Report of activity 2005-2006

Transcript

Istituto Pasteur
Fondazione Cenci Bolognetti
Report of activity
2005-2006
© 2007 - Università degli studi di Roma La Sapienza
P.le Aldo Moro, 5 - 00185 Roma
Edited by Teresa Ariaudo
Photos by Emilio D’Itri
w3.uniroma1.it/pasteur
Contents
Forward
by Maurizio Brunori, President, and Ernesto Di Mauro, Scientific Director
Boards and Staff
V
VI
Fellowships awarded in 2005 and 2006
Fellowships awarded for two years for training in foreign laboratories
Fellowships awarded to students who had a two-year experience abroad
Fellows working on the research programmes of the Foundation
VII
Seminars
IX
Meetings
X
Scientific Reports
Research area 1: Molecular biology of viruses and microrganisms
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 ..................
Bianca COLONNA - Involvement of nucleoid proteins in the transcriptional control of virulence genes
in Shigella and enteroinvasive Escherichia coli ..................................................................................................
Alberto FAGGIONI - Control of latency and replication of Epstein-Barr virus ..............................................
Anna TRAMONTANO - Computational analysis of the Hepatitis C virus proteome ........................................
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Research area 2: Molecular genetics of eukaryotes
Fiorentina ASCENZIONI - Development and analysis of chromosome vectors ................................................
Gabriella AUGUSTI-TOCCO - Signalling systems in neuronal differentiation and their possible mechanism
of action ......................................................................................................................................................................
Paola BALLARIO - Chromatin remodelling, histone code and signal transduction in Ascomycetes ............
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 - Is poly-ADPribose polymerases inhibition responsible for anomalous oncosuppressor
gene hypermethylation? ..........................................................................................................................................
Giorgio CAMILLONI - Molecular interactions at the rDNA locus in Saccharomyces cerevisiae ........................
Paolo COSTANTINO - The Dof transcription factors in Arabidopsis development ........................................
Ernesto DI MAURO - Internal and external determinants of nucleosome positioning and modifications
in the regulatory architecture of chromatin ......................................................................................................
Laura FRONTALI - New complex mitochondrial functions in cell biology ........................................................
Maurizio GATTI - Relationships between the central spindle and the contractile ring during Drosophila
cytokinesis ....................................................................................................................................................................
Giuseppe MACINO - Molecular mechanisms of transgene-induced post-transcriptional gene
silencing ......................................................................................................................................................................
Franco MANGIA - Chromatin remodelling and transcriptional activation of the zygotic genome in
preimplantation embryos of the mouse ................................................................................................................
Sergio PIMPINELLI - Heterochromatin, telomeres and modifiers of position effect variegation (PEV) in
Drosophila melanogaster ..............................................................................................................................................
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Research area 3: Molecular recognition in biomolecules
Maurizio BRUNORI - Evolutionary pressure on folding mechanisms: the case of cytochrome c and globin
protein families ............................................................................................................................................................
Felice CERVONE - Molecular signalling and recognition in plant defense mechanisms ................................
III
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REPORT OF ACTIVITY 2005-2006
Fabrizio EUSEBI - Molecular and functional approaches to investigate the physiopathological role of the
chemokines and their receptors in the central nervous system ....................................................................
Francesco GASPARRINI - Molecular and enantioselective recognition by receptors and proteins studied
in the gas phase, in free solution and at solid-liquid interfaces ......................................................................
Clara NERVI - Functional role of the interaction between transcriptional regulators, nuclear receptors
and leukemia-associated fusion proteins ..............................................................................................................
Paola PAGGI - Neuronal response to experimental interruption of the neural circuit: a molecular and
structural study in autonomic ganglia in vivo ....................................................................................................
Maria SAVINO - The role of DNA sequence in the organization of telomeric chromosomal domains ......
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Research area 4: Cellular and molecular immunology
Donatella BARRA - Defense mechanisms in innate immunity ..............................................................................
Massimo FIORILLI - Interaction of Hepatitis C virus with the immune system: cryoglobulinemia and
lymphomagenesis ........................................................................................................................................................
Enza PICCOLELLA - Identification of CD28 signals leading to chemokine and survival gene expression
through NF-kB activation ........................................................................................................................................
Angela SANTONI - Receptor-triggered signals leading to activation of NK cell migration and
functions ......................................................................................................................................................................
Isabella SCREPANTI - Analysis of the role of preTCR-triggered NF-kB in T cell leukemogenesis:
relationship with activated Notch signaling ........................................................................................................
Elio ZIPARO - Molecular mechanisms regulating the immune response in the testis: a balance between
immune privilege, tissue homeostasis and autoimmune disorders ................................................................
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Research area 5: New antimicrobial and antiviral agents
Marino ARTICO - New perspectives in the design for anti-HIV-1 agents targeted to reverse transcriptase
(RT) and integrase (IN): synthesis and in vitro evaluation ............................................................................
Antonello MAI - Synthesis and biological evaluation of aroyl-pyrrolyl-hydroxy-amides as new histone
deacetylase inhibitors ................................................................................................................................................
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Research area 6: Biology of malaria
Mario COLUZZI - Interactions between semiochemicals and olfactory-mediated behaviour of
Afrotropical malaria vectors for the development of new monitoring and control tools ......................
David MODIANO - Interferon regulatory factor-1 promoter polymorphism is associated with the control of
Plasmodium falciparum infection ................................................................................................................................
Bibliography
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IV
Forward
The Istituto Pasteur-Fondazione Cenci Bolognetti is a non-profit organization of the Sapienza
University of Rome, whose mission is to support scientific research in the area of biology and
medicine, along the lines of the Institut Pasteur à Paris. The Foundation is an official member of
the world-wide network of Pasteur Institutes, called Réseau International des Instituts Pasteur et
Instituts Associés.
Our mission is pursued through the funding of research projects, the award of fellowships and
the organization of scientific meetings and seminars. Training young researchers has always
been a top priority, which is achieved by providing fellowships to work abroad as well as at the
Sapienza-University of Rome; moreover, the Foundation supports the international Ph.D. Course
in Scienze Pasteuriane at the Sapienza. In the years 2005 and 2006, we supported 75 young fellows.
Over the same period, the Foundation organized (or participated in the organization of) 6 scientific meetings and 14 seminars and conferences.
At a time when attention of granting agencies and science policy makers in Italy and Europe is
largely focused towards application, it should not be forgotten that important breakthroughs in
biology and medicine often originated from curiosity driven research. The Nobel Prize 2006 for
Physiology or Medicine represented a striking confirmation of this policy. 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. In this domain we therefore
followed the legacy of Louis Pasteur who stated that "Il y a la science et les applications de la science,
liées entre elles comme le fruit à l’arbre qui l’a porté ". Our scientific programmes reflect this philosophy. The research projects illustrated in this Report of activity, were selected by a peer review
system based on anonymous referees chosen from the Institut Pasteur à Paris, EMBO and other
institutions, 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.
The results obtained during the years 2005 and 2006, documented in this volume, witness the
enthusiasm and productivity of the scientists involved. The financial contribution of the
Foundation is acknowledged in 203 full papers published in 2005 and 2006 in peer-reviewed journals; among these, many have been published in outstanding journals.
Ernesto Di Mauro
Scientific Director
Maurizio Brunori
President
V
Boards and Staff
Administrative Board
Scientific Council
The President is nominated by the Rector
of the University of Rome "La Sapienza".
The Board includes four professors
appointed as representatives of the
Faculties of Medicine, Natural Sciences,
and Pharmacy. Other members of the
Board are administrative officials of the
University of Rome "La Sapienza" and
three Auditors, appointed by the
University "La Sapienza", the Ministry of
Economics, and the Ministry of
Education, University and Research,
respectively.
The Scientific Council is composed of
seven renowned scientists, appointed by
the Faculties of Medicine, Natural
Sciences and Pharmacy. The Scientific
Director, elected within the Scientific
Council, is ex officio a member of the
Administrative Board. The Scientific
Council sets the guidelines and exerts
responsibility for running all the
scientific activities of the Foundation.
Scientific Director
Ernesto Di Mauro (Natural Sciences)
President
Maurizio Brunori (Medicine)
Members
Paolo Amati (Medicine), Laura Frontali (Natural
Sciences), Anna Teresa Palamara (Pharmacy),
Angela Santoni (Medicine), Anna Tramontano
(Medicine), Carlo Turano (Pharmacy)
Members
Mario Coluzzi (Medicine), Paolo Costantino
(Natural Sciences), Raffaele D’Amelio (Medicine),
Ernesto Di Mauro (Natural Sciences), Romano
Silvestri (Pharmacy)
Secretariat
Secretary
Emanuela Gloriani
Teresa Ariaudo, Maria Pia Lorenzoni, Lynda
Romani, Nicoletta Silvestri
Administrative Expert
Daniela Cavallo
Consultants
Tommaso De Dominicis (legal affairs); Anna Maria
Pivetti (architectural supervision); Barbara Hell
(financial affairs)
Auditors
Carlo Messina, Simona Ranalli, Valeria Valerio
VI
Fellowships awarded in 2005 and 2006
Gianluca CANETTIERI, at the Department of
Experimental Medicine and Pathology, Rome,
from the Salk Institute for Biological Studies, La
Jolla, USA
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
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
Fellowships awarded for two years for
training in foreign laboratories
Alejandro BORGIA, at the Department of
Chemistry, University of Cambridge, UK, from
the Department of Biochemical Sciences, Rome
Francesca FARINA, at the Institute of Genetics
and Microbiology, University Paris-Sud, Orsay,
France, from the Department of Cell and
Developmental Biology, Rome
Giuseppe LA REGINA, at the Welsh School of
Pharmacy, Cardiff University, Cardiff, UK, from
the Department of Medicinal Chemistry, Rome
Eugenia PICCINNI, at the Laboratory of
Molecular Genetics, Ecole Normale SupérieureCNRS, Paris, France, from the Department of Cell
and Developmental Biology, Rome
Edvin PRIFTI, at the Department of Surgery,
Sunnybrook Women's Medical College,
University of Toronto, Canada, from the
Department of Experimental Medicine and
Pathology, Rome
Fabiana RENZI, at the Laboratory of Structural
and Computational Biology, EMBL, Heidelberg,
Germany, from the Department of Biochemical
Sciences, Rome
Massimiliano RENZI, at the Department of
Pharmachology, University College, London,
UK, from the Department of Pharmachology and
Human Physiology, Rome
Ivan TATTOLI, at the Department of Immunology,
University of Toronto, Canada, from the
Department of Cell and Developmental Biology,
Rome
Simona TORCIA, at the Department of Obstetrics
and Gynecology, Stanford University School of
Medicine, USA, from the Department of Histology
and Medical Embryology, Rome
Barbara XELLA, at the Signalling and
Development Laboratory, Marie Curie Research
Institute, Oxted, UK, from the Department of
Genetics and Molecular Biology, Rome
Fellows working on the research
programmes of the Foundation
Monica BALLARINO, at the Department of Genetics
and Molecular Biology
Germana BANCONE, at the Department of Public
Health Sciences
Marialuisa BARBAGALLO, at the Department of
Cell and Developmental Biology
Manuel BENEDETTI, at the Department of Plant
Biology
Claudia BERTONATI, at the Department of
Biochemical Sciences
Claudia BERDINI, at the Department of Genetics
Claudia BONACCINI, , at the Department of
Marina BORRO, at the Department of Biochemical
Sciences
Fabio CANDURA, at the Department of Public
Health Sciences
Cristina CAPUANO, at the Department of
Experimental Medicine and Pathology
Maddalena CARUSO, at the Department of Cell
Biotechnology and Hematology
Cristina CERBONI, at the Department of
Saula CHECQUOLO, at the Department of
Francesco CHIANI, at the Department of Genetics
Raffaella CIPRIANI, at the Department of Human
Physiology and Pharmacology
Fellowships awarded to students who
had a two-year experience abroad
Maria Giulia BIGOTTI, at the Department of
Biochemical Sciences, from the Department of
Biochemistry of the University of Bristol, UK
VII
Antonio COLUCCIA, at the Department of
Pharmaceutical Studies
Donatella D'ELISEO, at the Department of
Maria DE LUCA, at the Department of Genetics and
Molecular Biology
Ilaria DE MARIA, at the Department of Cell and
Developmental Biology
Valeria DE TURRIS, at the Department of Cell and
Enea Gino DI DOMENICO, at the Department of
Francesca DI FELICE, at the Department of
Genetics and Molecular Biology
Luca FACCHINELLI, at the Department of Public
Health Sciences
Daniela FIOCCO, at the Department of Biochemical
Sciences
Fedra FRANCOCCI, at the Department of Plant
Biology
Valerio FULCI, at the Department of Cell
Biotechnology and Hematology
Alessia GAMBADORO, at the Department of Cell
and Developmental Biology
Giovanna GENTILE, at the Department of
Alejandro GIORGETTI, at the Department of
Silvia IAFRATE, at the Department of Genetics and
Molecular Biology
Rosanna LA ROCCA, at the Department of
Maria Carmela LATELLA, at the Department of
Lucia LEONE, at the Department of Cell and
Francesca MALERBA, at the Department of
Ludovica MARCELLINI, , at the Department of
Marcella MARCHETTI, at the Department of
Paolo MERCATILI, at the Department of
Giulia NIGRO, at the Department of Cell and
Romina OLIVA, at the Department of Biochemical
Sciences
Christian OLIVIERO, at the Department of
Prisca ORNAGHI, at the Department of Genetics
Raffaella PAPARCONE, at the Department of
Emanuela PASCUCCI, at the Department of
Claudia PELLACANI, at the Department of Genetics
Andrea PERRONE, at the Department of
Lucia PIACENTINI, at the Department of Genetics
Daniela PONTIGGIA, at the Department of Plant
Biology
Marco POMBI, at the Department of Public Health
Sciences
Arianna PROVENZA, at the Department of
Jessica Diana ROSATI, at the Department of
Francesca SALANI, at the Department of Cell and
Francesca Maria SCANDURRA, at the Department
of Biochemical Sciences
Giuliano SCIARA, at the Department of Biochemical
Sciences
Helena STABILE, at the Department of
Donatella STARACE, at the Department of
Histology and Medical Embryology
Raffaele STRIPPOLI, at the Department of
Domenico TARANTINO, at the Department of
Leila TILIA, at the Department of Experimental
Medicine and Pathology
Lucia TUFANO, at the Department of Plant Biology
Alessandra ZINGONI, at the Department of
VIII
Seminars
October 31, 2006 • Membrane receptors:
allosteric control - Prof. Stuart J. Edelstein,
Department of Biochemistry, University of
Geneva, Switzerland
December 2, 2005 • The molecular basis of
anautogeny in mosquitoes - Prof. Alexander S.
Raikhel, Department of Entomology and
Institute for Integrative Genome Biology,
University of California at riverside, USA
April 26, 2006 • Louis Pasteur and the discovery
of molecular chirality - Prof. Joseph Gal,
University of Colorado School of Medicine,
Denver, USA
November 15, 2005 • MicroRNA genes and cancer
- Prof. Carlo M. Croce, Department of
Molecular Virology, Immunology and Medical
Genetics, Ohio State University, Columbus, USA
April 20, 2006 • How neutrophils kill microbes:
free radicals out, ions in (and out)! - Prof.
Anthony W. Segal, Center for Molecular
Medicine, University College, London, UK
November 11, 2005 • Apical-basal pattern
formation in embryogenesis
November 10, 2005 • Growing up green - from
embryogenesis to the adult plant - Prof. Gerd
Juergens, Center of Plant Molecular Biology,
University of Tubingen, Germany
March 17, 2006 • Yeast glyoxalase 1: two active
sites in a single polypeptide - Prof. Per Jemth,
Department of Medical Biochemistry and
Microbiology, Uppsala University, Sweden
October 21 and 26, 2005 • Telomere structure Dr. Daniela Rhodes, Medical Research Council,
Laboratory of Molecular Biology, Cambridge, UK
March 10, 2006 • Patterns of retinal light
absorption related to retinitis pigmentosa
mutants from in silico model structures of
rhodopsin - Prof. Luis Alberto Montero
Cabrera, Faculty of Chemistry, University of
Havana
June 28, 2005 • Cheaters sometimes prosper: the
story of the Segregation Distorter system of
meiotic drive in D ro sop hi l a mel a n og a ste r Prof. Terrence Lyttle, University of Hawaii,
USA
March 7, 2006 • The amazing chromatoid body
- Dr. Paolo Sassone-Corsi, Institut de Génétique
et de Biologie Moléculaire et Cellulaire CNRSInserm, Université louis Pasteur, Illkirch,
Strasbourg, France
May 13, 2005 • A role of telomere repeat factors
at an origin of DNA replication
May 11, 2005 • Chromatin regulation of
Gammaherpesvirus latency - Prof. Paul
Lieberman, Winstar Institute of Philadelphia, USA
IX
Meetings
Giornate Scientifiche della Fondazione
Hosts, Symbionts and Parasites : Molecular
and Pharmacological Approaches
Rome - October 26-27, 2006
Bacteria and hosts: multifaceted interactions
Chairs: F. Bossa, E. Di Mauro
P.J. Sansonetti - Rupture, invasion and inflammatory destruction of the intestinal epithelium by
Shigella. The Yin and Yang of innate immunity
B. Colonna - Virulence gene expression in Shigella:
hovering between acquisition and loss of transcriptional activators
D. De Biase - The molecular mechanisms controlling the activity of glutamate decarboxylase, the
structural key component of acid resistance in
Escherichia coli
M.L. Bernardini - Manipulation of Shigella PAMPs:
turning Dr Jekyll into Mr Hyde
Promoting Committee: P. Amati, E. Di Mauro, L.
Frontali, A.T. Palamara, A. Santoni, A.
Tramontano, C. Turano
Scientific Committee: M.L. Bernardini, A.T.
Palamara
Cross-talk between innate and adaptive immunity
Chairs: L. Frati, R. D’Amelio
M. Rescigno - Epithelial cell-dendritic cell crosstalk at mucosal surfaces in bacterial handling
A. Santoni - NK cells in viral infections: mechanisms
of resistance and immunoevasion
V. Barnaba - Chronic immune activation by crosspresentation of caspase-cleaved self-antigens during
viral infections
I. Screpanti - Notch signaling in lineage fate decision of T cells
Mycetes: models and pathogenetic mechanisms
Chairs: V. Ziparo, L. Frontali
A. Zychlinsky - Innate immune effectors in infections
P. Ballario - Light signal transduction in an hypogeous symbiotic fungus: the truffle Tuber borchii
C. Mazzoni - Yeast as a model for the study of aging
and apoptosis
G. Simonetti - New perspectives on histone deacetylase inhibitors: interference with the main virulence
traits of Candida albicans
Virus-host cell interactions
Chairs: F. Chimenti, A.T. Palamara
S. Ludwig - Exploited defense: how influenza virus
misuses antiviral cellular signaling responses
P. Amati - The role of the major capsid protein of
Polyoma in virus - cell interactions: replication, tissue tropism and tumorigenesis
A. Faggioni - Virus - host interactions following
Epstein-Barr virus infection
A.T. Palamara - Intracellular factors drive the outcomes of Influenza infection in different cell populations
EMBO Workshop
Hedgehog-Gli Signaling in Cancer and
Stem Cells
Rome - Sept 30-Oct 4, 2006
with the contribution of the Foundation
Organisers: A. Gulino (Italy), Ariel Ruiz i Altaba
(Switzerland), F. Watt (UK), H. Hahn (Germany), I.
Guerrero (Spain), P. Therond (France), R.
Toftgard (Sweden)
Parasites, vectors and hosts
Chairs: M. Brunori, M.L. Bernardini
Award of the Gaia Luoni Prize 2006 on Genetics of
susceptibility to malaria
D. Modiano - Intra and inter-ethnic approaches in the
study of the genetics of the susceptibility to
Plasmodium falciparum malaria
B. Arcà - At the interface between parasite and host: the
salivary glands of the malaria vector Anopheles gambiae
A. Bellelli - Biosynthesis of prostaglandins by
Schistosomes
D. Cioli - Mechanism of action of anti-schistosomal
drugs
Hedgehog Signalling I
Chair: A. Gulino
A. Ruiz i Altaba - HH-GLI signaling regulates cancer stemness
I. Guerrero - Extracelular components as modulators of Hedgehog gradient formation
J. Taipale - Shh signal transduction
Hedgehog Signalling II
Chairs: A. Ruiz i Altaba, J. Taipale
X
K. Nybakken - Functional genomic dissection of the
Hedgehog signaling pathway
F. van Eeden - Analysis of ptc1 and ptc2 mutants in
zebrafish
G. Haussman - Dissection of the Hedgehog pathways by RNA interference in Drosophila cells
J. Reiter - Hedgehog signaling at the cell’s antenna:
Smoothened and the primary cilium
C. Emerson Jr - Signaling cross talk in the
Hedgehog pathway
J. Jiang - Regulation of Hh/Ci signaling by dual
ubiquitination systems
J. Eggenschwiler - Genetic analysis of mammalian
Hedgehog signal transduction
A. Gallet - Roles of the proteoglycans Dally and
Dally-like for the regulation of Hh movement and
reception
P. Mehlen - Patched as a dependence receptor: death
signaling and in vivo relevance
J. Briscoe - Graded Shh signaling and the control of
neural cell fate
C-C. Hui - Suppressor of fused regulates dorsoventral neural progenitor cell identity through modulation of Gli activator and repressor activities
C. Sanchez-Camacho - Shh controls the growth of
mouse retina ganglion cell axons
V. Wallace - Harnessing the Hh pathway for cell
therapy to the eye
I. Aifantis - Hedgehog signaling as an essential regulator of early hematopoiesis
K. Dittman - Targeted inactivation of the Hh receptor Ptch abrogates lymphocyte development in mice
E. Drakopoulou - Gli1 transcription factor in thymocyte development.
Hedgehog Signalling in Development and Cancer
I: Brain Tumorigenesis
Chairs: I. Guerrero, J. Briscoe
N. Dahmane - Regulation of SHH signaling during
forebrain development
R. Wechsler-Reya - Stem cells, progenitors and the
origins of medulloblastoma
J. Olson - N-myc is an essential downstream effector
of Shh signaling during both normal and neoplastic
cerebellar growth
B. Stecca - Conditional mis-expression of GLI1 in
the brain increases proliferation of neural precursors
and neural stem cells
A. Gulino - Negative regulation of Hedgehog-Gli
pathway and brain tumorigenesis
A. Kenney - IRS1 is a node of Shh-Insulin-like
Growth Factor cross-talk
S. Marino - The role of Bmi1 in linking the Shh
pathway to the cell cycle
O. Becher - SHH expression and Gli activity correlate with grade in gliomas
Hedgehog Signalling III
Chairs: R. Toftgard, J. Jiang
P. Therond - Revisiting fused and Costal2 functions
in Hh signaling
C. Soula - Specification of oligodendrocyte precursors in the ventral spinal cord: involvement of
Sulfatase 1 in modulating Shh signaling
B. Wang - A protein processing determinant domain
that controls the differential processing of Gli2 and
Gli3 transcription factors
L. Milenkovic - Smoothened localization in the primary cilium and Hedgehog pathway activation are
induced by oxysterols
M. Peppelenbosch - Repression of smoothened by
patched-dependent (Pro-)vitamin D3 secretion
R. Krauss - Cdo and Boc: multifunctional cell surface
proteins that regulate several signaling pathways,
including the Hedgehog pathway
Hedgehog Signalling in Development and Cancer
II: Skin Cancer
Chairs: F. Watt, D. Robbins
F. Watt - Pathways that act downstream of Wnt in
regulating the epidermal stem cell compartment
A. Oro - Spatial and temporal regulation of Shh signal reception in hair progenitors
F. Aberger - Cross-talk of EGFR and
Hedgehog/GLI signaling in human epidermis
V. Vidal - What is the role of Sox9, a new target of
the Shh pathway, during cutaneous basal cell carcinoma development?
