the nuclear level. We showed
that in M14 cells, where STAT3 is constitutively
activated, ERp57 binds with STAT3 to a subset of
well-characterized STAT3-associated promoters/
enhancers. The presence of ERp57 in these sites
appears to be essential for the regulatory activity of
STAT3 on transcription, since inhibition of ERp57
expression by RNA interference is accompanied by
the decrease of expression of several STAT3 dependent
genes. Moreover, the structure and/or composition
of DNA-bound STAT3 multiprotein complexes
may not be the same in the various promoters/
80
enhancers, even in the same cell type. ERp57 is a
redox-sensitive and stress-responsive protein, whose
expression is regulated by a variety of stimuli. One
of these is the oxidative stress, which is known to
lead also to the activation of STAT3 by an increased
phosphorylation. It is conceivable that the functional
interaction of ERp57 with STAT3 could reinforce
the response of STAT3 to the oxidative stress conditions.
It is interesting to note that another member
of the protein disulfide isomerases, the PDI, is
involved at the nuclear level in the transcriptional
regulation, in this case by influencing the conformation
of the estrogen receptor and by modulating its
affinity for the estrogen-responsive element.
The interaction between ERp57 and Ref1, both
expressed in E. coli and purified, was further analyzed
in vitro. Using a thiol probe able to modify
the electrophoretic mobility of proteins we confirmed
the reducing activity of ERp57 on oxidized
Ref1. Reduced form of ERp57 was generated by the
NADPH/NADPH-dependent thioredoxin reductase
system, as previously demonstrated. In addition,
ERp57 is able to remove the glutathione moiety on
Ref1 oxidized with a biotin-labeled glutathione. By
means of the ERp57 mutant above described, we
also observed the transferring of glutathione from
Ref1 to ERp57, thus confirming the redox activity of
ERp57 towards Ref1.
Publications
Chichiarelli S, Gaucci E, Ferraro A, Grillo C, Altieri
F, Cocchiola R, Arcangeli V, Turano C, Eufemi M.
Role of ERp57 in the signaling and transcriptional
activity of STAT3 in a melanoma cell line. Arch
Biochem Biophys. 2010, 494: 178-83. doi: 10.1016/j.
abb.2009.12.004.

Principal investigator: Maurizio Brunori
Professor of Chemistry and Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910544; Fax: (+39) 06 4440062
maurizio.brunori@uniroma1.it
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How proteins recognize their biochemical partners: ligand binding
and folding pathways of PDZ domains
Participants:
Carlo Travaglini-Allocatelli, professor; Stefano Gianni, CNR
researcher; Ylva Ivarsson, post-doc fellow; Rajanish Giri,
Angela Morrone, PhD students.
Collaborations:
University of Uppsala, Sweden (Prof. Per Jemth); University of
Cambridge, UK (Prof. Michele Vendruscolo).
Report of activity
The intricate nature of chemical reactions taking
place in living systems demands these reactions to be
highly specific. In general, such specificity is achieved
by displaying a geometrical precision in molecular
recognition with two interacting molecules displaying
complementary features; for example, a substrate
and an enzyme fit together like a key fits a lock. It is
therefore clear that the process of protein folding is
vital for any living organism.
Recent studies have shown that changes in the cellular
milieu or mutations may affect the stability of
proteins, resulting in a totally or partially denatured
state under physiological conditions; this event may
be involved in many life threatening diseases ranging
from Cystic Fibrosis to Alzheimer’s syndrome.
Moreover, residual structure in the denatured state,
even if minimal, can dictate nucleation and propagation
that is carried through the folding pathway.
This is exactly what we observed in the case
of nucleophosmin, a protein crucially involved in
human hematological malignancies: the characterization
of its folding pathway allowed us to show that
the protein folds through a compact denatured state
(Scaloni et al., 2009, 2010), a finding which is pertinent
to the properties of the mutated and thereby
pathological variants.
In the research project we employed extensive protein
engineering and exploited circularly permuted
proteins to probe the role of sequence connectivity,
rather than composition, in dictating folding pathways.
In particular:
(i) we have characterized the folding pathway of
an engineered variant based on the sequence of
a canonical PDZ domain, cpPDZ (Ivarsson et al.,
2009). To understand the role of sequence connectivity,
we carried out a complete Φ-value analysis
for an engineered circularly permuted PDZ domain,
which displays a peculiar symmetrical distribution
of secondary structure elements (i.e. ββαββαββ)
while maintaining the canonical tertiary interactions.
Reliable Φ values were obtained for both a
low-energy intermediate and the late rate-limiting
transition state, allowing a description of both early
and late events in folding. A comparison with the
wild-type PDZ2 reveals that while the structure
of the late transition state is robust and unaffected
by circular permutation, the folding intermediate is
stabilized by a different nucleus involving residues
located at the new N- and C-termini. This novel
result suggested that folding is driven by competing
nuclei whose stabilities may be selectively tuned by
circular permutation.
(ii) we have obtained for the first time the 3D
structure of a misfolded intermediate populated
in the folding pathway of a naturally occurring
PDZ domain from the D1p protease of the green
alga Scenedesemus obliquus (Gianni et al., 2010).
Incorrectly folded states transiently populated during
the protein folding process are potentially prone
to aggregation and have been implicated in a range
of devastating misfolding disorders that include
Alzheimer’s and Parkinson’s diseases. Despite their
medical importance, however, the structures of these
states and the mechanism of formation have largely
escaped detailed characterization because of their
short-lived nature. We have been able to obtain the
structures of all the major states involved in the
folding process of this PDZ domain, including an offpathway
“abnormal” intermediate. By using a com-

Maurizio Brunori
bination of transient kinetics, protein engineering
and computational techniques, we have shown that
although the productive folding of D1pPDZ is similar
to that observed in a canonical PDZ domain, the
off-pathway intermediate is less compact and characterized
by an “abnormal” docking of the N-terminal
β-hairpin, though its overall topology largely resembles
that of the native state. These results, that we
published in Nature SMB (Gianni et al., 2010), suggest
that misfolded intermediates may often display
an unexpected high degree of native-like structure,
mis-folding being dictated by the local mis-assembly
of a few structural elements (a swapped β-hairpin in
the case of D1pPDZ).
Publications
Gianni S, Brunori M, Jemth P, Oliveberg M, Zhang
M. Distinguishing between smooth and rough free
energy barriers in protein folding. Biochemistry. 2009,
48: 11825-30. doi: 10.1021/bi901585q.
Ivarsson Y, Travaglini-Allocatelli C, Brunori M,
Gianni S. Engineered symmetric connectivity of secondary
structure elements highlights malleability of
82
protein folding pathways. J Am Chem Soc. 2009, 131:
11727-33. doi: 10.1021/ja900438b.
Scaloni F, Gianni S, Federici L, Falini B, Brunori
M. Folding mechanism of the C-terminal domain of
nucleophosmin: residual structure in the denatured
state and its pathophysiological significance. FASEB
J. 2009, 8: 2360-5. doi: 10.1096/fj.08-128306.
Travaglini-Allocatelli C, Ivarsson Y, Jemth, P, Gianni
S. Folding and stability of globular proteins and implications
for function. Curr Opin Struct Biol. 2009,
19: 3-7. doi: 10.1016/j.sbi.2008.12.001.
Gianni S, Ivarsson Y, De Simone A, Travaglini-
Allocatelli C, Brunori M, Vendruscolo M. Structural
characterization of a misfolded intermediate populated
during the folding process of a PDZ domain. Nat Struct
Mol Biol. 2010, 12: 1431-7. doi:10.1038/nsmb.1956.
Scaloni F, Federici L, Brunori M, Gianni S.
Deciphering the folding transition state structure
and denatured state properties of nucleophosmin
C-terminal domain. Proc Natl Acad Sci USA. 2010,
107: 5447-52. doi: 10.1073/pnas.0910516107.

Plant innate immunity: cell wall-mediated signalling and
recognition in plant defense
Principal investigator: Felice Cervone
Professor of Plant Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912517; Fax: (+39) 06 49912446
felice.cervone@uniroma1.it
Participants:
Giulia De Lorenzo, Daniela Bellincampi, professors; Simone
Ferrari, Benedetta Mattei, researchers; Alexander Brutus,
Manuela Casasoli, Roberta Galletti, Vincenzo Lionetti,
Francesca Sicilia, Francesco Spinelli, post-doc fellows; Fedra
Francocci, Lorenzo Mariotti, Manuel Benedetti, Daniel
Savatin, PhD students; Daniela Pontiggia,Giovanni Salvi,
Isabel Santori, Lucia Tufano, technicians.
Report of activity
Plants are continually exposed to pathogens and,
in most cases, successfully defend themselves. They
are capable of detecting the presence of pathogens
and activating the signalling pathways that lead to
defence responses. This is part of the “plant innate
immunity” mechanism that is based on recognition
of the so-called pathogen-associated molecular patterns
(PAMPs) present in different microbes. The
cell wall plays an important role in plant innate
immunity since cell wall-derived oligolacturonides
(OGs) are formed during attempted attacks of pathogens
and trigger the typical responses elicited by
PAMPs. OGs may be regarded as DAMPs (Damage-
Associated Molecular Patterns).
The molecular basis of the plant innate immunity
elicited by DAMPs is the focus of this project. In
Arabidopsis the accumulation of OGs takes place
during the degradation of pectin by microbial polygalacturonases
(PGs) and is favoured when PGs interact
with specific plant inhibitors (polygalacturonaseinhibiting
proteins or PGIPs). OGs activate the
expression of defense genes through a signal transduction
pathway that has been partially elucidated
during this project. We have achieved the important
result of identifying the receptor of OGs.
Members of the Wall-Associated Kinase (WAK)
family have been thought for a long time to be candidate
receptors of OGs, due to their ability to bind
in vitro these oligosaccharides. Because lethality and
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AREA 4
redundancy have hampered the study of WAKs by
reverse genetics, we have adopted a chimeric receptor
approach to elucidate the role of Arabidopsis
WAK1. In a test-of-concept study, we first defined
the appropriate chimera design and demonstrated
that the Arabidopsis pattern recognition receptor
(PRR) EFR is amenable to the construction of functional
and resistance conferring chimeric receptors
carrying the ectodomain of another Arabidopsis
PRR, FLS2. After, we analyzed chimeras derived
from EFR and WAK1 and demonstrated that, upon
stimulation with OGs, the WAK1 ectodomain is
capable of activating the EFR kinase domain. On the
other hand, upon stimulation with the cognate ligand
elf18, the EFR ectodomain activates the WAK1
kinase, triggering defense responses that mirror
those normally activated by OGs and are effective
against fungal and bacterial pathogens. Finally, we
demonstrated that transgenic plants overexpressing
WAK1 are more resistant to the fungal pathogen
Botrytis cinerea.
Mitogen-activated protein kinases (MAPKs) are
important components of the plant innate immune
system. In Arabidopsis, the single kinases AtMPK3,
AtMPK6 and AtMPK4 are activated by pathogens
and elicitors; however, their specific contribution to
the regulation of defense responses is not completely
defined. We have shown that overexpression of the
protein phosphatase AP2C1 impedes the phosphorylation
of AtMPK3, AtMPK4 and AtMPK6 triggered
by OGs and by flg22, a well-known microbeassociated
molecular pattern (MAMP). The simultaneous
dephosphorylation of the three MAPKs
impairs the activation of responses to these elicitors
such as the expression of a subset of marker genes
and induction of resistance against Botrytis cinerea. In
contrast, production of reactive oxygen species and
the expression of a MAPK-independent gene are not
affected by AP2C1. Single mutant analysis showed
that only atmpk6, but not atmpk3 or mekk1, a triple
kinase upstream of AtMPK4 activation, has reduced

Felice Cervone
OG- and flg22-triggered gene activation. Our results
indicate that AtMPK3 acts as a negative regulator of
specific responses to DAMPs and MAMPs important
for plant immunity.
A plant protein (PGIP) localized in the cell wall
modulates the accumulation of OGs by interacting
with microbial PGs. PGIPs are leucine-rich repeat
(LRR) proteins and represent a useful model system
to study ligand-receptor coevolution dynamics at
the protein level. We have applied codon substitution
models to PGIP sequences of different eudicotyledonous
families to identify putative positively
selected sites and then compared these sites with
the propensity of protein surface residues to interact
with protein partners, based on desolvation energy
calculations. The two approaches remarkably correlated
in pinpointing several residues in the concave
face of the LRR domain. These residues were mutated
into alanine and their effect on the recognition of
several PGs was tested, leading to the identification
of unique hotspots for the PGIP-PG interaction.
We have obtained a low resolution structure of the
complex formed by PG from Fusarium phyllophylum
and PGIP from Phaseolus vulgaris as determined
by small-angle x-ray scattering (SAXS) analysis.
The inhibitor interacts with the enzyme through
its concave surface of the LRR domain. Both sides
of the enzyme active site cleft are recognized by the
84
inhibitor, accounting for the competitive mechanism
of inhibition observed. The structure is in agreement
with site-directed mutagenesis data and subsequent
assay of the inhibitory activity.
Publications
Casasoli M, Federici L, Spinelli F, Di Matteo A,
Vella N, Scaloni F, Fernandez-Recio J, Cervone F,
De Lorenzo G. Integration of evolutionary and desolvation
energy analysis identifies functional sites in
a plant immunity protein. Proc Natl Acad Sci USA.
2009, 106: 7666-71. doi: 10.1073/pnas.0812625106.
Lionetti V, Francocci F, Ferrari S, Volpi C,
Bellincampi D, Galletti R, D’Ovidio R, De Lorenzo
G, Cervone F. Engineering the cell wall by reducing
de-methyl-esterified homogalacturonan improves
saccharification of plant tissues for bioconversion.
Proc Natl Acad Sci USA. 2010, 107: 616-21. doi:
10.1073/pnas.0907549107.
Brutus A, Sicilia F, Macone A, Cervone F, De
Lorenzo G. A domain swap approach reveals a role of
the plant wall-associated kinase 1 (WAK1) as a receptor
of oligogalacturonides. Proc Natl Acad Sci USA.
2010, 107: 9453-7. doi: 10.1073/pnas.1000675107.

Spontaneous formation and evolution of informational nucleic
polymers
Principal investigator: Ernesto Di Mauro
Professor of Molecular Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912880; Fax: (+39) 06 4440812
ernesto.dimauro@uniroma1.it
Participants:
Giovanna Costanzo, researcher; Fabiana Ciciriello, post-doc
fellow; Samanta Pino, PhD student; Silvia Lopizzo, collaborator.
Collaborations:
Dipartimento di Agrobiologia e Agrochimica, Università della
Tuscia, Viterbo (Prof. Raffaele Saladino); Università Tor
Vergata, Rome (Dr. Claudia Crestini); INAF, Osservatorio
Astrofisico di Arcetri, Firenze (Prof. John R. Brucato); University
of Haifa, Israel (Prof. Edward N. Trifonov).
Report of activity
The general goal of our research has been the determination
of a plausible and experimentally verifiable
ensemble of chemical processes allowing the spontaneous
organization of informational polymers in abiotic
conditions. Once identified, this system could provide
invaluable information both on the origin of informational
polymers and on the persistence capacity of the
extant genetic systems in terrean and non-terrean environments.
In this frame of reference we have analyzed:
Synthesis of nucleic precursors
The reaction yielding nucleic bases from formamide
in the presence of catalysts but in the absence of
any biotic (cellular or enzymatic) effector has been
carefully analyzed. The analyses that have been performed
pertain to a series of sulphur and iron containing
minerals, of zirconium-based minerals and
of a large panel of boron-containing compounds. The
rationale for the first type of analyses is that iron and
sulphur minerals are involved in the hypothesized
initial chemical energy-transfer mechanisms and
that zirconia formed only in the presence of water.
The protective function of borates of various kinds
towards nucleosides has been recently identified.
Hence the interest of the exploration of their synthetic
capacity. The overall analysis has provided a
complete picture for the syntheses of nucleic bases
85
AREA 4
in the presence of the mineral compounds which are
major constituents of the solar system.
Quite interestingly the products obtained are precursors
of nucleic acids and related compounds, namely
purine, (1H)-pyrimidinone, isocytosine, adenine,
2-aminopurine, carbodiimide, urea, oxalic acid. The
stability properties of nucleic acids in the presence of
these minerals were analyzed. The results clearly show
that iron-based catalysts have strongly degradative
capacity and that the origin of informational polymers
with these catalysts only can be based upon the evolution
of specific protective mechanisms of the polymers
(“encapsulation” in lipids, interaction in the clays, etc.).
The results obtained with zirconium-based catalysts
have provided a similar set of results: components of
the nucleic acid bases are synthesized from formamide
in the presence of minerals which were present in the
early formation of planet. Earth Synthesis of nucleic
acid precursors has been obtained also in the presence
of a large panel of boron-containing minerals
(unpublished) and of powdered minerals of the meteorite
Murchison (unpublished). Also in these cases a
cornucopia of nucleic acid precursors was obtained,
along with a large number of carboxylic acids and of
formylated glycine and formylated alanine.
Taken together, these results show that:
- Formamide is the compound that may explain the
abiotic origin of nucleic acids, both for the production
in moderate-to-high yield of the necessary
building blocks and for the relative independence
upon a specific catalyst.
- Formamide may have created the conditions for the
activation (namely: phosphorylation) of abiotically
synthesized nucleosides and, as described in the following
section, of their polymerization.
- Stability assays have been developed and carried
out with all catalysts studied. The stability of RNA
in their presence has been measured, allowing to
define the conditions and the environments in which
the abiotically generated RNA polymers could have
been able to survive.

Ernesto Di Mauro
Polymerizations and stability
After the synthesis of nucleic bases and their possible
transformation in nucleosides, next step towards
complexity is the activation by phosphorylation of
abiotically synthesized nucleosides. This problem
has been solved by our group, as reported (1).
This observation has allowed us to answer the question:
may an ensemble of monomers give rise to
informational molecules endowed with pre-genetic
possibilities? We have previously determined the
physical-chemical conditions in which this may occur
(2,3). An extended set of analyses has determined
the properties of RNA molecules allowing the possibility
of evolution in abiotic environments and
the conditions in which this may occur. The results
are reported in (4). Starting from these findings, we
have focused on the determination of the resilience
properties of nucleic polymers in various prebiotic
scenarios, in both terrestrial and space-wise conditions.
Namely, experiments have been conducted to
answer the questions:
- How does the structure of nucleic acids affect their
resistance to the hydrolytic process?
- Do different environmental conditions affect
sequence evolution? Conditions were determined for
the polymerization of prebiotically formed monomers
into oligomers and increasingly complex informational
structures. Three levels of abiotic polymerization
were attained:
1) from 3’,5’ cyclic nucleotides: 3’,5’ cAMP and 3’,5’
cGMP. In the first case, short chains of RNA long
up to 8 nucleotides were obtained, which undergo
successive ligations, finally resulting in the production
of RNA chains up to 200 nucleotides long. In the
second case, serial growth of oligomers resulted in
the production of oligoG fragments up to 30 nucleotides
long.
2) Terminal ligation was observed for both polyA
oligonucleotides and sequence-complementary
polyC and polyG oligonucleotides. The mechanism
of reaction for these non-enzymatic ligations was
determined. These ligation reactions provide an
explanation to the production of RNA chains whose
size exceeds that expected from a synthesis/degradation
quickly reacing steady-state that would limit the
size of the observed polymers to molecules shorter
than a few tens of nucleotides.
3) Terminal growth of preformed oligonucleotides.
We have reported that 3’.5’ cyclic GMP can polymerize
on the 3’ tail of preformed oligoC polymers.
The net result is in the non-enzymatic formation of
mixed sequences molecules, pointing to the possible
86
mechanism for sequence-complexity evolution.
These results provide the proof-of-principle that
abiotic polymerizations are obtained spontaneously.
In addition, a set of conditions are established which
may provide the test systems for the analysis of the
evolution of genetic information in acellular, non.
biotic conditions. The results obtained are instrumental
to the formulation of a chemical theory of the
origin of informational polymers on this planet and
for the search of life in non-terrean environments.
References
1) G. Costanzo et al. Nucleoside phosphorylation
by phosphate minerals. J Biol Chem (2007) 282,
16729-35.
2) R. Saladino et al. Origin of informational polymers:
differential stability of 3’- and 5’-phosphoester
bonds in deoxy monomers and oligomers. J
Biol Chem (2005) 280, 35658-69.
3) R. Saladino et al. Origin of Informational
Polymers: Differential Stability of Phosphoester
Bonds in Ribo Monomers and Oligomers. J Biol
Chem (2006) 281, 5790-96.
4) F. Ciciriello, G. Costanzo, S. Pino, C. Crestini,
R. Saladino, E. Di Mauro. Molecular complexity
favors the evolution of ribopolymers. Biochemistry
(2008) 47, 2732-42.
Publications
Costanzo G, Pino S, Ciciriello F, Di Mauro E.
Generation of long RNA chains in water. J Biol Chem.
2009, 284: 33206-16. doi: 10.1074/jbc.M109.041905.
Saladino R, Crestini C, Ciciriello F, Pino S, Costanzo
G, Di Mauro E. From formamide to RNA: the roles
of formamide and water in the evolution of chemical
information. Res Microbiol. 2009, 160: 441-8. doi:
10.1016/j.resmic.2009.06.001.
Barks HL, Buckley R, Grieves GA, Di Mauro E, Hud
N, Orlando T. Guanine, Adenine, Hypoxanthine
production in UV-irradiated Formamide solutions:
relaxation of the requirements for prebiotic purine
nucleobase formation. Chembiochem. 2010, 11: 1240-
43. doi: 10.1002/cbic.201090037.
Saladino R, Neri V, Crestini C, Costanzo G, Graciotti
M, Di Mauro E. The role of the formamide/zirconia
system in the synthesis of nucleobases and biogenic
carboxylic acid derivatives. J Mol Evol. 2010, 71:
100-10. doi: 10.1007/s00239-010-9366-7.

Synthesis of pyridoxal phosphate in the vitamin B6 salvage
pathway and targeting of the cofactor to apo-enzymes
Principal investigator: Martino Luigi di Salvo
Researcher in Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49917684; Fax: (+39) 06 49917566
martino.disalvo@uniroma1.it
Participants:
Francesco Bossa, professor; Roberto Contestabile,
Sebastiana Angelaccio, researchers; Rita Florio, post-doc
fellow; Mirella Vivoli, PhD student.
Collaborations:
Department of Medicinal Chemistry, Virginia Commonwealth
University, USA (Prof. Martin Safo); Department of Medical
Laboratory Science and Biotechnology, National Cheng Kung
University, Taiwan (Prof. Tzu-Fan Fu).
Report of activity
In the 2009-2010 period, our research activity has
focused on the following topics:
The mechanism of PLP addition to apo-SHMT
during protein folding
Several mutant forms of E. coli SHMT, localized in
the so-called third hydrophobic cluster, have been
characterized for their enzymatic activity, spectroscopic
features and PLP binding capability. The
results show that the hydrophobic cluster is essential
for enzyme assembly and formation of the correct
quaternary structure, even though it is not localized
in the subunit interface. The cluster actually stabilizes
a bridging structural element, which is essential
for the overall enzyme structure; this element connects
the two protein domains, is involved in the
modeling of the active site and participates in the
quaternary assembly. Moreover, we have shown that
the final slow step of cofactor addition to SHMT
requires either free PLP or PLP-substrate (glycine or
L-serine) complex; other PLP-amino acid complexes
do not trigger the needed conformational change.
We also carried out a structural and functional study
on a conserved cation-π interaction (Arg-Tyr) in the
active site of SHMT. Both residues involved in this
cation-π interaction seem to have a catalytic role in
the transaldimination reaction, a common step in all
PLP-dependent catalyzed reactions.
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Studies on the putative PLP channelling
between PNPOx, PLK and SHMT
The channelling mechanism requires a direct interaction
between the donor enzyme and the acceptor
enzyme, through the formation of a protein complex.
In order to detect such interaction by means of
fluorescence anisotropy, we produced PNPOx and
PLK forms labelled with maleimide fluorophore.
All apo-B6 enzymes used in the experiments were
members of the Fold-Type I group. The experiments
demonstrated that PNPOx and PLK interact with
all apo-B6 enzymes tested. The calculated dissociation
constants ranged from 60 nM to 400 nM in the
case both donor and acceptor enzymes were from
E. coli. Dissociation constants with either human
PLK or PNPOx and E. coli apo-B6 enzymes ranged
from 1 to 20 µM. These results showed that the
transfer of PLP takes place more efficiently when
donor and acceptor enzymes from the same source
are employed, in agreement with the hypothesis of
the channeling mechanism. We have also developed
a new methodology, based on the employment of
a chemical quenched-flow instrument, that allows
rapid kinetic measurements of the PLP transfer from
either PNPOx or PLK to the apo-B6 enzymes.
Studies on PNPOx
We have produced and characterized the R229W
mutant forms of human PNPOx, known to cause
neonatal epileptic encephalopathy. This variant is
850-fold less efficient than the wild-type enzyme;
there is also a 50-fold reduction in the affinity for the
FMN cofactor. The crystal structure of the R229W
shows that the substitution of Arg229 has led to a
loss of hydrogen-bond interactions between FMN
and active site residues. Additionally, the mutation has
led to an alteration of the configuration of a β-strandloop-β-strand
structure at the active site, which is
important for substrate binding and orientation for
catalysis. These results provided a molecular basis for
the phenotype associated with the R229W mutation,

Martino Luigi di Salvo
allowing for a better understanding of the pathophysiological
consequences of PNPOx mutations.
Studies on PLK
For what PLK is concerned, we first focused on the
mechanism of action of the enzyme, and particularly
on the role of the active site residue Asp235, which
we showed to be involved in the deprotonation of the
substrate, an essential step for catalysis. During the
crystallographic studies on Asp235, we realized the
presence of a PLP molecule tightly bound to the active
site of the enzyme, with the formation of a high affinity
ternary complex between PLK, PLP and the substrate
MgATP. Then, PLP might work as a slow tight binding
inhibitor of PLK. Understanding the mechanism of
regulation of PLK is particularly important considering
the negative impact of several commonly used natural
products and drugs on the activity of the PLK (and,
consequently on the homeostasis of PLP in the cell).
For this reason, we have determined the inhibition
constants and mechanism of some of these compounds,
such as theophylline, gingkotoxin, lamotrigine, roscovitine.
The three-dimensional structure determination
of PLK in complex with the above inhibitors has been
determined and a manuscript is under submission.
88
Publications
Gandhi AK, Ghatge M, Musayev FN, Sease A,
Aboagye SO, di Salvo ML, Schirch V, Safo MK.
Kinetic and structural studies of the role of the active
site residue Asp235 of human pyridoxal kinase.
Biochem Biophys Res Commun. 2009, 381: 12-15. doi:
10.1016/j.bbrc.2009.01.170.
Musayev FN, Di Salvo ML, Saavedra MA,
Contestabile R, Ghatge MS, Haynes A, Schirch V,
Safo MK. Molecular basis of reduced pyridoxine
5’-phosphate oxidase catalytic activity in neonatal
epileptic encephalopathy disorder. J Biol Chem. 2009,
284: 30949-56. doi: 10.1074/jbc.M109.038372.
Vivoli M, Angelucci F, Ilari A, Morea V, Angelaccio
S, di Salvo ML, Contestabile R. Role of a conserved
active site cation-π interaction in Escherichia coli
serine hydroxymethyltransferase. Biochemistry. 2009,
48: 12034-46. doi: 10.1021/bi901568b.
Wilkins BJ, Marionni S, Young DD, Liu J, Wang Y,
Di Salvo ML, Deiters A, Cropp TA. Site-specific incorporation
of fluorotyrosines into proteins in Escherichia
coli by photochemical disguise. Biochemistry. 2010; 49:
1557-9. doi: 10.1021/bi100013s.

