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Istituto Pasteur 

Fondazione Cenci Bolognetti 

Report of activity 

2007-2008

© 2009 - 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 Ernesto Di Mauro, Scientific Director 

Boards and Staff 

Fellowships awarded in 2007 and 2008 

Fellowships awarded for two years for training in foreign laboratories 

Fellowships awarded to students who had a two-year experience abroad 

Fellows working on research programmes of the Foundation 

Seminars by outstanding invited speakers 

Meetings and Conferences organized or supported by the Foundation 

Scientific Reports 

Research area 1: Molecular biology of microorganisms and viruses 

Paolo AMATI, Polyomavirus-host interaction: role of the early phases of infection in determining virus 

permissivity and role of PARP-1 in the regulation of immediate early gene expression ........................ 

Alberto BOFFI, Escherichia coli strains overexpressing flavohemoglobins for the production of novel, 

biologically active compounds derived from phospholipid post-biosynthetic modifications ..................... 

Daniela DE BIASE, The glutamate-based acid resistance system of Escherichia coli: the molecular basis of 

the activation of its regulatory and structural components ............................................................................. 

Alberto FAGGIONI, Control of latency and replication of Epstein-Barr virus .................................................. 

Paola LONDEI, Translational regulation: from the archaea to the eukarya ......................................................... 

Research area 2: Pathogenetic mechanisms of microbially associated diseases 

Andrea BELLELLI, A structural biology study of schistosomiasis .......................................................................... 

Maria Lina BERNARDINI, Role of Shigella surface components in immunomodulation of the inflammatory 

response .......................................................................................................................................................................... 

Bianca COLONNA, Deciphering the complexity of virulence gene networks in Shigella and enteroinvasive 

Escherichia coli (EIEC): the interplay among nucleoid proteins, promoter DNA structure and 

regulatory factors ......................................................................................................................................................... 

Annateresa PALAMARA, Redox mediated mechanisms involved in the influenza virus replication and in 

the pathogenesis of influenza associated diseases ............................................................................................... 

Paolo SARTI, Nitric oxide detoxification in pathogenic protozoa: role of flavodiiron proteins ...................... 

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, Development and analysis of chromosome vectors .................................................... 

Paola BALLARIO, Molecular machines and effectors involved in the regulation of light response and cell 

cycle in simple Eukaryotes ......................................................................................................................................... 

Irene BOZZONI, RNA-RNA and RNA-protein interactions in the cell nucleus: structure, function and 

biosynthesis of a novel class of small non-coding RNAs ................................................................................. 

Paola CAIAFA, Crosstalk between poly(ADP-ribosyl)ation and DNA methylation in the regulation of 

gene expression ............................................................................................................................................................ 

Giorgio CAMILLONI, DNA topoisomerases as global controller of DNA transactions. Study of DNA 

topoisomerase IB and its functional partners ....................................................................................................... 

Paolo COSTANTINO, The role of the DAG transcription factors in Arabidopsis seed germination ............. 

Maria D’ERME, Epigenetic modifications in neurodegenerative diseases ............................................................ 

Patrizio DIMITRI, Drosophila melanogaster as a model organism for functional genomic analyses of 

essential genes resident in heterochromatin ......................................................................................................... 

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REPORT OF ACTIVITY 2007-2008 

Lucia FABIANI, DNA replication mechanisms and genome stability in Saccharomyces cerevisiae ..................... 

Claudio FALCONE, New tools in deciphering aging and apoptotic pathways ..................................................... 

Laura FRONTALI, New complex mitochondrial functions in cell biology ............................................................. 

Maurizio GATTI, The role of membrane trafficking in Drosophila cytokinesis ................................................... 

Alessandro GIACOMELLO, Biology and physiology of adult cardiac stem/progenitor cells with a view to 

optimizing their utility for autologous cell transplantation .............................................................................. 

Alberto GULINO, Regulation of the Hedgehog signaling in neural cell development and disease ............... 

Massimo LEVRERO, Histone Acetyltransferase (HAT) autoregulatory loops in the regulation of DNA 

damage responses ......................................................................................................................................................... 

Franco MANGIA, Molecular regulation of cell proliferation and apoptosis in early embryo blastomeres 

and granule neuron 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 presumptive human transcription factor Gas41 and of its S. cerevisiae 

homologue Yaf9 in chromatin organization and gene expression ................................................................... 

Sergio PIMPINELLI, Heterochromatin, gene silencing and “RNA interference” in Drosophila ........................ 

Mario STEFANINI, Biological characterization and in vitro culture of spermatogonial stem cells ................ 

Marco TRIPODI, Molecular mechanisms of the epithelial to mesenchymal transition in hepatocyte ........... 

Carlo TURANO, Roles of ERp57 in DNA repair and in the regulation of gene expression ........................... 

Giuseppe ZARDO, Identification of novel genetic and epigenetic targets in Leukemia by genome wide 

approaches ...................................................................................................................................................................... 

Research area 4: Molecular recognition in biomolecules 

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 B 6 salvage pathway and 

targeting of the cofactor to apo-enzymes ............................................................................................................. 

Francesco GASPARRINI, Molecular and enantioselective recognition by receptors and proteins studied in 

the gas phase, in free solution and at solid-liquid interfaces ............................................................................. 

Cristina LIMATOLA, Molecular and functional approaches to investigate the physiopathological role of the 

chemokines and their receptors in the central nervous system ....................................................................... 

Paola PAGGI, Neuronal response to experimental interruption of the neural circuit: a molecular and 

structural study in autonomic ganglia in vivo ....................................................................................................... 

Maria SAVINO, Structural and superstructural features of human telomeric chromatin ................................ 

Research area 5: Cellular and molecular immunology 

Vincenzo BARNABA, Innovative strategies eliciting CD8 T cell responses by exploiting proteomic 

analysis and improving cross-presentation ............................................................................................................ 

Roberto FOA’, Potential role of miRNAS in IgM-mediated signal transduction in normal and neoplastic 

B cells .............................................................................................................................................................................. 

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, Molecular mechanisms underlying the activation of NK cell regulatory and effector 

functions ......................................................................................................................................................................... 

Isabella SCREPANTI, Analysis of the role of preTCR-triggered NF-kB in T cell leukemogenesis: 

relationship with activated Notch signaling .......................................................................................................... 

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-κB 

activation ........................................................................................................................................................................ 

Elio ZIPARO, The role of Toll Like Receptors in immune responses to infections and in inflammation 

associated pathologies of the male reproductive system ................................................................................... 

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Research area 6: New antimicrobial and antiviral agents 


Donatella BARRA, Peptide effectors of innate immunity ......................................................................................... 

Nicoletta DESIDERI, New antipicornavirus flavanoids: synthesis, biological evaluation and structureactivity 

relationship studies ....................................................................................................................................... 

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 ............................................................ 

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, Indole nucleus as a selected pharmacophore for the design of novel highly potent 

anti-viral agents active against HIV-1 (RT and IN inhibitors) and also capable to inhibit HCV and 

tumor cell replication .................................................................................................................................................. 

Research area 7: Biology of malaria and other vector-borne diseases 

Alessandra DELLA TORRE, Vincenzo PETRARCA, Bionomical, genetical and molecular characterization of 

populations of the Anopheles gambiae complex of sibling species (Diptera: Culicidae), malaria vector in 

sub-Saharan Africa ....................................................................................................................................................... 

Anna TRAMONTANO, Computational Analysis of the gene products of the Plasmodium falciparum genome 

and their interaction with human proteins ............................................................................................................ 

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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 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 form 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 programmes 

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) cofunding 

the international Ph.D. Course in Scienze Pasteuriane at the Sapienza and iii) from 2009 

starting a program 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 2007 and 2008, documented in this volume, witness the 

enthusiasm and productivity of the scientists involved. 

Paolo Amati 

President 

VII 


Ernesto Di Mauro 

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 Medicine I and II, Natural 

Sciences, and Pharmacy. 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 (Medicine II) 

Members 

Mario Coluzzi (Medicine I), Paolo Costantino 

(Natural Sciences), Raffaele D'Amelio (Medicine II), 

Ernesto Di Mauro (Natural Sciences), Romano 

Silvestri (Pharmacy) 

Secretary 

Emanuela Gloriani 

Administrative Expert 

Daniela Cavallo 

Auditors 

Carlo Messina, Simona Ranalli, Valeria Valerio 

VIII 

Scientific Council 

The Scientific Council is a board of seven 

scholars in the field of the pasteurian 

sciences. Members are elected to a fouryear 

term by the Faculties of Medicine I 

and II, Natural Sciences, and Pharmacy. 

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 

Ernesto Di Mauro (Natural Sciences) 

Members 

Laura Frontali (Natural Sciences), Anna Teresa 

Palamara (Pharmacy), Angela Santoni (Medicine I), 

Anna Tramontano (Medicine I), Marco Tripodi 

(Medicine II), Carlo Turano (Pharmacy) 

Secretariat 

Teresa Ariaudo † , 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) 

† deceased on February 17 th , 2008

Fellowships awarded in 2007 and 2008 

Fellowships awarded for two years for 

training in foreign laboratories 

Alessandro BORGIA, at the Department of 

Chemistry, University of Cambridge, UK, from the 

Department of Biochemical Sciences 

Daniela CECCARELLI, at the Département de Biologie, 

Université de Sherbrooke, Québec, Canada, from the 

Department of Cell and Developmental Biology 

Fabiana CICIRIELLO, at the Department of 

Biochemistry, McGill University, Montreal, Canada, 

from the Department of Genetics and Molecular Biology 

Antonio COLUCCIA, at the Welsh School of Pharmacy, 

Cardiff University, UK, from the Department of 

Pharmaceutical Studies 

Giuseppe LA REGINA, at the Welsh School of 

Pharmacy, Cardiff University, UK, from the 

Department of Pharmaceutical Studies 

Anna OLIVIERI, at the National Institute for Medical 

Research, London, UK, from the Department of Public 

Health Sciences 

Claudia PALLADINO, at the Hospital General 

Universitario G. Marañón, Madrid, Spain, from the 

Department of Cell Biotechnology and Hematology 

Eugenia PICCINNI, at the Laboratory of Molecular 

Genetics, Ecole Normale Supérieure, CNRS, Paris, 

France, from the Department of Cell and Developmental 

Biology 

Sabrina PISANO, at the Laboratoire de Biologie 

Moléculaire de la Cellule, Ecole Normale Supérieure 

de Lyon, France, from the Department of Genetics and 

Molecular Biology 

Fabiana RENZI, at the Laboratory of Structural and 

Computational Biology, EMBL, Heidelberg, 

Germany, from the Department of Biochemical Sciences 

Chiara SOLDATI, at the Centre for the Cellular Basis 

of Behaviour, King's College, University of London, 

UK, from the Department of Cell and Developmental 

Biology 

Ivan TATTOLI, at the Department of Immunology, 

University of Toronto, Canada, from the Department 

of Cell and Developmental Biology 

Italo TEMPERA, at the The Wistar Institute, 

Philadelphia, USA, from the Department of 

Biochemical Sciences 

Simona TORCIA, Department of Obstetrics and 

Ginecology, Stanford University School of Medicine, 

USA, from the Department of Histology and Medical 

Embryology 

IX 


Laura VALERIO, at the Department of Entomology, 

University of California, Davis, USA, from the 

Department of Public Health Sciences 

Barbara XELLA, at the Marie Curie Research Institute, 

Oxted, UK, from the Department of Genetics and 


Fellowships awarded to students who 

had a two-year experience abroad 

Claudia BERTONATI, at the Department of 

Biochemical Sciences, from the Department of 

Biochemistry and Molecular Biophysics and Center for 

Computational Biology and Bioinformatics, Columbia 

University, New York, USA 

Maria Giulia BIGOTTI, at the Department of 

Biochemical Sciences, from the Department of 

Biochemistry of the University of Bristol, UK 

Daniele MARCHESE, at the Department of 

Experimental Medicine and Pathology, from the 

Institut fur Zellbiologie, University of Tubingen, 

Germany 

Giacomo Maria PAGANOTTI, at the Department of 

Public Health Sciences, from the Institute of Cell, 

Animal and Population Biology of the University of 

Edinburgh, UK 

Grazia Daniela RAFFA, at the Department of 

Genetics and Molecular Biology, from the 

Department of Molecular Biology, The Scripps 

Research Institute, La Jolla, USA 

Massimiliano RENZI, at the Department of Human 

Physiology and Pharmacology, from the Department 

of Pharmacology, University College London, UK 

Francesca SICILIA, at the Department of Plant 

Biology, from the Department of Biochemistry of the 

University of Cambridge, UK 

Alessandra SORIANI, at the Department of 

Experimental Medicine and Pathology, from the 

Department of Hematology-Oncology of the 

University of California-San Diego, USA 

Fellows working on research 

programmes of the Foundation 

Viviana ANNIBALI, at the Department of Biochemical 

Sciences 

Matteo BARBA, at the Department of Experimental 

Medicine and Pathology 

Emanuela BASTONINI, at the Department of 

Experimental Medicine and Pathology


Dario BENELLI, at the Department of Cell 

Biotechnology and Hematology 

Alexandre BRUTUS, at the Department of Plant 

Biology 

Maria CALZETTA, at the Department of Public Health 

Sciences 

Fabio CANDURA, at the Department of Public Health 

Sciences 

Sonia CANTERINI, at the Department of Psychology 

Sara CAPRODOSSI, at the Department of 

Experimental Medicine and Pathology 

Nicoletta CARUCCI, at the Department of Cell 


Viviana CASAGRANDE, at the Department of Cell and 

Developmental Biology 

Samantha CIALFI, at the Department of Experimental 


Alberto CIOLFI, at the Department of Cell 


Rossana COCCHIOLA, at the Department of 


Domenico COZZETTO, at the Department of 


Danilo D'AMBROSIO, at the Department of Genetics 

and Molecular Biology 

Enrico FERRARO, at the Department of Biochemical 

Sciences 

Roberto GAETANI, at the Department of Experimental 


Caterina GRILLO, at the Department of Biochemical 

Sciences 

Dominik GRONT, at the Department of Biochemical 

Sciences 

Tiziana GUASTAFIERRO, at the Department of Cell 


Federica LO SARDO, at the Department of Cell and 


Loredana LOMBARDI, at the Department of Cell and 


Simona LORETI, at the Department of Cell and 


Laura MAGGI, at the Department of Human 

Physiology and Pharmacology 

Adriana MAGNACCA, at the Department of Cell and 


Armando MAGRELLI, at the Department of Cell 


Marcella MARCHETTI, at the Department of Genetics 

and Molecular Biology 

Simone MARINELLI, at the Department of Cell and 


Valeria MICHELACCI, at the Department of Cell and 


X 

Federica MICUCCI, at the Department of 


Arianna MONTANARI, at the Department of Cell and 


Claudia MORICONI, at the Department of Cell and 


Giuseppe NATIVIO, at the Department of Biochemical 

Sciences 

Giulia NIGRO, at the Department of Cell and 


Francesca PAGANI, at the Department of Human 

Physiology and Pharmacology 

Giacomo Maria PAGANOTTI, at the Department of 

Public Health Sciences 

Gianna PANETTA, at the Department of Biochemical 

Sciences 

Giovanna PERUZZI, at the Department of 


Lucia PIACENTINI, at the Department of Genetics and 


Sabrina PISANO, at the Department of Genetics and 


Alessandro ROSA, at the Department of Genetics and 


Fabrizio ROSSI, at the Department of Genetics and 


Erika ROSSI, at the Department of Public Health 

Sciences 

Federica SANTOLAMAZZA, at the Department of 

Public Health Sciences 

Daniel SAVATIN, at the Department of Plant Biology 

Mauro SAVINO, at the Department of Cell 


Flavio SCALONI, at the Department of Plant Biology 

Francesca Maria SCANDURRA, at the Department of 


Stefano SECHI, at the Department of Genetics and 


Graziella SENIS, at the Department of Public Health 

Sciences 

Alessandra SORIANI, at the Department of 


Cristina SORINO, at the Department of Genetics and 


Lucia TUFANO, at the Department of Plant Biology 

Daniela UCCELLETTI, at the Department of Cell and 


Koudraogo Bienvenue YAMEOGO, at the Department 

of Public Health Sciences 

Adama Franck YAO, at the Department of Public 

Health Sciences 

Michele ZAMPIERI, at the Department of Cell 

Biotechnology and Hematology

Seminars by outstanding invited speakers 

2007 

May 8 • DNA methylation and genome defense in 

Neurospora crassa 

Prof. Eric U. SELKER, Institute of Molecular 

Biology, University of Oregon, Eugene, USA 

July 3 • Bordetella pertussis/adenylate cyclase: a 

Trojan horse for antigen delivery 

Prof. Agnes Ullmann, Institut Pasteur, Paris, 

France 

July 19 • Ultrafast protein folding 

Prof. William A. EATON, Laboratory of Chemical 

Physics, National Institutes of Health, Bethesda, 

USA 

November 14 • New genome sequences in the genus 

Drosophila 

Prof. Thomas C. KAUFMAN, Department of 

Biology, Indiana University, Bloomington, USA 

2008 

March 17 • The functions of antibiotics in nature 

Prof. Julian DAVIES, Department of 

Microbiology and Immunology, University of 

British Columbia, Vancouver, Canada 

May 26 • SHIP and immune dysfunction: multiple 

mechanisms 

Prof. William G. KERR, H. Lee Moffitt 

Comprehensive Cancer Center and Research 

Institute, Tampa, USA 

June 13 • Regulation of gene expression by the activity 

of the Shigella flexneri type III secretion apparatus 

Prof. Claude PARSOT, Pathogénie Microbienne 

Moléculaire, Institut Pasteur, Paris, France 

XI 


June 27 • Leucyl-tRNA synthetase quality control 

mechanisms for protein synthesis 

June 30 • Evolutionary adaptation of aminoacyl-tRNA 

synthetases for non-canonical RNA splicing activities in 

the cell 

Prof. Susan A. MARTINIS, Department of 

Biochemistry, University of Illinois, Roger Adams 

Laboratory Urbana, USA 

July 1 • 1) Single-molecule biology: principles; 2) Singlemolecule 

biology: applications to the study of 

transcription 

Prof. Jordanka ZLATANOVA, Department of 

Molecular Biology, University of Wyoming, USA 

October 17 • Heme protein induced oxidative stress 

Prof. Michael T. WILSON, Department of 

Biological Sciences, University of Essex, Colchester, 

UK 

November 13 • Protein folding triggered by electron 

transfer 

November 15 • Pathways for internal electron transfer 

in proteins 

Prof. Harry B. GRAY, California Institute of 

Technology, Pasadena, USA 

Novembre 19 • The genomic code for nucleosome 

positioning 

Novembre 20 • A code beyond genetics in DNA 

Prof. Jonathan WIDOM, Department of 

Chemistry, Northwestern University, Evanston, 

USA 

December 18 • Cross-talk between CTCF and PARP in 

Epstein-Barr Virus 

Dr. Italo TEMPERA, Wistar Institute, University 

of Philadelphia, USA


Meetings and Conferences organized or supported by the Foundation 

International Meeting 

The World of Small Non-Coding RNAs: 

from Basic to Applied Science 

Rome, June 11-12, 2007 

• Biogenesis and function of small non-coding 

RNAs 

• RNAi and therapy 

• Small non-coding RNA families in plants 

• miRNA and target identification 

• Regulatory role of miRNA in cell 

proliferation/differentiation 

• miRNAs and therapy 

Third Joint Workshop 

History of Medical Entomology 

Rome, October 11-12, 2007 

National Conference 

Dall’Origine della Terra alla Comparsa 

della Vita 

Rome, March 6, 2008 

Lincei - Sapienza International Meeting 

Stem Cells in Tissue Homeostasis, Repair 

and Cell Therapy 

Rome, May 28-30, 2008 

• The evolution of regenerative mechanisms 

• Stem cell biology 

• The tissue niche as an entity of action for stem-cell 

• From bench-to-bedside research 

Institut Pasteur - Department of Developmental 

Biology Meeting 

Departmental Retreat 

Rome, June 9-11, 2008 

• Mouse Molecular Genetics - CNRS: URA 2578 

• Epigenetic Regulation - CNRS: URA 2578 

• Macrophages & Development of Immunity - 

CNRS: URA 2578 

XII 

• Molecular Biology of Development - CNRS: 

URA 2578 

• Mouse Genetics Engineering Center 

• Mouse Functional Genetics - CNRS: URA 2578 

• Molecular Genetics of Development - CNRS: 

URA 2578 

• Stem Cells and Devel opment - CNRS: URA 2578 

• Morphogenesis Molecular Genetics - CNRS: URA 

2578 

• Developmental Genetics of Drosophila - CNRS: 

URA 2578 

• Drosophila Genetics and Epigenetics - CNRS: 

URA 2578 

• Human Developmental Genetics 

• Gene Expression, Development and Disease - 

CNRS: URA 2578 

Philip Avner with participants from Istituto 

Pasteur: Framework for our future collaborations 

General Discussion and Summing up: closing 

remarks by P. Avner 

Istituto Pasteur Cultural Day 

Le Tre Dimensioni della Genomica: 

Molecole, Geni e Genomi 

Rome, June 13, 2008 

A. Lazcano Araujo, Life without genes? Prebiotic 

evolution and the emergence of genetic material 

G. Bernardi, La selezione naturale nell’evoluzione 

del Genoma 

A. Tramontano, Dai genomi alle proteine e ritorno 

6th National Conference SIICA 

Rome, June 11-14, 2008 

• Novel mechanisms in antigen presentation 

• BD Biosciences 

• Immunotherapy of cancer: translation of 

knowledge, Istituto Superiore di Sanità, 

Alleanza Contro il Cancro 

• Dermatologia e reumatologia

• Dendritic cells and leukocyte recruitment 

• Tumor immunology I 

• Leukocyte activation 

• Panomics 

• Th17: molecular, cellular and clinical aspects 

• Effector cells and regulatory molecules in allergy 

• Innate and adaptive immune interactions 

• Tumor Immunology II 

• Regulation of inflammatory response 

• Autoimmunity and regulation of immune 

response 

• Innate Immunity 

Istituto Pasteur Science Days 

The Contribution of Basic Science 

to Therapeutic Innovation 

Rome, October 23-24,2008 

Scientific Committee: L. Frontali, R. Silvestri, C. Turano 

• Gene therapy and stem cells 

Chairs: F. Chimenti, L. Frontali 

I. Bozzoni, RNA as a new therapeutic target: the 

case of Duchenne muscular dystrophy 

A. Giacomello, Adult heart stem cells 

F. Ascenzioni, Therapeutic artificial chromosomes 

• New vaccines 

Chairs: L. Frati, A. Santoni 

M. Pizza, From the genome to the identification of 

XIII 


antigens for the development of new vaccines 

M.L. Bernardini, The vaccine against shigellosis: 

an ongoing challenge through hope and failure 

A. Saul, Developing vaccines for neglected diseases 

Award of the Gaia Luoni Prize 2008 on Genetics 

of susceptibility to malaria 

• New antitumoral drugs 

Chairs: V. Ziparo, C. Turano 

L. Pinna, Protein kinase CK2 as a potential target 

in the “Signal Transduction Therapy” 

R. Silvestri, Compounds that interact with 

tubulina: drugs today and future prospects 

R. Di Santo, Terminal transferase as a new 

therapeutic target against cancer: developing 

DKHA as specific inhibitors of TDT 

A. Brancale, The use of molecular modelling in 

designing new antitumoral agents 

A. Mai, Small molecules as epigenetic modulators 

of genic expression 

• Neurodegenerative diseases: towards 

therapeutical innovation 

Chairs: C. Falcone, A. Oliverio 

P. Puri, Drugs against muscular dystrophy 

F. Altieri, Neurodegenerative disease and 

oxidative stress: the involvement of proteindisolfuro-isomerasi 

L. Frontali, Mitochondrial models 

T. Rinaldi, Involvement of UPS system in 

neurodegenerative disease 

E.M. Valente, Pathogenetic mechanisms in 

Parkinsons disease: mitochondrions and more

AREA1 

Molecular 

biology of 

microorganisms 

and 

viruses

Molecular biology of microorganisms and viruses - AREA 1 

Polyomavirus-host interaction: role of the early phases of 

infection in determining virus permissivity and role of PARP-1 

in the regulation of immediate early gene expression 

P a r t i c i p a n t s : 

Rossella Maione, professor; Michaela Cavaldesi, research 

fellow; Rosaria Carbone, Maddalena Caruso, Rocco 

Figliola, post-doc fellows; Marianna Rossi, Anna Busanello, 

PhD students; Olga Sthandier, technician. 

C o l l a b o r a t i o n s : 

Dipartimento di Biotecnologie Cellulari ed Ematologia, Sapienza- 

Università di Roma (Prof. Paola Caiafa); Dipartimento di 

Medicina Sperimentale, Sapienza-Università di Roma (Dr. 

Massimo Gentile); The Burnham Institute, La Jolla, CA, USA 

(Prof. Robert Liddington); Department of Microbiology, 

University of Buenos Aires School of Medicine, Buenos Aires, 

Argentina (Prof. Norberto Sanjuan). 


Since many years we have been interested in the 

study of the biology of the murine Polyomavirus 

(Py). Py has been successfully used as a model system 

for the study of the mechanisms regulating replication, 

gene expression, tumor induction and, more 

recently, the initial virus-host cell membrane interactions. 

Our research has been focused on the central 

role played by the major capsid protein VP1 in the 

early phases of viral infection, including cell recognition, 

entry, formation of early transcription complexes 

and cell cycle activation. A second subject, 

originally branched from the study of the modifications 

of viral chromatin during the early response, 

concerns the role of Poly(ADP-ribose)polymerases 

(PARPs) in cell cycle re-entry. A third subject, initially 

stemmed from our interest in the oncogenic 

properties of Py, is related to the interdependence of 

cell cycle and differentiation in the muscle model. 

The most significant results concern the following 

topics: 

Principal investigator: Paolo Amati 

Professor of Molecular Genetics 

Dipartimento di Biotecnologie Cellulari ed Ematologia 

Sezione di Genetica Molecolare 

Tel: (+39) 06 490393; Fax: (+39) 06 4462891 

amati@bce.uniroma1.it 

3 

Role of virus-host cell receptor(s) interaction 

in determining the virus tissue tropism and 

tumorigenicity 

We have previously shown that Py cell entry into 

fibroblasts is a multi-step process involving the 

attachment to primary sialic acids-containing cell 

receptor(s) and post-binding interaction with secondary 

receptor(s), such as the α4β1 integrin, 

through the VP1-LDV motif. However, the role of 

Py-receptor(s) interaction in virus tissue-tropism 

and tumorigenicity in vivo remained to be more 

clearly understood. By studying a recombinant 

mutant, PyLNV, with a single aa substitution in the 

VP1-LDVmotif (D138N), we have shown that 

mutation in this motif affects Py infectivity and conditions 

virus tissue tropism in vivo. Indeed, although 

not critical for virus viability, the D138N substitution 

abrogates the post-attachment Py−α4β1 interaction, 

rendering the PyLNV mutant virus twofold 

less infectious than the Py wild type (Wt) in α4β1positive 

fibroblasts. Moreover, when inoculated in 

newborn C57BL/6 mice, PyLNV showed an altered 

spectrum of in vivo replication compared with 

PyWt, particularly in the skin and in the kidney 

(Caruso et al., 2007). 

Further analysis of the role of Py- α4β1 interaction 

in tissue tropism and tumorigenicity has been performed 

in collaboration with Prof. N. Sanjuan. 

Newborn C3H Bittner-Dawe mice were sub-cutaneously 

inoculated with PyLNV or PyWt, sacrificed 

at different days post infection to analyse the distribution 

of VP1 in different organs/tissues. No differences 

were observed in the ability of dissemination 

of both strains, neither in the involved organs, nor 

in the intensity of the immunolabeling. 

Surprisingly, both viruses infected the kidneys 

throughout the experiment, initially in the interstitial 

cells of the medulla and then in the tubules. The 

incidence of neoplasia for both viruses was about

P. Amati - Role of the early phases of infection in determining virus permissivity 

85% of the animals. Interestingly, a crucial difference 

was observed in kidney sarcomas induction 

ability. Indeed, the mutant induced this neoplasia in 

only 25% of mice in contrast to 65% of the PyWtinoculated 

mice (p=0.02). 

These results suggest that the interaction of Py VP1 

and α4β1 integrin is involved in tissue-tropism, during 

primary infection, and in kidney tumorigenesis. 

The meaning of these findings and the correlation 

between the Py replication and Py tumour induction 

will be further investigated. 

Role of PARP-1 the induction of growthregulated 

genes during cell cycle re-activation 

The interaction of Py with the cell membrane promotes, 

through the VP1 protein, cell cycle progression 

of quiescent fibroblast cells, by stimulating 

transcription of several early-response genes such as 

c-myc, c-fos, and c-jun. On the basis that i) an early 

event in VP1-host cell interaction, occurring in concomitance 

with the induction of early growthresponse 

genes, is the stimulation of PARP-1 activity; 

and that ii) PARP-1 has been recently shown to 

be involved in controlling cell cycle, we decided to 

investigate the possible role of PARP-1 in the exit 

from quiescence, a potentially important regulatory 

step, not only in the context of viral infections but 

also in the abnormal cell cycle of transformed cells. 

ADP-ribose polymerases (PARPs) lead to the transfer 

of ADP-ribose units from NAD + to target proteins, 

forming homopolymers of different sizes. 

This modification alters their functional and physico-chemical 

properties. The modified proteins loose 

their affinity for DNA and dissociate from it increasing 

the accessibility of protein complexes to chromatin. 

More recently it has been discovered that 

poly(ADP-ribosyl)ation is implicated in a rich variety 

of physiological processes, including mitotic 

functions, cell death, transcriptional regulation, differentiation 

and aging. 

The exit of cells from a quiescent state (G0 phase) is 

a multistep process that begins with the immediate 

early response to mitogens and extends into a specialized 

G1 phase. We have demonstrated that 

PARP-1 is required in G0-G1 transition of resting 

cells. Indeed, the examination of early times following 

stimulation of quiescent cells in the absence of 

PARP activity confirmed that poly(ADP-ribosyl)ation 

is necessary for G0 exit. We showed that 

PARP activity is involved in this step through the 

regulation of immediate early response genes, such 

4 

as c-Fos and c-Myc. This was supported by the finding 

that exogenous Myc expression substantially 

restores cell cycle re-activation in the absence of 

polymer synthesis. Furthermore, using siRNAs, we 

demonstrated that PARP-1 is the PARP family member 

involved in the initial step of cell cycle re-activation 

(Carbone et al., 2008). 

It is reasonable that PARP-1 activity participates in 

the regulation of chromatin organization modulating 

the accessibility of transcription factors. In line 

with this function we plan to study the direct implication 

of PARP-1 in the chromatin structure at the 

IEGs promoters. 

Interplay between muscle regulatory factors 

and cell cycle inhibitors 

The appropriately timed induction of cdk inhibitors 

and their sustained expression are critical for the 

induction and the maintenance of the postmitotic 

state in muscle cells as well as in other differentiating 

cell types. This research has been focused on the 

mechanisms regulating the expression of p57kip2, a 

cdk inhibitor with unique properties, devoting particular 

attention to the epigenetic modifications participating 

in the transcriptional control of this gene at 

the onset of differentiation. Our recent results showed 

that in muscle cells p57 is subject to a complex regulation 

involving a DNA demethylation process besides 

the induction of trans-acting factors (Figliola et al., 

2008). Work is in progress to further investigate the 

mechanisms regulating the methylation status of p57 

promoter during muscle differentiation and their relationship 

with transcriptional activation. 

Selected publications 

Caruso M, Busanello A, Sthandier O, Cavaldesi M, 

Gentile M, Garcia MI, Amati P. Mutation in the 

VP1-LDV motif of the murine polyomavirus affects 

viral infectivity and conditions virus tissue tropism 

in vivo. J Mol Biol. 2007, 367:54-64. 

Carbone M, Rossi MN, Cavaldesi M, Notari A, 

Amati P, Maione R. Poly(ADP-ribosyl)ation is implicated 

in the G0-G1 transition of resting cells. 

Oncogene 2008, 27:6083-92. 

Figliola R, Busanello A, Vaccarello G, Maione R. 

Regulation of p57(KIP2) during muscle differentiation: 

role of Egr1, Sp1 and DNA hypomethylation. J 

Mol Biol. 2008, 380:265-77.


Alessandra Bonamore, Alberto Macone, post-doc fellows. 


Dipartimento di Chimica, Università di Firenze (Prof. Giulietta 

Smulevich), CPC Biotech srl, Napoli (Dr. Fabio Arenghi). 


The main target of the project is to attribute biochemical 

and physiological functions to the vast family 

of bacterial globins and exploit their versatile 

catalytic properties in biotransformation processes 

for the synthesis of valuable intermediates for biopharmaceutical 

applications. To this end, the 

research project entails the identification of novel 

bacterial hemoglobins and their cloning into engineered 

Escherichia coli strains bearing oxygen 

dependent enzymes of biotechnological interest. In 

the first part of the project (see previous report of 

activity), the alkylhydroperoxide reductase of flavohemoglobins 

has been exploited for the modification 

of phospholipid acyl chains in order to produce novel 

substituted fatty acids. Moreover, this enzymatic 

activity of bacterial globins, flavohemoglobins in 

particular, has been demonstrated to be a major 

determinant in the physiological response to oxidative 

stress in bacteria. This finding has been recognized 

as a key biochemical clue for the understanding 

the physiological role of flavohemoglobins. In the 

last year of the project, the research has been focused 

mainly on truncated hemoglobins and in particular 

to their functional role. 

Novel bacterial globins 

Novel chimeric proteins made of a globin domain 

fused with a “cofactor free” monooxygenase domain 

have been identified within the Streptomyces avermitilis 

and Frankia sp. genomes by means of bioinfor- 

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 

5 


Escherichia coli strains overexpressing flavohemoglobins for the 

production of novel, biologically active compounds derived from 

phospholipid post-biosynthetic modifications 

matics methods. Structure based sequence alignments 

show that the globin domains of both proteins 

can be unambiguously assigned to the truncated 

hemoglobin family, in view of the striking similarity 

to the truncated hemoglobins from Mycobacterium 

tuberculosis, Thermobifida fusca and Bacillus subtilis. In 

turn, the non-heme domains belong to a family of 

small (about 100 aminoacids) homodimeric proteins 

annotated as antibiotic biosynthesis monooxygenases, 

despite the lack of a cofactor (e.g., a metal, a 

flavin or a heme) necessary for oxygen activation. 

The chimeric protein from S. avermitilis has been 

cloned, expressed and characterized. The protein is a 

stable dimer in solution based on analytical ultracentrifugation 

experiments. The heme ligand binding 

properties with oxygen and carbonmonoxide resemble 

those of other truncated hemoglobins. In addition, 

an oxygen dependent redox activity has been 

demonstrated towards easily oxidizable substrates 

such as menadiol and p-aminophenol. These findings 

suggest novel functional roles of truncated hemoglobins, 

which might represent a vast class of multipurpose 

oxygen activating/scavenging proteins 

whose catalytic action is mediated by the interaction 

with cofactor free monooxygenases. 

Understanding active site dynamics in 

truncated hemoglobins 

The heme binding pocket of truncated hemoglobins 

is shaped by a triad of aminoacids, tipically a tyrosine 

(TyrB10), a tryptophane (TrpG8) and a phenylalanine 

(PheCD1). The specific role of these 

aminoacids in heme ligand binding and catalysis has 

not been unveiled as yet. Thus, the active site of the 

oxygen-avid truncated hemoglobin from Bacillus subtilis 

has been characterized by infrared absorption 

and resonance Raman spectroscopies, and the 

dynamics of CO rebinding after photolysis has been 

investigated by picosecond transient absorption 

spectroscopies. The very low C-O stretching fre-

A. Boffi - Escherichia coli strains overexpressing flavohemoglobins for the production of novel compounds 

quency at 1888 cm-1 (corresponding to the extremely 

high RR stretching frequency at 545 cm-1) indicates 

unusually strong hydrogen bonding between 

CO and distal residues. On the basis of a comparison 

with other truncated hemoglobins it is envisaged 

that the two CO conformers are determined by specific 

interactions with the TrpG8 and TyrB10 

residues. Mutation of TrpG8 to Leu deeply alters 

the hydrogen-bonding network giving rise mainly to 

a CO conformer characterized by a Fe-CO stretching 

band at 489 cm-1 and a CO stretching band at 1958 

cm-1. Picosecond laser photolysis experiments carried 

out on the CO bound adduct revealed dynamical 

processes that take place within a few nanoseconds 

after photolysis. Picosecond dynamics is largely 

dominated by CO geminate rebinding and is consistent 

with strong H-bonding contributions of 

TyrB10 and TrpG8 to ligand stabilization. These 

data indicate that the structure and dynamics of the 

active site in bacterial globins is not designed to bind 

reversibly oxygen, as in vertebrate hemoglobins. 

Towards novel functional properties 

During the course of this investigation, it has been 

demonstrated that truncated hemoglobins from 

Bacillus subtilis and Thermobifida fusca are endowed 

with an unusual high affinity for hydrogen sulfide. 

The physiological relevance of this finding has been 

further demonstrated by the observation that, within 

the bacterial cells, these proteins are saturated with 

6 

hydrogen sulfide produced in at least two metabolic 

steps of the bacterial cell, namely cysteine degradation 

and synthesis of iron-sulfur centers. This finding 

point to a specific role of bacterial globins within 

the complex and still largely ununderstood metabolism 

of sulfur in bacteria. In our view, the physiological 

relevance (are bacterial globin sulfide sensing 

proteins?) and evolutionary perspective (are globins 

evolved from sulfide binding proteins in archea?) of 

this preliminary finding warrants further dedicated 

research on the subject. 

Selected Publications 

Bonamore A, Attili A, Arenghi F, Catacchio B, 

Chiancone E, Morea V, Boffi A. A novel chimera: 

the "truncated hemoglobin-antibiotic monooxygenase" 

from Streptomyces avermitilis. Gene 2007, 

398:52-61. 

Di Giulio A, Bonamore A. Globin interactions with 

lipids and membranes. Methods in Enzymol. 2008, 

436:239-52. 

Feis A, Lapini A, Catacchio B, Brogioni S, Foggi P, 

Chiancone E, Boffi A, Smulevich G. Unusually 

strong H-bonding to the heme ligand and fast geminate 

recombination dynamics of the carbon monoxide 

complex of Bacillus subtilis truncated hemoglobin. 

Biochemistry 2008, 47:902-10.


Francesco Bossa, professor; Angela Tramonti, CNR 

researcher; Maria Giulia Bigotti, post-doc fellow; Eugenia 

Pennacchietti, PhD student; Daniela Bastianelli, Marco 

Scacchi, graduate students. 


School of Biosciences, University of Wales, Cardiff, UK (Prof. Robert 

A. John); Biochemiches Institut, University of Zurich, Switzerland 

(Dr. Guido Capitani and Prof. Markus G. Gruetter). 


The purpose of our research project is to provide 

insight into the molecular mechanisms which in 

Escherichia coli control the expression of the glutamate-based 

acid resistance (GDAR) system, the most 

potent acid resistance system in E. coli and other 

enteric bacteria, as well as into the mechanism of 

action of its structural constituents. The elucidation 

of the mechanisms responsible for colonization of 

the mammalian host under low pH conditions will 

help to spot useful targets for therapeutics or vaccines. 

The GDAR system requires the intracellular protonconsuming 

activity of two glutamate decarboxylase 

isoforms, GadA and GadB, as well as the antiport 

activity of an integral membrane protein, GadC, 

which 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, which 

encodes an activator of the GDAR system. The gadA 

gene belongs to the so-called acid fitness island 

(AFI), a 15-kb genome region repressed by H-NS 

(the histone-like nucleoid-structuring protein) and 

controlled by RpoS, (the stationary phase σ factor of 

the RNA polymerase). The AFI contains 13 genes 

7 


The glutamate-based acid resistance system of Escherichia coli: 

the molecular basis of the activation of its regulatory and 

structural components 

Principal investigator: Daniela De Biase 

Professor of Biochemistry 

Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" 

Tel: (+39) 06 49917692; Fax: (+39) 06 49917566, 06 4440062 

daniela.debiase@uniroma1.it 

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 acid 

resistance in different ways. 

The interplay of the regulators in the gadA and 

gadBC promoter regions is very complex. The essential 

regulator GadE, a LuxR-like protein which is 

encoded by the AFI gene gadE, is under the control 

of three different activation circuits. The 

GadX/GadW-dependent circuit is required for gadE 

activation upon entry into the stationary phase and 

involves the transcriptional regulators GadX and 

GadW, the genes of which are located downstream 

of gadA and gadX respectively. This circuit is the 

most complex because it involves also the global regulators 

H-NS and CRP, which act as transcriptional 

repressors, and RpoS, which positively affects gadA 

and gadBC transcription. GadX and GadW are both 

members of the AraC/XylS family of transcriptional 

regulators that bind a 17–21-bp sequence at target 

promoters via two HTH motifs localized within the 

C-terminal domain. Interestingly, both regulators 

are capable of up-regulating the gadA and gadBC 

target genes both indirectly and directly via gadE 

activation and binding to the gadA and gadBC promoter 

regions respectively. 

GadY, the small RNA encoded downstream of gadX 

in the opposite direction, is unique among small regulatory 

RNAs because base-pairing with the monocistronic 

gadX mRNA, the only target known to date, 

occurs at the 3’- UTR of the gadX message, and the 

outcome is stabilization of the target mRNA instead 

of its degradation. 

In the period 2007-2008 we carried out a detailed biochemical 

characterization of GadB site specific 

mutants with an altered pH dependence (manuscript 

in preparation). In the present report we briefly 

describe the recently published findings which shed 

light on the regulation of the GadX/GadW-dependent 

circuit. In particular, we provided evidence for a 

new regulatory loop that operates in the gadY-gadW

D. De Biase - The glutamate-based acid resistance system of Escherichia coli 

intergenic region. In fact, the data from gadX::lacZ 

transcriptional fusions indicate that gadX transcription 

from its own promoter is not significantly affected 

by either the pH of the growth medium or any of 

the regulators of the GDAR system. This reinforces 

our previous findings that the intracellular levels of 

gadX mRNA, and consequently of the GadX protein, 

are mainly controlled at the transcriptional level by 

H-NS at the PgadX and PgadAX promoters. We 

showed that the GadX/GadW-dependent circuit is 

primarily controlled at the level of the gadX-gadW 

intergenic region, where the gadY gene is located and 

transcribed divergently from gadW. The gadY promoter 

responds positively to the acidic pH in the stationary 

phase and requires GadX for maximal activity. 

Indeed, we identified a GadX/GadW binding site 

in the gadY-gadW intergenic region. This binding 

site, spanning position -61 to -21 with respect to the 

+1 of gadY, positively affects gadY transcription and 

negatively affects gadW transcription from one of its 

promoters. Our analysis of gadW transcription 

revealed that although this regulator has a minor role 

in the GDAR system, its expression indirectly affects 

the gadX mRNA levels by controlling transcription 

from the gadY promoter. In fact, binding of the RNA 

polymerase to the gadY promoter favours the transcription 

of the divergent gadW gene, whereas gadW 

transcription has the opposite effect on the activity of 

the gadY promoter. 

Another important finding was the identification of 

a GadX/GadW consensus sequence. By aligning the 

GadX/GadW-binding site on the gadY promoter 

with the GadX/GadW-binding sites previously identified 

in the gadA and gadBC 5’ regulatory regions, 

we generated a 42-bp GadX/GadW consensus 

sequence. DNAse I footprinting analyses confirmed 

8 

that a 42-bp GadX/GadW-binding site is also present 

in the AFI regulatory regions of the slp-yhiF, 

hdeAB and gadE-mtdEF operons. The 42-bp long 

GadX/GadW-binding site clearly arises from the 

tandem arrangement of two 21-bp sequences, in 

which nine positions are highly-to-strictly conserved. 

The distance of the GadX/GadW-binding 

site from the transcriptional start site of the target 

genes varies significantly. 

In a previous study we showed that at least in vitro 

GadX is able to upregulate the gadA gene by itself 

and can also displace H-NS from the gadA promoter, 

thereby allowing its transcription even when H-NS 

is already bound. It was therefore suggested that in 

vivo, the most important role played by GadX 

(GadW) on the gadA promoter is as a H-NS countersilencer. 

Remarkably, we could demonstrate that the 

5’ regulatory regions of the transcriptional units slpyhiF, 

hdeAB-yhiD, hdeD, gadE-mtdEF, gadY and 

gadAX in the AFI were direct targets of the activators 

GadX and GadW. Molecular analysis in the 

presence of GadX (GadW) and H-NS is necessary to 

establish whether there is functional interplay 

between these proteins in the AFI. Overall, our findings 

suggest a possible role for GadX and GadW in 

the AFI as antirepressors of H-NS. 


Tramonti A, De Canio M, De Biase D. 

GadX/GadW-dependent regulation of the 

Escherichia coli acid fitness island: transcriptional 

control at the gadY-gadW divergent promoters and 

identification of four novel 42-bp GadX/GadW-specific 

binding sites. Mol Microbiol. 2008, 70:965-82.


Giuseppe Ragona, Antonio Angeloni, Pankaj Trivedi, 

professors; Roberta Santarelli, Mara Cirone, researchers; 

Antonella Farina, Roberta Gonnella, post-doc fellows; 

Marisa Granato, Eleni Anastasiadou, PhD students; 

Claudia Zompetta, technician. 


In herpesviruses, viral production results from a 

coordinated process requiring sequential waves of 

protein synthesis (immediate-early, early and late 

genes) that enable newly replicated DNA to be packaged 

into the pre-assembled capsid and its internal 

nucleocore. These immature viral particles are then 

submitted to several rounds of envelopment-deenvelopment 

that allow sequential crossings of intraand 

extracellular membranes to reach the extracellular 

milieu. The first egress results in a crossing of 

the nuclear membrane. Previous work using mutants 

of the HSV1 and pseudorabies virus has shown that 

this process requires interaction between the two 

conserved among herpesvirus membrane proteins 

UL34 and phosphoprotein UL31. These two proteins 

are sufficient to induce formation of primary 

envelopes derived from the nuclear membrane in 

which nucleocapsids are enwrapped after primary 

egress and genetic mutants lacking one of these proteins 

are sequestered in the nucleus. We suggested 

recently a similar scenario also for EBV, based of the 

identified interaction between the UL34 positional 

homolog BFRF1 and the UL31 positional homolog 

BFLF2 and on the observation that most BFRF1 

mutant viruses (∆BFRF1) remain trapped in the 

nucleus (Farina et al., J Virol. 2005; Gonnella et al., J 

Virol. 2005). However, the weak homology at the protein 

level between UL31 and BFLF2, their different 

timing of expression, with BFLF2 being expressed 

early and UL31 expressed late during lytic replication, 

suggest functional divergence between both 

Principal investigator: Alberto Faggioni 

Professor of General Pathology 

Dipartimento di Medicina Sperimentale 

Tel: (+39) 06 4461500; Fax: (+39) 06 4454820 

alberto.faggioni@uniroma1.it 

9 


Control of latency and replication of Epstein-Barr virus 

proteins. To clarify these issues, we have generated a 

mutant virus devoid of the BFLF2 gene and analyzed 

its phenotypic traits during lytic replication, 

infection and B cell immortalization in vitro. 

To construct a recombinant virus devoid of the 

BFLF2 gene we used the insertion mutagenesis technology. 

This is based on homologous recombinations 

events which allow to disrupt or delete the gene of 

interest. To this purpose, we used: 1) a BAC vector, 

named p2089, where the entire genome of EBV 

derived from B95-8 cell line was cloned. This vector 

contains the chloramphenicol resistence gene, a bacterial 

origin of replication needed for its replication 

in E. coli, the GFP gene and the gene conferring 

resistence to hygromycin, the selection marker for 

eukaryotic cells; 2) pKD46 plasmid, which contains 

the three genes gam, bet and exo used by λ phage during 

homologous recombination events; 3) a linear 

targeting vector containing the kanamycin 

resistence gene, flanked by sequences of approximately 

40 nucleotides and homologues to the regions 

upstream and downstream the BFLF2 gene. In addition, 

the kanamycin cassette is flanked by FRT 

sequences (Flip Recombination Target) which are 

recognized by the enzyme Flip recombinase during 

the excision process of the cassette. In the next step, 

the viral genome of the recombinant virus deleted of 

the BFLF2 gene has been stably transfected in the 

EBV negative cell line 293, which is known to be permissive 

to EBV replication. To analyze if BFLF2 

deletion alters the expression of other lytic viral 

genes, the line 293/∆BFLF2, and the control cell 

lines 293/WT (containing the entire viral genome 

wild type) and 293/∆BFLF2+BFLF2 (recomplemented, 

where BFLF2 is furnished in trans) have 

been transfected with BZLF1 to induce viral replication, 

and analyzed at 96 hrs post transfection by 

western blot and indirect immunofluorescence to 

verify the expression of early (BFRF1 and EA-D) 

and late (gp350/220 and BLRF2) lytic genes. As 

expected, BFLF2 was not expressed in

A. Faggioni - Control of latency and replication of Epstein-Barr virus 

293/∆BFLF2 cells, but was expressed in the BFLF2 

recomplemented cells. On the contrary, no variations 

in the expression of the other genes was observed, 

confirming that BFLF2 deletion does not modify the 

lytic gene cascade, which is completed with the 

expression of the structural late proteins gp350/220 

and BLRF2. Furthermore, to analyze if the absence 

of BFLF2 might influence one of the earliest phases 

of the lytic cycle, the viral DNA replication, we performed 

a Gardella gel analysis which discriminates 

between the two viral DNA forms, the episomal typical 

of latent infection, and the linear present during 

viral replication. The results showed that the recombinant 

virus present in the 293/∆BFLF2 cells is able 

to replicate, since newly synthesized linear genomic 

DNA is produced to levels comparable to that from 

other control cell lines. Finally, to analyze the infectivity 

of ∆BFLF2 virus, supernatants from cells containing 

the mutant virus were used to superinfect 

Raji cells or to infect B lymphocytes from healthy 

donors. The infection rate has been measured quantifying 

GFP expression, present in the recombinant 

virus genome. Approximately 400 fold less virus was 

produced by ∆BFLF2 virus-infected cells, in comparison 

to the control cell lines, wild type and recomplemented. 

This suggests a reduced production of 

virions. To analyze the intracellular maturation of 

the mutant virus and to evaluate at which level this 

process is blocked, electron microscopy evaluation of 

the infected cells was performed. The ultrastructural 

analysis showed in ∆BFLF2 cells numerous type B 

capsids, devoid of viral DNA, which accumulate 

within the nucleus and do not reach the cytoplasm. 

In cells where BFLF2 function has been recomplemented, 

the presence of numerous mature type C 

virions, which fully completed the viral replication 

cycle, has been consistently observed. Overall, the 

above results suggest that, in absence of BFLF2, the 

encapsidation of viral DNA is defective, and the 

egress of virions from the nucleus to cytoplasm is 

blocked, similarly to what we described previously in 

cells infected with the virus deleted of the other 

early lytic gene, BFRF1 (Farina et al., J Virol. 2005). 