E. Epstein - Basal cell carcinomas in man and mouse
A. Dlugosz - Hedgehog signaling in tumor initiation
Hedgehog Signalling in Development and Stem
Cell Behaviour
Chairs: P. Therond, R. Wechsler-Reya
R. Toftgard - GLI-mediated regulation of progenitor cells in the skin and the mammary gland
V. Palma - Role of SHH-Gli signaling in midbrain
development
T. Ellis - Patched1 regulates both neocortical
growth and lamination by controlling cell cycle
kinetics in neuronal precursors
E. Traiffort - Analysis of oligodendroglial progenitor proliferation induced by Sonic Hedgehog in the
cerebral cortex and corpus callosum
XI
telomeric chromatin to the nuclear periphery via
interaction with yKu
S. Cacchione (Italy) - The role of DNA sequence in
telomeric chromatin features
H. Lipps (Germany) - Telomere end-binding proteins control the formation of G-quadruplex DNA
structures in vivo
K. Lemke (Poland) - Interaction of antitumor triazoloacridone C-1305 with guanine-rich DNA: induction of structural changes in guanine triplets and
stabilization of G-quadruplexes
H. Renauld (USA) - Interstitial (sub)telomeric
repeats: punctuation marks of genome dynamics?
and maintenance
M. Philpott - The human forkhead transcription
factor FOXM1b, a downstream target of Sonic
Hedgehog enhances DNA repair and suppresses
UVB-induced apoptosis
C. Missero - Foxe1 transcription factor, a target of
sonic hedgehog signaling, regulates hair follicle morphogenesis
Hedgehog Signalling in Development and Cancer III
Chairs: M. Hebrok, F. de Sauvage
S. Pazzaglia - Dissecting the mechanisms of radiation-induced tumorigenesis in the skin and brain of
Ptc1 knockout mice
D. Robbins - Frequent requirement of Hedgehog
signaling in non-small cell lung carcinoma.
D. Bushman - Hedgehog signaling in prostate development and prostate cancer
H. Hahn - Pathogenesis of myogenic tumors in Ptch
mutant mice
Telomere Replication and Telomere Length
Regulation
Chair: V. Zakian (USA)
V. Zakian (USA) - Pif1p family helicases: effects on
telomerase
E. Gilson (France) - New pathways of telomere
length regulation in yeast
A. LondoÀo-Vallejo (France) - Telomere homeostasis in humans: length control at cell and single
telomere levels
S. Bekaert (Belgium) - Reference values and basic
determinants of telomere length in a large middleaged population
D. Baird (UK) - Telomere dynamics and instability
M.A. Cerone (Canada) - A human cell line that
maintains telomeres in the absence of telomerase
and of key markers of ALT
C.M. Counter (USA) - Loss of hPot1 function leads
to telomere instability and a cut-like phenotype
Hedgehog Signalling in Development and Cancer IV
Chairs: H. Hahn, E. Epstein
M. Hebrok - Hedgehog signaling in pancreatic cancer
G. van den Brink - Hedgehog signaling and colorectal
carcinogenesis
J. Xie - Hedgehog signaling in gastrointestinal cancers
M. Lauth - Identification and characterization of
small-molecule GLI antagonists
F. de Sauvage - Targeting the Hedgehog pathway in
cancer
EMBO Workshop
Chromosome Structural Elements: from
DNA Sequence to Function
Villa Mondragone, Monte Porzio Catone, Rome Sept 29- Oct 3, 2005
Chromosome Replication and Disjunction
Chair: H. Lipps (Germany)
M. Debatisse (France) - Dynamics of DNA replication in mammalian somatic cells
S. Riva (Italy) - Functional analysis of a human
DNA replicator
S. Smith (USA) - Telomeres require special mechanisms for their compaction and resolution at mitosis
P. Meister (Switzerland): Spatial and temporal separation of replication and recombination requires the
intra-S checkpoint
M. Cornfort (USA) - Excess of radiation-induced
dicentrics involving homologous chromosomes
B. Meier (USA) - The C. elegans catalytic subunit of
telomerase TRT-1 and DNA damage signaling
S. Bailey (USA) - Telomeres and double-strand
breaks: tying up loose ends
Centromeres, DNA Sequences and Chromatin
Plasticity
Organiser: F. Ascenzioni (Italy)
Co-organisers: S. Bacchetti (Italy); G. Novelli (Italy);
M. Savino (Italy)
Telomeres: Structure, Proteins and Positioning
Chair: P. Donini (Italy)
L. Luzzatto (Italy) - Chromosomal and molecular
basis of cancer
P. Kaminker (USA) - Dual function for a telomereassociated protein?
D. Rhodes (UK) - Towards the structure of the “30
nm” chromatin fiber
H. Schoiber (Switzerland) - Telomerase helps tether
XII
Chair: B. Grimes (USA)
B.A. Sullivan (USA) - Centromeric chromatin is a
continuous, dynamic domain that permits gene
expression
C. Farr (UK) - Topoisomerase II: untangling its contribution at centromere
G. Della Valle (Italy) - Dissecting Cenp-C function
in mammalian centromeres
W.C. Earnshaw (UK) - Genetic analysis of chromosome segregation in vertebrate cells
C. Desmaze (France) - Chromatin structure and
dynamics in the formation of chromosome aberrations
C. Woo (USA) - Long-range nucleosome mapping of
the HOX gene cluster
J. Fajkus (CZ) - Minisatellite telomeres in the family
Alliacae are lost with divergence of the genus allium
International Symposium
Pasteur a “La Sapienza”
Rome - June 23-25, 2005
within the celebrations for the 700th anniversary of the
University of Rome “La Sapienza”
De novo Centromeres Assembly and Chromosome
Engineering
Chairs: W.C. Earnshaw (UK), C. Farr (UK)
B. Grimes (USA) - Segregation of human artificial
chromosomes in mouse cells
T. Voet (Belgium) - Optimisation and exploitation of
a chromosomal vector
N. Mestrovic (Croatia) - The MEL-172 satellite
DNA library in parthenogenetic meloidogyne
species: conserved pattern of nucleotide variability
along the monomers
H. Masumoto (Japan) - Chromatin assemblies
required for de novo human artificial chromosome
formation
F. Ascenzioni (Italy) - Human minichromosomes:
expression vectors for human genes
G. Bernardi (Italy) - Chromatin structure in interphase nuclei of vertebrates and the formation GCrich isochores
The malaria challenge*
Chairs: F.C. Kafatos (Germany), P. Arese (Italy), O.
Mercereau-Puijalon (France), D. Taramelli (Italy)
A. Scherf (France) - Plasmodium falciparum antigenic variation: the first pieces of the jigsaw puzzle
D. Kwiatkowski (UK) - Malaria and the human
genome
X. Su (USA) - Plasmodium falciparum: worldwide
genome diversity and recent African population
expansion
O. Mercereau-Puijalon (France) - Plasmodium falciparum: population diversity
J.-F. Trape (Senegal) - A new look at holoendemic
malaria during infancy and childhood
F.C. Kafatos (Germany) - Immunogenomics of
Anopheles/Plasmodium interactions
R. Sinden (UK) - A molecular and genetic analysis of
the malarial development in the mosquito vector:
implications for intervention
M. Coluzzi (Italy) - Chromosomal speciation: from
mosquitoes to humans
* dedicated to Gaia Luoni (1970-2004)
Promoting Committee: R. Guarini (Rector of the
University of Rome “La Sapienza”); M. Brunori
(President of the Foundation); F. Bossa, L. Frati, D.
Misiti, A. Vecchione (Faculties’ Deans); M. Coluzzi,
P. Costantino, R. D’Amelio, F. Manna
(Administrative Council of the Foundation); P. Amati,
M. Artico, E. Di Mauro, L. Frontali, A.
Giacomello, A. Santoni, A. Tramontano (Scientific
Council of the Foundation)
Organising Committee: M. Brunori, M. Coluzzi, P.
Costantino, E. Di Mauro, M. Piccoli, A. Santoni
Chromosomes Organization and Gene Evolution
Chair: G. Novelli (Italy)
M. Cremer (Germany) - Nuclear architecture in the
light of conserved higher order chromatin arrangements.
M. Rocchi (Italy) - Centromere repositioning in evolution.
G. Novelli (Italy) - The role of the nuclear envelope
in genome organisation.
Immunology and therapeutic vaccines
Chairs: P.-A. Cazenave (France), A. Santoni (Italy),
M. Sela (Israel), V. Barnaba (Italy)
D.T. Golenbock (USA) - Initiation of the innate
immune response by TLRs
A. Mantovani (Italy) - Complexity and complementarity of innate immune recognition
V. Barnaba (Italy) - Proteomic approach of crosspresentation of apoptotic cells by dendritic cells
J. Di Santo (France) - Cellular redundancy in innate
immunity against intracellular infections.
XIII
P. Vieira (France) - Control of immune responses by
regulatory T cells
K. Dellagi (Tunisia) - Of mice and men: cor-relates
of protection in human and experimental models of
Leishmania major infection
F.K. Stevenson (UK) - DNA fusion vaccines: versatile tools to activate specific immunity
G. Forni (Italy) - Vaccines for tumor prevention
R. Arnon (Israel) - Towards antigen-specific therapeutic vaccines for infectious, autoimmune and neoplastic diseases
Chairs: R. Kopan, F. Schweisguth
S. Blacklow - Notch subunit heterodimerization and
prevention of ligand-independent proteolytic activation depend respectively on a novel domain and the
LNR repeats
A. Pintar - Structural studies of NOTCH ligands
U. Lendahl - Cross-talk between Notch and other signaling mechanisms
G.P. Dotto - Crosstalk between Notch1, p21waf/cip1
and calcineurin signaling pathways in control of keratinocytes self renewal and differentiation
N. Carlesso - Regulation of the G1-S phase transition by Notch Signaling: an alternate mechanism for
modulating cell fate decisions
C. Brou - Monoubiquitination, endocytosis and
gamma-secretase cleavage.
E. Hansson - Assembly and regulation of the
gamma-secretase complex
M. McGill - Regulation of Notch receptor trafficking by the cell fate determinant Numb
M. Vooijis - Genetic mapping of Notch1 activity in
vivo
Science and Society
Chairs: F. Gros (France), P. Costantino (Italy)
M. Morange (France) - The challenges for biologists
at the beginning of the 21st century.
F. Barré-Sinoussi (France) - HIV/AIDS, a unique
opportunity to change the course of history in global health equity
G. Corbellini (Italy) - The science-society dialogue.
The role of education and communication
G. Novelli (Italy) - Human reproduc-tion: a modern
paradigm in biomedicine (scientific advances, medical
perspectives and societal concerns)
C. Perrey (France) - Introduction to the general discussion
Notch and Development
Chairs: S. Artavanis-Tsakonas, G. Weinmaster
L. Bally Cuif - E(Spl) factors in zebrafish neurogenesis
S. Bray - Notch signaling in Drosophila: inputs and
outputs.
J. Knoblich - Asymmetric Rab11 endosomes regulate delta recycling and specify cell fate in the
Drosophila nervous system
T. Gridley - Notch signaling during embryonic
development in mice
EMBO Workshop
N o t c h S i g n a l i n g i n D e v e l o p me n t a n d
Cancer
Rome, Italy - April 21-24, 2005
Organisers: I. Screpanti (Italy), S. Krishna (India),
B.A. Osborne (USA), U. Lendahl (Sweden), L.
Miele (USA)
Local Organising Committee: I. Screpanti, A. Vacca,
M.P. Felli, D. Bellavia, A.F. Campese, C. Talora
Notch and Cancer I
Chairs: G.P. Dotto, F. Radtke
T. Capobianco - Defining the molecular and genetic interactions in Notch induced-neoplastic disease
D. Hayward - EBNA2, the Epstein-Barr virus Notch
mimic
S. Krishna - Notch-PI3K signaling in human epithelial cancers: phenotypes, mechanisms and signal integration
Notch Signaling I
Chairs: L. Miele, U. Lendahl
S. Artavanis-Tsakonas - Keynote address: Notch
signaling and cell fate control in evolution
G. Weinmaster - Delta and Notch get tubular
R. Kopan - Is there a proteolysis-independent Notch
signal or a Notch-independent presenilin activity in
mice? lessons from the somite address both questions
F. Schweisguth - Regulation of ligand endocytosis
in Notch receptor signaling
Notch and Cancer II
Chairs: S. Krishna, T. Capobianco
L. Miele - Live and let die: Notch signaling as a therapeutic target in breast cancer and melanoma
F. Ratke - Notch: lineage specifier, oncogene, tumor
suppressor and stem cell gate keeper?
J. Luis De La Pompa Minguez - New insights on
the Notch pathway in cardiac development and
Notch Signaling II
XIV
tumor progression
F. Esni - Notch inhibits PTF1 function and acinar
cell differentiation in developing pancreas.
O. Basak - Hes5 in neural stem cells
C. Eberhart - Notch pathway blockade promotes
either apoptosis or differentiation in medulloblastoma
D. Subramanyan - bFGF is a modulator of
SCFFbw7 mediated degradation of Notch1 in
human epithelial tumorigenesis
S. Pece - Loss of negative regulation by Numb over
Notch is relevant to human breast carcinogenesis
in thymic tumors from Ikaros-deficient mice
I. Screpanti - Notch3 and pre-TCR signalings meet
at the crossroads of T cell development and leukemogenesis
EMBO Workshop
Structural Basis of Papovavirus Biology
Abbazia di Pontignano, Siena, Italy - April 11-16,
2005
Notch and Hematopoiesis
Chairs: M. Toribio, T. Honjo
H. Neves - The effects of Delta1 and Jagged1 in
human myeloid progenitors
A. Zeuner - Notch2 is a regulator of basal and
cytokine-modulated erythropoiesis
A. Robert - RBPJk-dependent notch1 function regulates gata-2 expression to generate intraembryonic
hematopoietic cells
I. Bernstein - The density of Delta 1 determines the
generation of T, B and repopulating cells by multipotent hematopoietic precursors
C. Bleul - A recent thymic immigrant maps to the
branching point of the T versus B lineage decision
Structure
Chair: R.L. Garcea (USA)
B. Trus (USA) - Localization of the HPV16 minor
capsid protein L2 by difference imaging
R. Liddington (USA) - An evaluation of recent
structure-function studies of polyomavirus and
SV40
H. Kasamatsu (USA) - Structural rationalization for
intracistronic complementation of SV40 VP1 temperature sensitive mutants
G. Wadell (Sweden) - Receptors for non-car binding
adenoviruses
X. Chen (USA) - Mechanisms of conformational
change for the motor function of SV40 large T antigen
R.H. Cheng (USA) - Structure and assembly of
polyoma BK virus-like particles
Organisers: P. Amati (Italy), R.L. Garcea (USA),
A.H. Helenius (Switzerland)
Notch and the Immune System I
Chairs: I. Screpanti, H. von Boehmer
T. Honjo - Regulation of lymphocyte development
by Notch/RBP-J
J.C. Zuniga-Plucker - Early T cell development, is
Notch enough?
W. Pear - Notch in T cell commitment and function
M. Toribio - Notch1 signaling in human thymocyte
development
M. Dallman - Notch signaling: control of T cell differentiation in the periphery
B. Osborne - Notch regulation of CD4+T cell function
G. Mckenzie - Notch and PI3K signalling integrate
to regulate expression of the murine tribbles
homolog Trb2
Entry, Uncoating 1
Chair: R.E. Streeck (Germany)
W. Atwood (USA) - Receptor mediated events controlling human polyomavirus invasion of cells
M. Caruso (Italy) - Study of the role of the VP1LDV motif in polyomavirus infectivity
L.C. Norkin (USA) - The caveolae-mediated SV40
entry pathway bypasses the Golgi complex en route
to the endoplasmic reticulum
J. Forstová (Czech Rep) - Trafficking of mouse
polyomavirus towards the cell nucleus
A.E. Smith (Switzerland) - Dynamics of polyomavirus endocytosis
M. Sapp (Germany) - Identification of surfaceexposed amino acids of HPV16 L1 essential for cell
binding and infection
M. Schelhaus (Switzerland) - HPV16 pseudoviruses
exhibit directed retrograde movement along filopodia
H. Selinka (Germany) - Post-entry neutralization of
HPV pseudovirions by an anti-L1 antibody
Entry, Uncoating 2
Chair: A.H. Helenius (Switzerland)
Notch and the Immune System II
Chairs: B. Osborne, W. Pear
H. von Boehmer - Notch1 in T cell development and
T-ALL
J. Aster - Notch signaling in acute leukemia: new
insights
B. Kee - Activation of Notch1 is essential for survival of E2A-/- T cell lymphomas
P. Kastner - Early activation of the Notch pathway
XV
J.T. Schiller (USA) - Papillomavirus infection
requires furin cleavage of the minor capsid protein
L2
M. Kann (Germany) - Travelling through the
nuclear pore with hepatitis B viruses
U.F. Greber (Switzerland) - Corralled and unilinear
receptor and coreceptor mediated cell surface motilities of adenovirus type 2
G. Nemerow (USA) - Analyses of adenovirus-mediated membrane penetration
D. Majhen (Croatia) - Adenoviruses bearing NGR
motifs in the HI-loop of adenovirus fiber protein
bind aminopeptidase N and ?v‚3 integrins
J. Kleinschmidt (Germany) - Activation of phospholipase A2 on the AAV-2 capsid
M. Vihinen-Ranta (Finland) - Cytoplasmic and
nuclear events of parvovirus entry
S. Kirjavainen (Finland) - Canine parvovirus entry
and the role of viral PLA2
L. Gissmann (Germany) - Immunogenicity of
HPV16 L1/E7 chimeric particles in mice
P. Coursaget (France) - Characterization of the FG
loop domains of the papillomavirus type 16 and type
31 L1 proteinS associated with shared and type-specific epitopes
R.B.S. Roden (USA) - A broadly cross-neutralizing
epitope of human papillomavirus type 16 L2
overlapping a cell surface-binding motif involved in
infection
M. Stanley (UK) - T-cell responses to E2, E6 and E7
protein HPV 16 in women with low grade cervical
intra-epithelial neoplasia
E.J. Kremer (France) - Memory immunity to adenoviridae in humans: CD4+RO+ T cells, neutralising
and opsonising antibodies, and activation of dendritic cells
Immunology 2
Chair: L. Gissmann (Germany)
T. Benjamin (USA) - Cell entry pathway of polyoma
virus and interacions of VLPS with antigen presenting cells
A. Lukacher (USA) - Does polyoma virus infection
generate memory CD8 T cells?