Integrating advanced functional materials and chromatographymass
spectrometry to examine protein-ligand interactions
Principal investigator: Francesco Gasparrini
Professor of Organic Chemistry
Dipartimento di Chimica e Tecnologie del Farmaco
Tel: (+39) 06 49912776; Fax: (+39) 06 49912780
francesco.gasparrini@uniroma1.it
Participants:
Bruno Botta, Claudio Villani, professors; Ilaria D’Acquarica,
Marco Pierini, researchers; Alessia Ciogli, Deborah
Subissati, Laura Nevola, post-doc fellows; Giovanna
Cancelliere, technician.
Report of activity
The covalent immobilization of carbohydrates
on solid supports has recently been developed by
employing copper-catalyzed azide-alkyne cycloaddition
(CuAAC), which is in agreement with the
demonstrated potential of this reaction in surface
modification. In “click saccharides”, prepared by
Liang using the click chemistry approach, two-step
azido-modified silica gels were coupled to sugar
alkynes in the presence of a Cu catalyst to yield the
above packing materials, in which the linked carbohydrates
retained their inherent configuration and
properties. In this context, we prepared novel sugarbased
silica gels by exploiting the CuAAC of two
sugar alkynes with new single-step azido-activated
silica gels. The fully characterised stationary phases
were generally used for hydrophilic interaction chromatography
(HILIC), with particular application in
the stereoselective separation of monosaccharides.
Dynamic HILIC (DHILIC) experiments were performed
to evaluate the influence of anomerization on
the chromatographic peak shapes. The potential of
such materials was shown in the separation of other
highly polar compounds, including amino acids and
flavonoids.
We recently reported a new hybrid organic/inorganic
chiral stationary phase (CSP) for HPLC synthesized
by the grafting-from (g-from) radical polymerization
of an enantiopure diacryloyl derivative of trans-
1,2-diaminocyclohexane (DACH) in the presence of
mesoporous, azo-activated silica micro-particles. This
was the first example of application of the g-from
approach to the synthesis of a CSP for HPLC applica-
89
AREA 4
tions. However, the thermal grafting process could
not be surface confined only, since concomitant solution
polymerization may lead to ungrafted polymer
chains. Thus, we moved to study another way
to induce polymerization of the above monomer,
and focused our attention on photo-induced polymerization,
which has been obtaining much attention
in recent years because of its numerous industrial
applications. Two hybrid polyacrylamide CSPs
were thus prepared by the surface-initiated photoinduced
radical polymerization of enantiopure N,N’diacryloyl
derivatives of (1R,2R)-DACH and (1R,2R)diphenylethylenediamine
(DPEDA), initiated by the
system consisting of trichloroacetyl groups on mesoporous
silica particles and dimanganese decacarbonyl
Mn 2 (CO) 10 under UV irradiation. Physico-chemical
properties and chromatographic performances of the
two phases were evaluated in comparison with those
exhibited by the homologue CSPs obtained by the
g-from thermal-induced process. The new photopolymerization
approach yielded higher grafting density
than the thermal-induced one, especially in the case of
the less reactive monomer (the diacryloyl derivative
of (1R,2R)-DPEDA), good chromatographic efficiency
and a broad application field under normal phase and
polar organic mode conditions.
With regard to our previous studies of biorecognition
events monitored by Fourier-Transform Ion
Cyclotron Resonance (FT-ICR) mass spectrometry,
we investigated the nature of the non-covalent interactions
between ethanolamine neurotransmitters
and some chiral resorc[4]arene receptors in the
gas phase, under conditions mimicking the extensive
desolvation complementary to the uptake of
neurotransmitters inside their biological receptors.
The kinetic experiments, supported by molecular
mechanics calculations and molecular dynamics
simulations, indicate that the relative stability of
the diastereomeric proton-bound adducts between
ethanolamine neurotransmitters and the chiral hosts

Francesco Gasparrini
depends on (i) the relative position and orientation
of the hydroxyl- and amino-functionalities on the
neurotransmitter side chain, (ii) the presence of the
methyl substituent on its nitrogen atom, and (iii) the
specific group of the receptor proton-bonded to the
amino group of the ethanolamine.
Publications
Moni L, Ciogli A, D’Acquarica I, Dondoni A,
Gasparrini F, Marra A. Synthesis of sugar-based
90
silica gels by copper-catalysed azide-alkyne cycloaddition
via a single-step azido-activated silica intermediate
and the use of the gels in hydrophilic
interaction chromatography. Chem Eur J. 2010, 16:
5712-22. doi: 10.1002/chem.201000106.
Ciogli A, D’Acquarica I, Gasparrini F, Molinaro C,
Rompietti R, Simone P, Villani C, Zappia G. Hybrid
polyacrylamide chiral stationary phases for HPLC prepared
by surface-initiated photopolymerization. J Sep
Sci. 2010, 33: 3022-32. doi: 10.1002/jssc.201000355.

91
AREA 4
Molecular and functional approaches to investigate the
physiopathological role of the chemokines and their receptors in
the central nervous system
Principal investigator: Cristina Limatola
Professor of Physiology
Dipartimento di Fisiologia e Farmacologia
Tel: (+39) 06 49690243; Fax: (+39) 06 49910851
cristina.limatola@uniroma1.it
Participants:
Sergio Fucile, Francesca Grassi, Eleonora Palma, Davide
Ragozzino, professors; Flavia Trettel, Laura Maggi,
researchers; Myriam Catalano, Raffaela Cipriani, Clotilde
Lauro, post-doc fellows; Giuseppina d’Alessandro, Maria
Rosito, Mariella Scianni, PhD students; Giuseppina Chece,
technician.
Collaborations:
Istituto Mario Negri, Milano (Dr. Maria Grazia De Simone, Dr.
Pia Villa); Dipartimento di Fisiologia e Farmacologia, Sapienza-
Università di Roma (Dr. Letizia Antonilli, Dr. Valentina
Brusadin); Karolinska Institute, Stockholm, Sweden (Dr. Bertil
Fredholm); Università di Camerino (Prof. Gloria Cristalli);
Università di Perugia (Prof. Fabio Franciolini).
Report of activity
Chemokines are proteins endogenously expressed
in the central nervous system, where they play different
roles during development modulating the
timing of neuronal migration in different brain area
and in the adulthood, regulating glia-neuron communication
and leukocyte entry across the blood
brain barrier under physiological and pathological
conditions. The present research project aims to
investigate the many functions of chemokines and
their receptors in the central nervous system, with
particular interest on the cross talk between glial
cells and neurons and the molecular mechanisms
adopted by these molecules to mediate neuroprotection,
neuromodulation and cell migration. In
particular, in the last year, we focused our attention
on 1) the neuroprotective activity of the chemokine
CX3CL1 in in vitro models of excitotoxicity and
in in vivo model of cerebral ischemia; 2) the neuromodulatory
activity of CX3CL1 on glutamatergic
neurotransmission; 3) the chemotactic activity
induced by CXCL12 on glioblastoma cells and the
involvement of K+ channels.
Neuroprotective and neuromodulatory
activities of CX3CL1: involvement of
adenosine receptors
CX3CL1 is constitutively expressed in the nervous
system, but levels in the brain can be modulated under
diverse pathological conditions. The presence and the
stimulation of the CX3CL1 receptor CX3CR1 has
been correlated with a reduced release of interleukin-
1-b (IL-1-b) and tumor necrosis factor-a (TNF-a) from
microglial cells and a lower rate of neuronal degeneration
in different experimental models of neuropathologies
such as experimental autoimmune encephalomyelitis,
1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine
hydrochloride striatal injection, lipopolysaccharide
administration, and superoxide dismutase (SOD1)
mutation. These data attest to a role of the pair
CX3CL1/CX3CR1 in reducing neuronal degeneration
on several types of brain injury. The neurotrophic
activity of exogenously administered CX3CL1 has
been related to the simultaneous production of protective
factors from microglial cells and in particular with
the activation of adenosine receptors (ARs, Lauro et
al., 2008). We showed earlier that A1R are probably
involved in the neurotrophic activity of CX3CL1 as it
was blocked by a relatively selective antagonist (Lauro
et al., 2008). In the last year we strongly supported
this conclusion in experiments where the protective
effect of CX3CL1 is absent in A1R-/- mice. In addition
we performed evidence that extracellularly produced
adenosine mainly derived from the activity of ectonucleotidases,
suggesting a predominant involvement of
glial cell-dependent nucleotide release in this process.
Furthermore, since it is proposed that microglia has
a prominent role in mediating the neuroprotective
effects of CX3CL1 and we have recently shown that
CX3CL1-stimulated microglia releases neuroprotective
substances that reduce Glu-induced cell death
(Lauro et al., 2008) we further investigated this aspect
showing that the selective ablation of microglia from
hippocampal cultures, using clodronate-encapsulating

Cristina Limatola
liposomes, has the effect to fully abolish the neuroprotective
activity of CX3CL1 toward excitotoxic death
of hippocampal neurons, confirming that these cells
represent the first target, which primes the functional
effects of CX3CL1 (Lauro et al., 2010).
CX3CL1 is known to modulate glutamatergic neurotransmission
with a mechanism that requires GluR1
AMPA-type Glu receptors in hippocampal neurons
(Ragozzino et al., 2006). We previously demonstrated
that adenosine receptor type 1 (A1R) are involved in
CX3CL1-induced depression of glutamatergic currents
in hippocampal neurons and transfected HEK cells
(Lauro et al., 2008). In the last year we characterized the
role of different ARs in CX3CL1-induced modulation of
hippocampal glutamatergic transmission, using cultured
neurons and brain slices, demonstrating that CX3CL1
action requires the activation of A1A, A2AR and A3R,
by locally released or tonic adenosine. Specifically,
CX3CL1 causes glutamatergic depression through A1R
and A3R and potentiation through A2AR stimulation.
Thus, CX3CL1 might affect glutamatergic synapses in
opposite way, depending on the prevailing functional
expression of AR subtypes (Piccinin et al., 2010).
Modulation of glioblastoma chemotaxis by
CXCL12: requirement of IK channel activity
Growing evidence shows a role for the chemokine
CXCL12 and its receptor CXCR4 in the migratory
behavior of glioblastoma cells: CXCL12 expression in
low-grade glioma has been associated with a shorter
time to tumor progression, whereas CXCR4 is widely
expressed in glioblastoma tissues, overexpressed in
angiogenic areas and in infiltrating glioblastoma
cells in vivo, and a phosphorylated/activated form
of CXCR4 is largely found in astrocytomas. It has
been further shown that the expression of CXCR4
in highly malignant gliomas is associated with poor
prognosis and that CXCL12 stimulates the proliferation
of glioma progenitor cells. A chemotactic role of
CXCL12/CXCR4 axis has been demonstrated in several
glioblastoma cell lines in vitro, whereas a role in
tumor progression has been proved in animal models,
thus confirming its potentially active role in glioblastoma
invasiveness in vivo. Despite the large body of
information available on CXCL12/CXCR4 expression
and promigratory activity in glioblastoma, the
underlying mechanisms are still poorly understood.
In the last year we investigate the signaling mechanisms
involved in the chemotactic action of CXCL12
on highly invasive human glioblastoma cells and
compare them with those induced by EGF, a growth
factor deeply involved in glioblastoma progression.
We found that both CXCL12 and EGF induce IKCa
92
channel activation but, only in case of CXCL12, IKCa
channels undergo a long-term functional upregulation
and their activity is required for chemotaxis. These
findings suggest the possibility that IKCa channel
mediates the in vivo promigratory capability of
CXCL12 in invasive subpopulations of glioblastoma
cells overexpressing CXCR4 (Sciaccaluga et al., 2010).
Publications
Maggi L, Trettel F, Scianni M, Bertollini C, Eusebi
F, Fredholm BB, Limatola C. LTP impairment by
fractalkine/CX3CL1 in mouse hippocampus is mediated
through the activity of adenosine receptor type
3 (A3R). J Neuroimmunol. 2009, 215: 36-42. doi:
10.1016/j.jneuroim.2009.07.016.
Roseti C, Palma E, Martinello K, Fucile S, Morace
R, Esposito V, Cantore G, Arcella A, Giangaspero
F, Aronica E, Mascia A, Di Gennaro G, Quarato PP,
Manfredi M, Cristalli G, Lambertucci C, Marucci G,
Volpini R, Limatola C, Eusebi F. Blockage of A2A
and A3 adenosine receptors decreases the desensitization
of human GABA(A) receptors microtransplanted
to Xenopus oocytes. Proc Natl Acad Sci USA.
2009, 106: 15927-31. doi: 10.1073/pnas.0907324106.
Lauro C, Cipriani R, Catalano M, Trettel F, Chece
G, Brusadin V, Antonilli L, Van Rooijen N, Eusebi
F, Fredholm BB, Limatola C. Adenosine A recep-
1
tors and microglial cells mediate CX3CL1-induced
protection of hippocampal neurons against Gluinduced
death. Neuropsychopharmacol. 2010, 35: 1550-
59. doi:10.1038/npp.2010.26.
Sciaccaluga M, Fioretti B, Catacuzzeno L, Pagani
F, Bertollini C, Rosito C, Catalano M, D’Alessandro
G, Santoro A, Cantore G, Ragozzino D, Castigli E,
Franciolini F, Limatola C. CXCL12-induced glioblastoma
cell migration requires intermediate-conductance
Ca2+ -activated K + channel activity. Am J
Physiol Cell Physiol. 2010, 299: C175-84 doi: 10.1152/
ajpcell.00344.2009.
Trettel F, Di Angelantonio S, Limatola C, Ransohoff
RR. Conference report: Chemokines and chemokine
receptors in the nervous system, Rome, 24/25
October, 2009; 2nd workshop. J Neuroimmunol. 2010,
224: 1-7. doi:10.1016/j.jneuroim.2010.05.001.
Piccinin S, Di Angelantonio S, Piccioni A, Volpini
R, Cristalli G, Fredholm BB, Limatola C, Eusebi F,
Ragozzino D. CX 3 CL1-induced modulation at CA1
synapses reveals multiple mechanisms of EPSC modulation
involving AR subtypes. J Neuroimmunol. 2010,
224: 85-92. doi:10.1016/j.jneuroim.2010.05.012.

Principal investigator: Paola Paggi
Professor of Physiology
Dipartimento di Biologia e biotecnologie “Charles Darwin”
Tel: (+39) 06 49912323; Fax: (+39) 06 49912351
paola.paggi@uniroma1.it
93
AREA 4
Neurone response to experimental injury and lack of dystrophin:
a molecular, functional and structural study in autonomic ganglia
in vivo and in vitro
Participants:
Maria Egle De Stefano, professor; Silvia Di Angelantonio,
researcher; Loredana Lombardi, post-doc fellow; Valentina
Annese, Valerio Licursi, PhD students.
Collaborations:
Istituto Superiore di Sanità, Laboratorio di Biologia Cellulare,
Roma (Dr. Tamara Petrucci); Centro di Farmacologia Cellulare
e Molecolare, CNR, Dipartimento di Farmacologia Medica,
Università di Milano (Dr. Cecilia Gotti); Dipartimento di
Biologia e Biotecnologie “Charles Darwin”, Sapienza-Università
di Roma (Prof. Irene Bozzoni, Prof. Ernesto Di Mauro, Prof.
Andrea Mele, Prof. Rodolfo Negri, Prof. Alberto Oliverio);
Dipartimento di Scienze Biologiche, Università di Napoli Federico
II (Prof. Carla Perrone Capano); Erasmus Medical Center,
Department of Neuroscience, Rotterdam, The Netherlands (Dr.
Casper Hoogenraad); Paracelsus University of Salzsburg,
Austria (Dr. Falk Schroedl).
Report of activity
Aim of this project is the characterisation, in rodent
superior cervical ganglion (SCG), of the molecular
mechanisms and structural changes involved in the
establishment, maintenance and plasticity of the reciprocal
interactions between pre- and post-ganglionic
neurones and between post-ganglionic neurones and
their target organs. Damage of the ganglionic connectivities,
consequent to pre- and post-ganglionic nerve
crush, or to the lack of dystrophin, are used as a tool
to perform our investigation. Moreover, on the basis
of our previous data (De Stefano et al., 2007, Mol Cell
Neurosci 36: 174-184), showing the involvement of the
plasminogen activators/plasmin enzymatic system
in the synaptic plasticity induced by postganglionic
nerve crush in the rat SCG, we collaborated to a work
by Prof. A. Oliverio and Prof. A. Mele, investigating
whether this enzymatic complex is also involved in
the ventral striatal plasticity associated to the spatial
memory in mice.
Specifically, we focused on: (i) the modulation of
voltage-gated K+ channel expression after pre- and
post-ganglionic neuron axotomy in the rat SCG; (ii)
the role of dystrophin on nicotine and acetylcholineevoked
currents through the nicotinic acetylcholine
receptors (nAChRs) present in mouse SCG neurons
in vitro; (iii) levels of tissue plasminogen activator
(tPA) in the ventral striatum during consolidation of
spatial memory.
(i) Voltage-gated K + channels (Kv) are constituted
by four alfa subunits and a cytoplasmic regulatory
subunit, important for channel dynamics. As Kv are
responsible for K + inward current (I A ), a reduction of
which has been demonstrated after axotomy of rat
SCG neurons (Sacchi et al., 2005, Neuroscience 135:
31-45; Sacchi et al., 2006, J Neurophysiol 95: 2832-44)
we investigated whether I A reduction was associated
to changes in Kv expression. Therefore, we analyzed
mRNA and protein levels of members of the Kv4
and Kv1 families and of their respective regulatory
proteins, β2 and KChIP, known to be expressed in
rat SCG. Ganglion neurons of adult male rats
were either axotomized or denervated by crushing
the post- and pre-ganglionic nerves, respectively.
Animals were killed 8h (hour), 1d (day), 3d and 6d
after surgery for mRNA level evaluation and up to
20 d for protein level evaluation. After axotomy, all
mRNA examined decrease significantly, compared to
control, by 1d and, except for KChIP, remain lower
in the following dates. Differently from its mRNA,
Kv1.4 protein levels increase significantly by 1d and
remain higher than control up to 6d, while those
of its β2 subunit decrease. The axotomy-induced
decrease in mRNA levels is in accord with the reduction
in gene expression of proteins not essential for
axonal regeneration. Since Kv1 channels are mainly
presynaptic and axonal, the increase in Kv1.4 protein
levels suggests its accumulation in the perikaryon
induced by reduced export of this protein consequent
to distal axon degeneration. As the b2 subunit is
required for channel insertion into membrane, its

Paola Paggi
reduction is also consistent with the degeneration
of axonal plasma membrane. Denervation does not
affect Kv1.4 mRNA levels, while those of Kv4.2,
which is mainly somatodendritic, are reduced, as
expected after functionally silencing the ganglionic
neurons induced by the removal of pre-synaptic
boutons (De Stefano et al., 2010, 7th FENS forum of
European Neuroscience. Abstract 013.10; manuscript
in preparation).
(ii) In the sympathetic superior cervical ganglion
(SCG), nicotinic acetylcholine receptors (nAChRs)
mediate fast synaptic transmission. We previously
demonstrated that in SCG neurons of mdx mice, an
animal model for Duchenne muscular dystrophy,
lack of dystrophin causes a decrease, compared to
the wild-type, in post-synaptic nAChRs containing
the α3 subunit associated with β2 and/or β4 (α3β2/
β4-nAChRs), but not of those containing the α7
subunit (α7-nAChRs). We showed, by whole cell
patch-clamp recordings from cultured SCG neurons,
that both nicotine and acetylcholine-evoked currents
through α3β2/β4-nAChRs are significantly reduced
in mdx mice compared to wild-type, while those
through α7-nAChR are unaffected. This reduction
associates with that of the protein levels of α3, β2
and β4 subunits. We suggest that, in mdx mouse SCG
neurons, lack of dystrophin, by specifically affecting
membrane stabilization of α3β2/β4-nAChRs, could
determine an increase in receptor internalization and
degradation, with consequent reduction in the fast
intraganglionic cholinergic transmission.
(iii) To determine whether tissue plasminogen
activator (tPA) activity in the ventral striatum is
required for spatial memory consolidation, we examined
the long-term object place association memory
in mice treated in the ventral striatum, immediately
after training, with the tPA inhibitor PAI-1.
94
Administration of PAI-1 to the ventral striatum
significantly and dose-dependently decreased spatial
memory recall 24 h (hour) after training, compared
with vehicle treated mice, as measured by relative
exploration of the displaced object. In a further
experiment, we assessed the effects of immediate
posttraining injections of PAI-1 into the ventral striatum
on spatial memory performance in the Morris
water maze task. Mice were trained in the water
maze, administered immediately after the final training
session with 0.5 µg PAI-1 per dose, and tested in
a probe trial 24 h later. Control mice spent significantly
more time in the correct quadrant compared
with the other three quadrants, thus demonstrating
correct memory for the platform location. PAI-1treated
mice failed to show a preference for any
quadrant during the probe trial. The specificity of
the effect observed in the spatial version of the water
maze after intra- ventral striatum administrations of
PAI-1 was evaluated in mice trained in the cue version
of the task and treated immediately posttraining
with 0.5 µg PAI-1 per side. In this case, PAI-1 did
not affect performance of the mice, with both groups
spending more time in the correct quadrant than in
the other three. These data demonstrate that tPA
activity is required immediately after spatial learning
for long-term, but not short-term, memory.
Publications
Ferretti V, Roullet P, Sargolini F, Rinaldi A, Perri
V, Del Fabbro M, Costantini VJA, Annese V, Scesa
G, De Stefano ME, Oliverio A, Mele A. Ventral
striatal plasticity and spatial memory. Proc Natl
Acad Sci USA. 2010, 107: 7945-50. doi: 10.1073/
pnas.0911757107.

Principal investigator: Maria Savino
Professor of Biophysical Chemistry
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912238; Fax: (+39) 06 4440812
maria.savino@uniroma1.it
95
AREA 4
Structural and superstructural features of human telomeric chromatin
Participants:
Stefano Cacchione, researcher; Sabrina Pisano, Alessandra
Galati, post-doc fellows; Emanuela Micheli, PhD student.
Collaborations:
Dipartimento di Chimica, Sapienza-Università di Roma (Prof.
Armandodoriano Bianco, Prof. Pasquale De Santis, Prof.
Giancarlo Ortaggi, Dr. Marco Franceschin, Dr. Anita Scipioni);
Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli
Federico II (Prof. Luciano Mayol, Dr. Michela Varra); Medical
Research Council, Laboratory of Molecular Biology, Cambridge,
UK (Dr. Daniela Rhodes, Dr. Linda Chapman); Laboratory of
Molecular and Cellular Biology, ENS, Lyon (Prof. Eric Gilson).
Report of Activity
The recent few years in Molecular Biology have been
characterized by the development of very powerful
tools, which makes possible the study of whole
genomes at molecular level. Nucleosome positioning,
along genomes, can be considered as the first necessary
step to clarify chromosomes organization. Recently,
a strongly increasing interest is developing in the
building of models capable of predicting nucleosomes
positioning along different genomes in vitro and then
to explore their correlations with the corresponding in
vivo systems (Segal, Trends in Genetics 2009).
These models are, generally, based on the identification
of some recurrent nucleotide sequence features,
obtained from the statistical analysis of very large
pools of nucleosomal DNA sequences, experimentally
obtained. The sequence dependent statistical
model of nucleosome positioning appears rather
efficient in predicting in vitro nucleosome positioning
and more importantly, also the correlation with
corresponding in vivo systems seems possible.
Very recently, a different outcome was reported
showing that the correlation between in vitro and in
vivo nucleosome postioning could be flawed and that
in vivo nucleosomes positioning in coding regions
arises primarily from statistical positioning from a
barrier, a poorly defined parameter at molecular level,
that should depend on chromatin assembly factors as
well as by RNA polymerase II (Zhang, Nature 2009).
This unsatisfactory situation enhances the necessity
to build satisfactory physical models of nucleosome
positioning, on the basis of sequence dependent physical
features of DNA such as curvature, flexibility and
local hydration. It is reasonable to consider that chromatin
remodeling complexes or polymerase could
only modulate nucleosome basic positioning, due to
DNA wrapping around histone octamer according to
sequence dependent DNA physical features. In fact
histone octamer concentration is dramatically higher
with respect to that of other chromatin proteins.
We have developed an original physical model to connect
sequence dependent DNA physical features to
nucleosome positioning. Based on statistical mechanical
approach, the model allows the calculation of the
sequence dependent canonical ensemble free energy
involved in nucleosome formation. The theoretical free
energy was evaluated for one hundred single nucleosome
tracts and successfully compared with those
obtained, experimentally, with nucleosome competitive
reconstitution. These results, obtained for single
nucleosomes, could in principle allow the calculation
of the intrinsic affinity of nucleosomes along DNA
sequences virtually opening the possibility of predicting
nucleosome positioning along genomes on physical
basis. The theoretical nucleosome distribution was
compared and validated with that of yeast and human
genomes experimentally determined (Fig. 1).
Theoretical nucleosome positioning on physical basis
represents the key to clarify, at molecular level, the
structural and superstructural features of chromatin
organization at telomeres which is important to clarify
the basic mechanisms of replication, transcription
and DNA repair.
Our research is aimed to explore the relationship
between telomeric nucleosomes and telomeric proteins.
In most eukaryotic genomes, telomeric nucleosomes
are characterized by the lowest thermodynamic sta-

Maria Savino
bility and by the highest mobility, as we, first, have
shown (Filesi, FEBS 1997). Recently, using an original
approach based on single molecule visualization, by
AFM, we found that the telomeric protein TRF1 is able
to significantly increase the mobility of telomeric nucleosomes,
connecting telomeric proteins interactions to
the dynamic features of telomeric chromatin.
An unresolved problem in telomeric chromatin organization
is the folding of the nucleosome array into the
fiber. The fiber modulates DNA accessibility and thus
is important both in interacting with telomeric proteins
and telomeric RNA, very recently discovered.
We approached the problem by integrating the experimental
findings by electron microscopy visualization
of reconstituted nucleosome arrays with geometrical,
conformational and topological restraints, under the
hypothesis of minimum distortion of the nucleosome
and linker DNA structures. The torsional energy of
the chromatin fiber appears crucial in determining
the kind of nucleosome packing for short nucleosome
repeat length as in telomeres. The presence of
high torsional energy and topological extra-energy
could represent an important basic mechanism for
the formation of telomeric G-quadruplex structure.
Telomeric DNA G-quadruplex represents one of the
more exciting findings in telomeres research in the
last few years. In fact, the possibility to induce and
stabilize this structure, by small organic molecules in
mammalian cells, could represent a powerful tool to
control pharmacologically cellular processes, ruled by
telomerase, such as cancer and aging.
We have developed two different approaches aimed to
inhibit telomerase in mammalian cells, based on the
stabilization of G-quadruplex structures either at 3’
telomeric DNA overhang or at the proximal promoter
of gene coding for the enzymatic moiety of telomerase,
hTERT. A new class of hydrosoluble perylene
derivatives have been developed in collaboration with
a group of organic chemists. We found that these
molecules are able to selectively induce G-quadruplex
structure and inhibit telomerase in cell free systems,
both in the case of telomeric G-quadruplex than in the
96
case of G-quadruplex in hTERT core promoter (Fig.
2). Their activity in vivo systems are actually under
investigation in our research group.
Publications
Micheli E, D’Ambrosio D, Franceschin M, Savino M.
Water soluble cationic perylene derivatives as possible
telomerase inhibitors: the search for selective
G-quadruplex targeting. Mini Rev Med Chem. 2009,
9: 1622-32. doi:10.2174/138955709791012274.
Micheli E, Lombardo CM, D’Ambrosio D, Franceschin
M, Neidle S, Savino M. Selective G-quadruplex ligands:
the significant role of side chain charge density in a
series of perylene derivatives. Bioorg Med Chem Lett.
2009, 19: 3903-8. doi: 10.1016/j.bmcl.2009.03.106.
Scipioni A, Morosetti S, De Santis P. A statistical
thermodynamic approach for predicting the sequencedependent
nucleosome positioning along genomes.
Biopolymers. 2009, 91: 1143-53. doi: 10.1002/bip.21276.
De Santis P, Morosetti S, Scipioni A. Prediction of
nucleosome positioning in genomes: limits and perspectives
of physical and bioinformatic approaches. J
Biomol Struct Dyn. 2010, 27: 747-64.doi:
Pisano S, Leoni D, Galati A, Rhodes D, Savino M,
Cacchione S. The human telomeric protein hTRF1
induces telomere-specific nucleosome mobility. Nucleic
Acids Res. 2010, 38: 2247-55. doi: 10.1093/nar/gkp1228.
Scipioni A, Turchetti G, Morosetti S, De Santis P.
Geometrical, conformational and topological restraints
in regular nucleosome compaction in chromatin. Biophys
Chem. 2010, 148: 56-67. doi: 10.1016/j.bpc.2010.02.010.
Micheli E, Martufi M, Cacchione S, De Santis P,
Savino M. Self-organization of G-quadruplex structures
in the hTERT core promoter stabilized by polyaminic
side chain perylene derivatives. Biophys Chem.
2010, 153: 43-53. doi: 10.1016/j.bpc.2010.10.003.