In another part of the project, we identified and 

characterized p33, the product of Kaposi’s sarcoma 

associated herpesvirus (KSHV) ORF69, positional 

homolog of EBV BFLF2 (UL31). p33 is expressed 

upon induction of viral lytic cycle with early kinetics. 

Immunofluorescence analysis revealed that, in 

infected cell lines, the protein is localized in the 

nucleus, both in dotted spots or along the nuclear 

10 

membrane. Nuclear fractionation experiments 

showed that p33 partitions with the nuclear matrix, 

and both immunoblotting of purified virions and 

immunoelectron microscopy indicated that the novel 

protein is not a component of the mature virus. 

Following ectopic expression in KSHV negative 

cells, the protein was never associated with the 

nuclear membrane, suggesting that p33 needs to 

interact with additional viral proteins to reach the 

nuclear rim. In fact, after cotransfection with the 

ORF67 gene, the KSHV positional homolog of 

UL34, the p33 intranuclear signal changed, and the 

two proteins colocalized on the nuclear membrane. A 

similar result was obtained when ORF69 was 

cotransfected with BFRF1, the Epstein-Barr virus 

(EBV) positional homolog of UL34 and ORF67. 

Finally, upon cotransfection, ORF69 significantly 

increased nuclear membrane reduplications induced 

by BFRF1. 

In conclusion, these results indicate that p33 shares 

many similarities with its EBV homolog BFLF2 and 

suggest that, in human γ-herpesviruses, cross-complementation 

is possible between KSHV and EBV 

proteins. EBV and KSHV coinfect the majority of 

PEL cell lines, and molecular interactions between 

the two viruses have been reported. Whether these 

interactions might be relevant for the pathogenesis 

of EBV and KSHV associated diseases will deserve 

further studies. 

Selected publications: 

Serafini B, Rosicarelli B, Franciotta D, Magliozzi R, 

Reynolds R, Cinque P, Andreoni L, Trivedi P, Salvetti 

M, Faggioni A, Aloisi F. Dysregulated Epstein-Barr 

virus infection in the multiple sclerosis brain. J Exp 

Med. 2007, 204:2899-912. 

Granato M, Feederle R, Farina A, Gonnella R, 

Santarelli R, Hub B, Faggioni A, Delecluse HJ. 

Deletion of Epstein-Barr virus BFLF2 leads to 

impaired viral DNA packaging and primary egress 

as well as to the production of defective viral particles. 

J Virol. 2008, 82:4042-51. 

Santarelli R, Farina A, Granato M, Gonnella R, Raffa 

S, Leone L, Bei R, Modesti A, Frati L, Torrisi MR, 

Faggioni A. Identification and characterization of the 

product encoded by ORF69 of Kaposi's sarcoma-associated 

herpesvirus. J Virol. 2008, 82:4562-72.


Dario Benelli, research fellow; Antimo Naspi, PhD student; 

Dorina Polinari, undergraduate student. 


University of Wien, Austria (Prof. Udo Blaesi); University J.W. 

Goethe, Frankfurt, Germany (Prof. Joerg Soppa); CNRS, 

Strasbourg, France (Dr. Bruno Klaholtz); Università Politecnica 

delle Marche, Ancona (Prof. Anna La Teana). 


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. As the speed and efficiency 

of translation are modulated mainly at the initiation 

stage, the origin of the regulatory mechanisms 

acting in present-day cells may be traced back to the 

common ancestor of Archaea and Eukarya. 

Therefore, unravelling the mechanism and regulation 

of translational initiation in Archaea, besides 

being interesting in its own right, will also lead to a 

better understanding of the corresponding eukaryal 

process. 

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 trace a meaningful picture of the early evolution 

of these interesting proteins, thus gaining 

valuable insight into the onset of eukaryotic-type 

translational regulation. 

11 


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 

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. It is a G-protein, active only in the 

GTP-bound form. In eukaryotes, after delivering 

met-tRNAi, eIF2 hydrolyzes its GTP and is ejected 

from the ribosome in the inactive, GDP-bound form. 

To participate in another round of initiation, eIF2 

must be reactivated by GTP/GDP exchange, 

catalyzed by the auxiliary factor eIF2B. In most conditions 

(such as stress) when a rapid shut-off of protein 

synthesis is desirable, eIF2 is inactivated by 

phosphorylation (at ser 51) of its α-subunit, carried 

out by certain stress-activated kinases. This modification 

converts the α-protein in a competitive 

inhibitor of eIF2B, thereby inhibiting GTP/GDP 

exchange and blocking the recycling of eIF2. This 

mechanism of translational control is essential and 

widespread in eukaryotic cells, but does not exist in 

Bacteria, which use a different factor, the monomeric 

protein IF2, for delivering tRNAi to the ribosome. 

Why the Archaea, as Eukarya, should use a trimeric 

protein as the met-tRNAi binding factor is as yet 

unclear. a/eIF2 cannot be regulated with the same 

mechanism as in eukaryotes; it has a similar affinity 

for both GTP and GTP and does not need an 

exchange factor to be reactivated after GTP hydrolysis. 

Since it has been reported that the α-subunit of 

a/eIF2 is phosphorylated as in eukarya, one of the 

aims of our project is to verify this finding and investigate 

the physiological role of α-subunit modifications 

in Archaea. 

The modification pattern of the a/eIF2 α-subunit in 

the archaeon Sulfolobus solfataricus is studied both in 

vitro and in vivo. We have cloned, expressed and 

purified an archaeal kinase reported to be involved 

in a/eIF2 phosphorylation. The recombinant protein 

efficiently phosphorylates the a/eIF2 α-subunit 

in vitro. The modified a/eIF2 α-subunit does not 

seem to differ from its native counterpart in a series

Paola Londei - Translational regulation: from the archaea to the eukarya 

of assays so far performed, including interaction 

with the β and γ subunits to form the intact trimer 

and capacity of binding met-tRNAi. To investigate 

in vivo phosphorylation, we have performed 

immunoprecipitation assays with anti α-subunit 

antibodies on whole cell lysates, followed by western 

blotting with anti-phosphoserine antibodies. 

Preliminary results indicate that the a/eIF2 α-subunit 

is indeed phosphorylated in vivo, and that the 

amount of phosphorylation increases following 

heat-shock, indicating that the modification may 

inhibit protein synthesis. Work is in progress to 

confirm and expand these results and to elucidate 

the underlying molecular mechanism. 

A still mysterious protein: IF6 

The factor known as aIF6 (archaea) and eIF6 

(eukarya) is a monomeric protein of about 25 kDa. 

Eukaryotic IF6 was originally described as a ribosome 

anti-association factor, which interacted with 

the large ribosomal subunits inhibiting the formation 

of active 80S ribosomes. Later experiments revealed 

that depletion of eIF6 in yeast depressed the biosynthesis 

of 60S ribosomal subunits, but had otherwise 

no specific effect on translation initiation, assigning 

to the factor a main role in ribosome synthesis. 

Further experiments in mammals, however, have 

confirmed a role for eIF6 in translational regulation, 

that may or may not co-exist with other functions. 

60S ribosomal subunit must release eIF6 to enter the 

translation cycle; eIF6 dissociation from the ribosome 

is triggered by the phosphorylation of the factor 

carried out by certain regulatory kinases. More 

12 

recently, it has been shown that heterozygous knockout 

mice for eIF6 have defective translational initiation. 

Finally, it has been reported that eIF6 depletion 

inhibits miRNA-induced gene silencing in both 

human cells and in nematodes. Despite the many 

functions reported, the mechanism of action of eIF6 

in any of them remains unknown. 

Archaeal IF6 shares a high degree of homology with 

eIF6, both in primary sequence and in three-dimensional 

structure. Our recent work has revealed that S. 

solfataricus aIF6 has a ribosome anti-associating 

activity and is a potent inhibitor of protein synthesis 

in vitro. We have mapped for the first time the ribosomal 

binding site of aIF6: it lies in the centre of the 

30S-contacting face of the large ribosomal subunit. 

Our model of the aIF6/50S complex reveals that the 

factor inhibits the formation of the 70S ribosome by 

masking a number of RNA and protein determinants 

that bridge the two subunits. These data confirm the 

identity of IF6 as a translation factor and rationalize 

its anti-associating activity in molecular detail. 

Work is in progress to unravel the mechanism whereby 

aIF6 dissociates from the 50S subunit. Preliminary 

data indicate that this may entail a novel type of protein 

modification, possibly in association with a ribosome-dependent 

GTPase activity, yet to be identified. 


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. 2008, Epub.

AREA2 

Pathogenetic 

mechanisms of 

microbially 

associated 

diseases

A structural biology study of schistosomiasis 


Adriana Erica Miele, professor; Francesco Angelucci, postdoc 

fellow; Daniela Dimastrogiovanni, PhD student; 

Giovanna Boumis, technician. 


Istituto di Biologia Cellulare, CNR, Monterotondo, Roma (Prof. 

Donato Cioli, Prof. Piero Liberti, Dr. Livia Pica Mattoccia). 

Schistosomiasis is a widespread human parasitic disease 

caused by helminth parasites of the genus 

Schistosoma. The life cycle of the parasite depends 

on an intermediate and a definitive host, a freshwater 

mollusc and a mammal respectively. Because of 

the requirements of the intermediate host, the infection 

is restricted to tropical and subtropical climates 

and to the proximity of freshwater sources; it has 

nevertheless a major impact on human health and 

development since it may frustrate the attempts to 

develop agriculture. Efforts directed to the control 

of the intermediate host and to develop a vaccine for 

humans have been scarcely successful, and therefore 

therapy of conclamate cases remains the strategy of 

choice. 

The therapy of schistosomiasis relies mainly on one 

chemotherapeutic agent, praziquantel, although 

several other compounds exert anti-parasitic 

effects. Thus, development of drug resistance is an 

impending danger, with serious implications for the 

health protection of an estimated 200 millions people. 

This rational and legitimate concern might 

now begin to be relieved thanks to the increased 

knowledge of the genome and the molecular biology 

of the schistosome, which has lead to the recent 

proposal of new classes of compounds that could 

represent novel sources of drugs against schistosomiasis. 

We focussed our studies on two main potential 

drug targets (Cioli et al., Trends Parasitol. 2008, 

24:379-82), namely the proteins thioredoxin glutathione 

reductase (TGR) and cyclophylin (Cyp) 

Pathogenetic mechanisms of microbially associated diseases - AREA 2 

Principal investigator: Andrea Bellelli 



Tel: (+39) 06 49910955; Fax: (+39) 06 4440062 

Andrea.bellelli@uniroma1.it 

15 

both from S. mansoni; other potential targets are 

also being considered. 

TGR is a key flavoenzyme expressed by schistosomes 

that bridges the two detoxification pathways 

of thioredoxin and of glutathione. The enzyme is 

crucial for the parasite survival in the definitive 

host's organism (Kuntz et al., PLoS Med. 2007, 4, 

e206).We solved the crystal structure of TGR from 

S. mansoni (SmTGR), deleted in the last two 

residues at 2.2 Å resolution (Angelucci et al., 

Proteins 2008, 72:936-45). 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 

(TR) 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. SmTGR 

is the target of two classical antischistosomal 

drugs, emetic tartrate and oltipraz, that are not any 

more employed in therapy because of their severe 

side effects. Moreover it has been demonstrated that 

SmTGR is the target of auranofin, a gold compound 

used for the treatment of rheumatoid arthritis 

but capable of killing the schistosomes in vitro 

and in vivo. Finally, some new compounds have 

been discovered that inhibit SmTGR, e.g. the derivatives 

of furoxane (Sayed et al., Nat Med. 2008, 

14:407-12). Our structure may provide the information 

necessary for improving any of these lead compounds, 

yielding substantial improvements in specificity 

and activity. 

The immunosuppressant cyclosporin A has been 

shown to significantly diminish worm burden in mice 

infected with S. mansoni (Khattab et al., Exp Parasitol. 

1998, 90:103). Given the well established interaction

A. Bellelli - A structural biology study of schistosomiasis 

between cyclosporin A and the cyclophilin superfamily 

of peptidylprolyl cis-trans isomerases, we solved 

the structure of cyclophilin A from S. mansoni 

(SmCypA) by x-ray crystallography in the reduced 

and oxidized states at 1.5 and 1.8 Å of resolution, 

respectively (Gourlay et al., J Biol Chem. 2007, 

282:24851-7). Oxidized SmCypA contains a disulfide 

bridge between two C-terminal cysteines (Cys-122 

and Cys-126). This is the first example of a 

cyclophilin containing this disulfide bridge. Parallel 

functional studies suggest a mechanism for regulation 

of SmCypA activity via oxidation of its thiol groups; 

in fact, whereas oxidized SmCypA is inactive, reduced 

SmCypA is an efficient isomerase active at nanomolar 

levels with a k cat /K M of 1.1 x 107 M -1 s -1 , and it is 

inhibited by cyclosporin A (IC 50 of 14 +/- 4 nM). 

The lack of conservation of this cysteine couple 

within the CypA superfamily, their close proximity to 

the active site, and the importance of thiol groups for 

peptidyl-prolyl cis-trans isomerase activity render 

this structural feature a challenge for the development 

of alternative and more effective anti-schistoso- 

16 

miasis inhibitors and may in addition imply an alternative 

function of SmCypA in the schistosome. 


Gourlay LJ, Angelucci F, Baiocco P, Boumis G, 

Brunori M, Bellelli A, Miele AE. The three-dimensional 

structure of two redox states of cyclophilin A 

from Schistosoma mansoni. Evidence for redox regulation 

of peptidyl-prolyl cis-trans isomerase activity. J 

Biol Chem. 2007, 282:24851-7. 

Angelucci F, Miele AE, Boumis G, 

Dimastrogiovanni D, Brunori M, Bellelli A. 

Glutathione reductase and thioredoxin reductase at 

the crossroad: the structure of Schistosoma mansoni 

thioredoxin glutathione reductase. Proteins 2008, 

72:936-45. 

Cioli D, Valle C, Angelucci F, Miele AE. Will new 

antischistosomal drugs finally emerge? Trends 

Parasitol. 2008, 24:379-82.


Luigi Lembo-Fazio, Giulia Nigro, Laura Curcuru’, 

Valeria Liparoti, PhD students. 


Department of Immunology, University of Toronto, Toronto, 

Canada (Prof. Dana J. Philpott); Facoltà di Medicina Veterinaria, 

Università di Camerino (Prof. Giacomo Rossi); Dipartimento di 

Chimica Organica e Biochimica, Università di Napoli “Federico II” 

(Prof. Antonio Molinaro). 


The purpose of this project was to contribute to the 

knowledge of the mechanisms relative to PAMP- 

PRM recognition and subsequent signaling. In particular, 

we planned to investigate on the interaction 

of the PRR Nods with their agonist PAMP, peptidoglycan 

(PGN). Our aim was to understand how to 

modulate the immune response triggered by the 

PAMPs of the enteropathogen Shigella in order to 

reduce inflammation. 

Results and perspectives 

A certain number of invariant bacterial structures, 

so-called pathogen-associated molecular patterns 

(PAMPs), are selectively recognized by the host via 

pattern recognition molecules (PRMs), like Toll-like 

receptors (TLRs) and nucleotide-binding oligomerization 

domain (Nod) molecules (Inohara N, Núñez 

G, Nat Rev Immunol. 2003, 3:371-82). Upon interaction 

between a specific PAMP and its cognate PRM, 

the innate immune system is alerted essentially 

through NF-κB activation and subsequent cytokine 

production. 

Nod proteins are intracellular PRMs that recognize 

pathogens in the cytosol through sensing PGN 

motifs. In particular, Nod1 recognizes the core 

dipeptide structure, (iE-DAP), contained in the 


Role of Shigella surface components in immunomodulation 

of the inflammatory response 

Principal investigator: Maria Lina Bernardini 

Researcher in Cellular Microbiology 

Dipartimento di Biologia Cellulare e dello Sviluppo 

Tel: (+39) 06 49917850; Fax: (+39) 06 49917994 

marialina.bernardini@uniroma1.it 

17 

PGN of Gram-negative bacteria (Girardin SE et al., 

Science 2003, 300:1584-87) and Nod2 recognizes 

muramyldipeptide present on PGN of both Gramnegative 

and Gram-positive bacteria (Girardin SE et 

al., J Biol Chem. 2003, 278:8869-72). Genetic variants 

of Nod proteins are associated with inflammatory 

disorders, thus reinforcing the link between 

bacterial sensing and inflammation. 

Dramatic colonic inflammation is the feature of 

shigellosis, a human infectious disease caused by the 

infection of as few as 100 bacteria of the enteroinvasive 

pathogen Shigella spp. Shigellae penetrate the 

baso-lateral pole of intestinal epithelial cells (IEC) 

through injection of Ipa proteins via a type III secretion 

system (TTSS) (Tran Van Nhieu G et al., Cell 

Microbiol. 2000, 2:187-93). In IEC, shigellae stimulate 

NF-κB activation (Philpott DJ et al., J Immunol. 

2000, 165:903-14) upon recognition of iE-DAP by 

Nod1 (Girardin SE et al., Science 2003, 300:1584-87). 

Following this step, inflammation mounts via the 

production of pro-inflammatory cytokines, primarily 

IL-8 that stimulates polymorphonuclear leukocyte 

recruitment (Philpott DJ et al., J Immunol. 2000, 

165:903-14). 

However, despite the great number of studies focused 

on the interaction of PRMs with PAMPs, it is still 

unclear how in epithelial cells, which are not normally 

committed to ingest and digest pathogens, Nod1 

could physically interact in vivo with PGN, a structure 

internal to Gram-negative bacteria envelope. 

Several studies have detailed that following PGN 

remodeling around 40-50% of PGN is released during 

each bacterial generation and approximately 

90% of this material accumulates in the periplasm, 

from where it is re-imported into the cytoplasm for 

recycling. 

This project started from the hypothesis that this 

process, leading to the release of minimal PGN 

products, could contribute to pathogen recognition 

by Nod1 during natural infection. 

In order to assess the biological role of these PGN

M. L. Bernardini - Role of Shigella surface components in immunomodulation of the inflammatory response 

derivatives we have rationally mutagenized S. flexneri 

5 in two genes, ampG and mppA, in the attempt to 

impair PGN recycling and to augment the release of 

muropeptides by shigellae in host cells and tissues. 

AmpG is a transmembrane protein that acts as a specific 

permease for intact muropeptides (tri or tetra) 

whereas the periplasmic binding protein MppA binds 

murein tripeptides and utilizes general oligopeptide 

permease (Opp) to transfer its bound ligand into the 

cytoplasm where tripeptides are recycled. 

Our aim was to analyze whether and how these 

released muramylpeptides might influence host cell 

sensing by PRMs and ultimately Shigella virulence. 

Our results demonstrate that Shigella spontaneously 

releases PGN fragments and that this process can be 

greatly increased by inactivating either ampG or 

mppA genes. 

We found that the increase of muramylpeptide shedding 

corresponds to significant Nod1 activation in 

epithelial cells. In fact, the Shigella ampG and mppA 

mutants trigger Nod1-mediated NF-κB activation to 

a greater extent than the wild-type strain. Likewise, 

sterile purified supernatants (SPS) added from 

“inside” to host cells were able to stimulate Nod1 to 

varying degrees, depending on their relative amount 

and composition. In particular, muramylpeptides 

shed by the S. flexneri mutant ∆mppA, whose composition 

differs significantly from that of the wild-type 

and the ampG mutant, were especially efficient in 

mediating Nod1 stimulation in HEK293 cells 

expressing ectopic Nod1. 

Our findings support the idea that muramylpeptides 

released by pathogens during natural infection could 

modulate the immune response through qualitative 

and quantitative differences in Nod protein activation. 

Shigella ∆mppA was strongly attenuated. In lungs of 

mice infected intranasally with this strain, IL-6 production 

was comparable to that induced by wild-type 

bacteria, while the level of IFN-γ was higher. 

Similarly, the liver of animals infected intravenously 

with Shigella ∆mppA contained larger microgranulomas 

(Martino MC et al., Cell Microbiol. 2005, 7:115- 

18 

27), than those induced by the wild-type, in which a 

significant presence of IFN-γ expressing cells might 

facilitate Shigella clearance. IFN-γ could contribute to 

pathogen clearance as shown during natural and 

experimental shigellosis (Way SS et al., Infect 

Immun.1998, 66:1342-8) thus supporting the attenuation 

of this strain. 

Recent studies have stressed the role of bacterial 

PGN composition in immune evasion of various 

pathogens and have unveiled bacterial strategies to 

modulate the PGN structure or to inject 

muramylpeptides into host cells (Viala J et al., C R 

Biol. 2004, 327:551-5). Our results show that the 

immunopotential of Shigella is significantly influenced 

by quantitative and qualitative alterations in 

muramylpeptide shedding, thus suggesting that the 

regulation of PGN release could represent a further 

sophisticated bacterial strategy to survive in host tissues 

and to evade immune defenses. 


Hachani A, Biskri L, Rossi G, Marty A, Menard R, 

Sansonetti P, Parsot C, Tran Van Nhieu G, 

Bernardini ML, Alloaoui A. IpgB1 and IpgB2, two 

homologous effectors secreted via the Mxi-Spa type 

III secretion apparatus, cooperates to mediate polarized 

cell invasion and inflammatory potential of 

Shigella flexneri. Microb Infect. 2008, 10:260-8. 

Nigro G, Lembo Fazio L, Martino MC, Rossi G, 

Tattoli T, Liparoti V, De Castro C, Molinaro A, 

Philpott D, Bernardini ML. Muramylpeptide shedding 

modulates cell sensing of Shigella flexneri. Cell 

Microbiol. 2008, 10:682-95. 

Tattoli I, Lembo Fazio L, Nigro G, Carneiro LAM, 

Ferraro E, Rossi G, Martino MC, de Stefano ME, 

Cecconi F, Girardin SE, Philpott D, Bernardini ML. 

Intracellular bacteriolysis triggers a massive apoptotic 

cell death in Shigella-infected epithelial cells. 

Microb Infect. 2008, 10:1114-23.


Gianni Prosseda, researcher; Monica Pompili, post-doc 

fellow; Marialuisa Barbagallo, Maria Letizia Di Martino, 

PhD students. 


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). 


Shigella is a facultative intracellular pathogen causing 

a severe enteric syndrome in humans, mainly in 

the developing countries. The pathogenicity mechanism 

of Shigella is based on the capacity to reach and 

invade colonic epithelial cells, multiply intracellularly 

and spread to adjacent cells with consequent cell 

death and destruction of the colonic mucosa. This 

process is highly complex and requires the coordinated 

expression of virulence factors, encoded not 

only by chromosomal genes but also by the plasmid 

genome. The transition of Shigella towards a pathogenic 

phenotype is a good model for understanding 

how the virulence phenotype is triggered in a commensal 

organism. Major mechanisms possibly 

accounting for this phenomenon are the acquisition 

of additional genes encoding virulence determinants 

and the inactivation or loss of pre-existing genes. 

The acquisition of the virulence plasmid (pINV) has 

been undoubtedly one of the most critical events in 

the evolution of the pathogenic lifestyle of Shigella. 

The pINV plasmid contains all the genes required 

for invasion and for intra- and inter-cellular spread, 

including their positive activators VirF and VirB. To 

obtain a proper sensing of the host environment 

and an adequate virulence response, the expression 

of virulence determinants is integrated in layers of 


Deciphering the complexity of the virulence networks in Shigella 

and enteroinvasive Escherichia coli (EIEC): the interplay among 

nucleoid proteins, promoter DNA structure and regulatory factors 

Principal investigator: Bianca Colonna 

Professor of General Microbiology 


Tel: (+39) 06 49917580, 06 49917582; Fax: (+39) 06 49917594 

bianca.colonna@uniroma.it 

19 

iterative regulative networks, ensuring a coordinated 

and on-time synthesis of all determinants necessary 

to induce invasivity. Like in other life-threatening 

human pathogens, also in Shigella the entry from 

the outer environment into the warmer host milieu 

is one of the crucial events triggering the expression 

of virulence factors. The plasmid regulatory 

cascade of Shigella represents an interesting system 

to understand how pathogenic bacteria have evolved 

to prevent wasteful expression of virulence factors 

while ensuring strong and appropriate activation 

when this is required. The arrival of additional regulators 

in the novel pathogen through the acquisition 

of the pINV might have altered the transcriptional 

program of the ancestral E. coli cell. By transcriptomic 

analysis we have investigated on which 

E. coli genes are up- or downregulated in response 

to an increasing level of the VirF protein, which is 

the first positive activator of the Shigella invasivity 

cascade. The results indicate that in E. coli several 

genes are up-regulated including those involved in 

biosynthesis of bacterioferritin, tryptophan and 

sperimidine. Surprisingly several E. coli VirF upregulated 

genes are partially or completed deleted in 

all four Shigella species, thus suggesting that overexpression 

of these genes might have a deleterious 

effect on the intracellular life of pathogenic Shigella. 

Analysis of the effect on the invasivity process of 

Shigella strains transformed with plasmids containing 

the E. coli VirF up-regulated genes will strongly 

contribute to the understanding of the molecular 

processes which determine the optimal expression 

of the virulence phenotype. 

The acquisition of new positive activators encoded 

by the pINV is counterbalanced by the loss of some 

ancestral regulators. In the novel habitat the new 

pathogen reaches optimal fitness by modifications of 

its genome which often redesign important metabolic 

properties. Besides the loss of traits important for 

the survival in the environment, but redundant for 

the life inside the host, the new pathogen may also

B. Colonna - Deciphering the complexity of the virulence networks in Shigella and enteroinvasive Escherichia coli (EIEC) 

undergo a series of convergent mutations in genes 

which negatively interfere with the expression or 

function of virulence factors required for the survival 

within the host. The case of the cad system 

exemplifies one of the most important mutations 

arising by loss of antivirulence genes in Shigella. 

The cad operon encodes one of the main decarboxylase 

system based on the activity of lysine decarboxylase 

and is crucial for the maintenance of pH 

levels suitable for cell survival. Despite the fact that 

the cad system may be important during the bacterial 

transit through the intestinal tract, in Shigella 

and EIEC such a system has been completely lost. 

By comparative analysis of molecular rearrangements 

inducing silencing of the cad operon we have 

observed that the lack of lysine decarboxylase activity 

has been attained through diverse strategies, and 

that the first silencing step of the cad locus might 

have been the inactivation of the cadC gene, followed 

by a spread of insertion sequences in cadB 

and cadA inducing deletions within the cad locus or 

extending to the flanking regions. The observed 

lack or inactivation of the cadC gene can be accounted 

for by considering that CadC, the regulator of 

the cadBA operon, might play additional roles in 

controlling cellular functions not directly related to 

lysine utilization. In order to understand whether 

the CadC regulator may play a direct or indirect role 

in the control of other genes involved in the adaptation 

of the bacterium to the host environment we 

investigated how the expression profile of E. coli has 

20 

been affected by the disappearance of CadC. 

Transcriptome analysis revealed that several genes 

where over-expressed in the absence of CadC, 

including genes encoding other systems involved in 

pH homoestasis like the acid stress response or the 

arginine /putrescine trasport systems. 

Since both, the loss and the acquisition of new 

genetic systems, are flanked by the arrival or by the 

lack of transcriptional regulators, we plan to further 

investigate on how the transcriptional profile of the 

ancestor cell has been modified in order to improve 

survival within the host tissues. 


Prosseda G, Latella MC, Barbagallo M, Nicoletti 

M, Al Kassas R, Casalino M, Colonna B. The two 

faced role of the cad genes in the virulence of pathogenic 

Escherichia coli. Res Microbiol. 2007, 158:487- 

93. 

Prosseda G, Casalino M, Colonna B. Regulation of 

virulence genes in Shigella hovers between gain and 

loss of transcriptioanl activators. Nova Acta 

Leopoldina 2008, 98:65-70. 

Roscetto E, Rocco F, Carlomagno MS, Casalino M, 

Colonna B, Zarrilli R, Di Nocera PP. PCR-based 

rapid genotyping of Stenotrophomonas maltophilia 

isolates. BMC Microbiol. 2008, 8:202-7.


Lucia Nencioni, Claudio Passariello, researchers; Rossella 

Sgarbanti, post-doc fellow; Donatella Amatore, Ignacio 

Celestino, Paola Checconi, PhD students; Felicia D’Auria, 

Costantino Pichezzi, technicians. 


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, Germany (Prof. S. Ludwig); 

Dipartimento di Patologia e Medicina di Laboratorio, 

Università di Parma (Prof. W. Magliani). 


Influenza A viruses continue to represent a severe 

threat worldwide, causing large epidemics and pandemics 

responsible for thousands of deaths and hospitalization 

every year. Recently, there has been an 

enormous increase in the circulation of influenza 

viruses that are resistant to licensed anti-influenza 

drugs directed towards viral proteins, namely neuroaminidase 

(NA) and matrix (M1). 

Targeting antiviral drug discovery to host cell factors 

that are essential for viral replication instead of 

against viral structures would decrease the likelihood 

of acquiring drug resistance and increase the 

effectiveness towards different virus types and 

strains. Several intracellular signaling pathways, 

(MAP kinases, NFkB, ER oxidoreductases), that are 

involved in viral replication, are finely regulated by 

small changes in intracellular redox state. An imbalance 

in intracellular redox state has been extensively 

described as occurring in several viral infections, 

including influenza. 

The virus induced pro-oxidant state may then contribute, 

through different mechanisms, both to 

influenza virus replication and to the pathogenesis of 

virus-induced diseases. 


Redox mediated mechanisms involved in the influenza virus 

replication and in the pathogenesis of influenza 

associated diseases 

Principal investigator: Annateresa Palamara 

Professor of Microbiology 

Dipartimento di Scienze di Sanità Pubblica “G. Sanarelli” 

Tel: (+39) 06 4468622, Fax: (+39) 06 4468625 

annateresa.palamara@uniroma1.it 

21 

The scientific activities of our group in 2008 were 

aimed at defining the role of important redox regulated 

intracellular pathways in the replication of 

influenza virus and in the activation of inflammatory 

responses. These activities have been organized in 

the following main topics: 1) the definition of 

p38MAPK role in influenza virus replication and 

viral induced apoptosis 2) the study of redox mediated 

pathways involved in hemagglutinin maturation 

3) the identification of mechanisms involved in the 

internalization of Staphylococcus aureus in influenza 

virus-infected cells. 

Role of p38MAPK in influenza virus 

replication and viral induced apoptosis 

Our previous reports have shown that various steps 

in the influenza A virus life cycle are impaired in 

cells that are characterized by high levels of glutathione 

(GSH) and the expression of the antiapoptotic 

protein Bcl-2. Over this year we have found 

that Bcl-2 does not prevent host cells from undergoing 

virally triggered apoptosis despite its negative 

impact on viral replication. The protein’s 

reduced antiapoptotic capacity was related to the 

phosphorylation of its threonine 56 and serine 87 

residues by virally activated p38MAPK. In contrast, 

in Bcl-2-negative cells, which are fully permissive 

to viral infection, p38MAPK activation contributed 

to the phosphorylation of viral nucleoprotein 

that is an essential step for its export from the 

nucleus and virus assembly. Consistently, treating 

infected cells with p38MAPK inhibitor was able to 

strongly decrease vRNP traffic and viral release 

from infected cells. Our data suggest that 

p38MAPK’s impact on the influenza virus 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. A paper reporting these results is in 

press (Nencioni et al.).

A. Palamara - Redox mediated mechanisms involved in the influenza virus replication 

Definition of redox mediated pathways 

involved in hemagglutinin maturation 

Folding of influenza hemagglutinin (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 virus, without producing toxic effects on 

uninfected cells. Such an inhibition was associated 

with a decrease in the apparent molecular weight of 

HA, and a significant reduction of the protein’s 

dimeric and trimeric forms. Moreover, HA extracted 

from GSH-C4 treated cells was sensitive to Endo 

H digestion, suggesting that the reducing conditions 

caused by GSH-C4 treatment promoted the 

retention of the protein in the ER. As a consequence, 

the insertion of HA into the cell membrane 

was strongly inhibited. Altogether, these results 

suggest that the slight decrease in the HA molecular 

weight induced by GSH-C4 reflects an accumulation 

of the immature HA and provide the evidence 

that GSH-C4 inhibits influenza virus replication 

by interfering with the maturation process of 

HA. A paper reporting these results is in preparation 

(Sgarbanti et al.) 

Mechanisms involved in the internalization 

of Staphylococcus aureus in influenza 

virus-infected cells 

Opportunistic bacterial infections frequently exacerbate 

viral infections of the respiratory tract. We 

have previously demonstrated that human rhinoviruses 

significantly enhance the capacity of S. 

aureus to internalize into cultured cells with a mechanism 

that involves the virus-induced release of 

inflammatory cytokines. Over this year we have 

investigated the cellular and molecular mechanisms 

involved in the cooperation between influenza virus 

22 

and S. aureus. Our data demonstrated that a previous 

infection of MDCK cells with influenza virus 

strongly increased the capacity of S. aureus to internalize 

into the infected cells. Such a phenomenon 

was inhibited when virus infected cells were treated 

with an antibody against influenza HA, suggesting a 

direct role of this protein in bacterial invasion. 

Treating these cells with GSH derivative, which 

blocked viral infections and prevented the exposure 

of mature HA at the cell surface, resulted in a significant 

reduction of the efficiency of internalization 

(EI) of S. aureus when compared to untreated 

controls. On the contrary, treating infected cells 

with BSO, an inhibitor of GSH neo-synthesis, that 

was able to increase the concentration of HA at the 

surface of infected cells, resulted in a significant 

increase of the EI of S. aureus. Overall, these results 

suggest that the inhibition of HA expression 

through GSH modulating molecules may also affect 

the emergence of secondary bacterial infections. A 

paper that reports these results has been submitted 

for publication (Passariello et al.). 


Conti G, Magliani W, Conti S, Nencioni L, 

Sgarbanti R, Palamara AT, Polonelli L. Therapeutic 

activity of an anti-Idiotypic antibody-derived killer 

peptide against Influenza A virus experimental infection. 

AAC 2008, 52:4331-7. 

Fraternale A, Paoletti MF, Casabianca A, Nencioni 

L, Garaci E, Palamara AT, Magnani M. GSH and 

analogs in antiviral therapy. Mol Aspects Med. 2008, 

Epub. 

Saladino R, Gualandi G, Farina A, Crestini C, 

Nencioni L, Palamara AT. Advances and challenges 

in the synthesis of highly oxidised natural phenols 

with antiviral, antioxidant and cytotoxic activities. 

Curr Med Chem. 2008, 15:1500-19.


Alessandro Giuffrè, CNR researcher; Elena Forte, researcher; 

Daniela Mastronicola, Francesca Maria Scandurra, postdoc 

fellows; Fabrizio Testa, PhD student. 


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); Dipartimento di Scienze Biochimiche, 

Sapienza-Università di Roma (Prof. Maurizio Brunori). 

Report of Activity 

During infection, pathogenic microorganisms are 

exposed to the toxic effects exerted by NO, O 2 and 

their reactive species, a condition currently referred 

to as “oxidative/nitrosative stress”. Reactive oxygen 

and nitrogen species play a key role in the 

human immune response against microbial infection; 

hence, it is not surprising that pathogens are 

able to cope with these harmful species. A number 

of wide spread severe human diseases, such as giardiasis, 

amoebiasis and trichomoniasis, are caused by 

anaerobic protozoa, whose infection peculiarly often 

evolves into chronic inflammatory states. These 

protists are somehow able to resist to the host 

NO/O 2 -mediated immune response, but the molecular 

mechanisms at the basis of their resistance are 

largely unknown yet. Notably, genes coding for 

flavodiiron proteins (FDP), have been recently identified 

in the genome of several human pathogenic 

anaerobic protozoa, including Giardia intestinalis, 

Trichomonas vaginalis and Entamoeba histolytica. 

FDPs are typical prokaryotic enzymes that are 

endowed with O 2 - and/or NO-reductase activity, 

and thus believed to be implicated in the defense of 

anaerobes against oxidative and nitrosative stress. 

The current project aims at exploiting the role of 


Nitric oxide detoxification in pathogenic protozoa: role of 

flavodiiron proteins 

Principal investigator: Paolo Sarti 

Professor of Chemistry and Biochemistry 


Tel: (+39) 06 49910944; Fax: (+39) 06 4440062 

Paolo.sarti@uniroma1.it 

23 

these enzymes in pathogenic protozoa, particularly 

in G. intestinalis. 

The flavodiiron protein from the human 

parasite Giardia intestinalis 

The FDP from G. intestinalis has been expressed in E. 

coli, purified and characterized from a structural and 

functional standpoint. This is the first eukaryotic 

member of the flavodiiron proteins family to have 

been investigated. The crystallographic structure of 

the enzyme, solved at 1.9 Å resolution, shows that 

the FDP is a dimer of homodimers, where each 

monomer encompasses a flavodoxin-like domain, that 

binds a FMN moiety, and a β-lactamase-like domain 

with a non-heme diiron site. The enzyme maintains a 

tetrameric structure also in solution. Monomers are 

arranged in a head-to-tail configuration, so that the 

redox cofactors bound to opposing monomers are at 

a close distance. Compared to bacterial FDPs, the 

enzyme from Giardia shows remarkable overall similarities 

and a highly conserved structure at the level 

of the redox cofactors. 

The functional properties of the G. intestinalis FDP 

have been investigated both by time-resolved spectrophotometry 

and by amperometry using Clarktype 

electrodes selective for O2 and NO. 

Experiments carried out with a stopped-flow instrument 

show that the enzyme in the fully reduced state 

reacts with O2 rapidly (ms) and with high affinity, 

whereas the reaction with NO is much slower. In 

agreement with these results, NO- and O2-ampero metric measurements led us to conclude that the G. 

intestinalis FDP is an efficient O2-scavenging enzyme 

forming H2O as the reaction product, whereas it is a 

poor NO-reductase. O2 is scavenged at high rate 

(> 40 s-1 , at room temperature) and with high affinity 

(KM ≤ 2 µM). Remarkably, these results may be 

relevant in terms of microbial physiology because, 

although Giardia intestinalis is a highly O2-suscepti ble anaerobic parasite, it preferentially colonizes a 

fairly aerobic tract of the human intestine, i.e., the

P. Sarti - Nitric oxide detoxification in pathogenic protozoa: role of flavodiiron proteins 

upper part of the small intestine. Thus, it may be 

expected that survival of the parasite in the host 

requires an efficient O 2 -scavenging function. Such a 

function is currently attributed to a H 2 O-producing 

NADH oxidase identified and characterized a few 

years ago, but based on our results, FDP may also 

contribute significantly, if not predominantly, to O 2 - 

detoxification in Giardia cells, a hypothesis that 

needs to be validated yet. These findings may acquire 

clinical relevance too, because if the FDP function 

were shown to be essential for parasite survival in 

the human host, the enzyme (not expressed in 

humans) may become a suitable target for innovative 

anti-giardial pharmacological treatments. 

In a comparative study, we investigated also the 

flavodiiron proteins from Escherichia coli. This 

enzyme, named flavorubredoxin (FlRd) from a rubredoxin-type 

domain fused to each monomer, was previously 

shown to be an efficient NO-reductase under 

anaerobic conditions, while being essentially inactive 

towards O 2 . By stopped-flow spectroscopy, we found 

that E. coli NADH:flavorubredoxin oxidoreductase 

(FlRd-reductase) shuttles electrons between NADH 

and FlRd very rapidly; at 5 °C, the protein is reduced 

by NADH at k = 5.5 ± 2.2 x 10 6 M -1 s -1 and it 

reduces in turn FlRd at k~1 x 10 7 M -1 s -1 , the reaction 

being highly dependent on pH and ionic 

strength. We established that electrons are first 

donated by FlRd-reductase to the FeS center in the 

24 

rubredoxin-domain of FlRd and then rapidly equilibrate 

intramolecularly with the FMN and Fe-Fe site, 

where the NO chemistry occurs. 

In summary, our data support the idea that NADH 

→ FlRd-reductase → FlRd is an efficient electron 

transfer pathway that ensures a fast detoxification 

of NO in E. coli. Since in the genome of G. intestinalis, 

recently completely sequenced, apparently 

there are no genes coding for rubredoxins, the 

physiological redox partner protein of the FDP has 

to be identified yet. 


Vicente JB, Scandurra FM, Rodrigues JV, Brunori 

M, Sarti P, Teixeira M, Giuffrè A. Kinetics of electron 

transfer from NADH to the Escherichia coli 

nitric oxide reductase flavorubredoxin. FEBS J. 

2007, 274:677-86. 

Di Matteo A, Scandurra FM, Testa F, Forte E, Sarti 

P, Brunori M, Giuffrè A. The O 2 -scavenging flavodiiron 

protein in the human parasite Giardia intestinalis. 

J Biol Chem. 2008, 283:4061-8. 

Vicente JB, Scandurra FM, Forte E, Brunori M, 

Sarti P, Teixeira M, Giuffrè A. Kinetic characterization 

of the Escherichia coli nitric oxide reductase flavorubredoxin. 

Methods in Enzymol. 2008, 437:47-62.


Maurizio Alimandi, Deborah French, Patrizia Mancini, 

Vincenzo Visco, professors; Francesca Belleudi, researcher; 

Salvatore Raffa, post-doc fellow; Giulia Bolasco, Valerio 

Nobili, Paola Pisano, Danilo Ranieri, PhD students; Antonio 

Sabatucci, technician. 


Laboratorio di Virologia, Istituto Regina Elena IFO-IRE, Roma 

(Dr. Aldo Venuti). 


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 skin carcinogenesis, since its 

expression in vivo induces epidermal hyperplasia, 

aberrant differentiation and skin tumors. The 16E5 

protein transforms epithelial cells by deregulating 

cell growth, survival and differentiation through the 

modulation of growth factor receptors, such as the 

epidermal growth factor receptor (EGFR). 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 bet- 


Role of the keratinocyte growth factor receptor on the molecular 

and cellular alterations induced by the expression 

of HPV16 E5 oncoprotein 

Principal investigator: Maria Rosaria Torrisi 



Tel: (+39) 06 4468450 

mara.torrisi@uniroma1.it 

25 

ter elucidate the molecular events involved in the 

pathological effects induced by HPV infection and 

UVB exposure of human epidermis, our research 

project will attempt: 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 and the cellular 

response to UVB. 

During the first year of the project, to investigate if 

16E5 expression would be able to modulate KGFR 

in vitro, we used human keratinocytes, grown at different 

cell densities and expressing a modulated 

number of KGFRs following transfection or differentiation, 

as a model to study the 16E5 effects on the 

receptor activation and modulation and on the related 

epithelial proliferation/differentiation. First, we 

utilized the human immortalized keratinocytes 

HaCaT: stable transfectants of HaCaT cells 

expressing 16E5, HaCaT E5 (pMSG) and HaCaT 

E5 (RXR), were kindly provided by Dr. Venuti 

(Regina Elena Cancer Institute of Roma). E5 and 

KGFR transcript levels were analyzed by RT-PCR 

and we found that the induced expression of E5 

under the control of dexamethasone-inducible 

pMSG promoter was able to down-modulate KGFR 

mRNA. According to this KGFR modulation, by 

quantitative immunofluorescence we observed that, 

while treatment with KGF of control cells could 

trigger, as expected from our previous studies, 

HaCaT cell differentiation, testified by an increase of 

the expression of the early differentiation marker 

K1 and by keratinocyte stratification, the induction 

of E5 expression appeared to abolish the KGF differentiative 

effects. In addition, although HaCaT E5 

cells appeared to grow more rapidly than HaCaT 

control cells transfected with the empty vectors, the 

proliferative response to KGF, assessed by Ki67 pos-

M. R. Torrisi - Role of the keratinocyte growth factor receptor on the molecular and cellular alterations 

itivity to identify cycling cells, was much less pronounced 

in HaCaT cells expressing E5 compared to 

the control cells. These results indicate that E5 

expression down-modulates KGFR transcription 

and the consequent KGF-induced proliferation and 

differentation of human keratinocytes. 

Since 16E5 is known to inhibit the EGFR endocytic 

degradative pathway and to enhance receptor recycling, 

we performed also a series of experiments to 

investigate if the E5 protein would be able to interfere 

with the two alternative KGFR endocytic pathways. 

In fact, we have demonstrated that 

FGF10/KGF2 induces receptor recycling, whereas 

KGF/FGF7 stimulates receptor degradation 

(Belleudi et al., 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 would play a similar role in targeting 

KGFR to the recycling route. To this aim, first we 

performed transient transfection of HaCaT cells 

with a pCIneo-HA E5 expression vector (kindly provided 

by Dr. Venuti) and, 48 hours post-transfection, 

we immunostained E5 protein with anti-HA antibodies. 

Immunfluorescence and confocal analysis showed 

that the E5 signal colocalized with the endoplasmic 

reticulum marker calreticulin, as expected, while 

only a minor colocalization was observed with the 

marker of the Golgi complex giantin and no colocalization 

was detected with the mitochondrial marker 

MitoTracker. Next, to analyze if E5 expression 

could affect KGFR endocytosis, we studied the intracellular 

endocytic traffic followed by KGFR during 

internalization induced by the two ligands KGF and 

FGF10 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 in 

detail at immunofluorescence and confocal 

microscopy and the intracellular endocytic structures 

and compartments involved in the KGFR inter- 

26 

nalization process induced by the ligands were identified 

using the following markers: EEA1 for early 

endosomes, LysoTracker for lysosomes and internalized 

Transferrin for the recycling compartment. 

HaCaT KGFR/E5 cotrasfected and HaCaT KGFR 

transfected cells were treated with the ligands and 

with the anti-KGFR antibody at 4°C and warmed for 

1h at 37°C. Cells were also incubated in the presence 

of LysoTracker to identify lysosomes or in the presence 

of Tf-Tx to identify the juxtanuclear recycling 

compartment. The results showed that, upon KGF 

treatment of cells expressing E5, most of KGFRs 

did not reach the lysosomes, as demonstrated by the 

very low percentage of colocalization with 

LysoTracker. In contrast, in agreement with our previous 

results (Belleudi et al., 2007), the receptor 

extensively colocalized with LysoTracker in cells 

overexpressing KGFR alone. After FGF10 treatment, 

the KGFR signal colocalized with internalized 

Tf, but not with LysoTracker in both cotransfeted or 

singly transfected cells. suggesting that E5 expression 

does not inhibit receptor recycling to the plasma 

membrane. Thus, E5 appears to interfere with the 

KGF-induced KGFR transport to the degradative 

pathway, but not with the FGF10-induced receptor 

traffic through the recycling compartment (Belleudi 

et al. in preparation). 


Belleudi F, Leone L, Nobili V, Raffa S, 

Francescangeli F, Maggio M, Morrone S, Marchese 

C, Torrisi MR. Keratinocyte growth factor receptor 

ligands target the receptor to different intracellular 

pathways. Traffic 2007, 8:1854-72. 

Lotti LV, Rotolo S, Francescangeli F, Frati L, 

Torrisi MR, Marchese C. AKT and MAPK signaling 

in KGF-treated and UVB-exposed human epidermal 

cells. J Cell Physiol. 2007, 212:633-42. 

Cardinali G, Bolasco G, Aspite N, Lucania G, Lotti 

LV, Torrisi MR, Picardo M. Melanosome transfer 

promoted by keratinocyte growth factor in light and 

dark skin derived keratinocytes. J Invest Dermatol. 

2008, 128:558-67.

AREA3 

Molecular 

genetics 

of 

eukaryotes

Development and analysis of chromosome vectors 


Cristina Auriche, Enea Gino Di Domenico, Francesca 

Tilesi, post-doc fellows; Lucia Rocchi, PhD student. 


Cystic fibrosis (CF) is a recessive congenital disease, 

caused by mutations in the gene coding for CFTR 

(cystic fibrosis transmembrane conductance regulator), 

a chloride channel, which results in impaired 

anion secretion and hyper absorption of sodium 

across epithelia. Since localisation of the CFTR gene 

on the long arm of chromosome 7 in 1989, a huge 

effort was put into the development of gene therapy 

for this life threatening disease. Because effective 

therapy by in vivo delivery of therapeutic DNA 

requires physiological levels of expression and longterm 

maintenance, it was hypothesized that this can 

be achieved by using genomic fragments that contain 

all the long-range controlling elements that allow tissue-specific 

gene expression at physiological level. 

This is particularly important for CF since CFTR is 

only expressed in specialized epithelial cells of gut, 

airways, pancreas, sweat gland ducts and the male 

reproductive tract. The complex pattern of expression 

is dictated by regulatory elements, not fully elucidated 

yet, which have been claimed to be located 

upstream and downstream of the coding region and 

in some introns. We have isolated for the first time to 

our knowledge, the entire CFTR locus into a bacterial 

DNA molecule which can be used to engineer 

genomic vectors containing all the regulatory elements 

necessary for spatial and temporal regulated 

expression of CFTR in human epithelial cells. 

The aims of the project are as follows: 

1) delineate the minimal sequences required for spatial 

and temporal regulated expression of CFTR in 

human epithelial cells; 

2) compact this information into a DNA fragment 

that can be inserted into gene therapy vectors (lipid, 

29 

Molecular genetics of eukaryotes - AREA 3 

Principal investigator: Fiorentina Ascenzioni 

Professor of Microbiology 


Tel: (+39) 06 499917577, 06 49917614; Fax: (+39) 06 49917594 

fiorentina.ascenzioni@uniroma1.it 

cationic polymer and dendrimer- based vectors) to 

achieve full levels of controlled and tissue-specific 

expression of the CFTR gene; 

3) Assembly of a CFTR-containing de novo chromosome 

into human cell lines. 

Functional analysis of the CFTR locus in 

mammalian cells 

The CFTR protein is involved in different activities, 

the best known is the transport of the Clthrough 

the membrane, other activities include 

interaction with bacteria at the plasma membrane. 

To asses the functional activities of the CFTR 

locus cloned in the BAC vectors we have transferred 

the BACs into FRT epithelial cells (Fisher 

Rat Thyroid) which are routinely used to study 

CFTR activity. Large scale DNA preparations from 

cCFTR5A and cCFR∆12 were first analysed for 

DNA integrity and subsequently used to transfect 

FRT cells. Two versions of the BAC have been 

used, the cCFTR5A co-transfected with pcDNAzeo 

for selection, and cCFTRD12, which contains the 

zeo marker. We obtained 2 cCFTR5A/clones, both 

of them did not survive further growth, and 12 

cCFTRD12/clones. All the cCFTR12D clones, 

analysed by PCR, showed the presence of the 

cCFTRD12 vector. Subsequently, CFTR mRNA 

was detected by real time quantitative PCR in 7 out 

of the 12 clones, although at different level. 

Next, we evaluated CFTR activity by direct measurement 

of electrogenic Cl- transport. The 7 clones 

showing CFTR expression were seeded on 

Snapwell and at 4-7 days from plating the filters 

were mounted in a Ussing chamber and the 

transepithelial Cl- currents were determined. 