E. Szomolanyi-Tsuda (USA) - Humoral immune
responses to polyomavirus in mice: essential role for
innate immune signals
T. Dalianis (Sweden) - Murine polyomavirus VP1
virus-like particles immunise against some polyomavirus induced tumours
T. Ramqvist (Sweden) - Vaccination with polyomavirus VP1/VP2HER-2 virus-like particles
(VPLs) prevents outgrowth of HER-2/neu expressing tumours
N. Sanjuan (Argentina) - Progression of experimental
infection with high and low tumorigenic strains of
polyomavirus in mice
R. Ulrich (Germany) - Virus-like particles based on
major capsid protein VP1 of hamster polyomavirus
tolerate foreign insertions of different size and origin
A. Zvirbliene (Lithuania) - Immunogenic properties
of yeast-expressed hamster polyomavirus major
capsid protein VP1: chimeric VP1-based virus-like
particles are efficient immunogens to generate monoclonal antibodies of desired specificity
Assembly
Chair: P. Amati (Italy)
H. Handa (Japan) - The SV40 minor capsid proteins
promote in vitro assembly of Vp1 pentamers and
their DNA encapsulation
R.L. Garcea (USA) - Chaperoning papovavirus
assembly and disassembly
O. Ben-nun-Shaul (Israel) - Search for post-translational modification in the connector of SV40 VP1
D. Enderlein (Germany) - Application of avian
polyomavirus expressing histidine-tailed VP4 in
order to assess the localization of this protein within the viral capsid and to identify interacting partners
M.J. Imperiale (USA) - Viral factors involved in adenovirus assembly and encapsidation
L. Laimins (USA) - Life cycle of human papillomaviruses in differentiating epithelia
A. McBride (USA) - Maintenance and segregation
of papillomavirus genomes by the E2 protein
J.M. Almendral (Spain) - Regulatory signals in the
nuclear assembly and exit of the parvovirus MVM
capsid proteins
Immunology 1
Chair: J. Schiller (USA)
I.H. Frazer (Australia) - Overcoming ineffective
immune responses to papillomavirus antigens to
generate therapeutic immunity
W.M. Kast (USA) - HPV can escape immune recognition through Langerhans cell PI3-kinase activation
C. Buck (USA) - Alpha defensinins inhibit papillomavirus infection
Entry, Uncoating 3
Chair: L.A. Laimins (USA)
M. Carbone (Italy) - Poly(ADP-ribosyl)ation modulates chromatin structure determining polyomavirus
early gene transcription
H. Kasamatsu (USA) - Controlled and selective
XVI
structural alteration of virions containing minor
capsid proteins VP2/3 is required for the nuclear
import-competent state of infecting SV40
J. Moroianu (USA) - The positively charged termini
of L2 minor capsid protein required for bovine papillomavirus infection function separately in nuclear
import and DNA binding
Vaccines, Detection
Chair: R. Schlegel (USA)
M. Mueller (Germany) - DNA vaccination against
multiple papillomavirus types for cross-protective
immunity
R. Viscidi (USA) - Virus like particle (VLP) based
ELISA for detection of antibodies to human and animal polyomaviruses
R. Johne (Germany) - Detection of novel polyomaviruses by nested broad-spectrum PCR
M. Pawlita (Germany) - Reactivity of 83 VLP-specific monoclonal antibodies and of human sera with
bacterially expressed L1 GST fusion proteins of 15
HPV types
R. Kirnbauer (Austria) - Serological relationship of
HPV92, a new type isolated from a basal cell carcinoma, to high.risk HPV EV-types 5 and 8
D. Galloway (USA) - Characterization of HPV antibodies following incident infection: persistence and
mapping epitopes
Tumorigenesis
Chair: T.L. Benjamin (USA)
P.F. Lambert (USA) - Mouse models for HPV-associated cancers
M. Tognon (Italy) - SV40 transformation of human
cells as a model for human oncogenesis steps
M.J. Imperiale (USA) - Association of BKV with
early stages of prostate cancer
K. Doerries (Germany) - Association of infection.
Intracellular localization of human polyomavirus
DNA in peripheral blood cell subpopulations
C. Morelli (Italy) - SV40 and human fibroblasts:
short and long term transformation
T.-C. Wu (USA) - Identification of a new mechanism
for tumor evasion
R. Schlegel (USA) - Dihydroartemisinin selectively
kills papillomavirus-expressing cells in vitro and in
vivo
A. Gedvilaite (Lithuania) - Formation of virus-like
particles by hamster polyomavirus carboxy-terminally truncated VP1 mutants expressed in yeast
Gene therapy
Chair: B.E. Griffin (USA)
J. Forstová (Czech Rep) - Polyomavirus-like particles (PyVLPs): can we make them work for us?
P. Hobson (UK) - The use of scanning surface confocal microscopy to study the interaction between
the cell surface and virus-like particles
H. Lilie (Germany) - Cell-type specific gene transfer
using virus-like particles of PyVP1, an antibody
fragment, and listeriolysin O
P. Beard (Switzerland) - Role of the cellular DNA
damage response in transduction by adeno-associated virus vectors with single-stranded or self-complimentary DNA
A. Oppenheim (Israel) - Gene delivery to the lungs
for the treatment of acute respiratory distress syndrome using SV40-based vectors
I. Kopatz (Israel) - Studies on the nuclear entry and
localization of wild type SV40 and of SV40-based
vectors
P. Fortes (Spain) - Recombinant SV40 vectors
expressing therapeutic genes efficiently delay liver
cirrhosis progression and regress colon carcinoma
tumor nodules
N. Krauzewicz (UK) - Plasmid DBA delivered by
mouse virus-like particles localises to centrmeres
resulting in rapid but reversible transgene silencing
XVII
XVIII
AREA
1
Molecular
biology of
viruses
and
microrganisms
Molecular biology of viruses and microrganisms - 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
Principal investigator: Paolo Amati
Professor of Molecular Genetics
Dipartimento di Biotecnologie Cellulari ed Ematologia
Tel.: (+39) 06 490393, Fax: (+39) 06 4462891
[email protected]
Another part of the program, originally stemmed
from our interest in the oncogenic properties of Py
early functions, concerns the interdependence of cell
cycle and differentiation, in the muscle model.
Participants:
Rossella Maione, professor; Rosaria Carbone, Maddalena
Caruso, Rocco Figliola, post-doc fellows; Michaela
Cavaldesi, Marianna Rossi, Anna Busanello, PhD students;
Olga Sthandier, technician.
The role of polyomavirus VP1 in determining
viral growth and host specificity
In the last two years we made significant progress in
Collaborations:
Dipartimento di Biotecnologie Cellulari ed Ematologia, SapienzaUniversità di Roma (Prof. Paola Caiafa); Dipartimento di
Medicina Sperimentale e Patologia, 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).
the comprehension of the possible involvement of
VP1 in controlling early transcription. In fact, the
research on the structure of the mature virions (performed in collaboration with Prof. Turano’s group;
Carbone et al., J Virol 2004, 78:513-9) allowed us to
determine that VP1 is associated all around the viral
genome; however, in the enhancer region (that
appears free of nucleosomes) the amount of bound
VP1 is twice that in other regions. Analysis of crosslink with cis-platinum allowed us to confirm previous
reports that the virus is decapsidated in the nucleus.
This finding, in connection with the association of
VP1 (that replaces H1 in the viral chromatin structure) preferentially with the enhancer region,
brought us to look for possible chromatin modificators that could interact with VP1 to activate viral
early transcription.
Since Poly(ADP-ribose)polymerases (PARPs) are
involved in fundamental cellular events and in some
stages of viral infection, we investigated, in collaboration with Prof. Caiafa’s group, the possible role of
these enzymes. Viral early transcription was reduced
by competitive inhibitors of PARPs in Swiss 3T3
cells and almost abolished in PARP-1-knockout
fibroblasts. In vivo ChIP assays showed that
poly(ADP-ribosyl)ation facilitates the release of the
capsid protein VP1 from the chromatin of infecting
virions. In vitro experiments demonstrated that VP1
stimulates the enzymatic activity of PARP-1 and
binds free (ADP-ribose)-polymers non-covalently.
Our studies, reporting that PARP-1 promotes viral
Report of activity
Elucidation of the molecular mechanisms determining the early phases of virus interaction with the
host cell appears of relevant interest for a better
understanding of the general strategies adopted by
pathogens to infect specific target cells. The murine
Polyomavirus (Py) has been successfully used as a
model system for the study of the mechanisms regulating replication, gene expression, tumor induction
and, more recently, for the study of those determining initial virus-host cell membrane interactions. Py
is a non-enveloped small DNA tumor virus whose
genome - a double strand DNA molecule of 5297 bp,
coding for six proteins - is encapsidated in a shell
formed by only three proteins (VP1, VP2 and VP3).
Our research is 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. We will also analyse the role of
Poly(ADP-ribosyl)ation in relation to cell cycle reactivation.
3
P. Amati - Polyomavirus early functions and the capsid protein VP1
through a 165 bp promoter region. The levels of p73
a, b‚ and d isoforms are up-regulated during MyoDinduced differentiation and each of them, even when
exogenously over-expressed, is capable to induce p57
in fibroblasts lacking p21. Our finding that the
expression of a p73 dominant negative mutant interferes with the ability of MyoD to activate both the
endogenous gene and the exogenous promoterreporter construct strongly supports the requirement
of p73 in the pathway linking MyoD to p57. We have
reported evidence that p73 is involved in regulating
p57 also in spontaneously differentiating C2
myoblasts, thus suggesting a physiological role of
this pathway in skeletal muscle differentiation.
In conclusion, our finding that MyoD induces p21
and p57 through distinct molecular mechanisms, suggests that in muscle cells the two cdk inhibitors may
be activated following a different spatial and/or temporal expression pattern, in order to exert nonredundant
functions
during
myogenesis.
Interestingly, we have found that the ability to induce
p57 is restricted to specific cell types or to a subset of
cells within the same population. By using several
approaches, we have demonstrated that the responsiveness of p57 to MyoD-dependent regulation is
affected by the DNA methylation status of its promoter (Figliola et al., manuscript in preparation). We
are currently analyzing the relationship between
DNA methylation, chromatin conformation and transcriptional regulation. It will be interesting in the
future to investigate how DNA methylation of p57
promoter and its interplay with other epigenetic factors is regulated in response to differentiation signals.
early transcription by chromatin remodeling, have
been reported in Carbone et al., 2006.
Significant results have been obtained also from the
study of the role of VP1 in cell membrane recognition and internalization (Cavaldesi et al., 2003;
Caruso et al., 2004a, 2004b). In fact we have shown
that attachment and internalisation are two different
steps of viral entry into the cell. Furthermore, we
have shown that integrin alfa4/beta1 is one of the
secondary receptors for internalization and that
virus or pseudocapsids change the conformation of
their VP1 after binding to the sialic acid residues (the
primary receptor). In particular we have recently
reported (Caruso et al., 2006) that a Py-VP1 mutant
defective in integrin recognition displays a modified
infectivity and tissue tropism in vivo.
To what concerns the role of PARP-1 in the modification of the viral chromatin, much has to be still
elucidated. Fundamental will be to determine if
PARP-1, in specific cases, simply plays a role in the
mechanism of exposure of the regulatory region or
also participates in the organization of the transcription complex. The finding that PARP-1 interacts
directly with the transcription factor YY1, together
with our previous finding that VP1 also interacts
with this factor, makes likely that the role PARP-1 is
not limited to the epigenetic modification but it also
participates directly in the complex that regulates
viral gene expression. The work regarding the cell
recognition by Polyoma is at present the subject of a
detailed study in vivo, through the analysis, in collaboration with Prof. N. Sanjuan, of the growth and
oncogenicity of the integrin less-mutant.
Furthermore, we will pursue the study by XR diffraction (in collaboration with Prof. Liddington) of
the structural changes observed after viral interaction with sialic acid residues.
Selected publications
Vaccarello G, Figliola R, Cramerotti S, Novelli F,
Maione R. p57Kip2 is induced by MyoD through a
p73-dependent pathway. J Mol Biol, 2006;
356(3):578-88. Epub 2005 Dec 27.
Interdependence between cell cycle control,
muscle differentiation and cell survival
The cyclin-dependent-kinase inhibitors p21 and p57
are highly expressed in skeletal muscle where they
redundantly control cell cycle arrest during differentiation. We have recently shown that p57 is a target of
the myogenic factor MyoD in cells lacking p21
(Figliola & Maione, 2004). More recently, we have
reported (Vaccarello et al., 2006) that MyoD induces
p57 at the transcriptional level through a mechanism
different from that involved in p21 regulation, since it
is E-box-independent and requires new synthesized
protein(s). We have identified p73 family members as
the factors which mediate the activation of p57
Carbone M, Reale A, Di Sauro A, Sthandier O,
Garcia MI, Maione R, Caiafa P, Amati P. PARP-1
interaction with VP1 capsid protein regulates polyomavirus early gene expression. J Mol Biol, 2006;
363(4):773-85. Epub 2006 Jun 30.
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. Epub 2006 Dec 28.
4
Escherichia coli strains overexpressing flavohemoglobins for the
production of novel, biologically active compounds derived from
phospholipid post-biosynthetic modifications
Principal investigator: Alberto Boffi
Professor of Molecular Biology
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel. (+39) 06 4991 0990, Fax (+39) 06 4440062
[email protected]
between the C5-C6 of fatty acids. The natural substrates for desaturases are membrane phosholipids
whose acyl chains can be unsaturated in the presence
of oxygen as electron acceptor and ferredoxins as
electron donors. The D5 desaturase has been cloned
into a plasmid for overexpression in Escherichia coli
and its activity has been monitored by measuring (by
GC-MS) the amount of D5 unsaturated fatty acids
within the host membrane under different growth
conditions. Coexpression with flavohemoglobin, low
growth temperature (20-25 °C) and forced aereation
led to a spectacular increase in the desaturase activity such that more than one third of all membrane
fatty acids were transformed into D5 unsaturated
derivatives. The outcome in terms of pure products
(i.e. D5 unsaturated fatty acids) is satisfactory
although the presence of a considerable amount of
cyclopropanated fatty acids, common products in
stressed E. coli, must be overcome before scaling up
the process to the preparative scale.
Participants:
Rosa Marina Matarese, professor; Laura Giangiacomo,
post-doc fellow; Alessandra Bonamore, Alberto Macone,
PhD students.
Collaborations:
ACS Dobfar, Tribiano, Milano (Dr. Fabio Arenghi); Dipartimento
di Chimica, Università di Firenze (Dr. Giulietta Smulevich).
Report of activity
The main target of the present project is to exploit
the catalytic properties of bacterial hemoglobins in
order to produce novel molecular synthons 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.
The strategy underlining this approach is based on
the observation that bacterial hemoglobins counteract the oxidative stress induced by the high oxygenation required to sustain the catalytic activity of oxygen dependent enzymes such as desaturases.
Moreover, flavohemoglobins, one of the most studied subfamilies among bacterial hemoglobins, are
endowed with an efficient alkylhydroperoxide reductase activity. This enzymatic activity has been
demonstrated to be a major determinant in the physiological response to oxidative stress but also offers a
precious tool to perform hydroxylations not only on
alkyl- or acyl- substrates, but also on isoprenylated
compounds.
Novel bacterial hemoglobins
The bacterial hemoglobins family is continuously
growing in size as novel genomes are sequenced and
annotated. Genes codifying for hemoglobin-like proteins have been in fact identified within most microbial species, including several Archaea. Despite the
lack of consensus on the physiological role of these
proteins, their use as cofactors for oxidative biotransformations is widespread. Nevertheless, most
novel
accessions,
expecially
those
from
extremophiles, have not been investigated in terms of
functional properties and potential biotechnological
applications. In this framework, a novel thermostable
hemoglobin from the actinobacterium Thermobifida
fusca has been cloned, overexpressed in E. coli cells
and characterized in terms of thermal stability, ligand binding properties and tridimensional structures
(submitted). These unusual hemoglobin has been
Desaturase engineered E. coli strains
The D5 desaturase from Bacillus subtilis is a nonheme iron, integral membrane enzyme capable of
introducing stereo and regio selective insaturations
5
A. Boffi - Escherichia coli strains overexpressing flavohemoglobins for the production of novel compounds
demonstrated to be capable of exchanging reversibly
gaseous ligands up to 65-70 °C and (at least in the
case of T. fusca Hb) to afford a fast growth and
increased cell density in transformed E. coli strains
under high oxygenation. Moreover, the high expression levels and high stability of the protein prompted its use as a tag for overexpression of fusion proteins (see below).
co-expressing flavohemoglobins with desaturases.
Engineering E. coli strains by cloning novel bacterial hemoglobins fused with aromatic prenyltransferases further broadened the scope of the work thus
allowing a novel, synthetic biology based strategy to
the synthesis of prenylated aromatic molecules.
Bonamore A, Ilari A, Giangiacomo L, Bellelli A,
Morea V, Boffi A. A novel thermostable hemoglobin
from the actinobacterium Thermobifida fusca. FEBS
J, 2005; 272(16):4189-201.
Aromatic prenyltransferases
Two novel aromatic prenyltransferases from
Streptomyces species (NovQ, belonging to the operon of novobiocin biosynthesis and Orf2, involved in
the pathway of naphterpin biosynthesis) have been
cloned and expressed in E. coli cells as fusion proteins with the thermostable T. fusca Hb. The yield of
recombinant protein expression is highly increased
in the presence of the hemoglobin tag and both
chimeric enzymes exhibited sustained prenyltransferase activity with phenolic substrates.
In conclusion, the project has demonstrated that an
efficient production of D5 unsaturated fatty acids on
the technical scale can be obtained in E. coli strains
Botta B, Delle Monache G, Menendez P, Boffi A.
Novel prenyltransferase enzymes as a tool for
flavonoid prenylation. Trends Pharmacol Sci, 2005;
26(12):606-8. Epub 2005 Oct 17.
Bonamore A, Macone A, Colotti G, Matarese RM,
Boffi A. The desaturase from Bacillus subtilis, a
promising tool for the selective olefination of phospholipids. J Biotechnol, 2006; 121(1):49-53. Epub
2005 Aug 18.
6
Involvement of nucleoid proteins in the transcriptional control of
virulence genes in Shigella and enteroinvasive Escherichia coli
Principal investigator: Bianca Colonna
Professor of General Microbiology
Dipartimento di Biologia Cellulare e dello Sviluppo
Tel.: (+39) 06 49917580, 06 49917582, Fax: (+39) 06 49917594
[email protected]
keeping functions, as well as for virulence functions.
Several studies on virulence gene regulation in pathogenic bacteria have stressed the role of H-NS in the
thermoregulatory response. This phenomenon is
particularly evident in Shigella and enteroinvasive
Escherichia coli (EIEC), the causative agents of bacillary dissentery, since all the genes involved in adhesion, penetration and intra-/intercellular spreading
of the bacteria into the host cells are silenced by HNS at 30°C. The genes determining the invasivity
process are located on a large virulence plasmid and
are activated by two plasmid-encoded proteins, VirF
and VirB. During the last few years the activity of
our group has been mainly devoted to the understanding of the molecular mechanisms controlling
the activation of the virF promoter at the transcriptional level. Transcription of virF is strictly temperature dependent and is antagonistically mediated by
the nucleoid proteins H-NS and FIS which bind to
specific sites on the virF promoter. In particular we
have demonstrated that the curved DNA tract within the virF regulatory region operates as thermosensor and that its structural alterations affect the binding of H-NS molecules to their target sites, thus
controlling the transcriptional activity. Besides binding to the promoters of the regulatory genes virF
and virB, we have shown that H-NS is able to bind
also to the regulatory region of the icsA gene, which
encodes a structural protein involved in the reorganization of the cytoskeleton upon penetration of bacteria into host cells. After having demonstrated that
the icsA promoter is endowed with a strong bent
structure we are studying the molecular mechanisms
exploited by H-NS and by the positive regulator
VirF to activate the icsA promoter, as well as the possible contribution of other nucleoid proteins. So far
our results suggest that icsA is submitted to a multifactorial environmental control: the complexity of
the icsA regulatory region is not surprising, considering that the expression of the IcsA protein is very
critical for the invasivity process and must not occur
Participants:
Gianni Prosseda, researcher; Maria Carmela Latella,
Marialuisa Barbagallo, PhD students.
Collaborations:
Istituto di Biologia e Patologia Molecolari, CNR, Roma (Dr.
Gioacchino Micheli); Dipartimento di Biologia Molecolare,
Cellulare e Animale, Università di Camerino (Dr. Maurizio
Falconi); Dipartimento di Biologia, Università di Roma-Tre (Prof.
Mariassunta Casalino).
Report of activity
The protein composition of the nucleoid is dominated by a small group of polypeptides, known as
nucleoid or histone-like proteins, which are able to
profoundly affect the local as well as the global structure of the chromosome. Besides organizing the bacterial chromatin, in E.coli the major nucleoid proteins
exert regulatory influences on recombination, DNA
replication and transcription. In this respect nucleoid
proteins have been shown to function directly or
indirectly, and often in combination, to control the
expression of a wide variety of genes that are essential for cell viabilitily, such as those involved in protein synthesis and metabolism. Frequently they also
regulate other important genes whose expression
varies in response to environmental stimuli, as well
as genes affecting virulence. In this way they link
under a global control system a large number of
genes coding for disparate functions which, when
appropriately expressed, enhance the chances that
the cell will survive and prosper in its new environmental circumstances. In particular, this kind of control mechanism should improve the fitness of pathogenic bacteria when they encounter novel environments during infection of their host. Among
nucleoid proteins H-NS is by far the most important
global modulator, since it affects the expression of a
large number of unrelated genes coding for house-
7
B. Colonna - Nucleoid proteins in the transcriptional control of virulence genes in Shigella and E. coli
before the bacterium has accessed the cytoplasm of
the epithelial cells.
In the evolutionary route of Shigella from commensal E.coli ancestors towards a pathogenic lifestyle,
the critical events have been the acquisition, through
horizontal gene transfer, of the virulence plasmid
(pINV) which has conferred on these microrganisms
the ability to invade intestinal epithelial cells and to
multiply within them through the expression of a
very sophisticated and complex pathogenicity mechanism. Besides acquiring the virulence plasmid, these
enteroinvasive bacteria underwent a convergent
series of mutations, mainly mediated by insertion
elements in chromosomal genes, which redesigned
important metabolic properties and have allowed the
full expression of invasiveness. During the search
for optimal host conditions ensuring efficient and
coordinated expression of the newly acquired genes,
virulence genes have been inserted into complex regulatory networks submitted to the control of several regulatory factors. Cadaverine, a small polyamine
resulting from decarboxylation of lysine, has been
shown to hamper the full expression of Shigella invasiveness.We recently find that in Shigella and EIEC
silencing of the cad operon, encoding the lysine
decarboxylase, relies on a mutation in the cadC regulatory gene, suggesting that the CadC protein may
play a direct or indirect role in the control of other
genes or operons involved in the adaptation of the
bacterium to the host environment .These observations prompt a deeper understanding of which regulatory changes have followed, in the transition from
E.coli to Shigella, the uptake of virulence genes so as
to insert them into a regulatory network allowing
for an efficient expression mediated by the host environment.
Casalino M, Latella MC, Prosseda G, Ceccarini P,
Grimont F, Colonna B. Molecular evolution of the
lysine decarboxylase-defective phenotype in Shigella
sonnei. Int J Med Microbiol, 2005; 294(8):503-12.
Prosseda G, Latella MC, Casalino M, Nicoletti M,
Michienzi S, Colonna B. Plasticity of the P junc promoter of ISEc11, a new insertion sequence of the
IS1111 family. J Bacteriol, 2006; 188(13):4681-9.