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Fig. 1 - Comparison between the profiles of the experimental nucleosome distribution (dashed line) and the theoretical free energy difference
(full line) for a 6000 bp region of chromosome III of S. cerevisiae genome.
Fig. 2 - Representative model of POL-3 (a hydrosoluble perylene derivative)
with human telomeric G-quadruplex.

AREA 5
Cellular and
molecular
immunology

Interplay amongst chronic immune activation, apoptosis,
cross-presentation, immune-regulation, and autoimmunity
Principal investigator: Vincenzo Barnaba
Professor of Internal Medicine
Dipartimento di Medicina Interna e Specialità Mediche
Tel: (+39) 06 4453994; Fax: (+39) 06 49383333
vincenzo.barnaba@uniroma1.it
Participants:
Marino Paroli, professor; Daniele Accapezzato, researcher;
Vittorio Francavilla, Pisana Moroni, Antonella Propato,
post-doc fellows; Melissa Videtta, Tiziana Donato, Debora
Franceschini, Francesca Meloni, Laura Altieri, PhD students.
Report of activity
In the first year of our project, we addressed the role of
regulatory T (Treg) cells in the establishment of a state
of chronic low-level inflammation that is instrumental
to limit immunopathology, to limit the pathogen spread,
and ultimately to ensure a long-lasting survival of the
host. Treg cells expressing the transcription factor
forkhead box P (Foxp)3 develop either in the thymus
(natural) or in the periphery from conventional CD4 +
T cells (induced). As a result of the expression of the
Il-2 gene-inhibitory Foxp3 transcription factor, Treg
cells do not produce IL-2 and are unable to respond to
antigens (anergy). However, Foxp3 activity maintains
high levels of IL-2 receptors (CD25 hi ) on Treg cells,
hence compensating the incapacity of producing IL-2.
Indisputably, CD25 hi Treg cells promptly proliferate
both in vitro and in vivo in response to relevant antigens
in the presence of paracrine IL-2, which is mainly
produced by responder (effector) T lymphocytes, but it
is dominantly absorbed by Treg cells expressing higher
CD25 levels than responder T cells. This appears to
represent a key suppression mechanism.
Treg cells are induced and proliferate in response
to hepatitis C virus (HCV) and seem to modulate
liver inflammation in the course of chronic infection.
Therefore, the model of HCV infection supports the
idea that Treg cells participate in the establishment
of a fine equilibrium between immunopathology and
immune protection, ultimately resulting in the longlasting
survival of the host during chronic infections.
It is unclear how Treg cells control unwarranted
inflammation without completely suppressing the
protective immune responses.
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We demonstrated that PD-1 is over-expressed on
Foxp3 + Treg cells and limits Treg cell proliferation
and function during chronic HCV infection. We first
observed substantial Treg cell accumulation in the
inflamed liver of HCV patients, and that some of
this accumulation was caused by increased proliferation
(as calculated by Ki67 expression) occurring
selectively at the site of infection in vivo. Moreover,
accumulation and proliferation of Treg cells were
synchronized with those of T effector (Teff) cells
at the site of infection, but at a level significantly
and constantly lower than the parallel expansion of
intra-hepatic (IH)-Teff lymphocytes. Nonetheless,
IH-Treg cells were intrinsically “professional”,
because they exerted efficient suppression function
that was directly related with the level of Foxp3
expression, the decline of IH-Teff cells (as well as of
Ki67 + cells), and the clinical consequences of downregulation
of anti-viral (i.e., increased viral load), and
immunopathological responses (i.e., decreased liver
inflammation) in vivo. Because we were able to demonstrate
a notable production of IL-2 by Teff cells
derived from inflamed liver (known to be the most
important source of HCV antigens), the capacity of
Treg cells to proliferate in vivo is likely due to the
sustained amounts of viral antigens and paracrine
IL-2 at the site of infection. Our antigen-specific
Treg cell proliferation assay in vitro supported this
view because the combination of viral antigens and
IL-2 efficiently rescued Treg cells from anergy in the
majority of HCV patients tested, indicating a key role
of HCV in the generation of functional Treg cells.
To understand why the expansion and proliferation
of IH-Treg cells were (although parallel) constantly
overwhelmed by those of IH-Teff cells in all HCV
patients tested, we investigated the possibility that
Treg cells were more susceptible than the effectors to
the programmed death (PD-1) dependent exhaustion
mechanism in the inflamed tissue. The lower Treg
cell expansion correlated with a significantly higher
expression of PD-1 on Treg than on Teff cells infil-

Vincenzo Barnaba
trating the inflamed liver. Further in vivo observations
revealed that the PD-1/Foxp3 cell ratio correlated
inversely with the viral load, and directly with the
histological activity index (HAI) score, suggesting
that PD-1 expression on Treg cells contraregulates
the suppression of both the anti-viral and the
immunopathological responses. Data showed that the
PD-1/PD-L1 blockade significantly increased both
the expansion and the relative suppression function of
Treg cells when the latter were stimulated with either
specific or non-specific TCR ligands plus paracrine
IL-2 in the presence of a blocking mAb to PD-ligand1
(PD-L1) in vitro, thus providing evidence for the
hypothesis stating that the inhibition of suppression
is PD-1-dependent. The potential role of PD-1dependent
inhibitory signals in controlling Treg cell
proliferation is emphasized by the observation that
PD-1/PD-L1 blockade increased the expansion of
Ki67 + cells in the Treg cells upon TCR-dependent
stimulation plus IL-2 in the presence of anti-PD-L1
in vitro, a finding consistent with the inverse correlation
observed between Foxp3 + and Ki67 + Treg cells in
vivo. Notably, the PD-1-mediated inhibition of Treg
cell function is dependent on its capacity to decrease
expansion and, ultimately, the number of Treg cells
rather than the intrinsic Treg cell suppression capacity.
This finding is consistent with both the evidence
that Foxp3 expression dictates and fixes the suppression
function of the single Treg cells, irrespective of
the presence of inhibitory signals, and the hypothesis
that the decreased expansion of Treg cells by PD-1
can, however, result in a decreased suppression function
in a given inflammatory context. By contrast, the
observation that no correlation was evident when we
considered the peripheral Treg cells further demonstrates
that peripheral immune settings do not necessarily
reflect the local immune responses present at a
given inflammatory site.
High CD25 expression by Treg cells drives a positive
feedback loop, as the dominant IL-2 capture
increases STAT-5 phosphorylation (pSTAT-5) that
in turn drives Treg cell proliferation and function.
We demonstrated that pSTAT-5 was spontaneously
overexpressed in IH-Treg cells (significantly more
than in IH-Teff cells), and paralleled the expression
of both CD25 hi and PD-1 in vivo. In addition, kinetic
studies in patients with severe hepatitis reactivation,
showed that pSTAT-5 and PD-1 expression in Treg
cells were up-regulated in an alternate fashion in
vivo: pSTAT-5 upregulation followed the peak and
anticipated the dramatic suppression of disease activity,
whereas PD-1 upregulation preceded the return
of both pSTAT-5 and disease activity to steady lev-
102
els. Finally, PD-1 blockade increased both proliferation
and pSTAT-5 expression by fresh IH-Treg cells
significantly more than by IH-Teff cells ex vivo.
Taken together, these data provide evidence for
the hypothesis that PD-1, upon the contact with its
own ligands, inhibits pSTAT-5 via the activation of
Src homology 2–containing tyrosine phosphatases
(SHPs) (Fig. 1). As a consequence, responder T cells
can escape from excessive expansion of Treg cells
and render them available for responding to possible
novel waves of infection (likely leading to recovery).
This negative feedback loop assumes a different
significance during chronic infections, such as HCV.
The incapacity to clear HCV by the immune system
maintains a vicious spiral, whereby responder T cells
are chronically stimulated to produce IL-2 that will
be dominantly adsorbed by CD25 hi Treg cells that in
turn will continuously suppress the effector responses.
The PD-1 upregulation limits the excessive
expansion of Treg cells by controlling pSTAT-5 and
fine-tunes the Treg function in order to minimize
the immunopathology without completely switch off
those intended to limit excessive viral spread (Fig.
1). This may represent a critical contra-suppression
mechanism that has evolved to control that Treg
cells have a limited suppression. Homeostatic balance
participates in establishing a status of chronic
low-level liver inflammation that is in turn instrumental
to ensure a long-lasting survival of the host.
Publications
Franceschini D, Paroli M, Francavilla V, Videtta M,
Morrone S, Labbadia G, Cerino A, Mondelli MU, Barnaba
V. PD-L1 negatively regulates CD4+CD25+Foxp3+
Tregs by limiting STAT-5 phosphorylation in patients
chronically infected with HCV. J Clin Invest. 2009, 119:
551-64. doi: 10.1172/JCI36604.
Curtale G, Citarella F, Carissimi C, Goldoni M, Carucci
N, Fulci V, Franceschini D, Meloni F, Barnaba V,
Macino G. An emerging player in the adaptive immune
response: microRNA-146a is a modulator of IL-2
expression and AICD in T lymphocytes. Blood. 2010,
115: 265-73. doi: 10.1182/blood-2009-06-225987.
Fulci V, Scappucci G, Sebastiani GD, Giannitti C,
Franceschini D, Meloni F, Colombo T, Citarella F,
Barnaba V, Galeazzi M, Macino G. miR-223 is overexpressed
in Tlymphocytes of patients affected by
rheumatoid arthritis. Hum Immunol. 2010; 71: 206-
11. doi: 10.1016/j.humimm.2009.11.008.

Beauvillain C, Meloni F, Sirard JC, Blanchard S,
Jarry U, Scotet M, Magistrelli G, Delneste Y,
Barnaba V, Jeannin P. The scavenger receptors
SRA-1 and SREC-I cooperate with TLR2 in the
recognition of the hepatitis C virus non-structural
protein 3 by dendritic cells. J Hepatol. 2010, 52: 644-
51 doi: 10.1016/j.jhep.2009.11.031.
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Barnaba V. Hepatitis C virus infection: a “liaison a
trois” amongst the virus, the host, and chronic lowlevel
inflammation for human survival. J Hepatol.
2010, 53: 752-61. doi:10.1016/j.jhep.2010.06.003.
Fig. 1 - PD-1 controls Treg cells in HCV infection. (a) Responder (CD25 low Foxp3 – ) T effector memory (T EM ) cells proliferate in
response to HCV antigens, and produce IL-2, which through the IL-2R (CD25) signaling, induces pSTAT-5. This leads to the development
of the genetic program dictating their effector phenotype. In parallel, the same phenomena occur for the (CD25 hi Foxp3 + ) Treg cells that do
not proliferate to viral antigens alone because of the expression of the Il-2 gene-inhibitory Foxp3. Their proliferation is dependent on the
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
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,
the death receptors intrinsically deliver negative signals to activated T cells (including Fas, CTLA-4, and PD-1) in order to terminate the T
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.
This mechanism results in limiting both T EM cell responses and excessive Treg cell function. Under conditions resulting in the HCV resolution,
this loop is self-limited because of the disappearance of the viral antigenic stimuli. During a chronic HCV infection, in which responder T
cells have been unable to clear HCV, the negative loop is maintained by the persisting HCV antigens that chronically stimulate IL-2 producing
responder T cells. The chronic PD-1 expression on both T EM and Treg cells modulate the potential excessive pSTAT-5-dependent cell
proliferation. The resulting contra-regulation of Treg cells will have an important role to limit excessive suppression of immune responses
allowing the control of the viral spread, at the cost of the maintenance of the chronic low-level liver immunopathology. This mechanism
establishes a long-lasting survival of the host.

Potential role of miRNAS in IgM-mediated signal transduction in
normal and neoplastic B cells
Principal investigator: Roberto Foà
Professor of Hematology
Dipartimento di Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 85795753; Fax: (+39) 06 85795792
rfoa@bce.uniroma1.it
Participants:
Anna Guarini, Franca Citarella, Sabina Chiaretti,
researchers; Simona Tavolaro, post-doc fellow; Marilisa
Marinelli, Monica Messina, Nadia Peragine, PhD students.
Report of activity
Background
Chronic lymphocytic leukemia (CLL) is the most
common leukemia in the Western countries. It is
characterized by a clonal accumulation of small,
mature-appearing lymphocytes in the blood, marrow
and secondary lymphoid tissue. The disease presents
a highly variable clinical course, with some patients
surviving for many years without requiring treatment
and others who witness a rapidly progressing
disease, associated with a short life expectancy,
despite aggressive treatment. Several biological and
genetic properties of the leukemic cells – e.g. the
mutational status of the Ig heavy (H) chain variable
(V) genes, cytogenetic alterations, CD38 and ZAP-70
antigen expression, and p53 dysfunctions – that bear
an important prognostic value have been identified
and have enabled to stratify patients into risk categories.
These aberrations are important independent
predictors of disease progression and survival.
MicroRNAs (miRNAs) are endogenous, non-coding
small RNAs that negatively regulate gene expression
in a sequence specific manner via translational
repression and/or mRNA degradation. The important
role of miRNAs has been demonstrated in
normal and neoplastic hemopoietic cells. Our group
has shown the different expression of some miRNA
in CLL cells. We have identified 4 miRNAs (miR-
21, miR-150, miR-155 and miR-92) as differentially
expressed between CLL samples and healthy donors
(Fulci et al., Blood 2007). Furthermore, we and others
observed an overexpression of miR-150, miR-223,
and miR-29b and miR-29c in the IGHV mutated
CLL cases compared to the IGHV unmutated cases.
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Primary aim of the project
The primary aim of this project was an extension of
the study previously carried out by our group following
IgM stimulation of CLL cases. It was focused in
particular on the characterization of the gene expression
profile of leukemic cells after B-cell receptor
(BCR) stimulation and the importance of the role of
miRNA in the behavior of CLL cells. The support of
the Istituto Pasteur - Fondazione Cenci Bolognetti also
contributed to complete other related studies.
Results
In CLL, gene expression profiling analysis upon IgM
ligation has shown an upregulation of genes involved
in signal transduction, cell cycle and cytoskeleton
organization exclusively in IGHV unmutated
patients (poor prognosis patients), and functional
studies confirmed these findings, showing a significant
increase of cells in G1-phase only in IGHV
unmutated patients.
With regard to IgD stimulation, cells have been
evaluated at two different time-points (24 and 48
hours). At 24 hours, gene expression profiling highlighted
a downmodulation of a set of genes, most
involved in signal transduction; in contrast, at 48
hours, an activation of genes involved mainly in
gene expression regulation and transcription was
observed. Furthermore, genes encoding for BCR
signaling molecules were modulated. Finally, genes
involved in differentiation and apoptosis were overrepresented
at both time points.
These findings were corroborated at the functional
level in 17 patients (12 IGHV mutated and 5 IGHV
mutated), by evaluating cell cycle changes and apoptotic
rate upon IgD stimulus. At both 24 and 48
hours and in all cases, we did not record any proliferation,
coupled to an increased apoptotic rate. There
were no differences between IGHV mutated and
unmutated patients.

Roberto Foà
By flow cytometry, in agreement with the gene
expression profiling results, there was a downmodulation
of the CD79a, CD79b, CD52, CD27
and CD62L molecules. The downmodulation was
observed in all cases, regardless of the IGHV mutational
status and the clinical behavior of the disease,
at both 24 and 48 hours.
Overall, gene expression, functional and flow cytometry
data have shown substantial differences between
IgM and IgD stimulation: in fact, while CLL cell activation
upon IgM cross-linking is strictly associated
to the IGHV mutational status, IgD cross-linking
results in similar changes in all the CLL examined,
independently of the IGHV mutational status, thus
indicating that IgM and IgD stimuli induce different
patterns of response, which might play a variable role
in disease progression (manuscript in preparation).
Finally, extensive miRNA expression profiling is
currently ongoing using the Affymetrix platform at
our center: so far, 4 patients and 2 pools from healthy
donors have been evaluated upon IgM and IgD
stimulation. The data are currently been analyzed.
Further studies completed with the support
of the Istituto Pasteur - Fondazione Cenci
Bolognetti
The support of the Istituto Pasteur - Fondazione Cenci
Bolognetti has helped to complete other related studies.
The profile of miRNAs expression represents a key
of interpretation to some peculiar characteristics of
gene profile of leukemic cells. We have demonstrated
in a subgroup of adult T-lineage acute lymphocytic
leukemia (ALL) that the overexpression of myeloid
genes (MPO, CEBPA, CEBPB, GRN and IL8) was
correlated to the expression levels of miRNA-223,
a miRNA involved in myeloid differentiation. These
cases had significantly higher levels of miR-223 than
the other cases of T-ALL, with values comparable to
those observed in acute myeloid leukemia (Chiaretti
et al., Haematologica 2010).
We have completed the analysis of the gene profile
of 100 CLL with particular attention to the tyrosine
kinase (TK) pathway expression. We performed gene
expression profile on 505 PK genes. The analysis
highlighted an homogeneous upmodulation of several
PKs, 16 also overexpressed in two additional CLL
cohorts. Q-PCR analysis confirmed these findings.
No differences were observed in the main prognostic
subclasses, indicating that PK overexpression in
CLL appears to be disease-associated. Tests in vitro
have shown that the multi-TK inhibitor Dasatinib
partially reduces CLL cell viability, mostly in IGHV
germline patients (Tavolaro et al., Leukemia Research
106
2010). These findings suggest that treatment with
second generation TK inhibitors may represent an
attractive therapeutic strategy for CLL patients.
In view of the clinical results obtained in BCR/
ABL+ ALL patients treated with TK inhibitors, the
above gene expression analysis has been investigated
in adult ALL, subdivided on the basis of the genetic
features. The results obtained have documented an
altered expression profile of various protein kinases
in the different ALL subgroups, indicating that TK
inhibitors may be considered for the management
of ALL, beyond BCR/ABL+ cases (Messina et al.,
Cancer 2010).
We have also investigated the p53 gene status in
CLL because alterations of this gene represent
the worse prognostic parameter in CLL. We utilized
a novel non time-consuming microarray-based
resequencing assay and investigated exons 2-11.
Furthermore, we performed microarrays analysis on
TP53 mutated and/or deleted cases. This study has
allowed to confirm a high percentage of TP53 mutations
in CLL with unfavorable outcome and a significant
association between TP53 aberrations and
del(17p). Finally, specific gene expression profiles
are recognized for TP53 alterations (Chiaretti et al.,
Genes Chromosomes and Cancer, in press).
Publications
Tavolaro S, Chiaretti S, Messina M, Peragine N, Del
Giudice I, Marinelli M, Santangelo S, Mauro FR,
Guarini A, Foà R. Gene expression profile of protein
kinases reveals a distinctive signature in chronic
lymphocytic leukemia and in vitro experiments support
a role of second generation protein kinase inhibitors.
Leukemia Res. 2010, 34: 733-41. doi:10.1016/j.
leukres.2009.11.005.
Chiaretti S, Messina M, Tavolaro S, Zardo G, Elia L,
Vitale A, Fatica A, Gorello P, Piciocchi A, Scappucci G,
Bozzoni I, Fozza C, Candoni A, Guarini A, Foà R. Gene
expression profiling identifies a subset of adult T-cell
acute lymphoblastic leukemia with myeloid-like gene
features and over-expression of miR-223. Haematologica.
2010, 95: 1114-21. doi: 10.1002/cncr.25113.
Messina M, Chiaretti S, Tavolaro S, Peragine N,
Vitale A, Elia L, Sica S, Levis A, Guarini A, Foà
R. Protein kinase gene expression profiling and in
vitro functional experiments identify novel potential
therapeutic targets in adult acute lymphoblastic
leukemia. Cancer. 2010, 116: 3426-37. doi: 10.1002/
cncr.25113.

Signalling events negatively regulating FcepsilonRI expression
and mast cell functional responses
Principal investigator: Rossella Paolini
Professor of Immunology
Dipartimento di Medicina Molecolare
Tel/Fax: (+39) 06 4468448
rossella.paolini@uniroma1.it
Participants:
Stefania Morrone, professor, Rosa Molfetta, researcher;
Francesca Gasparrini, Alessandra Porzia, post-doc fellows.
Report of activity
Mast cells are key effectors in allergic diseases and
play essential roles in host defense against certain
types of parasitic infections. They release a wide
array of pro-inflammatory mediators after activation
by the ligation of several receptors, being the high
affinity receptor for IgE (FceRI) the most efficient
and best characterized.
FceRI is a multisubunit complex consisting of an IgEbinding
α-chain, a four transmembrane-spanning
β-chain with cytoplasmic N-terminal and C-terminal
edges, and a disulphide linked γ-γ homodimer. The
β and γ subunits have no role in ligand binding, but
they share a conserved immunoreceptor tyrosinebased
activation motif (ITAM) within their long
cytoplasmic tails that, upon FceRI aggregation, is
rapidly phosphorylated on tyrosines by the Src family
kinase Lyn, thus allowing the recruitment and
activation of the cytoplasmic kinase Syk, responsible
for signal propagation (1).
The full activation of FceRI requires the recruitment
of aggregated receptors into lipid rafts, membrane
domains rich in cholesterol and sphingolipids that
form ordered but dynamic structures floating in the
less ordered surrounding membrane.
Upon FceRI engagement, membrane rafts coalesce
into larger and more stable structures where
engaged receptors are concentrated (2) and can
more easily interact with signaling molecules, such
as active Lyn (3), favouring phosphorylation events.
The outcome of this activated state is the release of
preformed mediators and the de novo synthesis of
eicosanoids, cytokines and chemokines that result
in both immediate and late-phase immune reactions.
Besides these positive signals, FceRI engagement
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has recently been understood to generate negative
intracellular signals capable of limiting the rate
and the extent of mast cell functional responses (4).
Relevant to this, others and we have demonstrated
that the Cbl family proteins control the amplitude
of FceRI-generated signals by specific ubiquitin
modification of activated receptor subunits and associated
protein tyrosine kinases (5,6). Concurrently,
engaged receptors trigger their own endocytosis to
extinguish signaling through removal of activated
FceRI complexes from the cell surface and delivery
to lysosomes for degradation (7).
The cytosolic effectors of positive signalling have
been analyzed extensively, but very little is known
about the pathways and effectors involved in negative
signalling.
The present study is aimed at identifying the molecular
mechanisms ensuring the clearance of antigenstimulated
FceRI complexes from the cell surface,
thus contributing to the termination of mast cell
functional program.
Our first objective was to investigate the relationship
occurring between receptor ubiquitination, lipid
rafts, and receptor endocytosis.
We initially demonstrated that FceRI β and γ
subunits are mainly monoubiquitinated by c-Cbl at
multiple sites upon antigen stimulation, and provided
evidence that this modification controls receptor
internalization: under conditions of impaired
receptor ubiquitination we reproducibly observed a
decrease of FceRI down-regulation (8).
We have then shown that the recruitment of engaged
FceRI subunits into lipid rafts precedes their ubiquitination,
and that the integrity of these membrane
microdomains is required to allow both receptor
ubiquitination and endocytosis (8).
Thus, a cohesive model is one in which lipid rafts,
in addition to assemble the machinery needed for
the downstream propagation of the signal from the
engaged FceRI complexes, also serve as platforms to
recruit the necessary machinery for their endocytosis.