CFTR-dependent Cl- current was detected in two 

clones, FRT-cl7 and FRT-cl2, the former showed 

higher current with respect to the latter; three of 

the clones (FRT-cl1, -cl3 and -cl4) did not show 

detectable current while the remaining two (-cl5 

and -cl6) showed a moderate current.

F. Ascenzioni - Development and analysis of chromosome vectors 

The different amount of the CFTR mRNA and the 

different activity of the Cl- channel observed 

among the FRT clones analysed, could be due to 

differences in the copy number of the integrated 

locus and/or to epigenetic effects due to the nearby 

chromatin. To elucidate this point we determined 

the copy number of the human CFTR locus in the 

two most active clones, FRT-cl2 and FRT–cl7. By 

real time PCR we detected two copies of the human 

CFTR locus in FRT-cl7 and one copy in CFTR-cl2 

cells. FRT-cl7 cells were further analysed by FISH 

which could detect only one integration site into a 

big host chromosome, suggesting that both copies 

of the locus become integrated in the same site of 

the host genome. 

Finally the CFTR protein was detected by immunofluorescence 

analysis in FRT-cl7, which showed the 

highest CFTR mRNA level and Cl- current. For this 

purpose FRTcl7 cells were seeded in transwell (Costar) 

and let polarized for 5-7 days until the transepithelial 

resistance was in the range 1-2 KΩ cm 2 . CFTR protein 

was immunolocalized by anti-CFTR antibody and confocal 

microscopy. The result of this analysis showed 

the classical honey-bee distribution of the CFTR in 

the xy plane, and compartimentalization above the 

nucleus in the xz plane, strongly suggesting that the 

CFTR protein was properly transported to the plasmamembrane 

in FRT-cl7 cells. 

In conclusion these results clearly demonstrated that 

the human CFTR locus isolated in the BAC molecule 

is fully functional and provide good material for the 

remaining aims of the project. 

De novo chromosome assembly with the 

human CFTR locus 

One possibility to complement a genetic defect by 

using an entire locus is de novo assembly of an arti- 

30 

ficial chromosomes containing the gene of interest. 

We have performed preliminary experiments to 

determine the feasibility of this approach using the 

cCFTR5A BAC as a donor of the CFTR gene. For 

this purpose cCFTR5A and alphoid-PAC DNAs 

were co-transfected in HT1080 cells and after 

selection for markers present in the alphoid construct 

we have identified, among the positive clones, 

three clones that contained also the cCFTR5A construct. 

One of these clones showed the presence of 

a de novo artificial chromosomes as revealed by 

FISH with CFTR and alphoid probes. This result 

provided proof of principle for delivery into mammalian 

cells of the CFTR locus by de novo chromosome 

assembly. 


Conese M, Boyd AC, Di Gioia S, Auriche C, 

Ascenzioni F. Genomic context vectors and artificial 

chromosomes for cystic fibrosis gene therapy. Curr 

Gene Ther. 2007, 7:175-87. 

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. 2008, Epub. 

Pirone L, Bragonzi A, Farcomeni A, Paroni M, 

Auriche C, Conese M, Chiarini L, Dalmastri C, 

Bevivino AM, Ascenzioni F. Burkholderia cenocepacia 

strains isolated from cystic fibrosis patients are 

apparently more invasive and more virulent than 

rhizosphere strains. Environ Microbiol. 2008, 

10:2773-84.


Patrizia Filetici, CNR researcher; Andrea Brenna, Federica 

Tosi, PhD students; Eleonora Muggiano, Stefania Federico, 

Alberto Gualdieri, undergraduate students. 


Dipartimento di Biochimica e Biologia Molecolare, Università di 

Parma (Prof. Simone Ottonello); Dipartimento di Biologia 

Vegetale e Biotecnologie Agroambientali e Zootecniche, 

Università di Perugia (Dr. Leonardo Baciarelli Falini); Istituto 

per la Protezione delle Piante del CNR c/o Dipartimento Biologia 

Vegetale, Università di Torino (Dr. Raffaella Balestrini); INRA, 

Nancy, France (Dr. Francis Martin); UNAM, México DF, México 

(Dr. Alicia González). 


In Eukaryotes the association between DNA and histones 

does not represent a mechanical way of condense 

in a small volume thousands of genes, but constitutes 

a real regolative structure. Cells differentiation 

and cellular cross-talks are achieved through 

specific modifications of the chromatin. In particular 

histone tails are subject to several convalent posttranslation 

modifications as acetylation, methylation, 

phosphorylation, ubiquitinylation, sumoylation and 

ADP-ribosylation. More recently a large number of 

trascription factors have been observed also to be 

modified (mainly by acetylation and phosphorylation) 

by the same proteins operating on histones. 

These proteins are associated to specific domains 

(bromodomain, chromodomain, etc.) able to recognize 

the modifications, therefore assuming the role of 

effectors of the pathways of signal transduction. We 

have mainly dedicated our attention to the acetylation, 

as part of signal tranduction pathways in the 

model system of Ascomycetes. We have studied, 

among this class of simple Eukaryotes, three species: 

S. cerevisiae, N. crassa and Tuber borchii in order to 

understand the role of the HAT Gcn5 in eukaryotic 

31 


Molecular machines and effectors involved in the regulation 

of light response and cell cycle in simple Eukaryotes 

Principal investigator: Paola Ballario 

Researcher in Molecular Genetics 

Dipartimento di Genetica e Biologia Molecolare 

Tel: (+39) 06 49912318, 06 49912235; Fax: (+39) 06 4440812 

paola.ballario@uniroma1.it 

replication and transcription: a) S. cerevisiae the classical 

budding yeast has been used in order to investigate 

the role of one of the best characterized histone-acetyltransferase 

(HAT) Gcn5 in kinetochore 

(KT) and in centrosome assembly and function; b) N. 

crassa a filamentous fungus that is a classical model 

for the study of photobiology was the object of a 

previous study (Pasteur-Cenci Bolognetti Project, 

2005-2006) that demonstrated the correlation 

between light induction of transcription and histone 

H3 transient acetylation in the promoter of light 

dependent genes. Data obtained recently demonstrated 

that not only histones are substrate for 

acetyltransferase but also WC-1 itself (the photoreceptor 

organized as a vertebrate nuclear receptor) is 

subject to acetylation by an HAT; c) Tuber spp, an 

hypogeus filamentous fungus is well known since 

Roman Empire for its gastronomical value, but its 

life cycle is still misterious. Genetical and molecular 

tools begin to be developed only now. We are analyzing 

the influence of the environment, in particular of 

light on T. borchii life cycle. We have settled the conditions 

for truffles molecular transformation by A. 

tumefaciens and we are involved in T. melanosporum 

genomic sequence. 

Results 

A) The centromere and the KT are large complexes 

constituted by more then 60 proteins evolutivelly 

conserved. These complexes assembled on the 

centromeric DNA exert a control on chromosome 

attach by the mitotic spindle. Checkpoint activity 

that blocks cellular proliferation in presence of missegregation 

is linked to KT. An anomalous activity 

of the KT produces chromosomal misaggregation 

and aneuploidies, associated in humans to tumors 

and genetic pathologies (i.e. Down and Turner syndromes). 

Aim of this section of our project is to 

understand the role of Gcn5 (an HAT protein) in 

chromosomes segregation and, in centromere and 

kinetocore structural organization in S. cerevisiae. In

P. Ballario - Molecular machines and effectors involved in the regulation of light response and cell cycle in simple Eukaryotes 

the paper by Vernarecci et al. (2008), we report that 

Gcn5 is physically associated to the centromere and 

genetically interacts with several components of 

the kinetochore, controlling chromosomes segregation 

during cell cycle. Based on these results we 

want now to identify the molecular mecchanism 

used by Gcn5 for the regulation of structure and 

function of the centromeric region/kinetochore. 

Next steps will be: i) mapping of genetic interactions 

among Gcn5 and centromere/kinetochore 

elements, ii) analysis of the level of Gcn5 control 

(transcriptional or post translational), iii) validation 

of the model emerging for yeast cells in human culture 

cells, iv) clarification of the role of Gcn5 in 

the activation of the mitotic chekpoint associated 

to the spindle. 

B) In 2006 (Grimaldi et al., Mol Biol Cell) we demonstrated 

the important role of the Neurospora NGF-1 

HAT (the homologous of S. cerevisiae Gcn5) in the 

transcriptional activation of light regulated genes. 

In particular we have observed the transient acetylation 

of histone H3 residue K14 in the promoter 

regions of light regulated genes (i.e. Al3 and Vvd). In 

the last two years we have focused our investigation 

on the process of reversible acetylation of the main 

protein involved in light transduction: White 

Collar1. We have demonstrated an in vitro interaction 

between WC-1 and ScGcn5, and also, by ChIP 

analysis, that WC-1 is acetylated in vivo both in dark 

and in light conditions of growth. However the in 

vivo acetylation of WC-1 does not seem to be NGF- 

1 dependent because the protein is acetylated also in 

an ngf-1 mutant strain. The in vitro acetylation 

assays suggest that WC-1 contains an acetyltransferase 

activity (in prep.). Now we will search the 

other proteins present in the WC-1WC-2Gcn5 complex 

by Maldi-Toff and two hybrid analyses. 

32 

C) We are also interested in comparing our data on 

light transduction in Neurospora (WCC white collar 

complex) with the light system utilized by Tuber 

borchii a filamentous fungus, characterized by an 

hypogeous style of life. In the period 2005-2006 we 

isolated WC-1 and WC-2 from T. borchii and characterized 

light responses in the mycelium but we need 

some molecular-genetic tools like transformation 

and homologous recombination. We therefore set up 

the protocol for Tuber borchii transformation based 

on Agrobacterium tumefaciens binary vector system. 

We are also participating to an italian-french consortium 

for Tuber melanosporum genome sequencing, 

annotating genes involved in light transduction 

pathways. Several interesting data have been 

obtained. We have found the entire velvet system of 

red light transduction (previously characterized in A. 

nidulans) in T. melanosporum, this finding was quite 

unexpected, in fact, characterizing the action spectra 

of Tuber spp no red light response has been observed 

however the level of response to white and blue light 

is not the same in term of mycelia growth. This suggest 

the existence of a blue-red cross talk. 


Bizzarri B, Chimenti F, Bolasco A, Maccioni E, 

Chimenti P, Fioravanti R, Secci D, Ballario P, 

Vernarecci S, Filetici P. A novel histone acetyltransferase 

inhibitor modulating Gcn5 network: 

cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2yl)hydrazone. 

J Med Chem. 2008, Epub. 

Vernarecci S, Ornaghi P, Bâgu A, Cundari E, 

Ballario P, Filetici P. Gcn5p plays an important role 

in centromere kinetochore function in budding yeast. 

Mol Cell Biol. 2008, 28:988-96.


Elisa Caffarelli, Alessandro Fatica, Carlo Presutti, 

researchers; Michela Denti, Fernanda De Angelis, 

Mariangela Morlando, post-doc fellows; Monica Ballarino, 

Pietro Laneve, Alessandro Rosa, PhD students. 


Dipartimento di Medicina sperimentale, Sapienza-Università di 

Roma (Prof. Alberto Gulino); Dipartimento di Istologia ed 

Embriologia Medica, Sapienza-Università di Roma (Prof. Antonio 

Musarò); TIGEM, Napoli (Prof. Alberto Auricchio); City of 

Hope, Duarte, USA (Prof. John Rossi). 


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 the study of their 

functions in cell growth, differentiation and diseases. 

In a parallel line of research, we intend to exploit 

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. 

The miRNA “factory” 

miRNA are generated from a longer RNA polymerase 

II derived transcript by a stepwise process involving 

two RNaseIII enzymes: Drosha acting on nuclear primiRNA 

and then Dicer cleaving pre-miRNAs in the 

cytoplasm. We demonstrated that, in human HeLa 

Principal investigator: Irene Bozzoni 



Tel: (+39) 06 49912202; Fax: (+39) 06 49912500 

irene.bozzoni@uniroma1.it 

33 


RNA-RNA and RNA-protein interactions in the cell nucleus: 

structure, function and biosynthesis of a novel class 

of small non-coding RNAs 

cells, Drosha processing, as the major RNA-processing 

activities acting on pre-mRNAs, occurs during 

transcription on both dedicated and intronic miRNA 

genes. In this latter case we showed that the two 5'-3' 

and 3'-5' RNA exonucleases activities colocalized 

with the Microprocessor complex on chromatin 

associated with intronic miRNA genes. We also 

demonstrated that Drosha cleavage occurs before the 

host intron is spliced out and that co-transcriptional 

pre-miRNA processing does not affect the accumulation 

of the host spliced mRNA. These results allowed 

us to predict that both mature miRNAs and mRNAs 

derive from a common nascent transcript. 

Mammalian intronic-miRNAs are transcriptionally 

linked to the expression of their host genes. As the 

expression of miRNA-dedicated genes is dependent 

from their own promoter we are investigating 

whether specific miRNA cis-acting signals are 

required for the control of their biosynthesis. 

Role of miRNAs in hematopoietic 

differentiation 

In consideration of the important role played by 

miRNA in hematopoiesis and leukaemia, we have 

proceeded to the identification and characterisation 

of miRNAs with potential role in myeloid differentiation 

(reviewed in Fatica et al., Biochem Soc Trans. 

2008, 36:1201-5). 

In the direction of studying the molecular circuitries 

regulated by miRNAs and involved in the control of 

specific differentiation lineages, we have identified a 

new pathway by which the master hematopoietic 

transcription factor PU.1 regulates human monocytic 

differentiation. This includes the lineage–specific 

miR-424 and the transcriptional factor NFI-A. We 

have shown that PU.1 and these two components are 

linked to each other in a finely tuned temporal and 

regulatory circuitry: PU.1 activates the transcription 

of miR-424 and this up-regulation appears to be 

involved in stimulating monocyte differentiation 

through miR-424 dependent translational repression

I. Bozzoni - RNA-RNA and RNA-protein interactions in the cell nucleus 

of NFI-A. In turn, the decrease in NFI-A levels is 

important for the activation of differentiation-specific 

genes such as M-CSFr. In line with these data, 

both RNAi against NFI-A and ectopic expression of 

miR-424 precursor cells enhance monocytic differentiation. 

The interplay among these three components 

was found in APL cell lines as well as in culture of 

human CD34 + progenitors differentiating selectively 

through the monocyte/macrophage lineage. These 

data point to the important role of miRNAs in conjunction 

to master transcription factors in 

hematopoietic differentiation and indicate the crucial 

role of NFI-A in counteracting the monocyte differentiation 

programme (Rosa et al., 2007). 

Role of miRNAs in neuronal tumorigenesis 

mir-9, mir-125a and mir-125b have been shown to 

be up-regulated upon in vitro differentiation of 

neuroblastoma cell lines (Laneve et al., Proc Natl 

Acad Sci USA 2007, 104:7957-62). By two complementary 

approaches, ectopic expression and knockdown 

experiments, the role of these miRNAs in 

molecular circuitries contributing to neuronal cancerogenesis 

was clarified. We showed that the overexpression 

of the three miRNAs caused a strong 

reduction of cell growth; accordingly, microRNAs 

were down-regulated in human primary neuroblastomas, 

suggesting a tumor suppressor activity for 

these molecules. 

By bioinformatics and experimental data, we dissected 

the molecular mechanisms involving such 

miRNAs, and found a new regulatory circuitry controlling 

neuroblastoma cell growth. We have identified 

the truncated, enzimatically inactive, isoform of 

the neurotrophin-3 receptor trkC as their target 

gene and demonstrated that miRNA-mediated downregulation 

of this protein was crucial for controlling 

neuroblastoma cell growth. 

Furthermore, the first miRNA expression profiling 

of human primary medulloblastomas was performed 

by high throughput analysis; we found 78 miRNAs 

displaying differential expression between tumors 

34 

and controls, indicating that specific miRNA signatures 

may distinguish tumors from normal tissues. 

Use of sncRNA for the gene 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 strategy 

in restoring the synthesis of dystrophin in vivo. 

In the last year we have been able to show that persistent 

exon-skipping in mdx mice is maintained for 

the entire life of the animal through the use of 

Adeno-associated viral (AAV). The transduced muscles 

rescued dystrophin expression and displayed a 

significant recovery of function towards the normal 

values at single muscle fibre level. Therefore, this 

approach provides solid bases for a systemic use of 

AAV-mediated antisense-U1 snRNA expression for 

the therapeutic treatment of DMD. 

Antisense constructs specific for human mutations 

are currently under construction and testing in 

myoblasts from patient biopsies. 


Rosa A, Ballarino M, Sorrentino A, Sthandier O, De 

Angelis FG, Marchioni M, Guarini A, Fatica A, 

Peschle C, Bozzoni I. The interplay between the master 

transcription factor PU.1 and miR-424 regulates 

human monocyte/macrophage differentiation. Proc 

Natl Acad Sci USA 2007, 104:19849-54. 

Denti MA, Incitti T, Sthandier O, Nicoletti C, De 

Angelis FG, Rizzato E, Auricchio A, Musarò A, 

Bozzoni I. Long-term benefit of adeno-associated 

virus/antisense-mediated exon skipping in dystrophic 

mice. Hum Gene Ther. 2008, 19:601-8. 

Morlando M, Ballarino M, Gromak N, Pagano F, 

Bozzoni I, Proudfoot NJ. Primary microRNA transcripts 

are processed co-transcriptionally. Nat Struct 

Mol Biol. 2008, 15:902-9.


Anna Reale, professor; Michele Zampieri, Tiziana 

Guastafierro, post-doc fellows; Maria Giulia Bacalini, PhD 

student. 


Istituto di Biologia e Patologia Molecolari, CNR, Roma (Dr. 

Claudio Passananti). 


Over the past decade our laboratory has accumulated 

evidence that links PARylation with DNA methylation 

suggesting that PARylation regulates gene 

expression through its control over DNA methylation 

pattern. A series of different experimental 

strategies suggests that blockage of PARylation 

induces in vivo DNA hypermethylation, both in 

genomic DNA and in some CpG island regions 

(Zardo and Caiafa, 1998; de Capoa et al., 1999; Zardo 

et al., 1999). These observations suggested that 

ADP-ribose polymers (PARs) protect DNA from 

methylation. Recent data reinforce the hypothesis 

that a physiological level of PARylation and the balance 

between unmodified and PARylated PARP-1 are 

crucial in maintaining the genomic methylation pattern. 

We found that cells with hyperactive PARP-1, 

and hence increased PAR levels, are characterized by 

a widespread DNA hypomethylation (Guastafierro et 

al., 2008). 

We suggested a mechanism (Reale et al., 2005) in 

which PARP-1 in its PARylated form makes DNA 

methyltransferase 1 (Dnmt1) catalytically inactive 

and, thus, inefficient in DNA methylation. We 

hypothesize that during normal cell growth Dnmt1, 

having a higher affinity for ADP-ribose polymers 

than for DNA, is associated with PARylated PARP-1, 

forming PARylated PARP-1/Dnmt1 complex. This 

interaction precludes Dnmt1 binding to DNA, thus 

preventing unwanted DNA methylation. The right 

35 


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 


Tel: (+39) 06 49976530; Fax: (+39) 06 44231961 

caiafa@bce.uniroma1.it 

nuclear balance between unmodified and PARylated 

form of PARP-1 - which depends on the correct 

dynamics of PARPs/PARG activities - determines 

the maintenance of DNA methylation pattern (Caiafa 

et al., 2008). In the absence of PARylated PARP-1, the 

Dnmt1 is free to methylate DNA, whereas in condition 

of persistently high level of PARylated PARP-1, 

the stable inhibition of Dnmt1 would prevent its 

methylation maintenance activity at replicative forks. 

According to our data, decreased or increased levels 

of PARylated PARP-1 are responsible for diffuse 

hypermethylation or hypomethylation of DNA, 

respectively. 

CTCF, the highly conserved and ubiquitously 

expressed nuclear factor involved in imprinting and 

insulator processes (Ohlsson et al., 2001), attracted 

our attention as it brings together the two epigenetic 

events in which we are interested: DNA methylation 

and PARylation. As an enhancer-blocking insulator, 

CTCF interacts with specific sequences located 

in imprinting control regions; importantly, CTCF is 

able to interact with these regions only if they are 

unmethylated (Bell and Felsenfeld, 2000). 

Furthermore, CTCF binding protects them from de 

novo methylation in somatic cells (Bell et al., 2001). 

Recent studies have linked CTCF function in 

imprinting control to PARylation of its N-terminal 

domain (Yu et al., 2004). A PARylated form of CTCF 

has been found in cells; this form is capable of binding 

imprinting control regions in vitro. Furthermore, 

treatment of cells with 3-aminobenzamide, a competitive 

inhibitor of PARP activity, affects the insulator 

function of most CTCF target sites. 

These results were interpreted to mean that 

PARylation of CTCF is responsible for its imprinting 

function and this without altering the methylation 

pattern of the DNA regions involved in the control 

of imprinting. 

To gain further insight into the mechanism of action 

of PARylated CTCF, we performed a series of in vivo 

and in vitro experiments (Guastafierro et al., 2008).

P. Caiafa - Crosstalk between poly(ADP-ribosyl)ation and DNA methylation in the regulation of gene expression 

Coimmunoprecipitation and pull-down experiments 

indicated direct interaction between CTCF and 

PARP-1. Furthermore, in vitro PARP activity assay 

demonstrated that CTCF, by itself, activates PARP- 

1. Importantly, PARP-1 activation occurs even in 

absence of “nicked” DNA, suggesting that the 

CTCF-induced PARP1 activation may not be 

involved in DNA repair. 

We also provided, for the first time, evidence that 

CTCF is involved in the crosstalk between 

PARylation and DNA methylation. We found that 

DNA methyltransferase activity is decreased by 

about 70% in cells overexpressing CTCF vs cells 

transfected with empty vector. A more thorough 

examination of Dnmt1 indicated that the inhibition 

is not due to a decreased protein level. An in vitro 

approach excluded direct inhibition of Dnmt1 activity 

by CTCF, while confirmed our previous observation 

that PARs present on modified PARP-1 inhibit 

the enzyme (Reale et al., 2005). The persistence of 

high PAR levels induced by CTCF affects the DNA 

methylation machinery. Dnmt1 activity is inhibited, 

leading to wide hypomethylation of the genome, 

involving both centromeric and B1 repeats. Ectopic 

over-expression of CTCF in cells lacking PARP-1 

demonstrates that the functional relationship 

between CTCF and PARP-1 is specific. The fact that 

PARylated PARP-1 inhibits Dnmt1 activity and that 

CTCF is, by itself, capable of inducing PARylation of 

PARP-1, allow us to suggest a mechanism to explain 

36 

the control exerted by PARylation over the expression 

of imprinted genes (Caiafa et al., 2008). In this 

model the inhibition of Dnmt1 activity in cells with 

active PARylation is attributed to noncovalent interactions 

between PARs on either PARylated CTCF or 

PARylated PARP-1 and Dnmt1. Independently of 

the exact mechanism involved, it is clear that the 

proper balance between unmodified and PARylated 

PARP-1 is of paramount importance for, at least in 

this case, the functioning of gene imprinting. 


Chevanne M, Calia C, Zampieri M, Cecchinelli B, 

Caldini R, Monti D, Bucci L, Franceschi C, Caiafa P. 

Oxidative DNA damage repair and parp 1 and parp 2 

expression in Epstein-Barr virus-immortalized B 

lymphocyte cells from young subjects, old subjects, 

and centenarians. Rejuvenation Res. 2007, 10:191-204. 

Caiafa P, Guastafierro T, Zampieri M. Epigenetics: 

poly(ADP-ribosyl)ation of PARP-1 regulates 

genomic methylation patterns. FASEB J. 2008, 

23:672-8. 

Guastafierro T, Cecchinelli B, Zampieri M, Reale A, 

Riggio G, Sthandier O, Zupi G, Calabrese L, Caiafa P. 

CCCTC-binding factor activates PARP-1 affecting 

DNA methylation machinery. J Biol Chem. 2008, 

283:21873-80.


Francesco Chiani, Francesca Di Felice, post-doc fellows; 

Elisa Cesarini, Anna D’Alfonso, Francesca Romana 

Mariotti, PhD students. 


Department of Biological Chemistry, University of California, 

Irvine, USA (Dr. Masayasu Nomura); Department of Biological 

Chemistry, University of California, Los Angeles, USA (Dr. Michael 

Grunstein). 


By fine-tuning the level of DNA supercoiling, DNA 

topoisomerases are enzymes involved in DNA replication, 

transcription, recombination and chromatin 

remodeling. They represent the molecular machines 

that manage the topological state of the DNA in the cell. 

The interest in DNA topoisomerases derives not 

only from their crucial role in managing the DNA 

topology, but also from a wide variety of topoisomerase-targeted 

drugs that have been identified and 

utilized as antimicrobials and anticancer drugs, some 

of which are currently in widespread clinical use. 

In spite of a low number of reported proteins, we 

may still expect that other proteins interacting with 

DNA topoisomerase I (Topo I) remain yet unknown. 

Our aim is to identify the main functional and/or 

physical partners of Topo I by studying the processes 

in which Topo I is involved like transcription, 

recombination and transcriptional silencing. 

Altogether the data will contribute to better clarify 

the Topo I activity inside the cell and its possible 

interaction with DNA and/or any other partner. 

Nucleosomes as physical barriers for cleavage 

activity of Topo I in vivo 

To further characterize the Topo I activity in vivo, 

we wanted to evaluate the possible interference of 

Principal investigator: Giorgio Camilloni 



Tel: (+39) 06 49912808; Fax: (+39) 06 49912500 

giorgio.camilloni@uniroma1.it 

37 


DNA topoisomerases as global controller of DNA transactions. 

Study of DNA topoisomerase IB and its functional partners 

nucleosomal structures on its capability to react with 

the physiological state of DNA: chromatin. 

It is generally accepted that all sequences in DNA 

can be substrates for Topo I relaxing activity, even 

though Topo I seems to have a higher reactivity 

towards some particular sequences showing structural 

features like DNA bending. In order to verify 

whether nucleosomes affect Topo I site-specific 

cleavages, we studied a natural DNA sequence 

repeating 35 times the TTA trinucleotide. Such a 

sequence provides: the locally bent TA step, presumably 

a good substrate for local Topo I cleavage induction 

and an intrinsic flexibility potentially useful to 

efficiently assemble nucleosome. 

The TTA trinucleotide repeat has proved to be efficiently 

cleaved in vitro by Topo I in the repeated tract 

and also in its surrounding regions, confirming the 

preference of the enzyme for this sequence. 

Conversely, an in vivo approach showed a reduced 

reactivity of the same sequence towards Topo I. 

Thus, it is conceivable to hypothesize that chromatin 

structure affects Topo I cleavages. Recently it has 

been reported that DNA topoisomerase II is more 

efficient than Topo I in releasing topological stress 

from nucleosomal substrates; this supports the 

hypothesis that nucleosomes may represent a barrier 

for Topo I activity. 

At this purpose a distinction between the global 

relaxing activity and the local site specific cleavage 

reaction, should be taken under consideration. In fact 

a different chromatin organization in a given substrate 

could be not determinant in the whole relaxing 

activity of Topo I because the enzyme can 

release torsional stress acting on different sites in the 

substrate. Conversely, when a given sequence is analyzed 

in terms of cleavage activity exerted by Topo 

I, the absence/presence of a nucleosome could be 

very relevant and this latter point was investigated 

by our experimental system. 

In order to verify this hypothesis, we studied the in 

vivo chromatin organization of the (TTA)35 tract. A

G. Camilloni - DNA topoisomerases as global controller of DNA transactions 

positioned nucleosome occupies the (TTA)35 region 

possibly hindering the Topo I cleavage activity. Then 

we employed a yeast strain carrying a plasmid in 

which the H4 gene is under the GAL1 promoter. 

This allowed us to switch on/off the synthesis of 

this histone, depending on the carbon source. In the 

GAL condition the presence of nucleosomes impairs 

Topo I approach to the (TTA)35 sequence. 

Conversely, in the GLU condition, when H4 production 

is repressed, no organized chromatin is observed 

on the TTA repeat and Topo I increases its sequence 

specific activity, particularly on the (TTA)35 tract. 

In order to evaluate the cleavage difference of Topo 

I in vivo, in vitro and when chromatin is destructured 

(H4 no more synthesized in Glucose medium), we 

quantified the Topo I digestion profiles. When chromatin 

is regularly organized about 5-6% of the 

digested material is represented by the TTA repeat. 

Conversely the relative digestion of the TTA repeat 

when samples are reacted in vitro with Topo I reaches 

values near to 30%. Also the dissolution of regularly 

organized chromatin allows Topo I to digest 

the TTA repeat in vivo, but with an efficiency of 15% 

(about three times higher than on the regular chromatin). 

Thus we can conclude that: i) Topo I efficiently 

reacts with the TTA repeat; ii) the (TTA)35 

sequence, the longest and most stable among the 

simple repeated sequences in S. cerevisiae, is organized 

in a positioned nucleosome and possibly this can 

account for its high stability: in fact each nucleosome 

stores one negative supercoil, thus preventing DNA 

denaturation and induction of conformational alter- 

38 

ations responsible for genetic instability; iii) the positioned 

nucleosome on the (TTA)35 sequence represents 

a hindrance to the Topo I activity. 

Partners of DNA topoisomerase IB and 

functional assays 

Many works on protein partners of DNA topoisomerase 

IB were aimed at the identification of specific 

proteins, either by using approaches of molecular 

genetics like two hybrids, or by specific antibodies to 

reveal bound protein partners or carrying out experiments 

with isolated proteins. Looking for novel protein 

partners, we set up the following functional assays 

to evaluate: i) the capability of DNA topoisomerase IB 

to specifically cleave the DNA at selected genetic loci; 

ii) the production of extrachromosomal rDNA circles 

(ERCs) directly connected with rDNA units recombination 

events; iii) the non coding RNAs amount at 

rDNA region where transcriptional silencing occurs. 

Employing these assays (all Topo I dependent) we 

could find out functional partners involved in these 

processes. 


Cioci F, Di Felice F, Chiani F, Camilloni G. DNA 

Protein Interactions at the rRNA of Saccharomyces 

cerevisiae. Ital J Biochem. 2007, 56:81-90. 

Di Felice F, Chiani F, Camilloni G. Nucleosomes 

represent a physical barrier for cleavage activity of 

Topo I in vivo. Biochem J. 2008, 409:651-6.


Paola Vittorioso, professor; Sabrina Sabatini, researcher; 

Maura Cardarelli, CNR researcher. 


Dipartimento di Biologia Vegetale, Sapienza-Università di Roma 

(Prof. M.M. Altamura); Institute for Chemical Research, Kyoto 

University, Japan (Prof. Takashi Aoyama). 


The Dof proteins are transcription factors present 

only in plants and characterized by a strongly conserved 

single zinc finger domain. We have shown 

that two of these genes, DAG1 and DAG2 are 

involved in regulating the germination of seeds in 

Arabidopsis. 

Another Dof gene, NtBBF1, is involved in auxininducible 

gene expression in plant meristems. 

Currently our interests are focused on a) defining the 

role of DAG1 and DAG2 in regulating phytochrome 

B-mediated seed germination; b) defining the role of 

auxin in the development of the floral stamen; c) 

defining the role of auxin and cytokinin, and of 

genes responsive to these hormones in the maintenance 

of the root meristem. 

DAG1 

Previous data showed that inactivation of the gene 

encoding the Arabidopsis thaliana transcription factor 

DOF AFFECTING GERMINATION 1 (DAG1), 

renders seed germination more sensitive to both 

Phytochrome B (PhyB) and gibberellins (GA). dag1 

mutant seeds require less red (R) light fluence and a 

lower GA concentration than wild type to germinate. 

In the last year we found that inactivation of the 

gene PHYTOCHROME INTERACTING FACTOR 

3-LIKE 5 (PIL5) results in down-regulation of 

DAG1. Moreover, inactivation of PIL5 in the dag1 

mutant background further increased the germina- 

39 


The role of the DAG transcription factors in Arabidopsis seed 

germination 

Principal investigator: Paolo Costantino 



Tel: (+39) 06 4455344, 06 49912411; Fax: (+39) 06 4440812 

paolo.costantino@uniroma1.it 

tion potential of dag1 mutant seeds, confirming that 

DAG1 is under the positive control of PIL5. On the 

other hand, germination of dag1 phyB double mutant 

seeds showed a reduced requirement of gibberellins 

(GA) as compared to phyB mutant seeds, both in the 

presence and in the absence of endogenous GA 

biosynthesis. Furthermore, the GA biosynthetic 

gene AtGA3ox1 is upregulated in dag1 seeds as compared 

to the wild type, and DAG1 inactivation also 

affects the expression of different ABA metabolic 

genes. These data suggest that DAG1 is a negative 

regulatory element acting downstream of PIL5 in 

the PhyB signal transduction pathway that regulates 

GA and ABA metabolic genes during seed germination. 

In addition, based on the analysis of hypocotyls 

of dag1 and phyB mutant plantlets, of plantlets overexpressing 

PhyB in the dag1 mutant as well as of 

dag1 phyB double mutants, we suggest that DAG1 

may act as a negative regulatory element downstream 

of PhyB also in hypocotyl elongation. 

Hormones & stamen development 

Different hormones are involved in different stages 

of the development of the different floral organs. We 

analyzed the localization, synthesis, transport, and 

effects of auxin on the processes occurring late in the 

development of the Arabidopsis stamen: anther 

dehiscence, pollen maturation, and preanthesis filament 

elongation. Expression of auxin-sensitive 

reporter constructs suggests that auxin effects begin 

in anthers between the end of meiosis and the bilocular 

stage in the somatic tissues involved in the first 

step of dehiscence as well as in the microspores and 

in the junction region between anther and filament. 

In situ hybridizations of the auxin biosynthetic 

genes YUC2 and YUC6 suggest that auxin is synthesized 

in anthers. In agreement with the timing of 

auxin effects, the TIR1, AFB1, AFB2, and AFB3 

auxin receptor-encoding genes are transcribed in 

anthers only during late stages of development 

starting at the end of meiosis. We found that in tir1

P. Costantino - The role of the DAG transcription factors in Arabidopsis seed germination 

afb triple and quadruple mutants, anther dehiscence 

and pollen maturation occur earlier than in the wild 

type, causing the release of mature pollen grains 

before the completion of filament elongation. We 

also assessed the contribution of auxin transport to 

late stamen developmental processes. Our results 

suggest that auxin synthesized in anthers plays a 

major role in coordinating anther dehiscence and 

pollen maturation, while auxin transport contributes 

to the independent regulation of preanthesis filament 

elongation. 

Root meristem 

Plant postembryonic development takes place in 

the meristems, where stem cells self-renew and 

produce daughter cells that differentiate and give 

rise to different organ structures. For the maintenance 

of meristems, the rate of differentiation of 

daughter cells must equal the generation of new 

cells: how this is achieved is a central question in 

plant development. In the Arabidopsis root meristem, 

stem cells surround a small group of organizing 

cells, the quiescent center. Together they form 

a stem cell niche, whose position and activity 

depends on the combinatorial action of a small 

number of genes as well as on polar auxin transport. 

In contrast, the mechanisms controlling 

meristematic cell differentiation remain unclear. 

We demonstrated that cytokinins control the rate 

of meristematic cell differentiation and thus determine 

root meristem size via a two-component 

receptor histidine kinase-transcription factor signaling 

pathway. Analysis of the root meristems of 

cytokinin mutants, spatial cytokinin depletion, and 

exogenous cytokinin application indicated that 

cytokinins act in a restricted region of the root 

40 

meristem, where they antagonize a noncellautonomous 

cell-division signal, and we provided 

evidence that this signal is auxin. 

Subsequently, by means of a comprehensive genetic 

and molecular analysis, we showed that a primary 

cytokinin-response transcription factor, ARR1, activates 

the gene SHY2, a repressor of auxin signaling 

that negatively regulates the PIN auxin transport 

facilitator genes: thereby, cytokinin causes auxin 

redistribution, prompting cell differentiation. 

Conversely, auxin mediates degradation of the SHY2 

protein, sustaining PIN activities and cell division. 

Thus, the cell differentiation and division balance 

necessary for controlling root meristem size and root 

growth is the result of the interaction between 

cytokinin and auxin through a simple regulatory circuit 

converging on the SHY2 gene. 


Dello Ioio R, Scaglia Linhares F, Scacchi E, 

Casamitjana-Martinez E, Heidstra R, Costantino P, 

Sabatini S. Cytokinins determine Arabidopsis root 

meristem size by controlling cell differentiation. Curr 

Biol. 2007, 17:678-82. 

Cecchetti V, Altamura MM, Falasca G, Costantino 

P, Cardarelli M. Auxin regulates Arabidopsis anther 

dehiscence, pollen maturation and filament elongation. 

Plant Cell 2008, 20:1760-74. 

Dello Ioio R, Nakamura K, Moubayidin L, Perilli S, 

Taniguchi M, Morita M, Aoyama T, Costantino P, 

Sabatini S. A genetic framework for the control of 

cell division and differentiation in the root meristem. 

Science 2008, 322:1380-4.


Luciana Mosca, researcher; Italo Tempera, Alessandra 

Masci, post-doc fellows. 


Dipartimento di Farmacologia, Sapienza-Università di Roma (Dr. 

Luciano Saso); Université Paris 6 Pierre et Marie Curie, Paris, 

France (Prof. Francesco Visioli); Istituto di Biologia e Patologia 

Molecolari, CNR, Roma (Dr. Gianni Colotti). 


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. 

Amyloid beta peptide (Abeta), a 39-42 residue 

polypeptide derived from the proteolytic processing 

of the Amyloid precursor protein by secretases, is a 

major component of senile plaques and is considered 

to have a central role in the pathogenesis of 

Alzheimer’s disease. 

α-Synuclein can assume various conformations from 

monomeric to fibrillar, from unfolded in solution to 

alpha-helical in the presence of lipid-containing vesicles, 

to a beta-pleated sheet or amyloid structure in 

the fibrils that compose the Lewy bodies. 

It is well known that both Abeta and α -synuclein can 

rapidly undergo fibrillization giving rise to aggregated 

forms (Uversky, Curr Alzheimer Res. 2008, 

5:260) that is associated to the generation of oxygen 

free radicals (Smith et al., BBA 2007, 1768:1976). 

Oxidative stress is responsible of DNA damage 

which, in turn, is a prime activator of the enzyme 

poly(ADP-ribose)polymerase (PARP). 

PARP-1 is a zinc-binding nuclear enzyme which catalyzes 

the covalent addition of the ADP-ribose moiety 

of nicotinamide adenine dinucleotide (NAD + ) to 

proteins and the subsequent elongation step of the 

41 


Epigenetic modifications in neurodegenerative diseases 

Principal investigator: Maria d’Erme 



Tel: (+39) 06 49910923; Fax: (+39) 06 4440062 

maria.derme@uniroma1.it 

polymer, which is involved in many physiological 

processes such as gene expression, maintenance of 

genomic stability and cell death. A causal relationship 

between PARP and neurodegeneration is 

demonstrated by the findings that parkinsonian neurotoxins 

and amyloid-beta potently activate PARP in 

dopaminergic neurons and hippocampus, respectively. 

In addition, poly(ADP-ribosylated) nuclear proteins 

were showed in the neurons of Alzheimer's 

patient autopsy brain (Love et al., Brain 1999, 

122:247). Further evidence of an involvement of 

PARP in neuronal death is based on the finding of 

Outerio et al. (BBRC 2007, 357:596) showing that 

PARP-1 inhibitors significantly counteract neurotoxicity 

in experimental models of neurodegenerative 

diseases. 

The aim of the present research is to elucidate the 

contribution of poly(ADP-ribosylation) process in 

neurodegeneration. To achieve this objective, the 

dopaminergic neuroblastoma cell line SH-SY5Y was 

treated: i) with Abeta 25-35 fragment (Abeta 25-35 ), in 

the presence or absence of a new PARP inhibitor, the 

4-chinazolinonic derivative MC2050. Abeta 25-35 is 

able to preserve all the properties of its full-length 

counterparts, in particular the neurotoxicity; ii) with 

5-S-cysteinyldopamine (CysDA), or 6-hydroxydopamine 

(OH-DA). CysDA has been recently indicated 

as a putative endogenous parkinsonian neurotoxin 

(Spencer et al., J Neurochem. 2002, 81:122; 

Mosca et al., J Neuroscience Res. 2008, 86:954) but its 

effects in vivo have not been deeply investigated so far. 

CysDA is not commercially available and is routinely 

synthesized in our laboratory by a slight modification 

of the procedure for 5-S-cysteinyldopa described by 

Chioccara and Novellino (Synthetic Commun. 1996, 

16:967). OH-DA is a well known parkinsonian neurotoxin 

and was used as a positive control. 

First objective 

We assessed the PARP activity both in vitro and in 

cells, in the presence or absence of Abeta25-35 frag-

M. d’Erme - Epigenetic modifications in neurodegenerative diseases 

ment. All the experiments were carried out with an 

Abeta 25-35 fragment dissolved in PBS and left at 37 

°C overnight (aggregated form) or dissolved immediately 

before use (not aggregated form). 

The results indicate that aggregated Abeta 25-35 

fragment activates PARP around 30-40% after 1h 

incubation. It is of note that the not aggregated form 

activates the enzyme activity up to 60% in the same 

assay conditions. The activation of the enzyme is 

reduced in the presence of the new PARP inhibitor 

MC2050. 

A similar enzyme activation is seen when SH-SY5Y 

cells are treated with aggregated or not aggregated 

Abeta 25-35 which enhance endogenous PARP activity 

up to 30 or 40%, respectively, within 4 hours of 

treatment. The increase in PARP activity is prevented 

by pre-treatment with MC2050. 

In order to verify whether PARP activation is mediated 

by Reactive Oxygen Species (ROS) generated by 

Abeta fibrils formation, SH-SY5Y cells were treated 

with aggregated or not aggregated Abeta 25-35 and 

the intracellular ROS generation assessed by flow 

cytometry. The Abeta peptide was able to induce a 

marked rise in intracellular ROS production within 

the first two hours of treatment, particularly in the 

not aggregated form. These data allow to hypothesize 

that the ROS production induced by the fibrillization 

process is the main responsible for PARP 

activation. 

This study also demonstrate that MC2050 is highly 

active in the micromolar range (IC 50 = 25 mM) 

compared to the well known inhibitor 3-ABA. Cell 

viability assay revealed that this compound is not 

cytotoxic at the tested concentrations. 

42 

Second objective 

Recent studies show that MPP + and rotenone 

induce α-synuclein overexpression and aggregation 

(Lee et al., J Biol Chem. 2002, 277:5411) and this latter 

event is considered to be one of the pathological 

hallmarks of PD. Hence preliminary experiments 

were devoted to investigate whether the treatment 

with CysDA affects α-synuclein expression. Western 

blots of cell lysates show an increase of the level of 

this protein 8-24 hrs after CysDA treatment. 

Confirming previous data, the accumulation of 

α-synuclein has also been demonstrated by fuorescence 

microscopy. 

Since oxidative DNA damage is able to alter the 

enzymatic methylation at the CpG sites, thereby 

playing an important role in the regulation of gene 

expression, we investigated the methylation status of 

the α-synuclein promoter. Methylation level was 

assessed by the bisulfite genomic sequencing, which 

revealed no change of DNA methylation pattern 

under the experimental conditions. 

Further experiments will be dedicated to assess the 

effect of Poly(ADP-ribosylation) on the Abeta signal 

transduction and on the expression and aggregation 

of α-synuclein. This goal will be achieved with the 

use of the new PARP inhibitor MC2050. 


Mattiussi S, Tempera I, Matusali G, Mearini G, 

Lenti L, Fratarcangeli S, Mosca L, D'Erme M, 

Mattia E. Inhibition of Poly(ADP-ribose)polymerase 

impairs Epstein-Barr Virus lytic cycle progression. 

Infect Agent Cancer 2007, 2:18.


Nicolay Junakovic, CNR researcher; Melina Accardo, 

Nicoletta Corradini, Fabrizio Rossi, post-doc fellows. 


Dipartimento di Anatomia Patologica e Genetica, Università di 

Bari (Prof. Ruggiero Caizzi); Dipartimento di Genetica, Biologia 

Generale e Molecolare, Università di Napoli “Federico II” (Dr. 

Ennio Giordano). 


The main aim of our project was to study the functions 

of heterochromatin genes of D. melanogaster. 

We found that the 15 genes tested are expressed 

throughout all development, despite their location in 

a supposedly silent region of the genome. In addition, 

some of them are required for important functions 

such has chromosome condensation and cytokinesis. 

Future analyses will be carried out to delucidate 

the role played by CG40218 protein and its 

human hortolog CFDP1 in chromosome organization 

and function. 

Heterochromatin gene expression 

throughout development 

Using Northern blotting, we investigated transcription 

throughout developmental stages in wild-type 

Oregon-R strain of the vital gene Nipped-A and of 

15 putative genes: CG12567 and CG40042 of 2Lh, 

CG40218, CG41063, CG17691, CG40080, CG40130, 

CG40129, CG17665, CG2944, CG2682, CG30440, 

CG17514, CG10837 and CG17419. Notably, the protein 

products encoded by 13 of these gene models 

are conserved in humans. A Nipped-A-homologous 

transcript of about 11 kb was detected in all stages 

suggesting that the expression of this essential gene 

is required throughout all development. A similar 

picture emerges from the analysis of the putative 

genes tested: transcripts of all 15 genes are present 

Principal investigator: Patrizio Dimitri 

Professor of Genetics 


Tel/Fax: (+39) 06 49917948 

Patrizio.dimitri@uniroma1.it 

43 


Drosophila melanogaster as a model organism for functional 

genomic analyses of essential genes resident in heterochromatin 

at different stages of development, with the exception 

of CG17514 transcript not seen in embryos. 

Insertional mutagenesis of vital 

heterochromatic genes with P-elements 

Insertional mutagenesis (see original program) was 

performed with 3 different P{w+} inserts located in 

chromosome 2 heterochromatin (Corradini et al., 

2003). We screened 5899 chromosomes and found 23 

lethal mutations induced by P-element transposition. 

Among the recovered mutations we found insertional 

alleles in the following vital genes of 2Rh: 

l(2)41Aa (3 alleles), l(2)41Ae (2 alleles), l(2)41Af (2 

allele), Nipped-A (2 alleles) and Nipped-B (4 alleles). 

Linking l(2)41Aa vital gene and CG40218 

putative gene 

Using inverse PCR and molecular sequencing of 

LP1 insertional lethal allele, we identified the putative 

gene CG40218 (971bp) which is candidate to 

correspond to l(2)41Aa. Sequencing of CG40218 on 

genomic DNA from larvae homozygous for EMS-31, 

a lethal allele of l(2)41Aa, revealed that the EMS-31 

allele carries a single base pair substitution that gives 

rise to a stop codon in the aminoacid sequence. A 

GC40218-homologous transcript of the expected 

size (0.9 Kb) is present during all stages of development 

in the wild-type, but is absent in the LP1 

mutants. These results indicate that CG40218 corresponds 

to l(2)41Aa. 

RNAi mediated inactivation of 

heterochromatin genes in S2 cells 

Cytological analysis of metaphases after RNAi 

mediated inactivation of l(2)41Aa-CG40218 in 

Drosophila S2 tissue culture cells revealed that chromosome 

condensation is highly defective upon 

depletion of the CG40218 gene product. This defect 

is consistent with that observed in l(2)41Aa mutants 

(Cenci et al., 2003). We also performed doublestranded 

RNA-mediated interference of a group of

P. Dimitri - Drosophila melanogaster as a model organism for functional genomic analyses of essential genes 

300 heterochromatin genes. Among the genes studied, 

interesting results were obtained by inactivation 

of CG17528 which encodes a putative microtubule 

associated protein. After inactivation of CG17528, 

several defects occurred: 1) aberrant anaphases, 2) 

binucleate cells and 3) abnormal shape cells. The frequency 

of these defects was significantly higher 

than that found in the controls. These data are compatible 

with a role of CG17528 in spindle and 

cytoskeleton organization. RNAi analyses also 

revealed 5 genes (CG17168, CG40461, CG41281 

and concertina) whose protein product may be 

required for cytokinesis or proper cell shape. 

The GC40218 protein 

The CG40218 protein belongs to the conserved family 

of BCNT-like proteins found in animals and plants, 

whose functions is still unknown. Immuno-staining 

and western blotting experiments were carried out 

using antibody against the mouse CP27 protein which 

share homology with the CG40218 protein. In western 

blotting, the antibody recognizes a band of about 

27 Kda absent in CG40218 mutants. On polytene 

chromosomes of wild-type larvae, the immuno-staining 

is distributed all over the euchromatic arms and 

the chromocenter, but disappears in CG40218 

mutants. We also found that on polytene chromosomes 

deposition of H2Av, HP1 and H3trimetk9 

chromatin markers is affect in CG40218 mutants. 

RNAi mediated inactivation of CFDP1, the 

human ortholog of CG40218, in HeLa cells 

CFDP1 (craniofacial development protein 1), the 

human ortholog of CG40218, encodes a 299 a. a. 

belonging to the BCNT family, whose function is still 

unknown. RNAi mediated inactivation of CFDP1 in 

HeLa cells using a specific siRNA-expressing vector 

called psiUx (Denti et al. 2004). Expression of 

CFDP1 after RNAi was monitored by RT PCR and 

immunoblotting using policlonal antibodies against 

CFDP1 protein. The cytological analysis of treated 

cells revealed the presence of metaphase chromosomes 

with aberrant morphology and condensation 

defects similar to those observed after inactivation of 

CG40218 in Drosophila. 

44 

In vivo RNAi in S2 Drosophila cells 

We tested the RNAi-transgene in vivo silencing of 

endogenous heterochromatin gene targets by monitoring 

the presence of visible phenotypes and verifying 

transcript reduction of the endogenous gene by 

Northern blots. 

Ory gene: Inactivation of Ory, one of the two Ylinked 

genes (Carvalho et al., 2001) of the collection, 

induces a complete male sterile phenotype with 

testes of mutant males that lack mature spermatids 

and show empty seminal vesicles. Since mutant 

testes do not show any apparent defects in cells of 

the earlier stage of spermatogenesis we speculated 

that in Ory mutants a potential disruption of the 

individualization process occurs. 

CG17528 gene: inactivation of CG17528 generates 

in adult flies clear phenotypes characterized by 

defects in wing, notum and abdomen development. 

Since most of the observed defects perfectly matches 

the phenotype caused by disruption of wingless 

pathway in the wing disc (Morata and Lawrence, 

1977), this may represent a good starting point to 

study the role of CG17528 and the opportunity to 

use wing disc development as model. 


Corradini N, Rossi F, Giordano E, Caizzi R, 

Verní F, Dimitri P. Drosophila melanogaster as a 

model for studying protein-encoding genes that 

are resident in constitutive heterochromatin. 

Heredity 2007, 98:3-12. 

Hoskins RA, Carlson JW, Kennedy C, Acevedo D, 

Evans-Holm M, Frise E, Wan KH, Park S, Mendez- 

Lago M, Rossi F, Villasante A, Dimitri P, Karpen 

GH, Celniker SE. Sequence finishing and mapping of 

Drosophila melanogaster heterochromatin. Science 

2007, 316:1625-8. 