8
Control of latency and replication of Epstein-Barr virus
Principal investigator: Alberto Faggioni
Professor of General Pathology
Dipartimento di Medicina Sperimentale e Patologia
Tel.: (+39) 06 4461500, Fax: (+39) 06 4454820
[email protected]
estradiol did not express EBNA2 but Bjab K3+estradiol were high EBNA2 expressors as judged by using
PE2 anti-EBNA2 monoclonal antibody. BCL6, TCL1
and MTA3 were all found downregulated in high
EBNA2 expressing BJAB K3+estradiol cells as seen
by immunoblotting. The DLBCLs and BLs generally
express HLA class II genes. More differentiated B
cells cease to express this molecule on the cell-surface. In addition, we analysed EBNA2 expressing
U2932 clones for HLA DR expression by flow cytometry. The results revealed that the U2932 EBV
clone that expressed intermediate levels of EBNA2
had moderate downregulation of HLA DR. The high
EBNA2 expressor clones showed a strong decrease
in HLA DR expression. (Boccellato et al., J. Virol.,
2006).
The influence of the restricted type I viral gene
expression as seen in multiple myeloma and primary
effusion lymphomas was exactly the opposite. We
found that the GC hallmark genes BCL6, TCL1 and
MTA3 were upregulated in several MM and PEL
EBV infected clones. The expression of these genes
was verified by immunoblotting and real time PCR.
A surface marker analysis showed that syndecan-1
(CD138) and BLIMP-1, typical plasmacytoid markers were strongly downregulated in EBV carrying
MM and PEL clones, whereas the expression of
HLA class II was upregulated. All these suggests
that the restricted viral latent gene expression
induces dedifferentiation and underlines the tumorigenic potential of the virus. In essence, we found
that latency I (pro-tumorigenicity) and latency III
(anti-tumorigenicity) have an opposite effect on the
cellular phenotype. (Trivedi et al., manuscript in
preparation).
Regarding the part of the project dealing with viral
replication, we have characterized the promoter
region of the BFRF1 gene to understand the mechanisms of regulation of the expression of this gene
to add a further element to the knowledge of the
EBV lytic program.
Participants:
Maria Rosaria Torrisi, Giuseppe Ragona, Antonio
Angeloni, Pankaj Trivedi, professors; Roberta Santarelli,
Maria Cirone, researchers; Alessandro Farina, Roberta
Gonnella, post-doc fellows; Massimo Granato, Elias
Anastasiadou, PhD students; Claudia Zompetta, technician.
Report of activity
In order to investigate how the Epstein-Barr viral
gene expression influence the cell phenotype, we
continued to infect different cell lines derived from
different stages of B cell differentiation with recombinant EBV. In both DLBCLs and BLs, where the
infected cell lines showed a latency II/III phenotype,
there was a profound intereference with the germinal
center phenotype. BCL6, the master reglator of the
GC, was strongly downregulated in those DLBCLs
and BLs where EBNA2 was highly expressed. High
EBNA2 expressing clones showed almost complete
abrogation of BCL6 expression, suggesting a dosedependent effect. In parallel, two other GC associated genes TCL1 and MTA3 were negatively influenced by latency III and in particular EBNA2. Since
our DLBCLs and BL convertants expressed other
latency III associated proteins, it is possible that the
negative modulation of BCL6, TCL1 and MTA3
could be due to other latency III proteins and not
EBNA2. To address this question, we chose Bjab cell
line transfected with an EBNA2 expression vector
containing p554-3 encoding EBNA2/Estrogen
receptor, where EBNA2 expression is regulated by
estrogen. The transfected cells were selected in 1
mg/ml G418. Without estrogen, these cells do not
express EBNA2 and two days of 1 micromolar estrogen treatment led to a strong EBNA2 expression.
The untransfected parental Bjab, Bjab K3 cells
without estrogen and Bjab K3+estradiol were then
tested by immunoblotting for both EBNA2 and
BCL6 expression. As expected, Bjab K3 without
9
A. Faggioni - Control of latency and replication of Epstein-Barr virus
introduced in the ZV site (CAGGTc) the same single
nucleotide substitution described by Kraus. We
observed that the luciferase activity of the -679pF1
MV, which presented this point mutation, was
increased compared to the vector containing the no
mutated motif. Although the mutation, that we introduced, was not sufficient to restore the same level of
induction observed in -429pF1 plasmid.
Finally, to define the role of RRE-F1 we generated a
partial deletion of this domain. The RRE-F1 of the
BFRF1 promoter is not a canonical RRE since the
consensus sequence usually found is GNCC-N9GGNG: however it is similar in the 3’ core to an
RRE motif contained in the EBV BHLF1 promoter
which has been shown to be very active in responding to RTA expression. The RTA factor transactivated the -679pF1 construct, whereas the deletion
induced a decrease of 6 fold of the expression of
plasmid. These all luc-assays were also performed in
the DG 75 EBV-negative cell line. These results indicated that in the context of reporter gene assay, the
canonical ZIIIA and the novel RRE-F1 elements are
the motif that drive the responsiveness to ZEBRA
and RTA respectively (Granato et al., Virology,
2006).
The 5’-rapid amplification of cDNA ends (RACE)
method indicated that the transcription start site of
the BFRF1 mRNA is located at -306bp upstream of
the ATG of the gene. We performed a computer
analysis of the promoter region of BFRF1 gene,
searching for TPA (TRE), ZEBRA (ZRE) or RTA
(RRE) responsive elements. We found a canonical
ZRE (known as ZIIIA) and a putative RRE (that we
called RRE-F1) respectively at -373 bp and -518
upstream of the ATG start codon.
To further define the involvement of ZEBRA and
RTA in the activation of BFRF1 and to better understand the role played by the consensus sequences we
set up experiments of gene reporter. We generated a
reporter plasmids in which functional deletions of
the promoter region were linked to luciferase gene: 679pF1 contains the ZIIIA and RRE-F1 motifs,
whereas -429pF1 contains only the ZEBRA responsive element. Next we carried out a site-directed
mutagenesis of the consensus domains: we generated
-679pF1 ZIIIA(c,a) and -429pF1 ZIIIA (c,a) containing a mutated ZIIIA and -679pF1 RRE¢ in which a
deletion in the RTA responsive site (RRE-F1) was
introduced. The constructs were then transiently
transfected in a EBV-negative epithelial cell line,
known as 293 cells in the presence of the transactivators. A substitution of two bases in the ZRE
sequence (TGcGaCA) affected the capacity of
ZEBRA factor to induce the expression of the
reporter plasmid. The stimulation of -429pF1 ZIIIA
(c,a) was significantly impaired: the activity of this
construct was reduced by 5 fold compared to the wild
type -429pF1. Furthermore, ZEBRA was not able to
drive the expression of the -679pF1 construct, which
carries a long region of the promoter containing the
ZRE site, at similar level of the -429pF1: the mutation of the ZIIIA was almost ineffective. To verify the
presence of sequences that rule negatively the
expression of this vector we looked for known
repressor motifs by computer analysis in -679 fragment: we found in this region the presence of a
sequence called ZV (CAGGTG) which is also identified in the promoter of BZLF1. It knows that this site
is recognizes by a cellular factor indicated as ZEB: we
Anastasiadou E, Boccellato F, Cirone M, Kis LL,
Klein E, Frati L, Faggioni A, Trivedi P. Epigenetic
mechanisms do not control viral latency III in primary effusion lymphoma cells infected with a recombinant Epstein-Barr virus. Leukemia, 2005;
19(10):1854-6.
Boccellato F, Anastasiadou E, Rosato P, Kempkes B,
Frati L, Faggioni A, Trivedi P. EBNA2 interferes
with the germinal center phenotype by downregulating BCL6 and TCL1 in non-Hodgkin’s lymphoma
cells. J Virol. Epub 2006 Dec 6.
Granato M, Farina A, Gonnella R, Santarelli R, Frati
L, Faggioni A, Angeloni A. Regulation of the expression of the Epstein-Barr virus early gene BFRF1.
Virology, 2006; 347(1):109-16. Epub 2006 Jan 9.
10
Computational analysis of the Hepatitis C virus proteome
Principal investigator: Anna Tramontano
Professor of Biochemistry
Tel: (+39) 06 49910556, Fax: (+39) 06 49910717
[email protected]
Participants:
Sequence variability analysis
As described in our previous report, we collected all
available HCV polyprotein sequences and constructed a multiple sequence alignment that forms the
basis of our variability analysis of all the viral proteins. We subsequently extracted the position and
type of sequence variations from the alignment and
stored them in a local database.
The database is useful to map the viral variability
onto the three-dimensional models of HCV proteins,
thereby helping to assess their accuracy, but it can
also be used for attempting to correlate clinical data
with the specific genotype of the infectious agent.
HCV infection is frequently associated with extrahepatic manifestations, including non-malignant and
malignant B-cell lymphoproliferative disorders. It
has been reported that specific changes or recurring
motifs in the amino acid sequence of the HCV hypervariable region 1 (HVR1) may be associated with
cryoglobulinaemia, although this hypothesis is
rather controversial, being based on patient data
derived from a sample of moderate size.
Claudia Bonaccini, Romina Oliva, Alejandro Giorgetti,
Domenico Raimondo, post-doc fellows; Domenico
Cozzetto, PhD student.
Collaborations:
IRCS “Ospedale San Matteo”, Pavia (Prof. Mario Mondelli);
CASPUR, Sapienza-Università di Roma (Dr. Tiziana
Castrignanò).
Report of activity
The aim of the project is to use computational biology
tools to produce models of the structures of HCV proteins that have not been experimentally elucidated, to
analyse the genome variability of the virus in order to
produce testable hypotheses on the function and interactions of the viral proteins and to develop the appropriate computational tools for addressing these issues.
Hepatitis C virus (HCV) is responsible for both parenterally transmitted and sporadic non-A and non-B
hepatitis affecting 1-3% of the world population. HCV
is a positive stranded RNA virus encoding at least ten
unique structural and non-structural proteins. The
HCV viral genome contains a single open reading
frame which encodes a polyprotein of between 3008
and 3037 amino acids, depending on the isolate and
genotype, and is flanked by UTRs at both the 5’ and 3’
termini. The structural proteins lie in the N-terminal
portion of the polyprotein, whilst the non-structural
proteins form the remainder. Polyprotein processing
occurs via a combination of host and viral proteinases
and this gives ten unique proteins: C, E1, E2, p7, NS2,
NS3, NS4A, NS4B, NS5A and NS5B. C, E1 and E2
form the structural components of the virus whilst the
other proteins are non-structural.
In the last two years we concentrated on the analysis of
the viral variability and on the development of novel
techniques that can be applied to this viral system.
HCV and cryglobulinemia
In collaboration with Prof. Mario Mondelli from San
Matteo Hospital in Pavia, we carried out an analysis of
HCV-specific motifs potentially associated with the
development of cryoglobulinaemia as a prototype of
B-cell lymphoproliferative disorder by analysing a
large cohort of patients with HCV infection caused by
genotype 1 and 2 with and without detectable cryoglobulins. We analyzed the viral sequences obtained
from patients for the presence of specific motifs within
HVR1 and within the entire E2 sequence in a subset of
patients. Given the large sample size (111 patients
enrolled in the study) our results cannot be ascribed to
sampling limitations and should help settle the issue
about whether or not HCV-specific motifs are associated with cryoglobulinaemia.
Our results suggest that the initially non-neoplastic
11
A. Tramontano - Computational analysis of the Hepatitis C virus proteome
monoclonal expansion characteristic of HCV-associated B-cell lymphoproliferative disorders may be a
consequence of protracted antigenic stimulation, as
frequently observed in several chronic viral infections.
Mapping conformational epitopes on models
of HCV proteins
We also devoted efforts in developing a method for
identifying conformational epitopes of proteins,
given the high importance of the immune response
in HCV infection.
We developed a strategy, implemented in a server
named MEPS to map epitopes on the model of the
structure of the HCV E2 protein that we have previously produced. The method is able to find the surface region of a protein that can be effectively mimicked by a peptide, given the structure of the protein
and the maximum number of side chains deemed to
be required for recognition. It can also find and
report all peptide sequences of a specified length
that can mimic the surface of a given protein and
store them in a database.
Bioinformatics methods validating HCV
protein models
The problem of validating the quality of a model is an
important one as it increases the usefulness of a model
by dictating its appropriate usage. We have addressed
the problem in the context of the Critical Assessment
of Techniques for Protein Structure Prediction
(CASP) experiment, where we introduced a new category: Model quality evaluation.
The CASP experiment provides protein targets for
predictions by human groups and servers. In the latter
case, the sequences of the proteins are automatically
sent to participating servers and their reply collected
within 48 hours. The models produced by the servers
are immediately made available to the prediction community, as to avoid unnecessary overloading of the
servers, since their results are often used as starting
point for human predictions.
This strategy has been, for the first time, exploited also
to assess the ability of present methods to evaluate the
quality of the models. Participating groups were asked
to analyse the available server models before the structure of the target protein was available and assign
them a reliability value by giving a number comprised
between 1 (best model) and 0 (worse model). This prediction category was named Qmode1. They were also
asked to predict a “per-residue” estimate of the error
(Qmode 2). The results of two groups stood out as
best and statistically significant from the others: group
556 (Lee) and Group 634 (Wallner).
Using models to facilitate the experimental
resolution of protein structures
The predicted structure of a protein can be used in
molecular replacement when the computational
model is sufficiently accurate for a reasonably large
fraction of the structure in the crystal. A generally
accepted “rule of thumb” was that molecular replacement is effective if the model is reasonably complete
and shares at least 40%-50% sequence identity with a
protein of known structure. We decided to investigate more quantitatively the relationship between the
quality of models of protein structure and their usefulness as search models in molecular replacement.
We used the available models submitted to CASP to
verify in which cases they can be automatically used
as search templates for molecular replacement.
Our results show that, in this specific application of
modelling, what really counts is the overall ability of
the model to produce interpretable electron density
maps of the protein, which helps building most of it,
rather than the details of the less well predicted parts.
The Protein Model Database
The Protein Model Database (PMDB) is a public
resource aimed at storing manually built 3D models of
proteins. The database is designed to provide access to
models published in the scientific literature, together
with validating experimental data. It is a relational
database and it currently contains around 74 000 models for about 240 proteins. The system is accessible at
http://www.caspur.it/PMDB and allows predictors to
submit models along with related supporting evidence
and users to download them through a simple and
intuitive interface. Users can navigate in the database
and retrieve models referring to the same target protein or to different regions of the same protein. Each
model is assigned a unique identifier that allows interested users to directly access the data.
Giorgetti A, Raimondo D, Miele AE, Tramontano A.
Evaluating the usefulness of protein structure models
for molecular replacement. Bioinformatics, 2005;
21(s2):ii72-ii76.
Castrignanò T, De Meo PD, Cozzetto D, Talamo IG,
Tramontano A. The PMDB Protein Model Database.
Nucleic Acids Res, 2006; 34:D306-9.
Raimondo D, Giorgetti A, Bosi S, Tramontano A. An
automatic procedure for using models of proteins in
molecular replacement. Proteins, 2006; 66:689-696.
12
AREA
2
Molecular
genetics
of
eukaryotes
Molecular genetics of eukaryotes - Area 2
Development and analysis of chromosome vectors
Principal investigator: Fiorentina Ascenzioni
Professor of Microbiology
Tel.: (+39) 06 49917577, 06 49917614, Fax: (+39) 06 49917594
[email protected]
lished. More recently we have assembled a human
minichromosome (MC1-CFTR) containing the
entire human CFTR locus (Auriche et al., 2002,
EMBO Rep 3:862-8). The CFTRp produced by this
molecule in recipient cells functions properly as a
chloride channel. Nonetheless artificial chromosome
assembly and engineering still require complex and
time consuming techniques that limit their exploitation as carriers of exogenous genes both in vitro and
in vivo.
We proposed Cystic Fibrosis (CF) as a model disease
for chromosomal vector-based gene therapy for a
number of reasons: CF is caused by mutations of the
CF transmembrane conductance regulator (CFTR)
gene, which encodes a cAMP dependent chloride
channel whose expression is finely tuned in space
and time; gene therapy approaches to CF lung disease have demonstrated partial efficacy and shortlived CFTR expression in the airways providing
proof-of-principle of functional gene transfer; the
CFTR gene and the corresponding mRNA, are both
relatively large, the gene (comprised of 27 exons)
spanning 250 kb and the mRNA 6.5 kb, of which
4440 bases is amino acid coding sequence; CFTR
exhibits complex pattern of tissue-specific expression and its expression depend on a number of control elements located upstream and downstream of
the coding region and in various introns.
Participants:
Cristina Auriche, Piera Fradiani, Elisabetta Testa,
Francesca Tilesi, post-doc fellows; Enea Gino Di Domenico,
Lucia Rocchi, PhD students.
Collaborations:
Istituto Sperimentale per il Trattamento della Fibrosi Cistica,
DIBIT, Milano (Dr. Massimo Conese); Istituto “Giannina
Gaslini”, Genova (Dr. Olga Zegarra de Moran).
Report of activity
The use of genomic loci containing therapeutic
genes is in principle preferable to cDNA constructs
since they ensure the presence of the control elements, such as locus control regions, alternative
splicing signals, intronic control elements, normally
associated with chromosomal genes which dictate
the proper spaciotemporal expression of the genes.
But using such DNA fragments in gene therapy
approaches is hampered by their size, which exceeds
the capacity of conventional virus-based vectors.
Although mammalian loci containing entire genes
have been cloned into prokaryotic vectors, such as
PAC (P1 Artificial chromosomes) and BAC (Bacterial
Artificial Chromosomes), these molecules have no
known mammalian origins of replication, and their
fate on entry into the nucleus is unknown.
Drawbacks in the use of classical gene transfer vectors include immune response to viral proteins or to
unmethylated CpG motifs contained in bacteriallyderived vector DNA, and shut-off of viral promoters. Artificial chromosomes (AC) are undoubtedly
the ideal platform for large DNA fragments as they
can be engineered with very large DNA fragment,
they are maintained at low copy number and segregate faithfully into daughter cells. Their use as vectors for therapeutic genes has been proposed since
1997, when the first de novo chromosome was pub-
Functional assay of MC1-CFTR in cultured
human epithelial cells from CF and non CF
donors
One of the major challenge working with AC is the
transfer of the engineered molecules from host cells
into cellular models resembling the disease under
investigation. This is an important step toward the
validation of the chromosomal vector of gene therapy. Waiting for the development of methods based
on the physical separation of the chromosomal vectors from the host chromosomes and their use in
15
F. Ascenzioni - Development and analysis of chromosome vectors
combination with transfection reagents such as
lipids, dendrimers, the only procedure available is
based on cell fusion, which can be performed either
with whole cell or with microcells, the latter are produced by prolonged incubation of the donor cells
with agents that inhibit mitosis. In the last two years
working we have used both procedures with different
immortalized cells isolated from the airway epithelial
of CF patients, but only once we got one hybrid
clone containing MC1-CFTR. Although we demonstrated the presence of the minichromosome by
FISH, western blots and electrophysiological measurements failed to reveal the presence of an active
CFTR in this clone suggesting that amount of wt
CFTRp produced in the hybrid clone is not sufficient
to functionally complement the CF defect. We can
hypothesize that: a low fraction of the cells contain
an active minichromosome; CFTR expression is
downregulated by epigenetic effects due to chromatin composition and/or by minichromosome position within the nucleus. We are going to respond to
some of these questions by determining the copy
number of the minichromosome-CFTR gene and the
level of expression of the gene with respect to the
endogenous CFTR and to internal controls by real
time quantitative PCR.
topo-vector from further use.
Assembly of YAC/BAC vectors containing the entire
human CFTR locus. To use the reduced minichromosomes as a vector for the human CFTR gene, and to
allow insertion of the CFTR locus with its own regulatory elements, we produced a series of bacterial
constructs containing the human CFTR locus isolated in the YAC 37AB12 by Anand (1991, Genomics,
124-30). This fragment of about 300 kb, was shown
to complement CF null mice demonstrating that it
contains the complete set of sequences necessary to
allow a physiological expression of the CFTR gene
in vivo. The YAC 37AB12 was characterized by
restriction and hybrization analyses which suggested
the presence of the entire gene and about 70 kb 5’
upstream region, any indication was provided about
the 3’ end of the gene furthermore, any sequencing
was published. To allow a fine mapping of this
CFTR fragment and to provide an easy way to
manipulate and to purify this potentially therapeutic
DNA, we have engineered a YAC/BAC vector following this strategy: first, the YAC 37AB12 was circularized in yeast by using the retrofitting vector
pNKG418 which replaced both YAC arms with a
fragment containing a BAC module; subsequently,
the circular vector was transferred into E. coli cells
by using intact DNA extracts. The resulting bacterial clones were analyzed for the presence of the
expected molecule by PCR, PFGE electrophoresis
and finally sequencing (Auriche et al., manuscript in
preparation). We identified clones containing circular YAC/BAC-CFTR of the expected structure
(cCFTR). We found that cCFTR, as well as the original YAC 37AB12, contains the entire human CFTR
locus and adjacent sequences which, conversely to
published data, consists of 55 kb 5’ upsteam and 30
kb 3’ downstream. Thus the CFTR locus is now
available in a DNA molecule that can be easily modified, (we successfully used the RecE/T recombination) and isolated from bacterial cells. The cCFTR
molecule will provide a unique opportunity to perform deletion analysis the 5’ and 3’ region of the
CFTR gene and to develop a new class of vectors
based on the genomic locus.
Assembly of smaller version of the
minichromosome MC1
Fragmentation of the minichromosome MC1. To generate
smaller derivatives of MC1 we used telomere fragmentation in the CHO-MC1 hybrid. For this purpose
pBluHCMV plasmid (kindly provided by Dr. Gilson),
which contains 1.6 Kb of human telomeric sequences,
a selectable marker (hygromycin) and enhanced GFP
marker was used to engineer two fragmentation vectors : pSat2-tel, containing a Sat2-alphoid (350 bp)
fragment to tag recombination within the corresponding region of MC1; pSat2-DGFP-tel, equal to pSat2tel but without the GFP gene. CHO-MC1 cells were
transfected with each construct giving raise to about
40 clones. Three pSat2-DGFP-tel/clones contained a
fragmented MC1, as detected by PFGE and FISH,
whereas no reduction was observed in the clones
obtained with the other construct. Structural and functional analysis of the reduced minichromosomes are
under investigation.