Rossella Paolini
Our second objective was to identify the main endocytic
adapters involved in receptor endocytosis.
Likely candidates for coupling ubiquitinated FceRI
to the endocytic machinery are the ubiquitous adapter
proteins harboring Ub interacting motifs (UIMs),
namely Eps15, Eps15R, epsin and Hrs. We found
that all of them co-immunoprecipitate with endogenous
FceRI in an antigen-dependent fashion and
within a time-course compatible with function(s) at
different steps of the endocytic route (8).
Eps15, Eps15R and Epsin display similar functions:
they control the early steps of the endocytic route
coupling ubiquitinated receptors with components
of the budding vesicles (9,10). The key role of Hrs
is, instead, the delivery of ubiquitinated proteins to
the outer membrane of the late endosomes and the
sorting of the cargo into internal vesicles of multivesicular
bodies for lysosomal degradation (11).
Although we failed to observe a significant decrease
of FceRI entry in early endosomes upon individual
depletion of Eps15, Eps15R or Epsin, the simultaneous
depletion of all of them impaired ligand-induced
receptor endocytosis (8), suggesting a partial overlapping
function of these adapters in ubiquitinated
FceRI uptake.
Notably, we have also demonstrated a critical role
for Hrs in controlling the fate of internalized receptor
complexes: Hrs depletion retains ubiquitinated
receptors into early endosomes and partially prevents
their sorting into lysosomes (8).
In summary, our data support a key role for the ubiquitin
pathway to ensure proper endocytic trafficking
of an immune receptor to the lysosomal compartment
where degradation of the complexes can take
place (8,12).
The outcome of our proposal would be a better
comprehension of the molecular mechanisms that
functionally regulate the endocytic adaptor Hrs, thus
ensuring a proper delivery of internalized FceRI
complexes to lysosomes for degradation.
Elucidating the molecular mechanisms able to downregulate
the main activating receptor of mast cell
may be critical for the future development of effective
therapeutic strategies in the treatment of disorders
that involve mast cell activation.
108
References
1) Nadler MJ, Matthews SA, Turner H, Kinet JP.
Adv Immunol. 76: 325-355, 2000.
2) Field KA, Holowka D, Baird B. J Biol Chem. 272:
4276-4280, 1997.
3) Sheets ED, Holowka D, Baird B. J Cell Biol. 145:
877-887,1999.
4) Molfetta R, Peruzzi G, Santoni A, Paolini R. Arch
Immunol Ther Exp. 55: 219-229, 2007.
5) Paolini R, Molfetta R, Beitz LO, Zhang J,
Scharenberg AM, Piccoli M, Frati L, Siraganian R,
Santoni A. J Biol Chem. 277: 36940-36947, 2002.
6) Kyo S, Sada K, Qu X, Maeno K, Miah SM,
Kawauchi-Kamata K, Yamamura H. Genes Cells.
8: 825-836, 2003.
7) Molfetta R, Belleudi F, Peruzzi G, Morrone S,
Leone L, Dikic I, Piccoli M, Frati L, Torrisi MR,
Santoni A, Paolini R. J Immunol. 175: 4208-4216,
2005.
8) Molfetta R, Gasparrini F, Peruzzi G, Vian L,
Piccoli M, Frati L, Santoni A, Paolini R. PLoS
One. 4:e5604, 2009.
9) Carbone R, Fré S, Cannolo G, Belleudi F, Mancini
P, Pelicci PG, Torrisi MR, Di Fiore PP. Cancer
Res. 57: 5498-5504, 1997.
10) Chen H, Fre S, Slepnev VI, Capua MR, Takei K,
Butler MH, Di Fiore PP, De Camilli P. Nature.
394: 793-797, 1998.
11) Bache K., Brech A, Mehlum A, Stenmark H. J Cell
Biol. 162: 435-442, 2003.
12) Molfetta R, Gasparrini F, Santoni A, Paolini R.
Mol Immunol. 47: 2427-2434, 2010.
Publications
Molfetta R, Gasparrini F, Peruzzi G, Vian L, Piccoli
M, Frati L, Santoni A, Paolini R. Lipid raft-dependent
FcepsilonRI ubiquitination regulates receptor
endocytosis through the action of ubiquitin binding
adaptors.PLoS One. 2009, 4:e5604. doi: 10.1371/
journal.pone.0005604.
Molfetta R, Gasparrini F, Santoni A, Paolini R.
Ubiquitination and endocytosis of the high affinity
receptor for IgE. Mol Immunol. 2010, 47: 2427-34.
doi: 10.1016/j.molimm.2010.06.003.

Principal investigator: Enza Piccolella
Professor of Immunology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49917584; Fax: (+39) 06 49917584
enza.piccolella@uniroma1.it
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AREA 5
Analysis of the molecular mechanisms regulating FOXP3 gene and
protein expression in TCR- and CD28-activated CD4+CD25-T cells
and their influence on regulatory functions
Participants:
Cristina Camperio, Marzia Soligo, Cristiano Scottà, postdoc
fellows.
Collaborators:
Dipartimento di Dermatologia, Università di Tor Vergata, Roma
(Prof. Antonio Costanzo); Center of Allergy and Environment
(ZAUM), Technical University and Helmholtz Center Munich,
Germany (Prof. Carsten B Schimdt-Weber).
Report of activity
The CD4 + CD25 + FOXP3 + regulatory T cells (Treg)
are a specialized subset of CD4 + T cells that can
suppress the responses of effector T cells. Treg
maintain peripheral immune tolerance and prevent
chronic inflammation as well as autoimmune diseases.
Treg are characterized by the expression of
the a-chain of the interleukin-2 receptor (CD25)
and by the transcription factor forkhead box P3
(FOXP3). Peripheral CD4 + CD25 - T cell could be
converted into FOXP3-expressing Treg (induced
Treg) by signaling through T-cell antigen receptor
(TCR) and accessory molecules such as TGFb1 and
IL-2. Moreover, co-stimulation through the interaction
of CD28 with its ligand B7 is strictly required
for the activation of FOXP3 and for the maturation
and differentiation of Treg either in vivo or in vitro.
However, the molecular mechanisms whereby CD28
alone or associated with TCR can regulate FOXP3
expression remain unknown.
We have explored this function by using our previously
described CD28/B7 selective stimulation
system. Our results have shown that CD28 signals
independent from TCR and dependent on NF-kB
pathways are sufficient to induce the transcription
of FOXP3. Starting from this data, in this project
we have clarified the following unresolved issues:
1) the characterization of the NF-kB/Rel members
recruited on FOXP3 promoter after CD28 activation
and their interaction with NFAT to trans activate
FOXP3 promoter; 2) the determination whether, in
CD28-stimulated T cell, the region of FOXP3 promoter
is in an active chromatin conformation, and
whether epigenetic changes occur in specific binding
sites; 3) the ascertainment whether CD28- and TCRmediated
biochemical pathways function in independent
or dependent manner in regulating FOXP3
protein expression in activated T cells.
1. First, we have explored whether FOXP3
-1657/+176 promoter construct presented NF-kB
binding motifs. To this aim, HEK 293 cells were
co-transfected with FOXP3 -1657/+176 construct
and empty vector or with expression vector encoding
RelA or cRel. The results have shown that both
RelA and cRel homodimers interacted with DNA.
Furthermore, we have shown that DNA region
between -936 and -646 nucleotides of the human
FOXP3 promoter is characterized by at least three
functional and not redundant kB sites for the NF-kB
subunit RelA. However, these sites could not be
required for c-Rel-mediated FOXP3 activation, since
their deletion did not modify the effect of c-Rel on
FOXP3 promoter. We have also investigated the
effect of the co-expression of NFAT and RelA on
the activation of human FOXP3 promoter. In the
absence of the binding of RelA to FOXP3 promoter,
NFAT failed to mediate trans activating function,
suggesting that RelA and not NFAT is primarily
required to trans activate FOXP3 gene, and that the
occupancy of kB sites favours the recruitment of
NFAT on the specific binding sites.
2. FOXP3 promoter shows a stronger association
with acetylated histones in Treg than in conventional
T cells, suggesting that in order to induce
the activation of the FOXP3 gene, the histones that
cover FOXP3 DNA have to be acetylated. Therefore
CD28 could mediate the recruitment of RelA on the
specific binding sites and the induction of epigenetic
modification of the FOXP3 gene at the level
of DNA region distinct. Therefore, to verify this, we

Enza Piccolella
performed chromatin immunoprecipitation (ChIP)
assays with anti-RelA and anti-acetyl histone H4 in
CD4 + CD25 - T cells cultured with Dap3 or Dap3/
B7. The presence of RelA on the identified kB sites
and the histone acetylation of this region allowed
us to conclude to have identified a region on human
FOXP3 promoter where RelA can be recruited following
CD28 signaling and can contribute to the
transcription of FOXP3 gene by inducing histone
H4 acetylation and subsequent loading of RNA
polymerase II. This region is distinct from that
where AP-1 and Rel-NFAT binding sites have been
characterized.
3. To investigate the influence of TCR and CD28
signals on the activation of FOXP3 promoter, we
analysed the transcriptional activation of FOXP3
promoter construct expressed in CD4 + CD25 - T
cells activated by anti-CD28 and anti-CD3 mAbs.
Interestingly, the activation of TCR and CD28
signaling pathways increased the trans activation of
FOXP3 promoter, impling that TCR and CD28 may
induce the sequential and independent activation of
both RelA and c-Rel by using different pathways that
110
synergize in the activation of FOXP3. Therefore,
CD28 unique signals could mediate a fast engagement
of kB sites by RelA for initiating FOXP3 transcription,
but maximal transcription will occur only
upon c-Rel recruitment. Moreover, since TCR signaling
in the absence of CD28 costimulation did not
result in any epigenetic change, RelA could favor the
promoter occupancy by other transcription factors.
The evidence that siRNA-mediated knockdown of
RelA inhibited trans activation of FOXP3 promoter
in CD4 + CD25 - T cells full activated by TCR and
CD28 sustains this hypothesis.
In conclusion, the current study reveals a new
mechanism of FOXP3 gene regulation, and supports
a scenario where CD28-mediated recruitment of RelA
on FOXP3 gene is necessary for the induction of
FOXP3 transcription in CD4 + CD25 - T cells, while
supplementary signals such as those mediated by
TCR are required to allow sustained and maximal
FOXP3 synthesis. On the basis of our data it will be
possible to explore receptors and signaling partners
that may give access to specific interventions aiming
at regulatory T cells.

Anti-tumor effector functions of NK cells in tumor
microenvironment
Principal investigator: Angela Santoni
Professor of Immunology
Dipartimento di Medicina Molecolare
Tel/Fax: (+39) 06 44340632
angela.santoni@uniroma1.it
Participants:
Angela Gismondi, Marco Cippitelli, professors; Giovanni
Bernardini, Alessandra Zingoni, Cristina Cerboni,
Alessandra Soriani, researchers; Francesca Di Rosa,
CNR researcher; Cinzia Fionda, Helena Stabile, Michele
Ardolino, Giuseppe Sciumè, post-doc fellows; Maria Luisa
Iannitto, PhD student.
Collaborators:
Dipartimento di Biotecnologie ed Ematologia, Sapienza-Università
di Roma (Prof. Maria Teresa Petrucci, Prof. Maria Rosaria
Ricciardi, Prof. Anna Guarini, Prof. Robin Foà).
Report of activity
Natural Killer (NK) cells represent one of the main
effectors of the immunosurveillance against tumors
by exhibiting cytotoxic functions and secreting
a number of cytokines and chemokines. They are
extremely heterogeneous both in BM and periphery,
as acquisition of NK cell receptor repertoire, signalling
ability, and functional competence are developmentally
regulated. The specificity of NK cells relies
on the interplay between inhibitory receptors for
MHC class I molecules, and a wide array of activating
receptors that act in concert to induce efficient
elimination of tumor cells. Among the non MHC
I-recognizing activating receptors whose ligands
have been defined so far, particularly relevant for
tumor cell recognition are NKG2D, the receptor for
the MHC I-related molecules MICA/B and ULBPs,
and DNAM-1, the receptor for the nectin/nectinlike
family. NKG2D and DNAM-1 ligands are by no
means tumor-specific molecules, and the mechanisms
responsible for their expression regulation are still
rather obscure.
This research proposal is aimed at identifying the
molecular mechanisms underlying the NK cell recognition
of tumor cells, and the defects of NK cell
maturation and functional competence promoted
111
AREA 5
by tumor microenvironment. To this end, a tumor
type particularly suitable is the multiple myeloma
(MM), a clonal B cell malignancy characterized by
an excess of mature plasma cells mainly in the BM
where NK cell maturation occurs. We have previously
shown that treatment of MM cells with low
doses of therapeutic agents, such as doxorubicin,
melphalan and bortezomib, up-regulate DNAM-1
and NKG2D ligands. Accordingly, drug treatment
of MM cells increases NK cell degranulation, being
the NKG2D and DNAM-1 receptors the major
triggering molecules. Drug-induced DNAM-1 and
NKG2D ligand expression was abolished following
treatment with the pharmacological inhibitors (caffeine
and KU-55933) of the DNA Damage Response
(DDR)-initiating kinases ATM and ATR, and
was preferentially associated with senescent cells
arrested in the G2 phase of the cell cycle (Soriani et
al., Blood 2009).
More recently, we have investigated the signaling
pathways underlying NK cell recognition of druginduced
senescent MM cells with particular attention
to the role played by the generation of reactive
oxygen species (ROS) in the control of senescenceassociated
NKG2D and DNAM-1 ligand expression.
We have found that up-regulation of MICA and PVR
expression on MM cells by low doses of doxorubicin
and melphalan shown to induce ROS generation,
is inhibited by the potent scavenger of ROS NAC
(N-acetyl-L-cysteine), both at protein and mRNA
levels. Moreover, NAC completely abrogated drugtriggered
activation of ATM and of some signaling
components of DDR including the phosphorylation
of the ATM substrates H2AX and p53. Finally,
we found that ROS scavenging resulted in impairment
of cell cycle arrest in G2-M phase and of the
p14Arf senescent marker expression. Based on the
results obtained in vitro with MM cell lines, we
also assessed whether drug-induced increased ROS
production was responsible for the up-regulated
expression of NKG2D and DNAM-1 ligands on

Angela Santoni
patient-derived malignant plasma cells treated with
melphalan, doxorubicin or bortezomib, a proteasome
inhibitor largely used in the therapy of MM.
We observed that NAC pretreatment was able to
completely block the increased surface expression of
NKG2D and DNAM-1 ligands.
Overall our findings demonstrate that low doses
drug-induced NKG2D and DNAM-1 ligand upregulation
on Multiple Myeloma cells is triggered
by ROS-dependent activation of DNA Damage
Response and that the redox-dependent MICA
and PVR expression is preferentially enhanced on
p14ARF senescent cells.
Our study suggests a model in which the senescence
program promotes tumor cell recognition and elimination
by NK cells (Fig. 1).
Our attention was also focused on the analysis of in
vivo NK cell-mediated tumor cell recognition and
elimination upon treatment with chemotherapeutic
drugs. To this end we have established a model
of syngeneic orthotopic MM in C57BL/KaLwRij
mice using the 5TGM1 cell line a subclone of the
5T33 tumor cell line. NK cells from this mouse
strain express NKR-P1C recognized by anti-NK1.1
mAb and display similar maturation markers (our
unpublished observations). Disease progression was
followed by measurement of IgG2b serum titers
and analysis of paraplegia, and in live mice by using
5TGM1 cells constitutively expressing firefly luciferase
and the Xenogen IVIS Optical In Vivo Imaging
System. Preliminary data show that NK cell depletion
by administration of anti-NK1.1 mAb results in
enhanced tumor growth in the BM already at twothree
weeks after tumor challenge. The results were
also confirmed by evaluation of tumor burden by
intracellular IgG2b staining and FACS analysis on
cell samples from spleen and BM at three-four weeks.
In addition, we found that ip injection of suboptimal
doses of melphalan enhanced NKG2DL expression
on MM cells, suggesting that drug-induced enhancement
of NKG2D-dependent NK cell recognition of
tumor cells can also occur in vivo.
Delineating the receptors and functional competence
of discrete NK cell populations on one hand and
the phenotypic analysis of ligand expression on the
other, would allow prediction of tumor cell sensitivity
to NK cell-mediated killing and thus the efficacy
of NK cell-mediated immunotherapy.
112
Publications
Cerboni C, Ardolino M, Santoni A, Zingoni A.
Detuning CD8+ T lymphocytes by down-regulation
of the activating receptor NKG2D: role of NKG2D
ligands released by activated T cells. Blood. 2009,
113: 2955-64. doi: 10.1182/blood-2008-06-165944.
Fionda C, Soriani A, Malgarini G, Iannitto ML,
Santoni A, Cippitelli M. Heat shock protein-90
inhibitors increase MHC class I-related chain A
and B ligand expression on multiple myeloma cells
and their ability to trigger NK cell degranulation.
J Immunol. 2009, 183: 4385-94. doi: 10.4049/jimmunol.0901797.
Soriani A, Zingoni A, Cerboni C, Iannitto ML,
Ricciardi MR, Di Gialleonardo V, Cippitelli M,
Fionda C, Petrucci MT, Guarini A, Foà R, Santoni
A. ATM-ATR-dependent up-regulation of DNAM-
1 and NKG2D ligands on multiple myeloma cells
by therapeutic agents results in enhanced NK-cell
susceptibility and is associated with a senescent
phenotype. Blood. 2009,113: 3503-11. doi: 10.1182/
blood-2008-08-173914.
Kettner A, Di Matteo M, Santoni A. Insulin potentiates
FcepsilonRI-mediated signaling in mouse bone
marrow-derived mast cells. Mol Immunol. 2010, 47:
1039-46. doi:10.1016/j.molimm.2009.11.013.
Porzia A, Lanzardo S, Citti A, Cavallo F, Forni G,
Santoni A, Galandrini R, Paolini R. Attenuation of
PI3K/Akt-mediated tumorigenic signals through
PTEN activation by DNA vaccine-induced anti-
ErbB2 antibodies. J Immunol. 2010, 184: 4170-7. doi:
10.4049/jimmunol.0903375.
Sciumè G, Santoni A, Bernardini G. Chemokines and
glioma: invasion and more. J Neuroimmunol. 2010,
224(1-2): 8-12. doi:10.1016/j.jneuroim.2010.05.019.
Stabile H, Carlino C, Mazza C, Giliani S, Morrone
S, Notarangelo LD, Notarangelo LD, Santoni A,
Gismondi A.Impaired NK-cell migration in WAS/
XLT patients: role of Cdc42/WASp pathway in
the control of chemokine-induced beta2 integrin
high-affinity state. Blood. 2010, 115: 2818-26. doi:
10.1182/blood-2009-07-235804.

Fig.1 - NK cell-mediated recognition of tumor cells undergoing genotoxic stress.
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AREA 5

Dissection of Notch signaling-dependent pathways involved in
the progression of T cell leukemia
Principal investigator: Isabella Screpanti
Professor of General Pathology
Dipartimento di Medicina Sperimentale;
Tel: (+39) 06 44700816; Fax: (+39) 06 4464129
isabella.screpanti@uniroma1.it
Participants:
Maria Pia Felli, professor; Diana Bellavia, Antonio F.
Campese, researchers; Paola Grazioli, Gaia Scafetta, postdoc
fellows; Maria Pelullo, Roberta Quaranta, PhD students.
Collaborations:
Department of Immunology, Imperial College of London, UK
(Prof. Guido Franzoso); Molecular Oncology Research Program,
Division of Surgical Oncology, University of Miami, FL, USA (Prof.
Anthony J. Capobianco).
Report of activity
The role of Notch signaling in the development of
T cell leukemia is sufficiently established, whereas
its role in promoting and sustaining its progression
remains undefined. The triggering of the canonical
pathway of the transcription factor NF-kB,
sustained by the constitutive surface expression of
an active preTCR, that induces in turn, the activation
of several anti-apoptotic and pro-proliferative
signals (Bcl2-A1, IL2, Ciclina D1) appears to exert
an essential role during the expansion phase of the
disease. In this context, it has been recently suggested
that the combined use of pharmacological
compounds that inhibit Notch signaling and NF-kB
activation, is able to improve the therapy response
in mouse models of Notch-induced T-ALL. We previously
demonstrated that the deletion of PKCtheta
kinase, that plays an important role in signal
transduction ending in the triggering of NF-kB
canonical pathway, significantly reduces the disease
aggressiveness in the Notch3-induced T-ALL
mouse model, and induces an important delay in the
progression of the disease.
To clarify the Notch/NF-kB relationships in the
onset and development of T cell leukemia, we decided
to inhibit NF-kB canonical pathway in N3-tg mice.
We generated double mutant mice, deleted of
the NF-kB/p50 subunit in a Notch3 transgenic
115
AREA 5
background (N3-tg/p50 -/- ). The follow-up of
N3-tg/p50 -/- versus N3-tg mice was carried out to
analyze the potential effects of NF-kB inhibition on
the aggressiveness and progression of T-ALL.
The immunophenotypic analysis of hematopoietic
cell populations was performed at different age
and in multiple tissues from the indicated animals
(thymus, spleen, bone-marrow and blood), by flowcytometry
tecniques.
As expected, p50 deletion virtually abolished the
onset of T-ALL in N3-tg/p50 -/- mice, as defined
primarily by the development of splenomegaly and
the peripheral expansion of immature CD4 + CD8 +
T cells. However, the mortality curves evidenced
that double mutant mice succumb earlier than
N3-tg counterparts. Surprisingly, moribund N3-tg/
p50 -/- mice display the trait of a myeloproliferative
disease, with the dramatic expansion of Mac1 + Gr1 +
myeloid cells in both spleen, peripheral blood and
Bone Marrow. Preliminary data indicate that these
cells do not express Notch3, suggesting that in the
absence of p50 expression, Notch3 is able to mainly
influence the equilibrium of the myeloid compartment
in trans.
Our results suggest that the ablation of NF-kB
canonical pathway may strongly impact on the outcomes
of a T cell specific deregulation of Notch signaling.
Thus, providing a useful experimental model
to extend our understanding of Notch/NF-kB interplay
and to unravel novel strategies for the therapy
of different hematological malignancies.
Publications
Canettieri G, Coni S, Della Guardia M, Nocerino
V, Antonucci L, Di Magno L, Screaton R, Screpanti
I, Giannini G, Gulino A. The coactivator CRTC1
promotes cell proliferation and transformation via
AP-1. Proc Natl Acad Sci USA. 2009, 106: 1445-50.
doi: 10.1073/pnas.0808749106.

Isabella Screpanti
Ferretti E, De Smaele E, Po A, Di Marcotullio
L, Tosi E, Espinola MS, Di Rocco C, Riccardi R,
Giangaspero F, Farcomeni A, Nofroni I, Laneve P,
Gioia U, Caffarelli E, Bozzoni I, Screpanti I, Gulino A.
MicroRNA profiling in human medulloblastoma. Int
J Cancer. 2009, 124: 568-77. doi: 10.1002/ijc.23948.
116
Checquolo S, Palermo R, Cialfi S, Ferrara G, Oliviero
C, Talora C, Bellavia D, Giovenco A, Grazioli P,
Frati L, Gulino A, Screpanti I. Differential subcellular
localization regulates c-Cbl E3 ligase activity
upon Notch3 protein in T-cell leukemia. Oncogene.
2010, 1129: 1463-74. doi: 10.1038/onc.2009.446.

The role of HLA-B27 in autoimmunity: from the genetics to the
function
Principal investigator: Rosa Sorrentino
Professor of Pathology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49917706; Fax: (+39) 06 49917594
rosa.sorrentino@uniroma1.it
Participants:
Maria Teresa Fiorillo, researcher; Fabiana Paladini, Adriana
Magnacca, post-doc fellows; Elisa Cocco, Elisa Nurzia, PhD
students.
Collaborations:
Istituto di Biologia Cellulare, CNR, Roma (Dr. Isabella
Cascino); Department for Crystallography, FU Berlin, Germany
(Prof. Wolfang Saenger); Institut für Immungenetik,
Universitätsklinikum Charité Humboldt-Universität zu Berlin,
Germany (Prof. Andreas Ziegler, Dr. Barbara Uchanska-
Ziegler); Dipartimento di Scienze Mediche, Università di Cagliari
(Prof. Alessandro Mathieu).
Report of activity
The association between HLA-B27 gene and
Spondyloarthropathies has been known since many
decades. However the role of these molecules is still
controversial. The main contribution of our research
group has been the identification of a novel allele
in Sardinia, HLA-B*2709, which does not confer
susceptibility to Ankylosing Spondylitis (AS). This
allele differs for a single amino acid change, H116D
from the B*2705 the most common and AS associated
allele, and the functional consequences have
been analysed in detail by us. Crystallographic studies
have shown how the two molecules, the ancestral
B*2705 and the B*2709, can differ in the way they
bind and present to T lymphocytes some peptides
rich in Arginines. We have identified two peptides
from self-proteins, pVIPR1 and pGlucR, that can
be bound in a dual mode: pVIPR1 when bound to
B*2705 and pGlucR when bound to either subtype.
Interestingly, this observation correlates with the
presence of autoreactive T cells: pVIPR-specific
present in B*2705 positive patients only whereas
pGlucR-specific in both patients and B*2709 positive
healthy controls. This suggests that the presence
of more than one conformation, possibly with
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AREA 5
a dynamic behaviour, can hamper the correct T cell
selection in the thymus interfering with the recognition
by TCR. The crystallographic structure of the
three party, TCR, HLA-B27 molecules and peptide
could give insights on how the recognition is influenced
by the double structure. A second hypothesis
that could explain the differential association is that
different haplotypes can harbour the two HLA-B27
alleles and therefore additional HLA genes can
co-operate to confer susceptibility to AS. In this
context, our observation that patients with AS in
Sardinia show a higher frequency of homozygous
markers in the region between HLA-A and HLA-B
compared to HLA-B27 controls, seems to support
this second, not mutually exclusive, hypothesis. A
possible candidate gene is the HLA-E mapping in
between HLA-A and HLA-B. The HLA-E gene
encodes for the ligand of NKG2A receptor and is stabilized
by peptides derived from the leader sequence
of HLA-class I genes. This represents an inhibitory
signal for the Natural Killer cells and, interestingly,
the signal sequence of the HLA-B27 allele shows a
polymorphism that interferes with the binding of the
HLA-E molecules. A peak of disease association has
been reported by us with a functional polymorphism
of the HLA-E gene. Experiments are in progress to
verify this hypothesis.
We have also characterized an interesting alternative
pathway for antigen cross-presentation which is
exploited by the HLA-B27 and not by the HLA-A2
molecules and takes place in the TGN, a cellular
compartment in which the molecules go through
their way to the cell surface. In this compartment
the HLA-B27 molecules can load peptides embedded
in chimeric proteins even in not professional antigen
presenting cells. This appears as particularly interesting
given the specificity for the HLA-B27 molecules
and might have important implications in cells
that are not equipped to perform cross-presentation.
Other investigated genes are in the frame of a metaanalysis
of the IL-1 gene and the regulation of the

Rosa Sorrentino
gene encoding for VIP receptor 1 (VPAC1) whose
interest for us was twofold: this is the gene from
which the pVIPR peptide derives and the VIP-
VPAC1 system plays a relevant regulatory role during
immune-mediated inflammation. A correlation
between some polymorphisms in the 3’-UTR regulatory
region of VPAC1 and its down-modulation in
the presence of an inflammatory stimulus such as the
bacterial LPS was observed. We therefore postulated
that a possible regulatory mechanism could be due
to the action of a microRNA whose complementary
sequence maps in the 3’-UTR region of the VPAC1
gene. The hypothesis was found to be correct and
the microRNA, MiR 525-5p, was shown to be upregulated
by LPS and to target the region in close
proximity of a SNP. The experimental protocol did
not allow such a fine modulation to verify subtle
quantitative variations eventually due to the polymorphism.
This is something that will be verified in
the next future.
A second line of research has been more theoretical
and it is based on the observation that the distribution
of the HLA-B27 molecules in general and B*2705
in particular follows a clear north to south gradient
along the world and is opposite to that of malaria. On
the contrary, the establishment of the HLA-B27 subtypes
derived by point mutations from the ancestral
B*2705 type and associated or not with AS, shows
a gradient coincident with that of malaria. This has
allowed us to hypothesize, based on several observations
reported by others, that HLA-B27, although
being an efficient antigen presenting molecule that
protects from infections such as those due to HIV or
Hepatitis C, has indeed being negatively selected by
malaria and the enrichment of some HLA-B27 sub-
118
types in particular areas where the malaria is endemic,
could be due to a selective pressure driven by the parasite.
The association or not with AS, is likely to be a
secondary effect of this variation which might be due
to either the functional consequences of these mutations
or to other genes belonging to the haplotypes
harbouring the different B27 alleles.
Publications
Mathieu A, Paladini F, Vacca A, Cauli A, Fiorillo
MT, and Sorrentino R. The interplay between the
geographic distribution of HLA-B27 alleles and their
role in disease: an unifying hypothesis. Autoimmun Rev.
2009, 8: 420-25. doi: 10.1016/j.autrev.2009.01.003.
Paladini F, Belfiore F, Cocco E, Carcassi C, Cauli
A, Vacca A, Fiorillo MT, Mathieu A, Cascino I,
Sorrentino R. HLA-E gene polymorphism associates
with Ankylosing Spondylitis in Sardinia. Arthritis Res
Ther. 2009, 11: R171. doi: 10.1186/ar2860.
Nurzia E, Panimolle F, Cauli A, Mathieu A,
Magnacca A, Paladini F, Sorrentino R, Fiorillo M T.
CD8+ T-cell mediated self-reactivity in HLA-B27
context as a consequence of dual peptide conformation.
Clin Immunol. 2010, 135: 476-82. doi: 10.1016/j.
clim.2010.01.009.
Cocco E, Paladini F, Macino G, Fulci V, Fiorillo
MT, Sorrentino R. The expression of vasoactive
intestinal peptide receptor 1 is negatively modulated
by microRNA 525-5p. PLoS One. 2010, 105:e12067.
doi:10.1371/journal.pone.0012067.