Rossi F, Moschetti R, Caizzi R, Corradini N, 

Dimitri P. Cytogenetics and molecular characterization 

of heterochromatin gene models in Drosophila 

melanogaster. Genetics 2007, 175:595-607.

DNA replication mechanisms and genome stability in 

Saccharomyces cerevisiae 


Federica Lo Sardo, graduate student. 


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). 


Different biological processes are involved in the 

maintenance of genome integrity 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. Complete and 

accurate DNA replication is integral to the maintenance 

of the genetic integrity of all organisms. 

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 Saccharomyces 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” chromosome fragments, 

mutants defective in the maintenance of an “originless” 

chromosome fragment were isolated (originless 

fragment maintenance mutants). Only three of them, 

OFM1, ofm6 and ofm14, meet the ofm criterion 

because they are defective only in the maintenance of 

the “originless” fragment but proficient in the maintenance 

of the same fragment that carries its normal 

complement of replicators. The study of ofm mutants 

can help to define the mechanisms responsible of the 

Principal investigator: Lucia Fabiani 

Researcher in Molecular Biology 


Tel: 06 49912243; Fax: (+39) 06 49912351 

lucia.fabiani@uniroma1.it 

45 


replication of the “originless” fragment and a more 

detailed analysis of ofm2 and ofm5 mutants will allow 

us to understand also the mechanisms and the genes 

involved in chromosome stability both being mutants 

characterized by a high loss rate of the wildtype 

derivative of chromosome III. We have recently identified 

the gene able to complement the ofm5 mutation. 

CHL1 (Chromosome Loss 1) is the mutated gene. 

Chl1p (861 aa) is a very interesting protein, member 

of DEAH helicases family-ATP dependent, its helicase 

activity is important for the establishment of sister 

chromatid cohesion (SCC) during S phase but its 

role is poorly understood. Chl1p interacts physically 

and genetically with Eco1p (Establishment cohesion 

1) involved in the regulation of SCC establishment. 

The sister chromatid cohesion is an essential mechanism 

for proper chromosome segregation and DNA 

double strand breaks repair by homologous recombination. 

The SSC required the coordinated activites of 

several factors: cohesins, deposition and cohesion 

establishment proteins. 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. Recently it has been shown (A) a 

correlation between DNA replication and sister chromatid 

cohesion, proteins important for DNA replication 

interact directly with proteins involved in sister 

chromatid cohesion, and (B) the establishment of 

SCC is not restricted to S phase but occurs also after 

DNA damage. 

Our interest is to understand the role of Chl1p in the 

establishment of sister chromatid cohesion during 

DNA replication in S. cerevisiae as a model system. 

Chl1p shares a high similarity with human BACH1 

(BRCA1 Associated C-terminal Helicase) protein 

that directly interact with the tumor suppressor 

BRCA1 (BReast CAncer 1); mutations in BACH1 are 

associated to breast cancer and genetically linked to 

the bone marrow disease Fanconi Anemia (FA). 

Chl1p, in normal condition, could be recruited at the

L. Fabiani - DNA replication mechanisms and genome stability in Saccharomyces cerevisiae 

level of the fork to give the correct conformation 

through the cohesin ring. In replication stress condition 

and in the presence of DNA damage Chl1p could 

contribute to protect the fork from the formation of 

ricombinogenic intermediates and to promote the 

DNA replication restart. 

During these first months of financial support we 

identified the mutation present in ofm5 by the gap 

repair technique to recover and finally sequence the 

mutated copy of CHL1 gene in ofm5. The sequencing 

of CHL1 in both ofm5 and wildtype strains 

showed the presence of a point mutation in chl1 ofm5 

allele, a transition of G to A at the level of 

nucleotide 275 of the coding sequence, converting a 

Trp codon (TGG) at the aminoacid 92 of the protein 

to a stop codon (TAG). This premature stop codon 

cause a null allele: all the important helicase domains 

are lost. We obtained the chl1 deletion mutant and 

analyzed its sensitivity to DNA-damaging agents. 

chl1 is sensitive to hydroxyurea (HU) (depletion of 

dNTP pools), methylmethanesulfonate (MMS) (alkylation 

of bases) and UV radiation (pyrimidine dimer 

formation). We analyzed then the correct activation 

of the S phase checkpoints, the replication and intra- 

S checkpoints, after synchronization in G2 with 

46 

nocodazole and release, respectively, in the presence 

of HU (S phase arrest in response to replication 

blocks) and MMS (slowing of replication in response 

to DNA damage in S phase). Both checkpoint mechanisms 

are efficiently activated. 

In the future, with the purpose to understand the 

potential role of Chl1p at the replication fork, we will 

analyze, by two-dimensional gel electrophoresis, the 

replication intermediates in both wildtype and chl1 

deletion mutant, at the level of chromosome III, 

before and after treatment with DNA damaging 

agents (HU, MMS), and at the level of the region of 

chromosome XII containing the cluster of rDNA. 

rDNA is a good model to study the different steps of 

DNA replication and genome stability beeing one of 

the most fragile sites of the genome. It will be then 

interesting to verify the presence of Chl1p at the 

level of the replication fork by ChIP (Chromatin 

Immuno-Precitation) experiments by adding a tag 

sequence at the C-terminus of the protein. The presence 

of a tag will allow us to check the presence of 

post-translational modifications (phosphorylation, 

sumoylation, ubiquitinylation and acetylation) of 

Chl1p under normal and in response to DNA damage 

conditions.


Cristina Mazzoni, researcher; Vanessa Palermo, post-doc fellow; 

Mirko Torella, PhD student; Michele Saliola, technician. 


Institute of Molecular Biosciences, University of Graz, Austria 

(Prof. Frank Madeo); Dipartimento di Medicina e Patologia 

Sperimentale, Sapienza-Università di Roma (Prof. Patrizia 

Mancini). 


The aim of the present project concerns the isolation 

of anti-apoptotic genes and the study of their suppression 

mechanisms. 

Apoptosis is a form of programmed cell death 

required for health, homeostasis and embryonic 

development in metazoans. Apoptosis is required for 

the removal of autoreactive immune cells, virusinfected 

cells and cells with DNA damage which can 

undergo transformation, playing an important role in 

different diseases such as cancer, neurodegenerative 

disorders, or stroke. A simple model system, such as 

yeast, has been proved to be very useful to clarify the 

regulatory network underlying this phenomenon. 

In fact, during the past years, scientists were successful 

in identifying new cell-death regulators of 

humans, plants and fungi using the yeast 

Saccharomyces cerevisiae. Moreover, yeast analogues of 

some crucial components of the apoptotic cascade in 

mammals have also been described, (i.e., caspase, 

Omi, AIF and EndoG) suggesting that the basic 

machinery of apoptosis is indeed present and functional 

also in this unicellular organisms. 

The biological significance of apoptosis in an unicellular 

organism, like yeast, could be related to the possibility 

in nature of discarding individual aged and 

damaged cells from the whole population and allow 

the survival of young and healthy cells. 

Principal investigator: Claudio Falcone 

Professor of Industrial Microbiology 


Tel: (+39) 06 49912278; Fax: (+39) 06 49912256 

claudio.falcone@uniroma1.it 

47 


New tools in deciphering aging and apoptotic pathways 

Among the physiological conditions that can induce 

programmed cell death in yeast there are the presence 

of small quantities of the conjugation 

pheromone (mating type pheromone), and aging. 

In the first case, it has been demonstrated that only 

those cells that are unable to mate will undergo apoptosis, 

confirming that apoptosis acts as a selection for 

the fitness. 

Two different kinds of aging processes can be studied 

in yeast: RLS (Replicative Life Span), that measures 

the number of generation performed by a single 

cell in the life, and CLS (Chronological Life Span), 

which measures cell viability when cells stop dividing 

(stationary phase). In both replicative and 

chronological aging, the most aged cells die following 

an apoptotic pathway. 

In our laboratory, we constructed a viable S. cerevisiae 

mutant with reduced longevity and showing all 

markers of apoptosis. This mutant, namely Kllsm4∆1, 

presents a delayed mRNA turnover in consequence of 

defects in decapping, one of the first step in the 

degradation of messenger RNAs. 

This is a very upstream mutant for the onset of 

apoptosis and we used it for the isolation of downstream 

suppressors and the identification of different 

apoptotic pathways. 

One of the suppressors that we identified was HIR1, 

the gene that encodes a transcriptional co-repressor 

of histone genes. 

We have shown that the over-expression of HIR1 

reduced the transcription of histone genes, but also 

of other genes, suggesting that the over-expression 

of HIR1 would lead to the constitution of a 

repressed chromatin. We postulated that the reduction 

of transcription might compensate the 

increased stability of mRNAs observed in the 

Kllsm4∆1 mutant. 

To better understand this relationship, we checked 

during the cell cycle the expression of the histone 

genes, in that their trascription is tightly regulated

C. Falcone - New tools in deciphering aging and apoptotic pathways 

and limited to the S phase. Hydroxyurea (HU) 

inhibits DNA replication and causes cell cycle arrest 

in S-phase through the inhibition of the ribonucleotide 

reductase (RNR), an enzyme that catalyzes 

the rate-limiting step in the production of dNTPs. 

Cells treated with HU showed low levels of histones 

transcripts and synchronized at the beginning of S 

phase. In wild type cells, histone transcription peaks 

after about 45 min following the release from the HU 

block. On the contrary, in the Kllsm4∆1 mutant, histone 

transcription starts as soon as HU is removed 

suggesting. This result suggests, beside the defect in 

mRNA degradation, the existence of an impaired control 

of histones genes transcription during cell-cycle. 

Following the over-expression of HIR1, histones transcription 

is repressed and, although delayed compared 

to the wild type, the cyclic expression is restored. 

These results suggest that some S phase entry 

checkpoints, which repress the transcription of his- 

48 

tone genes before S phase, could be lost in the 

Kllsm4∆1 mutant. 

It is known that a lag in S phase, caused either by 

genetic (i.e. swi6) or chemical means (i.e. hydroxyurea), 

enhances silencing. 

We observed that the presence of low doses of 

hydroxyurea could suppress the premature loss of 

viability of the Kllsm4∆1 mutant during chronological 

aging, confirming that in this mutant a delay of 

entry in S phase recovers some cellular defects. 


Mazzoni C, D’Addario I, Falcone C. The C-terminus 

of the yeast Lsm4p is required for the association 

to P-bodies. FEBS Letters 2007, 581:4836-40. 

Mazzoni C, Falcone C. Caspase-dependent apoptosis 

in yeast. Biochem Biophys Acta 2008, 1783:1320-7.

New complex mitochondrial functions in cell biology 


Claudio Falcone, professor; Silvia Francisci, Teresa Rinaldi, 

Cristina Mazzoni, researchers; Cristina De Luca, Vanessa 

Palermo, post-doc fellows; Michele Saliola, technician. 


Laboratory of Molecular Genetics, Université Paris Sud, Orsay, 

France (Prof. Monique Bolotin-Fukuhara); Department of 

Biology, The Technion, University of Haifa, Israel (Dr. Michael 

Glickman). 


Our work was planned to connect three aspects of 

mitochondrial functions in cell life, apoptosis and 

disease. 

The first research line investigates the role of the 

proteasome in regulating mitochondrial fusion and 

fission events, which are essential in maintaining the 

mitochondrial shape. This highly dynamic system is 

conserved in evolution and is altered in some human 

diseases. We have shown that the proteasomal 

mutant (rpn11-m1) exhibits fragmented mitochondria 

and a very peculiar cell cycle defect. Since the 

mpr1 protein lacks the last 31 aminoacids of the wt 

Rpn11 protein, which is the deubiquitinating 

enzyme of the proteasome, we investigated in detail 

the function of these aminoacids. As previously suggested 

by the genetic analysis of the rpn11 extragenic 

revertants, the cell cycle and mitochondrial 

defects can be separated, so we have performed a site 

specific mutagenesis in order to identify the 

aminoacids involved in maintaining the correct 

mitochondrial morphology and those necessary for 

the correct cell cycle. We have identified a putative 

α-helix necessary for the maintenance of the correct 

cell cycle, while a very short region of the C terminal 

part of Rpn11 was found to be essential for the 

maintenance of the tubular mitochondrial morphology. 

Furthermore we have shown that expression of 

Principal investigator: Laura Frontali 

Professor of Microbial Chemistry 


Tel: (+39) 06 4453950, Fax: (+39) 06 4461980 

laura.frontali@uniroma1.it 

49 


the C-terminal part of Rpn11 is able to complement 

in trans all the pleiotropic phenotypes of the rpn11m1 

mutant; this result suggests that the Rpn11 protein 

could act in controlling the mitochondrial shape 

even independently from the proteasome. Finally we 

have investigated the mechanisms by which Rpn11 

controls the mitochondrial shape and show that 

Rpn11 may regulate the mitochondrial fission and 

tubulation processes. 

In the second line of research, we have concentrated 

our analysis on aging and apoptosis. Mitochondrial 

morphology changes from a network shape to a 

punctuate one during aging and following an apoptotic 

stimulus. The significance of these alterations 

is not known, but it is clear that an excess of fission 

events in mitochondria and/or a defective mitochondrial 

fusion results in destruction of the mitochondrial 

tubular network with consequent respiratory 

defect, accumulation of ROS, and apoptosis in mammalian 

cells. We found that the deletion of the 

DNM1, known to be protective from apoptotic triggers, 

leads also to extended longevity. The F-box 

MDM30/DSG1 is known to affect mitochondrial 

morphology, but its role in aging and apoptosis has 

never been investigated. We showed that the mdm30 

null mutant presents extended longevity and high 

resistance to oxidative stress, suggesting a role for 

this gene in both apoptosis and aging. Yme1p is a 

subunit, together with Mgr1p, of the mitochondrial 

inner membrane i-AAA protease complex, which is 

responsible for degradation of unfolded or misfolded 

mitochondrial proteins. Yme1p shows 42% and 33% 

homology with the human proteins YME1L and 

paraplegin (SPG7), respectively. We demonstrated 

that the deletion of YME1 leads to premature loss of 

viability during chronological aging and to increased 

sensitivity to apoptotic stimuli. 

Lastly we have confirmed the possibility of using 

yeast mitochondria as a flexible and versatile tool to

L. Frontali - New complex mitochondrial functions in cell biology 

investigate open problems in mitochondrial disease, 

such as. elucidation of molecular mechanisms; differentiation 

of pathogenic mutations from non-pathogenic 

polymorphisms; explanation for the existence 

of homoplasmic mutations; understanding of the 

reason for the variability of penetrance; and unravelling 

of the basis of different pathogenic effects of 

the same mutation in different individuals. 

We have previously shown that introduction in yeast 

mt tRNAleuUUR gene, by biolistic procedure, of base 

substitutions equivalent to 3243, 3256 and 3291 found 

in MELAS patients, produces very severe growth 

defective phenotype in glycerol, with loss of mt DNA. 

We have now developed this research line by studying 

several new mutations, including the C25Tbase 

substitution in tRNAVal, equivalent to the homoplasmic 

human mutation 1624 having different penetrance 

in various members of the same family. 

The phenotypes we observed (growth in glycerol, 

respiration, rho° formation, analysis of tRNAs by 

Northern blotting on acidic sequencing gels) were 

found to be different in different laboratory strains 

and interestingly, the severity of all phenotypes varied 

with the same trend for all mutations. 

Another important aspect of this research is the possibility 

of correcting the defects due to the mutations 

by the overexpression of some nuclearly 

encoded mitochondrial factors which interact with 

the mutated tRNA and probably stabilize their structure. 

We have previously shown that among these 

suppressors there are the mitochondrial protein 

elongation factor EF-Tu and the cognate aminoacyltRNA 

synthetase. We have now extended these 

results to new mutations and we have shown that the 

suppression is dependent on the amount of suppres- 

50 

sor available. We also observed that the nuclear context 

as well as the endogenous expression level of 

the suppressors, affect dramatically the defective 

phenotype of the analyzed mutants. 

A variable amount of suppressors might be important 

for the understanding of the tissue specificity of 

cell damage observed in patients and a possible perspective 

basis for the correction of the defects. 

Actually our results on the suppressive effects of 

the above mentioned tRNA interactors have suggested 

to several important groups working on 

human patients to verify the same effect in human 

cells (myoblasts from patient biopsies). Results have 

been positive, thus confirming the usefulness of the 

yeast model. 


Palermo V, Falcone C, Mazzoni C. Apoptosis and 

aging in mitochondrial morphology mutants of S. 

cerevisiae. Folia Microbiologica 2007, 52:479-83. 

Montanari A, Besagni C, De Luca C, Morea V, Oliva 

R, Tramontano A, Bolotin-Fukuhara M, Frontali L, 

Francisci S. Yeast as a model of human mitochondrial 

tRNA base substitutions: investigation of the 

molecular basis of respiratory defects. RNA 2008, 

14:275-83. 

Rinaldi T, Hofmann L, Gambadoro A, Cossard R, 

Livnat-Levanon N, Glickman MH, Frontali L, 

Delahodde A. Dissection of the carboxyl-terminal 

domain of the proteasomal subunit rpn11 in maintenance 

of mitochondrial structure and function. Mol 

Biol Cell 2008, 19:1022-31.


Silvia Bonaccorsi, Maria Grazia Giansanti, Patrizia 

Somma, Fiammetta Vernì, researchers; Elisabetta 

Bucciarelli, Gianluca Cestra, post-doc fellows; Claudia 

Pellacani, PhD student; Giorgio Belloni, technician. 


Stanford University, USA (Prof. Margareth Fuller); Cornell 

University, USA (Prof. Michel L. Goldberg). 


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 abscisssion, 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 at the advancing 

cleavage furrow of Drosophila spermatocytes. We 

have recently identified mutations in six genes 

required for furrow ingression during meiotic 

cytokinesis of Drosophila males. All these genes 

encode products involved in membrane trafficking: 

Zw10, Rab1, the Exocist complex components 

Exo84 and Sec8, and the ortholog of PACS-1. We 

plan to define the roles of these proteins in furrow 

Principal investigator: Maurizio Gatti 



Tel: (+39) 06 49912842; Fax: (+39) 06 4456866 

maurizio.gatti@uniroma1.it 

51 


The role of membrane trafficking in Drosophila cytokinesis 

ingression, and investigate their functional relationships 

with other proteins involved in membrane traffic 

during cytokinesis. 

In the past two years we have carried out three main 

research projects. 

The role for of bond in furrow ingression 

during cytokinesis in Drosophila 

spermatocytes 

Recent work has shown that plasma membrane lipids 

influence membrane biophysical properties such as 

membrane curvature and elasticity and play an active 

role in cell function. We have 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) is required to regulate 

Rab11 behavior during meiotic cytokinesis in 

Drosophila males 

We have found that successful furrow ingression during 

meiotic cytokinesis in Drosophila males requires 

function of the gene brunelleschi (bru), which encodes 

the ortholog of the yeast transport protein particle 

(TRAPP) II complex subunit, Trs120p. Dividing male 

meiotic cells from bru mutants assemble a normal contractile 

ring that initiates constriction but fails to con-

M. Gatti - The role of membrane trafficking in Drosophila cytokinesis 

strict fully during the course of furrow ingression. 

Consistent with an essential role for membrane trafficking, 

cytokinesis during male meiosis is blocked by 

the Golgi trafficking inhibitor brefeldin A (BFA). A 

Bru-GFP fusion protein localizes to Golgi organelles 

with a substantial amount dispersed throughout the 

cytoplasm as well. In post-meiotic spermatids, Bru 

also localizes to the acroblast, a developmentally regulated 

Golgi derivative. Acroblasts fail to form in bru 

mutant spermatids, implying a requirement for 

TRAPPII in coordinating the organization of Golgi 

membranes during acroblast formation. bru genetically 

interacts with Rab11, which encodes a small 

GTPase that regulates late stages of membrane trafficking, 

and with the phosphatidylinositol 4-kinase β 

(PI4Kβ)-encoding gene four wheel drive (fwd). 

Consistent with this, localization of Rab11 protein to 

the cleavage furrow of male meiotic cells requires 

wild-type function of bru. The genetic interactions 

between the Drosophila genes encoding Bru, PI4Kβ, 

and Rab11 are consistent with the known genetic relationships 

between their orthologs in budding yeast, 

suggesting that membrane trafficking processes that 

are essential to the growth and viability of budding 

yeast support cleavage furrow ingression during male 

meiosis in Drosophila. 

Zw10 is required for cytokinesis with a role in 

membrane addition during cleavage furrow 

ingression 

ZW10, along with the other member 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 required for the 

association of dynein with Golgi membranes, with 

reduced levels of Zw10 resulting in disruption of 

ER to Golgi transport. We have found that the male 

meiotic divisions of zw10 mutants display defects in 

cytokinesis, where the acto-myosin ring forms and 

begins to ingress but then regresses. Surprisingly, 

52 

this cytokinesis failure is observed only in zw10 

mutants even though all three members of the RZZ 

complex localize across the mid-zone of the spindle 

envelope during telophase. In zwilch and rod 

mutants Zw10 no longer localizes to the spindle 

envelope mid-zone but concentrates in the ER suggesting 

an involvement of Zw10 in the ER-Golgi 

membrane traffic required for cytokinesis. Although 

no defects were detected in the structure of ER or 

Golgi stacks, acroblast formation is defective in 

zw10 mutants. These results suggest Zw10, besides 

its well-known role in the SAC machinery, is also 

involved in membrane dynamics during cytokinesis 

and acroblast formation. 

Perspectives 

We plan to continue our work on Rab1, the Exocist 

complex components Exo84 and Sec8 and the 

Drosophila ortholog of PACS-1 encoded by smeagol 

(sgo). We believe that the analysis of the role of these 

proteins during 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. 


Giansanti MG, Belloni G, Gatti M. Rab11 is 

required for membrane trafficking and actomyosin 

ring constriction in meiotic cytokinesis of Drosophila 

males. Mol Biol Cell 2007, 18:5034-47. 

Giansanti MG, Bucciarelli E, Bonaccorsi S, Gatti 

M. Drosophila Spd-2 is an essential centriole component 

required for PCM recruitment and astral microtubule 

nucleation. Curr Biol. 2008, 18:303-9. 

Szafer-Glusman E, Giansanti MG, Nishihama R, 

Pringle J, Gatti M, Fuller MT. Specialized lipids are 

required to couple actomyosin ring to the plasma 

membrane during meiotic cytokinesis in Drosophila 

males. Curr Biol. 2008, 23:1426-31.


Fabio Miraldi, Giacomo Frati, professors; Elisa Messina, 

medical manager; Lucio Barile, post-doc fellow; Roberto 

Gaetani, Isotta Chimenti, Elvira Forte, PhD students; 

Vittoria Ionta, Francesco Angelini, Silvia Turi, undergraduate 

students. 


Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma (Dr. 

Settimio Grimaldi); Istituto di Tecnologie Biomediche, CNR, Pisa 

(Dr. Cristina Magli); Dipartimento di Biotecnologie e Bioscienze, 

Università di Milano-Bicocca (Prof. Sergio Ottolenghi); School of 

Medicine, Division of Cardiology, Johns Hopkins University, 

Baltimore, USA (Prof. Eduardo Marbán). 


Aim 

The project aims at characterizing the cell biology 

and physiology of cardiac stem cells (CSCs) and cardiospheres 

(CSps), with particular focus on optimizing 

their utility for cardiac cell therapy clinical translation. 

The aims are grouped into three categories, as 

follows: 1)to improve and optimize cell culture methods 

and conditions for CSCs, that is to develop culture 

conditions that favor CSC expansion and differentiation 

towards the cardiac lineage in vitro; 2) to 

verify the efficacy of CSCs in infarct repair in vivo 

using animal models; 3) to assess the cell biology of 

CSCs and CSps, that is: a) to determine the origin of 

human CSCs under physiologic and pathologic conditions; 

b) to characterize the physiological properties 

of CSCs at various stages of differentiation, in 

terms of commitment and potency. 

Effects of electromagnetic fields exposure 

of CSCs 

One of the major limitations to cardiac cell therapy 

applicability is the low specific cardiomyogenic 

Principal investigator: Alessandro Giacomello 

Professor of Pathology 


Tel/Fax: (+39) 06 4461481 

alessandro.giacomello@uniroma1.it 

53 


Biology and physiology of adult cardiac stem/progenitor cells 

with a view to optimizing their utility for autologous 

cell transplantation 

potential of the candidate cells. Electromagnetic 

fields (EMFs) are known to interfere with many 

cellular functions, such as proliferations and differentiation, 

most likely by altering membrane currents, 

in particular through Ca 2+ channels. 

Recently, extremely low frequency (ELF) EMFs 

have been shown to drive cardiac specific differentiation 

in embryonic stem cells. Therefore, we 

exposed up to 5 days CSps and CDCs to ELF- 

EMFs, tuned at the resonance frequency of the 

Ca 2+ ion, inside an a-magnetic room, in order to 

guarantee full reproducibility. Exposed CSCs displayed 

increased metabolic activity and higher proliferation 

rates, compared to unexposed controls. 

Transcriptional and translational analysis after 5 

days of ELF-EMF exposure revealed increased 

expression of cardiac markers, such as Nkx2.5, 

TnI, MHC and Cx43, while markers of vascular 

lineage, like KDR and SMA, were down-regulated. 

Exposure to a control frequency, far from the resonance 

of biologically relevant ions, did not elicit 

any detectable effect. Chronically exposed cells displayed 

marked intracellular Ca 2+ accumulation, as 

assessed by Orange-Green fluorescence intensity. 

Furthermore compartmentalized analysis of Rhod- 

2 fluorescence allowed the detection of Ca 2+ fluxes 

among intracellular storage compartments in 

exposed CDCs, while no effect was detectable in 

unexposed cells or in cells exposed to the control 

frequency. In conclusion, the modulation of cell 

proliferation and specific cardiac differentiation 

elicited by our system through ELF-EMFs could 

represent an effective and safe biotechnological tool 

to improve their cardiac regenerative potential. 

The origin of CSCs 

In order to investigate whether bone marrow (BM)derived 

cells can contribute to the endogenous c-kit + 

CSC pool, we transplanted BM cells from transgenic 

mice, expressing GFP under the c-kit promoter, in 

wild-type lethally-irradiated syngeneic mice. After 4-

A. Giacomello - Biology and physiology of adult cardiac stem/progenitor cells 

5 months the reconstituted mice with transgenic BM 

were subjected to surgical LAD ligation. After 3 

weeks hearts were excised and plated as primary 

explants, and CSps were isolated. CSps from transplanted, 

infarcted mice were GFP + , indicating that 

GFP + BM-derived cells had been mobilized and had 

migrated to the infarcted heart. Much less CSps were 

obtained from transplanted, non infarcted mice, and 

they were GFP - . To assess the biology and commitment 

of kit/GFP + CSps, 1x10 5 CSp-derived cells 

(CDCs) were acutely injected into the infarct border 

zone of wild-type mice. Three weeks later GFP + 

cells had survived and engrafted in the infarct area, 

and most of them had differentiated into cardiomyocytes 

and vessels. Our data show that, under local 

CSC depletion and tissue damage, BM cells can 

migrate to the heart and apparently adopt the phenotype 

and functional characteristics of the endogenous 

CSC pool. 

The “paracrine hypothesis” for CSps and CDCs 

Many recent pre-clinical and clinical studies, observing 

beneficial effects after cardiac cell therapy without 

direct massive cardiac regeneration, brought 

along the hypothesis that other mechanisms could be 

involved. Transplanted cells might produce beneficial 

humoral factors, promoting endogenous cardiomyocytes 

survival and angiogenesis. Therefore 

we investigated the paracrine abilities of CSps and 

CDCs. Both stages are able of secreting in vitro 

VEGF and HGF in significant amounts, compared to 

a control cell line of dermal fibroblasts. CSps also 

secrete IGF1, that is not detectable anymore after 

this stage, despite many different culture conditions 

tested. However both CSps and CDCs express the 

corresponding mRNA for VEGF, HGF and IGF1, 

and also their receptors. Since so far an in vivo functional 

improvement has been demonstrated only for 

CDC injection, we assessed whether CDC-conditioned 

media (CM) has paracrine effects in vitro in 

ischemic-like conditions, that is serum and glucose 

54 

starvation. CDC-CM was able to significantly reduce 

the percentage of apoptotic neonatal rat ventricular 

myocytes after 72 hours of hypoxic culture, compared 

to fibroblast-CM and control basal media. 

Furthermore CDC-CM was able to recover 

HUVECs ability to form complex tube networks in 

an in vitro angiogenesis assay; this ability was lost in 

the basal media and in the fibroblast-CM. The preincubation 

with neutralizing antibodies for VEGF 

and/or HGF partially but significantly reduced both 

the anti-apoptotic and the pro-angiogenic effects, 

suggesting that at least in part these beneficial effects 

depend on the secreted growth factors. CDCs are 

also able to secrete VEGF, HGF and IGF1 in vivo, as 

detected by RT-PCR and WB in the same SCID 

infarction model where they have been previously 

demonstrated to mediate improvement in LV function. 

Thus, CSps and CDCs are able to secrete significant 

amounts of pro-survival and pro-angiogenic 

growth factors. This could be a key mechanism, 

together with their spontaneous commitment to the 

cardiac lineage, contributing to the beneficial effects 

observed in cardiac cell therapy settings. 


Barile L, Chimenti I, Gaetani R, Forte E, Miraldi F, 

Frati G, Messina E, Giacomello A. Cardiac stem 

cells: isolation, expansion and experimental use for 

myocardial regeneration. Nat Clin Pract Cardiovasc 

Med. 2007, 4 Suppl 1:S9-S14. 

Barile L, Messina E, Giacomello A, Marbán E. 

Endogenous cardiac stem cells. Prog Cardiovasc Dis. 

2007, 50:31-48. 

Smith RR, Barile L, Cho HC, Leppo MK, Hare JM, 

Messina E, Giacomello A, Abraham MR, Marbán E. 

Regenerative potential of cardiosphere-derived cells 

expanded from percutaneous endomyocardial biopsy 

specimens. Circulation 2007, 115:896-908.


Gianluca Canettieri, Elisabetta Ferretti, professors; Enrico De 

Smaele, Lucia Di Marcotullio, Alessandra Ianari, researchers. 

Subversion of cerebellar neural progenitor cell development 

leads to neoplastic transformation into medulloblastoma, 

an aggressive brain malignancy with high 

incidence in childhood. Current treatment approaches 

have limited efficacy and severe side effects. Therefore, 

new risk-adapted therapeutic strategies based on 

molecular classification are required. A crucial master 

regulator of cerebellar neural progenitor cell development 

is represented by the Hedgehog pathway which 

displays, when deregulated, a critical role in the pathogenesis 

of medulloblastoma. Cross-regulation between 

Hedgehog signalling and several tumorigenic pathways 

(e.g. the oncogene products of the AP1/jun/fos complex) 

plays a role in cell development and tumorigenesis. 

Furthermore, deregulation of tumor suppressor 

gene function (i.e. Rb) is critical for the formation of 

Hedgehog-dependent medulloblastoma. We have investigated 

a number of molecular mechanisms involved in 

the formation of medulloblastoma (Gulino et al., J Clin 

Invest. 2009, in press; Gulino et al., Curr Opin Oncol. 

2008, 20:668-75; Gulino et al., Psychoneuroendocrinology 

2007, 32:S55-56; Farioli-Vecchioli et al., FASEB J. 2007, 

21:2215-25). We have also further dissected the molecular 

steps of the regulation of the Hedgehog pathway 

as well as of the control of the transcriptional function 

of AP1 and Rb proteins. Specific aspects of the 

research are described below. 

MicroRNA control of cerebellar neural 

progenitors and medulloblastoma cells via 

targeting of the Hedgehog pathway 

MicroRNAs are crucial post-transcriptional regulators 

of gene expression and control cell differentiation and 

proliferation. MicroRNA expression analysis has 

emerged as a powerful tool to identify candidate molecules 

playing an important role in a large number of 

malignancies. However little is known about their targeting 

of specific developmental pathways and, more 

specifically, no data are yet available on human primary 

medulloblastomas. We have performed a high through- 

Principal investigator: Alberto Gulino 

Professor of Molecular Pathology 


Tel: (+39) 06 4464021; Fax: (+39) 06 4461974 

alberto.gulino@uniroma1.it 

55 


Regulation of the Hedgehog signaling in neural cell development 

and disease 

put microRNA expression profile analysis in human 

primary medulloblastoma specimens to investigate 

microRNA involvement in medulloblastoma carcinogenesis. 

We identified specific microRNA expression 

patterns which distinguish medulloblastoma differing 

in histotypes (anaplastic, classic and desmoplastic), in 

molecular features (ErbB2 or c-Myc over-expressing 

tumors) and in disease risk stratification. MicroRNAs 

expression profile clearly differentiates medulloblastoma 

from either adult or fetal normal cerebellar tissues. 

Only a few microRNAs displayed up-regulated 

expression, while most of them were downregulated in 

tumor samples, suggesting a tumor growth inhibitory 

function. This property has been addressed for miR-9 

and miR-125a whose rescued expression promoted 

medulloblastoma cell growth arrest and apoptosis 

while targeting the pro-proliferative truncated isoform 

of TrkC neurotrophin receptor (Ferretti et al., Int J 

Cancer 2009, 124:568-77; Laneve et al., Proc Natl Acad 

Sci USA 2007, 104:7957-62). 

Our microRNA high-throughput profile screening also 

revealed a downregulated microRNA signature in 

human medulloblastomas with high Hedgehog signaling. 

Specifically, we identify miR-125b and miR-326 as 

suppressors of the pathway activator Smoothened 

together with miR-324-5p which also targets the 

downstream transcription factor Gli1. Downregulation 

of these microRNAs allows high levels of Hedgehogdependent 

gene expression leading to tumor cell proliferation. 

Interestingly, the downregulation of miR-324- 

5p is genetically determined by medulloblastoma-associated 

deletion of chromosome 17p. Of note, we also 

report that while microRNA expression is downregulated 

in cerebellar neuronal progenitors, it increases 

along differentiation, thereby allowing cell maturation 

and growth inhibition. These findings identify a novel 

regulatory circuitry of the Hedgehog signaling pathway 

and suggest that misregulation of specific 

miRNAs, leading to its aberrant activation, sustains 

cancer development. 

In conclusion, specific microRNA deregulation signatures 

characterize medulloblastomas and discriminate 

tumor subsets with distinct properties thus representing 

potential targets for novel therapeutic strategies.

A. Gulino - Regulation of the Hedgehog signaling in neural cell development and disease 

Identification of novel Hedgehog-target genes 

in cerebellar neural progenitors and 

medulloblastoma 

Cerebellar neural progenitor development occurs during 

the first two weeks of post-natal life, under the 

activity of Shh. Therefore, we first analyzed gene 

expression profles in mouse cerebella during the first 

two weeks of development by means of Gene-Chip 

Murine microarray (Affimetrix), to identify subsets of 

genes specifically upregulated in the time window in 

which Hedgehog pathway is strongly activated. Then, 

we have selected several of these genes and verified if 

they are induced in cultured cerebellar GCPs treated 

with Shh. Finally, we analyzed mouse and human medulloblastoma 

samples for the expression levels of these 

genes. Through this approach, we have identified Insm1 

and Nhlh1/NSCL1 as new Hedgehog target genes, that 

are induced by Shh in cultured GCPs. We have identified 

and isolated Nhlh1 promoter and found that it is bound 

and activated by Gli1 transcription factor. Remarkably, 

the expression of these genes is also upregulated in 

mouse and human Hedgehog–dependent medulloblastomas, 

suggesting that they may be either a part of the 

Hedgehog-induced tumorigenic process or a specific 

trait of Hedgehog-dependent tumor cells (De Smaele et 

al., Neoplasia 2008, 10:89-98). 

Dissecting transcriptional function of AP-1 

The AP-1 complex mediates the transcriptional 

response to mitogens and its deregulation causes developmental 

defects and tumors by cross-talking with a 

number of signaling pathways including Hedgehog. 

The molecular mechanisms of this cross-regulation are 

poorly understood. We have observed that the coactivator 

TORC1 (Transducer Of Regulated CREB 1, also 

called CRTC1) is a potent and indispensable modulator 

of AP-1 function. Following exposure of cells to the 

AP-1 agonist TPA, TORC1 is recruited to AP-1 target 

gene promoters and associates with c-Jun and c-Fos to 

activate transcription. Consistently, TORC1 synergizes 

with the proto-oncogene c-Jun to promote cellular 

growth, whereas AP-1-dependent proliferation is abrogated 

in TORC1-deficient cells. Remarkably, we demonstrate 

that TORC1-Maml2 fusion oncoprotein binds 

and activate both c-Jun and c-Fos. As a consequence, 

ablation of AP-1 function disrupts cellular transformation 

and proliferation mediated by this oncogene. Taken 

together, these data illustrate a novel mechanism 

required to couple mitogenic signals to the AP-1 gene 

regulatory program (Canettieri et al., PNAS 2009). 

A novel pro-apoptotic transcriptional function 

of the retinoblastoma tumor suppressor protein 

The function of the retinoblastoma tumor suppressor 

protein (pRB) is disrupted in many human tumors 

through either inactivation of the Rb gene or alter- 

56 

ations in its upstream regulators. pRB’s loss of function 

has been reported to cooperate with Hedgehog pathway 

for cerebellar tumorigenesis via unelucidated mechanisms. 

pRB’s tumor suppressive activity is at least partially 

dependent upon its ability to arrest cells through 

E2F transcription factors inhibition. This inhibition is 

relieved through mitogen-induced phosphorylation of 

pRB, triggering E2F release and activation of cell cycle 

genes. We have previously reported that E2F1 can also 

activate pro-apoptotic genes in response to genotoxic or 

oncogenic stress, via acetylation-dependent protein 

modification (Pediconi et al., Nat Cell Biol 2003, 5:552; 

Ianari et al., J Biol Chem. 2004, 279:30830). However, 

pRB's role in this context has not been established. We 

have observed that DNA damage and E1A-induced 

oncogenic stress promotes formation of a pRB-E2F1 

complex even in proliferating cells. Moreover, pRB is 

bound to pro-apoptotic promoters that are transcriptional 

active and pRB is required for maximal apoptotic 

response in vitro and in vivo. Together, these data 

reveal a previously unappreciated function for pRB in 

the induction of apoptosis in response to genotoxic or 

oncogenic stress. Our data now establish a second role 

for pRB as a stress-induced activator of apoptosis. 

Notably, pRB’s ability to promote either arrest versus 

apoptosis seems to be context dependent, with apoptosis 

being favored in proliferating cells. This finding has 

the potential to explain why cells are typically more 

resistant to apoptosis when in the arrested state. Most 

importantly, our observations suggest that Rb status 

will influence tumor response to chemotherapy by 

impairing both the arrest and apoptotic checkpoint 

responses (Ianari et al., Cancer Cell 2009). 


De Smaele E, Fragomeli C, Ferretti E, Pelloni M, Po 

A, Canettieri G, Coni S, Di Marcotullio L, Greco A, 

Moretti M, Di Rocco C, Pazzaglia S, Maroder M, 

Screpanti I, Giannini G, Gulino A. An integrated 

approach identifies Nhlh1 and Insm1 as Sonic 

Hedgehog-regulated genes in developing cerebellum 

and medulloblastoma. Neoplasia 2008, 10:89-98. 

Ferretti E, De Smaele E, Miele E, Laneve P, Po A, 

Pelloni M, Paganelli A, Di Marcotullio L, Caffarelli E, 

Screpanti I, Bozzoni I, Gulino A. Concerted microRNA 

control of Hedgehog signalling in cerebellar neuronal 

progenitor and tumour cells. EMBO J. 2008, 27:2616-27. 

Ferretti E, Tosi E, Po A, Scipioni A, Morisi R, 

Espinola MS, Russo D, Durante C, Schlumberger M, 

Screpanti I, Filetti S, Gulino A. Notch signaling is 

involved in expression of thyrocyte differentiation 

markers and is downregulated in thyroid tumors. J Clin 

Endocrinol Metab. 2008, 93:4080-7.


Laura Belloni, Rossana De Iaco, Natalia Pediconi, 

Barbara Testoni, post-doc fellows; Francesca Guerrieri, 

Valeria Schinzari, Cecilia Scisciani, PhD students. 


Istituto Tumori Regina Elena and AIRC, Rome Oncogenomic 

Center (Dr. Giovanni Blandino, Dr. Maurizio Fanciulli); 

NIAMS, NIH, Bethesda, USA (Dr. Vittorio Sartorelli). 


Individual cellular programs (i.e. proliferation, differentiation, 

senescence, apoptosis) are executed at 

the transcriptional level by the selective expression 

of subset of genes, which specify the cell phenotype. 

The acetyltransferases p300/CBP and PCAF 

play an important role in the positive control of 

transcription due to their ability to acetylate the eamino 

group of lysine residues of both histones 

and nonhistone proteins. Their interaction with 

HDAC1 and Sir2/Sirt1 supports a model in which 

acetyltransferases and deacetylases may be simultaneously 

present on the same gene regulatory loci. 

p300, CBP and PCAF are subjected to a variety of 

covalent modifications, including acetylation, sumolation, 

methylation and phosphorylation, that contribute 

to their regulation. p300 undergoes 

autoacetylation upon activation of its acetyltransferase 

activity whereas, in the case of PCAF, both 

auto- and p300-mediated acetylation promote its 

acetyltransferase activity. 

Our research has focused on the DNA damage 

response as a prototype cellular response that is 

known to involve the recruitment of both p300 

and PCAF and aimed to determine the role of 

p300 and PCAF acetylation in the specification of 

the repertoire of target genes coactivated namely 

by E2F1 and p73. 

Principal investigator: Massimo Levrero 

Professor of Internal Medicine 

Dipartimento di Medicina Interna 

Tel: (+39) 06 49970892; Fax: (+39) 06 49383333 

massimo.levrero@uniroma1.it 

57 


Histone Acetyltransferase (HAT) autoregulatory loops in the 

regulation of DNA damage responses 

hSirT1-dependent regulation of the PCAF- 

E2F1-p73 apoptotic pathway in response to 

DNA damage 

The E2F family of transcription factors have critical 

roles in the control of cell proliferation and apoptosis. 

E2F1 upregulates transcription of several genes 

involved in the activation or execution of apoptosis, 

including the Apaf-1, caspase 7 and p73 genes. The 

E2F1/p73 pathway is thought to play a major role in 

DNA damaging drugs-induced apoptosis and tumor 

chemosensitivity. In response to DNA damage E2F1 

is phosphorylated by Chk2 and acetylated by PCAF. 

These post-translational modification potentiate 

E2F1 apoptotic activity and direct its selective 

recruitment onto the P1p73 promoter. Pre-existing 

and newly synthesized TAp73 is then phosphorylated 

by the nuclear tyrosine kinase cAbl, acetylated by 

p300 and assembled with the WW domain protein 

YAP to activate transcription of downstream apoptotic 

target genes. The importance of the E2F1/p73 

pathway is reinforced by the strong reduction of 

p53-independent apoptosis when either PCAF, p73 

or YAP expression is abrogated by specific siRNAs in 

cells exposed to DNA damage. 

We found that the interaction between hSirT1, the 

human homologue of the nicotinamide adenine dinucleotide 

(NAD)-dependent class III deacetylase Sir2 

(silent information regulator 2) and PCAF controls 

the E2F1/p73 apoptotic pathway. hSirT1 represses 

E2F1-dependent P1p73 promoter activity in untreated 

cells and inhibits its activation in response to DNA 

damage. hSirT1, PCAF and E2F1 are co-recruited 

on the P1p73 promoter. hSirT1 deacetylates PCAF in 

vitro and modulates PCAF acetylation in vivo. In cells 

exposed to apoptotic DNA damage nuclear NAD+ 

levels decrease and inactivate hSirT1, without altering 

hSirT1 interaction with PCAF and hSirT1 binding 

to the P1p73 promoter. Reactivation of hSirT1 

by pyruvate, that increases the [NAD + ]/[NADH] 

ratio, completely abolish the DNA damage-induced 

activation of TAp73 expression, thus linking the

M. Levrero - Histone Acetyltransferase (HAT) autoregulatory loops in the regulation of DNA damage responses 

modulation of chromatin-bound hSirT1 deacetylase 

activity by the intracellular redox state with P1p73 

promoter activity. Release of PCAF from hSirT1 

repression favours the assembly of transcriptionally 

active PCAF/E2F1 complexes onto the P1p73 promoter 

and p53-independent apoptosis. These results 

identify hSirT1 and PCAF as potential targets to 

modulate tumor cell survival and chemoresistance 

irrespective to p53 status. 

Chromatin Immunoprecipitation-based 

innovative reagents for a comprehensive 

evaluation of the E2F1 transcriptome 

Microarrays-based expression profiling studies have 

led to the identification of hundreds potential direct 

target genes activated and/or repressed by the different 

members of the E2F family of transcription 

factors. Genome-wide ChIP-chip experiments have 

confirmed that several hundreds putative E2F binding 

sites exist and are potentially bound throughout 

the genome. We combined an extensive in silico 

analysis with literature survey to select 327 putative 

E2F1 direct target genes containing highly conserved 

E2F/DP binding sites in their promoter or 

first intron (i.e. -2500 to +1000 respect to tss). To 

build up the E2F/DP dedicated microchip slide 3 different 

45-50mer oligos for each of the 327 target 

gene were designed. Each oligo was spotted in triplicate 

and the resulting array printed 4 times on the 

same slide for a total of 11772 spots. Gene ontology 

analysis of the 327 selected genes is in agreement 

with the notion that the spectrum of direct E2Fs target 

genes might be far more variated than previously 

thought: apoptosis (31), DNA damage (30), cell 

cycle (71) cell growth (16), signal transduction (29); 

transcription regulation (73), ubiquitination/Protein 

degradation (7), protein/cell metabolism (30); 

cytoskeleton/cell adhesion (38). 

The E2F-ChIP array has been validated by 

hybridization with anti-E2F1 and anti-Ac-H3 

immunoprecipitated chromatin derived from untreat- 

58 

ed and doxorubicin treated U2OS cells. The analysis 

of 2 independent ChIP-chip experiments has 

revealed that E2F1 is present on 48% of the genes in 

untreated cells. In cells exposed to apoptotic dosages 

of doxorubicin, E2F1 is released from 33 out of 157 

genes that were occupied in untreated genes (10% out 

of 48%) whereas E2F is recruited onto 118 additional 

target genes (36%) that were not bound in untreated 

cells. There results indicate that DNA damage 

results in a wide change in the spectrum of E2F1 

bound promoters. E2F1 is bound before and after 

treatment on 41% of the genes involved in apoptosis 

and DNA repair included in the array and it is 

recruited on additional 38% of these genes. 

Interestingly, among the genes that regulate cell 

cycle and whose expression is expected to be mostly 

down regulated in cells undergoing DNA damageinduced 

growth-arrest and apoptosis, only 17% loose 

E2F1 after exposure to doxorubicin whereas E2F1 

remains bound to 30% of these genes and is recruited 

on 39% additional genes. The analysis of the correlation 

between E2F1 promoter occupancy and gene 

expression indicate that multiple mechanisms are 

involved in the regulation of E2F target genes after 

DNA damage including the recruitment of repressive 

E2F1-containing complexes and the modulation 

of E2F1-bound complexes by the recruitment of corepressors 

and the release of coactivators. The E2F 

ChIP-on-chip reagent we have developed and validated 

and the knowledge that it generates will represent 

a powerful tool to define the spectrum of genes targeted 

by E2F1 to modulate cell death, senescence and 

proliferation and to functionally characterize the 

E2F transcriptome in vitro and in vivo. 


Blandino G, Fanciulli M, Levrero M, Piaggio G. 

The post-genomic era: workshop on chromatin 

immunoprecipitation-related techniques. Cell Death 

Differ. 2007, 14:1390-1.


Maria Teresa Fiorenza, Arturo Bevilacqua, professors; Sonia 

Canterini, researcher; Adriana Bosco, Valentina Carletti, 

Valentina De Matteis, Domenico Grillo, PhD students. 


Istituto Dermopatico dell’Immacolata, Roma (Dr. Giandomenico 

Russo, Dr. Maria Grazia Narducci); Ohio State University, 

Columbus, Ohio, USA (Prof. Carlo Croce); University of Turin (Dr. 

Annalisa Buffo); University of Antwerp, Belgium (Dr. Michele 

Giugliano). 


We have exploited two different model systems, the 

preimplantation mouse embryo development and the 

in vitro differentiation of cerebellum granule neurons, 

to investigate the control of early blastomere proliferation 

and the commitment to apoptosis, respectively, 

with particular reference to the functions of the oncogenic 

factor T-cell Leukemia Factor 1 (TCL1) and the 

putative tumor suppressor THG-1pit/Tsc22d4. 

Besides T-cell leukemias, TCL1 is physiologically 

expressed both in embryonic stem cells downstream 

from the Oct4 gene and in early preimplantation 

embryos, in which it enhances early blastomere proliferation. 

TCL1 is currently believed to promote normal/tumoral 

cell proliferation by binding and 

transphosphorylating AKT/PKB (AKT) at the level of 

plasma membrane and then mediating the phosphorylated 

AKT transfer to nucleus. However, the AKT isoform(s) 

that actually interacts with TCL1 and the 

TCL1 requirement for AKT nuclear transfer are still 

uncharacterized. We have directly approached these 

questions by depleting one-cell embryos by an intracytoplasmic 

microinjection of anti-AKT1, anti-AKT2 or 

anti-AKT3 antibodies and then following the in vitro 

development of injected embryos. Depletion of AKT2 

significantly delayed/blocked embryo development to 

blastocyst, as we had previously observed in Tcl1 KO 

Principal investigator: Franco Mangia 

Professor of General Biology 

Dipartimento di Psicologia, Sezione di Neuroscienze 

Tel: (+39) 06 49917784; Fax: (+39) 06 49917873 

franco.mangia@uniroma1.it 

59 


Molecular regulation of cell proliferation and apoptosis in early 

embryo blastomeres and granule neuron precursors of the mouse 

embryos (Narducci et al., PNAS 2002, 99:11712-7). In 

contrast, depletion of AKT1/AKT3 had no apparent 

effect on embryos, pinpointing the AKT2 isoform as 

the actual TCL1 interactor in preimplantation mouse 

embryos. Moreover, immunofluorescence experiments 

showed that AKT2, but not AKT1 nor AKT3, 

migrates to nucleus in concomitance with TCL1 

nuclear localization. The possibility that AKT 

Ser473/Thr308 phosphorylation depended on 

upstream factors/kinases, including PI3K, PDK1, and 

HSP90 protein was probed by treating embryos with 

specific inhibitors, showing that Ser473/Thr308-phosphorylated 

AKT2 is fully inherited from oogenesis and 

that, following fertilization, AKT does not undergo 

significant changes in the ratio between phosphorylated 

and dephosphorytlated conditions. This indirectly 

indicates that TCL1 is not required for AKT phosphorylation, 

whereas it represents an absolute requirement 

for phosphorylated AKT transfer to nucleus. In 

light of the well established finding that the AKT 

Ser473 residue is phosphorylated by the mTOR-Rictor 

complex (Sarbassov et al., Science 2005, 307:1098-101), 

we also investigated the expression of mTOR, Raptor 

and Rictor during preimplantation development by 

RT-PCR and, limitedly to the blastocyst stage, by 

western blot. Raptor mRNA appeared to be expressed 

during entire preimplantation development. In contrast, 

the mTOR message first appeared at 16-32 cell 

stage (suggesting a maternal origin of the protein) and 

Rictor mRNA was constantly lacking from fertilization 

to blastocyst, further supporting the hypothesis that 

AKT phosphorylation takes place during oogenesis, 

but not during preimplantation development. 