A second generation of MC1 tagging vectors was
produced by increasing the size of the Sat2/alphoid
tag. For this purpose we constructed a fragment of
about 2,5 kb by in vitro rolling of the original 350 bp
fragments, the resulting fragment was cloned in
Selected publication
Conese M, Boyd AC, Di Gioia S, Auriche C,
Ascenzioni F. Genomic context vectors and artificial
chromosomes for cystic fibrosis gene therapy. Current
Gene Therapy (in press).
16
Signalling systems in neuronal differentiation and their possible
mechanism of action
Principal investigator: Gabriella Augusti Tocco
Professor of Developmental Biology
Tel: (+39) 06 49912822, Fax: (+39) 06 49912351
[email protected]
of acetylcholine action on neuronal differentiation
we investigated the ability of ChAT-transfected
N18TG2 clones to release acetylcholine; analysis of
muscarinic receptors showed the presence of M1M4 subtypes and the activation of both IP3 and
cAMP signal transduction pathways. Since receptor
activation is followed by an increase in EGR-1 levels,
mainly dependent on M3 activation, the role of this
factor in the enhancement of differentiation was
investigated in transfection experiments of N18TG2
cells with a construct for EGR-1. From our data it
appears that Egr-1 transfected cells show increased
neurite extension as well as a decrease in REST
expression: both these features are also exhibited by
ChAT-transfected clones. The data reported confirm
that the progression of N18TG2 in the developmental program is dependent on ACh release and the
ensuing activation of muscarinic receptors, which in
turn modulate the level of neurospecific transcription factors, such as EGR-1 and REST.
Matrix metalloproteases. A major marker of neuronal
differentiation modulated by Ach is fiber extension,
as described in previous reports. Fiber extension is a
complex process, requiring a fine regulation of cell
interaction with ECM components and cytoskeleton
reorganization. In this process an important role is
played by matrix metalloproteases (MMPs), which
are released at the fiber tips. MMPs are a large family of proteins, synthesized as propeptide and then
converted to active enzyme; MMPs are released by
the cells in order to modify ECM components and
assist neurite elongation. We evaluated whether
MMPs expression or processing is modified in neuroblastoma cells, following activation of muscarinic
receptors. MMPs in culture medium was evaluated
by zymography and Western blot analysis. N18TG2
cell line and ChAT transfected clones were analyzed
under different culture conditions, leading to activation or inactivation of muscarinic receptors, which
are equally expressed in the two cell types.
The data obtained show that N18TG2 cells do not
Participants:
Stefano Biagioni, Ada M. Tata, professors; Emanuele Cacci,
Giancarlo Poiana, researchers; Monica Salani, Chiara
Soldati, PhD students.
Collaborations:
Dipartimento di Biologia Animale e dell’Uomo, Università di
Torino (Prof. I. Perroteau); Dipartimento di Fisiologia Generale
ed Ambientale, Università di Napoli ‘Federico II’ (Prof. C.
Perrone-Capano); Istituto di Istologia ed Analisi di Laboratorio,
Università Carlo Bo di Urbino (Dr. F. Mannello).
Report of activity
The aim of the project was to investigate the role of
neurotransmitters, as part of the signalling system
acting in nervous system development, particularly
in directing neuron differentiation. As described in
previous reports, we have focused our studies on
acetylcholine (Ach), using two experimental systems: mouse neuroblastoma lines defective for neurotransmitter synthesis and chick and rat dorsal root
ganglia (DRG). Evidences for the presence of an
Ach autocrine/paracrine loop in both systems has
been described in 2003-04 report. In 2005 efforts
have been devoted to further characterize the panel
of genes, whose expression is modulated by the
autocrine loop activated in ChAT transfected neuroblastoma cells and to characterize the Ach releasing mechanism in DRG.
ChAT transfected neuroblastoma cells
Muscarinic receptors. Neurotransmitters, including
acetylcholine, are considered part of the signalling
system which acts in nervous system development.
In this context we have previously reported that
acetylcholine is capable of enhancing neuronal differentiation through activation of muscarinic receptors in DRG primary cultures and in the neuroblastoma line N18TG2. In order to study the mechanism
17
G. Augusti Tocco - Signalling systems in neuronal differentiation and their possible mechanism of action
give origin to more than one transcripts. Their regulation appears to be rather complex and they can
also be independently regulated.
Analysis by real time-PCR demonstrated that chick
DRG express transcripts for both ChAT and VAChT.
VAChT is expressed at an early developmental stage
(E12) and its levels decreases during development
and in post hatching period. On the other hand
ChAT and mediatophore expression shows a progressive increase, suggesting that ACh synthesis and
release might play a key role during DRG development. However the release is likely mediated by both
VAChT and mediatophore at early developmental
stage (E12), while at a later stage (E18) and in the
post hatching period (P7) mediatophore becames the
main release mechanism. According to this hypothesis, VAChT expression appears to decrease also in rat
DRG development, while ChAT expression shows a
linear increase until 15d postnatal.
In rat DRG we have also analyzed the transcripts,
using selective primers for transcripts arising from
different promoter activity. RT-PCR analysis
revealed the presence of the splice variants V1/V2
for VAChT and R1, R2 and N for ChAT. Their transcript levels appear to be modulated during development. Altogether these results suggest that in DRG
VAChT and ChAT transcripts are independently
regulated.
constitutively secrete MMPs in the medium, whereas two members of the MMP family, MMP-2 and
MMP-9 are present in the medium of ChAT transfected cells, associated to the reported induction of
fiber extension. MMPs secretion can also be induced
in N18TG2 cells upon exposure to the muscarinic
agonist carbachol or in EGR-1 transfected N18TG2.
In conclusion MMPs appear to be among the genes,
whose expression is modulated by Ach release in
ChAT transfected cells, through the activation of the
neurospecific transcription factor EGr-1.
Dorsal root ganglia (DRG)
We have previously reported that DRG neurons,
although they do not use ACh as neurotransmitter,
express cholinergic markers at early developmental
stages and release acetylcholine. They are also able
to release ACh with a calcium dependent mechanism,
and to respond to cholinergic stimulation, since they
express both muscarinic and nicotinic cholinergic
receptors. Considering the non-cholinergic nature of
DRG neurotransmission, it appeared of interest to
study in more details the mechanism of Ach release
by DRG neurons. We have then studied a major component of the Ach release mechanism, the vesicular
Ach transporter (VAChT), which is responsible for Ach
transport into synaptic vesicles, and a protein
involved in an alternative non vesicular release, the
mediatophore, a proteolipid first described in Torpedo
electric organ. VAChT is an interesting case, since it
is localized together with the ACh biosynthetic
enzyme ChAT, on a common locus, designated as
cholinergic locus. ChAT and VAChT expression in
mammals is dependent on multiple promoters, which
Augusti-Tocco G, Biagioni S, Tata AM.
Acetylcholine and regulation of gene expression in
developing systems. J Mol Neurosci, 2006; 30(1-2):45-8.
18
Chromatin remodelling, histone code and signal transduction in
Ascomycetes
Principal investigator: Paola Ballario
Researcher in Molecular Genetics
Dipartimento di Genetica e Biologia Molecolare
Tel.: (+39) 06 49912318, Fax: (+39) 06 4440812
[email protected]
tails of histones H3 and H4. In addition the conserved domain (bromodomain) of Gcn5 is able to
recognize the residue K16 of histone H4 when acetylated. All these characteristics confers to this small
protein, member of multiprotein complexes, a pivotal function in many cellular functions although not
completely identified.
In addition we are in progress with the molecular
genetic characterization of T.borchii another filamentous fungus, belonging to the order of Pezizales This
order is ecologically significant because it includes
both hypogeous and epigeous fungi, with either a
saprotrophic or a symbiotic lifestyle, that colonize a
variety of forest and semidesertic ecosystems. The
multicellular fungus forming strongly flavored fruitbodies, Tuber spp. are highly prized as a natural food
complement in Italy but also in various parts of the
world and there is a fairly strong interest in improving their production. Fruitbody development only
takes place after the vegetative mycelium has established a productive, symbiotic interaction with a
compatible host plant. This association, called ectomycorrhiza, greatly improves plant mineral nutrition
as well as plant survival under a variety of conditions. The molecular biology and genetics of Tuber
spp. is in its infancy, therefore we are working at two
levels: one is a cellular and genomic characterization
of the organism, the other is the identification light
signal transduction pathways. Our results are
reported for each organism.
Neurospora crassa. Blue light-induced transcription in
this microrganism is regulated by the photoreceptor
White Collar-1 (WC-1). The aim of our project was
to investigate the role of chromatin remodelling in
the control of albino-3 (al-3) promoter by ChIP
analysis we seen it to become transiently acetylated
after photoinduction. This acetylation depends on
WC-1. The relevance of this chromatin modification
was directly evaluated in vivo by construction of a
Neurospora strain with a mutated histone H3 gene
(hH3K14Q). This strain phenocopies a wc-1 blind
Participants:
Patrizia Filetici, CNR researcher; Benedetto Grimaldi, postdoc fellow; Stefano Vernarecci, PhD student; Andrea
Brenna, Alessandra Gatti, Stefania Federico, graduate
students.
Collaborations:
Department of Biology and Institute of Molecular Biology,
University of Oregon, Eugene, OR, USA (Prof. Eric Selker);
Dipartimento di Biochimica, Università di Parma (Prof. Simone
Ottonello); Dipartimento di Biologia Vegetale e Biotecnologie
Agroambientali e Zootecniche (Dr. Leonardo Baciarelli Falini).
Report of activity
We have studied the relationships between signals
transduction and post-translation modification of
proteins (mainly histones) as a mechanism of epigenetic control of gene expression. The Ascomycetes:
Saccharomyces cerevisiae, Neurospora crassa and Tuber
borchii are our model systems for these studies.
The epigenetic regulation of gene expression operates through the modifications of N-terminal tails of
histones. By this mechanism a particular locus can be
maintained in a silenced or activated transcriptional
condition for many cellular generations, but can also
be quickly reversed, on the basis of new cellular or
environmental signals. Our principal objective is to
contribute to the understanding of the interrelationship between the “histone code” proposed by D.Allis
and signal transduction in eukaryotic model systems
We have studied in S.cerevisiae and N.crassa the
responses to two type of signals: to light as morphogenetic stimulus in filamentous fungi and to cellular factors controlling cell cycle progression in
yeast. An element in common among the different
systems is the histone acetyl transferase Gcn5p, a
well known coactivator with pleiotropic functions
whose main function (histone acetyltransferase HAT) is the acetylation of lysines of the N-terminal
19
P. Ballario - Chromatin remodelling, histone code and signal transduction in Ascomycetes
Tuber borchii.. We have previously identified in T.
borchii (Ambra et al., 2004) a response to blue light
and cloned a gene coding for a blue light photoreceptor (Tbwc-1), extremely conserved, in functional
domains and amino acidic sequence, in comparison to
the Neurospora WC-1. In order to demonstrate that
Tbwc-1 was responsible for light responses in Tuber
we would like to make gene disruption. Therefore we
developed the first protocol of genetic transformation of Tuber spp. We used an hypervirulent
Agrobacterium tumefaciens strain bearing the binary
plasmid pBGgHg for the transformation of Tuber
borchii mycelia growth in vitro. The ‘hygromycin
resistance’ and the ‘enhanced green fluorescent protein’ genes, both under the control of vector-borne
promoters, were employed as selection markers.
Patches of dark and fluorescent hyphae were
observed upon fluorescence microscopy examination
of hygromycin-resistant mycelia. The presence of
EGFP was confirmed by both confocal microscopy
and PCR analysis (Grimaldi et al., 2005). Therefore
we have now a protocol for Tuber borchii transformation, but we need that the transgenic DNA integrate
in the homologous genomic locus. We have now
developped two new Agrobacterium based plasmids
studied for homologous integration and we will try
to obtain the KO of Tbwc-1.
In addition, our efforts are concentrated on the possibility of obtaining the genome sequence of this
interesting hypogeous fungus, partecipating to a
consortium of several international groups.
mutant and shows a strong reduction of lightinduced transcriptional activation of both al-3 and
vivid (vvd), another light inducible gene. We mutated
ngf-1 (Neurospora GCN Five), which encodes a homologue of the yeast HAT Gcn5p, to generate a strain
impaired in H3 K14 acetylation and found that it was
defective in photoinduction. Our findings reveal a
direct link between histone modification and light signaling in Neurospora and contribute to the understanding of the molecular mechanisms operating in
light inducible gene activation (Grimaldi et al., 2006).
We are now studing the possible acetylation by
Gcn5p of other factors, as WC-1 and FRQ involved
in light signal translation.
Saccharomyces cerevisiae.. Acetylation of chromatin
mediated by Gcn5p activity is involved mainly in
transcriptional control, but also in other cellular
functions. In a delta gcn5 strain we observed defects
in mitosis, in particular in the steps of elongation
and nuclear division in addition to a high rate of
chromosomes lost. It is known that the correct
assembly of kinetochore on the centromeric region
ensures the correct function of the machinery for
chromosomes segregation. In order to understand if
a genetic interaction of Gcn5p with members of
multiprotein complexes involved in kinetocore
assembly exists we constructed double mutants
between delta gcn5 and mutant in single kinetocore
subunit mutants. Several interactions of proteins
have been identified. The most robust interaction
occurred between gcn5 and the yeast centromeric
specific histone H3 variant cse4-1 allele. Negative and
positive genetic synergies between mutation in Gcn5p
and in kinetochore components provide evidences of
functional interaction of these cellular components.
Remarkably, the cse4-1 mutant shows a significant
phenotypic convergence with that of gcn5-delta
strain.the analysis by accessibility to nuclease digestion and of nucleosome positioning over the core centromeric region provide evidences that Gcn5p directly contributes to the centromeric chromatin structure
(Manuscript submitted).
Chromosome segregation defects are a characteristic of human tumor progression; therefore we have
(Smith et al., 2006) and we will investigate the possibility of using Gcn5 as target for new molecules
of inhibitors. Future work will determine if and
how histone acetylase inhibitors could represent
valuable anti-mitogenic molecules.
Grimaldi B, Coiro P, Filetici P, Berge E, Dobosy JR,
Freitag M, Selker EU, Ballario P. The Neurospora crassa
White Collar-1 dependent blue light response requires
acetylation of histone H3 lysine 14 by NGF-1. Mol Biol
Cell, 2006; 17(10):4576-83. Epub 2006 Aug 16.
Pizzitutti F, Giansanti A, Ballario P, Ornaghi P,
Torreri P, Ciccotti G, Filetici P. The role of loop ZA
and Pro371 in the function of yeast Gcn5p bromodomain revealed through molecular dynamics and experiment. J Mol Recognit, 2006; 19(1):1-9.
Grimaldi B, de Raaf MA, Filetici P, Ottonello S,
Ballario P. Agrobacterium-mediated gene transfer and
enhanced green fluorescent protein visualization in the
mycorrhizal ascomycete Tuber borchii: a first step
towards truffle genetics. Curr Genet, 2005; 48(1):6974. Epub 2005 May 3.
20
RNA-RNA and RNA-protein interactions in the cell nucleus:
structure, function and biosynthesis of a novel class of small non
coding RNAs
Principal investigator : Irene Bozzoni
Tel.: (+39) 06 49912202, Fax (+39) 06 49912500
[email protected]
lying mechanisms.
To achieve these goals, integrated and multidisciplinary action was required including basic research
and development, genetic approaches, as well as cell
biology assessment.
Participants:
Elisa Caffarelli, Alessandro Fatica, Carlo Presutti,
researchers; Fernanda De Angelis, Mariangela Morlando,
Michela Denti, post-doc fellows; Monica Ballarino, Pietro
Laneve, Alessandro Rosa, PhD students.
Biosynthesis of small non-coding RNAs
Collaborations:
Many snoRNAs are encoded in introns of protein coding genes. In vertebrates, we have demonstrated that
the release of several of these snoRNAs requires
endonucleolytic cleavage inside the intron. In the last
years, we have been able to purify the endonucleolytic
activity (XendoU) and to study several biochemical
parameters that control its function (Gioia et al., 2005,
J Biol Chem, 280:18996-9002). More recently, we have
been also able to crystallize the protein and to study its
physical-chemical properties (Renzi et al., 2006, Acta
Cryst, F62:298-301; Renzi et al., 2006, Proc Natl Acad
Sci USA, 103:12365-70): the combination of biochemical and structural approaches allowed us to identify
the peculiar architecture of the XendoU active site,
that is conserved in the SARS-coronavirus homolog.
This knowledge, besides elucidating the enzymatic
activity, represents the first step towards the design of
antiviral drugs that may specifically interfere with the
viral replication.
At difference with vertebrates, in yeast many snoRNAs
are independently transcribed. Our recent results have
shown that the biogenesis of the snoRNPs belonging
to the box H/ACA class already starts during the transcription stage and that this assembly is required for
correct maturation of the snoRNP. We described that
the assembly factor Naf1p and the core components,
Cbf5p and Nhp2p, are recruited on H/ACA snoRNA
genes very early during transcription. We also showed
that the co-transcriptional recruitment of Naf1p and
Cbf5p is Ctk1p-dependent and that Ctk1p and Cbf5p
are required for preventing the readthrough into the
snoRNA downstream genes. All these data suggest
that correct co-transcriptional snoRNP assembly controls 3’-end formation and, consequently, the release of
Dipartimento di Scienze Biochimiche, Sapienza-Università di
Roma (Prof. Maurizio Brunori); TIGEM, Napoli (Prof. Alberto
Auricchio); University of Pavia (Prof. Roberto Bottinelli);
Dipartimento di Istologia ed Embriologia Medica, SapienzaUniversità di Roma (Prof. Antonio Musarò, Prof. Clara Nervi);
City of Hope, Duarte, USA (Prof. John Rossi).
Report of activity
The aim of this project was to exploit and further
develop the high potential of RNA-based methodologies for the comprehension of the molecular basis of
several processes of gene regulation and to apply this
knowledge to the study and cure of several genetic disorders, such as cancer or inherited diseases. One part of
the project was aimed at elucidating the general rules
which control several RNA maturative steps and, in
particular, to analyze the temporal and physical connection between the processing machineries and the
transcription apparatus: in the last years it has been
shown that many of the post-transcriptional modifications are mediated by factors that assemble very early
on pre-RNAs, eventually during the initial phases of
transcription. In a parallel line of research we have
studied the role that the newly discovered class of
microRNAs (miRNAs) plays in the control of gene
expression and we analyzed the function of specific
miRNAs in controlling the switch between proliferation and differentiation. Another part of the project
wanted to exploit the vast potential of different RNA
activities (antisense and interference) for human therapy, based on an advanced understanding of the under-
21
I. Bozzoni - RNA-RNA and RNA-protein interactions in the cell nucleus: a novel class of snoRNAs
towards the normal values at single muscle fibre
level (Denti et al., 2006, Proc Natl Acad Sci USA,
103:3758-63). Very interestingly, also muscular districts such as heart and diaphragm (particularly
affected in DMD patients) resulted efficiently colonized and able to produce high levels of dystrophin.
This approach provides solid bases for a systemic use
of AAV-mediated antisense-U1 snRNA expression
for the therapeutic treatment of DMD.
Vectors for siRNA expression. Several vectors for the
induction of RNA interference in mammalian cells
have been described, based mainly on polIII-dependent
promoters. They transcribe short hairpin RNAs
(shRNA) that, after being processed into short interfering RNAs (siRNAs), mediate the degradation of the
target mRNA We previously developed the siRNAexpressing vector psiUx based on the polII promoter
of the U1 small nuclear RNA gene. This construct has
proven to be very efficient in producing the knock
down of specific mRNAs and proteins in several different cell systems (Tessitore et al., 2006, Mol Therapy,
17:565-74). More recently, we converted such construct into an inducible system by inserting a loxPStuffer-loxP cassette upstream of the transcriptional
initiation site. This construct does not express the
shRNA until the two canonical loxP sites undergo
Cre-mediated recombination. Using a Cre recombinase
whose transcription is under the control of a tetOn
system, we were able to show the temporally controlled expression of an shRNA directed towards the
lamin A/C mRNA, as well as the regulated knockdown
of its target (Iovino et al., 2005, RNA Biology, 2:3).
a functional particle (Ballarino et al., 2005, Mol Cell
Biol, 25:5396-403).
Role of miRNA in differentiation
miRNAs are emerging as a very interesting and new
class of transcripts, of very limited size, that play
crucial functions in cell differentiation and proliferation by acting as translational inhibitors of specific
target genes. We have described that the transcriptional up-regulation of miR-223 is important for
human granulocytic differentiation and that this
transcriptional control is modulated by the competitive binding of NFI-A and C/EBPa transcriptional
factors to its own promoter. NFI-A maintains miR223 at low levels, while its replacement by C/EBPa
subsequent to the differentiation stimulus, induces
miR-223 up-regulation. The competition between
C/EBPa and NFI-A is alleviated by miR-223 itself
that negatively controls NFI-A translation. Notably,
ectopic expression of miR-223 in APL cells increases the percentage of granulocytic cells, while its
knock-down inhibits the differentiation response.
RNAi against NFI-A enhances RA-induced differentiation demonstrating that decreased levels of this
factor are required for granulocytic maturation.
Altogether, our data indicate that miR-223 plays a
crucial role during granulopoiesis and point to the
NFI-A repression as an important molecular pathway mediating gene reprogramming in this cell-lineage (Fazi et al., 2005, Cell 123:819-31; Fatica et al.,
2006, Cold Spring Harbor Symposia on Quantitative
Biology 71; Nervi et al., 2006, in: Acute promyelocytic Leukemia: Molecular Genetics, Mouse Models and
Targeted Therapy, Springer-Verlag GmbH Berlin).
Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis
ML, Nervi C, Bozzoni I. A minicircuitry comprised
of microRNA-223 and transcription factors NFI-A
and C/EBPalpha regulates human granulopoiesis.
Cell, 2005; 123(5):819-31.
Therapeutic RNAs
Antisense RNAs. Duchenne Muscular Dystrophy
(DMD) is a X-linked muscle disease characterized by
mutations in the dystrophin gene. Many of these can
be corrected at the post-transcriptional level by skipping the mutated exon. We have obtained persistent
exon-skipping in mdx mice by local injection (Denti
et al., 2006, Hum Gene Ther, 17:565-74) or tail vein
injection (Denti et al., 2006, Proc Natl Acad Sci USA,
103:3758-63) with an Adeno-associated viral (AAV)
vector expressing antisense sequences as part of the
stable cellular U1 snRNA. Systemic delivery of the
AAV-construct resulted in effective body-wide colonization, significant recovery of the functional properties in vivo and lower creatine kinase serum levels,
suggesting an overall decrease in muscle wasting.
The transduced muscles rescued dystrophin expression and displayed a significant recovery of function
Ballarino M, Morlando M, Pagano F, Fatica A,
Bozzoni I. The cotranscriptional assembly of
snoRNPs controls the biosynthesis of H/ACA
snoRNAs in Saccharomyces cerevisiae. Mol Cell Biol,
2005; 25(13):5396-403.
Denti MA, Rosa A, D’Antona G, Sthandier O, De
Angelis FG, Nicoletti C, Allocca M, Pansarasa O,
Parente V, Musaro A, Auricchio A, Bottinelli R,
Bozzoni I. Body-wide gene therapy of Duchenne
muscular dystrophy in the mdx mouse model. Proc
Natl Acad Sci USA, 2006; 103(10):3758-63. Epub
2006 Feb 24.
22
Is poly-ADPribose polymerases inhibition responsible for
anomalous oncosuppressor gene hypermethylation?
Principal investigator: Paola Caiafa
Professor of Clinical Biochemistry and Molecular Biology
Tel. (+39) 06 85795530, Fax: (+39) 06 44231961
[email protected]
vation that modified PARP-1 and Dnmt1 coimmunoprecipitate in vivo and on data showing that ADPribose polymers, present on modified PARP-1,
almost completely inhibit Dnmt1 activity in vitro.
Our recent findings (Zampieri et al. Poly(ADP-ribosyl)ation controls Dnmt1 expression, submitted) have
shown that poly(ADP-ribosyl)ation is involved in the
control of Dnmt1 expression, and therefore in control of level of the enzyme particularly responsible
for DNA methylation process. In fact, Western Blot,
RT-PCR and Real-Time PCR analyses, carried out to
verify whether level of Dnmt1 and/or expression of
Dnmt1 gene depend on the time of inhibition of
PARP activity, have shown that level of Dnmt1
increases of three-fold vs control in human fibroblast
cycling-cells after short times of treatment with
inhibitor while long times lead to its silencing. Thus
the decreased level of ADP-ribose polymers regulates over time the nuclear level of Dnmt1 and RTPCR and Real Time PCR data indicate that the
absence of Dnmt1 is consequence of decreased
expression of correlated gene.
To explain the silencing of Dnmt1 we examined if
the CpG island, which is located in the promoter
region of Dnmt1, could be the one that becomes
methylated in anomalous way after inhibition of
PARP activity. Following bisulphite reaction, fragments located inside the CpG island were analysed
for their level of methylation by methylation PCR
sensible method (MSP) and by DNA sequencing.
Through the reaction with bisulphite, which fixes the
memory of methylation pattern present on genomic
DNA, it was possible to see that, after prolonged
treatment with 3-ABA, all CpG dinucleotides under
examination were methylated unlike those from
respective controls where they were completely
unmethylated. Thus in this scenario which sees the
spreading of anomalous methylation on CpG island
regions, we have found that the island that is present
in the promoter of Dnmt1 undergoes methylation
leading to the silencing of the enzyme.
Participants:
Anna Reale, professor; Barbara Cecchinelli, post-doc fellow;
Tiziana Guastafierro, Michele Zampieri, PhD students.
Collaborations:
Istituto di Biologia e Patologia Molecolari, CNR, Roma (Dr.
Nicoletta Corbi, Dr. Claudio Passananti).
Report of activity
CpG islands represent 1-2% of genomic DNA, are
about 30,000 and are generally located in the 5’ promoter region of housekeeping genes, sometimes
overlapping the coding region to variable extents
(usually to the first exon). Although their sequence is
enriched in CpG dinucleotides, which are the best
substrates for DNA methyltransferase, CpG islands
are unmethylated and transcription of genes associated with them is active when these regions are
unmethylated while it is inhibited when these
regions undergo methylation. The mechanism by
which CpG islands are protected from methylation
during replication and in chromatin and also the
mechanism(s) whereby these DNA regions, which
remain protected from methylation in normal cells,
are susceptible to methylation in tumour cells is still
an open question.
Previous data of our research group have shown that
poly(ADP-ribosyl)ation and DNA methylation are
interconnected as it has been found that competitive
inhibition of poly(ADP-ribose) polymerases
(PARPs) leads to introduction of anomalous methyl
groups onto DNA. To explain this phenomenon two
possible molecular mechanisms have been proposed.
The first one based on the observation that inhibition
of PARP activity leads to over-expression of Dnmt1
in the anomalous G1/early S phase in vivo, thus
allowing the premature formation of PCNA-Dnmt1
active complex in the phase in which CpG islands
undergo replication. The second based on the obser-
23
P. Caiafa - Is poly-ADPribose polymerases inhibition responsible for anomalous oncosuppressor gene hypermethylation?
A parallel examination of bulk CpG islands showed
that inhibition of PARP activity is accompanied by a
decrease in the “tiny DNA fragments” which are
formed after HpaII digestion of genomic DNA, phenomenon which is reversed following prolonged treatment suggesting an unexpected demethylation phenomenon. As further confirmation for demethylation,
Southern-Blot analyses were carried out using two different probes against Alu-Sx elements since these,
enriched in CpG dinucleotides, can produce HpaII tiny
fragments when they undergo demethylation. Data
obtained have shown that DNA purified from 3-ABA
treated cells has undergone a more extensive digestion
by HpaII due to the presence of new demethylated cutting sites within the Alu sequences.
Experiments of poly(ADP-ribose) glycohydrolase
(PARG) over-expression - the enzyme responsible
for dynamic ADP-ribose polymer degradation in
nuclei - confirm that ADP-ribose polymers directly
controls Dnmt1 expression. Thus, this research has
shown that a deregulation of PARP activity induces
a deregulation of Dnmt1 activity and that, by modulating Dnmt1 expression, controls the methylation
pattern of some CpG islands.
Although, at this stage, our research does not provide a direct correlation between poly(ADP-ribo-
syl)ation and cancer, it demonstrates that the deregulation of PARP activity is able to modify the methylation state of some CpG islands, an event that
recalls what happens in promoter regions of tumour
suppressor genes which become methylated during
tumourigenesis in cancer cells. Above all, these data
suggest that the hypomethylation which is present
on genomic DNA of some tumour cells could be correlated to the observed spreading of methylation
which causes the silencing of Dnmt1.
Caiafa P, Zampieri M. DNA methylation and chromatin structure: the puzzling CpG islands. J Cell
Biochem, 2005; 94(2):257-65.
Reale A, Matteis GD, Galleazzi G, Zampieri M,
Caiafa P. Modulation of DNMT1 activity by ADPribose polymers. Oncogene, 2005; 24(1):13-9.
Carbone M, Reale A, Di Sauro A, Sthandier O, Garcia
MI, Maione R, Caiafa P, Amati P. PARP-1 interaction
with VP1 capsid protein regulates polyomavirus
early gene expression. J Mol Biol, 2006; 363(4):77385. Epub 2006 Jun 30.
24
Molecular interactions at the rDNA locus in Saccharomyces
cerevisiae
Principal investigator: Giorgio Camilloni
Tel.: (+39) 06 49912808, Fax: (+39) 06 49912500
[email protected]
to clarify the role of this enzyme in important events
occurring in the rDNA such as the control of
genome stability, transcriptional silencing and cellular aging.
Participants:
Francesca Di Felice, post-doc fellow; Francesco Chiani, PhD
student; Elisa Cesarini, Francesca Romana Mariotti,
Valentina Robbiati, graduate students.
Design, synthesis, and biological evaluation
of sirtinol analogues as class III
histone/protein deacetylase (sirtuin)
inhibitors
Collaborations:
Department of Biological Chemistry, University of CaliforniaIrvine, Irvine, CA, USA (Dr. Masayasu Nomura); Dipartimento di
Studi Farmaceutici, Sapienza-Università di Roma (Prof.
Antonello Mai); Department of Biological Chemistry, University
of California Los Angeles, Los Angeles, CA, USA (Dr. Michael
Grunstein); Department of Pathology, Harvard Medical School,
Boston, MA, USA (Dr. David A. Sinclair).
In a search for potent inhibitors of class III histone/protein deacetylases (sirtuins), a series of sirtinol analogues have been synthesized 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-naphthalenylmethylene)-amino]-N-(1-phenylethyl)benzamide (i.e., m- and p-sirtinol), were 2- to 10-fold
more potent than sirtinol against human SIRT1 and
SIRT2 enzymes. In yeast in vivo assay, these two
small molecules were as potent as sirtinol.
Compounds lacking the 2-hydroxy group at the
naphthalene moiety or bearing several modifications
at the benzene 2D-position of the aniline portion
(carbethoxy, carboxy, and cyano) were 1.3-13 times
less potent than sirtinol, whereas the 2D-carboxamido analogue was totally inactive. Both (R)- and (S)sirtinol had similar inhibitory effects on the yeast
and human enzymes, demonstrating no enantioselective inhibitory effect.
Report of activity
FOB1 affects DNA topoisomerase I in vivo
cleavages in the enhancer region of the S.
cerevisiae ribosomal DNA locus
In S. cerevisiae the FOB1 gene affects replication fork
blocking activity at the replication fork block (RFB)
sequences and promotes recombination events within the rDNA cluster. Using in vivo footprinting
assays we mapped two in vivo Fob1p binding sites,
RFB1 and RFB3, located in the rDNA enhancer
region and coincident with those previously reported
to be in vitro binding sites. We previously provided
evidences that DNA topoisomerase I is able to cleave
within this region in two sites. The results obtained
indicate that the DNA topoisomerase I cleavage specific activity at the enhancer region is affected by the
presence of Fob1p and independent on replication
and transcription activities. We thus hypothesize
that the binding to DNA of Fob1p itself may be the
cause of the DNA topoisomerase I activity in the
rDNA enhancer.
The finding of an involvement of Fob1p in the sitespecific activity of DNA topoisomerase I contributes
SIR2 modifies histone H4-K16 acetylation and
affects superhelicity in the ARS region of
plasmid chromatin in S.cerevisiae
The null mutation of the SIR2 gene in Saccharomyces
cerevisiae has been associated with a series of different phenotypes including loss of transcriptional
silencing, genome instability and replicative aging.
Thus, the SIR2 gene product is an important constituent of the yeast cell. SIR2 orthologues and paralogues have been discovered in organisms ranging
25
G. Camilloni - Molecular interactions at the rDNA locus in Saccharomyces cerevisiae
from bacteria to human, underscoring the pivotal
role of this protein. Here we report that a plasmid
introduced into sir2D cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells (Figure 1). This
effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not
depend on the number or positioning of nucleosomes on plasmids. Our results indicate that this new
phenotype is due to the lack of Sir2p histone
deacetylase activity in the sir2D strain, because only
the H4-K16 residue of the histone octamer undergoes an alteration of its acetylation state.
The overall conclusion is that Sir2p limits MCM
complex loading onto ARSs: in the sir2D mutant,
overloading of the complex increases the unwinding
of DNA, leading to negative supercoiling of plasmids. Based on our demonstration that the only
amino acid hyperacetylated in plasmid chromatin is
H4-K16, we hypothesize that a specific histone
deacetylase activity is involved in the reported phenotypes, and more specifically that H4-K16 hyperacetylation represents a signal for the recruitment of
additional factor(s), such as the MCM complex. This
is in agreement with previous data showing that loss
of Sir2p catalytic activity increases MCM loading on
ARS sequences. We cannot eliminate the possibility
that our results were due not only to the Sir2p histone-deacetylase activity on H4-K16, but also to its
putative ability to deacetylate different proteins. In
fact, Sir2p homologues in other species, including
mammals, have been demonstrated to deacetylate
non-histone proteins as well. A further interpretation of our data is that it might reflect direct
deacetylation activity of Sir2p on the MCM complex, with the consequent alteration of its function
in the assembly of the Pre-RC complex. In support
of this hypothesis, it has been demonstrated that
Mcm3p is acetylated in mammalian cells. Our
demonstration that the topology of plasmids is
altered in sir2D mutants reveals a new phenotype for
this gene; whether the effect is directly mediated by
the MCM complex or follows indirect pathways will
be the subject of future investigations.
Chiani F, Di Felice F, Camilloni G. SIR2 modifies
histone H4-K16 acetylation and affects superhelicity
in the ARS region of plasmid chromatin in
Saccharomyces cerevisiae. Nucleic Acids Res, 2006;
34(19):5426-37.
Mai A, Massa S, Lavu S, Pezzi R, Simeoni S, Ragno
R, Mariotti FR, Chiani F, Camilloni G, Sinclair DA.
Design, synthesis, and biological evaluation of sirtinol analogues as class III histone/protein deacetylase (Sirtuin) inhibitors. J Med Chem, 2005; 48
(24):7789-95.
Di Felice F, Cioci F, Camilloni G. FOB1 affects DNA
topoisomerase I in vivo cleavages in the enhancer
region of the Saccharomyces cerevisiae ribosomal
DNA locus. Nucleic Acids Res, 2005; 33(19):6327-37
Fig. 1 - Analysis of the topoisomers distribution in different yeast plasmids in WT and sir2D cells.
26
The Dof transcription factors in Arabidopsis development
Principal investigator: Paolo Costantino
Tel.: (+39) 06 4455344, 06 49912242, 06 49912411, Fax: (+39) 06
4455344, 06 4440812
[email protected]
active in the vascular system of the mother plant but
not in the embryo. In situ mRNA hybridizations confirm the presence of the transcripts of the two genes
in the phloem of the mother plant but not in the
embryo at any stage of development. These data are
confirmed by segregation analysis indicating that the
effect of the mutation of both DAG1 and DAG2 is
maternal. In contrast, we have shown that the major
phenotype of the mutants is in seed germination,
where mutant dag1 and dag2 seeds show a higher
and, respectively, lower germination potential than
wt seeds. We have subsequently shown that the
DAG1 protein, as a translational fusion of its cDNA
with the green fluorescent protein (GFP), is present
in cells where the DAG1 gene is not expressed. Our
data indicate that the DAG1:GFP protein is present
even in embryo cells. This indicates that either the
DAG1 protein is transported from the mother plant
to the embryo, or that the DAG1:GFP construct
lacks a crucial transcriptional/post-transcriptional
regulation that normally limits the presence of the
DAG1 mRNA to the mother plant. We are currently
investigating on this issue with several complementary approaches, including in situ mRNA hybridizations and the search for miRNAs. In addition, we are
analyzing the possible regulatory role of the first
intron of the DAG1 gene whose unusually large size
in the Arabidopsis genome (~800 bp) makes it a good
candidate for the presence of regulatory sequences.
With the aim of identifying the role of DAG1 with
respect to PhyB and other genes involved in PhyBdependent germination, we used a genetic approach
to isolate and characterize double mutants of dag1,
phyB and other genes known to be involved in PhyBdependent seed germination and other PhyB-mediated developmental processes. In particular, we have
crossed dag1 with both a null phyB allele (phyB-9)
and a leaky one (phyB-4); with pil5 and obp3 mutant
alleles, which are respectively a negative regulator of
PhyB-dependent seed germination, and a suppressor
Participants:
Paola Vittorioso, professor; Maura Cardarelli, Mirella
Pomponi, Giovanna Serino, researchers.
Collaborations:
Dipartimento di Biologia Vegetale, Sapienza-Università di Roma
(Prof. M.M. Altamura); Dipartimento di Biologia Evolutiva e
Funzionale, Università di Parma (Prof. L. Sanità di Toppi);
Department of Molecular Genetics, Utrecht University, The
Netherlands (Prof. R. Heidstra).
Report of activity
The Dof proteins are transcription factors present
only in plants and characterized by a strongly conserved single CX2CX21CX2C zinc finger domain. In
Arabidopsis, data from the complete genomic
sequence indicate the presence of 36 members of the
Dof gene family. We isolated and are currently
working on three closely related Dof genes. We have
shown that two of these genes, DAG1 and DAG2 are
involved in regulating the germination of seeds.
Another Dof gene was originally identified in our
laboratory as encoding the tobacco protein
(NtBBF1) responsible for the regulation of the oncogene rolB in plant meristems and for the responsiveness of rolB expression to the hormone auxin. Very
recently, we isolated the Arabidopsis hortolog of the
NtBBF1 gene (AtBBF1) and a T-DNA insertion
mutant allele of AtBBF1.
Objective of this research is to clarify the role of
these three Dof genes in Arabidopsis development
by: a) defining the function of DAG1 and DAG2 in
seed germination; b) defining the function of
AtBBF1 and rolB in inducing meristem formation
and in regulating hormonal response of plant genes.
DAG1. We have previously shown that the promoters of the DAG1 and DAG2 genes are specifically
27
P. Costantino - The Dof transcription factors in Arabidopsis development
of the phyB-4 mutation. At the moment all these
double mutants have been isolated by PCR, and their
characterization is underway, with respect to seed
dormancy and germination (in different environmental conditions), and to GA requirement (Dello Ioio et
al., Cytokinins determine Arabidopsis root meristem
size by controlling cell differentiation, in press).
pDMC1:rolB tobacco plants, containing the promoter
of the Arabidopsis gene DMC1, fused to the rolB
coding region, display shorter filaments as compared
to controls, a severe delay in anther dehiscence and
alterations in male and female meiosis (Cecchetti et
al, Plant J, 38: 512-525, 2004). We subsequently isolated a tobacco gene, ROX1, acting downstream of
rolB, overexpressed in pDMC1:rolB anthers at all
developmental stages. Plants with reduced levels of
ROX1 mRNA, due to the expression of a ROX1-antisense construct, have flowers with stamens and pistils longer than normal, due to an increased number
of cells. Longer stamens of antisense plants show a
delayed xylem differentiation while the shorter stamens of pDMC1:rolB plants show a precocious differentiation of xylem cells and a reduced number of
cells. In agreement with these data expression of
ROX1 in stamens is mostly localized in procambial
cells. The results of this study indicate a role for
ROX1 in procambial cell proliferation and xylem differentiation during stamen development (Cecchetti
et al, Plant J, 49:27-37, 2006).
AtBBF1. We characterized two T-DNA insertion
mutants in the AtBBF1 gene, that we named atbbf11 and atbbf1-3: atbbf1-1 still contains significant
amount of an AtBBF1 truncated trancript, while
atbbf1-3 is a complete loss-of-function allele. Because
our morphological and anatomical analysis of the
two alleles did not reveal any obvious phenotype, we
hypothesized a functional redundancy among other
Dof genes. To explore this possibility, we extended
our analysis to DOF24, the AtBBf1 closest homologue. DOF24 and AtBBF1 share 45,4% identity at
the aminoacid level on the overall sequence and
100% identity over the DOF domain. Interestingly,
DOF24 and AtBBf1 are both expressed in the vascular tissue of Arabidopsis seedlings. In the adult
plants, however, AtBBF1 and DOF24 show different
but complementary expression profiles in floral
organs: AtBBF1 is mostly expressed in the tapetum
tissue of the anthers and in the stigma only during
fertilization, while DOF24 is mostly expressed in
pollen grains. A double atbbf1/dof24 mutant is now
under study and will help us to clarify the relative
contribution of these two DOF factors to plant
development (Mattioli et al., Modulation of intracellular proline levels affects flowering and development in Arabidopsis, submitted).
Pomponi M, Censi V, Di Girolamo V, De Paolis A, di
Toppi LS, Aromolo R, Costantino P, Cardarelli M.
Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd(2+) tolerance
and accumulation but not translocation to the shoot.
Planta, 2006; 223(2):180-90. Epub 2005 Aug 20.
Cecchetti V, Altamura MM, Serino G, Pomponi M,
Falasca G, Costantino P, Cardarelli M. ROX1, a gene
induced by rolB, is involved in procambial cell proliferation and xylem differentiation in tobacco stamen.
Plant J, (in press).
rolB. By means of the localized expression of rolB,
we previously demonstrated a role for the hormone
auxin on stamen and pistil development.
28
Internal and external determinants of nucleosome positioning
and modifications in the regulatory architecture of chromatin
Pricipal investigator: Ernesto Di Mauro
Tel.: (+39) 06-49912880, Fax: (+39) 06 4440812
[email protected]
ester bonds in both deoxy monomers and deoxy
oligomers under a large set of conditions. The
results show a strong dependence of the relative stability of these bonds on the physico-chemical environment. In certain conditions, the stability of the
5’-phosphoester bond is higher in the polymer than
in the mononucleotide, thus indicating a plausible
route to the evolution of the genetic information.
As for the second research topic, we have focused on a
group of co-regulated genes from the yeast
Saccharomyces cerevisiae, all repressed by glucose and
derepressed in the presence of the transcription factor
Adr1. The aim is to uncover the determinants of
nucleosome positioning both in terms of specific signature elements, as for example the TATA box, present in the DNA sequence and of defined architectural
elements, as for example proteins involved in the stability and reorganization of chromatin, under different external stimuli. Nucleosome remodelling complexes play a key role in gene activation in response to
environmental changes by driving promoter chromatin to reach an accessible configuration. They also
mediate genome-wide chromatin organisation,
although their role in processes other than activationrelated chromatin remodelling are poorly understood.