Principal investigator: Loretta tuosto
Researcher in Molecular Immunology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49917595, Fax: (+39) 06 49917594
loretta.tuosto@uniroma1.it
119
AREA 5
CD28 co-stimulatory molecule as a key regulator of T lymphocyte
differentiation and survival: characterisation of the biochemical
pathways and molecules coupling CD28 to NF-κB activation
Participants:
Michela Muscolini, PhD student; Elisa Montagni, student,
Silvana Caristi, technician.
Collaborations:
Centro de Biología Molecular Severo Ochoa, Universidad
Autónoma de Madrid, Cantoblanco, Madrid, Spain (Prof. Balbino
Alarcón); Dipartimento di Biologia Cellulare, Università di Tor
Vergata, Roma (Prof. Mauro Piacentini); Dipartimento di
Dermatologia, Università di Tor Vergata, Roma (Dr. Antonio
Costanzo); Laboratorio di Oncogenomica Traslazionale, Istituto
Regina Elena, Roma (Prof. Giovanni Blandino), Hoddaido
University, Sapporo, Japan (Prof. Kazuyasu Sakaguchi).
Report of activity
The signalling pathway that leads from antigenreceptor
and/or co-receptor triggering to the activation
of transcription factors of the NF-κB family has
a crucial role in the regulation of cell survival, and
is controlled by highly similar molecular events in
B and T cells. The understanding of the molecular
mechanisms that control NF-κB activation in lymphocytes
has important implications for the design
of specific therapies for immune diseases.
CD28 is one of the most important co-stimulatory
receptors necessary for full T lymphocyte activation.
By binding its cognate ligands, B7.1/CD80 or B7.2/
CD86 on the surface of professional antigen presenting
cells (APC), CD28 plays a critical role in the
recruitment of key molecules to the T cell receptor
(TCR), thus lowering the activation threshold and
enhancing TCR-mediated signalling pathways.
The existence of autonomous CD28 signalling
has been proposed by Schwartz in 1990, when he
observed that CD28 was able to act as a TCRindependent
signalling unit and to deliver in trans
co-stimulation (Schwartz, 1992). The efforts made
in an attempt to characterize the signalling pathways
activated by CD28/B7 interaction in a TCR-
independent manner, led to the identification of
NF-kB as the most relevant CD28 biochemical
target. In this context, our research group evidenced
that CD28 engagement by B7.1/CD80 expressed on
the surface of APCs is, indeed, sufficient to induce
NF-kB activation in the absence of TCR. In particular,
CD28/B7 interaction alone leads to the activation
of a non-canonical IKKα/NF-kB2-like cascade
and to the nuclear translocation of p52/RelA dimers
(Marinari et al., 2004). IKKα is mainly involved in the
activation of the alternative NF-kB pathway, which
is characterized by IKKα-mediated phosphorylation
of p100, its degradation by proteasome and the
nuclear translocation of active of RelB/p52 or RelA/
p52 dimers. We found that RelA/p52 dimers control
the trans-activation and expression of IL-8, BAFF
and Bcl-xL genes in CD28-stimulated memory T
cells (Marinari et al., 2004). All these data evidence
the existence of an autonomous CD28 signalling
that, through the activation of an alternative-like
IKKα-dependent NF-kB pathway, selectively regulates
the expression of pro-inflammatory cytokines/
chemokines as well as of pro-survival proteins.
CD28 co-stimulation regulates T cell survival by upregulating
the anti-apoptotic Bcl-2 family member,
Bcl-xL through NF-kB (Boise et al., 1995; Kerstan
& Hunig, 2004; Khoshnan et al., 2000). However, the
up-regulation of Bcl-2 family anti-apoptotic genes, in
particular A1/Bfl-1 and Bcl-xL, may account only in
part for the protective effects exerted by NF-kB in T
cells. Recently, co-stimulation dependent activation
of NF-kB has been described to counteract TCRdriven
apoptosis by inhibiting p73 expression (Wan &
DeGregori, 2003). p73 is a member of the p53 family
that integrates stimuli from both DNA damage and
receptor-mediated apoptosis in T cells (Lissy et al.,
2000). Similarly to p53, p73 regulates apoptosis by
inducing the expression of several Bcl-2 family proapoptotic
proteins, including Bax (Melino et al., 2002).
We found that CD28 stimulation by B7 and in
the absence of TCR protects primary T cells from

Loretta Tuosto
IR-mediated apoptosis through two mechanisms. In
one hand, CD28 induces the nuclear translocation
of active RelA/p52 dimers, which together with the
histone acetylase p300/CBP co-activator trans-activate
the bcl-xL promoter. In the other hand, CD28
engagement also stimulates the formation of histone
deacetylase HDAC1/RelA co-repressor complexes,
which inhibit p73 transcriptional activation of bax.
The key clue associated to the nature of CD28 signalling
derives from the composition of its small cytoplasmic
domain (41 aa) and its interaction with intracellular
proteins. It contains a N-terminal YMNM
motif that following phosphorylation binds the p85
subunit of phosphatidylinositol 3-kinase (PI3K) and
Grb2 (Cai et al., 1995; Okkenhaug et al., 2001).
Downstream of the YMNM motif, CD28 has two
proline-rich regions, the N-terminal P 196 RRP that
binds the SH3 domain of the IL-2 inducible kinase
(Itk), and a C-terminal motif Y 206 QP 208 YAPP containing
two proline (P) residues, that binds the SH3
domain of Lck (Holdford et al., 1999; Tavano et al.,
2004), Grb2 (Kim et al., 1998) and FLNa (Tavano et
al., 2006), a large cytoplasmic protein that crosslinks
cortical actin.
By using the CD28-negative Jurkat T cells
(CH7C17) stably transfected with human wild-type
CD28 (CD28 WT), or with CD28 mutants in the
Y 191 MNM 194 motif (CD28Y 191 F or CD28M 194 Q), or in
the N-terminal P 196 RRP 199 motif (CD28AA 196-199 ) or
in the C-terminal proline-rich Y 206 QP 208 YAPP motif
(CD28-3A, or CD28FF 206/209 ), we found that mutation
of either proline (CD28-3A) or tyrosine residues
(CD28FF 206/209 ) in the C-terminal proline-rich motif
strongly reduced both CD28-mediated IKKα and
NF-kB activation.
The C-terminal Y 206 QP 208 YAPP motif of CD28
regulates the recruitment of FLNa to the membrane.
FLNa is a scaffold protein that binds several proteins
involved in cell signalling (Zhou et al., 2010) and
participates in T cell activation (Tavano et al., 2006).
In searching for specific proteins associating FLNa
and coupling CD28 to IKKα and a non-canonical
NF-kB2-like pathway, we looked at the IKKα activator
NIK (Senftleben et al., 2001). Our data evidence
that NIK constitutively associated with FLNa in
both Jurkat and primary T cells. Confocal analyses
revealed that in CD28WT cells NIK was efficiently
recruited to the membrane and co-localized with
FLNa in the T:APC contact zone, while CD28-3A
and CD28FF 206/209 mutants failed to do that. CD28
engagement by B7 significantly induced the kinase
activity of NIK-associated IKKα, without affecting
the amount of co-precipitated IKKα. Both CD28-3A
120
and FF 206/209 mutants failed to induce NIK-associated
IKKα kinase activity. Similar analyses performed
in primary T lymphocytes, revealed that CD28
stimulation induced the stabilization of NIK, its association
with IKKα and NIK-associated IKKα kinase
activity. Thus, the C-terminal proline-rich motif of
CD28 regulates the recruitment of both FLNa/NIK
complexes, thus leading to NIK/IKKα activation
and to the induction of the non-canonical NF-kB2
pathway.
Selected publications
Muscolini M, Montagni E, Caristi S, Nomura T,
Kamada R, Di Agostino S, Corazzari M, Piacentini
M, Blandino G, Costanzo A, Sakaguchi K, Tuosto L.
Characterization of a new cancer-associated mutant
of p53 with a missense mutation (K351N) in the
tetramerization domain. Cell Cycle. 2009, 8: 3396-405.
doi: 10.4161/cc.8.20.9910.

Principal investigator: Elio Ziparo
Professor of Embryology
Dip. di Scienze Anatomiche, Istologiche, Medico-Legali e dell’Apparato Locomotore
Tel: (+39) 06 49766586; Fax: (+39) 0649766340
elio.ziparo@uniroma1.it
121
AREA 5
The role of Toll Like Receptors in immune responses to infections
and in inflammation associated pathologies of the male
reproductive system
Participants:
Antonio Filippini, professor; Anna Riccioli, researcher;
Claudia Giampietri, post-doc fellow; Roberta Galli,
Alessio Paone, PhD students; Fabrizio Padula, Simonetta
Petrungaro, Donatella Starace, technicians.
Collaborations:
Dipartimento di Medicina Sperimentale, Università de L’Aquila
(Prof. Paola De Cesaris).
Report of activity
Hypoxia inducible factor 1 (HIF-1) is a transcription
factor, which regulates a number of genes required
for hypoxic response via binding specific regions of
their promoters, named hypoxia responsive elements
(HREs). HIF-1 is active only as a heterodimer of
HIF-1α and HIF-1β subunits. HIF-1β is constitutively
expressed in all cell types, whereas HIF-1α
levels are tightly controlled.
It has been clearly demonstrated that HIF-1 regulates
genes relevant to cancer progression, especially
as predictor of clinical outcome in patients
with adenocarcinomas (Hoffmann et al., 2008). In
particular, HIF-1α has emerged as a potential prognostic
biomarker in proteomic assessment of prostate
cancer (Kimbro and Simons, 2006) since clinical
observation of high-grade prostate intraepithelial
neoplasia (PIN) lesion (precursor of most prostate
adenocarcinoma (PCa), showed increased HIF-1α
expression (Zhong et al., 2004) and HIF-1α upregulation
in PCa as well as in prostate cancer bone
metastases has been observed (Zhong et al., 1999).
PCa is a prevalent tumour among elderly men and
survival benefit with current PCa therapies is often
limited (Gomella et al., 2009). Indeed standard pharmacological
therapy, consisting of withdrawal of
androgens, leads to only transient regression of the
disease and there is no cure for prostate cancer once
become androgen refractory. Although HIF-1α pro-
tein is mainly induced by hypoxic conditions, other
stimuli can strongly increase the HIF-1 complex in
normoxic conditions and modulate the transcription
of hypoxic genes. Interestingly, also a variety
of molecular components derived from bacteria
or viruses has been described to activate HIF-1α
in normoxia through specific Toll Like Receptors
(TLRs) (Blouin et al., 2004; Nicholas and Sumbayev,
2009; Ramanathan et al., 2009). TLRs are a group
of transmembrane proteins (11 in humans) that
recognize pathogen-associated molecular patterns
(PAMPs). Recent studies demonstrate that tumour
cells express functional TLRs and that TLR signaling
can promote opposite outcomes: tumour growth
and immune evasion, or apoptosis and cell cycle
arrest (Huang et al., 2005; Kelly et al., 2006; Paone et
al., 2008b; Salaun et al., 2006).
The TLR3-ligand poly(I:C) mimics the action of
double stranded RNA (dsRNA), the genetic material
of many viruses, and TLR3 engagement, directly
inhibits cell proliferation and induces tumour cell
death (Salaun et al., 2006; Salaun et al., 2007). We
have previously demonstrated that LNCaP cells, an
androgen-dependent human prostate cancer cell line,
are sensitive to poly(I:C)-induced apoptosis, whereas
PC3 cells, a more aggressive androgen-independent
prostate cancer cell line, show a weak sensitivity to
the same stimulus (Paone et al., 2008b). Recently, a
number of articles reported the ability of HIF-1 complex
to mediate the resistance to several apoptotic
stimuli (Chen et al., 2009; Peng et al., 2006). Based
on these data, we have hypothesized that the limited
response of PC3 cells to poly(I:C) could be due to the
induction of a parallel pro-tumoral signal involving
HIF-1 complex activation.
We described a novel pro-tumour machinery triggered
by TLR3 activation in PC3 cells consisting
in increased expression of the specific I.3 isoform of
HIF-1α and nuclear accumulation of HIF-1 complex
in normoxia, resulting in reduced apoptosis and in
secretion of functional VEGF. Moreover, we report

Elio Ziparo
that in the less aggressive LNCaP cells, TLR3 activation
fails to induce nuclear accumulation of HIF-
1α. However, the transfection of I.3 isoform of hif-1α
in LNCaP cells allows poly(I:C)-induced HIF-1 activation,
resulting in apoptosis protection and VEGF
secretion. Altogether, our findings demonstrate that
differences in basal level of HIF-1α expression in
different prostate cancer cell lines underlie their differential
response to TLR3 activation, suggesting a
correlation between different stages of malignancy,
hypoxic gene expression and beneficial responsiveness
to TLR agonists (Paone et al., 2010).
Mounting evidence shows that the enhancement of
innate and adaptive immunity represents the principal
mechanism by which TLR stimulation produces
anti-tumour activity. Apart from a direct effect of
TLR3 on prostate cells apoptosis, we studied also the
ability of TLR ligands to affect the recruitment of
different immune cell populations by human prostate
cancer cell lines and the underlying mechanisms. We
have investigated the expression of TLRs in different
human prostate cancer cell lines, LNCaP, DU-145
and PC3 and shown that only TLR3 and TLR5 are
shared by the three cell lines. Treatment with their
respective agonists poly(I:C) and flagellin rapidly
triggers NF-κB-dependent up-regulation of different
inflammatory molecules, as assayed by microarray
and ELISA. Furthermore, we demonstrate that conditioned
media from poly(I:C)- and flagellin-treated
LNCaP and DU-145 cells induce the recruitment of
different leukocyte subpopulations, suggesting that
TLR stimulation is able to activate the earliest step
of immune response mediated by soluble factors.
Interestingly, the more aggressive cancer cell line
PC3 was found to express TLR3 and TLR5, but
failed to respond to TLR agonists in terms of NF-κB
activation and ability to attract immune effectors
(Galli et al., 2010). Altogether, our data indicate that
both TLR3 and TLR5 are expressed and functionally
active in LNCaP and DU-145 prostate cancer
cells, whereas in PC3 cells TLR3/5 stimulation does
not affect NF-kB activation, cytokine/chemokine
expression and immune cell recruitment, suggesting
that PC3 cells, representing the most aggressive
castration-resistant stage of prostate malignancy, are
not able to activate an anti-tumor immune response.
In conclusion, the ability to mount an immune
response following TLR stimulation seems to be in
reverse correlation with neoplastic progression.
122
Overall, our data show that poly(I:C), by exerting
both a direct pro-apoptotic effect on LNCaP cells
(Paone et al., 2008a), and an immune-mediated effect
due to the recruitment of NK and cytotoxic CD8 cells
(Galli et al., 2010), is a potentially valid therapeutic
agent in prostate cancer. In addition to the known
anti-tumour effect for HIF-1 knock-down, our data
indicate that targeting HIF-1 in the most aggressive
PC3 cells has also the potential to improve the
efficacy of treatment with TLR3 agonists as direct
effectors of prostate cancer cell apoptosis (Paone et
al., 2010).
Selected publications
Antonangeli F, Giampietri C, Petrungaro S, Filippini
A, Ziparo E. Expression profile of a 400-bp Stra8
promoter region during spermatogenesis. Microsc
Res Tech. 2009, 72: 816-22. doi: 10.1002/jemt.20724.
Antonangeli F, Petrungaro S, Coluccia P, Filippini
A, Ziparo E, Giampietri C. Testis atrophy and
reduced sperm motility in transgenic mice overexpressing
c-FLIP(L). Fertil Steril. 2010, 93: 1407-14.
doi: 10.1016/j.fertnstert.2009.01.122.
Starace D, Muciaccia B, Morgante E, Russo MA,
Pensini S, D‘agostino A, De Cesaris P, Filippini A,
Ziparo E, Riccioli A. Peculiar subcellular localization
of Fas antigen in human and mouse spermatozoa.
Microsc Res Tech. 2009, 72: 573-9. doi: 10.1002/
jemt.20700.
Galli R, Starace D, Busà R, Angelini DF, Paone
A, De Cesaris P, Filippini A, Sette C, Battistini L,
Ziparo E, Riccioli A. TLR stimulation of prostate
tumor cells induces chemokine-mediated recruitment
of specific immune cell types. J Immunol. 2010,
15184: 6658-69. doi:10.4049/jimmunol.0902401.
Paone A, Galli R, Gabellini C, Lukashev D, Starace
D, Gorlach A, De Cesaris P, Ziparo E, Del Bufalo
D, Sitkovsky MV, Filippini A, Riccioli A. Toll-like
receptor 3 regulates angiogenesis and apoptosis in
prostate cancer cell lines through hypoxia-inducible
factor 1 alpha. Neoplasia. 2010, 7: 539-49. doi:
10.1593/neo.92106.

AREA 6
New
antimicrobial
and antiviral
agents

Peptide effectors of innate immunity
Principal investigator: Donatella Barra
Professor of Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 4456663; Fax: (+39) 06 4440062
donatella.barra@uniroma1.it
Participants:
Maurizio Simmaco, Giuseppina Mignogna, professors; M.
Luisa Mangoni, Rossella Miele, Alessandro Paiardini,
researchers; Marina Borro, Giovanna Gentile, post-doc
fellows; Ludovica Marcellini Hercolani Gaddi, Sara
Marsango, Vincenzo Luca, PhD students; Alessandra
Franco, Alessandra Giorgi, technicians.
Collaborations:
Institute of Molecular Biology, Austrian Academy od Sciences,
Salzburg, Austria (Prof. Günther Kreil); Centro de Investigaciones
Biológicas (CSIC) Madrid, Spain (Prof. Luis Rivas); Dipartimento
di Scienze e Tecnologie Chimiche, Università di Roma di Tor
Vergata (Prof. Lorenzo Sella); Dipartimento di Fisiologia e
Farmacologia “V. Erspamer”, Sapienza-Università di Roma (Prof.
Lucia Negri).
Report of activity
Because of the increasing emergence of microbes
which are resistant to conventional antibiotics, the
discovery of new antimicrobial agents with a new
mode of action is urgently needed. Naturally occurring
antimicrobial peptides (AMPs), which are produced
by almost all forms of life, represent promising
candidates for the development of new anti-infective
drugs. Amphibian skin is one of the richest sources
for such molecules. In contrast with conventional
antibiotics, most AMPs interact and increase the
permeability of the bacterial membrane as part of
their killing mechanism. However, before reaching
it, they need to cross the cell wall that, in Gramnegative
bacteria, is surrounded by the lipopolysaccharide
(LPS)-outer membrane, which forms a very
efficient barrier against a variety of hydrophilic and
hydrophobic molecules.
We had previously found that some isoforms of
temporins, produced within the same frog specimen
(temporins A and B), can synergize when combined
each with temporin L, to overcome the bacterial
125
AREA 6
resistance imposed by the LPS protective layer. In
addition, we discovered that these peptides can synergize
in the detoxification of LPS molecules, by suppressing
the release of TNF-α from LPS-activated
macrophages. Note that both synergisms inversely
depend on the length of the LPS-polysaccharide
chain. Studies on the underlying molecular mechanisms
are in progress.
In parallel, we carried out an in-depth study on
the in vitro and in vivo antimicrobial properties of
esculentin fragments. With regards to the in vitro
experiments, the mode(s) of action of Esc(1-18)
(GIFSKLAGKKLKNLLISG-NH 2 ) on Escherichia coli
was investigated. We found that the peptide rapidly
kills E. coli at the minimal inhibitory concentration
(MIC) and that the lethal event is concomitant with
the membrane perturbation. Note that many hostdefense
peptides do not destabilize membranes at
their MICs. Importantly, proteomic analysis revealed
that Esc(1-18) has a limited ability to modify the bacterium’s
protein expression profile, at both sub-lethal
and lethal concentrations. This suggests the bacterial
membrane as the major target for the killing
mechanism of this peptide, rather than intracellular
processes. In addition, Esc(1-18) displays a strong
synergism with conventional antibiotics, presumably
by increasing the intracellular influx of these drugs,
as a result of an increased membrane permeability.
With regards to the in vivo studies, we analyzed
the antimicrobial activity of Esc(1-18) and temporins
on the minihost model of Caenorhabditis elegans
infected by a multidrug resistant (MDR) strain of
Pseudomonas aeruginosa. The data showed that both
temporin B and Esc(1-18) can cause a drastic reduction
in the number of viable bacteria within the
worm’s gut, promoting survival of the infected nematodes.
The peptides’ effect on the bacterial colonization
was also visualized by fluorescence microscopy,
using a GFP-expressing strain of P. aeruginosa. After
90 min of exposure to a sub-lethal concentration of
Esc(1-18), the fluorescence intensity throughout the

Donatella Barra
gut of the infected worms decreased considerably.
Remarkably, this peptide is able to alter the permeability
of the membrane of Pseudomonas cells within
the animal. To the best of our knowledge, this is the
first case showing the ability of an AMP to permeate
microbial membranes within a living organism.
Concerning in vivo studies with more complex
animals, such as mammals, we tested the activity
of Esc(1-21) (GIFSKLAGKKIKNLLISGKG-NH 2 )
on cows affected by mastitis, the most common
and expensive illness of dairy cows throughout the
world. Although stress and physical injuries may
give rise to infiammation of the udders, infections
by bacteria or other microorganisms remain the
principal cause: infusion of antibiotics is the major
treatment approach. However, the increased emergence
of MDR pathogens and the production of
milk contaminated with antibiotics has become a
serious threat in the livestock. Our data indicated
that Esc(1-21) is highly potent against the most
common mastitis-causing microbes (e.g. Streptococcus
agalactiae). Most importantly, the peptide is active in
vivo, causing a visible regression of the clinical stage
of mastitis in dairy cows after 1 week of treatment.
Altogether, these results encourage further study of
esculentin peptides to clarify their in vivo antimicrobial
activity, pharmacokinetics and half-life.
Another interesting family of frog skin AMPs is
given by bombinins H, isolated from amphibia of
Bombina genus. They include isomers with a single
D-amino acid resulting from a post-translational
epimerization of the corresponding L-residue.
Specifically, we evaluated the antimicrobial activity
of the pair H2 (IIGPVLGLVGSALGGLLKKI-NH 2 )
and H4, differing by only the configuration of the
second amino acid (an L-isoleucine in H2 and a
D-alloisoleucine in H4), against a large panel of
reference and clinical isolates of Gram-negative and
-positive bacteria; we have performed membrane
permeation assays on both intact cells and model
membranes; used biochemical tools, such as trypsinencapsulated
liposomes and capillary electrophoresis,
to monitor the peptides’ ability to translocate
through the membrane of liposomes mimicking the
E. coli inner membrane. Our current results have
pointed out interesting relationships between the
presence of a single D-amino acid in the sequence
of an AMP and its target microbial cell selectivity/
membrane perturbing activity. Such findings will
contribute to the development of new peptide-based
therapeutic drugs.
Bv8/prokineticins are small proteins that regulate
diverse biological processes including gastro-
126
intestinal motility, angiogenesis, circadian rhythm
and innate immune response. The Bv8 gene from
Bombina orientalis has been cloned, sequenced and
its promoter has been functionally characterized.
Recombinant expression of Bv8 has been obtained in
Pichia pastoris.
Prokineticins bind two closed related G-protein coupled
receptors (GPCRs), PKR1 and PKR2. In general,
these receptors act as molecular switches to relay
activation to heterotrimeric G proteins and a growing
body of evidence points to the fact that GPCRs
exist as homo- or heterodimers. We have shown
by Western blot analysis that PKR2 has a dimeric
structure in neutrophils. By heterologous expression
of PKR2 in Saccharomyces cerevisiae, we examined the
mechanisms of intermolecular interaction of PKR2
dimerization. The potential involvement of three types
of mechanisms was investigated: coiled-coil, disulfide
bridges and hydrophobic interactions between transmembrane
domains. Characterization of differently
deleted or site-directed PKR2 mutants suggest that
dimerization proceeds through interactions between
transmembrane domains. We demonstrated that coexpressing
binding-deficient and signalling-deficient
forms of PKR2 can re-establish receptor functionality,
possibly through a domain-swapping mechanism.
Publications
Islas-Rodríguez AE, Marcellini L, Orioni B, Barra
D, Stella L, Mangoni ML. Esculentin 1-21: a linear
antimicrobial peptide from frog skin with inhibitory
effect on bovine mastitis-causing bacteria. J Pept Sci.
2009, 15: 607-14. doi: 10.1002/psc.1148.
Maisetta G, Mangoni ML, Esin S, Pichierri G, Capria
AL, Brancatisano FL, Di Luca M, Barnini S, Barra D,
Campa M, Batoni G. In vitro bactericidal activity of the
N-terminal fragment of the frog peptide esculentin-1b
(Esc 1-18) in combination with conventional antibiotics
against Stenotrophomonas maltophilia. Peptides.
2009, 30: 1622-6. doi: 10.1016/j.peptides.2009.06.004.
Mangoni ML, Shai Y. Temporins and their synergism
against Gram-negative bacteria and in lipopolysaccharide
detoxification. Biochim Biophys Acta. 2009,
1788: 1610-19. doi: 10.1016/j.bbamem.2009.04.021.
Marcellini L, Borro M, Gentile G, Rinaldi AC, Stella
L, Aimola P, Barra D, Mangoni ML. Esculentin-
1b(1-18)--a membrane-active antimicrobial peptide
that synergizes with antibiotics and modifies the
expression level of a limited number of proteins in

Escherichia coli. FEBS J. 2009, 276: 5647-64. doi:
10.1111/j.1742-4658.2009.07257.x.
Marsango S, Bonaccorsi di Patti M C, Barra D, Miele
R. The Bv8 gene from Bombina orientalis: molecular
cloning, genomic organization and functional
characterization of the promoter. Peptides. 2009, 30:
2182-90. doi: 10.1007/s00018-010-0601-6.
Simmaco M, Kreil G, Barra D. Bombinins, antimicrobial
peptides from Bombina species. Biochim
Biophys Acta. 2009, 1788: 1551-5. doi: 10.1016/j.
bbamem.2009.01.004.
Miele R, Lattanzi R, Bonaccorsi di Patti M C,
Paiardini A, Negri L, Barra D. Expression of Bv8
127
AREA 6
in Pichia pastoris to identify structural features for
receptor binding. Protein Expr Purif. 2010, 73: 10-4.
doi: 10.1016/j.pep.2010.04.012.
Marcellini L, Giammatteo M, Aimola P, Mangoni
ML. Fluorescence and electron microscopy methods
for exploring antimicrobial peptides mode(s)
of action. Methods Mol Biol. 2010, 618:249-66. doi:
10.1007/978-1-60761-594-1_16.
Uccelletti D, Zanni E, Marcellini L, Palleschi C,
Barra D, Mangoni ML. Anti-Pseudomonas activity
of frog skin antimicrobial peptides in a Caenorhabditis
elegans infection model: a plausible mode of action in
vitro and in vivo. Antimicrob Agents Chemother. 2010,
54: 3853-60. doi: 10.1128/AAC.00154-10.