THG-1pit expression was preliminarily determined 

during postnatal development by Northern/Western 

blot analyses of RNA/protein extracts from cerebellum 

and primary cultures of cerebellar granule neurons 

(CGN) at increasing days of in vitro culture 

(DIV1-6). Northern blot analysis of both cerebellum 

and CGN extracts showed the presence of a single 2.7 

kb transcript, corresponding to the length expected

F. Mangia - Molecular regulation of cell proliferation and apoptosis in early embryo blastomeres 

by cDNA coding sequence (Fiorenza et al., Gene 2001, 

278:125-30). In contrast, western blot analysis displayed 

the presence of 4 electrophoretic bands having 

apparent MW of 42, 55, 67 and 72 kDa, respectively, 

the relative abundance of which varied during cerebellum 

development and in vitro CGN culture. In 

detail, the 72 kDa band relatively increased in amount 

during postnatal cerebellum development, suggesting 

a functional role in the fully mature cerebellum, 

whereas, among other THG1-pit bands, the 42 kDa 

one corresponded to the MW expected by the encoded 

amino acid sequence (including a number of putative 

phosphorylation and/or O-glycosylation sites) 

and thus was likely to represent the precursor of 

higher MW bands. This possibility was investigated 

by digesting CGN protein extracts with alkaline 

phosphatase and/or a mixture of glycosidases, leading 

to the conclusion that the 42 kDa protein gave origin 

to the 67 kDa by O-glycosylation and that this band 

was then converted to the 72 kDa by phosphorylation. 

Because these findings suggested that THG1-pit functions 

were finely regulated, we investigated the effects 

of: i) TGF-β1 on CGN in vitro differentiation; and ii) 

external K + concentration (25 mM K + ext , depolarizing 

and maintaining cell viability; 5 mM K + ext , 

hyperpolarizing and committing CGN to apoptosis; 

D'Mello et al., Proc Natl Acad Sci USA 1993, 90:10989- 

93) on Thg-1pit transcritpion and intracellular THG- 

1pit localization. CGN incubation in the presence of 5 

60 

mM K + ext and TGF- β1 transiently elicited both an 

early (peaking at 30 min) and a late (peaking at 3 hr) 

transcriptional waves, rapidly resulting in a significant 

increase in THG-1pit content. We have recently 

investigated the function(s) of different THG-1pit 

forms also by biochemically fractionating CGN cytoplasmic, 

chromatin and nuclear matrix fractions, 

showing the specific presence of the 72 kDa band in 

the chromatin (indipendent of K + ext ) and the 67 kDa 

in the nuclear matrix (transiently increasing under 

apoptotic condition). The functional meaning of 72 

kDa and 67 kDa subnuclear localizations are currently 

under investigation. 

Selected Pubblications 

Puglisi R, Bevilacqua A, Carlomagno G, Lenzi A, 

Gandini L, Stefanini M, Mangia F, Boitani C. Mice 

overexpressing the mitochondrial phospholipid 

hydroperoxide glutathione peroxidase in male germ 

cells show abnormal spermatogenesis and reduced 

fertility. Endocrinology 2007, 148:4302-9. 

Fiorenza MT, Torcia S, Canterini S, Bevilacqua A, 

Narducci MG, Ragone G, Croce CM, Russo G, 

Mangia F. TCL1 promotes blastomere proliferation 

through nuclear transfer, but not direct phosphorylation, 

of AKT/PKB in early mouse embryos. Cell 

Death Differ. 2008, 15:420-2. 

Fig. 1 - Nuclear translocation of THG-1pit landmarks GN committment 

to apoptosis. DIV7 CGNs cultured on coverslips were 

maintained for 1 hr in the presence of 25 mM or 5 mM K+ext supplemented 

with TGF- 1 and were eventually processed for 

immunofluorescence detection of THG-1pit and the early apoptosis 

marker AIF. Nuclei were stained with Hoechst 33258. Note THG- 

1pit and AIF colocalization.


Gabriella Dobrowolny, Emanuele Rizzuto, post-doc fellows; 

Michela Aucello, PhD student; Carmine Nicoletti, technician. 


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). 


The aim of the project is to define the molecular 

mechanisms that modulate muscle atrophy, associated 

with several pathological conditions. Oxidative 

status has profound consequences on the function of 

skeletal muscle, where the oxidative level is the highest 

in the whole body. The anti-oxidant status of 

muscle decreases with age, and is affected in several 

other pathological conditions, such as sarcopenia, 

chronic fatigue syndrome, liver and kidney diseases, 

cancer, muscular dystrophy and amyotrophic lateral 

sclerosis (ALS), in which the delicate balance 

between oxidant production and antioxidant defense 

is severely compromised, leading to muscle wasting. 

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 diseases-related 

decrease of muscle performance and 

mass and whether skeletal muscle is a direct target 

of SOD1 mutation remains largely unknown. 

In this study we undertook a transgenic approach 

Principal investigator: Antonio Musarò 

Professor of Biotechnology 

Dipartimento di Istologia ed Embriologia Medica 

Tel: (+39) 06 49766956; Fax: (+39) 06 4462854 

antonio.musaro@uniroma1.it 

61 


Study of the molecular and cellular mechanisms of sarcopenia: 

role of mIGF-1 and oxidative stress 

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 (SOD1G93A) cDNA driven by skeletal 

muscle specific regulatory elements from the rat 

myosin light chain (MLC)-1/3 locus. Expression of 

the MLC/SOD1G93A 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 SOD1G93A 

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. 

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/SOD1G93A mice, suggesting that 

autophagy is the dominant pathway that mediates 

the atrophic stimulus of oxidative stress and that the 

modulation of the autophagy pathway can be a 

potential therapeutic mechanism to counteract muscle 

atrophy associated with oxidative stress.

A. Musarò - Study of the molecular and cellular mechanisms of sarcopenia: role of mIGF-1 and oxidative stress 

In addition, our study is the first that documents how 

the T-tubule might be the potential donor of membrane 

that forms sequestering autophagocytic vesicle. 

In fact one of the unresolved questions related to 

autophagy concerns the origin of the membrane that 

forms the sequestering vesicle. It has been proposed 

that autophagosomes might initiate from the modification 

of pre-existing structures. 

In our study we documented the sequence of events 

by which T-tubules, which normally run perpendicularly 

(transversely) to the long axis of the fiber and 

form junctions (or triads) with the SR terminal cisternae, 

curved into a L-like structure that progress 

to a vesicle-like structure encompassing amorphous 

cellular material. 

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. 

62 

We are currently analyzing the role of the growth 

factor mIGF-1 in ameliorating the disease in this 

new mouse model. Our working hypothesis is that 

mIGF-1 counteracts muscle wasting by the modulation 

of proteolytic systems and oxidative pathways, 

thus preserving the functional connection between 

muscle and nerve. 

The characterization of this new mouse model 

promises to significantly advance our understanding 

of the possible pathogenic mechanisms that lead to 

muscle wasting in human diseases and offers novel 

approaches for treatment of diseases associated with 

oxidative stress accumulation. 


Dobrowolny G, Aucello M, Rizzuto E, Beccafico S, 

Mammucari C, Bonconpagni S, Belia S, Wannenes F, 

Nicoletti C, Del Prete Z, Rosenthal N, Molinaro M, 

Protasi F, Fanò G, Sandri M, Musarò A. Skeletal 

muscle is a primary target of SOD1G93A-mediated 

toxicity. Cell Metab. 2008. 8:425-36.


Michele M. Bianchi, professor; Simona Loreti, Eugenia 

Piccinni, post-doc fellows; Angela Alagia, Viviana 

Casagrande, PhD students. 


Laboratoire de Génomique CNRS et Ecole Normale Supérieure, 

(Prof. Frédéric Devaux); Department of Genetics, Institute of 

Biochemistry and Biophysics, Polish Academy of Sciences (Prof. 

Anna Chelstowska); Dipartimento di Biologia Cellulare e dello 

Sviluppo, Sapienza-Università di Roma (Prof. Laura Frontali, 

Dr. Teresa Rinaldi, Prof. Gabriella Tocco). 


The glioma-amplified sequence (GAS) 41 gene was 

identified for the first time as an amplified sequence 

in the human chromosome region 12q13-15, a locus 

known to be involved in gene amplification in human 

gliomas. Abnormalities like this are frequently found 

in human tumors and are thought to be a manifestation 

of the intrinsic genome instability of cancer 

cells. In addition it is presumed that over-expression 

of amplified genes confers a selective advantage to 

cell clones. The goal of this scientific program is to 

define the role of the presumptive transcription factor 

Gas41 in chromatin organization and gene 

expression. To this purpose we take advantage from 

the extraordinary conservation of structure, and 

very likely of function, of this protein across different 

eukaryotic species. In particular the yeast 

Saccharomyces cerevisiae Yaf9, that we have recently 

characterized is highly homologous to Gas41 and 

appears to share similar functions. 

Yaf9 is structurally and functionally associated to 

two chromatin modification complexes in S. cerevisiae: 

the NuA4 histone acetyltransferase complex and 

the SWR complex, involved in the substitution of 

histone H2A with its isoform H2AZ. On the other 

hand, Gas41 is involved in the Tip60 complex 

Principal investigator: Rodolfo Negri 



Tel: (+39) 06 44917790; Fax: (+39) 06 44917594 

rodolfo.negri@uniroma1.it 

63 


Role of the presumptive human transcription factor Gas41 and 

of its S. cerevisiae homologue Yaf9 in chromatin organization 

and gene expression 

which appears to correspond to a near-perfect 

fusion of the two yeast complexes NuA4 and SWR. 

Since these complexes are involved in the formation 

of a chromatin structure favourable to gene transcription, 

it is very likely that Gas41 and Yaf9 transcription 

functions are linked to the recruitment of 

the complexes to regulated promoters in both yeast 

and mammals. 

The transcriptional role of Yaf9 

in S. cerevisiae 

Our group characterized yeast strains lacking Yaf9 

(Del Vescovo et al., 2008; Casagrande et al., 2008). 

These strains show genomic instability, a series of 

growth phenotypes, including cesium chloride sensitivity, 

as compared with its isogenic wild type, and 

defined defects in cell morphology and spindle body 

formation. These phenotypes are accompanied, and 

possibly caused, by the transcriptional modulation of 

several genes and by changes in the level of histone 

acetylation and/or histone H2AZ variant occupancy 

at genomic sites like regulated promoters and centromeres. 

We analyzed the global transcriptional 

response of S. cerevisiae cells exposed to different 

concentrations of CsCl added in the growth medium 

and at different times after addition. Early responsive 

genes were mainly involved in cell wall structure and 

biosynthesis. About half of the induced genes were 

previously shown to respond to other alkali metal 

cations in a fashion dependent on the HOG (High 

Osmolarity and Glycerol) signaling pathway. 

Western blot analysis confirmed that cesium concentrations 

as low as 100 mM activate phosphorylation 

of Hog1, the MAP kinase of the HOG pathway. 

Another important fraction of the cesium-modulated 

genes requires Yaf9 for full responsiveness, as shown 

by the transcriptome of a yaf9∆ strain in the presence 

of cesium. We showed that a cell wall-restructuring 

process promptly occurs in response to 

cesium addition, which is dependent on the presence 

of both Hog1 and Yaf9 proteins. Moreover, the sen-

R. Negri - Role of the presumptive human transcription factor Gas41 

sitivity to low concentration of cesium of the yaf9∆ 

strain was not observed in the hog1∆-yaf9∆ double 

mutant. Finally, we showed that the osmotic activity 

of cesium salt was detected and signaled by the two 

branches downstream of the Sln1 and Sho1 sensors 

of the HOG pathway, but the cesium-specific 

response mediated by Yaf9, that counteracted the 

efficiency of the HOG pathway, was not routed by 

these sensors. In conclusion, the cesium specific 

response of S. cerevisiae involved the Yaf9 protein 

and its activity of chromatin remodelling and transcription 

regulation. 

Genome-wide localization of Yaf9 

in S. cerevisiae 

We constructed a yeast strain carrying a C-terminal 

HA tag fused to Yaf9: the fusion protein could suppress 

the phenotypes of the yaf9∆ strain. We performed 

a genome-wide localization of Yaf9 by ChIP 

on chip analysis, using antibodies against the HA tag 

and control experiments on the wild type strain. The 

immunoprecipitated DNA was used to hybridize 

microarray slides containing the whole S. cerevisiae 

genome. Data were normalized and analysed with 

microarray-dedicated data mining softwares. The 

main conclusions of this analysis are the following: 

1) the genome-wide localization of Yaf9 is similar to 

that one of the NuA4 histone acetylation complex; 2) 

the localization is not limited to promoter regions 

but extends to ORFs; 3) there is a positive and significant 

correlation between ORF occupancy and 

transcription level; 4) Yaf9 binds significantly to centromeric 

regions. 

It should be noted that the experimental background 

was high. In fact conclusions 3 and 4 seem to be partially 

applicable also to the tag-less wild type control. 

We are now validating these results with real time 

PCR quantification. 

64 

Searching for Gas41 molecular interactors 

We performed a search of Gas41 molecular interactors 

in collaboration with Anna Chelstowska (I.B.B., 

Polish Academy of Sciences) by using the yeast twohybrid 

system. Gas41 has been used as a bait to screen 

pre-transformed library of the human fetal brain. The 

screening was performed following the yeast mating 

protocol, which improves the sensitivity and the reliability 

of the results. The level of stringency was 

adjusted by varying the number of the reporter genes 

in the primary selection, and this enabled us to identify 

weak and strong interactors of Gas41. 

Among putative interactors were N-myc, FERM 

D4A and RNF8. These proteins were able to interact 

also with Yaf9, suggesting that the interaction was 

mediated by a structurally conserved domain, possibly 

the N-terminal one. N-myc was certainly the 

most interesting interactor. The proteins of this 

family are very well known transcription factors containing 

helix-loop-helix-zipper domains. They are 

involved in the control of cell proliferation, differentiation 

and apoptosis. Recent data suggest that their 

action is mediated by chromatin remodelling complexes. 

Each interaction will be confirmed in vitro by 

biochemical methods (pull-down, co-immunoprecipitation), 

and in mammalian cells by co-transfection 

experiments. Validation of the N-myc interaction 

was obtained with Gas41 attached to sepharose that 

could pull-down both N-myc present in neuroblastoma 

cell extract and purified recombinant C-myc. 


Del Vescovo V, Casagrande V, Bianchi MM, Piccinni 

E, Frontali L, Militti C, Fardeau V, Devaux F, Di Sanza 

C, Presutti C, Negri R. Role of Hog1 and Yaf9 in the 

transcriptional response of Saccharomyces cerevisiae to 

caesium chloride. Physiol Genomics 2008, 33:110-20.


Laura Fanti, professor; Lucia Piacentini, researcher; 

Barbara Perrini, Marcella Marchetti, post-doc fellows; 

Enzo Marchetti, Eleonora Vitagliano, technicians. 


Martin-Luther University, Halle, Germany (Prof. Gunter Reuter); 

Università di Lecce (Prof. Maria Pia Bozzetti). 


The aim of this research is to systematically search, 

by a combined genetic, cytological and molecular 

approach, genes involved in RNAi, heterochromatin 

formation and gene silencing in Drosophila. 

Recently, a new and fascinating relationship between 

RNAi and heterochromatin has been discovered. In 

Drosophila, it has been found that mutations in genes 

that are components of the RNA interference 

machinery, are also involved in heterochromatin formation. 

These genes are expressed in the male and 

female germlines, are implicated in germline stem 

cell self-renewal and have crucial functions in the 

early stages of development. 

Intriguingly, it has also been found that these mutations 

induce testicular transcription of the repetitive 

Stellate DNA sequences causing a complex meiotic 

phenotype. This meiotic phenotype is characterized by 

the presence of star or needle-shaped crystals in spermatocytes, 

that are mainly composed by the STEL- 

LATE protein, abnormal chromosome condensation at 

first metaphase, and irregularities in the transmission 

of the chromosomes during meiosis such as nondisjunction 

and meiotic drive. Stellate sequences are 

repetitive sequences organized in one cluster at the 

crystal locus of the entirely heterochromatic Y chromosome 

and two other clusters, respectively located in 

the heterochromatin and euchromatin of the X chromosome, 

that are repressed by the crystal + in male 

testes via an RNAi mechanism that uses the production 

of rasiRNA (repeat-associated small interfering RNA), 

also called piRNA (piwi-associated RNA) (see Figure). 

Principal investigator: Sergio Pimpinelli 



Tel: (+39) 06 49912876; Fax: (+39) 06 4456866 

sergio.pimpinelli@uniroma1.it 

65 


Heterochromatin, gene silencing and “RNA interference” in 

Drosophila 

Search for gene involved in RNA interference 

To genetically dissect the RNAi machinery and its 

relationship with heterochromatin, we performed a 

screen of genes acting during spermatogenesis by 

using Stellate phenotype as reporter. This screen led 

to the identification of 13 genes and 3 new X-linked 

mutants that induce the expression of the Stellate 

sequences in presence of a normal Y chromosome 

carrying the crystal + locus. 

We tested, so far, six mutants that show a strong Ste 

phenotype (fs(1)h, CycB, snf, shu, Xst30 and Xst49) and 

we found that all these mutations affect the biogenesis 

of Piwi-associated interfering RNAs (piRNAs) 

corresponding to the Stellate sequences and to the 

sequences of several retrotransposons. Our results 

have permitted to isolate and identify new genes that 

are involved in RNAi mechanisms. 

We are now performing further experiments to assess 

the potential role of these genes in the various steps 

of RNAi-response pathway. The results of this work 

will be described in a manuscript in preparation. 

The Hsp90 is involved in piRNAs biogenesis 

in Drosophila 

The Hsp90 chaperone proteins are conserved proteins 

that are involved in several cellular processes and 

development pathways. It has been shown in Drosophila 

that mutations in the Hsp90 encoding gene are capable 

to induce the expression of the Stellate repeated 

sequences in testes of mutant males. These sequences 

are normally repressed by the Y-linked crystal locus by 

an RNA interference (RNAi) machinery that produces 

silencing small rasiRNAs (or piRNAs). 

We have found that mutations in Hsp83 locus, which 

encodes the Hsp90 protein, induce the activation of 

Stellate by affecting the production of rasiRNAs 

(piRNAs). In addition, we show that this mutation 

activates the transcription and the mobilization of 

diverse class of mobile elements by affecting the biogenesis 

of the complementary piRNAs. 

Both set of data demonstrate for the first time, that 

Hsp90 chaperone is involved in negative regulation

S. Pimpinelli - Heterochromatin, gene silencing and “RNA interference” in Drosophila 

of repetitive sequences and trasposons expression by 

playing a significant role in piRNAs-dependent 

RNAi silencing. The results of this work will be 

described in a manuscript in preparation. 


Minervini CF, Marsano RM, Casieri P, Fanti L, 

Caizzi R, Pimpinelli S, Rocchi M, Viggiano L. 

Heterochromatin protein 1 interacts with 5'UTR of 

66 

transposable element ZAM in a sequence-specific 

fashion. Gene 2007, 393:1-10. 

Fanti L, Perrini B, Piacentini L, Berloco M, 

Marchetti E, Palumbo G, Pimpinelli S. The trithorax 

group and Pc group proteins are differentially 

involved in heterochromatin formation in Drosophila. 

Chromosoma 2008, 117:25-39. 

Fanti L, Pimpinelli S. HP1: a functionally multifaceted 

protein. Curr Opin Genet Dev. 2008, 18:169-74. 

Fig. 1 - (a) Localization of stellate sequences by FISH. As reported also diagrammatically in (b) such sequences are arranged in two clusters 

on the X chromosome: one euchromatic and the other heterochromatic. One other cluster of Stellate-like sequences is located at the 

crystal locus of the Y chromosome. (c) Northern blot showing a mature coding stellate RNA that is present only in testes of X/0 males. (d) 

Two different rasiRNAs from the crystal sequences that are present only in testes of X/Y males. (e) Cristalline aggregates that present only in 

testes of X/0 males, and are decorated by an antibody against the stellate protein.


Carla Boitani, professor; Elena Vicini, researcher; Barbara 

Muciaccia, Rossella Pugliesi, post-doc fellows; Margherita 

Grasso, Laura Grisanti, PhD students; Stefania Fera, Tiziana 

Menna, technicians. 


The highly efficient process of sperm production is 

dependent on proper hormone balance, local cellular 

interactions and, more importantly, it relies on the 

biological activity of spermatogonial stem cells 

(SSCs), the stem cells of the germline. In the last 

years the activity of our group has focused on the 

isolation and characterization of SSCs. Our previous 

work has led us to the identification of SSCs in the 

testis “side population” (T-SP). Further characterization 

of T-SP SSCs by double vital staining with 

Hoechst and rhodamine, indicated that T-SP SSCs 

represent only a subset of SSCs. Of note, other 

groups were able to derive pluripotent stem cells 

from mouse and human germ cells in culture. 

Therefore, SSC subsets may be endowed with different 

functional properties and developmental potency. 

The aim of this project was to further characterize 

and exploit functional differences among SSC subsets. 

A complementary project carried out in our 

group was aimed to develop experimental strategies 

to achieve conditional gene inactivation in the 

germline. 

Identify spermatogonial stem cells 

heterogeneity in vivo 

Evidence for phenotypic and functional heterogeneity 

have been obtained after prospective isolation of 

SSC and their biological assay, namely germ cell 

transplantation. However there is no evidence for the 

presence of such stem subsets in vivo. In the accepted 

model, the SSCs are type Asingle spermatogonia 

(As). The As either self-renew by forming single 

Principal investigator: Mario Stefanini 

Professor of Histology and Embryology 


Tel: (+39) 06 49766570; Fax:(+39) 06 4462854 

mario.stefanini@uniroma1.it 

67 


Biological characterization and in vitro culture of spermatogonial 

stem cells 

cells or generate pairs of cells, named type Apaired 

spermatogonia (Apr), connected by an intercellular 

bridge, which are committed to differentiate. To test 

the hypothesis that, in adult mouse testis, As spermatogonia 

are phenotypically heterogeneous, we 

have analyzed GFRA1 expression in undifferentiated 

spermatogonia. GFRA1 is the co-receptor for GDNF 

a niche-derived factor essential for SSC self-renewal 

and differentiation. As spermatogonia can be easily 

identified in whole mounted seminiferous tubules 

after immunostaining for the nuclear repressor 

PLZF. By immunofluorescence, confocal microscopy 

and morphometric analysis, we were able to demonstrate 

that 10% of the As cell population did not 

express this receptor. A common mechanism to generate 

cell diversity is the asymmetrical inheritance of 

proteins that specify daughter cell fate. We therefore 

analyzed by morphometric analysis the distribution 

of GFRA1 in daughter cells. We found doublet of 

daughter cells asymmetric for GFRA1 expression 

(5% of clones). This suggest that generation of SSC 

subsets may hold on asymmetric germ stem cell division. 

We next analyzed cell cycle kinetics of the two 

subsets by in vivo bromodeoxyuridine (BrdU) administration. 

Our results indicated that both subsets are 

actively engaged in cell cycle. A surrogate markers 

largely employed in different tissues for stem cells 

identification has been their preferential BrdU label 

retention, labeling-retaining-cells (LRCs). In fact, 

stem cells are expected to divide more slowly than 

many of their cell progeny. Time-course analysis 

after BrdU pulse labeling showed some LRCs at one 

month of chase. However, at two months no LRCs 

were identified. All together this indicate that germ 

stem cells are actively engaged in cell cycle and no 

quiescent stem cells are present in the adult testis, 

regardless of their GFRA1 expression pattern. 

Complementary observation in human testis samples 

also revealed that the most primitive germ cells are 

also heterogeneous for the expression of GFRA1. 

This observation lend further support to germ stem

M. Stefanini - Biological characterization and in vitro culture of spermatogonial stem cells 

cell heterogeneity that may be a conserved feature in 

mammalian testis (Grisanti et al., submitted for publication). 

In vitro Cre recombinase transduction induces 

conditional gene inactivation in 

spermatogonia stem cells 

Gene inactivation often results in early embryonic 

lethal phenotype hampering the analysis of gene 

inactivation in later stages of development. To circumvents 

these drawbacks, inactivation of candidate 

genes have been accomplished by conditional mutagenesis. 

The expression of recombinase Cre in target 

cells, results in the deletion of the loxP-modified 

alleles. Recently, Cre fusion proteins have been developed 

that enter mammalian cells maintained in vitro 

and perform recombination with high efficiency. In 

order to obtain conditional mutagenesis in adult 

SSCs we analyzed the feasibility of the ex vivo Cre 

delivery to germ cells combined with their transplantation 

in recipient mice testis. To test for Creinduced 

gene recombination in SSCs, germ cells were 

obtained from ROSA26 Cre reporter mice (R26R), in 

which Cre recombination can be monitored by LacZ 

expression. Cre-transduced SSCs retained their ability 

to self-renew and differentiate in vivo. Cre-transduced 

SSCs originate colonies of donor-derived 

spermatogenesis, in which LacZ-expressing germ 

68 

cells were normally arranged along seminiferous 

tubules. These data demonstrated, the feasibility of 

in vitro conditional mutagenesis in stem cell 

germline. This experimental strategy allows genetic 

manipulation of postnatal SSCs and their germ cell 

progeny, and may prove useful for the study of genes 

whose inactivation is either embryonically lethal or 

interferes with prenatal germ cell development 

(Grisanti et al., 2008). 


Muciaccia B, Corallini S, Vicini E, Padula F, Gandini 

L, Liuzzi G, Lenzi A, Stefanini M. HIV-1 viral DNA is 

present in ejaculated abnormal spermatozoa of 

seropositive subjects. Hum Reprod. 2007, 22:2868-78. 



overexpressing the mithochondrial phospholipid 

hydroperoxide glutathione peroxidase in male germ 

cells show abnormal spermatogenesis and reduced fertility. 

Endocrinology 2007, 148:4302-9. 

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 2008, in press.


Laura Amicone, Carla Cicchini, Alessandra Marchetti, 

researchers; Alice Conigliaro, post-doc fellow; Marta Colletti, 

PhD student; Claudio Cavallari, technician. 


Istituto Nazionale per le Malattie Infettive “L. Spallanzani”, Roma 

(Dr. Tonino Alonzi, Dr. Veronica Bordoni, Dr. Carmine 

Mancone). 


The epithelial-to-mesenchymal transition (EMT), by 

which an epithelial cell undergoes a conversion to a 

mesenchymal cell, dissociates from initial contact 

and migrates to secondary sites, is a crucial process 

both in development and in late stage of tumor 

process. EMT requires loss of epithelial polarity, 

alteration in cellular architecture and acquisition of 

migration capacity. Hallmarks of EMT include 

increased expression of mesenchymal markers, 

nuclear localization of β-catenin and production of 

transcription factors able to inhibit E-cadherin 

expression, in particular Snai1 (Snail). The key role 

for Snail family members in triggering EMT has 

been well established in vitro and in vivo. 

Recently an important role for EMT have been proposed 

also in the onset and development of chronic 

liver diseases that result from derangements in the 

synthesis and degradation of extra-cellular matrix. 

In particular, the transition of hepatocytes and 

cholangiocytes to a mesenchymal fibrogenic cell 

may contribute to pathogenesis of liver fibrosis and 

cirrhosis. 

A reverse trans-differentiation event, the mesenchymal-to-epithelial 

transition (MET), occurs at the 

secondary site. During MET, all the EMT markers 

are inversely modulated. 

Continuous balance between EMT and MET characterizes 

the so-called “metastable phenotype”, 

69 


Molecular mechanisms of the epithelial to mesenchymal 

transition in hepatocyte 

Principal investigator: Marco Tripodi 



Sezione Genetica Molecolare 

Tel: (+39) 06 4461387; Fax: (+39) 06 4462891 

tripodi@bce.uniroma1.it 

expression of stem cell plasticity. Metastable stem 

cells in fact express both epithelial and mesenchymal 

traits, while the dynamic fine regulation of 

EMT/MET oscillations permits self-renewal as well 

as the generation of differentiating precursors. 

In the frame of this project we previously demonstrated 

that in hepatocytes i) EMT correlates with 

the down-regulation of HNF4α, a master regulator 

of hepatocyte differentiation, and ii) EMT “master 

gene” Snail is sufficient to reproduce the TGFβinduced 

down-regulation of several epithelial markers 

and to induce EMT in hepatocytes. Most relevantly, 

we found that Snail represses the transcription 

of the HNF4α gene through a direct binding to 

its promoter demonstrating that Snail control both 

epithelial morphogenesis and differentiation 

(Cicchini et al., J Cell Physiol. 2006, 209:230-8). 

During the last year of the project we collected the 

following results: 

Role of MAPK in TGFb-mediated EMT of the 

hepatocytes 

In the attempt to further define the role for signalling 

pathways activated by TGFβ in hepatocytes, we analyzed 

the MAPK cascade and, in particular, the extracellular 

signal-regulated protein kinase 5 (ERK5). 

We found that ERK5 is phosphorylated and activated 

by TGFβ with a rapid and sustained kinetic, 

through a Src-dependent pathway. Interference with 

ERK5 activation by means of a specific dominant 

negative mutant of MEK5 also allowed to uncover a 

role for ERK5 in the TGFβ-induced cellular 

responses. In fact, we demonstrated that ERK5 participates 

to Snail protein accumulation. We also 

found that ERK5 inactivation impedes the TGFβmediated 

glycogen synthase kinase-3β inactivation, 

thus suggesting this as mechanism responsible for 

Snail stabilization. Our results, demonstrating a 

novel pathway of TGFβ signalling are reported in 

the selected publication Marchetti et al., 2008.

M. Tripodi - Molecular mechanisms of the epithelial to mesenchymal transition in hepatocyte 

Current studies are aimed: i) to better define the contribution 

of ERK5 activation by TGFβ to EMT, by 

analyzing the modulation of already characterized 

EMT markers, including known target of Snail such 

as E-cadherin and HNF-4, and ii) to deeper characterize 

the mechanisms involved in the regulation of 

GSK-3β by ERK5. 

Role of Src/FAK signalling in TGFβ-mediated 

EMT of the hepatocytes 

As previously described, while Snail is sufficient to 

down-regulate epithelial markers is not sufficient for 

the up-regulation of mesenchymal and invasivity 

EMT markers. In order to unveil the signalling 

involved in the acquisition of mesenchymal phenotype, 

we recently showed that during EMT TGFb 

induces a Src-dependent activation of the focal adhesion 

protein FAK. Relevantly, we demonstrated that 

FAK signaling is required for i) transcriptional upregulation 

of mesenchymal and invasivity markers 

and ii) delocalization of membrane-bound E-cadherin. 

These data provide the first evidence for a FAK 

role in TGFb-induced EMT. Our results are reported 

in the selected publication Cicchini et al., 2008. 

Current studies are focused on the mechanisms of 

FAK-mediated regulation of the E-cadherin and its 

implication in tumor onset and metastasis. 

Isolation, characterization and establishment 

in line of a resident liver stem cell (RLSC) 

We recently reported the isolation, characterization 

and establishment in line of a murine resident liver 

stem cell (RLSC) with immunophenotype (Sca+, 

CD34-, CD45-, α-fetoprotein+, albumin-) and differ- 

70 

entiative potentiality typical of a pre-hepatoblast/liver 

precursor cell. RLSCs in fact, spontaneously 

differentiate into hepatocytes and cholangiocytes 

both in vivo and in vitro, and, when cultured in 

appropriate conditions, into mesenchymal and neuroectodermal 

cell lineages. 

RLSCs represent a versatile cellular model usable in 

studies regarding the mechanisms controlling the 

EMT/MET oscillations in stem cells. These results 

are described in the selected publication Conigliaro 

et al., 2008. 

Current studies are focused to the identification of 

microenviromental factors involved in stem cell maintenance, 

expansion and lineage-specific differentiation. 


Cicchini C, Laudadio I, Citarella F, Corazzari M, 

Steindler C, Conigliaro A, Fantoni F, Amicone L, 

Tripodi T. TGFβ-induced EMT requires Focal 

Adhesion Kinase (FAK) signaling. Exp Cell Res. 2008, 

314:143-52. 

Conigliaro A, Colletti M, Cicchini C, Guerra MT, 

Manfredini R, Zini R, Bordoni V, Siepi F, Leopizzi M, 

Tripodi M, Amicone L. Isolation and characterization 

of a murine liver resident stem cell. Cell Death 

Differ. 2008, 15:123-33. 

Marchetti A, Colletti M, Cozzolino AM, Steindler 

C, Lunadei M, Mancone C, Tripodi M. 

ERK5/MAPK is activated by TGFβ in hepatocytes 

and required for the GSK-3β-mediated Snail protein 

stabilization. Cell Signal 2008, 20:2113-8.


Anna Ferraro, Fabio Altieri, Margherita Eufemi, professors; 

Silvia Chichiarelli, researcher; Caterina Grillo, post-doc fellow; 

Manola Maceroni, Elisa Gaucci, Valentina Arcangeli, 

PhD students. 


ERp57 is a member of the protein disulfide isomerase 

family of proteins, whose roles in the endoplasmic 

reticulum as chaperon and disulfide-reshuffling protein 

are well understood. ERp57 is a stress-responsive 

protein, overexpressed in transformed cells. Its 

presence in other subcellular locations, i.e. the cell 

surface, the cytosol and the nucleus, has been extensively 

documented, and also its ability to interact with 

nuclear proteins and with DNA. However, its functions 

in these extra-ER locations are still obscure. 

Data from different laboratories suggest that ERp57 

participate in the regulation of transcription factors 

activity and in DNA repair. This suggestion is 

strengthened by some findings from our laboratory, 

such as the interaction of ERp57 with specific DNA 

sequences and its association with APE/Ref-1, which 

is a DNA-repair endonuclease and an activator of 

transcription factors, and with the nuclear STAT3, 

which is a transcription factor involved in inflammation, 

cell proliferation, cell survival and oncogenesis. 

The aim of the proposed research is to explore the 

functions of ERp57 in its non-ER locations and to 

verify its role when bound to specific DNA sites, 

which we have begun to identify. If the binding of 

ERp57 to DNA, which is redox-dependent, has a 

truly regulative role, then the protein would appear to 

be an oxidative stress-responsive factor capable of 

acting directly in the nucleus by regulating the 

expression of specific genes. 

We previously described the DNA-binding properties 

of ERp57 and identified its C-terminal region as 

being directly involved in the DNA-binding activity. 

71 


Roles of ERp57 in DNA repair and in the regulation of gene 

expression 

Principal investigator: Carlo Turano 



Tel: (+39) 06 49910598; Fax: (+39) 06 4440062 

carlo.turano@uniroma1.it 

In this research, we investigated in some detail the 

structural features of ERp57 responsible for this 

interaction with DNA. Since the fourth domain of the 

protein (called a’) is the one directly involved in DNA 

binding, we produced a recombinant isolated a’ 

domain and demonstrated that its DNA-binding 

properties are strongly dependent on its redox state. 

Site-directed mutagenesis on the first cysteine 

residue of the -CGHC-thioredoxin-like active site 

leads to a mutant domain (C406S) lacking DNA-binding 

activity. Biochemical studies on the recombinant 

domain revealed a conformational change associated 

with the redox-dependent formation of a homodimer, 

having two disulfide bridges between the cysteine 

residues of two a’ domain active sites. The formation 

of intermolecular disulfide bridges rather than 

intramolecular oxidation of active site cysteines is 

important to generate species with DNA-binding 

properties. Thus, in the absence of any dedicated 

motif within the protein sequence, this structural 

rearrangement might be responsible for the DNAbinding 

properties of the C-terminal domain. 

Moreover, NADH-dependent thioredoxin reductase 

is active on intermolecular disulfides of the a’ 

domain, allowing the control of dimeric protein content 

as well as its DNA-binding activity. A similar 

behavior was also observed for the entire ERp57. 

In order to explore the functions of ERp57, we investigated 

the binding of various ligands to the protein 

and their effects on the interaction with DNA and on 

the enzymatic reductase activity. We found that some 

antibiotics, which had already been tested on PDI, 

bound to ERp57 with an inhibitory action on its 

activities and with an affinity constant two to three 

orders of magnitude higher than that displayed 

towards PDI. Therefore these inhibitors provide a 

mean to differentiate the biological activities of these 

two protein-disulfide-isomerases, which share homologous 

structures and similar sub-cellular locations. 

The DNA-ERp57 interaction was then studied in 

vivo by means of chromatin immunoprecipitation

C. Turano - Roles of ERp57 in DNA repair and in the regulation of gene expression 

(ChIP) on intact HeLa, Raji and M14 cells. Cloning 

and sequencing of the DNA immunoprecipitated 

from ChIP experiments in HeLa cells allowed the 

identification of 37 sites on DNA as targets for 

ERp57, most of which were validated by PCR in all 

three cell types examined. Of these 37 sites, 15 were 

introns of known genes, 5 were on the 5’ or 3’ flanking 

regions of genes, and the others were in intergenic 

regions. The identified genes were involved in 

cellular regulatory processess (such as DNA repair, 

cell adhesion or vescicular traffic) rather that in 

metabolic pathways. In HeLa cells, three gene targets 

were present in DNase hypersensitive sites, 

which are most likely regulatory sites of gene 

expression. Among the genes targeted by ERp57 

are POLD1 (the catalytic subunit of the DNA polymerase 

δ) and MSH6, both involved in DNA repair, 

and LRBA, involved in response to lipopolysaccharide. 

The products of these genes are proteins 

whose functions are compatible with a response to 

cellular stress. Considering that ERp57 is a stress 

response protein itself, it is likely that ERp57 acts in 

part by regulating the expression of proteins 

involved in such response. 

The expression of one of the MSH6 gene was measured 

in conditions of a decreased amount of ERp57. 

This was achieved by RNA interference of ERp57, 

which caused a significant decrease in MSH6 expression 

both in HeLa and M14 cells. 

The intervention of ERp57 in gene expression regulation 

was confirmed by studying some STAT3-dependent 

genes in M14 cells. In five of these genes (A2M, 

72 

CRP, CDKN1A, CDC25A, MMP9), ChIP experiments 

demonstrated that ERp57 was bound directly to DNA 

in proximity of STAT3. The expression of the same 

five genes was clearly decreased upon siRNA-ERp57 

treatment of the cells. Inhibitors of ERp57 appeared 

to hinder the binding in vitro of STAT3 to its consensus 

sequence. Taken on the whole, these data provide 

conclusive evidence of an involvement of nuclear 

ERp57 in transcription regulation. 

At present, we are also investigating the function of 

ERp57 on the cell surface, where it seems to involved 

in the process of hormone- or growth factor-receptor 

interactions, with a possible intervention in internalization 

and/or nuclear import phenomena. 


Chichiarelli S, Ferraro A, Altieri F, Eufemi M, 

Coppari S, Grillo C, Arcangeli V, Turano C. The 

stress protein ERp57/GRP58 binds specific DNA 

sequences in HeLa cells. J Cell Physiol. 2007, 

210:343-51. 

Grillo C, D'Ambrosio C, Consalvi V, Chiaraluce R, 

Scaloni A, Maceroni M, Eufemi M, Altieri F. DNAbinding 

activity of the ERp57 C-terminal domain is 

related to a redox-dependent conformational change. 

J Biol Chem. 2007, 282:10299-310. 

Gaucci E, Chichiarelli S, Grillo C, Vecchio ED, 

Eufemi M, Turano C. The binding of antibiotics to 

ERp57/GRP58. J Antibiot. (Tokyo) 2008, 61:400-2.


Alberto Ciolfi, PhD student; Carmen Maresca, undergraduate 

student. 


UCSF Cancer Center, University of California, San Francisco, USA 

(Prof. Joseph F. Costello); Roswell Park Cancer Institute, 

Buffalo, NY, USA (Prof. Dominic J. Smiraglia). 


The main objective of our project is the identification 

of targets of aberrant DNA methylation in 

acute myeloid leukemia (AML) cell lines and primary 

blasts from AML patients by Restriction Landmark 

Genome Scanning (RLGS). RLGS has been widely 

used to identify imprinted genes, targets of DNA 

amplification, deletion, and gene amplification. When 

the RLGS is executed using methylation sensitive 

enzymes, it represents a genome wide quantitative 

approach that is uniquely suited for simultaneously 

assessing the methylation status of thousands of 

CpG islands within and between samples. The identification 

of a large number of aberrantly methylated 

loci in a variety of tumors via RLGS has been 

instrumental in showing that aberrant CpG island 

methylation patterns are tumor-type specific and non 

random. RLGS separates radiolabeled NotI fragments 

in two dimensions and allows distinction of 

single-copy CpG islands from multicopy CpG-rich 

sequences 2 . The methylation sensitivity of the 

endonuclease activity of NotI provides the basis for 

differential methylation analysis. The reduction in 

spot intensity or the appearance of “novel spots” in 

RLGS profiles indicates events of aberrant methylation 

or demethylation respectively. 

To date, we have produced a “master DNA methylation 

profile” to use as control profile in our experiments and 

comparisons. We obtained this normal “master DNA 

methylation profile” from the integration of methyla- 

73 


Identification of novel genetic and epigenetic targets in Leukemia 

by genome wide approaches 

Principal investigator: Giuseppe Zardo 

Researcher in Clinical Chemistry 


Tel: (+39) 06 80319026; Fax: (+39) 06 80319054 

zardo@bce.uniroma1.it 

tion data from RLGS profiles of purified human bone 

marrow CD34+HSC/HPCs (2 samples) and PB cells 

(2 samples) isolated from consenting healthy donors. 

This profile is composed of 1262 analyzable genomic 

loci. These selected loci are unmethylated and present 

in all analyzed RLGS profiles. 

We were able to assign the chromosomal location 

and corresponding DNA sequence to 757 out of 

1262 genomic loci. The identification of the chromosomal 

location and DNA sequence for the remaining 

loci will be performed upon the recognition of these 

loci as targets of aberrant DNA methylation events. 

Next, with the aim to evaluate targets of aberrant 

DNA methylation in human AML cell lines we have 

acquired the DNA methylation profiles of several 

human leukemia cell lines that differ for their FAB 

classification, block of differentiation, presence of 

oncogenic fusion proteins with epigenetic activity 

and sensitivity to the differentiating effect of retinoic 

acid. In detail, we have acquired RLGS DNA methylation 

profiles of the following AML cell lines: 

-HL-60, FAB M2, control and treated with 1 µM 

retinoic acid for 24/48/72/96 hours 

-Kasumi-1, FAB M2, AML1/ETO+ 

-SKNO-1, FAB M2, AML1/ETO+ 

-NB-4, FAB M3, PML/RAR+, control and treated 

with 1 µM retinoic acid for 72 hours 

-ML-2, FAB M4 

-ME-1, FAB M4eo 

-MV4-11, FAB M5 

-AML-193, FAB M5 

-HEL, FAB M6 

Still, in order to complete our RLGS experiments we 

need to obtain the RLGS DNA methylation profiles 

from MOLM-16, FAB M0 and UT-7, FAB M7. To 

date, a stringent comparison and cross-comparison has 

been carried out between the DNA methylation profiles 

of HL-60 FAB M2, NB-4 FAB M3 PML/RAR+ 

control and treated with retinoic acid for 72 hours and 

the normal “master DNA methylation profile”.

G. Zardo - Identification of novel genetic and epigenetic targets in Leukemia by genome wide approaches 

These comparisons have provided the following 

results: 

i) In HL-60, FAB M2, 249 out of 1188 (20.9%) analyzable 

loci were found to be hypermethylated when 

compared to the normal “master DNA methylation 

profile”. For 149 out of 249 (59.8%) hypermethylated 

loci, it was possible to assign the chromosomal 

localization and corresponding DNA sequence. 

ii) In NB-4 FAB M3 PML/RAR+, 386 out of 1188 

(32.5%) analyzable loci had a hypermethylated status 

when compared to the normal “master DNA methylation 

profile”. For 220 out of 386 (57%) hypermethylated 

loci, it was possible to assign the chromosomal 

localization and corresponding DNA sequence. 

iii) The cross-comparison between the DNA methylation 

profiles of HL-60 FAB M2 and NB-4 FAB M3 

PML/RAR+ showed common and diverse targets of 

hypermethylation. 183 hypermetylated genomic loci 

out of 1188 total loci (15.4%) were shared by the 

two different cell lines. 63 out of 249 (25.3%) loci 

were hypermethylated in HL-60 but not in the NB-4 

cell line. 196 out of 386 (50.8%) loci were hypermethylated 

in NB-4 but not in the HL-60 cell line. 

iv) Retinoic acid in combination with chemotherapy 

is the frontline treatment for the therapy of acute 

promyelocitic leukemia (APL). APL is characterized 

by the t(15;17) that generates the oncogenic fusion 

protein PML/RAR that holds epigenetic activity. 

74 

NB-4 is a human PML/RAR+ cell line considered to 

be an in vitro model for the study of APL. With the 

aim to identify targets of aberrant DNA methylation 

that might contribute to the onset of APL in 

humans, we treated the NB-4 cell line with retinoic 

acid to determine if the differentiation process 

induced by this drug might be associated to the 

demethylation of specific loci aberrantly methylated. 

We compared the RLGS DNA methylation profiles 

of control NB-4 and NB-4 cells treated for 72 hours 

with 1µM retinoic acid. In NB-4, PML/RAR+ cells, 

retinoic acid induced the demethylation of 10 

genomic loci (0.84%) out of 1188 analyzed. The 

chromosomal localization and DNA sequence was 

assigned to 5 out of 10 loci. The remaining 5 loci 

will be identified using the “in silico” procedure 

described in the main proposal. 

In conclusion, our data show that a portion of DNA 

methylation targets are shared by leukemic cells of 

different FAB and carrying fusion proteins, but 

above all, show specific DNA methylation signatures 

depending on the stage of differentiation block and 

presence of fusion proteins. Moreover, our data 

prove that the in vitro treatment with retinoic acid of 

NB-4 PML/RAR+ cells leads to the demethylation 

of aberrantly methylated genomic loci that might be 

relevant for the induction of the differentiation 

process and to prevail over the differentiation block.

AREA4 

Molecular 

recognition 

in 

biomolecules


Carlo Travaglini-Allocatelli, professor; Stefano Gianni, CNR 

researcher; Ylva Ivarsson, post-doc fellow; Nicoletta Calosci, 

PhD student. 


University of Uppsala, Sweden (Prof. Per Jemth); University of 

Cambridge, UK (Prof. Michele Vendruscolo). 


The main goal of the present project is to understand 

if and how the topological properties and the 

sequence connectivity between different elements of 

secondary structure affect the folding pathway and 

the molecular recognition process mediated by proteins. 

The model system employed is the PDZ 

domain, small globular protein of 90 - 100 a.a. 

residues, involved in a variety of cellular processes, 

from the organization of macromolecular complexes 

to the regulation of signalling cascades. We plan to 

take advantage of topological mutations (both engineered 

or naturally evolved circular permutants) to 

test the role of sequence connectivity between different 

elements of secondary structure in the 

(de)stabilization of metastable species, such as intermediate 

and transition states. The task is to unveil 

the molecular events involved in the folding and the 

binding reactions of PDZ domains, and the correlation 

between the two processes. Furthermore, the 

results are compared with the folding and binding 

reaction of recently identified naturally evolved circularly 

permutated variants (i.e. PDZ domains from 

bacteria and plants). The folding pathway of each 

protein is studied by employing an array of experimental 

methods, including protein engineering, 

innovative ultra-rapid mixing instruments in combination 

with stopped-flow equipment, and molecular 

dynamics simulations. Particular attention is devoted 

to the identification and characterization of mis-fold- 

77 

Molecular recognition in biomolecules - AREA 4 

How proteins recognize their biochemical partners: ligand binding 

and folding pathways of PDZ domains 

Principal investigator: Maurizio Brunori 

Professor of Chemistry and Biochemistry 


Tel: (+39) 06 49910544; Fax: (+39) 06 4440062 

maurizio.brunori@uniroma1.it 

ed states, such as off-pathway intermediates. 

Molecular dynamics simulations allow to model the 

structure of such mis-folded states, in an attempt to 

obtain structural information on species which are 

prone to aggregation, leading to the formation of 

amyloids, often implicated in neurodegenerative 

human diseases. 

The process of intermolecular recognition involving 

PDZ domains and their target proteins may be investigated 

to determine the mechanism of the complex 

formation and the regions of the PDZ domains 

involved in the control, over-and-above the binding 

pocket. By employing this overall strategy we aim at 

the identification of both the residues crucial in the 

protein folding process and those that occupy functionally 

important positions in molecular recognition 

events. In addition, the role of internal protein 

dynamics in controlling the folding mechanism and 

the specific recognition of protein targets of biological 

relevance is investigated. 

Results and Perspectives 

The energy landscape theory provides a general 

framework for describing protein folding reactions. 

However, since a large number of studies have 

focused on two-state proteins with single welldefined 

folding pathways and without detectable 

intermediates, the extent to which free energy landscapes 

are shaped up by the native topology at the 

early stages of the folding process has not been fully 

characterized experimentally. 

To obtain a glimpse of the width of the free energy 

landscape at the early stages of the folding, we compared 

the folding pathways of two homologous 

three-state proteins. The study of homologous proteins 

represents a powerful approach to obtain 

insight into the process of protein folding, especially 

when combined with structural information on intermediate 

events. Here we present an original illustration 

of the width of the upper regions of a free energy 

funnel by comparing the early and late transition

M. Brunori - How proteins recognize their biochemical partners: ligand binding and folding pathways of PDZ domains 

states of two homologous three-state proteins, PSD- 

95 PDZ3 and PTP-BL PDZ2 (33% sequence identity). 

The analysis was carried out by Φ-value analysis, 

a powerful experimental method to obtain structural 

information about transition states. In this technique 

residue-specific structural information is inferred by 

comparing the kinetics of folding of the wild-type 

protein with a series of conservative single mutants. 

The structural information provided by the measurement 

of Φ-values was used to build models of the 

transition state structures. 

Our results demonstrate that for the PDZ proteins 

the late folding transition states (TS2) are more 

similar to each other than the early transition 

states (TS1) (Fig. 1). Thus, taking two snapshots 

along each folding pathway illustrates the presence 

of a weak native bias at the early stages of the 

process, which results in alternative folding events. 

78 

By contrast approaching the native conformation 

the folding pathway is essentially dictated by the 

native topology. This result, which reflects the funnelling 

of the free energy landscape toward the 

native state, opens the way to new results on the 

structure of transition states and intermediates in 

the circular permutants, and to the definition of 

the role of topology in determining on-pathway or 

off-pathway misfolded intermediates. 