The S. cerevisiae ADH2 gene, the prototype of Adr1dependent genes, represents an excellent model for
understanding the role of chromatin structure and
remodelling in gene regulation. Following glucose
depletion, highly positioned promoter nucleosomes
are destabilised leading to strictly regulated kinetics of
transcription activation. Nevertheless, no chromatin
remodelling activities responsible for establishing or
remodelling ADH2 chromatin structure have been
identified to date. We show that the absence of the
Isw1 and Chd1 ATP-dependent chromatin remodelling activities delays the maximal expression of
ADH2 without impairing the chromatin remodelling
that occurs upon activation.
Instead, a destabilised chromatin structure on the
ADH2 coding and termination region is observed in
Participants:
Rodolfo Negri, Micaela Caserta, Giovanna Costanzo,
Sabrina Venditti, researchers.
Collaborations:
Laboratory of Molecular Biology, Medical Research Council,
Cambridge, UK (Prof. Andrew A. Travers); University of
Washington at Seattle, USA (Prof. Elton T. Young); University of
California at Los Angeles, USA (Prof. Michael Grunstein).
Report of activity
The global architecture of chromosomes and the
functions of the DNA-protein complexes at the
eukaryotic promoters are determined by a large
number of factors. A minimal list includes DNA
(both in its sequence and its topological properties),
the nucleosome, regulatory and enzymatic proteins.
The evolution of regulation in complex genomes is
related to the parallel evolution of DNA and of the
proteins which interact with a defined nucleotide
sequence. This evolution is very difficult to understand if it is not studied starting from its roots. In
order to contribute to the partial solution of some
aspects of this wide topic, we have undertaken:
(i) the in vitro analysis of the evolution and stability
of information in nucleic polymers;
(ii) the in vivo analysis of the position and stability of
defined nucleosomal particles, and of the effects of
their interactions with regulatory proteins.
The first research topic has lead to the identification
of the physico-chemical conditions in which the stability of polymers becomes comparable with that of
the precursor monomers. To survive, an informational macromolecule must solve the major problem
set by its very polymeric nature: instability. This is
especially true in prebiotic terms because of the presumed initial absence of protective structures (proteins, lipids, etc.). We have analyzed the stability of
the beta-glycosidic and of the 3’- and 5’-phospho-
29
E. Di Mauro - Internal and external determinants of nucleosome positioning in the architecture of chromatin
the absence of Isw1 or Chd1 in repressing conditions. The specific Isw1 complex involved in this
nucleosome repositioning is Isw1b because the deletion of Ioc2 and Ioc4, but not of Ioc3, causes the
same phenotype as the deletion of Isw1. Moreover,
the lack of Chd1 combined with the absence of Isw1
and Isw2 impairs nucleosome spacing along the
ADH2 gene, and genome-wide in S. cerevisiae. Thus,
the ISWI and Chd1 remodelling factors are not only
involved in transcription-related chromatin remodelling, but also are required to maintain a specific chromatin configuration across the yeast genome.
Besides ATP-dependent chromatin remodelling
complexes, enzymes involved in the covalent modification of histones and other key proteins play relevant roles in the control of nucleosome structure
and function. Many biological processes are regulated via post-translational modifications of histones.
Acetylation of lysine residues at the N-terminal histone tails is one of the most studied covalent modifications influencing gene regulation in eukaryotic
cells. Whether this is caused by a general increase in
nucleosome fluidity due to charge neutralisation or
by a more specific code is still matter of debate. By
using a set of glucose-repressed Adr1-dependent
genes of S. cerevisiae, whose transcription was previously shown to require both Gcn5 and Esa1, we
asked how changes of histone acetylation patterns at
the promoter nucleosomes regulate chromatin
remodelling and activation.
When the signal of glucose reduction reaches the
cells, H4 acetylation is kept constant while an
increase of H3 acetylation occurs, in an Adr1- and
Gcn5-dependent manner. In cells lacking Gcn5
activity, the H3 acetylation increase does not occur
and an unexpected increase of histone H4 acetylation is observed. Nevertheless, chromatin remodelling and transcription activation are impaired, suggesting that acetylation of H3 and H4 histones plays
different roles.
Xella B, Goding C, Agricola E, Di Mauro E, Caserta
M. The ISWI and CHD1 chromatin remodelling activities influence ADH2 expression and chromatin organization. Mol Microbiol, 2006; 59(5):1531-41.
Verdone L, Agricola E, Caserta M, Di Mauro E.
Histone acetylation in gene regulation. Brief Funct
Genomic Proteomic, 2006; 5(3):209-21. Epub 2006 Jul 28.
Agricola E, Verdone L, Di Mauro E, Caserta M. H4
acetylation does not replace H3 acetylation in chromatin remodelling and transcription activation of
Adr1-dependent genes. Mol Microbiol, 2006;
62(5):1433-46.
30
New complex mitochondrial functions in cell biology
Principal investigator: Laura Frontali
Professor of Microbial Chemistry
Tel.: (+39) 06 4453950, Fax: (+39) 06 4461980
[email protected]
all the pleiotropic phenotypes of the mutant; this
result suggests that the Rpn11 protein could act in
controlling the mitochondrial shape even independently from the proteasome. Thus we have identified
the morphology defect of the mpr1-1 strain as a deficiency in the mitochondrial fusion process.
In the second line of research, we have concentrated
our analysis on aging. Aging is accompanied in
eukaryotic organisms by alterations in mitochondrial
morphology, including a transition from a network
shape to a punctuate one. The significance of these
alterations with regard to aging is not known, but it is
clear that an excess of fission events in mitochondria
and/or a defective mitochondrial fusion results in a
destruction of the mitochondrial tubular network
with consequent respiratory defect, accumulation of
ROS, and apoptosis in mammalian cells. We found that
mutants of the LSM complex, that showed all phenotypic markers of apoptosis and a premature senescence, show heavy mitochondrial fragmentation.
Moreover, the destruction of the mitochondrial tubular network was also observed in wild type strains
after apoptotic stimuli. Mitochondrial fragmentation
after the apoptotic stimuli or in apoptotic mutants
was, at least in part, caspase dependent. We have also
analyzed a series of mutants in genes known to affect
mitochondrial morphology. Our results indicate that
MDM30 and DNM1, the genes encoding an F-box
protein and the dynamin-related GTPase respectively,
are involved in triggering aging and apoptosis. We
also found that FIS1, the mitochondrial fission gene,
might play a protective role after an apoptotic insult
while it seems to promote cell death in aging cells.
Lastly we are examining the possibility of using yeast
mitochondria as a flexible and versatile tool to investigate some open problems in mitochondrial disease. We
have previously shown by biolistic procedure, that
introduction into yeast mt tRNAleuUUR gene of base
substitutions equivalent to those found at positions
3243, 3256 and 3291 in MELAS patients, produces
very severe growth defective phenotype in glycerol
Participants:
Claudio Falcone, professor; Silvia Francisci, Cristina Mazzoni,
Teresa Rinaldi, researchers; Cristina De Luca, Vanessa
Palermo, post-doc fellows; Michele Saliola, technician.
Collaborations:
Laboratory of Molecular Genetics, University Paris Sud, Orsay,
France (Prof. Monique Bolotin-Fukuhara); Department of
Biology, The Technion, University of Haifa, Israel (Dr. Michael
Glickman).
Report of activity
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 it is altered in
some human diseases. We have shown that the proteasomal mutant (mpr1/rpn11) exhibits fragmented
mitochondria and a 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 mpr1 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 alpha-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
mitochondrial morphology. Finally, we have shown
that expression, in trans, of the C-terminal part of
Rpn11, in the mpr1-1 strain, is able to complement
31
L. Frontali - New complex mitochondrial functions in cell biology
with loss of mt DNA. We have developed this
research line by studying new mutations: the A29G
mutation, equivalent to A3260G, exhibits slow and
temperature sensitive growth and a general decrease
in mt tRNA transcripts; the T20C substitution, equivalent to T3250C, is responsible of a less drastic disease in human (MM-CPEO) and results in yeast in a
weak growth phenotype on respiratory substrates.
The G19A mutation (G3249A in humans) causes a
very strong defective phenotype in yeast: the diploid
does not grow on glycerol and accumulates rho ° cells
devoid of mtDNA. We have also constructed the
mutation C25T in the tRNAval gene equivalent to
C1624T which shows a variable penetrance in
humans. The yeast mutant exhibits a thermosensitive
phenotype in glycerol. 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 shown that the suppression was dependent on the amount of suppressor
available and we also observed that the nuclear context as well as the endogenous expression level of the
suppressors, affect dramatically the defective pheno-
type of the analyzed mutants. The amount of TUF1
transcript is variable in different yeast strains and its
level is affected by the mitochondrial status suggesting the existence of a sort of a retrograde regulation
as if the defective mitochondrial protein synthesis
might upregulate the expression of the TUF1 gene. 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.
Francisci S, DE Luca C, Oliva R, Morea V,
Tramontano A, Frontali L. Aminoacylation and conformational properties of yeast mitochondrial tRNA
mutants with respiratory deficiency. RNA, 2005;
11(6):914-27.
Mazzoni C, Herker E, Palermo V, Jungwirth H,
Eisenberg T, Madeo F, Falcone C. Yeast caspase 1
links messenger RNA stability to apoptosis in yeast.
EMBO Rep, 2005; 6(11):1076-81. Epub 2005 Sep 9.
De Luca C, Besagni C, Frontali L, BolotinFukuhara M, Francisci S. Mutations in yeast mt
tRNAs: specific and general suppression by nuclear
encoded tRNA interactors. Gene, 2006; 377:169-76.
Epub 2006 Apr 29.
32
Relationships between the central spindle and the contractile ring
during Drosophila cytokinesis
Principal investigator: Maurizio Gatti
Professor of Genetics
Tel.: (+39) 06 49912842, Fax: (+39) 06 4456866
[email protected]
Participants:
The role of the Lkb1 kinase in the
asymmetric cytokinesis of Drosophila
neuroblasts
Silvia Bonaccorsi, Maria Grazia Giansanti, Patrizia
Somma, Fiammetta Vernì, researchers; Elisabetta
Bucciarelli, post-doc fellow; Valeria Naim, PhD student;
Giorgio Belloni, technician.
We have isolated lethal mutations in the dlkb1 gene,
the Drosophila homologue of C. elegans par-4 and
human LKB1 mutated in Peutz-Jeghers syndrome.
We have found that these mutations disrupt spindle
formation, resulting in frequent polyploid cells in
larval brains. In addition, dlkb1 mutations affect
asymmetric division of larval neuroblasts (NBs);
they suppress unequal cytokinesis, abrogate proper
localization of Bazooka and Miranda, but affect neither Pins/Gai localization nor spindle rotation. Most
aspects of the dlkb1 phenotype are exacerbated in
dlkb1 pins double mutants, which exhibit more severe
defects than those observed in either single mutant.
This suggests that Dlkb1 and Pins act in partially
redundant pathways to control the asymmetry of
NB divisions. Our results also indicate that Dlkb1
and Pins function in parallel pathways controlling
the stability of spindle microtubules. The finding
that Dlkb1 mediates both the geometry of stem cell
division and chromosome segregation provides novel
insight into the mechanisms underlying tumor formation in Peutz-Jeghers patients.
Collaborations:
Stanford University, USA (Prof. Margareth Fuller); Cornell
University, USA (Prof. Michel L. Goldberg); Dipartimento di
Genetica, Biologia e Biochimica, Università di Torino (Prof.
Ferdinando Di Cunto).
Report of activity
During late anaphase, animal cells assemble the central spindle, a robust bundle of antiparallel microtubules that interdigitate at the cell equator. During
telophase, the central spindle midzone becomes
encircled by the acto-myosin ring, which constricts
the equator of the dividing cell until cytokinesis is
completed. We are interested in the molecular mechanisms underlying animal cell cytokinesis. Previous
work of our laboratory has shown that during
Drosophila cytokinesis the central spindle and the
contractile ring are interdependent structures: when
one of them is perturbed, the proper assembly of the
other is also disrupted. In addition, a large screen for
mutants defective in meiotic cytokinesis of males has
recently led us to the identification of 16 new genes
required for the process (Giansanti et al., Mol Biol
Cell, 2004, 15:2509-22). The discovery of these
genes has broadened our research perspectives and
raised a new interest in the mechanism of membrane
formation at the advancing cytokinetic furrow. In the
past two years, we have carried out three main projects aimed at the elucidation of the mechanisms of
Drosophila cytokinesis.
Rab11, Giotto and Fwd function in a
common pathway controlling formation of
new membrane during Drosophila cytokinesis
The Rab11 GTPase is a key component of the recycling endosome that regulates several aspects of
vesicular trafficking. In mammalian cells, Rab11
mediates the delivery of endosomes to the cleavage
furrow and is essential for completion of cytokinesis.
Previous work in Drosophila showed that Rab11 is
required for cellularization during embryogenesis
but did not demonstrate its direct involvement in
cytokinesis. We have found that Rab11 accumulates
33
M. Gatti - Relationships between the central spindle and the contractile ring during Drosophila cytokinesis
at the cleavage furrow of Drosophila spermatocytes
and is essential for meiotic cytokinesis. Mutant spermatocytes form regular actomyosin rings, but these
rings fail to constrict to completion, leading to
cytokinesis failures. rab11 spermatocytes also exhibit
an abnormal accumulation of Golgi-derived vesicles
at the telophase equator, suggesting a defect in membrane-vesicle fusion. These cytokinesis phenotypes
are identical to those elicited by mutations in giotto
(gio) and four wheel drive (fwd) that encode a PITP
(phosphatidylinositol transfer protein) and a phosphatidylinositol 4-kinase, respectively. Double
mutant analysis and immunostaining for Gio and
Rab11 indicated that gio, fwd and rab11 function in
the same pathway, with Gio and Fwd acting
upstream of Rab11. We propose that Gio and Fwd
mediate Rab11 recruitment at the cleavage furrow by
causing a local enrichment in phosphorylated PtdIns,
and that Rab11 facilitates membrane addition to the
furrow and/or targeted delivery of proteins required
for cytokinesis.
of a developmentally regulated Golgi derivative, the
acroblast, was defective in bru mutant post-meiotic
spermatids, implying a requirement for TRAPPII in
coordinating Golgi behavior. Additionally, genetic
interaction between bru and four wheel drive (fwd),
which encodes a phosphatidylinositol 4-kinase b
(PI4Kb), suggests that TRAPPII function may collaborate with or depend on the subcellular organization of membrane phosphoinositides to support
cleavage furrow ingression.
In the next years, we plan to continue to work on the
new mutants identified in our screen (Giansanti et al.,
Mol Biol Cell, 2004, 15: 2509-22). We have already
cloned seven of the genes specified by these mutants
(one of them is bru). We believe that the molecular
characterization of the remaining nine genes will permit identification of several additional functions
required for Drosophila cytokinesis. This will provide
essential information to unravel the molecular interactions underlying the interdependence between the central spindle and the contractile ring, and the mechanism of membrane addition at the cleavage furrow.
TRAPPII function is required for cytokinesis in
Drosophila male meiosis
Mutations in the brunelleschi (bru) gene prevent complete constriction of the actomyosin ring, resulting
in cytokinesis failures during meiotic division of
Drosophila spermatocytes. We have cloned the bru
gene and found that it encodes the fly orthologue of
the yeast TRS120p subunit of TRAPPII vesicle trafficking complex. Analysis of the Bru protein fused to
GFP revealed that a portion of the protein localizes
with Golgi organelles while a substantial amount
was dispersed throughout the cytoplasm. Assembly
Raffa GD, Cenci G, Siriaco G, Goldberg ML, Gatti
M. The putative Drosophila transcription factor woc
is required to prevent telomeric fusions. Mol Cell,
2005; 20(6):821-31.
Giansanti MG, Bonaccorsi S, Kurek R, Farkas RM,
Dimitri P, Fuller MT, Gatti M. The class I PITP
giotto is required for Drosophila cytokinesis. Curr
Biol, 2006; 16(2):195-201.
34
Molecular mechanisms of transgene-induced post-transcriptional
gene silencing
Principal investigator: Giuseppe Macino
Professor of Cell Biology
Tel-: (+39) 06 4452806, Fax: (+39) 06 4457731
[email protected]
shares some components with the RISC complex.
To test the hypothesis of the coexistence of both
these complexes in N.crassa (in which some proteins
that are homologue of components of the RITS of
S. pombe can be found), we analyzed the methylation
pattern of the endogenous albino-1 (al-1) locus in an
al-1 silenced strain. Even though we failed to observe
any significant change in the chromatin status of the
endogenous locus, we used ChIP to show that the
transgenic loci, bearing tandemly repeated al-1
copies that induce PTGS, display hyper-methylation
of Lys9H3. Moreover (as reported in the case of the
centromeric region of S. pombe), despite its hetherochromatic features, this hyper-methylated heterochromatin produces two classes of transcripts,
sense and anti-sense. Nevertheless, in opposition to
what happens in S.pombe, this transcriptional silencing is not triggered by PTGS; in fact, the same
analyses conducted on defective strains for Qde-1,
Qde-2 and Qde-3 showed the same methylation pattern observed in the wt strain.
According to these experiments, we could say that in
N.crassa the genes involved in PTGS are not necessary to induce the modification of the chromatin status of the endogenous locus. However, the opposite
is not true: in fact, a mutant in the lys9H3 methyltransferase dim-5 was unable to maintain PTGS,
with transgenic copies being rapidly lost, resulting
in reversion of the silenced phenotype. This happens
because the organism is not able to maintain anymore the tandem array of the transgenic locus, probably because, in the absence of histone methylation,
it undergoes recombination with the endogenous
locus. This destabilization cannot be observed in the
absence of a functional DNA-methylase dim-2, so
that we can assume that DNA methylation (whose
inhibition prevents recombination in other organisms) is not involved in this mechanism. RISC-mediated PTGS pathway and do not communicate with a
RITS complex to effect chromatin-based. We conclude that in Neurospora siRNAs produced from the
Participants:
Carlo Cogoni, professor; Caterina Catalanotto, Agustin
Chicas, Lisa Franchi, post-doc fellows; Emma Forrest, PhD
student; Annette Pickford, Gianluca Azzalin, technicians.
Collaborations:
Department of Environmental and Biomolecular Systems OGI,
School of Science and Engineering, Oregon Health and Science
University, Bearverton, USA (Dr. Paul ReFalo); Department of
Molecular Microbiology and Immunology, School of Medicine,
Oregon Health and Science University, Bearverton, USA (Dr.
Matthew S. Sachs); Whitehead Institute Center for Genome
Research, Massachusetts, USA (Dr. James E. Galagan).
Report of Activity
The name “Post-transcriptional gene silencing”
(PTGS) identifies a series of different mechanisms that
inhibit the translation of mRNAs. Even though this
phenomenon can be carried out in different ways in the
organisms which share this regulatory pathway (ranging from the degradation of the target RNA to its
sequestration in ribosome-free regions of the cytoplasm), a common feature is the presence of small
RNA (with a length of about 20-25 nucleotides) produced by an endonucleolitic cleavage of double-stranded precursors mediated by the RNAse III Dicer.
In this last part of the project our goal was to elucidate the epigenetic aspects of this phenomenon in
Neurospora crassa (where the gene silencing has been
named Quelling), and clearly analyze the molecular
and functional relationship between the siRNA producing enzyme Qde-1, Qde-2 and Qde-3, and those
enzymes who act by modifying the chromatin organisation (in particular, attention was paid to the
methylation of Lys9H3): in fact, many papers
showed that in Neurospora-related organisms such
as S.pombe and in plants, siRNAs can act by inducing
to the Lys9H3 methylation of the homologous locus
through a protein complex, called RITS, which
35
G. Macino - Molecular mechanisms of transgene-induced post-transcriptional gene silencing
transgenic locus are used exclusively in the silencing.
In plants PTGS works on transposons to induce histone and DNA methylation. In this way, siRNA can
direct either the degradation of transcripts arising
from these regions and transcriptional silencing. To
test the existence of this mechanism in Neurospora,
we observed the repressed status of a LINE1-like
transposone (tad) in the context of quelling-defective strains (Qde-1, Qde-2, Qde-3) . Tad methylation
(characteristic of wt strain) can be observed only
when the activity of Qde-2 and Dicer-like enzymes is
not disrupted, whatever Qde-1, Qde-3 or dim-2 are.
These results could imply the existence in Neurospora
of different silencing mechanisms for each type of
repetitive element. Anyway, we could not exclude that
the repeated sequences are methylated just after that the
inactivative mechanism of Point Mutation (RIP) acts on
them (since the majority of the methylated sequences
are formerly inactivated), coming ahead siRNA.
In the end, our data suggest that the biological function of RNAi in Neurospora is to control repeated
sequences by the means of the degradation of the
transcripts arising from these regions.
Nolan T, Braccini L, Azzalin G, De Toni A, Macino G,
Cogoni C. The post-transcriptional gene silencing
machinery functions independently of DNA methylation to repress a LINE1-like retrotransposon in
Neurospora crassa. Nucleic Acids Res, 2005; 33(5):1564-73.
Chicas A, Forrest EC, Sepich S, Cogoni C, Macino
G. Small interfering RNAs that trigger posttranscriptional gene silencing are not required for the
histone H3 Lys9 methylation necessary for transgenic tandem repeat stabilization in Neurospora crassa. Mol Cell Biol, 2005; 25(9):3793-801.
Franchi L, Fulci V, Macino G. Protein kinase C
modulates light responses in Neurospora by regulating the blue light photoreceptor WC-1. Mol
Microbiol, 2005; 56(2):334-45.
36
Chromatine remodelling and transcriptional activation of the
zygotic genome in preimplantation embryos of the mouse
Principal investigator: Franco Mangia
Professor of Neurobiology
Dipartimento di Psicologia, Sezione di Neuroscienze.
Tel.: (+39) 06 49917784, Fax: (+39) 06 49917873
[email protected]
ule cells, Purkinje cells, pyramidal neurons and
mitral cells, by an activity-dependent fashion, making this gene an interesting model of interneuronneuron functional interaction in the adult brain.
Participants:
Maria Teresa Fiorenza, Arturo Bevilacqua, professors;
Sonia Canterini, researcher; Adriana Bosco, Valentina De
Matteis, Domenico Grillo, Antonio Trabalza, PhD students.