New pyrrole derivatives of BM 212: a new class of
antimycobacterial agents. Design, synthesis, biological evaluation
and study of their mode of action
Principal investigator: Mariangela Biava
Professor of Medicinal Chemistry
Dipartimento di Chimica e Tecnologie del Farmaco
Tel: (+39) 06 49913812; Fax: (+39) 06 49913133
mariangela.biava@uniroma1.it
Participants:
Maurizio Botta, Alessandro De Logu, Edda De Rossi,
Raffaello Pompei, Giulio Cesare Porretta, professors;
Fabrizio Manetti, researcher; Giovanna Poce, post-doc fellow;
Claudio Battilocchio, PhD student; Roberto Torri, technician.
Collaborations:
Department of Medicinal Chemistry and Pharmacognosy,
University of Illinois at Chicago College of Pharmacy, Chicago,
IL, USA (Prof. Scott G. Franzblau); Department of Immunology
and Infectious Diseases, Harvard School of Public Health, Boston,
USA (Prof. Eric Rubin); CNRS, Immunologie et Embryologie
Moléculaires, Université Orléans, France (Prof. Bernhard Ryffel).
Report of activity
The recrudescence of Tuberculosis (TB) has led to a
situation that the World Health Organization describes
as having the potential to cause a “global catastrophe”. 1
The major factors explaining the explosion in numbers
of infections with Mycobacterium tuberculosis (MTB) are:
i) the deadly synergy with HIV, 2-4 ii) the resistance to
the majority of the common antitubercular drugs as
impermeable to these agents, 5 iii) the ability of mycobacteria
to establish a persistent phase, living in a slow
growing or not-growing state, 6,7 recalcitrant to treatment
by conventional anti TB drugs. 8
For all this reason, the aim of this project is to synthesize:
1) new antimycobacterial compounds endowed
with a mechanism of action different from that of
drugs employed in the current therapy, towards
which mycobacteria proved to be resistant; 2) bactericidal
molecules more active, an less cytotoxic than
the previously synthesized ones; 3) low-cost drugs,
that can be used not only in industrialized countries
but also in developing ones in which tuberculosis, in
the last years, is rapidly growing.
The work of this first year has been devoted to the synthesis
of new derivatives of the lead compounds previously
identified, BM212 and BM521 (see Figure), on
129
AREA 6
the basis of both structure-activity relationship (SAR)
and molecular modelling studies previously performed.
The rational approach started with the generation
of a first subclass 9 of compounds in which the parafluoro-phenyl
moiety was placed in turn at N1 or C5
of the central nucleus, while the remaining position
was arranged (ortho or para positions) with different
methyl-derived moieties (CF 3 , SCH 3 or OCH 3 ) to evaluate
the activity, when replacing the C5 methyl group
present in BM521, with different groups endowed with
different electronic properties, but bioisosteres, and the
following superposition on the pharmacophoric model.
Then, to verify the hypothesis that the presence of a
fluorine atom, an alkyl moiety or an electron-releasing
moieties at the para position of the phenyl rings were
crucial parameters for activity we develop a second
subclass 9 of compounds characterized by an electrondonating
group (methoxyl or methylsulphanyl) and
an alkyl one (ethyl, propyl and i-propyl) alternatively
placed at N1 and C5 of the pyrrole core, by maintaining
the thiomorpholino-methyl group positioned at C3.
Finally, in the attempt to conjugate lipophilic and
structural parameters, a further enlargement of SAR
was carried out developing a third subclass 10 of compounds
in which the methyl chain at C2 of the central
nucleus was replaced with an ethyl one. This was
accomplished to find out if the introduction of a more
lipophilic moiety at C2 of the pyrrole, instead of at the
phenyl rings in N1 or C5, was able to affect the activity,
being the C2 methyl group devoid of interactions
with the pharmacophore features, but involved in the
conformational modulation of the other substituents.
New derivatives were synthesized following the
scheme previously reported, characterized by only
three steps, very versatile and cheep, that allowed us
to assure their potential low-cost, satisfying the goal 3.
All the products have been evaluated for their cytotoxicity
and tested for their in vitro activity against
i) intra and extra-cellular MTB; ii) atypical mycobacteria,
such as M. avium, particularly incident in HIVimmunocompromised
patients; iii)clinical strains

Mariangela Biava
resistant to the conventional drugs. The study of the
possible target is in progress, and it is performed by
external research groups.
Microbiology
All compounds were assayed to assess their in vitro
activity towards Mycobacterium tuberculosis 10347. The
activities of compounds were evaluated as minimal
concentration that completely inhibits the bacteria
growth (MIC). Cytotoxicity was evaluated in a cellbased
assay employing Vero Cells and expressed as
50 % maximum non-toxic dose, MNTD or as 50%
50
cytotoxic concentration, CC . The protection index
50
(PI) was calculated as MNTD (or CC ) vs MIC ratio.
50 50
Compounds belonging to the first subclass, shared
good MICs. N1-halo-phenyl derivatives are endowed
with lower inhibitory concentrations with respect to
corresponding C5 analogs. One compound of this series
showed an outstanding activity in terms of cytotoxicity,
with a PI (>1000) higher than that shown by rifampin.
In general, chlorinated derivatives seem to generate
less cytotoxic effect and better biological profiles with
respect to the corresponding fluorine derivatives.
Furthermore, the para substitution leads to derivatives
more potent than corresponding ortho.
All derivatives belonging to the second subclass showed
very low inhibitory concentration and the results highlighted
that an increase in electronic density of the phenyl
rings at N1 and C5 translates into activity enhancement.
It can be clearly seen that N1/C5 alkyl/methoxy
or alkyl/methylsulphanyl subclasses of compounds are
endowed with very good potential and their activities
follow an almost resembling trend, with inhibitory
concentrations ranging from 0.125 to 2 µg/mL. On the
other hand, this subclass of molecules were characterized
by moderate cytotoxicity and only one compound
in the series proved to perform cytotoxic effects comparable
to that shown by reference compounds (PI >512).
In this sense, the substitution of the fluorine atom with
a para-methoxyl group and the methyl with a propyl
one does not seem to change the biological interactions
(in terms of MICs and cytotoxicity). It is also worthy
to note that an increase of lipophylicity in the alkyl/
methylsulphanyl subseries reflects in an improvement
130
of activity, both in terms of MICs and PIs.
Compounds bearing an ethyl chain at C2 of the
pyrrole core, belonging to the third subclass, are
characterized by MIC values two- to four fold higher
than those reported for methyl analogs. This result
shows that correlation between higher lipophilicity
and antimycobacterial activity may depend on
structural modifications mainly involving the phenyl
rings at N1 and C5, that are able to match the hydrophobic
features of the pharmacophoric model.
Perspective
The future work will be devoted to perform the
microsomial stability for the most active compounds,
and their pharmacokinetic study as preliminary evaluation
for the following in vivo test. Further modifications
to the structure will be performed as well, in
the attempt to improve the biological profile of these
molecules.
References
1) WHO Report 2009. Geneva, Switzerland.
2) Corbett EL et al., Arch Intern Med 2003, 163,1009.
3) Breen RA et al., Drugs 2006, 66, 2299.
4) Phillips KD, J Assoc Nurses AIDS Care 2007, 18, 75.
5) Inderlied CB et al., Clin Microbiol Rev, 1993, 6, 266.
6) Stewart GR et al., Nat Rev Microbiol 2003, 1, 97.
7) Dooley KE et al., Clin Chest Med 2006, 26, 313.
8) Wallis RS et al., Antimicrob Agents Chemother
1999, 43, 2600.
9) Biava M et al., Bioorganic & Medicinal Chemistry, 2010,
18, 8076.
10) Biava M et al., ChemMedChem, submitted.
Publications
Biava M, Porretta GC, Poce G, Battilocchio C,
Alfonso S, De Logu A, Serra N, Manetti F, Botta
M. Identification of a novel pyrrole derivative
endowed with antimycobacterial activity and protection
index comparable to that of the current
antitubercular drugs streptomycin and rifampin,
Bioorg Med Chem. 2010, 18: 8076–84. doi: 10.1016/j.
bmc.2010.09.006.

Principal investigator: Roberto Di Santo
Professor of Farmaceutical Chemistry and Toxicology
Dipartimento di Chimica e Tecnologie del Farmaco
Tel: (+39) 06 49913150; Fax: (+39) 06 491491
roberto.disanto@uniroma1.it
131
AREA 6
Pyrrolyl diketo hexenoic acid derivatives as novel anti-HIV agents
targeted to the ribonuclease H function of the HIV-1 reverse
transcriptase enzyme
Participants:
Roberta Costi, professor; Gaetano Miele, post-doc fellow;
Giuliana Cuzzucoli Crucitti, PhD student; Federica Rosi,
Alberto Iacovo, graduate students; Giovanni Santilli, technician.
Collaborations:
Università di Napoli “Federico II” (Prof. Ettore Novellino,
Dr. Luciana Marinelli); Università di Cagliari (Prof. Enzo
Tramontano); NCI at Bethesda, NIH, Bethesda, USA (Prof. Yves
Pommier); ChiefKatholieke Universiteit Leuven, Belgium (Prof.
Christophe Pannecouque); Politechnika Lodzka, Poland (Prof.
Grzegorz Bujacz).
Report of activity
The HIV-1 RT-associated ribonuclease H (RNase H)
function is a validated and very attractive new target
for HIV/AIDS drug development (1-3); up to today
no drug against the HIV-1 RT-associated RNase
H is i) approved for therapy, ii) under evaluation in
clinical trial, iii) under later stages of pre-clinical
evaluation. The current available information on the
3D structure of the HIV-1 RT (comprising its RNase
H domain) give a solid support for drug development
by both in silico screening and lead compound
optimization through docking studies and the first
crystal structure of the HIV-1 RT with an inhibitor
bound to the RNase H domain has been very recently
reported by an american academic team (3).
Recently, two interesting DKA derivatives have been
reported. The first derivative, 4-[5-(benzoylamino)
thien-2-yl]-2,4-dioxobutanoic acid (BTDBA), originally
synthesized for HIV-1 IN inhibition, has been
shown to inhibit the HIV-1 RT RNase H function
without affecting its polymerase activity (4). BTDBA
provided the proof of concept for direct inhibition of
the HIV-1 RT RNase H associated activity by DKAs,
even though it was not highly selective for RNase H
since i) it inhibited in the same concentration range
also the HIV-1 IN in enzyme assays and ii) it did not
block the viral replication in cell-based assays.
The second DKA derivative, 6-[1-(4-fluorophenyl)
methyl-1Hpyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid
ethyl ester (RDS 1643) was recently reported by
our research group, has been reported to have anti-
RNase H activity, as well BTDB (5). RDS 1643,
inhibited the HIV-1 RNase H activity in enzyme
assays with an IC 50 value of 13 µM, showing lower
potency if compared to BTDBA. It did not affect neither
the HIV-1 RDDP function nor the AMV and E.
coli RNase H activity, while it inhibited the HIV-1 IN
reaction (IC 50 = 90 µM). Differently from BTDBA,
RDS 1643 inhibited the replication of wild type
HIV-1 in cell-based assays showing an EC 50 value of
13 µM and a CC 50 value of 63 µM. Furthermore, RDS
1643 blocked the replication of three HIV-1 NNRTI
resistant viral mutants (RT mutations were Y181C;
K103N/Y181C; K103R/V179D/P225H) with EC 50
values ranging from 7 to 19 µM. Mode of action
studies demonstrated that the RDS 1643 maximum
adsorbance shifted in the presence of the Mg 2+ ions,
suggesting a mechanism of action similar to that
shown by BTDBA. Recent molecular modeling studies
supported the hypothesis that RDS1643 may bind
to the HIV-1 RNase H domain similarly to BTDBA
(7). However, RDS 1643 would not reach towards
the p51 subunit as far as BTDBA, showing therefore
a less favorable interaction with RT, consistently
with the lower potency of RDS 1643 inhibition as
compared to BTDBA inhibition. Therefore, even
though no straight demonstration that RDS 1643
inhibits the RNase H activity inside the cells has
been published yet, these docking results support the
claim that RDS 1643 inhibits the HIV-1 replication
through the selective inhibition of the RT-associated
RNase H function.
Since RDS 1643 was originally designed as HIV-1
IN inhibitor (8), a few RDS 1643 derivatives were
synthesized by our research group and assayed on
both HIV-1 RT-associated enzyme activities. Firstly,
20 derivatives (PDKAs) with different substituents
on the benzyl portion linked to the 1-position of

Roberto Di Santo
the pyrrole ring were synthesized and tested on i)
RT enzyme to determine their ability to inhibit its
RNase H function, and ii) infected cells with HIV-1
and HIV-2 (Table 1).
All tested compounds within this series were found
active against RNase H. Moreover, among the tested
DKA derivatives, both ester and acid derivatives
showed comparable anti-RNase H activities. The
compounds reported in Table 1 were all mono-substituted
on the benzyl ring. It is worthy to note that
the activities seem to be not highly dependent by
the nature or position of the substituent. In fact, the
IC 50 s are still contained within a fairly narrow range
(2.5-26 µM). The above compounds were tested also
in cell-based assays regarding their antiretroviral
activities against both HIV-1 and HIV-2 viruses,
to assess whether the enzyme inhibition also corresponded
to an ex vivo activity. It has been noted
that, in general, the anti-HIV activity is higher than
the inhibition of RNase H. So we hypothesize that
inhibition of further targets could be involved in the
antiretroviral activity, and decided to test these compounds
also against IN in enzyme assays. This was
conceived as a logical approach since many RNase H
inhibitors, including aryl diketo acids, showed activity
also against IN (Test by Pommier, NIH). The data
that we obtained, confirmed that these compounds
can be considered as dual inhibitors (Table 1).
Therefore, we designed and synthesized new PDKAs
to better understand SAR within this class of derivatives,
to obtain both dual and selective inhibitors.
The RNase H selective inhibitors will be particularly
useful to understand the mechanism of action
of this function of RNase H that is far to be totally
elucidated. Three of the most potent compound are
under investigation about their mechanism of action
and under studies to obtain crystal structure of the
complex RNase H-PDKHA inhibitor. This information
could be useful to define the structural elements
in this series that can move their activity to one or
other of the two targets. Thus, further 30 derivatives
mono- and di-substituted on the benzene ring have
been obtained by means of parallel synthesis (structure
A). After this step about 100 inhibitors have
been designed characterized by more pronounced
structural modifications, whose structures are shown
in Table 2 (selected derivatives).
These compounds have been designed also based
on information obtained by RX and MM studies
reported in literature, which described the interac-
132
tions between various RNase H inhibitors, including
RDS 1643, and the catalytic site of RNase H.
We obtained excellent dual inhibitors, in particular
derivative RDS 1610 active on both targets and
also in cell-based assays against HIV-1 and HIV-2
viruses. Additional studies are currently ongoing
that will certainly allow to design the next generation
of RNase H inhibitors to be considered as new
preclinical trial drug candidates.
Finally, due to the discovery of dual inhibitors
activity, we decide to test also more than 100 quinolonyl
diketo acid derivatives that were previously
described by us as IN inhibitors, and found a number
of hit with good anti-RNase H activities. These
compounds will be developed soon as novel RNase
H inhibitors. During this screening also an activator
of RNase H was identified, which is endowed of
antiretroviral activity.
References
1) De Clercq E. J Med Chem. 48: 1297-1313 (2005).
2) Tramontano E. Mini Rev Med Chem. 6: 727-737
(2006).
3) Himmel DM, Serafianos SG, Dharmasena S,
Hossain MM, McCoy-Simandle K, Ilina T, Clark
Jr AD, Knight JL, Julias JG, Clark PK, Krogh-
Jespersen K, Levy RM, Hughes SH, Parniak MA,
Arnold E. ACS Chem Biol. 1: 702-712 (2006).
4) Beutler J, Legrice SFJ, Budihas SR, Wamiru A,
Gardella R, Wilson J, Goncharova K. PCT Int
Appl. 2006026619 A2 (2006).
5) Tramontano E, Esposito F, Badas R, Di Santo R,
Costi R, La Colla P. Antiv Res. 65: 117-124 (2005).
6) Mizrahi V, Brooksbank R, Nkabinde N. J Biol
Chem. 269: 19245-49 (1994).
7) Klumpp K, Mirzadegan T. Curr Pharm Des. 12:
1909-22 (2006).
8) Di Santo R, Costi R, Artico M, Ragno R, Greco G,
Novellino E, Marchand C, Pommier Y. Farmaco.
60: 409–417 (2005).
Pubblications
Di Santo R. Natural products as antifungal agents
against clinically relevant pathogens. Nat Prod Rep.
2010, 27: 1084-1098. doi: 10.1039/b914961a.
Tramontano E, Di Santo R. HIV-1 RT-associated
RNase H function inhibitors: recent advances in drug
development. Curr Med Chem. 2010, 17: 2837-2851.

Cpd
RDS
R 1
R
IC 50 RH
(µM)
IC 50 IN
(ST)(µM)
133
IC 50 HIV-1
(µM)
IC 50 HIV-2
(µM)
CC 50
(µM)
1698 H Et - 15 1.46±0.03 2.32±0.53 24.00±7.58
1699 H H 15 0.09 0.83±0.49 2.11±0.33 66.55±7.45
1683 2-F Et 6.3 0.98 1.20±0.21 2.70±0.32 16.90±3.28
1684 2-F H 6.4 0.98
1743 3-F Et 9.0 11 5.39±4.75 ≥6.86 43.95±15.89
1744 3-F H - 0.92 2.61±1.77 9.52±1.81 64.30±4.85
1643 4-F Et 8.0 2.3 1.70±0.06 Borderline 16.78±5.64
1644 4-F H 2.5 0.026 1.71±0.42 3.09±0.66 60.33±9.13
1759 2-Cl Et 9.8 8.0 ≥2.86 ≥13.73 13.73±1.33
1760 2-Cl H 5.0 4.0 4.19±3.63 7.73±0.86 52.83±11.40
1713 4-Cl Et 8.0 42 ≥14.78 ≥14.78 14.78±1.37
1714 4-Cl H 5.0 ≥4.1 ≥59.28 ≥59.28 59.28±8.74
1687 2-Me Et 3.0 32 ≥2.23 ≥13.48 13.48±1.43
1688 2-Me H 26 0.17 1.56±0.31 3.19±1.00 14.43±2.52
1736 3-Me Et 9.6 8.0 2.17±0.36 ≥25.63 25.63±19.6
1737 3-Me H 4.6 1.3 4.66±2.84 8.35±1.08 72.53±2.96
1692 4-Me Et 7.3 ≥111 ≥12.30 ≥12.30 12.30±1.54
1693 4-Me H 17 1.2 ≥13.33 ≥13.33 13.33±1.52
1703 4-NO 2 Et 12 - ≥59.98 ≥59.98 59.98±5.57
1715 4-OMe Et 6.7 110 ≥13.28 ≥13.28 13.28±1.09
1716 4-OMe H 3.0 4.1
Table 1 - Anti-RNase H and antiretroviral activities of mono-substituted PDKHAs derivatives.
AREA 6
Cpd Str R1 R IC (µM)IN 50 IC (µM)RH 50 IC (µM)HIV1 50 IC (µM)HIV2 50 CC (µM) 50
1610 B =O Et 0.31 10 1.53 2.32 30
1711 C - Et ≥100 3.0 ≥13.80 ≥13.80 13.80
1712 C - H 0.73 7.0 7.30 11.00 43.05
1790 D Me H - 28 ≥13.23 ≥13.23 13.23
1791 D Me Et - 20 ≥13.45 ≥13.45 13.45
2400 E 4-F Et 90.00 55 >50
2401 E 4-F H 26.00 69 >50
2200 F 4-F Et 1.6 > 100 >50
2201 F 4-F H 0.024 41 >50
2600 G 4-F H >111 64 >50
2231 H 4-F Et 0.79 6 3.9
2232 H 4-F H 0.019 14 >50
Table 2 - Activities of selected new PDKA derivatives.

Design, synthesis and biological evaluation of small molecule
epigenetic modulators: a novel approach for anticancer,
antifungal, and antiviral chemotherapy
Principal investigator: Antonello Mai
Professor on Medicinal Chemistry
Dipartimento di Chimica e Tecnologie del Farmaco
Tel: (+39) 06 49913392; Fax: (+39) 06 491491
antonello.mai@uniroma1.it
Participants:
Giovanna Simonetti, professor; Rino Ragno, researcher;
Dante Rotili, Sergio Valente, Silvia Simeoni, post-doc
students; Antonia Caroli, Giorgia Botta, Domenico
Tarantino, PhD students.
Collaborations:
Università di Siena (Prof. Silvio Massa), University of Innsbruck,
Austria (Prof . Gerald Brosch), II Università di Napoli (Prof.
Lucia Altucci), Università di Milano & Istituto Europeo di
Oncologia, Milano (Prof. Saverio Minucci), University of Texas,
USA (Prof. M. T. Bedford, Dr. Donghang Cheng).
Report of Activity
HDAC inhibitors. In cancer cells, HDAC inhibition
causes histone hyperacetylation and leads to
transcriptional activation of genes associated with
growth arrest, terminal differentiation, and/or apoptosis,
both in vitro and in vivo. A number of HDAC
inhibitors is actually in >100 clinical trials, alone or
in combination, for the treatment of several types
of cancer, and two of them, vorinostat (SAHA) and
romidepsin (FK-228) have been approved by FDA
for the treatment of cutaneous T cell lymphoma
(CTCL). Since 2001 we reported various series of
hydroxamate compounds as HDACi, from pyrrole
derivatives to uracil-based and cinnamyl hydroxamates.
In this Project, we continued our searches focusing
on class I- and class II-selective HDAC inhibitors
(HDACi). A) Class I-selective HDACi. MS-275 is
a class I-selective HDACi from the Bayer Schering
AG Pharma group, that inhibits tumor growth in
vivo and promotes differentiation or apoptosis. Since
the main chemical difference between MS-275 and
vorinostat (a pan-HDACi) is the zinc-binding-group
(the 2-aminoanilide in MS and the hydroxamate
in vorinostat), we prepared a number of 2-aminoanilide
analogues in addition to new hydroxamates.
Among the newly synthesized aroylamino-cinnamyl
135
AREA 6
hydroxamates, the majority of them behaved as class
I-selective HDACi. Moreover, they showed high
apoptosis induction (higher than vorinostat) and low
differentiating activity (U937 cells). Differently, the
cinnamic 2-aminoanilides showed class I HDACselectivity
joined to low apoptosis and high cytodifferentiation
effects, similar to that of MS-275.
Among the uracil-based 2-aminoanilides, the most
potent derivatives inhibited class I and IIb HDACs
but not class IIa, displaying high antiproliferative
and dose-dependent granulocytic differentiation on
U937 cells. B) Class II-selective HDACi. In cancer
therapy, inhibition of class I HDACs is clinically
relevant, nevertheless a role of class II HDACs in
cancer cannot be ruled out. Our class II-selective
HDACi (MC1568 and MC1575) have been extensively
used to dissect and to elucidate the biological
role of class II HDACs in some conditions. In human
breast cancer, MC1568 and MC1575 displayed antiproliferative
activity without induction of apoptosis
or cytodifferentiation. In thyroid carcinoma,
MC1568 was ineffective whereas class I-selective
HDACi (MS-275) reduced TRAIL degradation by
interfering with the proteasomal pathway. In muscle
cells, MC1568 arrest myogenesis through reduction
of MEF2D expression and stabilisation of the
HDAC3-HDAC4-MEF2D complex. Other applications
involving MC1568 have been described by
us in HIV-1 induction from latency, inflammation
(TNF-α expression during LPS stimulation), RAR-
and PPARγ-induced adipocyte differentiation, chromatin
folding in HUVEC through the NO-PP2Aclass
II HDACs pathway, and muscular (DMD) and
neurodegenerative (Huntington) diseases.
SIRT modulators. In a search for potent sirtuin inhibitors
we prepared a series of sirtinol analogues by
studying the replacement of the benzamide linkage
of the prototype with other bioisoster such as anilide,
sulfonamide, sulfide, 1-oxopropyl groups. These new
compounds were evaluated against human SIRT1

Antonello Mai
and SIRT2, and their effects in nematodes expressing
a muscular dystrophy protein have been described.
Moreover, the new compounds were tested in U937
cells showing high apoptosis induction and/or cytodifferentiation.
One of them, salermide, was well tolerated
by mice at concentrations up to 100 µM, and prompted
tumor-specific apoptosis in a wide range of human
cancer cell lines. Genetic p53 knockdowns showed that
the SIRT1-dependent pro-apoptotic effect of salermide
is p53-independent, but it is due to the reactivation of
pro-apoptotic genes epigenetically repressed exclusively
in cancer cells by SIRT1. In addition, we designed
some barbituric and thiobarbituric acid analogues to
evaluate as SIRTi and anticancer agents. When tested
as SIRT1 and SIRT2 inhibitors, only the tetracyclic
compound MC2141 showed high and selective SIRT1
inhibiting activity joined to high apoptosis induction
on U937 cells. Searching new putative SIRTi based
on the nicotinamide scaffold, we identified some SIRT
activators, able to increase SIRT1 as well as SIRT2
activity. Cellular screening (on U937 at 50 µM) did
not show any significant apoptosis- or differentiationinducing
properties, but such new SIRT activators
showed anti-senescence effect as well as activation of
PGC1α and increase of mithocondrial effects similar
as resveratrol.
HMT and HD inhibitors. In addition to histone and
non-histone protein acetylation, histone methylation
has also been shown to be important in epigenetic
regulation of gene expression. Histone methylation
can occur on both Lys (K, by HKMTs) or Arg (R,
by PRMTs) residues. Several Lys residues have been
found to undergo mono-, di-, or tri-methylation, the
result of the chemical modification being transcriptional
activation as well as silencing. In 2008 we
prepared and tested some bis(3-bromo-4-hydroxy-
and 3,5-dibromo-4-hydroxyphenyl) compounds and
their analogues against PRMT1, CARM1/PRMT4,
SET7 (a HKMT), p300/CBP (an HAT enzyme),
SIRT1, and SIRT2. Depending on the extension of
bromination of the molecule and on the nature of the
linker connecting the two dibromophenol moieties
some of such compounds behaved as epigenetic multiple
ligands (epi-MLs), they being active against all
the tested enzymes. The H3K4 demethylase LSD1
has been shown to be up-regulated in high-risk
tumors. Very few molecules have been reported
as able to inhibit the LSD1 demethylating action.
Starting from the report of the LSD1 inhibiting
activity of the tranylcypromine (TCPA), we prepared
two series of TCPA derivatives by inserting
at its benzene ring an amide or amino acid function.
136
Among such compounds, MC2580 showed K i = 1.3
µM against LSD1 (~ 200-fold more potent than
TCPA) and was inactive against MAO-B. MC2580
also dramatically enhanced the efficacy of RA on
growth and differentiation of acute promyelocytic
leukemia cells, including primary murine APL blasts.
Publications
Cirilli R, Ferretti R, La Torre F, Borioni A, Fares
V, Camalli M, Faggi C, Rotili D, Mai A. Chiral
HPLC separation and absolute configuration of
novel S-DABO derivatives. Chirality. 2009, 21: 604-
12. doi: 10.1002/chir.20654.
Lara E, Mai A, Calvanese V, Altucci L, López-Nieva
P, Martínez-Chantar ML, Varela-Rey M, Rotili
D, Nebbioso A, Ropero S, Montoya G, Oyarzabal
J, Velasco S, Serrano M, Witt M, Villar-Garea
A, Imhof A, Mato JM, Esteller M, Fraga MF.
Salermide, a Sirtuin inhibitor with a strong cancerspecific
proapoptotic effect. Oncogene. 2009, 28: 781-
91. doi:10.1038/onc.2008.436.
Mai A, Altucci L. Epi-drugs to fight cancer: From
chemistry to cancer treatment, the road ahead. Int
J Biochem Cell Biol. 2009, 41: 199-213. 10.1016/j.
biocel.2008.08.020.
Mai A, Rotili D, Tarantino D, Nebbioso A, Castellano
S, Sbardella G, Tini M, Altucci L. Identification of
4-hydroxyquinolines inhibitors of p300/CBP histone
acetyltransferases. Bioorg Med Chem Lett. 2009, 19:
1132-5. doi: 10.1016/j.bmcl.2008.12.097.
Mai A, Rotili D, Valente S, Kazantsev AG. Histone
deacetylase inhibitors and neurodegenerative disorders:
holding the promise. Curr Pharm Des. 2009, 15:
3940-57.
Mai A, Valente S, Meade S, Carafa V, Tardugno M,
Nebbioso A, Galmozzi A, Mitro N, De Fabiani E,
Altucci L, Kazantsev A. Study of 1,4-dihydropyridine
structural scaffold: discovery of novel sirtuin activators
and inhibitors. J Med Chem. 2009, 52: 5496-
5504. doi: 10.1021/jm9008289.
Mai A, Valente S, Nebbioso A, Simeoni S, Ragno R,
Massa S, Brosch G, De Bellis F, Manzo F, Altucci
L. New pyrrole-based histone deacetylase inhibitors:
Binding mode, enzyme- and cell-based investigations.
Int J Biochem Cell Biol. 2009, 41: 235-47.
doi:10.1016/j.biocel.2008.09.002.