Calosci N, Chi CN, Richter B, Camilloni C, 

Engström A, Eklund L, Travaglini-Allocatelli C, 

Gianni S, Vendruscolo M, Jemth P. Comparison of 

successive transition states for folding reveals alternative 

early folding pathways of two homologous 

proteins. Proc Natl Acad Sci USA 2008, 105:19240-5. 

Fig. 1 - Representative structures of early (TS1) and late (TS2) transition states in the folding of two PDZ domains as obtained by - value 

analysis and restrained molecular dynamics simulations. 

(left) A representative structure of TS1 of PSD-95 PDZ3. (right) Superposition of representative structures of TS2 of PSD-95 PDZ3 and PTP-BL 

PDZ2. The TS1 structures of the two proteins were too different to be superimposed.


Giulia De Lorenzo, Daniela Bellincampi, professors; 

Simone Ferrari, Benedetta Mattei, researchers; 

Manuela Casasoli, Francesca Sicilia, Roberta Galletti, 

Vincenzo Lionetti, post-doc fellows; Francesco Spinelli, 

Fedra Francocci, Lorenzo Mariotti, Manuel Benedetti, 

Daniel Savatin, PhD students; Lucia Tufano, graduate student; 

Giovanni Salvi, Daniela Pontiggia, technicians. 


Plants are continually exposed to pathogens and, in 

most cases, successfully defend themselves. Knowledge 

of the mechanisms underlying their defense ability 

paves the way to improvement of crop resistance. A 

sophisticated surveillance system detecting the presence 

of pathogens is interconnected with the plant 

defense signalling pathways that lead to the activation 

of defense responses. The cell wall plays an instrumental 

role in the plant surveillance system by releasing 

oligosaccharide fragments (oligolacturonides=OGs) 

that trigger the typical responses elicited 

by PAMPs (Pathogen Associated Molecular Patterns). 

The formation of OGs takes place during the degradation 

of homogalacturonan by microbial polygalacturonases 

(PGs) and is favoured when PGs interact with 

specific plant cell wall proteins (polygalacturonaseinhibiting 

proteins or PGIPs). Micromolar concentrations 

of OGs activate the expression of defense genes 

via a signal transduction pathway that functions independently 

of the known pathways involving salicylic 

acid (SA), jasmonate (JA), and ethylene (Et). The characterization 

of this novel signalling pathway is a key 

feature of our project. Specific tasks of the project are 

i) the dissection of the OG signalling pathway and ii) 

the development of resistant plants. 

Dissection of the OG signalling pathway 

OGs induce a variety of plant defense responses and 

increase resistance to the necrotrophic fungal pathogen 

Principal investigator: Felice Cervone 

Professor of Plant Biology 

Dipartimento di Biologia Vegetale 

Tel: (+39) 06 49912517; Fax: (+39) 06 49912446 

felice.cervone@uniroma1.it 

79 


Plant innate immunity: cell wall-mediated signalling and 

recognition in plant defense 

Botrytis cinerea independently of JA-, SA-, and ETmediated 

signalling. Microarray analysis showed that 

about 50% of the genes regulated by OGs, including 

genes encoding enzymes involved in secondary metabolism 

have a similar change of expression during B. 

cinerea infection. In particular, expression of PHY- 

TOALEXIN DEFICIENT3 (PAD3) is strongly upregulated 

by both OGs and infection independently of 

SA, JA, and ET. OG treatments do not enhance resistance 

to B. cinerea in the pad3 mutant. Similarly to OGs, 

the bacterial flagellin peptide elicitor flg22 enhances 

resistance to B. cinerea in a PAD3-dependent manner, 

independently of SA, JA, and ET. Moreover a rapid 

induction of gene expression by OGs is also independent 

of salicylic acid, ethylene, and jasmonate. OGs 

elicit a robust extracellular oxidative burst that is generated 

by the NADPH oxidase AtrbohD. However, the 

burst is not required for the expression of OG-responsive 

genes or for OG-induced resistance to B. cinerea, 

whereas callose accumulation requires a functional 

AtrbohD. OG-induced resistance to B. cinerea is also 

unaffected in powdery mildew resistant4, despite the fact 

that callose accumulation is almost abolished in this 

mutant. These results indicate that the OG-induced 

oxidative burst is not required for the activation of 

defense responses effective against B. cinerea, leaving 

open the question of the role of reactive oxygen 

species in elicitor-mediated defense. 

Development of resistant plants 

PGs, enzymes that hydrolyze the homogalacturonan 

of the plant cell wall, are virulence factors of several 

phytopathogenic fungi and bacteria. On the other 

hand, PGs may activate defense responses by releasing 

OGs perceived by the plant cell. Tobacco 

(Nicotiana tabacum) and Arabidopsis (Arabidopsis 

thaliana) plants expressing a fungal PG (PG plants) 

have a reduced content of homogalacturonan, are 

more resistant to microbial pathogens and have constitutively 

activated defense responses. Interestingly, 

either in tobacco PG or wild-type plants treated with

F. Cervone - Plant innate immunity: cell wall-mediated signalling and recognition in plant defense 

OGs, resistance to fungal infection is suppressed by 

exogenous auxin, whereas sensitivity to auxin of PG 

plants is reduced in different bioassays. The altered 

plant defense responses and auxin sensitivity in PG 

plants may reflect an increased accumulation of OGs 

and subsequent antagonism of auxin action. 

Alternatively, it may be a consequence of perturbations 

of cellular physiology and elevated defense status 

as a result of altered cell wall architecture. 

A way of altering pectin susceptibility to hydrolysis 

by microbial endopolygalacturonases is the constitutive 

expression of specific protein inhibitors of 

pectin methylesterases (the genes AtPMEI-1 and 

AtPMEI-2 of Arabidopsis thaliana). The overexpression 

of the inhibitors decreases PME activity and 

increases the degree of pectin methylesterification by 

about 16%. Transformed plants show a slight but significant 

increase in root length and are more resistant 

to Botrytis cinerea. The reduced symptoms caused by 

the fungus on transgenic plants are related to its 

impaired ability to grow on methylesterified pectins. 

80 


Ferrari S, Galletti R, Denoux C, De Lorenzo G, 

Ausubel FM, Dewdney J. Resistance to Botrytis 

cinerea induced in Arabidopsis by elicitors is independent 

of salicylic acid, ethylene, or jasmonate signaling 

but requires PHYTOALEXIN DEFI- 

CIENT3. Plant Physiology 2007, 144:367-79. 

Lionetti V, Raiola A, Camardella L, Giovane A, Obel 

N, Pauly M, Favaron F, Cervone F, Bellincampi D. 

Overexpression of pectin methylesterase inhibitors 

in Arabidopsis restricts fungal infection by Botrytis 

cinerea. . Plant Physiology 2007, 143:1871-80. 

Ferrari S, Galletti R, Pontiggia D, Manfredini C, 

Lionetti V, Bellincampi D, Cervone F, De Lorenzo G. 

Transgenic expression of a fungal endo-polygalacturonase 

increases plant resistance to pathogens and 

reduces auxin sensitivity. Plant Physiology 2008, 

146:669-81.


Giovanna Costanzo, researcher; Fabiana Ciciriello, post-doc 

fellow; Samanta Pino, PhD student; Silvia Lopizzo, collaborator. 


Dipartimento di Agrobiologia e Agrochimica, Università della 

Tuscia, Viterbo (Prof. Raffaele Saladino); Università di Roma 

Tor Vergata, (Dr. Claudia Crestini); INAF, Osservatorio Astrofisico 

di Arcetri, Firenze (Prof. John R. Brucato). 


The general goal of our research is 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 human genetic system in terrean and nonterrean 

environments. 

In this frame of reference we are currently analyzing: 

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 is being carefully 

analyzed. Previous analyses have determined 

the syntheses from formamide and the catalysts: simple 

metal oxides, olivines, titanium oxides, clays, 

phosphate minerals, basalts, sulphur-containing minerals. 

The analyses being currently performed pertain 

to a series of zirconium-based minerals and a 

large panel of boron-containing compounds. The 

rationale for the first type of analyses is that zirconia 

formed only in the presence of water. Given that zirconia 

are the oldest minerals identified on Earth, the 

syntheses of nucleic acids occurring in their presence 

would be highly instructive on the relationship 

“catalysts: water: nucleic bases”. The protective func- 

Principal investigator: Ernesto Di Mauro 



Tel: (+39) 06 49912880; Fax: (+39) 06 4440812 

ernesto.dimauro@uniroma1.it 

81 


Spontaneous formation and evolution of informational 

nucleic polymers 

tion of borates of various kinds towards nucleosides 

has been recently identified. Hence the interest of 

the exploration of their synthetic capacity. The overall 

analysis will provide a complete picture for the 

syntheses of nucleic bases in the presence of the 

mineral compounds which are major constituents of 

the solar system. 

The syntheses yielding acyclonucleosides are being 

analyzed in mixed systems consisting of: [formamide 

+ formaldehyde] as building material, and mixtures 

of minerals as catalyst component. The reactions 

are being performed in terrestrial conditions and will 

be compared to similar reactions performed in spacewise 

conditions. 

Polymerizations and stability 

Next step towards complexity is the activation by 

phosphorylation of abiotically synthesized nucleosides. 

This problem has been solved by our group, as 

reported (Costanzo et al., J Biol Chem. 2007, 

282:16729-35). 

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 

(Saladino et al., J Biol Chem. 2005, 280:35658-69; 

Saladino et al., J Biol Chem. 2006, 281:5790-6). A 

recent set of analyses has determined the properties 

of RNA molecules that allow the possibility of evolution 

in abiotic environments and conditions. The 

results are in (Ciciriello et al., Biochemistry 2008, 

47:2732-42). Starting from these findings, we have 

focused on the determination of the resilience properties 

of nucleic polymers in terrestrial and spacewise 

conditions. Namely, experiments are being conducted 

to answer the questions: 

- How does the structure of nucleic acids affect their 

resistance to the hydrolytic process? 

- Are cleavages in nucleic polymers being repaired 

differently in low-gravity conditions?

E. Di Mauro - Spontaneous formation and evolution of informational nucleic polymers 

- Do these differential 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: 

i) from 2’-3’ cyclic and 3’-5’ cyclic nucleotides: 

oligomerizations up to 9-mers; 

ii) from 15-nucleotides-long oligomers: 3’-5’ phosphodiester 

bonds reshuffling and oligomers formation, 

resulting in chain elongation up to 25 mers; 

iii) form 15-nucleotides-long oligomers: multimerization 

to dimers, trimers and higher forms. 

These results provide the proof-of-principle that abiotic 

polymerization are obtained spontaneously. In 

addition, a set of conditions are established which 

may provide the test systems for the analysis of stability 

of genetic information in acellular, space-wise 

conditions. 

A large panel of new catalysts was tested in synthetic 

reactions from formamide: basalts, sulphurcontaining 

minerals, zircons. The results obtained 

are instrumental to the formulation of a chemical 

theory of the origin of informational polymers and 

for the search of life in non-terrean environments. 

Syntheses have been performed in the presence of 

FeS, Pyrrhotine, Fe (1-x) S, FeS 2 , Pyrite FeS 2 , 

Chalcopyrite FeCuS 2 , Bornite, FeCu 5 S 4 , 

Tetrahedrite, (Fe,Cu,Sb)S, Covellite, CuS, 

82 

Covellite:pyrite (2:1), Covellite:pyrite (3:1), 

Covellite:pyrite (4:1), starting from the substrate formamide. 

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 are in 

preparation. 


Ciciriello F, Costanzo G, Pino S, Crestini C, 

Saladino R, Di Mauro E. Molecular complexity 

favors the evolution of ribopolymers. Biochemistry 

2008, 47:2732-42. 

Pino S, Ciciriello F, Costanzo G, Di Mauro E. 

Nonenzymatic RNA ligation in water. J Biol Chem. 

2008, 283:36494-503. 

Saladino R, Neri V, Crestini C, Costanzo G, 

Graciotti M, Di Mauro E. Synthesis and degradation 

of nucleic acid components by formamide and iron 

sulphur minerals. J Am Chem Soc. 2008, 130:15512-8.


Francesco Bossa, professor; Roberto Contestabile, 

Sebastiana Angelaccio, researchers; Rita Florio, Mirella 

Vivoli, PhD students. 


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). 


Pyridoxal 5'-phosphate (PLP) was first identified in 

the mid forties as the cofactor required for the biochemical 

reaction of transamination. Since then, 

PLP-dependent enzymes have been the subject of 

extensive research. The interest aroused by these 

enzymes comes from their unequalled catalytic versatility 

and widespread involvement in cellular 

processes. Nowadays, more than 140 different 

enzyme activities based on PLP are classified by the 

Enzyme Commission and represent 4% of all known 

catalytic activities. PLP-dependent enzymes are not 

only involved in the synthesis, interconversion and 

degradation of amino acids (among which neurotransmitters) 

but also play key roles in the metabolism 

of one-carbon units, biogenic amines, 

tetrapyrrolic compounds, amino-sugars, modulation 

of steroid receptor-mediated gene expression and 

regulation of immune function. While bacteria, 

plants and fungi synthesize PLP, animals obtain this 

cofactor from B 6 vitamers acquired from the diet 

and recycled in a salvage pathway, in which two 

enzymes are essentially involved: pyridoxal kinase 

(PLK) and pyridoxamine phosphate oxidase 

(PNPOx). Once made available, PLP is somehow 

targeted to the dozens different apo-B 6 enzymes 

that are being synthesized in the cell. Crucial and 

poorly answered questions are how PLP availability 

83 


Synthesis of pyridoxal phosphate in the vitamin B 6 salvage 

pathway and targeting of the cofactor to apo-enzymes 

Principal investigator: Martino Luigi di Salvo 

Researcher in Biochemistry 


Tel: (+39) 06 49917684; Fax: (+39) 06 49917566 

martino.disalvo@uniroma1.it 

is regulated and how the cofactor reacts with the 

apo-enzymes while these are folding. Dietary deficiency 

of vitamin B 6 or inborn defects in the 

enzymes of the salvage pathway result in severe 

neurological dysfunctions. The study of inborn 

errors affecting PLP availability may provide a 

unique opportunity for studying the role of PLP as 

a regulator of enzyme activities in man. Autosomal 

recessive mutations in the gene encoding PNPOx 

cause neonatal epileptic encephalopathy (NEE). 

Individuals affected by NEE were found to be 

homozygote for missense, splice site and stop codon 

mutations. One of these mutations is R229, the last 

amino acid in a short sequence which is completely 

conserved in all known species. Structural studies on 

recombinant hPNPOx have shown that R229 might 

be involved in the tight binding of cofactor FMN to 

the active site of the enzyme, by direct interaction 

with the phosphate group of FMN. Substitution of 

Arg with Trp is predicted to disrupt this interaction 

and that of other amino acids in the vicinity. We 

have expressed, purified, functionally and structurally 

characterized human PNPOx R229W and 

R229Q mutants. Dissociation constants, kinetic and 

crystal structure analyses show that the enzyme is 

still able to bind the cofactor, but its catalytic activity 

is greately impaired mostly because of its low 

capability of binding the substrate. 

Another important aspect of B 6 related metabolic 

dysfunctions is that some patients with inborn errors 

affecting PLP-dependent enzymes are responsive to 

pyridoxine treatment, suggesting that the mutations 

may impair the binding of the cofactor. Moreover, 

PLP may play a role in assisting protein folding and 

stability. Although much work has been done on the 

mechanism and structure of B 6 enzymes, little is 

known on how the apo-forms are converted to the 

holo-forms while they are folding. According to some 

authors, PLP may be transferred by PNPOx directly 

to the apo-enzymes which require it as cofactor. 

Alternatively, PLP may react with the enzymes that

M. L. di Salvo - Synthesis of pyridoxal phosphate in the vitamin B 6 salvage pathway 

use it as cofactor in the form of a complex with the 

specific substrates. We have recently carried out 

experiments with both human and E. coli PNPOx and 

shown that the human enzyme is more efficient in 

transferring PLP to human PLP-dependent enzymes, 

whereas E. coli PNPOx transfers PLP better to bacterial 

enzymes. This results strongly suggest an 

enzyme-enzyme recognition step, in which PLP may 

be transferred directly to the apo-enzymes through a 

channelling mechanism. The encounter between PLP 

and apo-proteins in the cell may take place at different 

stages of the polypeptide folding process and 

affect it to a variable extent. Apparently, the role of 

PLP in folding and structural stabilization varies 

among the enzymes which use it as cofactor. This is 

not surprising, considering that there are at least five 

evolutionary unrelated families of such enzymes, corresponding 

to completely different protein folds. Our 

research group has focused attention on serine 

hydroxymethyltransferase (SHMT), which may be 

taken as a model for the whole fold type I family. The 

folding mechanism of E. coli SHMT has been investigated 

in detail and is understood better than that of 

any other fold-type I enzyme. Our studies on the 

mechanism of addition of PLP to folding apo-SHMT 

involve several aspects: the role of conserved 

hydrophobic clusters in the late phase of folding and 

PLP binding; the structural requisites involved in the 

recognition process between PLP and apo-SHMT; 

the detailed studies on the structural changes taking 

place in folding and PLP binding; and the characterisation 

of human S394N and L474F SHMT mutants 

84 

with respect to the kinetics of PLP addition. These 

two single nucleotide polymorphisms of the gene 

encoding cytosolic SHMT, may be associated with 

several disease states, such as neural tube defects, cardio 

vascular diseases and pregnancy complications, 

connected to hyperhomocysteinemia. 

Our research project also involves human PLK, the 

other enzyme involved in the vitamin B 6 salvage 

pathway. Recently, a direct inhibitory effect of several 

drugs on PLK has been shown, which results in 

vitamin B 6 deficiency and various side effects related 

to the central nervous system. Well known examples 

are theophylline, used to treat asthma, and progabide 

to treat epilepsy. A number of polymorphisms were 

also discovered in the PLK coding gene. Our work 

has focused on the delucidation of the mechanism of 

action of this enzyme, with particular interest on the 

residue D235 which was shown to play a major role 

in the deprotonation step of the substrate hydroxyl 

group prior to nucleophilic attack the γ-phosphate 

group of ATP. We are currently carrying out structural 

and functional requirement studies on the 

interaction between PLK and the several drugs that 

modutale its activity. 


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., in press.


Bruno Botta, Claudio Villani, professors; Ilaria 

D’Acquarica, Marco Pierini, researchers; Alessia Ciogli, 

Deborah Subissati, post-doc fellows; Laura Nevola, 

PhD student; Giovanna Cancelliere, technician. 


One of the greatest current challenges in modern 

bioanalysis is to gather some insights into the understanding 

of biochemical regulation and communication 

processes, most of which are based on non-covalent 

interactions between proteins and a variety of 

binding partners. Proteins and other macromolecules 

can associate with one another, and they can bind to 

low-molecular-weight ligands. Such interactions are 

fundamental for signalling, enzymatic activity regulation, 

immune response processes, and many other 

functions in living systems. In addition, these binding 

events play a key role in drug action mechanisms. 

There are numerous techniques currently available to 

detect the presence of binding interactions and for 

the determination of dissociation constants. For 

example, affinity chromatography involves the immobilization 

of either a protein target or a ligand on a 

column, such that binding partners are diversely 

retained. In the present project, the rational design of 

systems featuring molecular recognition abilities was 

focused on the surface linking of suitable proteins or 

ligands on solid supports. These materials are the 

cores of high performance liquid chromatography 

(HPLC), one of the most used techniques for the 

rapid and effective analysis of complex mixtures of 

biomolecules (such as proteins, polynucleotides, and 

polysaccharides) in biofluids, and for the isolation of 

suitable targets at preparative scale. A relatively 

recent advance in chromatographic devices is the 

development of monolithic supports based on either 

silica or polymer skeletons. These self-supporting 

columns do not require frits, have a continuous 

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 

85 


Molecular and enantioselective recognition by receptors 

and proteins studied in the gas phase, in free solution 

and at solid-liquid interfaces 

bimodal porosity that leads to high-plate numbers, 

and have high through-pore volumes, which provide 

low back-pressure and hence increased flow-rates relative 

to bead columns. Together, these factors lead to 

shorter separation times and more efficient separations. 

In this context, we prepared new polymerbased 

monolithic HPLC columns by means of gamma 

radiation-induced polymerization of monomers 

directly inside the final vessels (columns). For this 

purpose, a mixture of monomers and cross-linkers 

was irradiated in the presence of suitable solvents, 

directly inside the chromatographic column whose 

internal walls have adequately been pre-activated and 

functionalized. Activation was achieved by pre-treatment 

with a silane containing methacryloyl groups 

(the “grafting to the wall” process), or by introducing 

azo-groups onto the internal walls of the tubular 

support (“grafting from the wall” approach). These 

fragments are able to generate radical species that 

trigger the polymerization from the wall of the tubular 

support inwards, creating an “active” functionalization 

of the internal surfaces. 

In the way to find a new class of synthetic receptors 

endowed with the ability of recognizing complex 

frames such as dipeptide chains, we attached four dipeptide 

loops differing for composition and stereochemistry 

to the feet of cone resorc[4]arene octamethyl 

ethers through their terminal amino groups. The 

resulting N-linked peptidoresorc[4]arenes were shown 

to recognize the homologue dipeptides by means of 

hydrogen bonds, both in solution and in the gas phase. 

The complexes appeared to be quite stable, since hosts 

and guests could not be separated by column chromatography. 

Complexation phenomena were investigated 

by NMR methods, including DOSY experiments, 

for the detection of translational diffusion. 

Heteroassociation constants of 2030 and 186 M -1 were 

obtained by the Foster-Fyfe method for one homochiral 

complex and the corresponding heterochiral, respectively, 

the latter being comparable to the self-association 

constant of the dipeptide itself.

F. Gasparrini - Molecular and enantioselective recognition by receptors and proteins studied in the gas phase 

With regard to the studies of biorecognition events 

monitored by mass spectrometry, we investigated 

the nature of the non-covalent interactions 

between amphetamine enantiomers and some chiral 

resorc[4]arene receptors in the gas phase, under 

conditions mimicking the extensive desolvation 

complementary to the uptake of amphetamine 

inside its biological receptor. Since the mechanisms 

of action of amphetamine proceed through initial 

interaction with the N-terminal amino acids of 

human dopamine transporter (or their phosphorylated 

forms), we decided to focus our attention on 

chiral macrocyclic hosts, containing either flexible 

amino acidic or rigid bis(diamido)-bridged pendants. 

The kinetic and dynamic study performed 

represents a starting point for a deeper comprehension 

of the different affinities of D- and L-amphetamine 

towards chiral hosts, their selective binding to 

the monoamine transporters and their sensitivity to 

inorganic ions. 

86 


Botta B, D’Acquarica I, Delle Monache G, Subissati 

D, Uccello-Barretta G, Mastrini M, Nazzi S, 

Speranza M. Synthesis and host-guest studies of chiral 

N-linked peptidoresorc[4]arenes. J Org Chem. 

2007, 72:9283-90. 

Gasparrini F, Ciogli A, D’Acquarica I, Misiti D, 

Badaloni E, Giorgi F, Vigevani A. Synthesis and 

characterization of novel internal surface reversedphase 

silica supports for high-performance liquid 

chromatography. J Chromatogr A 2007, 1176:79-88. 

Botta B, Tafi A, Caporuscio F, Botta M, Nevola L, 

D’Acquarica I, Fraschetti C, Speranza M. Modelling 

amphetamine/receptor interactions: a gas-phase 

study of the complexes formed between amphetamine 

and some chiral amido[4]resorcinarenes. Chem 

Eur J. 2008, 14:3585-95.


Sergio Fucile, Francesca Grassi, Eleonora Palma, Davide 

Ragozzino, professors; Flavia Trettel, researcher; Myriam 

Catalano, Clotilde Lauro, post-doc fellows; Raffaela 

Cipriani, Fabrizia Sobrero, PhD students; Giuseppina 

Chece, technician. 


Istituto Mario Negri, Milano (Dr. Pietro Ghezzi, 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 Roma Tor Vergata (Dr. Angelo Spinedi). 


Chemokines and their receptors play important roles 

in the CNS both under physiological and pathological 

conditions. The main aim of our research project 

is to investigate the physiopathological activities of 

chemokines as modulators of neuronal and glial 

functions, identifying the molecular pathways 

involved. In the last two years, we have been mainly 

involved in the study of the neuromodulatory activity 

of two chemokines: fractalkine/CX3CL1 and 

IL8/CXCL8. 

Neuroprotective activity of CX3CL1 

The activities of CX3CL1 as neuroprotective substance 

has been deeply investigated in vitro in neurotoxicity 

models. Several evidences indicate that 

chemokines have both neurotrophic and neuroprotective 

activities and their production could also represent 

a defensive response toward toxic insults: the 

trophic and neuroprotective action of chemokines 

has been demonstrated in different neuronal systems, 

like hippocampal (Meucci et al., 1998; Araujo and 

Cotman, 1993), cortical (Bruno et al., 2000) and cerebellar 

granule cultures (Limatola et al., 2000a), as 

well as in oligodendrocytes precursors (Robinson et 

Principal investigator: Cristina Limatola 

Professor of Physiology 

Dipartimento di Fisiologia e Farmacologia 

Tel: (+39) 06 49690243; Fax: (+39) 06 49910851 

cristina.limatola@uniroma1.it 

87 


Molecular and functional approaches to investigate the 

physiopathological role of the chemokines and their receptors 

in the central nervous system 

al., 1998). The chemokine RANTES specifically protects 

cortical neurons from Aβ-induced toxicity 

(Bruno et al., 2000), while IL8/CXCL8 protects cerebellar 

granule neurons from apoptosis (Limatola et 

al., 2000). CX3CL1 is a unique chemokine belonging 

to the CX 3 C subfamily, that is present both as soluble 

and membrane anchored forms (Baggiolini et al., 

1998). Recent evidence indicates that this chemokine 

mediates microglial-neuron communications; in particular, 

neuron-derived fractalkine regulates 

microglial activation, affecting microglial survival 

(Streit et al., 2001). 

We have recently reported that CX 3 CL1 strongly 

reduces neuronal hippocampal cell death induced by 

glutamate (excitotoxicity), and that its neuroprotective 

effects are maintained even if the chemokine is 

administrated up to 8 hours after the excitotoxic 

insult (Limatola et al., 2005). Since there is a large 

debate in literature concerning the kind of cells that 

express the specific CX 3 CL1 receptor CX3CR1, we 

were interested in understanding if CX3CR1 activation 

responsible of the neuroprotective effect was of 

neuronal or glial origin. At this aim we used mice 

engineerized to express the fluorescent protein GFP 

in place and under the promoter of the CX3CR1 

gene (CX3CR1 GFP/GFP mice) and used hippocampal 

neurons obtained from these mice in experiments 

of excitotoxic death to analyse the neuroprotective 

properties of the medium conditioned by CX3CL1stimulated 

wild type microglial cells. In these experiments 

we demonstrated that CX3CL1 induce the 

release of neurotrophic factor(s) from microglial 

cells and identified adenosine as a putative neurotrophic 

factor, by HPLC analysis (Lauro et al., 

2007). Since adenosine can activate four different 

GPCRs, using subtype-specific receptor antagonists, 

we identified adenosine receptor type 1 (AR1) as the 

one involved in CX3CL1-mediated neuroprotection. 

The involvement of AR1 has been also confirmed 

using mice that do not express AR1 (AR1-/-, kindly 

provided by Dr. Bertil Fredholm, Stockolm). We are

C. Limatola - Molecular and functional approaches to investigate the physiopathological role of the chemokines 

now extending these results trying to define how 

general is the importance of AR1 in the neuroprotective 

mechanisms induced by different neurotrophins 

like brain-derived neurotrophic factor, 

erythropoietin, interleukin-6. 

Neuromodulatory activity of CXCL8 

Among the many chemokines and chemokine receptors 

discovered in the last few years, the chemokine 

CXCL8 and its receptor CXCR2 are particularly 

interesting, being expressed in the brain both on 

neurons and glial cells (Tran and Miller, 2003) and 

playing neuromodulatory roles in synaptic transmission 

(Meucci et al., 1998; Ragozzino et al., 1998; 

Giovannelli et al., 1998; Xiong et al., 2003; Lax et al., 

2002; Puma et al., 2001), important roles during 

oligodendrocyte development, proliferation and 

recruitment to MS lesions (Robinson et al., 1998; 

Omari et al., 2006) and protective activities towards 

neurons and astrocytes (Limatola et al., 2000; Saas et 

al., 2002; Wang et al., 2007; Watson and Fan, 2005). 

We have recently demonstrated that CXCR2 expression 

and activation mediates the modulation of 

AMPA-type glutamate receptors (GluR) properties, 

changing AMPAR affinity, binding site cooperativity, 

and channel opening probability (Lax et al., 2002). 

Furthermore, the mutual interaction between GluR1 

and CXCR2 determines the signaling properties of 

CXCR2, possibly due to alterations in the ability of 

CXCR2 to holigomerize (Trettel et al., 2003). 

AMPA receptors are responsible for the majority of 

excitatory synaptic transmission in the brain. It is 

well established that protein phosphorylation regulates 

ion channel functional properties of the 

tetrameric AMPARs (Song and Huganir, 2002). 

There are at least ten phosphorylation sites on the 

intracellular carboxy-terminal domain of GluR1- 

GluR4 subunits, two of which on the GluR1 subunit, 

serine 831 (S831) and serine 845 (S845) have been 

88 

extensively characterized. Specifically, S831 is phosphorylated 

by CAMKII and protein kinase C (PKC), 

while S845 is phosphorylated by cAMP-dependent 

protein kinase (PKA), and their phosphorylation 

potentiates the function of the homomeric GluR1 

(Banke et al., 2000). We investigate whether the activation 

of CXCR2, that yields a gain-of-function of 

the homomeric GluR1 (Ragozzino et al., 1998; 

Giovannelli et al., 1998; Limatola et al., 2002), was 

associated with receptor phosphorylation, and if the 

described physical and functional interactions 

between these two receptors could be mediated, at 

least in part, by the phosphorylation of GluR1 on 

S845 and/or S831. We have demonstrated that 

CXCL8 induces GluR1 phosphorylation on neurons 

and transfected cells and the importance of GluR1 

phosphorylation on S845 and/or S831 (assessed by S 

replacement with A or E) in modulating the physical 

and functional interaction between GluR1 and 

CXCR2 (Catalano et al., 2008). 


Limatola C, Massa V, Lauro C, Catalano M, 

Giovannetti A, Nuccitelli S, Spinedi A. Evidence for 

a role of glycosphingolipids in CXCR4-dependent 

cell migration. FEBS Letters 2007, 581:2641-6. 

Catalano M, Trettel F, Cipriani R, Lauro C, Sobrero 

F, Eusebi F, Limatola C. Chemokine CXCL8 modulates 

GluR1 phosphorylation. J Neuroimmunol. 2008, 

198:75-81. 

Lauro C, Di Angelantonio S, Cipriani R, Sobrero F, 

Antonilli L, Brusadin V, Ragazzino D, Limatola C. 

Activity of adenosine receptors type 1 is required for 

CX 3 CL1-mediated neuroprotection and neuromodulation 

in hippocampal neurons. J Immunol. 2008, 

180:7590-6.


Maria Egle De Stefano, professor; Arianna Del Signore, 

Lucia Leone, Loredana Lombardi, post-doc fellows. 


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 

Genetica e Biologia Molecolare, Sapienza-Università di Roma 

(Prof. Ernesto Di Mauro, Prof. Alberto Oliverio); 

Dipartimento di Scienze Biologiche, Università di Napoli “Federico 

II” (Prof. Carla Perrone Capano). 


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, will be used as a tool to 

perform our investigation. 

Specifically, we focused on: (i) the possible involvement 

of the plasminogen enzymatic cascade in the 

remodelling of the rat SCG intraganglionic synapses 

induced by pre- and post-ganglionic nerve crush; 

(ii) the role of dystrophin and SCG target organs in 

the maintenance of the structural and functional 

integrity of ganglionic circuits in mice. 

(i) Axotomy of SCG neurons is characterized by 

peripheral regeneration of injured axons and temporary 

disassembly of the intraganglionic synapses, 

necessary for synaptic silencing. Both events require 

remodelling of the extracellular matrix achieved 

through controlled proteolysis of its components by 

89 


Neuronal response to experimental interruption 

of the neural circuit: a molecular and structural study 

in autonomic ganglia in vivo 

Principal investigator: Paola Paggi 

Professor of Physiology 


Tel: (+39) 06 49912323; Fax: (+39) 06 49912351 

paola.paggi@uniroma1.it 

different enzymatic systems. Therefore, we investigated 

the involvement of the plasminogen enzymatic 

cascade in response to axotomy of rat SCG neurons. 

All the components of this enzymatic pathway: 

tissue plasminogen activator (tPA), plasminogen and 

their membrane receptor annexin II, as well tPA 

inhibitor-1 (PAI-1), are constitutively expressed in 

uninjured SCGs and increase significantly after SCG 

neuron axotomy. Immunolocalization of plasminogen, 

the key protein converted into the enzymatically 

active plasmin by tPA in both neuronal and nonneuronal 

cells, indicate a contribution by all cell 

types (Fig. 1). The time course of activation of 

tPA/plasmin enzymatic pathway suggests its 

involvement in both axonal regeneration and intraganglionic 

synapse remodelling (De Stefano et al., 

2007). 

(ii) We previously reported that in the SCG of dystrophic 

mdx mice, which lack full-length dystrophin, 

there is a loss of neurons projecting to SCG muscular 

targets, like the iris. Nonetheless, surviving neurons, 

innervating either iris or submandibular gland 

(SuGl), a SCG non-muscular target, underwent 

reduced axon defasciculation and terminal branching. 

Analysis of the components of the NGF signaling 

complex, during early post-natal development, 

revealed that levels of pro-apoptotic proNGF in mdx 

mouse iris, but not in the SuGl, are higher than in the 

wild-type. This increase, along with reduced levels of 

NGF receptors (TrkA and p75NTR) in SCG, may be 

partly responsible for the observed loss of neurons 

projecting to the iris. These alterations, combined 

with a reduction in polysialylated-NCAM and neurofilament 

protein levels in SCG, may also account for 

reduced axon defasciculation and terminal branching 

in mdx mouse SCG targets (Lombardi et al., 2008.). 


De Stefano ME, Leone L, Moriconi C, Del 

Signore A, Petrucci TC, Paggi P. Involvement of

P. Paggi - Neuronal response to experimental interruption of the neural circuit 

the plasminogen enzymatic cascade in the reaction 

to axotomy of rat sympathetic neurons. Mol Cell 

Neurosci. 2007, 36:174-84. 

90 

Lombardi L, De Stefano ME, Paggi P. Components 

of the NGF signaling complex are altered in mdx 

mouse superior cervical ganglion and its target 

organs. Neurobiol Dis. 2008, 32:402-11. 

Fig. 1 - Changes in plasmin(ogen) immunolabeling in control and injured (5 postoperative days) SCGs. (A-C and inset) Control SCG. (A) 

Plasmin(ogen) immunolabeling is scanty. At this magnification, blood vessels (arrows) are recognizable as the most immunopositive structures 

inside the ganglion. (B) At high magnification, immunopositive nerve fiber-like structures (arrowheads), bearing varicosities, are seen in 

between ganglionic neurons. (C and inset) Strongly immunopositive non-neuronal cells (double arrows) abut ganglionic neurons and emit 

long processes (arrowheads), which surround the neuronal perikaryon. (D) After post-ganglionic nerve crush, plasmin(ogen) immunolabeling 

increases notably, filling all the extracellular spaces. Scale bars: A, D: 50 µm; B, C and inset: 25 µm (De Stefano et al., 2007).


Stefano Cacchione, researcher; Sabrina Pisano, post-doc fellow; 

Alessandra Galati, Emanuela Micheli, PhD students. 


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). 


Telomeres are the special nucleoprotein structures 

that protect chromosome ends from both recombination 

and degradation. In humans, telomeres consist 

of TTAGGG repeats about 10 kbp long, ending in a 

3’ G-rich overhang. As a consequence of incomplete 

DNA replication at chromosome termini, telomeres 

progressively shorten in replicating cells, till they 

reach a critical length that leads to cellular senescence. 

In germ line cells, telomere loss is counteracted 

by the reverse transcriptase enzyme telomerase, 

which adds telomeric repeats to the 3’ ends of the 

chromosomes using as template its RNA moiety. 

Both telomere length regulation and the maintenance 

of a correct end-capping structure are essential 

for cell and organism survival. Most tumors 

show aberrant telomerase expression levels, that 

allow cells to divide indefinitely. 

The long human telomeres are organized in tightly 

packed nucleosomes separated by 10-20 bp of linker 

DNA. Two proteins, TRF1 and TRF2, bind to doublestranded 

TTAGGG repeats, whereas POT1 recognizes 

single-stranded G-rich overhangs. Telomere 

structure is still poorly defined. A sound model is the 

Principal investigator: Maria Savino 

Professor of Biophysical Chemistry 


Tel: (+39) 06 49912238; Fax: (+39) 06 4440812 

maria.savino@uniroma1.it 

91 


Structural and superstructural features of human telomeric 

chromatin 

t-loop, a closed protective structure in which the 3’ 

overhang circles back and insert into the upstream 

duplex telomeric DNA region. The G-rich singlestranded 

extension can also fold to form a structure 

named G-quadruplex, a four-stranded structure stabilized 

by cations at physiological concentrations. The 

emerging view is that telomeres are dynamic structures 

that interconvert among different structures 

along with the cell cycle and differentiation. 

Whereas the roles played by telomere-binding proteins 

in the regulation of telomere length and stability have 

been widely studied, little is known about the involvement 

of nucleosomes in telomere structure and functions, 

and about the interplay between nucleosomes 

and specific telomeric proteins. In the past years, we 

demonstrated that telomeric nucleosomes are the least 

stable nucleosomes so far studied and occupy multiple 

isoenergetic positions, as a consequence of the 6 bp 

telomeric DNA periodicity, which is out of phase with 

the DNA double helical repeat, Furthermore, AFM 

studies on nucleosomal arrays reconstituted in vitro 

showed that the very short spacing of human telomeric 

nucleosomes (157 bp) depends mainly on the intrinsic 

physical properties of telomeric DNA. Finally, we 

found that hTRF1 is able to specifically recognize 

telomeric binding sites located within nucleosomes, 

inducing alterations in nucleosome structure without 

dissociation of histone subunits. All these findings 

suggest that nucleosomes may play a relevant role in 

telomere function and dynamics. 

In the past two years we focused on two main issues: 

1) The role of nucleosomes in telomere dynamics 

and their interplay with TRF proteins; 2) Human 

telomere G-quadruplex structures as targets for 

anticancer strategies. 

Intrinsic and induced mobility of telomeric 

nucleosomes 

Chromatin remodeling is a major player in the regulation 

of biological processes, through the action of 

ATP-dependent remodeling complexes. By means of

M. Savino - Structural and superstructural features of human telomeric chromatin 

a restriction enzyme assay and of AFM imaging, we 

demonstrated that nucleosomes shift from telomeric 

DNA to an adjacent sequence in a temperature and 

ionic strength dependent manner (Pisano et al., 

2007). On the contrary, most nucleosomes formed on 

an average sequence remain in the starting position. 

Very recently, we found that TRF1 is able to induce 

nucleosome mobility (S. P, M. S, S. C., in preparation). 

This remodelling activity is specific for telomeric 

nucleosomes since TRF1 has no effect on nucleosomes 

formed on other sequences. Our findings 

support the hypothesis that inherent nucleosome 

mobility depends on DNA sequence, and reveal an 

unknown property of TRF1 with relevant implications 

in telomere structure and dynamics. 

Inhibition of telomerase by targeting human 

telomere G-quadruplex structures 

The characterization of the structure of telomeric 

chromatin represents a relevant research issue, not 

only as a matter of basic research but also for the 

implications in cancer research. In most tumors reactivation 

of telomerase allows to overcome the telomeric 

checkpoint and to maintain unlimited proliferative 

activity. Since telomerase is inactive in somatic 

cells, telomerase represents a specific target for anticancer 

strategies. 

The relationship between human telomere G-quadruplex 

structure and chromatin organization could represent 

an important way to regulate oncogene transcription. 

It has been recently shown that PQS (putative 

quadruplex forming sequences) are present in 

most proto-oncogenes regulative sequences. In particular, 

we have recently found a PQS about 100 bp 

upstream of hTERT, encoding the catalytic subunit 

92 

of telomerase, corresponding to a major DNaseI 

hypersensitive site. Targeting this site with polypurinic 

oligonucleotides gives rise to the formation of 

a triple helix structure (Rossetti et al., 2007). 

The stability of this unusual DNA structure was 

substantially increased by interacting with water 

soluble perylene derivatives, and represents a promising 

strategy to inhibit telomerase. We synthesized 

several polyamine side-chain perylene derivatives 

and showed that they are able to induce G-quadruplex 

structures and to inhibit telomerase in a cellfree 

assay (Franceschin et al., 2008). Our future aim 

is to test these molecules for their ability to inhibit 

telomerase in vivo. 


Pisano S, Marchioni E, Galati A, Mechelli R, Savino 

M, Cacchione S. Telomeric nucleosomes are intrinsically 

mobile. J Mol Biol. 2007, 369:1153-62. 

Rossetti L, D’Isa G, Mauriello C, Varra M, De 

Santis P, Mayol L, Savino M. A model for triple helix 

formation on human telomerase reverse transcriptase 

(hTERT) promoter and stabilization by specific 

interactions with the water soluble perylene derivative, 

DAPER. Biophys Chem. 2007, 129:70-81. 

Franceschin M, Lombardo CM, Pascucci E, 

D’Ambrosio D, Bianco A, Ortaggi G, Savino M. The 

number and distances of positive charges of 

polyamine side chains in a series of perylene 

diimides significantly influence their ability to induce 

G-quadruplex structures and inhibit human telomerase. 

Bioorg Med Chem. 2008, 16:2292-304.

AREA5 

Cellular and 

molecular 

immunology


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. 


The capacity of professional antigen-presenting 

cells (pAPCs) to present either exogenous antigens 

derived from other cells (usually necrotic or apoptotic 

cells), or soluble antigens on class I molecules 

to CD8 + T cells is defined as cross-presentation. 

This process is primarily carried out by dendritic 

cells (DCs) in vivo, and seems to be crucial for inducing 

both CD8 + T cell immunity (cross-priming) 

against allografts, tumors, or those pathogens that do 

not infect or impair pAPCs, and tolerance (cross-tolerance) 

against self-antigens. An additional support 

for the idea that DCs carry out opposing functions 

according to their maturational stage and the 

microenvironment in which they work is provided by 

studies investigating the complex interplay amongst 

cross-presentation, apoptotic cells, DCs and stimulatory 

signals. The current view suggests that, under 

steady state conditions, apoptotic cells are consistently 

captured by immature DCs that in turn induce 

tolerance (or cross-tolerance) of apoptotic cellderived 

antigen-specific T cells. Otherwise, DCs 

mature and cross-prime these T cells in the presence 

of sustained infectious/inflammatory mediators, 

necrotic cell products, or CD4 + Th cells inducing 

DC maturation via the CD40/CD40L interaction. In 

this project, we have studied more in depth the consequences 

of cross-presentation of apoptotic cells 

unveiling self-antigens during the course of inflammatory 

processes. In addition, we investigated the 

possibility to exploit this phenomenon in order to 

identify new self- or tumor-associated antigens that 

could be included for the design of innovative strate- 

Principal investigator: Vincenzo Barnaba 

Professor of Internal Medicine 

Dipartimento di Medicina Interna 

Tel: (+39) 06 4453994; Fax: (+39) 06 49383333 

vincenzo.barnaba@uniroma1.it 

95 

Cellular and molecular immunology - AREA 5 

Innovative strategies eliciting CD8 T cell responses by exploiting 

proteomic analysis and improving cross-presentation 

gies for the manipulation of immune responses in the 

related diseases. 

Principal results and perspectives 

As demonstrated by our previous data (Nature Med. 

2001, 7:807-13), apoptotic CD40L + T cells directly 

induce DC maturation and cross-priming of CD8 + T 

cells specific to apoptotic cell-associated self-antigens, 

irrespective of additional exogenous signals. In contrast, 

if apoptotic T cells are CD40L - (such as those 

derived from resting T cells), the help of a third party 

activated T cell or surrogate CD40L molecule is 

needed for priming. Thus, the balance between 

CD40L + (apoptotic) and CD40L - T cells during 

cross-presentation appears to dictate tolerance or 

induction of CD8 T cell responses against T cellassociated 

epitopes, and to maintain or to stop the 

related responses in the course of an inflammatory 

process. It is tempting to hypothesize that these 

responses have a critical role in the amplification of 

chronic inflammation via the continuous bystander 

effects of inflammatory cytokines produced by T cells 

specific for the apoptotic T cell-associated antigens. 

In this context, more recently we studied the intricate 

interplay amongst apoptotic cells, cross-presentation 

and the phenomenon of chronic immune activation 

(CIA). CIA is hallmark of several chronic 

immune-mediated (viral or autoimmune) diseases. It 

is characterized by: (a) hyper-reactivity of T and B 

cells resulting in (b) a continuous turnover of apoptotic 

cells derived from the former, and (c) hyper-production 

of cytokines and chemokines. It is only in 

minimal part due to self-antigen or virus-specific T 

cells, as only a small proportion of activated T cells 

are specific for the viruses or the self antigens related 

to the relevant diseases. The origin of CIA is not 

clear and has been attributed to various causes, 

including bystander T cell activation, homeostatic 

proliferation, T cell responses to a variety of selfantigens 

or gut microorganisms, progressive loss of 

regulatory T cells.

V. Barnaba - Innovative strategies eliciting CD8 T cell responses by exploiting proteomic analysis and improving cross-presentation 

In this project, we have examined the proteomic profile 

of apoptotic T cells by the combination of twodimensional 

electrophoresis (2DE) and matrix assisted 

laser desorption ionization-time of flight-mass 

spectrometry (MALDI-TOF-MS) analyses, and 

found that the apoptotic T cell proteome is represented 

by several fragments of cytoskeleton-associated 

proteins, which showed antigenic properties 

with respect to human CD8+ T cells in vivo. In addition, 

we have defined a new role of caspases by which 

they facilitate cross-presentation of apoptotic cellassociated 

antigens. Our data suggest that caspase 

cleavage allows the release of cell-associated antigens 

(possibly long-lived) from the cellular matrix 

and their subsequent delivery to the cross-presentation 

pathway. The unveiling of a new antigenic 

repertoire in apoptotic cells caused by caspasedependent 

cleavage may have a major role in inducing 

either cross-tolerance (in the steady state) or 

cross-priming (in pathological conditions) of selfreactive 

CD8 + T cells. This process may contribute 

to the improved caspase-dependent immunogenicity 

observed in some autoimmune or tumor models. 

A major finding of this study is that in patients with 

chronic HIV infection, apoptotic T cells seem to 

induce a wide repertoire of effector (ef)CD8 + T cells 

specific to apoptotic antigens in vivo. This is supported 

by the evidence showing a correlation 

between apoptosis and the strength of the efCD8 + 

T-cell responses directed against the apoptosis-associated 

peptides. The possible role of apoptotic epitope-specific 

CD8 + T cells in AIDS immunopathology 

is substantiated by the evidence that their magnitude 

was related with the decline of circulating 

CD4 + T cells, representing an accurate marker of 

disease progression in HIV infection. The observation 

that cross-presentation of apoptotic cells activate 

efCD8 + T cells in HIV patients ex vivo, suggests 

this mechanism might be operative in generating the 

great variety of apoptotic antigen-specific CD8 + T 

cells shown in these patients, as reported in several 

experimental models. 

In conclusion, it is tempting to envision from our 

data that CIA commonly observed during different 

chronic viral or autoimmune diseases results partly 

from a vicious cycle. In that cycle, the cross-presen- 

96 

tation of apoptotic T cells leads to the activation of 

a large repertoire of apoptotic antigen-specific T 

cells, which in turn undergo apoptosis after they 

have performed their effector functions, and so on. 

Depending on the amplification of this process, it 

ultimately may contribute to the irreversible impairment 

of the immune system, as in the case of HIV 

infection. This knowledge may be exploited to tune 

the immune homeostasis in diseases affected by 

excessive T-cell apoptosis. Conversely, our recent 

yet unpublished data suggest that the identification 

of caspase-cleaved proteins from apoptotic tumor 

cells may contribute to improve anti-tumor 

immunotherapy in combination with efficient adjuvants, 

appropriate chemotherapy, and even compounds 

enhancing antigen cross-presentation via 

inhibition of lysosomal acidification. Indeed, as documented 

in our previous study (J Exp Med. 2005, 

202:817-28,), efficient antigenic delivery for the 

cross-presentation requires neutral pH in phagoendosomes, 

in order that exogenous antigens are not 

degraded in those compartments, and can thus 

access the cytosolic processing pathway. 


Rawson PM, Molette C, Videtta M, Altieri L, 

Franceschini D, Donato T, Finocchi L, Propato A, 

Paroli M, Meloni F, Mastroianni CM, d'Ettorre G, 

Sidney J, Sette A, Barnaba V. Cross-presentation of 

caspase-cleaved apoptotic self antigens in HIV infection. 

Nat Med. 2007, 13:1431-9. 

Meloni F, Accapezzato D, Agresti C, Aloisi F, 

Ristori G, Salvetti M, Furlan R, Martino G, Barnaba 

V, Paroli M. Dendritic cells loaded with apoptotic 

oligodendrocytes as a source of myelin T-cell epitopes 

in multiple sclerosis. Clin Immunol. 2008, 

129:286-94. 

Morandi F, Raffaghello L, Bianchi G, Meloni F, 

Salis A, Millo E, Ferrone S, Barnaba V, Pistoia V. 

Immunogenicity of human mesenchymal stem cells 

in HLA-class I-restricted T-cell responses against 

viral or tumor-associated antigens. Stem Cells 2008, 

26:1275-87.


Anna Guarini, Franca Citarella, researchers; Sabina 

Chiaretti, post-doc fellow; Marilisa Marinelli, Monica 

Messina, Nadia Peragine, Simona Santangelo, PhD students. 


Background 

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 elevated 

tissue-specific expression of some miRNA genes 

suggests that they might be involved in tissue differentiation 

and maintenance of cell-type identity in 

animals; miRNAs would share such a role with tissue-specific 

transcriptional factors. Several studies 

have reported distinct patterns of miRNA expression 

in different hematopoietic cell lineages. 

Aims 

The aim of the study was the evaluation of the role 

of miRNAs as modulators of the signal transduction 

in neoplastic B cells, obtained from chronic lymphocytic 

leukemia (CLL) patients compared to normal B 

cells upon BCR stimulation and the correlation of 

these results with the gene profile expression of 

IgM stimulated neoplastic and normal B cells. The 

study is built on the hypothesis that BCR signaling 

plays an important role in the proliferation and 

maintenance of the malignant B-cell clone in 

patients with CLL. This implies that the key to the 

distinctive behavior of the neoplastic cell most likely 

relates with the differential ability of the BCR to 

respond to stimuli. It is, therefore, of primary relevance 

to investigate how the interactions of the BCR 

with environmental stimuli may contribute to the 

differential disease progression and prognosis that 

lead to an heterogeneous clinical course characteris- 

97 


Potential role of miRNAS in IgM-mediated signal transduction in 

normal and neoplastic B cells 

Principal investigator: Roberto Foà 

Professor of Hematology 


Sezione di Ematologia 

Tel: (+39) 06 85795753; Fax: (+39) 06 85795792 

rfoa@bce.uniroma1.it 

tic of CLL patients. All these events could be regulated 

by miRNAs that play a significant role in the 

proliferation and differentiation of hematopoietic 

cells, and it is well-known that changes in miRNA 

expression may contribute to cancer predisposition 

and progression in cells of the immune system. 

Patients and methods 

Eighteen patients with a diagnosis of CLL based on 

the presence of more than 5,000 peripheral lymphocytes/µL 

expressing a typical phenotype (CD5/ 

CD20/CD19/CD23+, weak surface immuno-globulin 

expression, CD10-), have so far been evaluated 

before any treatment. The implication that some biological 

predictor factors have a role with the progression 

of the disease suggested the investigation 

of these markers. Thus, CLL cells were evaluated for 

the expression of prognostic parameters: CD38 and 

ZAP-70 expression; deletion of the 13q14, 11q23, 

17p13 regions and chromosome 12 trysomy; the 

mutational status of the immunoglobulin variable 

genes (IgVH ). 

Twelve healthy donors have been utilized as controls. 

Normal and leukemic cells have been selected from 

peripheral blood using CD19+ microbeads and the 

obtained purity was greater than 98%. The purified 

cells were cultured in 96 well U bottom plates coated 

overnight at 4°C with 50µg/ml anti-goat F(ab’)2 

IgG developed in rabbit. BCR stimulation was performed 

by adding a goat F(ab’)2 anti-human IgM at 

a final concentrations of 10µg/ml for 24 and 48 

hours; stimulated and unstimulated cells were thereafter 

collected. Some cells were lysed and total RNA 

was extracted for gene profile analysis, for miRNA 

detection and for validation RQ-PCR tests. miRNA 

expression profiling was performed using a service 

provider (LC Sciences), while gene expression profiling 

analysis was performed using the Affymetrix 

HGU133 Plus 2.0 gene chips arrays. Stimulated and 

unstimulated CLL cells were evaluated also for apoptosis 

and cell cycle.

R. Foà - Potential role of miRNAS in IgM-mediated signal transduction in normal and neoplastic B cells 

Results 

We are analyzing preliminary data. In particular, 

gene expression profile results have highlighted an 

upregulation of genes involved in proliferation, cell 

cycle induction and apoptosis only in IgVH-unmu tated CLL cases, while no changes are observed in 

IgVH-mutated CLL cases. These data have been 

confirmed by evaluating cell cycle progression and 

apoptosis function in neoplastic cells following BCR 

engagement and suggest that BCR stimulation with 

IgM is effective only in IgVH-unmutated CLL. In 

particular, we analyzed 6 CLL cases and in 3 with 

unmutated IgVH we found an increased number of 

cycling cells after IgM stimulation; on the contrary, 

no significant differences were observed in mutated 

IgVH cases. All data concerning the gene expression 

modulation after 24 hours of stimulation are in 

agreement with our published data (Guarini et al., 

Blood 2008, 112:782-92). After 48 hours of stimulation, 

gene expression profile analysis showed a similar 

pattern. No significant changes were observed. 

The analysis of miRNA expression profile was performed 

in 4 CLL cases, 2 with unmutated IgVH and 

2 with mutated IgVH genes, and in a pool of 6 (normal) 

B cells samples obtained from healthy donors. 

The data are expressed as change fold of unstimu- 

98 

lated/stimulated cells. The first approach of statistical 

elaboration of the results shows that 47 miRNA 

are differentially expressed. The differences seem 

more significant after 48 hours of IgM stimulation 

compared to 24 hours, in particular for mutated 

IgV H CLL cells. Normal B cells show minimal 

response to IgM stimulation. 

The analysis of miRNA profile are preliminary and 

must be confirmed in further cases. 

During the coming year, the correlation between gene 

expression profile and miRNA expression will be 

examined and statistically correlated. The analysis of 

a positive correlation will evaluate several hypothesis: 

1) miRNAs and their host genes: a positive correlation 

should be observed in those cases in which the 

miRNA resides within the intron of a given gene. 

This hypothesis will be tested using the annotations 

included in the miRBase website; 

2) miRNA promoters: we aim at using computational 

tools as TRANSFAC for the search of putative 

transcription factor binding sites in the miRNA promoter 

regions. The presence of such motifs may be 

suggestive of a correlation that will be experimentally 

validated; 

3) the relationship between a miRNA and a mRNA is 

not a direct, but a secondary, target of that miRNA.


Marzia Soligo, Cristiano Scottà, post-doc fellows; Cristina 

Camperio, PhD student. 

C o l l a b o r a t o r s : 

Dipartimento di Dermatologia, Università di Roma Tor Vergata, 

(Prof. Antonio Costanzo). 


Both in mice and in humans CD4 + CD25 + T cells 

with regulatory functions are important components 

of peripheral tolerance that is responsible in the control 

of autoreactive T cells in normal individuals. 

FOXP3 transcription factor is a critical regulator of 

these cells. Among the signals necessary to generate 

CD4 + CD25 + FOXP3 + T cells, a pivotal role is 

played by CD28. However, while in mice it was possible 

to study the role played by the interaction of 

CD28 with its ligand B7 in the activation and maintenance 

of CD4 + CD25 + FOXP3 + T cells, in 

humans similar results are lacking. Therefore we 

verified whether CD28 signaling independently of 

TCR promoted FOXP3 expression and regulates 

CD4 + CD25 + FOXP3 + T cell functions in humans. 

Specific tasks of the project are: i) the analysis of the 

effects of the unique signal delivered by CD28 in 

CD4 + CD25 - T cells on FOXP3 gene activation and 

ii) the influence of FOXP3 on the phenotypic 

and functional properties of CD28-activated 

CD4 + CD25 - T cells. 

The analysis of the effects of CD28 signal provided 

for the first time the experimental evidence that 

CD28 signals independent from TCR and dependent 

on PI3K/Akt pathways are sufficient to induce the 

transcriptional activation of FOXP3, in primary 

CD4 + CD25 - T cells. We reached this conclusion by 

activating CD28 either by the natural ligand B7 or by 

cross-linking with specific mAb. However, despite 

FOXP3 was rapidly induced, less than 30% of the 

Principal investigator: Enza Piccolella 

Professor of Immunology 


Tel: (+39) 06 49917584; Fax: (+39) 06 49917584 

enza.piccolella@uniroma1.it 

99 


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 

CD4 + CD25 - T cells expressed FOXP3 after CD28mediated 

activation, supporting the view that among 

CD4 + CD25 - T cells only a small number of cells are 

pre-committed to express FOXP3. Starting from 

these results, we went on to study: i) the translocation 

of CD28-induced FOXP3 in the nucleus, ii) its binding 

to FOXP3 consensus regions on target genes, and 

iii) cell proliferation and regulatory functions of 

CD4 + CD25 - T cells become CD4 + CD25 + FOXP3 + . 

To verify whether CD28 signal could induce posttranslational 

modification of FOXP3, we prepared 

cytoplasmic and nuclear extracts from CD28-stimulated 

CD4 + C25 - T cells, and FOXP3 was revealed by 

immunoblotting. Figure 1 shows that the maximum 

expression of FOXP3 occurred in the cytoplasm 

after 24h from CD28-mediated activation, but only 

after 48h in the nucleus. 

The recruitment of FOXP3 on target genes has been 

analyzed by chromatin immunoprecipitation (ChIP) 

experiments. Indeed it has been demonstrated that 

FOXP3 could occupy CD25, Il-2 and Ctla4 promoters 

in both murine CD4 + CD25 + T regulatory cells 

and Jurkat T cells retrovirally transduced with 

FOXP3, and stimulated by TCR plus CD28 and/or 

ionomycin. To verify this phenomenon also in primary 

CD4 + CD25 - T cells, we have activated the 

cells for 24 and 48 h by Dap3/B7. The results 

reported in Figure 2 show for the first time, at the 

best of our knowledge, that the activatory pathways 

mediated by CD28 in CD4 + CD25 - T cells favour not 

only the transcription and translation of FOXP3 but 

also its occupancy of CD25, Il-2 and Ctla4 promoters. 

Interestingly, after 24 h FOXP3 was recruited on 

all studied promoters, but the occupancy of CD25 

and Il-2 promoters (Panels A, C) was maintained for 

all the observation time, while FOXP3 left Ctla4 promoter 

between the 24 and 48 h from the activation 

(Panel B). To analyze the sensitivity of FOXP3 

recruitment on Il-2 promoter to CsA, we stimulated 

CD4 + CD25 - T cells with anti-CD28 mAbs for 24 h

E. Piccolella - Analysis of the molecular mechanisms regulating FOXP3 gene 

in the presence and absence of CsA (Panel D). CsA 

induced a slight decrease of FOXP3 recruitment on 

Il-2 promoter suggesting that, although CD28 signaling 

regulates FOXP3 expression in a CsA resistant 

manner, signals from calcineurin may have a role 

in this system. 

Finally, we have analyzed whether CD28-mediated 

activation of FOXP3 and its recruitment on CD25, 

Il-2 and CTLA4 promoters converted a small subpopulation 

of CD4 + CD25 - T cells to CD4 + CD25 + 

T cells and acquired an anergic phenotype. Indeed in 

these cells T cell proliferation and IL-2 synthesis 

were impaired. However, we present evidence that 

this unresponsiveness was not maintained in the 

absence of FOXP3, and T cell proliferation could be 

easily reconstituted upon subsequent exposures to 

antigen. This suggests that the induction of FOXP3, 

at least in a small number of CD4 + CD25 - T cells 

committed to express FOXP3, in the absence of 

TCR signalling, represents a new mechanism by 

which FOXP3 can mediate a transient shut down of 

TCR pathways. We have also shown that the delivery 

of CD28 signal in CD4 + CD25 - T cells led to 

propensity to apoptosis. However, the evidence that 

Fig. 1 - CD4 + CD25 - T cells were stimulated with anti-CD28 for 

24 and 48 hours. Western blottings for the presence of FOXP3 

were performed on Cytoplasmic (C) and nuclear (N) extracts. Antitubulin 

and anti-PCNA were used as loading control. 

100 

exogenous IL-2 rescued CD4 + CD25 + T cells from 

apoptosis but did not affect the decrease of FOXP3 

and CD25, supports the view that FOXP3-driven 

enhancement of CD25 is important for T cells survival. 

In conclusion, we have not only confirmed in humans 

that CD28 provides an unique signal to promote 

FOXP3 expression necessary to convert human 

CD4 + CD25 - T cells to CD4 + CD25 + T cells, but we 

went on demonstrating that FOXP3 upregulation 

occurred independently of TCR. This suggests a 

scenario where the expression of B7 on professional 

antigen presenting cells may contribute significantly 

to the homeostasis of CD4 + CD25 - T cells expressing 

or committed to acquire FOXP3 and to induce in 

these cells a transient nonresponsiveness important 

for the maintenance of peripheral tolerance. 


Scottà C, Soligo M, Camperio C, Piccolella E. 

FOXP3 induced by CD28/B7 interaction regulates 

CD25 and anergic phenotype in human CD4 + CD25 - 

T lymphocytes. J Immunol. 2008, 181:1025-33. 

Fig. 2 - CD4 + CD25 - T cells were stimulated with adherent 

Dap3/B7 cells for different times. ChIP assays were performed by 

using anti-FOXP3 antibody or no antibody. Immunoprecipitated 

DNA was analyzed by PCR with CD25 (A), CTLA-4 (B) and IL-2 (C) 

promoter-specific primers. (D) CD4 + CD25 - T cells were activated 

for 24 h with anti-CD28 and treated or not with CsA.


Ricciarda Galandrini, Angela Gismondi, Stefania 

Morrone, Marco Cippitelli, Rossella Paolini, professors; 

Giovanni Bernardini, Alessandra Zingoni, Cristina 

Cerboni, Alessandra Soriani, researchers; Francesca Di 

Rosa, CNR researcher; Cinzia Fionda, Rosa Molfetta, 

Helena Stabile, post-doc fellows; Michele Ardolino, 

Giuseppe Sciumè, Elisabetta Parretta, PhD students. 


Principal investigator: Angela Santoni 

Professor of Immunology 


Tel/Fax: (+39) 06 44340632 

angela.santoni@uniroma1.it 

Our proposal is aimed at analyzing the molecular 

mechanisms underlying the activation of NK cell 

functional program. In particular in the last year 

(2007-2008), we focused our attention on: 

The analysis of the role of phosphatidylinositol 

4,5-bisphosphate (PIP2)-dependent pathways 

in the development of natural cytotoxicity 

Receptor-triggered activation of PI3K and PLC 

gamma and signalling components controlling 

cytoskeleton rearrangement are emerging as critical 

events for the development of NK cell cytotoxicity. 

We have recently demonstrated that the prototype of 

NK activating receptors CD16, is coupled to phosphatidylinositol 

4phosphate-5kinase type I (PI5KI) 

alpha (Galandrini et al., Blood 2005). More recently 

we have analyzed the requirement of PIP2 in the 

generation of NK cell cytotoxicity. By live confocal 

imaging of primary human NK cells expressing the 

chimeric protein GFP-PH we observed the consumption 

of a pre-existing PIP2 pool which is critically 

required for the activation of cytolytic machinery, 

during effector-target cell interaction. 

We identified type I phosphatidylinositol4phosphate- 

5kinase (PI5KI) α and γ isoforms as the enzymes 

responsible for PIP2 synthesis in NK cells. In addition, 

by means of shRNA-driven gene silencing we 

demonstrated that both enzymes are required for the 

proper activation of NK cytotoxicity and for inosi- 

101 


Molecular mechanism underlying the activation of NK cell 

regulatory and effector functions 

tol-3,4,5trisphosphosphate generation upon receptor 

stimulation. In an attempt to elucidate the specific 

step controlled by PI5KIs we found that lytic granule 

secretion but not polarization resulted impaired 

in PI5KI α- and γ-silenced cells. 

Collectively, our findings delineate a novel mechanism 

implicating PI5KIα and γ isoforms in the synthesis 

of PIP2 pools critically required for IP3dependent 

Ca 2+ response and lytic granule release. 

A paper containing these results has been published 

in Blood 2008, 111:4165-72, and was highlighted in 

the Blood preview by Dr. M. Colonna. 

The analysis of Cdc42/WASP pathway in the 

development of natural cytotoxicity 

In collaboration with Prof. Luigi Notarangelo 

(University of Brescia), using cultured NK cells from 

Wiskott-Aldrich patients as effector cells, we have 

previously reported a marked impairment of natural 

and CD16-mediated cytotoxic functions (Gismondi et 

al., Blood 2004). The inhibition of NK cell cytolytic 

activity was associated with reduced ability of WAS 

or XLT NK cells to form conjugates with susceptible 

target cells and to accumulate F-actin upon binding. 

More recently we have focused our attention on the 

molecular mechanisms that control WASp function 

in NK cells triggered through beta1 or beta2 integrins 

or chemokine receptors as it has been reported 

that chemokines can augment NK cell cytotoxicity 

and play a major role in the control of immunological 

synapse. 

We observed that receptor engagement rapidly 

results in activation of Cdc42 and induction of 

WASp tyrosine phosphorylation. Moreover, we 

have observed that upon beta1 or beta2 integrin or 

chemokine receptor ligation, WASp can associate 

with the Src kinase Fyn and the focal adhesion 

kinase Pyk-2. Finally, we found that the inhibition 

of NK cell functions observed in NK cells from 

WASp patients was associated with a reduced ability 

of WAS or XLT NK cells to up-regulate the

A. Santoni - Molecular mechanism underlying the activation of NK cell regulatory and effector functions 

CD18 neoactivation epitope expression upon 

chemokine stimulation. Similar results were also 

obtained when NK cells from normal donors were 

pretreated with wiskostatin, a specific pharmacological 

inhibitor of Cdc42 interaction with 

WASp/N-WASp family members, and then assayed 

for the expression of the CD18 neoactivation epitope 

upon chemokine stimulation. Pretreatment of 

normal donor NK cells with wiskostatin also resulted 

in a significant inhibition of integrin-supported 

NK cell response to chemokines. 

All together these data indicate that WASp plays a 

crucial role in the regulation of NK cell response to 

chemokines by acting as a critical component of the 

chemokine-induced inside-out signaling regulating 

LFA-1 function and suggest that upon integrin or 

chemokine receptor engagement WASp function is 

regulated by the coordinate action of both Cdc42 and 

tyrosine kinases. 

A paper contaning these information is in preparation 

(Gismondi et al.). 

The analysis of the role of chemokines on NK 

cell functional maturation and trafficking in 

the BM 

In regard to this task, we focused our attention on 

understanding the chemokine involvement in the 

selective trafficking of developing and mature 

mouse NK cells in the BM. We observed drastic 

changes of CCR1, CXCR3 and CXCR4 expression 

during the progression from NK cell precursors 

(pNK) to immature DX5- (iNK) and mature DX5+ 

(mNK) NK cells. pNK and mNK cells expressed all 

receptors, while only CXCR4 was detected on iNK 

cells. Correspondingly, mNK cells migrated in 

response to CXCL12, CXCL10 and CCL3, and 

pNK and iNK cells to CXCL12, while pNK cells 

migrated to CCL3 and CXCL10 only after 

CXCL12 stimulation. 

102 

When compared to BM mNK cells, spleen and 

peripheral blood mNK cells differently expressed 

CXCR4 and CXCR3 and were less responsive to 

chemokines. CXCR4 antagonist AMD-3100 administration 

to C57BL/6 mice promoted strong reduction 

of mNK and iNK cell numbers in BM and their 

increase in blood and spleen, while pNK cell distribution 

slightly varied. Recombinant CCL3 administration 

selectively mobilized mNK cells and this correlated 

with its ability to inhibit CXCL12-mediated 

mNK cell in vitro responses. 

Our results indicate that the combined action of 

chemokines may regulate selective localization of 

NK cell subsets in the BM and direct their maturation 

and migration to the periphery. In addition, 

understanding how NK cells are mobilized from BM 

could assist the development of novel NK cell-based 

immunotherapy for cancer and chronic infections. 

A paper containing these results has been published 

in Blood 2008, 111:3626-34. 


Cerboni C, Zingoni A, Cippitelli M, Piccoli M, Frati 

L, Santoni A. Antigen-activated human T lymphocytes 

express cell-surface NKG2D ligands via an 

ATM/ATR-dependent mechanism and become susceptible 

to autologous NK- cell lysis. Blood 2007, 

110:606-15. 

Bernardini G, Sciumè G, Bosisio D, Morrone S, 

Sozzani S, Santoni A. CCL3 and CXCL12 regulate 

trafficking of mouse bone marrow NK cell subsets. 

Blood 2008, 111:3626-34. 

Micucci F, Capuano C, Marchetti E, Piccoli M, Frati 

L, Santoni A, Galandrini R. PI5KI-dependent signals 

are critical regulators of the cytolytic secretory 

pathway. Blood 2008, 111:4165-72.


Alessandra Vacca, Maria Pia Felli, professors; Diana 

Bellavia, Antonio F. Campese, researchers; Claudio Talora, 

Saula Checquolo, post-doc fellows; Samantha Cialfi, Rocco 

Palermo, Giuseppina Di Mario, PhD students; Massimo 

Zani, technician. 


Department of Cell and Molecular Biology, Medical Nobel 

Institute, Karolinska Institute, Stockholm, Sweden (Prof. Urban 

Lendahl); Harvard Medical School, Dana-Farber Cancer Institute, 

Boston, MA, USA (Prof. Harald von Boehmer). 


We previously observed that thymocytes from 

Notch3-IC (N3-IC) transgenic (tg) mice display 

overexpression of pTα/pre-TCR chain and 

increased NF-κB activity (Bellavia et al., EMBO J. 

2000, 19:3337 ). Furthermore, we demonstrated that 

combined overexpression of pTα and Notch3 in tg 

mice, triggers important molecular events, some of 

which are NF-κB-mediated and cooperate in sustaining 

Notch3-induced lymphomagenesis (Felli et al., 

Oncogene 2005, 24:992; Talora et al., EMBO Rep. 

2003, 4:1067). 

The ability that we demonstrated of activated Notch3 

to lead to constitutive activation of NF-kB both, in 

vivo and in vitro, together with previous reports that 

demonstrated on one hand that Notch1 is able to bind 

to the p50 NF-kB subunit and on the other hand that 

NF-kB2, the precursor of p52, is a putative target 

gene of activated Notch1 suggest strict relationships 

between activated Notch signaling and different NFkB 

activation pathways. In accord with this hypothesis, 

we recently demonstrated that Notch3 is indeed 

able to activate canonical and alternative NF-kB pathways 

by regulating the assembling and activation of 

different IKK complexes, depending on the presence 

of pre-TCR (Vacca et al., EMBO J. 2006, 25:1000). 

Moreover, IKKalpha forms a complex with Notch3 

Principal investigator: Isabella Screpanti 



Tel: (+39) 06 44700816; Fax: (+39) 06 4464129 

isabella.screpanti@uniroma1.it 

103 


Analysis of the role of preTCR-triggered NF-kB in T cell 

leukemogenesis: relationship with activated Notch signaling 

even independently on the presence of pre-TCR , but 

only the presence of pre-TCR allows NIK to participate 

at the Notch3-IKKalpha complex. This last 

observation suggests that Notch3 may activate the 

alternative pathway through an additional direct 

mechanism. 

In order to genetically approach this issue, we 

planned to generate two lines of double transgenic 

mice, one overexpressing Notch3-IC and mutant for 

p50, a component of the canonical heterodimer, and 

the other one overexpressing Notch3 and mutant 

forp52, a component of the alternative heterodimer, 

to the purpose to obtain mice in which only one NFkB 

activation pathway is impaired. 

The following results have been obtained in the last 

two years: 

Generation of Notch3-IC/ p50-/- and Notch3- 

IC/p52-/- double transgenic mice 

We obtained p50-/- and p52-/- mice from Dr. G. 

Franzoso (University of Chicago, ILL, USA). These 

mice were generated by homologous recombination 

and are totally lacking of functional p50 or p52 protein. 

We have now established Notch3-IC/p50-/and, 

more recently, Notch3-IC/p52-/- double mutant 

mice and started their phenotypic characterization. 

Phenotypic and functional characterization 

of cell population in different lymphoid 

organs with respect to the kinetic of 

development of multicentric T cell lymphoma 

The T cell oncogenic potential of Rel/NFkB is well 

established. If the oncogenic role of Notch3 is mediated 

by the activation of NF-kB, we would predict 

that in double transgenic mice the lymphoma development 

would be affected. For this reason, the mice were 

clinically observed and mortality curves were drawn. 

a) Notch3-IC/p52-/- double mutant mice. We previously 

showed that the canonical pathway of NF-kB 

activation, mediated by the nuclear translocation of 

p50/p65 heterodimer, is mostly responsible of the

I. Screpanti - Analysis of the role of preTCR-triggered NF-kB in T cell leukemogenesis 

proliferation of lymphoma cells (Bellavia et al., 

EMBO J 2000, 19:3337). The analysis of Notch3- 

IC/p52-/- double mutant mice showed that the 

course of the T cell lymphomas was slightly delayed 

and 80% of the mice were dead at 15 weeks of age 

(vs 12 weeks of Notch3-IC single mutant animals). 

However, at the end point the phenotype of the mice 

was not significantly different from the Notch3-IC 

transgenics: lethargy, hunched posture and distended 

abdomen. At necroscopy the mice revealed a significant 

enlargement of the upper mediastinum, and 

increase in size and weight of spleen and lymph 

nodes. Thus, as expected from the results previously 

obtained, the inhibition of the alternative pathway of 

NFkB activation, does not affect significantly the 

development of the Notch3-induced T cell leukemia. 

Preliminary experiments to study the immunophenotype 

of lymphoid organs were done at the age of 

7 weeks, when most of the Notch3-IC transgenic 

mice display the characteristic features of Notch3induced 

T cell leukemia (i.e. presence of CD4+CD8+ 

double positive cells in the spleen, increased expression 

of CD25 and CD3 in both thymocytes and 

splenic lymphocytes) and we did not observe significant 

modifications in double mutant mice. 

Interestingly, as a peculiar feature, the double mutant 

mice displayed a virtual absence of B220+ cells 

(0.5% vs 12% in Notch3-IC transgenic) and a significant 

increase of Mac1+ (38% vs 4% in Notch3-IC 

transgenic) and Gr1+ (30% vs 1% in Notch3-IC 

transgenic) cells in the spleen. 

b) Notch3-IC/p50-/- double mutant mice. We previously 

described that the triggering of the canonical 

pathway of NFkB activation, was mainly responsible 

for the transcriptional activation of anti-apoptotic 

and pro-proliferative genes, thus mainly responsible 

104 

for the progression of T cell leukemia in Notch3-IC 

transgenic mice. Since the canonical pathway activity 

is sustained by the p50/p65 heterodimer, we would 

expect that deletion of p50 would delay or even abolish 

the development of T cell leukemia. Surprisingly, 

the mortality curve showed that most of the mice die 

earlier with respect to Notch3-IC single mutant mice. 

Indeed 80% of the double mutant mice were dead at 

8.5 weeks of age (vs 35% of Notch3-IC transgenic 

and none of p50-/- mice) and all of the mice are dead 

by 12 weeks of age. However, in any of the double 

mutant mice we were able to detect the features of 

leukemia/lymphoma. Rather, the mice appear significantly 

smaller in size with respect to wild type or single 

mutant mice of the same age and, while displaying 

a spleen of normal size, their thymus was significantly 

reduced, with a total thymocyte yield equivalent 

to less than 10% of the cellularity of the thymus 

from wild type or single mutant mice. Similarly to 

what observed in Notch3-IC/p52-/- mice, also 

Notch3-IC/p50-/- mice display a significant reduction 

in the spleen of B220+ cells and a concomitant 

increase of Mac1+ and Gr1+ cells. 


Bellavia D, Mecarozzi M, Campese AF, Grazioli P, 

Talora C, Frati L, Gulino A, Screpanti I. Notch3 and 

the Notch3-upregulated RNA-binding protein HuD 

regulate Ikaros alternative splicing. EMBO J. 2007, 

26:1670-80. 

Bellavia D, Checquolo S, Campese AF, Felli MP, 

Gulino A, Screpanti I. Notch3: from subtle structural 

differences to functional diversity. Oncogene 

2008, 27:5092-8.


Maria Teresa Fiorillo, researcher; Fabiana Paladini, post-doc 

fellow; Francesca Belfiore, Elisa Cocco, Adriana 

Magnacca, Elisa Nurzia, PhD students. 


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, 

Berlin, Germany (Prof. Andreas Ziegler, Dr Barbara 

Uchanska-Ziegler); Dipartimento di Scienze Mediche, Università 

di Cagliari (Prof. Alessandro Mathieu). 


The project plan was subdivided in three different 

parts: 

Analysis of T cell responses specific for 

peptides sharing homology with the viral 

peptide pLMP2 in patients with AS versus 

healthy controls 

The viral pLMP2 (RRRWRRLTV) and the self 

pVIPR (RRKWRRWHL) peptides are unusually 

rich in arginines. This amino acid can undergo 

post-translational modifications consisting in citrullination. 

These modifications could confer some 

new antigenic properties to these peptides making 

the immune system able to recognize them as 

novel antigens and mount a cytotoxic T cell 

response (Beltrami et al., J Biol Chem. 2008, 

283:27189-99). 

We therefore have synthesized new peptides possessing 

citrulline instead of arginine, either in single 

(p1; p5 and p6) or in multiple positions (p1 and 

p5; p1 and p6; p5 and p6). One of these peptides, 

pVIPR-U5 (RRKWURWHL; U = citrulline), has 

already been analysed both at functional and crystallographic 

levels to verify whether the exchange of 

Principal investigator: Rosa Sorrentino 

Professor of Pathology 


Tel: (+39) 06 49917706; Fax: (+39) 06 49917594 

rosa.sorrentino@uniroma1.it 

105 


The role of HLA-B27 in autoimmunity: from the genetics to the 

function 

arginine to citrulline affects the display of this peptide 

by two human major histocompatibility antigen 

class I subtypes, HLA-B*2705 and HLA-B*2709. 

The crystal structures show that a modified selfpeptide, 

pVIPR-U5 (RRKWURWHL; U = citrulline) 

is presented by the two HLA-B27 molecules 

in distinct conformations. These binding modes differ 

not only drastically from each other but also 

from the conformations exhibited by the non-citrullinated 

peptide in a given subtype. The differential 

reactivity of HLA-B27-restricted cytotoxic T cells 

with modified or unmodified pVIPR supports the 

structural findings and shows that the presentation 

of citrullinated peptides has the potential to influence 

immune responses. Furthermore, citrullinated 

peptides such as pVIPR-U5 and pVIPR-U5+U6 

have been tested for their ability to induce self-reactivity 

in B*2705 patients with AS. So far, we have 

observed CD8+ T cell responses in 2 out of 5 

patients and the responsive CTL lines have shown 

either specificity for the citrullinated forms of 

pVIPR or cross-reactivity with the native peptide. 

This result supports the possibility that some circustances 

such as chronic inflammatory conditions 

that occur in AS could favor the activity of PAD 

(peptidylarginine deiminases), the enzyme involved 

in the arginine to citrulline conversion. T cells from 

B*2709 positive individuals have also been stimulated 

with citrullinated forms of pVIPR and so far no 

reactivity has been detected. In progress is a large 

screening in which CTL lines primarily driven by 

pLMP2, pVIPR and pVIPR-U5 or pVIPR-U5+U6 

are assayed against the complete panel of citrullinated 

pVIPR and pLMP2 analogs (the arginine at 

each position substituted by citrullines). These 

immunological results will be combined with structural 

data assessing the ability of the citrullinated 

peptides to form a stable complex with either 

B*2705 and B*2709 molecules and with spettroscopic 

measurements,i.e. circular dichroism (CD) 

and differential scanning calorimetric (DSC).

R. Sorrentino - The role of HLA-B27 in autoimmunity: from the genetics to the function 

Assessment of the functional effect of SNP 

rs1264457 on the expression of HLA-E 

Due to the difficulties to find a monoclonal antibody 

specifically recognizing the HLA-E molecules, we 

have spent several months to validate the MEM/02 

antibody (a kind gift from Dr. Vaclav Horejsi) which 

shows some cross-reactivity with the classical HLAclass 

I molecules, usually expressed at much higher 

amount than HLA-E. Having set up the right conditions, 

we are now performing some functional experiments. 

Meanwhile, we have validated the association 

between Ankylosing Spondylitis and the SNP 

rs1264457, the functional polymorphism of the 

HLA-E molecules, analysing a larger cohort of 

patients and HLA-B27 controls from Sardinia than 

previously published (Cascino et al., Arthritis Rheum. 

2007, 56:2640-51). We have confirmed the strong 

association between AS and this functional polymorphism 

(allelic difference between patients with AS 

versus HLA-B27-matched controls p=0.0005). The 

same cohorts have been also analysed for 5 SNP 

mapping in the gene encoding for the VIPR1 gene 

which is involved both in the down-regulation of the 

inflammatory response and as donor of the pVIPR 

peptide. Very interestingly we found that the presence 

of HLA-B*2705 correlates with the presence of 

T at the SNP896 in the 3’UTR of the gene. In addition, 

this polymorphism correlates with a more 

prompt down-regulation of the mRNA for VIPR1, 

suggesting that subjects positive for HLA-B*2705, 

the allele associate with AS, might have a different 

106 

regulation of the inflammatory response associated 

with the VIP/VIPR1 signaling. These data have 

been published (Paladini et al., 2008). 

Characterization of an alternative route for 

antigen presentation 

The question we wanted to answer was whether an 

alternative pathway for antigen presentation previously 

described by us as active in the case of HLA- 

B27 molecules (Bettosini et al., J Virol. 2005, 

79:15537-46), was specific for these molecules or was 

of more general usage. We have therefore singled 

out epitopes specific for HLA-B27 and HLA-A2 and 

inserted them in the same chimeric proteins. The 

results clearly show that this pathway is not accessible 

to the HLA-A2 molecules. B27 mutants which 

are unable to recycle from the cell membrane also 

show that this pathway is not dependent from this 

mechanism but rather from nascent molecules. 

Experiments have been undertaken to verify the 

influence of a Cys at position 67 which is absent in 

all HLA class I molecules but HLA-B27. 


Paladini F, Cauli A, Cascino I, Vacca A, Belfiore F, 

Fiorillo MT, Mathieu A, Sorrentino R. A functional 

polymorphism of the vasoactive intestinal peptide 

receptor 1 gene correlates with the presence of 

HLA-B*2705 in Sardinia. Genes Immun. 2008, 

9:659-67.


Michela Muscolini, PhD student; Silvana Caristi, technician. 


Centro de Biología Molecular Severo Ochoa, Universidad Autónoma 

de Madrid, Cantoblanco, Madrid, Spain (Prof. Balbino Alarcón); 

Dipartimento di Biologia Cellulare, Università di Roma Tor Vergata, 

(Prof. Mauro Piacentini); Dipartimento di Dermatologia, 

Università di Roma Tor Vergata, (Dr. Antonio Costanzo). 


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. Genetic deficiencies in NFκB 

family members or signalling components that 

act upstream of NF-κB have been linked to 

immune deficiencies, whereas aberrant constitutive 

NF-κB activation has been associated with the 

development of autoimmune and neoplastic disorders. 

The understanding of the molecular mechanisms 

that control NF-κB activation in lymphocytes 

has therefore important implications for the 

design of novel and more specific therapies for 

immune diseases. 

CD28 is one of the most important co-stimulatory 

receptors necessary for full T lymphocyte activation. 

As a member of the immunoglobulin (Ig) supergene 

family, CD28 extra-cellular Ig-like domain binds to 

the cognate ligands, B7.1/CD80 or B7.2/CD86 on 

the surface of professional antigen presenting cells 

(APC), including macrophages, dendritic cells and 

activated B lymphocytes. As an integral component 

of the immunological synapse, 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 path- 

Principal investigator: Loretta Tuosto 

Researcher in Molecular Immunology 


Tel. : (+39) 06 49917595; Fax: (+39) 06 49917594 

loretta.tuosto@uniroma1.it 

107 


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 

ways. More recent data evidence that CD28 can also 

act as a TCR-independent signalling unit, by delivering 

specific signals, which induce NF-κB actvity 

and regulate the survival of primary T cells to several 

apoptotic stimuli. 

In the last years, we focused our research studies 

on the characterisation of the biochemical and 

molecular mechanisms coupling CD28 to the activation 

of NF-κB. We evidenced that CD28 stimulation 

by B7 activates, in memory CD4 + T cells, a 

non-canonical NF-κB2-like cascade leading to the 

selective recruitment of RelA/p52 and RelA/RelA 

dimers on the promoter of pro-inflammatory (i.e. 

IL-8 and BAFF) as well as anti-apoptotic (Bcl-xL) 

genes. We also found that CD28-induced NF-κB 

activation rescues T cells form ionising radiation 

(IR)-induced apoptosis by interfering with the 

binding of active p73, a member of p53 tumour 

suppressor family, and transcription of the proapoptotic 

gene bax. The analysis of the biochemical 

pathways regulating both transcription of antiapoptotic 

bcl-xL and repression of bax expression 

by RelA/NF-κB revealed a key role exerted by 

phosphatidylinositol 3-kinase (PI3K)/Akt. Indeed, 

CD28-induced activation of PI3K/Akt pathway 

leads to the recruitment of transcriptionally active 

complexes, containing RelA/NF-κB and the histone 

acetyltransferase p300/CBP, on the promoter 

of anti-apoptotic bcl-xL. At the same time, 

PI3K/Akt cascade induces the binding of corepressor 

complexes, formed by RelA and the histone 

deacetylase HDAC1, on the bax gene promoter 

thus inhibiting Bax expression and ensuring T 

cell survival in response to death stimuli. 

CD28 also plays a critical role in regulating actin 

remodelling by inducing the early cytoskeleton 

rearrangements events required for the recruitment 

and activation of signalling molecules to the membrane. 

We previously demonstrated that Vav-1, an 

exchange factor for Rac-1 and Cdc42 GTP-binding 

proteins, is a key upstream regulator of the sig-

L. Tuosto - CD28 co-stimulatory molecule as a key regulator of T lymphocyte differentiation and survival 

nalling cascades relating CD28 stimulation to both 

actin reorganization and NF-κB activation. Our preliminary 

data indicate that CD28 is also involved in 

the recruitment of a complex containing Vav-1 and 

Nck. Nck is an adapter that plays a key role in the 

remodelling of actin cytoskeleton following TCR 

stimulation, through the specific interaction with the 

CD3ε chain, thus favouring TCR/CD3 signalling 

and T cell activation. In particular, we observed that 

stimulation of T cells with B7 expressing APC 

induces the association of CD28 with Nck and the 

recruitment to CD28 of a complex containing Vav- 

1, Nck and actin (Fig. 1). 

Experiments are in progress to characterize the 

nature of CD28/Nck/Vav interaction, in order to 

identify the domains of CD28, Nck and Vav1 

involved in their reciprocal association. 

108 


Annibaldi A, Sajeva A, Muscolini M, Ciccosanti F, 

Corazzari M, Piacentini M, Tuosto L. CD28 ligation 

in the absence of TCR promotes RelA/NF-kappaB 

recruitment and trans-activation of the HIV-1 LTR. 

Eur J Immunol. 2008, 38:1446-51. 

Cianfrocca R, Muscolini M, Marzano V, Annibaldi A, 

Marinari B, Levrero M, Costanzo A, Tuosto L. 

RelA/NF-kappaB recruitment on the bax gene promoter 

antagonizes p73-dependent apoptosis in costimulated 

T cells. Cell Death Differ. 2008, 15:354-63. 

Muscolini M, Cianfrocca R, Sajeva A, Mozzetti S, 

Ferrandina G, Costanzo A, Tuosto L. Trichostatin A 

up-regulates p73 and induces Bax-dependent apoptosis 

in cisplatin-resistant ovarian cancer cells. Mol 

Cancer Ther. 2008, 7:1410-9. 

Fig. 1 - Jurkat JCH7C17 cells expressing CD28 WT were transfected with 15µg of Vav-GFP and 15µg HA-NCK for 6 h, then activated with 

5-3.1/B7 cells for 15 min and seeded on cover glasses. After fixing and permeabilization, Nck was stained with a rabbit anti-Nck antiserum 

followed by Alexa fluor 594-conjugated goat anti-rabbit secondary antibody. Slides were analyzed by using a Zeiss LSM510 META confocal 

microscope. Bar, 10 µm.


Antonio Filippini, professor; Anna Riccioli, researcher; 

Claudia Giampietri, Donatella Starace, post-doc fellows, 

Roberta Galli, Alessio Paone, PhD students; Giuseppina 

Avigliano, graduate student; Fabrizio Padula, Simonetta 

Petrungaro, technicians. 


Dipartimento di Medicina Sperimentale, Università de L’Aquila 

(Prof. Paola De Cesaris). 


Viruses are known to be capable of infecting the male 

reproductive tract resulting, in some instances, in 

impaired fertility. In the testis, it is well known that 

viruses can induce pathological conditions such as 

orchitis, decrease in semen quality and may participate 

in the etiology of testicular cancer (Kondho, J Virol. 

1991), however the molecular mechanisms involved 

are still under investigation. The major role of Sertoli 

cells in the antiviral defence system is confirmed by 

data showing that they constitutively express several 

IFN-induced antiviral proteins, such as 2’5’ A synthetase, 

Mx2 and Mx1 proteins and PKR. Toll-like 

receptors (TLRs) recognize pathogen-associated 

molecular patterns (PAMPs) and elicit antimicrobial 

immune responses. We have previously demonstrated 

that mouse Sertoli cells play a key role in the bactericidal 

testicular defence mechanism by expressing a 

panel of TLRs (Riccioli, J Immunol. 2006). In order to 

better characterize the potential role of Sertoli cells in 

the response against testicular viral infections, we 

investigated the presence of TLR3, TLR7, and TLR9 

in primary Sertoli cell cultures from young mice. We 

observed that Sertoli cells significantly express TLR3 

mRNA and protein, whereas TLR7 and TLR9 are not 

expressed, as assessed by RT-PCR. 

Upon ligand binding, TLR3 interacts with the adaptor 

protein, TRIF (Toll/IL1-receptor domain con- 

Principal investigator: Elio Ziparo 

Professor of Embryology 


Tel.: (+39) 06 49766586; Fax: (+39) 0649766340 

elio.ziparo@uniroma1.it 

109 


The role of Toll Like Receptors in immune responses to infections 

and in inflammation associated pathologies of the male 

reproductive system 

taining adapter inducing IFNB1), which mediates 

the activation of NF-KB, mitogen activated protein 

kinases (MAPKs), and IFN regulatory factor 3 

(IRF3) as well as the induction of type I IFNs, 

cytokines critical to the host defence against viral 

infections. We reported that Sertoli cells, under 

stimulation with specific TLR3 agonist, the synthetic 

dsRNA analogue poly (I:C), produce the proinflammatory 

molecule ICAM-1 and secrete the 

functionally active chemokine CCL2. By using both 

pharmacological and genetic approaches, we report 

that these effects are TLR3-dependent. Moreover, 

by using ELISA, we show that IFNA is constitutively 

produced and not further inducible, whereas 

IFNB is absent but is strongly inducible by poly 

(I:C) only if it is transfected into cells. These data 

are in agreement with those of previous reports on 

different cell types (Milhaud, Bioconjug Chem. 1992) 

showing that poly (I:C), either microinjected or 

delivered with acid-sensitive liposomes, induces IFN 

production. These data indicate different control 

mechanisms underlying IFNA and IFNB production. 

To conclude, our results demonstrate that poly 

(I:C) elicits both inflammatory and antiviral 

responses in Sertoli cells. 

Since chronic infection and inflammation are strongly 

linked with the development and progression of 

carcinogenesis, a further topic of this project concerns 

the potential role of TLRs within the development 

and etiology of prostate cancer, the most common 

tumour in the male reproductive tract. So far, 

regarding the linkage between TLR activation and 

prostate cancer, in addition to epidemiological data 

linking chronic inflammation to increased cancer 

risk, genetic link between TLR and cancer also 

exists. In fact, sequence variants in Tlr gene cluster 

(Tlr6–Tlr1–Tlr10) and in Tlr4 are related to 

increased risk of human prostate cancer (Sun, J Natl 

Cancer Inst. 2005; Chen, Cancer Res. 2005), clearly 

indicating a role of TLRs in its aetiology. Such association 

of gene sequence variants and prostate can-

E. Ziparo - The role of Toll Like Receptors in immune responses to infections and in inflammation associated pathologies 

cer risk provides evidence for a role of TLRs in 

prostate cancer. Moreover, conflicting reports have 

been published claiming pro- or anti-tumoral effects 

of TLR9 agonist on human prostate cancer cell lines. 

Mounting evidence shows that the enhancement of 

innate and adaptive immunity represents the principal 

mechanism by which TLR stimulation produces 

antitumour activity. However, a direct pro-apoptotic 

effect of TLR agonists on TLR3 + and TLR9 + 

tumour cells has been recently reported (Salaun, J 

Immunol. 2006; Salaun, Clin. Cancer Res. 2007). Based 

on this evidence, in order to elucidate the role of 

TLRs in the etiology and pathogenesis of prostate 

cancer, we used as experimental model three different 

cell lines: the prostatic benign human hyperplasia 

cell line BPH-1, the androgen responsive cell line 

LNCaP and the androgen unresponsive PC3 cell line. 

Firstly, we investigated the effect of the TLR3 agonist 

poly (I:C) on the proliferative and apoptotic rates 

of LNCaP and PC3. We demonstrated that poly (I:C) 

elicits inhibition of proliferation associated with a 

significant induction of TLR3-mediated apoptosis in 

both prostate cancer cell lines. However, we found 

that LNCaP cells are very sensitive to poly (I:C)induced 

apoptosis, whereas PC3 cells, a more aggressive 

prostate cancer cell line, shows a significant 

resistance to this apoptotic stimulus. Among differently 

expressed genes in two cell lines, WT p53 and 

androgen receptor (AR) are expressed in LNCaP 

cells, whereas PC3 cells, , are null for both p53 and 

AR. The dependence of prostate tumour on AR 

activity is exploited in treatment of disseminated 

prostate cancers, wherein ablation of AR function 

induces the regression of prostate tumours mainly 

through increased apoptosis. Tumour progression is 

associated with inappropriately restored AR function, 

despite sustained androgen ablation and/or the 

110 

use of AR antagonists (Hsieh, Lancet Oncol. 2007) 

Since PC3 cells lack AR and p53 expression, the differences 

in response to poly (I:C) between LNCaP 

and PC3 cells might result from variations in the AR 

and p53 status of these cells. To determine whether 

these differences may account for the minor susceptibility 

of PC3 cells to poly (I:C)-induced effects, we 

performed apoptosis assay on PC3 cells stably transfected 

with AR plasmid or transiently transfected 

with p53 plasmid (PC3–p53) and treated with poly 

(I:C). Our results indicate that forced expression of 

AR in androgen-independent prostate cancer cell 

lines confers a higher sensitivity to poly (I:C)induced 

apoptosis, whereas no increment of apoptosis 

was observed in PC3–p53 cells. 

Finally, we identified a new IFN-B-independent 

pathway that involves protein kinase C (PKC)-alpha 

activation as responsible for the poly (I:C)-mediated 

apoptosis in LNCaP cell line. 


Dal Secco V, Riccioli A, Padula F, Ziparo E, Filippini 

A. Mouse Sertoli cells display phenotypical and functional 

traits of antigen-presenting cells in response 

to interferon gamma. Biol Reprod. 2008, 78: 234-42. 

Paone A, Starace D, Galli R, Padula F, De Cesaris P, 

Filippini A, Ziparo E, Riccioli A. Toll like receptor 3 

triggers apoptosis of human prostate cancer cells 

through a PKC-alpha-dependent mechanisms. 

Carcinogenesis 2008, 29:1334-42. 

Starace D, Galli R, Paone A, De Cesaris P, Filippini 

A, Ziparo E, Riccioli A. Toll-like receptor 3 activation 

induces antiviral immune responses in mouse 

Sertoli cells. Biol Reprod. 2008, 79: 766-75.

AREA6 

New 

antimicrobial 

and antiviral 

agents

Peptide effectors of innate immunity 


Maurizio Simmaco, Giuseppina Mignogna, professors; 

M. Luisa Mangoni, Rossella Miele, researchers; Marina 

Borro, Giovanna Gentile, post-doc fellows; Alessandra 

Franco, technician. 


Institute of Physiology and Pathophysiology, Paracelsus Medical 

University, Salzburg, Austria (Prof. Günther Kreil); Centro de 

Investigaciones Biológicas (CSIC) Madrid, Spain (Prof. Luis 

Rivas); Department of Biological Chemistry, Weizmann Institute 

of Science, Rehovot, Israel (Prof. Yechiel Shai). 


Beside searching for novel antimicrobial peptides 

from amphibian sources, we intend to exploit in 

more detail the peptide parameters responsible for 

the target selectivity of the peptides available in our 

laboratory and to select natural peptides or design 

analogues that, for their spectrum of activity, mechanism 

of action, stability, lack of toxicity, low induction 

of microbial resistance, may represent ideal 

compounds for in vivo studies to better evaluate 

their potential antimicrobial/anticancer effects. 

Recent studies performed in our laboratory have 

suggested that minor changes in the sequence of 

even small peptides like temporins can significantly 

affect their target selectivity and that both the 

cationic character, amphipathicity and peptide 

length are important determinants for the antibacterial 

potency of these molecules. 

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. 

Principal investigator: Donatella Barra 


Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" 

Tel: (+39) 06 4456663; Fax: (+39) 06 4440062 

donatella.barra@uniroma1.it 

113 

New antimicrobial and antiviral agents - AREA 6 

Amphibian skin is one of the richest sources for such 

molecules. In contrast with conventional antibiotics, 

most AMPs interact with 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 Gram-negative 

bacteria, is surrounded by the lipopolysaccharide 

(LPS)-outer membrane, which forms a very efficient 

barrier against a variety of hydrophilic and 

hydrophobic compounds. 

We first compared the in vitro bactericidal activities 

of five AMPs from three different species of anurans 

against multidrug-resistant clinical isolates 

belonging to species often involved in nosocomial 

infections (Staphylococcus aureus, Enterococcus faecium, 

Pseudomonas aeruginosa, Stenotrophomonas maltophilia 

and Acinetobacter baumannii). The peptides tested 

were: the short and mildly cationic temporins A, B, 

and G from Rana temporaria (13-residues long with 

a net charge of +3); the 1-18 fragment of esculentin 

1b [Esc(1-18)] from Rana esculenta; and the 

20-residues bombinin H2 from Bombina variegata. 

All these peptides were able to kill microorganims at 

concentrations ranging from 0.5 to 48 microM, with 

only few exceptions. In particular, temporins were 

more active against Gram-positive bacteria, especially 

when assayed in human serum; Esc(1-18) showed 

a fast and strong bactericidal activity against the 

Gram-negative species, at concentrations of 0.5-1 

microM; bombinin H2 displayed a similar potency 

towards all isolates. Interestingly, while in 20% 

human serum temporins and bombinin H2 were 

almost completely inhibited against the Gram-negative 

species, Esc(1-18) partially preserved its antimicrobial 

efficacy also in the presence of 40% serum. 

This property renders the latter peptide an attractive 

molecule for the development of new compounds for 

the treatment of infectious diseases. 

To address the issue that more than 10 isoforms of 

temporins are produced within the same frog specimen, 

we tested temporins A, B and L in combination

D. Barra - Peptide effectors of innate immunity 

with each other and found that the isomers A and B, 

which are only weakly active on Gram-negative bacteria, 

could synergize when combined separately 

with temporin L, overcoming the bacterial resistance 

imposed by the LPS protective layer. This effect 

depends on the length of the LPS sugar chain on the 

target microorganism. 

To understand the underlying mechanism, we investigated 

the effect of LPS from two strains of E. coli 

with different LPS moiety on the structural organization 

of temporins, alone and when mixed one with 

each other. Our data have indicated that the synergistic 

effect is related to the ability of temporin L to 

prevent the oligomerization of the A and B isomers, 

thus allowing their translocation across the bacterial 

cell wall into the target cytoplasmic membrane. 

Overall such studies have revealed two important 

findings: 

(i) temporins A and B are not active on Gram-negative 

bacteria, because of their oligomerization when 

in contact with the outer membrane: their larger size 

should interfere with the peptide diffusion through 

the cell wall into the target inner membrane; 

(ii) the synergistic activity between temporins on 

Gram-negative bacteria is related to the ability of 

temporin L to assist A and B in traversing the LPS 

layer by preventing their oligomerization. This effect 

is highly dependent on the LPS structure. 

In addition, we have shown that the same temporin 

combinations suppress the endotoxin effects of LPS, 

by inhibiting TNF-α release (considered to be a primary 

mediator of endotoxemia) from macrophages. 