Naldi M, Calonghi N, Masotti L, Parolin C, Valente
S, Mai A, Andrisano V. Histone post-translational
modifications by HPLC-ESI-MS after HT29 cell
treatment with HDAC inhibitors. Proteomics. 2009,
9: 5437-45. doi: 10.1002/pmic.200800866.
Nebbioso A, Manzo F, Miceli M, Conte M, Manente
L, Baldi A, De Luca A, Rotili D, Valente S, Mai A,
Usiello A, Gronemeyer H, Altucci L. Selective class
II HDAC inhibitors impair myogenesis by modulating
the stability and activity of HDAC-MEF2 complexes.
EMBO Rep. 2009, 10: 776-82. doi: 10.1038/
embor.2009.88.
Rotili D, Simonetti G, Savarino A, Palamara AT,
Migliaccio AR, Mai A. Non-cancer uses of histone
deacetylase inhibitors: effects on infectious diseases
and beta-hemoglobinopathies. Curr Top Med Chem.
2009, 9: 272-91.
Binda C, Valente S, Romanenghi M, Pilotto S, Cirilli
R, Karytinos A, Ciossani G, Botrugno OA, Forneris
F, Tardugno M, Edmondson DE, Minucci S, Mattevi
A, Mai A. Biochemical, structural, and biological
evaluation of tranylcypromine derivatives as inhibitors
of histone demethylases LSD1 and LSD2. J Am
Chem Soc. 2010, 132: 6827-6833.
Borbone E, Berlingieri MT, De Bellis F, Nebbioso
A, Chiappetta G, Mai A, Altucci L, Fusco A. Histone
deacetylase inhibitors induce thyroid cancer-specific
apoptosis through proteasome-dependent inhibition
of TRAIL degradation. Oncogene. 2010, 29: 105-116.
doi:10.1038/onc.2009.306.
Castellano S, Milite C, Ragno R, Simeoni S, Mai
A, Limongelli V, Novellino E, Bauer I, Brosch G,
Spannhoff A, Cheng D, Bedford M T, Sbardella G.
Design, synthesis and biological evaluation of carboxy
analogues of arginine methyltransferase inhibitor
1 (AMI-1). ChemMedChem. 2010, 5: 398-414. doi:
10.1002/cmdc.200900459.
Colussi C, Banfi C, Brioschi M, Tremoli E, Straino
S, Spallotta F, Mai A, Rotili D, Capogrossi MC,
Gaetano C. Proteomic profile of differentially
expressed plasma proteins from dystrophic mice and
following suberoylanilide hydroxamic acid treatment.
Proteomics Clin Appl. 2010, 4: 71-83. doi:
10.1002/prca.200900116.
Colussi C, Berni R, Rosati J, Straino S, Vitale S,
Spallotta F, Baruffi S, Bocchi L, Delucchi F, Rossi S,
137
AREA 6
Savi M, Rotili D, Quaini F, Macchi E, Stilli D, Musso
E, Mai A, Gaetano C, Capogrossi MC. The histone
deacetylase inhibitor suberoylanilide hydroxamic
acid reduces cardiac arrhythmias in dystrophic mice.
Cardiovasc Res. 2010, 87: 73-82. doi: 10.1093/cvr/
cvq035.
Colussi C, Illi B, Rosati J, Spallotta F, Farsetti A,
Grasselli A, Mai A, Capogrossi M C, Gaetano C.
Histone deacetylase inhibitors: keeping momentum
for neuromuscular and cardiovascular diseases treatment.
Pharmacol Res. 2010, 62: 3-10. doi: 10.1016/j.
phrs.2010.02.014.
Musmuca I, Caroli A, Mai A, Kaushik-Basu N, Arora
P, Ragno R. Combining 3-D quantitative structureactivity
relationship with ligand based and structure
based alignment procedures for in silico screening of
new hepatitis C virus NS5B polymerase inhibitors.
J Chem Inf Model. 2010, 50: 662-76. doi:10.1021/
ci9004749.
Nebbioso A, Dell’Aversana C, Bugge A, Sarno R,
Valente S, Rotili D, Manzo F, Teti D, Mandrup S,
Ciana P, Maggi A, Mai A, Gronemeyer H, Altucci
L. HDACs class II-selective inhibition alters nuclear
receptor-dependent differentiation. J Mol Endocrinol.
2010, 45: 219-28. doi: 10.1677/JME-10-0043.
Palacios D, Mozzetta C, Consalvi S, Caretti G,
Saccone V, Proserpio V, Marquez V E, Valente S,
Mai A, Forcales S V, Sartorelli V, Puri P L. TNF/
p38α/polycomb signaling to Pax7 locus in satellite
cells links inflammation to the epigenetic control of
muscle regeneration. Cell Stem Cell. 2010, 7: 455-69.
doi: 10.1016/j.stem.2010.08.013.
Pasco M Y, Rotili D, Altucci L, Farina F, Rouleau G
A, Mai A, Néri C. Characterization of sirtuin inhibitors
in nematodes expressing a muscular dystrophy
protein reveals muscle cell and behavioral protection
by specific sirtinol analogues. J Med Chem. 2010, 53:
1407-11. doi: 10.1021/jm9013345.
Quinti L, Chopra V, Rotili D, Valente S, Amore A,
Franci G, Meade S, Valenza M, Altucci L, Maxwell
MM, Cattaneo E, Hersch S, Mai A, Kazantsev A.
Evaluation of histone deacetylases as drug targets
in Huntington’s disease models. Study of HDACs
in brain tissues from R6/2 and CAG140 knock-in
HD mouse models and human patients and in a neuronal
HD cell model. PLoS Curr. 2010, Sep 22. pii:
RRN1172. doi: 10.1371/currents.RRN1172.

Antonello Mai
Rotili D, Tarantino D, Carafa V, Lara E, Meade S,
Botta G, Nebbioso A, Schemies J, Jung M, Kazantsev
AG, Esteller M, Fraga MF, Altucci L, Mai A.
Identification of tri- and tetracyclic pyrimidinediones
as sirtuin inhibitors. ChemMedChem. 2010, 5: 674-77.
doi: 10.1002/cmdc.201000030.
Ruotolo R, Tosi F, Vernarecci S, Ballario P, Mai
A, Filetici P, Ottonello S. Chemogenomic profiling
of the cellular effects associated with histone H3
acetylation impairment by a quinoline-derived compound.
Genomics. 2010, 96: 272-80. doi:10.1016/j.
ygeno.2010.08.005.
Spallotta F, Rosati J, Straino S, Nanni S, Grasselli A,
Ambrosino V, Rotili D, Valente S, Farsetti A, Mai
A, Capogrossi MC, Gaetano C, Illi B. Nitric oxide
determines mesodermic differentiation of mouse
138
embryonic stem cells by activating class IIa histone
deacetylases: potential therapeutic implications in a
mouse model of hindlimb ischemia. Stem Cells. 2010,
28: 431-42 doi: 10.1002/stem.300.
Thaler F, Varasi M, Colombo A, Boggio R, Munari
D, Regalia N, Rozio MG, Reali V, Resconi AE, Mai
A, Gagliardi S, Dondio G, Minucci S, Mercurio C.
Synthesis and biological characterization of amidopropenyl
hydroxamates as HDAC inhibitors
ChemMedChem. 2010, 5: 1359-1372. doi: 10.1002/
cmdc.201000166.
Zhu H, Shan L, Schiller PW, Mai A, Peng T. Histone
deacetylase-3 activation promotes TNF-{alpha}
expression in cardiomyocytes during lipopolysaccharide
stimulation. J Biol Chem. 2010, 285: 9429-9436.
doi: 10.1074/jbc.M109.071274.

Principal investigator: Elena Mattia
Professor in Microbiology
Dipartimento di Scienze di Sanità Pubblica
Tel: (+39) 06 49914608; Fax: (+39) 06 49914626
elena.mattia@uniroma1.it
139
AREA 6
Effects of resveratrol on Epstein-Barr Virus latent and lytic phases
of infection
Participants:
Livia Di Renzo, researcher; Alessandra De Leo, Giulia
Matusali, PhD students; Giuseppe Arena, graduate student;
Egidio Lacanna, Claudia Stecca, Giorgio Oliviero,
undergraduate students.
Collaborations:
Dipartimento di Biologia e Biotecnologie “C. Darwin”, Sapienza-
Università di Roma (Prof. Enzo Marchetti); The Wistar Institute,
Philadelphia, USA (Prof. Paul M. Lieberman).
Report of activity
EBV, the causative agent of infectious mononucleosis,
is associated with a variety of different human
tumors including Burkitt’s lymphoma (BL), nasopharyngeal
carcinoma (NPC), Hodgkin’s desease
(HD) and post transplant lymphoproliferative disorders
(PTLDs) occurring in immunocompromized
patients. In all these malignancies the virus establishes
a latent infection characterized by different
EBV gene expression profiles. Three latency programs
have been described depending on the selective
expression of six nuclear (EBNAs) and three
membrane antigens (LMPs). EBV lytic cycle activation
which results in the production of infectious
particles, is initiated by the two immediate early proteins
BZLF1 (ZEBRA or Zta) and BRLF1 (Rta) that
function as transcriptional activators of EBV early
genes. In vitro, latency can be disrupted by a variety
of different agents such as phorbol esters, sodium
butyrate, TGFβ, anti-immunoglobulins (anti-IgG)
and calcium ionophores.
Resveratrol (3,4’,5-trihidroxystilbene; RV), a polyphenolic
phytoalexin has been reported to have cardioprotective,
anti-oxidant, anti inflammatory, antibacterial
and anti-viral activity. In addition, RV was
found to inhibit proliferation and induce apoptosis in
several human tumor cells.
Because EBV infection might confer additional sur-
vival potential to malignant B cells, in this study, we
have examined the antiproliferative activity of RV on
BLs with different forms of restricted virus latency.
Thus, in vitro systems enforcing the full Latency III,
Latency II-like and Latency I program in a BL cell
background, we have investigated the contribution
that different patterns of EBV gene expression give
to RV-induced susceptibility to apoptosis and studied
the molecular mechanisms underneath. In addition,
the antiviral effects of resveratrol on viral replication
have been studied. EBV lytic cascate was activated
in Raji cells by introduction of the BZLF1 expression
vector while in Akata cells EBV lytic cycle was
induced by cross-linking surface immunoglobulin G
(IgG) with anti-Ig , a method which mimics in vivo
activation of the B-cell receptor.
Resveratrol and EBV latent infection
As we aimed to determine whether EBV gene expression
played a role on the susceptibility of BL cells to
RV, we tested two EBV (-) (2A8 and Ramos) and
three EBV(+) BL cell lines, the latter expressing different
patterns of EBV latency genes. In particular,
Raji cells express all EBV latency genes (latency III),
Jijoye M13, a subline of Jijoye cells which is classified
as group II-like do not express LMP1, and Akata
cells show a Latency I phenotype. All cell lines were
exposed to concentrations of the polyphenol varying
from 20 to 300 µM for different periods of time.
We observed that that RV inhibited proliferation of
each cell line in a dose and time-dependent manner.
To investigate the antiproliferative effect of RV on
BL cells, we analyzed the cell cycle distribution after
incubation with the polyphenol and found that a 12
hours treatment with RV increased significantly the
percentage of cells in the G1 phase with a reduction
of those in S and G2/M phases. Cell cycle arrest
by RV treatment was associated with apoptosis as
revealed by Annexin V binding and PARP cleavage
tests. However, our data indicated that EBV(-)
BL cells and latency I Akata were more prone to

Elena Mattia
RV-induced apoptosis than Latency II and Latency
III cell lines. We then examined the expression of
several cell cycle and apoptosis-related proteins by
Western blot analysis. Exposure to RV increased
the expression of the cyclin dependent kinase (cdk)
inhibitor p27 and that of cyclin E in all cell lines
while the analysis of the levels of antiapoptotic proteins
revealed a strong down-regulation of the c-myc
oncogene but also that of IAP1, FLIP and XIAP.
Furthermore, RV treatment of BL cell lines strongly
activated p38 and dramatically inhibited ERK1/2,
two MAPKs pathways important in determining
cell proliferation or apoptosis; in addition, it caused
a marked inhibition of NFkB activity in Latency I
and Latency II cell lines but not in Latency III cells.
Therefore, we looked for EBV genes expressed in
the Latency III program that would confer additional
survival advantage against the effects of the
polyphenol. LMP1 is the principal EBV oncogene
with the potential to antagonize apoptosis as well as
to promote cellular survival and proliferation. We
demonstrated that silencing LMP1gene by siRNA
dramatically increased the sensitivity of Latency III
Raji cells to RV.
Resveratrol and EBV lytic infection
Raji cells were electroporated with BZLF1 plasmid
to induce EBV lytic cycle and exposed to RV at concentrations
of 130 or 300 mM for 24 and 48 hours
to evaluate the expression of the immediate early
antigen (IEA) BZLF1 as well as that of different
EBV early genes expressed in the lytic cycle. We
found that at the lower concentration of RV, BZLF1
expression was stimulated while it was inhibited at
the higher one. Nevertheless, both RV concentrations
were effective in suppressing the expression
of several EBV early antigen (EA) examined, such
as BFRF1, BFLF2 and BALF5. Interestingly, RV
inhibited EBV EA expression also when added 4
hours after EBV activation, confirming that the
polyphenol is able to block lytic gene cascade also
downstream of IEA expression.
The inhibition of EBV lytic cycle in Akata cells
was concentration dependent and RV, at 130 mM,
completely suppressed EBV activation as judged
by immunological detection of IEA and EA expression.
Analysis of the corresponding transcripts by
RT-PCR revealed that RV down-regulated EBV
lytic gene expression at the transcriptional level.
Our study also demonstrated that RV inhibition of
EBV lytic cycle, actually decreased the production
140
of mature viral particles. We examined the activity
of transcription factors involved in regulating EBV
gene expression. The results obtained by EMSA
carried out on Akata cells treated with IgG, revealed
that RV negatively affects the binding activity of
NFkB, SP1 and AP1. Moreover, evaluation of the
levels of reactive oxygen species (ROS), reported to
increase during EBV lytic cycle, revealed a drastic
decrement indicating a rapid intracellular uptake of
the compound and a strong reducing activity. The
decrement of ROS appears to be largely responsible
for RV-mediated inhibition of EBV lytic cycle.
Conclusions
The results of this research project show that RV
induces cell cycle arrest and apoptosis of EBVinfected
B cells; however, each form of EBV infection
is associated with a specific degree of protection from
apoptosis, more extended as a larger set of viral genes
is expressed. Moreover, the finding that silencing the
viral oncogene LMP1 decreased the protection of
Latency III cells to RV-induced apoptosis suggests
that the combined use of RV and siRNA directed to
viral genes may configure a novel therapy for the
treatment of EBV-positive B cell malignancies with
a known pattern of viral gene expression.
We found that RV inhibits the transcription of EBV
immediate early and early genes, the expression of
the viral lytic proteins and decreases the production
of viral particles.
The molecular mechanisms underlying RV inhibition
of EBV lytic cycle include a significant reduction
in the activity of transcription factors as well as
redox-dependent epigenetic events involved in the
activation of viral lytic genes expression.
Publications
Matusali G, Arena G, De Leo A, Di Renzo L, Mattia
E. Inhibition of p38 MAP kinase pathway induces
apoptosis and prevents Epstein Barr virus reactivation
in Raji cells exposed to lytic cycle inducing compounds.
Mol Cancer. 2009, 8: 18. doi: 10.1186/1476-
4598-8-18.
De Leo A, Matusali G, Arena G, Di Renzo L,
Mattia E. Epstein-Barr virus lytic cycle activation
alters proteasome subunit expression in Burkitt’s
lymphoma cells. Biol Chem. 2010, 391: 1041-46. doi:
10.1515/BC.2010.107.

Principal investigator: Romano Silvestri
Professor of Medicinal Chemistry
Dipartimento di Chimica e Tecnologie del Farmaco
Tel: (+39) 06 49913800; Fax: (+39) 06 491491
romano.silvestri@uniroma1.it
141
AREA 6
Drug design and synthesis of non-nucleoside inhibitors of both
HIV-1 wild type and resistant mutant strains reverse transcriptase,
and Coxsackie B4 virus
Participants:
Giuseppe La Regina, researcher; Antonio Coluccia,
Francesco Piscitelli, post-doc fellows; Valerio Gatti, PhD
student.
Collaborations:
CNR, Istituto di Genetica Molecolare, Pavia (Dr. Giovanni
Maga); Rega Institute for Medical Research, K.U., Leuven,
Belgium (Dr. Jean Balzarini); Welsh School of Pharmacy,
Cardiff University, UK (Dr Andrea Brancale); IrsiCaixa, Hospital
Germans Trias i Pujol, Badalona, Spain (Prof. José A. Esté).
Report of activity
Human immunodeficiency virus (HIV) was identified
as the causative agent of acquired immunodeficiency
syndrome (AIDS) in 1983. Although significant progress
has been made in the prevention and treatment
of AIDS/HIV infection over the past twenty years,
the number of people living with HIV is constantly
growing. Worldwide, AIDS-related diseases are still
one of the leading causes of death, and are predicted
to cause significantly premature mortality in the
coming decades. Highly active antiretroviral therapy
(HAART) which combines three (recommended) or
four different antiretroviral drugs, can slow down
viral replication and reduce plasma viremia below
the detection level, resulting in a substantial delay
of disease progression. However, long-term HAART
treatments lead to the emergence of complications
due to drug resistance, toxicity and adverse effects.
Therefore, new NNRTIs which display a broad spectrum
of activity against clinically relevant HIV-1
mutant strains are needed.
Our research activity has been focused towards
the development of indolylarylsulfone (IAS) HIV-1
NNRTIs. Structure-activity relationship (SAR)
studies led the identification of three regions of the
IAS scaffold: (A) the spectrum of activity against
mutant HIV-1 strains was significantly expanded by
introduction of two methyl groups at positions 3’ and
5’ of the 3-phenylsulfonyl moiety; (B) potent HIV-1
inhibitors were obtained by coupling the indole-
2-carboxamide with either natural or unnatural
amino acids; (C) IASs bearing the 5-chloro-4-fluoro
substitution pattern at the indole ring were potent
inhibitors of RT WT and RTs carrying the K103N,
Y181I, and L100I mutations.
We synthesized new IASs characterized by (a) natural
and unnatural amino acids at the 2-carboxamide
and (b) different electron-withdrawing substituents
at the positions 4 and 5 of the indole. These compounds
proved to be highly potent against HIV-1
replication in human T-lymphocyte (CEM) cells and
showed inhibitory potencies in the low/subnanomolar
range of concentrations that were comparable
with those of the previously reported lead compounds.
Against HIV-1 WT, the inhibitory activity
seemed only marginally affected by the substituent
introduced on the indole nucleus. In general, none of
the IASs proved to be markedly cytostatic. Against
the L100I and K103N RT HIV-1 strains, five IASs
were always superior to EFV and had similar activity
to that reported for MK-49652 and TMC-120. These
IASs were also equipotent to EFV against the mutant
Y181C RT HIV-1 strain. Several compounds were
inhibitory to Coxsackie B4 virus at EC50 = 3-5 μM
but were much less selective in their anti-Coxsackie
virus activity than observed for HIV. In view that
for picornaviruses, no single antiviral drug has ever
been approved, these agents may serve as basis for
the development of drugs endowed with both anti-
HIV-1 RT and Coxsackie B4 virus inhibitory activities.
In L100I mutated RT, the mutation of leucine
to isoleucine does not affect the binding mode, and
it was consistent with the interactions reported for
the WT strain. The lower antiviral activity against
the mutant K103N and Y181I RT HIV-1 strains
could be the consequence of the reduced interaction
with the mutated residues. For the Y181I RT HIV-1
strain, the limited anti-HIV efficiency could also be

Romano Silvestri
caused by the loss of the favorable protein-ligand π-π
interactions, while in the K103N mutant, the loss of
the hydrophobic interaction between the ligand and
the side chain of the lysine 103 might be responsible
for the reduced activity.
The di-halo-IAS 5-chloro-3-[(3,5-dimethylphenyl)
sulfonyl]-4-fluoro-1H-indole-2-carboxamide showed
improved activity towards the Y181I mutant enzyme
and comparable activity towards the K103N mutant
RT, in comparison with the previously characterized
IAS derivatives, which preferentially associated with
the free enzymatic form of HIV-1 RT. In addition,
this compound showed much slower dissociation rates
from both mutant enzymes with respect to HIV-1 RT
WT and showed a superior activity profile towards
the K103N, L100I and Y181I mutants with respect to
the reference compounds NVP and EFV. The results
obtained by this study, as well as the excellent activity
demonstrated by these inhibitors in blocking the viral
replication in infected cells, suggest a possible use of
these compounds for the therapy of HIV.
Second generation NNRTIs currently under development
are potent inhibitors of HIV-1 WT and the
most clinically relevant HIV-1 mutant strains, and
show inhibitory activities in the nano- or subnanomolar
range of concentration. Despite all compounds
are endowed with potent and (more or less)
similar antiretroviral potency, they do not share a
common binding mode to the NNBS of the RT. In the
past five years, NNRTIs based on the “butterfly-like”
model seemed obsolete with respect to the newer
“horseshoe-like” inhibitors. However, new drug candidates
renewed the interest for the “butterfly-like”
active conformation. In addition, a new concept of
binding interaction with the HIV-1 drug-resistant
mutant strains emerged: the “flexibility hypothesis”.
According to this idea, a powerful inhibition of the
142
mutant variants of the HIV-1 should be correlated
to the ability of the molecule to adopt variable binding
conformations which would be not affected by
the interchanges of the amino acids residues into the
NNBS of the mutant RT. These findings support
the opinion that a newer era for the HIV-1 NNRTIs
is just begun. Future NNRTIs should be capable to
adopt multi binding conformations for a powerful
inhibition of HIV-1 WT and the most relevant drug
resistant mutant strains.
Publications
Piscitelli F, Coluccia A, Brancale A, La Regina G,
Sansone A, Giordano C, Balzarini J, Maga G, Zanoli
Z, Samuele A, Cirilli R, La Torre F, Lavecchia A,
Novellino E, Silvestri R. Indolyl aryl sulfones bearing
natural and unnatural aminoacids. Discovery
of potent inhibitors of both HIV-1 non-nucleoside
wild type and resistant mutant strains reverse transcriptase,
and coxsackie B4 virus. J Med Chem. 2009,
52: 1922-34. doi: 10.1021/jm801470b.
Samuele A, Kataropoulou A, Viola M, Zanolia S, La
Regina G, Piscitelli F, Silvestri R, Maga G. Nonnucleoside
HIV-1 reverse transcriptase inhibitors
di-halo-indolyl aryl sulfones achieve tight binding
to drug-resistant mutants by targeting the enzymesubstrate
complex. Antivir Res. 2009, 81: 47-55. doi:
10.1016/j.antiviral.2008.09.008.
La Regina G, Coluccia A, Silvestri R. Looking for an
active conformation of the future HIV-1 non-nucleoside
reverse transcriptase inhibitors. Antiviral Chem
Chemoth. 2010, 20: 231-7. doi: 10.3851/IMP1607.