Furthermore, by means of spectroscopic and thermodynamic 

studies, we have demonstrated that the 

synergism of temporins in the anti-endotoxin activity 

relies on the peptide’s ability to dissociate LPS 

aggregates to smaller sized vesicles and that this 

property is inversely related to the length of the 

polysaccharide region of LPS. 

Another interesting family of frog skin AMPs is 

given by bombinins H, isolated from amphibia of 

Bombina genus and containing isomers with a single 

D-amino acid, resulting from a post-translational 

epimerization of the corresponding L-residue. Indepth 

studies on the biological activity of these molecules 

and the underlying mode/s of action have 

shown that bombinin H2 (IIGPVL- 

GLVGSALGGLLKKI-NH 2 ) and H4, differing by 

114 

only the configuration of the second amino acid (an 

L-isoleucine in H2 and a D-alloisoleucine in H4) display 

antibacterial and anti-parasitic activities, with a 

stronger potency and faster killing kinetic of the Damino 

acid-containing bombinin H4. This peptide 

was also found to have a higher haemolytic capacity 

on human erythrocytes. In all cases, the isoforms H2 

and H4 were able to perturb the membrane of the 

target cells, causing leakage of large cytosolic components 

(e.g. proteins) or diffusion of smaller molecules 

through local membrane disruptions. 

We have therefore demonstrated the importance of a 

single L- to D-epimerization as a new approach 

developed by nature to modulate not only the bioavailability 

(e.g. higher solubility) and biostability 

(i.e. protection from proteolytic degradation) of 

AMPs, but also their biophysical properties (peptide 

structure and organization within membranes) and 

antimicrobial activities. 

These studies help getting insight into the molecular 

basis accounting for the quantitative difference in the 

biological activities of H2 and H4 epimers and the 

role of the D-amino acid in their different behaviour, 

and in general to have a deeper understanding of the 

mechanisms of action of these new potentially useful 

weapons to fight infections. 


Mangoni ML, Maisetta G, Di Luca M, Marcellini 

HGL, Esin S, Florio W, Brancatisano FL, Barra D, 

Campa M, Batoni G. Comparative analysis of bactericidal 

activity of amphibian peptide analogues 

against multidrug-resistant nosocomial bacterial 

strains. Antimicrob Agents Chemother. 2007, 52:85-91. 

Mangoni ML, Marcellini HGL, Simmaco M. 

Biological characterization and modes of action of 

temporins and bombinins H, multiple forms of short 

and mildly cationic anti-microbial peptides from 

amphibian skin. J Pept Sci. 2007, 13:603-13. 

Mangoni ML, Epand RF, Rosenfeld Y, Peleg A, 

Barra D, Epand RM, Shai Y. Lipopolysaccharide, a 

key molecule involved in the synergism between 

temporins in inhibiting bacterial growth and in 

endotoxin neutralization. J Biol Chem. 2008, 

283:22907-17.


Gustavo Portalone, professor; Cinzia Conti, Paola Goldoni, 

researchers. 


Istituto Superiore di Sanità, Roma (Prof. Maria Giovanna 

Quaglia, Dr. Elena Bossù). 


Picornaviruses, in particular enteroviruses (EVs) and 

rhinoviruses (HRVs), are responsible for several 

human viral diseases ranging from mild upper respiratory 

infections to fatal neurological or cardiacbased 

illnesses. Due to the widespread nature of the 

diseases associated with picornaviruses and the difficulty 

of vaccine development for the majority of 

these viruses, extensive efforts have been expended 

in the search for effective anti-picornavirus agents. 

However, despite the in vitro activity of several specific 

compounds, to date only few drugs have shown 

efficacy in humans and none have been approved for 

clinical use. 

The target of this search is to identify new lead compounds 

with potent activity, low cytotoxicity and 

broad spectrum of antipicornavirus activity. The 

study of the mechanism of action of compounds 

selected during the antiviral screening is a further 

aim of the project. 

In continuation of our search on antipicornavirus 

activity of flavanoids and related classes of 

inhibitors, we designed and synthesized new series 

of (Z)-3-benzylidenechromans, 3-benzylchromans 

and 3-benzyl-2H-chromenes related to the most 

active synthetic 3(2H)-isoflavenes and homoisoflavones 

previously studied by the participant of this 

project. The antiviral potency of the new synthesized 

compounds was evaluated against HRVs in a 

plaque reduction assay, starting from the maximum 

non-cytotoxic concentration (MNTC). The exis- 

Principal investigator: Nicoletta Desideri 

Professor of Drug Analysis 


Tel: (+39) 06 49913892; Fax: (+39) 06 491491 

nicoletta.desideri@uniroma1.it 

115 


New antipicornavirus flavanoids: synthesis, biological evaluation 

and structure-activity relationship studies 

tence of two groups (A and B) of HRVs with contrasting 

susceptibility for antiviral compounds suggests 

the selection of at least one serotype from 

each antiviral group. Group B contains twice as 

many serotypes as group A, and accounts for five 

times as many colds as group A serotypes. We utilized 

HRV 1B and 14 as representative serotypes for 

group B and A, respectively. 

With the exception of 3-benzyl-2H-chromene, all 

chromenes and chromans showed high antiviral 

activity against HRV 1B within micro or submicromolar 

range (IC 50s ranging from 0.11 µM to 6.62 

µM). Generally, the potent inhibitory activity on 

HRV 1B coupled with the low cytotoxicity resulted 

in compounds with high therapeutic index (TI). In 

contrast, only a modest inhibition of HRV 14 replication 

was observed up to the MNTC. 

(Z)-3-(4-chlorobenzylidene)chroman (1) 

On the basis of both high anti-HRV1B activity and 

therapeutic index (IC 50 = 0.12 µM and TI = 625), 

(Z)-3-(4-chlorobenzylidene)chroman (1) was chosen 

to clarify the mechanism of antiviral action by evaluating 

the effects produced either on virus particles or 

multiplication. Initially, the direct effect of 1 on 

HRV1B infectivity and stability was investigated. 

Afterwards, the antiviral activity of 1 (12 µM or 36 

µM) towards different stages of HRV1B multiplication 

was investigated under one-step growth conditions. 

Compound was continuously present during the 

entire time of virus replication, during virus binding 

to the cell membrane only or added or removed at different 

time intervals after virus adsorption in the cold. 

The overall analysis of data from inactivation and stabilization 

studies of HRV infectivity are consistent

N. Desideri - New antipicornavirus flavanoids: synthesis, biological evaluation and structure-activity relationship studies 

with 1 acting as a capsid-binder, although binding was 

reversible by dilution. Previous research on capsidbinding 

compounds demonstrated that optimum 

activity is associated with a high degree of occupancy 

of a pocket within the capsid protein VP1. Therefore, 

the difference in activity observed against HRV 1B 

and 14 could be attributed to the relative size of the 

binding site. In fact, the viral group B binding site 

accommodates shorter molecules, while longer compounds 

were routinely more active against serotype 

14. In agreement with the capsid-binder hypothesis, 

data from time of addition/removal studies indicate 

that 1 interferes with very early event(s) of virus 

infection process (Figure 1), as already reported for 

several structurally related compounds such as 3(2H)isoflavene. 

Moreover, the finding that an equal efficacy 

is achieved when 1 is present during the entire replication 

time or during virus binding only (Figure 2), 

strongly suggests that this compound exerts its 

antiviral effect by a direct interaction with virus parti- 

Fig. 1 - Effect of varying the time of removal of 1 (12 µM or 36 

µM) on the inhibition of HRV1B replication under one-step growth 

conditions. Virus yield was determined by plaque assay. Virus control 

titre was 3.8 . 10 5 PFU/mL. Compound was added after virus 

adsorption period (1 h at 4 °C, time 0) and removed at different 

lengths of incubation (0.5, 1, 2, 4 h). 

116 

cles during HRV attachment to the cell surface. This 

observation is further supported by the absence of a 

direct action on cell membrane receptors for virus in 

pre-treatment studies. 

To verify if the introduction of longer chains 

between phenyl and heterocyclic ring can produce a 

more efficient occupation of VP1 pocket, we synthesized 

new series of chromans and chromenes with 

linkers containing 2-4 carbon atoms. The antiviral 

tests are actually in progress. 


Desideri N. An efficient synthesis of 3-benzyl-2Hchromenes 

as potential antipicornavirus agents. 

Letters in Organic Chemistry 2007, 3:546-8. 

Conti C, Desideri N. Synthesis and antirhinovirus 

activity of new 3-benzyl chromene and chroman 

derivatives. Bioorg Med Chem, in press. 

Fig. 2 - Effect of 1 on HRV1B attachment to the cell surface and on 

membrane receptors for virus. Virus yield was determined by plaque 

assay after one cycle of virus growth (1 h at 4 °C and 10 h at 33 °C). 

CV: Virus control. 

A (12 µM) and D (36 µM): Cells exposed to 1 for 1 h at 4 °C 

before virus attachment (1 h at 4 °C). 

B (12 µM) and E (36 µM): Cells exposed to 1 during the time of 

virus attachment (1 h at 4 °C). 

C (12 µM) and F (36 µM): Cells exposed to 1 during virus attachment 

(1 h at 4 °C) and replication (10 h at 33 °C).


Roberta Costi, professor; Gaetano Miele, post-doc fellow; 

Giuliana Cuzzucoli Crucitti, PhD student; Federica Rosi, 

Alberto Iacovo, graduate students, Giovanni Santilli, technician. 


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); Katholieke Universiteit Leuven, Belgium (Prof. 

Christophe Pannecouque); Politechnika Lodzka, Poland (Prof. 

Grzegorz Bujacz). 


The HIV-1 RT-associated RNase H function is a validated 

and very attractive new target for HIV/AIDS 

drug development (De Clercq, J Med Chem. 2005, 

48:1297-1313; Tramontano, Mini Rev. Med Chem. 

2006, 6:727-37; Himmel et al., ACS Chem Biol. 2006, 

1:702-12); up to today no drug against the HIV-1 RTassociated 

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 (Himmel et al., ACS Chem 

Biol. 2006, 1:702-12). 

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 (Beutler et 

al., PCT Int. Appl. 2006, 2006026619 A2). BTDBA 

117 


Pyrrolyl diketo hexenoic acid derivatives as novel anti-HIV agents 

targeted to the ribonuclease H function of the HIV-1 reverse 

transcriptase enzyme 

Principal investigator: Roberto Di Santo 

Professor of Farmaceutical Chemistry and Toxicology 


Tel: (+39) 06 49913150; Fax: (+39) 06 491491 

roberto.disanto@uniroma1.it 

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-1H-pyrrol-2-yl)]-2,4-dioxo-5hexenoic 

acid ethyl ester (RDS1643) was recently 

reported by our research group. In enzyme assays, 

RDS1643 inhibited the HIV-1 RNase H activity with 

an IC 50 value of 13 µM, it did not affect neither the 

HIV-1 RDDP function nor the AMV and E. coli 

RNase H activity, while it slightly inhibited the HIV- 

1 IN reaction (IC 50 value of 92-98 µM) 

(Tramontano et al., Antiv. Res. 2005, 65:117-24). 

Noteworthy, in cell-based assays it was able to block 

the replication of wild type HIV-1, showing an EC 50 

value of 13 µM and a CC 50 value of 63 µM, and the 

replication of three HIV-1 non-nucleoside RT 

inhibitor (NNRTI) resistant viral mutants 

(RT mutations were Y181C; K103N/Y181C; 

K103R/V179D/P225H) showing EC 50 values of 7- 

19 µM (Tramontano et al., Antiv. Res. 2005, 65:117- 

24). Mode of action studies demonstrated that the 

RDS1643 maximum adsorbance shifted in the presence 

of the Mg 2+ ions. This results suggested that, 

similarly to BTDBA (Mizrahi et al., J. Biol. Chem. 

1994, 269: 19245-49), RDS1643 may sequestrate the 

active site divalent metals having a specific binding 

site on the HIV-1 RNase H domain (Tramontano et 

al., Antiv. Res. 2005, 65:117-24). 

More recently, BTDBA has been modeled into the 

HIV-1 RNase H active site assuming that the DKA 

triple-oxygen motif may interact with the protein 

active site metal ions (Klumpp & Mirzadegan, Curr. 

Pharm. Des. 2006, 12:1909-22). According to this 

model, its aromatic moiety may extend towards the 

W266, L422 and W426 amino acid residues on the 

p51 subunit. Consistently, further modeling studies 

proposed that RDS1643 may bind to the HIV-1 RNase

R. Di Santo - Pyrrolyl diketo hexenoic acid derivatives as novel anti-HIV agents 

H domain similarly to BTDBA (Klumpp & 

Mirzadegan, Curr. Pharm. Des. 2006, 12:1909-22). 

However, RDS1643 would not reach towards the p51 

subunit as far as BTDBA, showing therefore a less 

favourable interaction with RT, consistently with the 

lower potency of RDS1643 inhibition as compared to 

BTDBA inhibition (Tramontano et al., Antiv. Res. 

2005, 65:117-24; Klumpp & Mirzadegan, Curr. Pharm. 

Des. 2006, 12:1909-22). Other DKA derivatives, structurally 

similar to BTDBA and RDS1643, but lacking 

inhibitory activity against the HIV-1 RNase H function, 

were also modelled into the RNAse H active site 

showing steric hindrance with its domain; this data fitted 

with the lack of their biological activity. 

Since RDS1643 was originally designed as HIV-1 

IN inhibitor (Di Santo et al., Farmaco 2005, 60:409- 

17), a few RDS1643 derivatives were synthesized by 

our research group in the last year and were assayed 

on both HIV-1 RT-associated enzyme activities. The 

data coming form the enzyme assays showed that 

introduction of substituents in 4-position of the 

benzyl ring of RDS1643 did not affect the interaction 

with the biological target giving compounds 

with inhibitory potency comparable to that of 

RDS1643. This is consistent with the proposed 

118 

model of interaction between RDS1643 and the 

HIV-1 RNase H active site. 

Selected pubblications 

Di Santo R, Costi R, Roux A, Miele G, Crucitti GC, 

Iacovo A, Rosi F, Lavecchia A, Marinelli L, Di 

Giovanni C, Novellino E, Palmisano L, Andreotti M, 

Amici R, Galluzzo CM, Nencioni L, Palamara AT, 

Pommier Y, Marchand C. Novel quinolinonyl diketo 

acid derivatives as HIV-1 integrase inhibitors: 

design, synthesis, and biological activities. J Med 

Chem. 2008, 51:4744-50. 

Jegede O, Babu J, Di Santo R, McColl DJ, Weber J, 

Quiñones-Mateu M. HIV type 1 integrase inhibitors: 

from basic research to clinical implications. AIDS 

Rev. 2008, 10:172-89. 

Terrazas-Aranda K, Van Herrewege Y, Hazuda D, 

Lewi P, Costi R, Di Santo R, Cara A, Vanham G. 

Human immunodeficiency virus type 1 (HIV-1) integration: 

a potential target for microbicides to prevent 

cell-free or cell-associated HIV-1 infection. 

Antimicrob Agents Chemother. 2008, 52:2544-54.


Giovanna Simonetti, professor; Rino Ragno, researcher; 

Dante Rotili, Sergio Valente, Silvia Simeoni, post-doc fellows; 

Antonia Caroli, Giorgia Botta, Domenico Tarantino, 

PhD students. 


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). 


Design, synthesis, and biological validation of 

novel, class-selective HDAC inhibitors (HDACi) 

Class I-selective HDACi. Following our searches on 

class I-selective HDACi bearing a cinnamyl moiety 

we prepared some new cinnamyl hydroxamates as 

well as 2-aminoanilides (MS-275 analogues) by 

replacing the benzene ring with a heteroaromatic 

ring, and by adding a (hetero)aryl substituent at the 

2-aminoanilide group at the right head of the molecules. 

From preliminary data, they should be class Iselective 

inhibitors, and some of them were highly 

active as apoptotic and/or cytodifferentiating agents. 

Moreover, a new series of uracil-based 2aminoanilides 

as analogues of the corresponding 

hydroxamates have been prepared and characterized 

as HDACi. 

Class II-selective HDACi. About reactivation of HIV- 

1 expression in latent cellular reservoirs, we have no 

data for assessing a specific involvement of class I or 

class II HDACs for silencing. It has been reported 

that NF-κB p50-HDAC1 complexes constitutively 

bind the latent HIV LTR and induce histone deacetylation 

and repressive changes in chromatin structure 

Principal investigator: Antonello Mai 

Professor on Medicinal Chemistry 


Tel: (+39) 06 49913392; Fax: (+39) 06 491491 

antonello.mai@uniroma1.it 

119 


Design, synthesis and biological evaluation of small molecule 

epigenetic modulators: a novel approach for anticancer, 

antifungal and antiviral chemotherapy 

of the HIV LTR, changes that impair recruitment of 

RNA polymerase II and transcriptional initiation. 

Similarly, chromatin immunoprecipitation (ChIP) 

assays revealed that c-Myc, Sp1, and HDAC1 coexist 

in the same DNA-protein complex at the HIV promoter, 

and are absent from the promoter after VPA 

treatment in concert with histone acetylation, RNA 

polymerase II recruitment, and LTR expression. 

Thus, HDAC1 (a class I HDAC) seems to be surely 

involved in HIV-1 expression silencing at the promoter, 

but an involvement of class II HDACs can not 

be ruled out. In cancer therapy also, for example, the 

fact that class I HDAC enzymes are clinically relevant 

is still controversial and not absolute. Indeed, in 

2008 we have shown a specific regulation and activity 

of class II HDACs in human breast cancer cells, 

and in another paper we will propose a regulatory 

role for class II HDACs on class I enzyme activity in 

muscle cells. Thus, also in HIV-1 expression reactivation 

we could have somehow an involvement of 

class II HDACs and/or a regulation of the class I 

HDAC functions through the class II enzymes. To 

date, we screened a library of our HDACi showing 

different degrees of class/isoform selectivity against 

HIV-1 to reactivate the virus from its latent reservoirs, 

and we are publishing a brief report about the 

first obtained data. These data were also filed in a 

patent with the ISS. 

Sirtuin inhibitors. The human Sir2 ortholog, SIRT1, 

is a NAD+-dependent deacetylase implicated in a 

variety of important disease-related processes 

including silencing of p53, inflammatory response, 

cell defence and survival, and fatty acid metabolism. 

In a search for potent sirtuin inhibitors as apoptotic 

and/or cytodifferentiating agents, we prepared a 

series of sirtinol analogues, and the degree of inhibition 

was assessed in vitro using recombinant yeast 

Sir2, human SIRT1, and human SIRT2, and in vivo 

with a yeast phenotypic assay. Two analogues, namely 

3- and 4-[(2-hydroxy-1-naphthalenylmethyl-

A. Mai - Design, synthesis and biological evaluation of small molecule epigenetic modulators 

ene)amino]-N-(1-phenylethyl)benzamide (ie, metaand 

para-sirtinol) were from 2- to 10-fold more 

potent than sirtinol against human SIRT1 and 

SIRT2 enzymes. Among the prepared compounds, 

obtained by replacement of the benzamide linkage of 

the prototype with other bioisoster groups such as 

anilide, sulfonamide, sulfide, 1-oxopropyl groups, 

salermide (N-{3-[(2-hydroxynaphthalen-1-ylmethylene)amino]phenyl}-2-phenylpropionamide) 

emerged as a sirtuin inhibitor with a strong cancerspecific 

proapoptotic effect, due to the reactivation of 

proapoptotic genes epigenetically repressed exclusively 

in cancer cells by SIRT1. 

Design, synthesis, and biological evaluation 

of histone methyltransferase inhibitors 

Protein arginine N-methyltransferases (PRMTs) and 

histone lysine N-methyltransferases (HKMTs) have 

been implicated in a variety of processes, including 

biosynthesis, nuclear receptor-regulated transcription, 

signal transduction, chromatin regulation, gene 

silencing, protein repair, and protein trafficking. 

PRMTs are known coactivators for nuclear receptors, 

so they may represent likely candidates to be overexpressed 

in the hormone-dependent prostate and breast 

cancers. Among HKMTs, SET7/9 has been reported 

to act not only on histones, but it also methylates the 

tumor suppressor p53 leading to gene silencing. 

In an effort to find small molecules that could represent 

lead compounds MT inhibitors, we designed 

and synthesize a series of compounds bearing two 1hydroxy-2,6-dibromophenyl 

moieties connected by a 

spacer. We hypothesized that the 1-hydroxy-2,6dibromophenyl 

group could act as a pharmacophore 

in these molecules; in fact the same or similar groups 

are present in eosin, recently reported as PRMTi, 

and in psammaplins, a series of natural compounds 

endowed with anti-HDAC and anti-DNMT activi- 

120 

ties. Some of the prepared compounds were effectively 

active against PRMTs, others were able to 

selectively inhibit a limited range of MTs (for example 

against CARM1 or EZH2), and others behaved as 

epigenetic multiple ligands, by inhibiting at the same 

time and in the same range sirtuins (SIRT1 and -2), 

HAT (p300), PRMTs and HKMTs. These last derivatives 

displayed high apoptotic and/or cytodifferentiating 

properties, higher than those shown by the 

related single-target inhibitors. 


Mai A, Jelicic K, Rotili D, Di Noia A, Alfani E, 

Valente S, Altucci L, Nebbioso A, Massa S, 

Galanello R, Brosch G, Migliaccio AR, Migliaccio 

G. Identification of two new synthetic histone 

deacetylase inhibitors that modulate globin gene 

expression in erythroid cells from healthy donors 

and patients with thalassemia. Mol Pharmacol. 2007, 

72:1111-23. 

Colussi C, Mozzetta C, Gurtner A, Illi B, Rosati J, 

Straino S, Ragone G, Pescatori M, Zaccagnini G, 

Antonini A, Minetti G, Martelli F, Piaggio G, 

Gallinari P, Steinkulher C, Clementi E, Dell'Aversana 

C, Altucci L, Mai A, Capogrossi MC, Puri PL, 

Gaetano C. HDAC2 blockade by nitric oxide and histone 

deacetylase inhibitors reveals a common target 

in Duchenne muscular dystrophy treatment. Proc 

Natl Acad Sci USA 2008, 105:19183-7. 

Mai A, Cheng D, Bedford MT, Valente S, Nebbioso 

A, Perrone A, Brosch G, Sbardella G, De Bellis F, 

Miceli M, Altucci L. Epigenetic multiple ligands: 

mixed histone/protein methyltransferase, acetyltransferase, 

and class III deacetylase (sirtuin) 

inhibitors. J Med Chem. 2008, 51:2279-90.


Livia Di Renzo, researcher; Giulia Matusali, PhD student; 

Alessandra De Leo, Giuseppe Arena, graduate students; 

Egidio Lacanna, Claudia Stecca, Valentina Scalise, undergraduate 

students. 


The Wistar Institute, Philadelphia, USA (Prof. Paul M. Lieberman). 


Resveratrol (3,4’,5-trihydroxy-trans-stilbene), a 

polyphenolic natural product present in grapes and 

other fruits, has been shown to possess chemopreventive 

properties against several cancers, heart diseases, 

ischemic injuries and inflammation. Moreover, 

increasing evidence in the literature indicate that 

resveratrol is able to exert anti-viral activity by interfering 

with several intracellular signaling and to synergistically 

enhance the effects of antiviral drugs. 

In this study we are investigating the effects of 

resveratrol on Epstein Barr Virus (EBV) infection. 

EBV, the ethiologic agent of infectious mononucleosis, 

is associated with several types of malignancies 

of epithelial and lymphoid origin including nasopharyngeal 

carcinoma (NPC), Hodgkin’s lymphoma, 

Burkitt’s lymphoma and many lymphoproliferative 

diseases arising in immunocompromised individuals. 

In each of these tumors, the selective expression of 

six nuclear antigens (EBNA1, 2,3A, 3B, 3C and LP) 

and three membrane associated proteins 

(LMP1,LMP2a and 2b ) characterize different forms 

of latency programs (I, II and III). The lytic phase, 

ending with the production of new viral particles, 

expands the pool of infected cells favoring the 

spread of viral infection. 

For the purposes of this project, EBV-infected cell 

lines, showing different latency programs, have been 

treated for 24 hours with 30 µg/ml of resveratrol, a 

concentration chosen because able to affect cell pro- 

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 

121 


Effects of resveratrol on Epstein-Barr Virus latent and lytic phases 

of infection 

liferation without causing significant loss of cell viability 

or marked morphological alterations. Burkitt’s 

lymphoma-derived Raji and Jijoye cells (latency III), 

Akata (latency I) and Hodgkin’s derived RPMI6666 

cells (latency II) have been incubated with resveratrol 

and the effects on the cell cycle have been studied 

by analyzing in flowcitometry the distribution of 

the cell population in the different phases, as well as 

by measuring in Western blot the expression levels 

of molecules functioning as regulators of the cell 

cycle. We have found that the polyphenol affects cell 

cycle progression by blocking Raji and Jijoye cells in 

the G1 phase and by inducing apoptosis (sub G1 

events) in Akata and RPMI 6666 cells. To evaluate 

the extent of apoptosis, the percentages of annexin 

V-positive cells have been determined; we found that 

after 24 hours incubation, resveratrol induced apoptosis 

in all cell lines with percentages varying 

between 30 to 60%. In agreement with the cytofluorimetric 

data, the levels of the cell cycle regulators 

CDK1, CDK2 and cyclin A dramatically decreased 

while cyclin-dependent-kinase inhibitors p27 was 

strongly up-regulated. To investigate whether the 

resveratrol exerts a direct effect on EBV expression, 

we measured the levels of transcription of EBV 

latent genes in the cells treated with the polyphenol. 

The results of RT-PCR experiments carried out on 

four EBV-positive cell lines incubated for up to 24 

hours with resveratrol indicated that latent EBV 

genes were strongly down-regulated independently 

of the latency program, while the levels of the b2microglobulin 

remained unchanged. From these 

results it is possible to conclude that resveratrol is 

able to affect both cell cycle progression of the host 

cell, as well as EBV latent gene expression. 

While a very restricted subset of tumor cells produces 

virions by lytic infection, EBV productive cycle 

represents the main EBV infection pattern in the 

plasmacells of patients with infectious mononucleosis. 

In vitro, EBV lytic cycle can be induced by the 

addition to the cells of TGFb, sodium butyrate,

E. Mattia - Effects of resveratrol on Epstein-Barr Virus latent and lytic phases of infection 

P(BU)2 or anti Ig. Following EBV activation, two 

major viral genes are expressed: BZLF1 and BRLF1. 

BZLF1 product is a transcriprion factor and together 

with BRLF1 product starts the lytic cascade that 

leads to viral DNA replication and production of 

viral particles. 

We examined the effects of resveratrol on EBV lytic 

infection by exposing to the polyphenol cells treated 

with EBV lytic cycle-inducing compounds. We found 

that EBV activation was largely prevented by resveratrol 

as judged by immunofluorescence detection 

of EBV early antigens expression. This result was 

confirmed by the drastic reduction of the immediate 

early antigen BZLF1 expression detected by western 

blot analysis; moreover, the increment of the latent 

protein LMP1, usually over-expressed during the 

early phases of EBV lytic cycle activation, was significantly 

reduced (Fig.1). In several types of cancer 

cells, treatment with resveratrol resulted in cell cycle 

arrest, induction of p53 tumor suppressor protein 

and apoptosis. Similarly, resveratrol up-regulated the 

tumor suppressor p53 protein both in latently-infected 

cells, as well as in cells treated with EBV lytic cycle 

activators. Cumulatively, these data indicate that 

resveratrol (i) arrests cell cycle and induces apoptosis 

of EBV-infected cells (ii) impairs expression of latent 

EBV genes (iii) suppresses EBV lytic cycle activation. 

Based on these results, experiments are currently carried 

out to gain insights into the molecular mechanisms 

by which resveratrol affects EBV expression in the two 

phases of infection and to identify the viral and cellular 

factors involved in cell cycle arrest and apoptosis. 


Fig.1 - Effects of resveratrol on EBV lytic cycle activation. 

122 

Galletti R, Masciarelli S, Conti C, Matusali G, Di 

Renzo L, Meschini S, Arancia G, Mancini C, Mattia E. 

Inhibition of Epstein Barr Virus LMP1 gene expression 

in B lymphocytes by antisense oligonucleotides: 

uptake and efficacy of lipid-based and receptor-mediated 

delivery systems. Antiviral Research 2007, 74:102-10. 

Matusali G, De Leo A, Gavioli R, Bertelli L, Di 

Renzo L, Mattia E. Down-regulation of proteolytic 

complexes following EBV activation in BL cells. 

Biochem Biophys Res Commun. 2007, 352:947-52. 

Mattiussi S, Tempera I, Matusali G, Mearini G, 

Lenti L, Fratarcangeli S, Mosca L, D’Erme M, 

Mattia E. Inhibition of Poly(ADP ribose)polymerase 

impairs Epstein Barr Virus lytic cycle progression. 

Infect Agents Cancer 2007, 2:18.


Marino Artico, Roberto Di Santo, Roberta Costi, professors; 

Giuseppe La Regina, Rino Ragno, researchers; 

Gabriella De Martino, post-doc fellow; Antonio Coluccia, 

Francesco Piscitelli, PhD students. 


Università di Roma Tor Vergata (Prof. Alberto Bergamini, Dr. 

Chiara Ciaprini, Dr. Anna Sinistro); Istituto di Genetica 

Molecolare, CNR, Pavia (Dr. Giovanni Maga, Dr. Emmanuele 

Crespan); NCI at Bethesda, NIH, USA (Prof. Yves Pommier); 

Rega Institute for Medical Research, K.U., Leuven, Belgium (Dr. 

Jean Balzarini); Welsh School of Pharmacy, Cardiff University, 

UK (Dr. Andrea Brancale, Dr. John Disson, Dr. Maria Chiara 

Barbera); NCI at Frederick, NIH, Maryland, USA (Prof. Ernest 

Hamel, Dr. Michael C. Edler). 


Anti-HIV-1 Agents 

Human immunodeficiency virus (HIV) is the etiological 

agent of acquired immunodeficiency syndrome 

(AIDS). More than 20 antiretroviral drugs 

are available and fall into six classes: nucleoside and 

nucleotide reverse transcriptase inhibitors (NRTIs), 

non-nucleoside reverse transcriptase inhibitors 

(NNRTIs), protease inhibitors (PIs), fusion 

inhibitors (FIs), integrase inhibitors (INIs), and 

CCR5 coreceptor antagonist. Three (recommended) 

or four antiretroviral drugs are combined in the 

highly active antiretroviral therapy (HAART) that 

proved to be effective in reducing morbidity and 

mortality of HIV-infected people. However, 

HAART is unable to eradicate the viral infection; 

the needed long-term or permanent treatments 

favor the emergence of drug resistance, toxicity, and 

unwanted side effects. 

Principal investigator: Romano Silvestri 

Professor of Medicinal Chemistry 


Tel: (+39) 06 49913800; Fax: (+39) 06 491491 

romano.silvestri@uniroma1.it 

123 


Indole nucleus as a selected pharmacophore for the design of novel 

highly potent anti-viral agents active against HIV-1 (RT and IN 

inhibitors) and also capable to inhibit HCV and tumor cell replication 

Non-Nucleoside Reverse Transcriptase 

Inhibitors. 

NNRTIs have received a huge attention because of 

low toxicity and favorable pharmacokinetics properties, 

being four NNRTIs approved for AIDS 

treatment (nevirapine, delavirdine, efavirenz, and 

etravirine). However, the emergence of drug 

resistance remains a pressing problem. Our studies 

on sulfone NNRTIs led to the development of 

potent indolylarylsulfones (IASs). Continuing our 

efforts to develop IASs with improved activity 

against the viral mutants, we planned the synthesis 

of new derivatives bearing two halogen atoms at 

the indole ring. New IASs were obtained starting 

from the appropriate ethyl pyruvate phenylhydrazone, 

obtained according to the method of Japp- 

Klingemann, that was treated with polyphosphoric 

acid to furnish the corresponding ethyl 1H-indole- 

2-carboxylate (Fischer’s indole synthesis). 

Reaction of the indole ester with the appropriate 

phenylthiosuccinimide in the presence of boron 

trifluoride diethyl etherate afforded the corresponding 

ethyl 3-(phenylthio)-1H-indole-2-carboxylate, 

that was oxidated to sulfone and transformed 

into carboxamide by treating with 3-chloroperoxybenzoic 

acid and ammonium hydroxide, respectively. 

IASs bearing the 4,5-difluoro or 5-chloro-4-fluoro 

substitution pattern at the indole ring were 

potent inhibitors of HIV-1 WT and the NNRTIresistant 

strains Y181C and K103N-Y181C. These 

compounds were highly effective against the 112 

and the AB1 strains in lymphocytes and inhibited 

at nanomolar concentration the multiplication of 

the IIIBBa-L strain in macrophages. 5-Chloro-3- 

(3,5-dimethylphenylsulfonyl)-4-fluoro-1H-indole- 

2-carboxamide was exceptionally potent against 

RT WT and RTs carrying the K103N, Y181I, and 

L100I mutations. The synthesis of new derivatives 

characterized by natural and unnatural aminoacids 

at the 2-carboxamide and different electron-with-

R. Silvestri - Indole nucleus as a selected pharmacophore for the design of novel highly potent anti-viral agents active against HIV-1 

drawing substituent(s) at position(s) 4 and 5 of the 

indole is currently in progress. 

Inhibitors of Tubulin Polymerization. 

Microtubules (MTs) are essential cytoskeletal polymers 

that are made of repeating α, β-tubulin (TB) heterodimers 

and are present in all eukaryotes. The crucial 

role of MTs in vital cellular functions for tumor 

cells, including mitosis, motility and cell-cell contacts, 

has made of MT interfering agents (MIAs) one of the 

best classes of cancer chemotherapeutic drugs available 

to date. A large number of chemically diverse 

compounds are able to bind TB or MTs and inhibit 

proliferation by acting on the mitotic spindle. Some of 

these compounds (vinca alkaloids, colchicine) inhibit 

MT polymerization, whereas others (taxanes) stabilize 

MTs. While drugs that act on the vinca and taxane 

sites have well-established roles in the treatment 

of human cancers, the therapeutic potential of the 

colchicine site in cancer treatment has yet to be realized. 

Recently, we have reported arylthioindoles 

(ATIs) as a new class of potent inhibitors of tubulin 

polymerization and the growth of some carcinoma 

cells that bind to colchicine site on β-tubulin close to 

its interface with α-tubulin. Fist generation ATIs 

were characterized by (i) an ester function at position 

2 of the indole nucleus; (ii) a 3-arylthio group; (iii) an 

appropriate substituent on the indole nucleus. 

Focusing our attention on amino derivatives related to 

combretastatin A-4, we designed and synthesized second 

generation of ATI derivatives, bearing a hydrogen 

atom or a methyl group at position 2 of the indole 

nucleus. New ATIs were synthesized by a two-step 

procedure. O-Ethyl-S-(3,4,5-trimethoxyphenyl) carbonodithioate 

was transformed into 3,4,5trimethoxythiophenol 

by heating at 65 °C in aqueous 

ethanol in the presence of sodium hydroxide. This 

mixture was made acidic with 6 N HCl and treated at 

25°C with the appropriate indole while adding dropwise 

an aqueous iodine-potassium iodide solution. The 

new compounds strongly inhibited tubulin polymerization, 

with activity in the low micromolar range, 

comparable to the effects of colchicine and combretastatin 

A-4. Derivatives bearing a halogen atom or a 

small alkyl or ether group at position 5 of the indole 

nucleus, were also potent inhibitors of MCF-7 

124 

cell growth. For example, 5-bromo-3-[(3,4,5trimethoxyphenyl)thio]-1H-indole 

showed tubulin 

polymerization IC 50 = 1.6 µM and growth of MCF- 

7 cells IC 50 = 43 nM, and inhibited [ 3 H]colchicine 

binding at 65%. Cell cycle analysis revealed an accumulation 

of HeLa cells in the G2/M phase at 24 h and 

polyploidization at 48 h. At 24 h, inhibition of tubulin 

polymerization had not caused extensive apoptosis, 

suggesting that impaired cell viability might occur 

through a “mitotic catastrophe”. Molecular modelling 

studies showed that, despite the absence of the ester 

moiety, the second generation ATIs bind into the 

colchicine site in the same orientation as the first generation 

ones. Binding to β-tubulin involved formation 

of a hydrogen bond between the indole and Thr179 

and positioning of the trimethoxy phenyl group in a 

hydrophobic pocket near Cys241. The synthesis of 

new derivatives obtained by bioisosteric replacement 

of the sulfur bridge with either carbonyl or methylene, 

and some related groups, and the indole nucleus 

with differently substituted pyrrole and imidazole 

rings is currently in progress. 


La Regina G, Coluccia A, Piscitelli F, Bergamini A, 

Sinistro A, Cavazza A, Maga G, Samuele A, Zanoli S, 

Novellino E, Artico M, Silvestri R. Indolyl aryl sulfones 

as HIV-1 non-nucleoside reverse transcriptase 

inhibitors: role of two halogen atoms at the indole 

ring in developing new analogues with improved 

antiviral activity. J Med Chem. 2007, 50:5034-8. 

La Regina G, Edler MC, Brancale A, Kandil S, 

Coluccia A, Piscitelli F, Hamel E, De Martino G, 

Matesanz R, Díaz JF, Scovassi AI, Prosperi E, 

Lavecchia A, Novellino E, Artico M, Silvestri R. 

Arylthioindole inhibitors of tubulin polymerization. 

3. Biological evaluation, structure-activity relationships 

and molecular modeling studies. J Med Chem. 

2007, 50: 2865-74. 

Ragno R, Coluccia A, La Regina G, Silvestri R. 

Indolyl aryl sulphones as HIV-1 reverse transcriptase 

inhibitors: docking and 3D QSAR studies. Exp 

Opin Drug Discovery 2007, 2:87-114.

AREA7 

Biology of 

malaria 

and other 

vector-borne 

diseases


Maria Angela Di Deco, researcher; Beniamino Caputo, 

Emiliano Mancini, Marco Pombi, Federica Santolamazza, 

post-doc fellows; Maria Calzetta, Erika Rossi, graduate students. 


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); 

Department of Ecology and Evolutionary Biology, Yale University, 

New Haven CT, USA (Prof. Jeffrey Powell, Prof. Adalgisa 

Caccone); Département de Biologie Animale, Faculté des Sciences et 

Techniques, Université de Dakar, Sénégal (Dr. Lassana Konate); 

Department of Biochemistry, Virginia Polytechnic Institute and State 

University, Blacksburg VA, USA (Prof. Zhijian Tu); 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); Malaria 

Research and Training Center, University of Bamako, Mali (Dr. Sékou 

Traoré); Medical Research Council Laboratories, Fajara, Banjul, The 

Gambia (Prof. David Conway, Dr. Davis Nwakanma); Medical 

Entomology Unit, Pasteur Institute, Dakar, Sénégal (Dr. Ibrahima 

Dia); Ministry of Health, National Program of Malaria Control, 

Luanda, Angola (Dr. Filomeno Fortes); Natural Resources Institute, 

University of Greenwich, Chatham, UK (Dr. Gabriella Gibson, Dr. 

Steve J. Torr); Vector Group, Liverpool School of Tropical Medicine, 

Liverpool, UK (Dr. Martin J. Donnelly). 


The following bionomical, genetic and molecular 

studies on Anopheles gambiae populations have been 

carried out. 

Geographical distribution of species and 

molecular forms of the A. gambiae complex 

The geographical distribution of species and molecular 

forms of the A. gambiae complex has been 

127 

Biology of malaria and other vector-borne diseases - AREA 7 

Bionomical, genetical and molecular characterization of 

populations of the Anopheles gambiae complex of sibling species 

(Diptera: Culicidae), malaria vector in sub-Saharan Africa 

Principal investigators: Alessandra della Torre - Vincenzo Petrarca 

Professors of Parasitology 

Dipartimento di Scienze di Sanità Pubblica; Dipartimento di Genetica 

e Biologia Molecolare 

Tel: (+39) 06 49694268; 06 49914932; Fax: (+39 ) 06 49914653 

ale.dellatorre@uniroma1.it; vincenzo.petrarca@uniroma1.it 

analysed, particularly in 2 geographical areas at the 

extremes of A. gambiae s.s range in West/Central 

Africa, where pertinent literature data were scarce 

and dated. i) 1,336 specimens were sampled across 

Angola from 2001 to 2005: M-form predominated in 

localities of the tropical dry and semi-desertic belts, 

where unexpectedly no A. arabiensis was found, while 

the S-form predominated in comparatively more 

humid and less anthropized sites; A. melas was found 

in northern coastal sites. ii) Over 4,000 specimens 

were collected and identified in dry season 2005 and 

rainy season 2006 along the Gambia river in The 

Gambia and in Senegal; M-form was mainly found in 

sympatry with A. melas and S-form in the western 

part of the transect, and with A. arabiensis in the central 

part; S-form was found to prevail in rural 

Sudano-Guinean savanna areas of Eastern Senegal, 

in sympatry with A. arabiensis; A. melas and A. arabiensis 

relative frequencies were generally lower in the 

rainy season samples, while no large seasonal fluctuations 

were observed for M and S-forms; in areas 

where both M and S were recorded, an unusual high 

frequency (1%-7%) of MxS hybrids was observed. 

Chromosomal characterization of species and 

molecular forms of the A. gambiae complex 

Half-gravid female samples from Angola and 

Senegambia were scored for inversion polymorphisms. 

In Angola, A. gambiae M and S forms were 

characterized by a low degree of polymorphism, 

based solely on inversion 2La, a pattern usually associated 

with populations from forested and humid 

savanna areas. In Senegambia, the populations were 

characterized by: i) 2Rb, 2Rd and 2La polymorphisms 

in both forms in the coastal area; ii) higher 

frequencies of 2Rb and 2La inverted arrangements 

in M-form from flooded and rice field areas in the 

central part of the transect, where S-form was not 

found; iii) presence of S-form populations in eastern 

sites characterised by higher genetic complexity due 

to additional inversions on chromosome-2R.

Della Torre - Petrarca - Bionomical, genetical and molecular characterization of populations of the Anopheles gambiae complex 

Furthermore, we analysed a database of all A. gambiae 

s.s. karyotyped by the group in the last 30 years, to 

evaluate evolutionary patterns based on rare chromosomal 

inversions (RCIs), which were recorded but not 

specifically analysed so far. Among >7,300 females 

from 16 Afrotropical countries, 82 RCIs were recorded 

in 160 specimens: 23% were found repeatedly, in 

the same sample and/or at the same sampling location 

across different years and/or in different sampling 

locations, while the others only once in single 

specimens. The analysis of breakpoint distribution of 

RCIs showed that these, like common inversions, are 

disproportionately clustered on 2R, which may indicate 

that this chromosomal arm is especially prone to 

breakages. However, RCIs were equally frequent 

across biomes and on both sides of the Great Rift 

Valley (GRV), whereas common inversions predominated 

in arid ecological settings and west of the GRV. 

We propose that RCIs are subject mainly to drift 

under unperturbed ecological conditions, but may 

represent an important reservoir of genetic variation 

for A. gambiae in response to environmental changes. 

Molecular karyotyping of chromosomal 

inversions in A. gambiae 

We developed 2 PCR-based approaches allowing the 

identification of the alternative arrangements of 2La 

and 2Rj polymorphic inversions of A. gambiae s.s. The 

first method was validated on 765 specimens sampled 

across Africa and was shown to be specific and efficient, 

thus providing groundwork for future studies 

on the non-random distribution of 2La-carriers. The 

second method was validated on >700 field specimens: 

it was shown to be robust and accurate on 2R+ j and 

2Rj homozygotes, thus providing the first molecular 

approach for the rapid identification of the BAMAKO 

form across developmental stages and sexes, opening 

new perspectives for studies on the bionomics of this 

A. gambiae chromosomal form. 

Molecular characterization of A. gambiae 

molecular forms 

We analysed the insertion polymorphism of a nearly 

200 bp-long SINE (SINE200) within genome areas 

of high differentiation (i.e. “speciation islands”) of M 

and S A. gambiae molecular forms. SINEs (Short 

INterspersed Elements) are homoplasy-free and codominant 

genetic markers which represent useful 

tools for population genetic studies. Eight loci were 

successfully amplified and found to be specific for A. 

gambiae s.s.: 5 on chromosome 2L and one on Xchromosome 

turned out to be monomorphic, while 

two loci were found to be polymorphic. 1) S200 

128 

2R12D was homozygote for the insertion in most Sform 

samples, while intermediate levels of polymorphisms 

were shown in M-form, resulting in an overall 

high degree of genetic differentiation between 

molecular forms (Fst=0.46, P


Paolo Marcatili, Domenico Cozzetto, post-doc fellows, 

Emanuela Giombini, PhD student. 


Istituto Superiore di Sanità, Roma (Dr. Enrica Pizzi, Dr. Pietro 

Alano) 


The aim of the project is to use computational biology 

tools to predict the function and structure of 

Plasmodium proteins involved in key biochemical 

processes, predict the structure of the malaria and 

human proteins involved in erythrocyte invasion, 

model their mode of interaction. 

In this first year we concentrated on predicting the 

interaction of the PfEMP1 Malaria Protein and the 

Human ICAM-1 Receptor and on predicting the 

structure of human glycophorins A and B, known to 

play a role in Plasmodium infection. 

Glycophorins are a group of transmembrane sialoglycoproteins 

expressed on the surface membrane of 

human cells. There are 5 different glycophorins in 

humans, all very similar, and likely to derive by 

duplications of an ancestral gene. 

GPA and GPB are the glycophorins most expressed 

on erythrocytes, they are responsible for the different 

blood antigen present in different people and are the 

proteins more directly involved in the interaction 

with plasmodium. 

GPA is a protein of 131 aa, while GPB is shorter, 

about 91 aa. Both proteins are formed by three structural 

domains (an extra-cellular, a transmembrane 

and a cytoplasmatic domain) and both undergo posttranslational 

modification: the proteolytic cleavage of 

a signal peptide of 19 aa and the addition of specific 

sugars. GPA has 16 oligosaccharides chain, 15 O-glycosidic 

bond linked to threonine/o/serine, and one 

linked to asparagine with C-glycosidic bond 3 . It is 

129 

Biology of malaria and other vector-borne diseases - AREA 7 

Computational Analysis of the gene products of the Plasmodium 

falciparum genome and their interaction with human proteins 

Principal investigator: Anna Tramontano 



Tel: (+39) 06 49910556; Fax: (+39) 06 49910717 

anna.tramontano@uniroma1.it 

known that GPA binds EBA-175 (erythrocyte binding 

protein-175) a transmembrane protein of plasmodium, 

while this is not the case for GPB. 

Plasmodium recognition in mediated by the extracellular 

region where GPA and GPB are very similar, 

except for the 4th exon. It is therefore likely that 

binding regions are mostly located in this exon. 

Binding of GPA with EBA-175 seems to be mainly 

mediated by Sialic Acid, since it is inhibited when 

cells are treated with neuramidase, an enzyme that 

cleaves the sugar from the protein, however the protein 

moiety is expected to be involved in the recognition 

process with its fourth exon. There is a crystallographic 

structure for the GPA transmembrane 

region (PDB id: 1AFO), but this region is clearly not 

involved in plasmodium recognition. Clearly the sugars 

are the most important players in GPA-EBA-175 

binding. Unfortunately, it is very difficult to predict 

their precise conformation with our present knowledge. 

However, they are bound to the proteic moiety 

and a 3D model of the latter can help investigating 

the possible modes of interaction. Consequently, we 

built 3D models for GPA and GPB. 

GPA and GPB three-dimensional models 

GPA and GPB, even though sharing a high sequence 

identity, show some remarkable differences from a 

functional and molecular point of view. We inferred 

the three-dimensional structures for GPA and GPB 

independently, and then compared them in order to 

highlight structural dissimilarities that could at least 

partially explain the different behavior of the two 

proteins. Solved structures of the cytoplasmatic and 

membrane domains of GPA and GPB are available, 

so we focused only on the extracellular domain. Most 

of the differences between GPA and GPB are located 

in this domain, which is responsible for the 

EBA175 binding as well. 

Using a de novo prediction protocol, we were able to 

produce models for both GPA and GPB. These models, 

even if with a different degree of reliability, are

A. Tramontano - Computational Analysis of the gene products of the Plasmodium falciparum genome 

consistent with most of the experimental data reported 

in the literature, and can be a starting point to 

unravel the specificities and the mechanisms adopted 

by P. falciparum proteins to invade the erythrocytes. 

GPA has no detectable similarity with any protein of 

known structure and therefore the standard homology-modeling 

protocols cannot be applied in this case. 

An analysis of the predicted secondary structure 

(PSIPRED) and of the disordered regions (DISO- 

PRED) of such proteins did not reveal the presence 

of unstructured or disordered regions. Therefore 

we decided to adopt a de novo strategy to predict the 

three-dimensional structure of the proteins. To this 

aim we used the Rosetta suite with the following 

strategy: 

1. We generated 10.000 decoys for each protein (de 

novo modeling followed by a full-atom “relax” 

refinement); 

2. selected the 1.000 decoys with the lowest score; 

3. clustered the 1.000 decoy subset, 

4. Selected the lowest-scoring decoy in the largest 

cluster as a structural candidate. 

This strategy produced excellent result for GPB. We 

found a very large cluster (250 decoys, RMSD of 

1.72 Å) with several low-score decoys. In the Figure 

we report the lowest score model, which is a single 

beta sheet formed by three antiparallel strands. This 

model is coherent with literature data (most of the 

glycosilation sites are located on loops and exposed 

to the solvent) and the deleted region (with respect 

to GPA) is on the loop that connects the second and 

the third strands. 

Fig. 1 - Predicted structure of GPA 

130 

The same protocol, when applied to GPA, did not 

produce a single, reliable model. No cluster could be 

detected with the given threshold, so we decided to 

analyze the three lowest-score models. Even though 

some of the strands identified in GPB are conserved, 

there are regions of the GPA models without 

a clear secondary structure. Such a big difference 

between the models for the two proteins was 

unexpected, given the high sequence identity 

between GPA and GPB, the short dimension of the 

insertion present in GPA (approx. 30 residues) and 

the quality of the GPB model prediction and 

deserves further studies. 


Cozzetto D, Kryshtafovych A, Ceriani M, 

Tramontano A. Assessment of predictions in the 

model quality assessment category. Proteins 2007, 

8:175-83. 


R, Tramontano A, Bolotin-Fukuhara M, Frontali L, 

Francisci S. Yeast as a model of human mitochondrial 

tRNA base substitutions: Investigation of the 

molecular basis of respiratory defects. RNA 2008, 

14:275-83. 

Soro S, Orecchia A, Morbidelli L, Lacal PM, Morea 

V, Ballmer-Hofer K, Ruffini F, Ziche M, D'Atri S, 

Zambruno G, Tramontano A, Failla CM. A proangiogenic 

peptide derived from vascular endothelial 

growth factor receptor-1 acts through alpha5beta1 

integrin. Blood 2008, 111:3479-88.

2007-2008 

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