AREA 7
Biology of
malaria
and other
vector-borne
diseases

Genetic and phenotypic characterization of species and
“molecular forms” of the Anopheles gambiae complex (Diptera:
Culicidae), afrotropical malaria vectors
Principal investigators: Alessandra della torre - Vincenzo Petrarca
Professors of Parasitology
Dipartimento di Sanità Pubblica e Malattie Infettive - Dipartimento di Biologia e
Biotecnologie “Charles Darwin”
Tel: (+39) 06 49694268, 06 49914932; Fax: (+39 ) 06 49914653
ale.dellatorre@uniroma1.it - vincenzo.petrarca@uniroma1.it
Participants:
Marco Pombi, researcher, Beniamino Caputo, Emiliano
Mancini, Federica Santolamazza, post-doc fellows; Federica
Tammaro, PhD student; Maria Calzetta, graduate student.
Collaborations:
Center for Global Health and Infectious Diseases, Department of
Biological Sciences, University of Notre Dame, IN, USA (Prof. Nora
J. Besansky); Centro de Malária e outras Doenças Tropicais,
Instituto de Higiene e Medicina Tropical, Lisbon, Portugal (Dr.
João Pinto); Dipartimento di Biologia Animale e Genetica,
Università di Firenze (Dr. Francesca Dani, Prof. Stefano
Turillazzi); Institut de Recherche pour le Développement, (France:
Prof. Didier Fontenille; Burkina Faso: Dr. Frédéric Simard;
Cameroun: Dr. Carlo Costantini); Medical Research Council
Laboratories, Fajara, Banjul, The Gambia (Prof. David Conway,
Dr. Davis Nwakanma); Division of Cell and Molecular Biology,
Imperial College London, UK (Prof. F. Catteruccia); Department
of Entomology, Virginia Tech, Blacksburg, VA, USA (Prof. I.
Sharakhov); Lab. Réponse Immunitaire et Développement Chez
les Insectes, CNRS, Strasburg, France (Prof. EA. Levashina);
Vector Group, Liverpool School of Tropical Medicine, Liverpool,
UK (Dr. H. Ranson, Dr. MJ. Donnelly); IRSS/Centre Muraz,
Laboratoire de Parasitologie/Entomologie, Bobo-Dioulasso,
Burkina Faso (Dr. Roch Dabiré); Dipartimento di Biologia
delle Piante Agrarie, Università di Pisa, (Prof. P. Pelosi, Dr.
A. Felicioli); Dipartimento di Medicina Sperimentale e Scienze
Biochimiche, Università di Perugia, Terni (Prof. F. Catteruccia.
Dr. F. Baldini); Dipartimento di Scienze Biochimiche, Sapienza-
Università di Roma, Rome, (Prof. A. Tramontano, Dr. D.
Raimondo, Dr. A. Via).
Report of Activity
Analysis of the genetic and ecological
differentiation of Anopheles gambiae s.s. M
and S molecular forms
Molecular genotyping of M and S forms in area of high
hybridization at the western extreme of their geographical
145
AREA 7
range. The physically unlinked centromeric regions
on all 3 chromosomes of M and S molecular forms
contain fixed nucleotide differences which have been
found in nearly complete linkage disequilibrium in
geographic areas of no or low M-S hybridization.
We applied recently developed diagnostic assays
for detection of SNPs and structural differences
between M and S forms in the 3 centromeric regions
in samples from the western extreme of their range
of sympatry, the only area where high frequencies
of putative M/S hybrids have been reported. The
results revealed a level of admixture not observed in
the rest of the range. In particular, we found: i) heterozygous
genotypes at each marker at frequencies
lower than expected under panmixia; ii) virtually all
possible genotypic combinations between markers
on different chromosomes, although genetic association
was nevertheless detected; iii) discordant M and
S genotypes at two X-linked markers near the centromere,
suggestive of introgression and inter-locus
recombination. These results suggest a secondary
contact zone between M and S, although it is not
possible to completely rule out the hypothesis of
maintenance of ancestral polymorphisms. Overall,
the above findings highlight the complexity and the
variability of the biological and genetic differentiation
of these incipient species. In order to get a more
complete picture of this speciation model, we collected
samples from a west to east transect in Guinea
Bissau in October 2010 which are being currently
identified and genotyped.
Chromosomal characterization of M and S forms in area
of high hybridization at the western extreme of their
geographical range. Floating paracentric inversions
on chromosome-2, probably involved in adaptation
to sub-niches, are shared by the M and S forms,
although with different frequencies of alternative
inverted arrangements. Little is currently known
about the distribution and chromosomal inversion
patterns of molecular forms in the western limits of

Alessandra della Torre – Vincenzo Petrarca
their geographical range. We completed the karyotyping
of A. gambiae s.s. collected in The Gambia in
2005-06, finding that the M form is mostly characterized
by frequencies of 2Rb, 2Rd, 2La inversions
increasing from 40% to 80% from the coast eastwards.
The S form coastal populations are characterized
by karyotypes similar to those of M, while the
eastward populations by more complex arrangements
also based on inversions 2Rj and 2Rbk. These results
are currently being correlated with those from an
extensive microsatellite analysis. Additionally, the M
and S samples collected in October in Guinea Bissau
are currently being identified and karyotyped.
Analysis of genetic/phenotypic differentiation
involved in pre/post-mating behaviour in the
Anopheles gambiae complex
Analysis of A. gambiae antennal proteins. Odourant
Binding Proteins (OBPs) and Chemo-Sensory
Proteins (CSPs) are proteins involved in the detection
of odour molecules and in the activation of odorant
receptors. Based on the published genome of A.
gambiae, 57 genes encoding for putative OBPs and 7
encoding for CSPs have been identified. So far, the
expression pattern of these genes has been almost
exclusively investigated by genomic approaches:
some OBPs have been found ubiquitously, while
others have been shown to be specific of the olfactory
organs. The later genes are likely to encode
for OBPs relevant to olfactory processes involved
in behaviour, such as the mating partner choice or
the search for sugar-sources and, for females, the
search and selection of hosts for blood-meal or
of appropriate oviposition sites. Traditional proteomic
approaches have been proved difficult to
be performed on mosquito antennae, due to low
concentration of OBPs and CSPs, and difficulties in
obtaining sufficient material for 2-dimensional gels.
We have exploited an innovative shotgun proteomic
approach to analyse OBPs and CSP expression in A.
gambiae antennae. The sensitivity and efficiency of
this approach is significantly higher than that based
on the traditional 2-dimensional gel analysis: we
identified 2.390 proteins in male and female antennal
samples, 28 of which were olfactory soluble proteins,
and showed that some OBPs are significantly more
expressed in females than in males.
Moreover, to clarify the role of some OBPs in A.
gambiae, we have expressed six of these proteins
146
and investigated their ligand-binding properties.
Ligand-binding experiments revealed marked differences
between these OBPs. In situ hybridisation has
revealed that some OBPs are co-expressed in antennal
sensilla of A. gambiae, suggesting that OBPs also
in mosquitoes might form complexes with novel
ligand specificities, thus amplifying the repertoire of
OBPs and the number of semiochemicals that could
be discriminated.
Analysis of differentiation of genes involved in postmating
behaviour in the An. gambiae complex. In A.
gambiae male accessory glands (MAG) products are
transferred to females upon mating as a mating plug
that induces a series of physiological post-mating
responses in the female lower reproductive tract
(LRT). We analysed the molecular evolution of
two clusters of 3 LRT- and 3 MAG- specific genes
potentially involved in post-mating mechanisms in 5
species of the A. gambiae complex.
MAG-specific paralog genes Agap009369 and
Agap009370 were shown to be highly conserved
among those 5 species. Moreover, we identified at
3rd gene, clustering with the former two, which is
not annotated in the A. gambiae genome: intriguingly,
this paralog gene shows signs of positive
selective pressure and purifying selection maintaining
lineage-specific products.
The 3 LRT-specific genes analyzed encode serineproteases
that are down-regulated after mating, 2
of which are expressed in the atrium and interact
with the mating plug and 1 in the spermatheca. The
analysis of polymorphisms revealed a high level of
replacement polymorphisms consistent with relaxed
evolutionary constraints of duplicated genes, allowing
to rapidly fix novel replacements to perform new
or more specific functions. Adaptive evolution was
detected in several codons of the 3 genes and evidence
of episodic selection were also found. In addition,
the structural modeling of these proteases highlighted
some important differences in their substrate
specificity, and provided evidence that a number of
sites evolving under selective pressures lie relatively
close to the catalytic triad and/or on the edge of the
specificity pocket, known to be involved in substrate
recognition or binding. The observed patterns suggest
that these proteases may interact with factors
transferred by males during mating and that differently
evolved in independent A. gambiae lineages.

Immune responses to malaria and autoimmune disorders:
investigating common gene-regulatory networks
Principal investigators: David Modiano
Professor of Parasitology
Dipartimento di Sanità Pubblica e Malattie Infettive
Tel: (+39) 06 49914933, 06 33775629; Fax: (+39 ) 06 49914653
david.modiano@uniroma1.it
Participants:
Valentina Mangano, Pamela Avellino, Cinzia Rizzo, postdoc
fellows.
Collaborations:
Wellcome Trust Center for Human Genetics, Oxford, UK (Dr.
Dominic Kwiatkowski); London School of Tropical Medicine
and Hygiene, London, UK (Dr. Eleanor Riley); Centre National de
Recherche et Formation sur le Paludisme, Ouagadougou, Burkina
Faso (Dr. Bienvenu Sodiomon Sirima).
Report of Activity
Background
The Fulani of West Africa have been shown to
mount stronger immune responses to P. falciparum
antigens and to be less susceptible to infection and
mild disease than sympatric populations (Modiano
et al., 1996). The Fulani also show a higher response
to other pathogens, and both their Th1 and Th2
responses are enhanced, suggesting that their resistance
to malaria could result from a generally stronger
immune activation. Indeed key genes related to T
regulatory cell function are indeed down-regulated
in the Fulani (Torcia et al., 2008). This disorder of
immune homeostasis could be driven by genetic
factors positively selected by P. falciparum and may
underlie the higher susceptibility of the Fulani to
diseases with autoimmune pathogenesis reported in
the literature. To investigate this hypothesis, we are
conducting a large-scale epidemiological and immunogenetic
study in Burkina Faso supported also by
the Malaria Genomic Epidemiology Network (2008).
Serological analyses
Anti-Nuclear Antibodies (ANA) are markers of systemic
autoimmune diseases. Their prevalence in the
European population is about 5% while it ranges
from 30 to 100% in European patients with systemic
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AREA 7
autoimmune diseases. Little is known on the prevalence
of ANAs in African populations. We measured
IgG against a combination of 11 self nuclear antigens
(dsDNA, U1-snRNP, Sm, SS-A/Ro, SS-B/La, Scl-
70, CENP, Jo-1, Histone, PM-Scl, Rib-P) in plasma
samples including 474 Fulani and 900 Non Fulani
using a commercial ELISA Kit (Varelisa, Phadia).
We then validated the results by IFAT. We found
a higher prevalence of ANA in Fulani (63%) than
Non Fulani (54%) (P=0.002). These values are much
higher than that of European controls (6%) and suggest
that the response to self nuclear antigens could
be highly enhanced in malaria endemic countries,
for reasons yet to be investigated. Furthermore, this
response is higher in the population with the lower
susceptibility to malaria. The levels of ANA were
also higher in Fulani than Non Fulani (P=0.001).
In the same samples, in order to confirm previous
observations, we measured IgG against four P. falciparum
antigens (CSP, MSP1, MSP2, AMA1) by ELISA.
As expected we observed higher prevalences and levels
in Fulani than Non-Fulani (P

David Modiano
ated with protection from infection and/or clinical
malaria in both populations. We compared
the Minor Allele Frequency (MAF) of 168 Single
Nucleotide Polymorphisms (SNPs) - genotyped by
the MalariaGEN Resource Centre in 2225 DNA
samples - between Fulani and Non Fulani. For all
SNPs showing a significant difference in allele frequency
between the two populations (62 SNPs after
Bonferroni correction) we performed the following
allele-based (minor vs major allele) association analyses
at the intra-population level: association with P.
falciparum infection (yes/no) in 5 cross-sectional surveys
in all age groups (Mantel-Haenszel Chi Square
test stratified by survey); association with clinical
malaria (T>37.5 and P. falciparum infection, yes/no)
in 2 longitudinal surveys in the 0-5 years old age
group (Mantel-Haenszel Chi Square test stratified
by survey). We found no SNPs associated with either
infection or clinical malaria in both populations.
We then selected 60 loci with a reported role in
autoimmunity for genetic association analysis with
humoral response and susceptibility to malaria, as
well as with immunological markers of autoimmune
diseases. The following selection criteria were used:
immune system function; association with 1 or more
autoimmune diseases in GWAS or in meta-analysis
based on at least 5 papers (HuGE Navigator v3.1, Yu
et al., 2008); differential expression in Treg cells of
Fulani vs Mossi (Torcia et al., 2008). Polymorphisms
for each gene (around 120 SNPs in total) have been
selected based on their functional consequences
(e.g. non-synonymous coding, transcription factor
binding site, splicing site) and on their MAF in the
Yoruba population from Nigeria (HapMap data).
148
These SNPs are currently being genotyped in collaboration
with the WTCHG in Oxford.
References
- Modiano et al., Different response to Plasmodium
falciparum malaria in West African sympatric ethnic
groups. PNAS USA 1996, 93: 13206-13211.
- Torcia et al., Functional deficit of T regulatory
cells in Fulani, an ethnic group with low susceptibility
to Plasmodium falciparum malaria. PNAS
USA 2008, 105: 646-51.
- Malaria Genomic Epidemiology Network,
-
Genome-wide and fine-resolution association analysis
of malaria in West Africa. Nature Genetics 2009.
Malaria Genomic Epidemiology Network. A global
network for investigating the genomic epidemiology
of malaria. Nature 2008; 456: 732-7.
- Yu et al., A Navigator for Human Genome
Epidemiology. Nature Genetics 2008, 40: 124-5.
Publications
Lulli P, Mangano VD, Onori A, Batini C, Luoni G,
Sirima BS, Nebie I, Chessa L, Petrarca V, Modiano D.
HLA-DRB1 and -DQB1 loci in three west African ethnic
groups: genetic relationship with sub-Saharan African
and European populations. Hum Immunol 2009, 42:
903-9. ISSN: doi: 10.1016/j.humimm.2009.07.025.
Gouagna LC, Bancone G, Yao F, Yameogo B,
Dabiré KR, Costantini C, Simpore J, Ouedraogo JB,
Modiano D. Genetic variation in human HBB is associated
with Plasmodium falciparum transmission. Nat
Genet. 2010, 42: 328-31. doi: 10.1038/ng.554.

Computational Analysis of the gene products of the Plasmodium
falciparum genome and their interaction with human proteins
Principal investigator: Anna tramontano
Professor of Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910556; Fax: (+39) 06 49910717
anna.tramontano@uniroma1.it
Participants:
Paolo Marcatili, Domenico Raimondo, Allegra Via, Mauro
Chinappi, researchers; Alba Lepore, Le Pera Loredana, PhD
students.
Collaborations:
Istituto Superiore di Sanità, Roma (Dr. Elisabetta Pizzi, Dr.
Pietro Alano); Universita’ di Siena (Prof. Campiani), CRS4
(Dr. Matteo Floris, Dr. Massimiliano Orsini), University
of California Davis, USA (Dr. Krzysztof Fidelis, Dr. Andriy
Kryshtafovych), CARB (Dr. John Moult), The Hebrew Univeristy
of Jerusalem, Israel (Dr. Yaniv Lowenstein, Dr. Michal Linial),
University College of London, UK (Dr. Christine Orengo), EBI
(Dr. James Watson, Dr. Janet Thornton), Helmholtz Center,
Munchen, Germany (Dr. Dmitrij Frishman).
Report of activity
This project aimed at exploiting the knowledge of
the genomes and proteomes of Plasmodium, the etiological
cause of the deadly parasitic malaria disease
to understand the structure and function of their
proteins, in particular those involved in host pathogen
interaction and in the development of resistance.
We focused on three parasite proteins, PfEMP1,
EBA-175 and PfCRT. The first two interact with the
human proteins ICAM-1 and Glycophorin A (GPA),
respectively. The third is involved in resistance to
chloroquine. They represent different challenges of
increasing difficulty for computational methods as
discussed below.
The strategy we used in the first case (complex
between PfEMP1 and ICAM-1) was to model the
interacting plasmodium protein by comparative modelling
techniques (i.e. using as first approximation the
structure of an evolutionary related protein) and to
use docking methods to predict the orientation of the
protein in its complex with ICAM-1 (whose structure
is experimentally known). The result was very convincing
and could explain a number of experimental
149
AREA 7
observations, which convinced us that we could use
the model of the complex to identify regions of the
human protein that could be mimicked by peptides in
order to interfere with the interaction.
The second case (complex between EBA-175 and
GPA) was much more challenging for at least two
reasons. We could not model the structure of GPA
(and also of the related GPB protein known not to
interact with EBA-175) by comparative modelling
and had to recur to the more computer expensive
and somewhat less reliable fragment based methods.
Once again, however, the model was very convincing
because it would fit with known experimental
data. However, the protein is heavily glycosylated
and sugar moieties are known to be involved in its
interaction with the Plasmodium protein. Because of
their high flexibility, modelling of the structure of
sugars is an unsolved problem and we are developing
methods to do so in order to be able to obtain the
model of the complex.
The third case is also very challenging since the
involved protein (PfCRT) is a transmembrane protein
and, because of the paucity of known structures
of transmembrane proteins, their prediction is still
very difficult. Our main question in the case of PfCRT
regarded the details of the function of the protein.
In its erythrocyte stage, P. falciparum invades the
red blood cells where it forms a lysosomal isolated
acidic compartment known as the digestive vacuole.
Chloroquine is a diprotic weak base and, at physiological
pH (~7.4), can be found in its un-protonated,
mono-protonated and di-protonated forms.
The uncharged chloroquine is the only membrane
permeable form of the molecule and it freely diffuses
into the erythrocyte up to the vacuole. In this compartment,
chloroquine molecules become protonated
and, since membranes are not permeable to charged
species, the drug accumulates into the acidic digestive
vacuole where it is believed to bind haematin, a
toxic byproduct of the haemoglobin proteolysis, preventing
its incorporation into the haemozoin crystal

Anna Tramontano
and thereby causing damage to the Plasmodium
membranes. Chloroquine sensitive parasites (CQS)
accumulate much more chloroquine in the vacuole
than chloroquine resistant strains (CQR) and this
has been associated with point mutations in the
gene encoding for the transmembrane P. falciparum
chloroquine resistance transporter (PfCRT) protein.
There are two alternative models for the function
of PfCRT: 1) the channel model (i.e. a passive channel
that enables charged chloroquine to leak out of
the food vacuole down its electrochemical gradient)
or 2) the carrier model (i.e. an active efflux carrier
extruding chloroquine from the food vacuole).
Understanding the molecular basis of the mutated
PfCRT mechanism of action is obviously important
and it would open the road to new strategies for
circumventing, and perhaps suppressing its role in
determining resistance.
The question we addressed is whether available data,
together with a combination of analytical and modeling
approaches can provide insights into the mechanism
of action of chloroquine and PfCRT.
Our approach consisted in developing an analytical
model and test the consistency of several plausible
hypotheses about the binding mode of the drug to
the heme related species inside the vacuole and the
mechanism of action of PfCRT with experimental
data. We could demonstrate that some of the hypotheses
can be excluded on the basis of the available
data, but also that the latter are compatible with
both models of PfCRT being a passive channel and
a transporter. Evolutionary analysis of the protein
was therefore required to solve the issue. We demonstrated
that sequence analysis, advanced database
searching methods and structural prediction taken
together strongly support the hypothesis that the
protein acts as an active carrier and, based on the
conclusions derived from the mathematical model,
the carried molecule is either or both the mono and
di-protonated forms of chloroquine.
In the course of this project we used available computational
tools, but we also needed to develop new
ones. We set up a tool for mapping mutations from
genomes to proteomes and vice versa, a method to
analyse the three-dimensional context of post-translational
modification, a system for identifying extracellular
regions of proteins and, obviously, tools to build
and assess the quality of three-dimensional models of
150
proteins. The scope of application of these methods
is not limited to the specific problem of malaria and
therefore they were also made available to the community
in the form of publicly accessible web server.
Publications
Cozzetto D, Kryshtafovych A, Fidelis K, Moult J,
Rost B, Tramontano A. Evaluation of templatebased
models in CASP8 with standard measures.
Proteins. 2009, 77: 18-28. doi: 10.1002/prot.22561.
Cozzetto D, Kryshtafovych A, Tramontano A.
Evaluation of CASP8 model quality predictions.
Proteins. 2009, 77: 157-66. doi: 10.1002/prot.22561.
Loewenstein Y, Raimondo D, Redfern OC, Watson
J, Frishman D, Linial M, Orengo C, Thornton J,
Tramontano A. Protein function annotation by
homology-based inference. Genome Biol. 2009, 10:
Article 207. doi: 10.1186/gb-2009-10-2-207.
Maullu C, Raimondo D, Caboi F, Giorgetti A, Sergi
M, Valentini M, Tonon G, Tramontano A. Sitedirected
enzymatic PEGylation of the human granulocyte
colony-stimulating factor FEBS J. 2009, 276:
6741-50. doi: 10.1111/j.1742-4658.2009.07387.x.
Moult J, Fidelis K, Kryshtafovych A, Rost B,
Tramontano A. Critical assessment of methods of
protein structure prediction - Round VIII. Proteins.
2009, 77: 1-4. doi: 10.1002/prot.22589.
Chinappi M, Via A, Marcatili P, Tramontano A.
On the mechanism of chloroquine resistance in
Plasmodium falciparum. PLoS One 2010, 5:e14064.
doi: 10.1371/journal.pone.0014064.
Giombini E, Orsini M, Carabino D, Tramontano A.
An automatic method for identifying surface proteins
in bacteria: SLEP. BMC Bioinformatics. 2010, 11: 39.
doi: 10.1186/1471-2105-11-39.
Le Pera L, Marcatili P, Tramontano A. Picmi: mapping
point mutations on genomes. Bioinformatics.
2010, 26: 2904-5. doi: 10.1093/bioinformatics/
btq547.

Start-Up
Specification
and
maintenance
of retinal
stem cells

Specification and maintenance of retinal stem cells
Principal investigator: Giuseppe Lupo
Researcher in Development Biology
Dipartimento di Biologia e Biotecnologie “Charles Darwin”
Tel: (+39) 06 49912206; Fax: (+39) 06 49912351
giuseppe.lupo@uniroma1.it
Participants:
Nicoletta Carucci, post-doc fellow.
Collaborations:
Dipartimento di Biologia e Biotecnologie “C. Darwin” (Dr.
Emanuele Cacci, Dr. Maria Elena Miranda Banos);
Department of Physiology, Development and Neuroscience,
University of Cambridge, UK (Prof. William Harris); Anne
McLaren Laboratory for Regenerative Medicine, University
of Cambridge, UK (Prof. Roger Pedersen); Department of
Developmental Neurobiology, Kumamoto University, Japan (Prof.
Kunimasa Ohta).
Report of activity
Background
The retina is a favoured system to study formation of
the central nervous system (CNS), due to its accessible
position, and to its simple and well-characterized
structure. The mature retina consists of only six cell
classes, identifiable by their morphology and molecular
markers. Several vision-impairing diseases, such
as retinitis pigmentosa and macular degeneration,
are due to the loss of retinal cells. Studies in animals
suggest that they may be treatable by transplantation
of healthy retinal cells, but obtaining a sufficient
number of suitable donor cells remains a major problem.
This has propelled stem cell based approaches
to generate pure populations of retinal cells in vitro,
to be used for cell-replacement therapy. Retinal stem
cells (RSCs) are the embryonic progenitors that give
rise to the retina during embryogenesis and early
post-natal life. RCSs are specified early in development
as a result of three consecutive steps. First, the
neuroectoderm (NE) is induced in the dorsal part of
the embryo. Subsequently, the NE is patterned along
the embryonic antero-posterior (AP) axis, specifying
forebrain (FB), midbrain, hindbrain and spinal cord
at different AP levels. Finally, a subset of the FB is
153
START-UP
specified as the eye field (EF), containing the RSCs.
Although most of the RSCs eventually differentiate
to form retinal neurons and glia, RSCs are retained
at the periphery of the retina, at least during early
post-natal life. The signals that promote self-renewal
of RSCs and prevent them from differentiating prematurely
are still largely unclear.
Objectives
1. The elucidation of the signalling systems that
control specification of RSCs during early NE development,
using pluripotent stem cell lines as a model
system.
2. The comparison of RSCs obtained from pluripotent
cells in vitro, with RSCs in the embryonic retina
or the post-natal eye.
3. The comparison of RSCs with neural stem cells
(NSCs) derived from other embryonic NE regions,
in order to understand the mechanisms controlling
regionalization of progenitor cells during CNS
development.
4. The identification of factors that can support proliferation
and self-renewal of RSCs, while preventing
loss of retinal identity and differentiation.
Results
In collaboration with Dr Cacci at the University
of Rome “La Sapienza”, and confirming previous
results by Dr Chiara Soldati, Prof. Biagoni and Prof.
Augusti-Tocco at the same University, we have generated
a repertoire of NSC lines, derived from various
region of the E13.5 mouse CNS: the cerebral cortex,
the lateral ganglionic eminence, the dorsal and
ventral mesencephalon, the spinal cord. In all this
cases, is has been possible to isolate and propagate
in vitro NSCs that efficiently self-renew in the presence
of FGF and Epidermal Growth Factor (EGF).
Furthermore, these different NSC lines appear to
retain in vitro a positional identity consistent with
their region of derivation in vivo. For example, we

Giuseppe Lupo
have confirmed that dopaminergic neuron differentiation
can be activated in vitro in NSCs derived
from the lateral ganglionic eminence or the ventral
mesencephalon, in agreement with the presence of
dopaminergic neurons in these areas in vivo.
However, retinal progenitors isolated from the E13.5
mouse retina stopped proliferating and completely
differentiated in the same culture conditions and
hence could not be propagated in vitro. These results
are very interesting as they suggest that FGF and
EGF are sufficient for self-renewal of NSCs located
in the embryonic neural tube (brain and spinal cord),
while RSC self-renewal requires alternative or additional
factors.
In collaboration with Prof. Ohta at Kumamoto
University, Japan, we have also investigated the signals
supporting proliferation of RSCs located at the
periphery of the mouse retina. Our work has shown
a crucial role for the secreted molecule Tsukuchi
(TSK), belonging to the small leucine-rich proteoglycan
(SLRP) family, in regulating RSC proliferation.
TSK is expressed in the peripheral retina from
embryonic stages to adulthood and TSK knock-out
mice show increased proliferation in this region.
Activation of the Wnt pathway also stimulates RSC
proliferation and we found that TSK inhibits Wnt
signalling by competing for Wnt ligands to receptor
binding. These results suggest that RSC proliferation
in the peripheral retina is modulated by the antagonistic
interaction between TSK and Wnt ligands and
they also raise the interesting possibility that TSK
activity may be responsible for the limited or absent
RSC self-renewal in the adult peripheral retina.
Future plans
Objective 1. We have obtained a Material Transfer
Agreement with the University of Cambridge to
import and use mouse pluripotent stem cell lines
(mouse embryonic stem cells (ESCs) and epiblast
stem cells (EpiSCs)) in our Department in Rome.
We aim to apply to these cells the same protocol that
154
we previously devised in human ESCs in the labs of
Prof. Harris and Prof. Pedersen at the University
of Cambridge, UK, to obtain specification of FB
and retinal progenitors. The use of mouse cells will
offer the advantage of using cells lines derived from
genetically modified mice. Of particular interest will
be the use of cell lines bearing fluorescent reporter
proteins (eg GFP) inserted into the loci of RSC
markers for flow cytometry analysis and sorting of
RSCs. Furthermore, the use of mouse pluripotent
stem cells for generation of RSCs in vitro will allow a
direct comparison of these cells with RSCs and NSCs
derived from the embryonic CNS within the same
species. Our experimental priorities are: i) the identification
of the molecular mechanisms influencing
retinal progenitor specification in ESCs and EpiSCs;
ii) the sorting of retinal progenitors produced in
vitroto obtain pure populations of RSCs; iii) the
comparison (eg by transcriptome analysis) of ESC-
or EpiSC-derived RSCs with RSCs derived from the
embryonic retina.
Objectives 2-4. We will focus on the derivation of
RSC lines from the mouse embryonic retina by
employing a systematic screening approach where
retinal progenitor cells will be cultured in the presence
of various combinations of growth factors and
cytokines. We will especially concentrate on molecules
working in the Wnt signalling pathway, since
our results on the study of the TSK molecule indicate
a critical role for this pathway in RSC proliferation
and self-renewal. Embryo-derived RSCs will be compared
with ESCs- or EpiSC-derived RSCs in terms
of both their gene expression profiles and growth
factor requirement for self-renewal. Furthermore,
we will also compare the differentiation potential of
RSCs with NSCs derived from other CNS regions, in
order to understand the developmental mechanisms
controlling the specification of regional identities in
CNS progenitors in vivo and the plasticity of NSCs
of different origins cultured in vitro with regards to
their regional identity.

2009-2010
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