Promotion of mitotic early blastomere
proliferation by TCL1
Collaborations:
Istituto Dermatologico dell’Immacolata, Roma (Dr.
Giandomenico Russo); Dipartimento di Sanità Pubblica e
Biologia Cellulare, Università di Roma “Tor Vergata” (Prof.
Pellegrino Rossi, Prof. Claudio Sette); Dipartimento di
Istologia ed Embriologia Medica, Sapienza-Università di Roma
(Prof. Carla Boitani); Dipartimento di Medicina Sperimentale e
Patologia, Sapienza-Università di Roma (Prof. Alberto Gulino).
TCL1 is a 14 KDa, non enzymatic b-barrel protein
that localizes in both nucleus and cytoplasm of lymphoid cells. It is commonly accepted that TCL1 promotes cell proliferation by enhancing the activity of
AKT/PKB, a serine/threonine kinase having a central role in the signaling pathways that control cell
proliferation and survival. In fact, TCL1 heterodimerizes with AKT and mediates both AKT
transphosphorylation at Ser472/473 residue and
AKT transfer to nucleus. However, it is still unclear
if these TCL1 functions depend on each other and
how they are actually relevant to the promotion of
normal/neoplastic cell growth exerted by TCL1. We
have exploited the TCL1 expression in early preimplantation embryos to ask if this regulation depends
on the TCL1 ability to enhance AKT transphosphorylation at the level of cell membrane and/or to
mediate the phosphorylated AKT transfer to the
nucleus. Immunofluorescence experiments and injection of anti-AKT1, -AKT2 or -AKT3 antibodies in
one-cell embryos pinpointed AKT2, and not
AKT1/AKT3, as the major mediator of TCL1 function during preimplantation embryo development.
Accordingly, embryos deficient in AKT2 displayed
abnormal blastomere division similar to that previously observed in Tcl1-/- embryos. The regulation of
AKT phosphorylation was studied in one/two-cell
embryos by immunofluorescence with anti-phosphorylated AKT antibodies. Nuclear AKT appeared to
be constantly phosphorylated at both Ser473 and
Thr308 residues, independently of PI3 Kinase and
PDK1 activity. In fact, AKT Ser473-Thr308 phosphorylation of two-cell embryos was not apparently
affected by a continuous embryo exposure to the spe-
Report of activity
The major aim of this research project was to analyze the molecular regulation of early embryo development in the mouse, with particular reference to the
role played by the product of the proto-oncogene
Tcl1 in the promotion of mitotic blastomere division.
Tcl1 is of interest to both early embryo development
(Narducci et al., 2001, PNAS 99, 11712-7) and the
pathogenesis of prolymphocytic T cell leukemia in
humans (Virgilio et al., 1994, PNAS 91: 12530-4).
This gene, in fact, is expressed and plays similar
functions in both B and T cell lymphopoiesis and
early preimplantation embryos. Thus information
gained on embryos may also elucidate oncogenetic
mechanisms acting in lymphoid cells. In addition, we
report on results recently obtained on a recently discovered mouse gene, Thg-1pit, originally cloned by
one of us (Fiorenza et al., 2001, Gene 278, 125-30),
the functional characterization of which in cerebellar
granule cells and Purkinje cells is ongoing in our
laboratory since a few years. This gene is expressed
in both embryonic and adult granule cells of cerebellum, hippocampus, rostral migratory stream and
olfactory bulb of mouse brain. In addition, it is also
expressed in the mature synaptic partners of gran-
37
F. Mangia - Chromatine remodelling and transcriptional activation of the zygotic genome in preimplantation embryos
cific PI3 Kinase inhibitors LY294002 (10-20 mM) or
Wortmannin (6-24 nM), neither it was when the
embryos were treated with the specific PKCa
inhibitor LY33353 at doses (10-20 mM) that also
inhibit PDK1 activity. To evaluate whether AKT was
protected from phosphatase by a complex with
endogenous Hsp90, embryos were treated with the
Hsp90 inhibitor 17-AAG. Treatment did not modify
AKT phosphorylation, while PP1 and PP2A phosphatases had very low activities during preimplantation development, suggesting that embryo’s AKT is
not subject to de novo phosphorylation or dephosphorylation. In line with this hypothesis, no quantitative difference was found between the amounts of
Ser473/Thr308 phosphorylated AKT in unfertilized
eggs and one-/two-cell embryos, suggesting that
early mouse embryos inherit fully phosphorylated
AKT molecules from oogenesis. The lack of TCL1
did not affect the level of AKT Ser473/Thr308
phosphorylation, but completely suppressed the
transfer of phosphorylated AKT to nuclei in twocell embryos. We conclude that TCL1 is dispensable
for AKT phosphorylation and represents an absolute
requirement for phosphorylated AKT transfer to
nucleus.
factor AIF (PDCD8), and the mRNA stabilization
factor PCBP1. We have recently developed a number
of experimental approaches to investigate the role of
this factor in vitro using primary granule cell cultures from postnatal (PN6) cerebella and N1E-115
neuroblastoma cells. Antibodies directed to the
human homolog THG-1 specifically revealed the
presence of the THG1-pit protein in the soma and
neurite terminals of granule and Purkinje cells.
Similar results were also obtained with transfected
N1E-115 cells expressing a GFP-Thg-1pit chimeric
construct, suggesting tht THG-1pit is involved in
synaptic function. The effect of TGF-b2 and FGF-2
factors on Thg-1pit transcriptional activation was
investigated using primary cultures of cerebellar
granule cells from PN6 mice. These cells were isolated and in vitro cultured for 7 days, then continuously exposed to 2 ng/ml of either FGF-2 or TGFb2 for increasing times, and eventually processed for
semiquantitative RT-PCR determination of Thg-1pit
transcripts. Both factors activated Thg-1pit expression, with a transient, 10-20 fold increase of Thg-1pit
transcripts after a 1-3 hour treatments, but with different kinetics. The functional significance and differential kinetics of Thg-1pit activation by morphogens need to be further investigated.
Functional characterization of THG-1pit
factor in mouse granule cells
Thg-1pit, the murine homolog of human THG-1,
belongs to the gene family TSC-22/DIP/bun,
including genes that are typically regulated by morphogens as TGF-b. Thg-1pit codes for a protein provided with a leucine zipper domain that may homodimerize and/or heterodimerize with other leucine
zipper-containing factors. This may allow Thg-1pit
to form a variety of homo-/heteromultimers having
unique and specific regulatory properties that are different from those of individual multimer components. Interest on this factor has recently been raised
by a study aimed at identifying the protein-protein
interaction network for human inherited ataxias by a
two-hybrid system, showing that Thg-1pit binds a
number of inherited ataxia-related factors, including
Sacsin, the prion protein PRNP, the antiapoptotic
Luconi M, Torcia S, Grillo D, Fiorenza MT, Forti
G, Mangia F, Baldi E. Enhancement of mouse sperm
motility by the PI3-kinase inhibitor LY294002 does
not result in toxic effects on preimplantation embryo
development. Hum Reprod, 2005; 20(12):3500-4.
Epub 2005 Aug 26.
Puglisi R, Tramer F, Carlomagno G, Gandini L,
Panfili E, Stefanini M, Lenzi A, Mangia F, Boitani C.
PHGPx in spermatogenesis: how many functions?
Contraception, 2005; 72(4):291-3.
Canterini S, Mangia F, Fiorenza MT. Thg-1 pit gene
expression in granule cells of the developing mouse
brain and in their synaptic targets, mature Purkinje,
and mitral cells. Dev Dyn, 2005; 234(3):689-97.
38
Heterochromatin, telomeres and modifiers of position effect
variegation (PEV) in Drosophila melanogaster
Principal investigator: Sergio Pimpinelli
Professor of Genetics
Tel.: (+39) 06 49912876, Fax: (+39) 06 4456866
[email protected]
involvement in the activation of several euchromatic
genes in Drosophila. By immunostaining experiments
using a HP1 antibody, we found that HP1 is associated with developmental and heat shock induced puffs
on polytene chromosomes. Since the puffs are the
cytological phenotype of intense gene activity, we
did a detailed analysis of the heat shock induced
expression of HSP70 encoding gene in larvae with
different doses of HP1 and we found that HP1 is
positively involved in Hsp70 gene activity (Piacentini
et al., 2003, J Cell Biol., 161: 707-714). Since we found
that, besides the puffs, HP1 is located in a total of
200 euchromatic sites (Fanti et al., 2003, Genetica,
117:135-147), we compared mRNAs from wild-type
and Su(var)2-5 mutants lacking HP1 by microarray
analysis. The results of this analysis revealed that
284 genes show a twofold or greater increase in
expression and 261 genes show a twofold or greater
decrease in expression in the Su(var)2-5 mutant compared with wild-type. The immunoprecipitation
analysis of three genes that require HP1 for their
expression showed that such genes associate with
HP1.
The results of this work have been published on
Developmental Dynamics (Cryderman et al., 2005).
In conclusion, all the results above described, suggest a positive role for HP1 in euchromatic gene
expression and significantly broaden the current
views of the roles of HP1 in vivo by demonstrating
that this protein has multifunctional roles.
Participants:
Laura Fanti, professor; Enzo Marchetti, Eleonora
Vitagliano, researchers; Marcella Marchetti, Barbara
Perrini, Lucia Piacentini, post-doc fellows.
Collaborations:
University of Utah, USA (Dr. Kent Golic); University of
Washington, USA (Dr. Barbara Wakimoto); University of Iowa,
USA (Dr. Lori Wallrath); Simon Fraser University, Canada (Dr.
Barry Honda); Università di Bari (Dr. Maria Berloco);
Dipartimento di Scienze Biochimiche, Sapienza-Università di
Roma (Prof. Carlo Turano, Prof. Anna Ferraro).
Report of activity
The long range goal of our research is the definition
of the components of Drosophila heterochromatin
and telomeres and the understanding of their biological roles. Our major objective is the identification
and the functional analysis of proteins that bind heterochromatin and telomeres. A large collection of
mutations that act as modifiers of position effect variegation (PEV) provide the starting tools for a systematic dissection of the molecular interactions controlling heterochromatin and telomere formation
and their relationship with euchromatin. We have
planned to classify the modifier mutations of PEV
into functionally related groups using genetic and
cytogenetic criteria. As a first step, we have initiated
a serie of studies on the Su(var)2-5 gene that encodes
the heterochromatin protein 1 (HP1) and other heterochromatic genes.
The nature of binding of HP1 to telomeres
We have previously shown that HP1 is a telomere capping protein since when mutated induces telomeric
fusions in Drosophila (Fanti et al., 1998, Mol Cell, 2:120). We extended these studies and we found that HP1
is involved in telomere capping and in controlling
telomeric DNA transcription and elongation by two
different types of binding to the telomeres. The
telomere capping depends on the direct binding of
HP1 to telomeric sequences while the transcriptional
HP1 is associated with induced gene
expression in Drosophila euchromatin
HP1 is a conserved nonhistone chromosomal protein, firstly discovered in Drosophila, which is
involved in heterochromatin formation and gene
silencing in many organisms. We have shown a novel
striking feature of such protein demonstrating its
39
S. Pimpinelli - Heterochromatin, telomeres and modifiers of position effect variegation (PEV) in Drosophila melanogaster
species. The genus Drosophila is ideal for a comparative study of heterochromatic genes: it consists of a
large number of well-studied species and possesses a
relatively stable karyotype, while nevertheless
exhibiting exceptionally malleable evolutionary
dynamics at the intrachromosomal level.
We have recentely performed an evolutionary analysis of two genes, Dbp80 and RpL15, which are adjacent in proximal 3L heterochromatin of D.
melanogaster. DmDbp80 is typical of previously
described heterochromatic genes: large, with repetitive sequences in its many introns. In contrast,
DmRpL15 is uncharacteristically small. The
orthologs of these genes were examined in D.
pseudoobscura and D. virilis. In situ hybridization and
whole-genome assembly analysis show that these
genes are adjacent, but not centromeric in the
genome of D. pseudoobscura, while they are located on
different chromosomal elements in D. virilis. Dbp80
gene organization differs dramatically among these
species, while RpL15 structure is conserved. A bioinformatic analysis in five additional Drosophila species
demonstrates active repositioning of these genes
both within and between chromosomal elements.
This study shows that Dbp80 and RpL15 can function in contrasting chromatin contexts on an evolutionary timescale. The complex history of these
genes has also provided unique insight into the
dynamic nature of genome evolution. The results of
this work have been published on Genetics (Schulze et
al., 2006).
control of such sequences depends on the interaction
of HP1 chromodomain with H3-Me3K9. These data
have suggested a simple model for HP1 function at the
telomeres. According to our model, HP1 first directly
binds telomeric DNA also targeting a yet unknown
specific HMTase. The enzyme would then methylate
H3-K9, thus creating an additional binding site for
HP1. The spreading of HP1, HMTase and H3Me3K9 interactions would form the telomeric repressive chromatin (Perrini et al., 2004, Mol Cell, 15:467476). The results of our work on Drosophila telomeres
have been reviewed in the CSHL Book “Telomeres” (S.
Pimpinelli, 2006, pp 433-463).
The genomic organization of telomeric
sequences in Drosophila
Drosophila melanogaster telomeres contain arrays of
two non-LTR retrotransposons called HeT-A and
TART. Previous studies have shown that HeT-A- and
TART-like sequences are also located at non-telomeric sites in the Y chromosome heterochromatin. To
assess a possible heterochromatic location of TART
and HeT-A elements in other Drosophila species, we
performed in situ hybridization experiments, using
both TART and HeT-A probes, on mitotic and polytene chromosomes of D. simulans, D. sechellia, D.
mauritiana, D. yakuba and D. teissieri. We found that
TART and HeT-A probes hybridize at specific heterochromatic regions, including those that are pericentromeric, of the Y chromosome in all Drosophila
species that we analyzed.
These data suggest that in Drosophila, the centromere of the Y chromosome was probably derived
from telomeres by intra-chromosomal rearrangements. The results of this work have been published
on Cytogenet Genome Res (Berloco et al., 2005)
Berloco M, Fanti L, Sheen F, Levis RW, Pimpinelli
S. Heterochromatic distribution of HeT-A- and
TART-like sequences in several Drosophila species.
Cytogenet Genome Res, 2005; 110(1-4):124-33.
Heterochromatic genes in Drosophila
Although heterochromatin is considered a suppressor of gene activity, surprisingly, there are genes
naturally resident in heterochromatin, which appear
to require this environment for optimal activity. One
way to address how genes come to reside and function in such a transcriptionally repressive environment, is to elucidate the evolutionary history of heterochromatic genes by examination of the structure
and position of these genes in a set of related
Cryderman DE, Grade SK, Li Y, Fanti L, Pimpinelli
S, Wallrath LL. Role of Drosophila HP1 in euchromatic gene expression. Dev Dyn, 2005; 232(3):767-74.
Schulze SR, McAllister BF, Sinclair DA, Fitzpatrick
KA, Marchetti M, Pimpinelli S, Honda BM.
Heterochromatic genes in Drosophila: a comparative
analysis of two genes. Genetics, 2006; 173(3):143345. Epub 2006 Apr 30.
40
AREA
3
Molecular
recognition
in
biomolecules
Molecular recognition in biomolecules - Area 3
Evolutionary pressure on folding mechanisms: the case of the
cytochrome c and globin protein families
Principal investigator: Maurizio Brunori
Professor of Chemistry and Biochemistry
Tel.: (+39) 06 4450291, Fax: (+39) 06 4440062
[email protected]
Participants:
Cavities in globins and cytochromes
Myoglobin is the paradigm to investigate conformational dynamics by time-resolved Laue crystallography, a powerful and advanced structural approach.
We have shown (2) that after rapid initiation of the
reaction, the nonequilibrium population of protein
structures generated by photolysis relaxes over a
broad time range, extending from picosec to millisec;
this process is associated with migration of the ligand to internal cavities in the protein matrix. We
have proposed that the extended relaxation of the
globin moiety reflects re-equilibration among conformational substates known to play an essential role
in controlling protein function and basic to the conception of the funnel theory of protein folding.
Within this scenario it should be possible to energetically tune the folding pathway of a protein by selectively (de)stabilizing intermediate and transition
states. We therefore focused our attention on the possible role of internal cavities in the folding process of
c-type cytochromes and produced site-directed
mutants of cyt c551, designed in order to fill, or
introduce, some packing defects in the protein
matrix. The cyt c551 F7A mutant, which is thermodynamically stabilized with respect to the wild-type
protein, is characterized by a folding intermediate
sufficiently stabilized to be kinetically detectable (3).
The 3D structure of F7A showed that replacement
of Phe with Ala produces a big cavity (Fig. 1B); surprisingly, such packing defect is associated to an over
stabilization. An explanation for this apparent contradiction may reside (i) in the presence of three
ordered water molecules in the cavity and (ii)
removal of steric constraint which allows the main
chain atoms to properly fit the H-bond network of
the N-terminal helix (Fig. 1A). The ~ 4-fold
enhancement of the helical propensity of the N-terminal helix increases the probability for the intermediate state to be populated, in agreement with our
general hypothesis (Travaglini-Allocatelli et al.,
Trends Biochem Sci, 2004, 29:535-41). In conclusion,
Francesca Cutruzzolà, Carlo Travaglini-Allocatelli,
Beatrice Vallone, professors; Stefano Gianni, researcher;
Alessandro Borgia, Nicoletta Calosci, PhD students.
Collaborations:
University of Rochester, USA (Dr. Kara Bren); Fox Chase Cancer
Center, Philadelphia, USA (Dr. Heinrich Roder); ESRF, Grenoble,
France (Dr. Dominique Bourgeois).
Report of activity
Protein folding is considered the most important
problem in structural biochemistry, because of its
potential role in predicting 3D structures from
sequences and in molecular medicine, since a variety
of diseases are caused by misfolding events occurring in vivo. Comparison of the folding process for
different members of a family is a powerful strategy
which allow to discover the essential determinants of
a specific fold, with the identification of a consensus
mechanism. A necessary complementary approach to
the Ê-value analysis requires understanding the role
of the protein matrix cavities in function and folding.
c-type cytochromes
This large family of globular proteins is widely used for
folding studies. By comparing the folding kinetics of
cyt c551 from the mesophilic bacterium P. aeruginosa
with that of cyt c552 from the thermophilic bacteria T.
thermophilus and H. thermophilus, we demonstrated (1)
that the folding transition states of these proteins share
fundamental structural similarities, in spite of large differences in sequence and thermodynamic stability. In a
nutshell, we have demonstrated that the folding mechanism of all c-type cytochromes involves the accumulation of an obligatory intermediate with similar structural features, and that a minimum three-state scheme,
characterized by similar intermediate and transition
states, is of general validity.
43
M. Brunori - Evolutionary pressure on folding mechanisms: the case of the cytochrome c and globin protein families
we have acquired kinetic and structural evidence that
even a single rational mutation can significantly
change the shape of the energetic landscape characteristic of a protein folding process, resulting not only in
stabilization of the native state but also in the unveiling of a consensus folding intermediate, otherwise
energetically inaccessible.
folding pathway of cytochrome c552 from
Hydrogenobacter thermophilus. J Biol Chem, 2005;
280(27):25729-34. Epub 2005 May 9.
Borgia A, Bonivento D, Travaglini-Allocatelli C, Di
Matteo A, Brunori M. Unveiling a hidden folding
intermediate in c-type cytochromes by protein engineering. J Biol Chem, 2006; 281(14):9331-6. Epub
2006 Feb 1.
Bourgeois D, Vallone B, Arcovito A, Sciara G,
Schotte F, Anfinrud PA, Brunori M. Extended subnanosecond structural dynamics of myoglobin
revealed by Laue crystallography. Proc Natl Acad Sci
USA, 2006; 103(13):4924-9. Epub 2006 Mar 17.
Travaglini-Allocatelli C, Gianni S, Dubey VK, Borgia
A, Di Matteo A, Bonivento D, Cutruzzola F, Bren
KL, Brunori M. An obligatory intermediate in the
Fig. 1 - (A) Structural superimposition of the N-terminal ·-helix of wt Pa cyt c551 (light grey) and F7A mutant (dark grey). The H-bond in
the F7A structure between main chain atoms is shown. (B) Electron density map at 1.86 Å resolution showing the cavity generated by the
mutation and the three H-bonded water molecules
44
Molecular signalling and recognition in plant defense
mechanisms
Principal investigator: Felice Cervone
Professor of Plant Biology
Dipartimento di Biologia Vegetale
Tel.: (+39) 06 49912517, Fax: (+39) 06 49912446
[email protected]
isoform 2 from Phaseolus vulgaris (PvPGIP2) by
means of inhibition assays, homology modeling, and
molecular docking simulations. Our results indicate a
mixed mode of inhibition. This is compatible with a
model for the interaction where PvPGIP2 binds the
N-terminal portion of BcPG1, partially covering its
active site and decreasing the enzyme affinity for the
substrate. The structural framework provided by the
docking model is confirmed by site-directed mutagenesis of the residues that distinguish PvPGIP2 from
the isoform PvPGIP1. The finding that PvPGIP2
inhibits BcPG1 with a mixed-type kinetics further
indicates the versatility of PGIPs to evolve different
recognition specificities.
PGIPs are members of the leucine-rich repeat (LRR)
protein family. The crystal structure of PGIP
reveals a negatively charged surface on the LRR concave face, likely involved in binding PGs. A cluster of
four arginine and lysine residues (R183, R206, K230,
and R252), protruding into the solvent, is responsible for the interaction of PGIP with pectin. The
four residues were mutated and the protein variants
were expressed in Pichia pastoris. The ability of both
wild-type and mutated proteins to bind pectins was
investigated by affinity chromatography. Single
mutations impaired the binding and double mutations abolished the interaction, thus indicating that
the four clustered residues form the pectin-binding
site. Remarkably, the binding of PGIP to pectin is
displaced in vitro by PGs, suggesting that PGIP
interacts with pectin and PGs through overlapping
although not identical regions.
Pectin is secreted in a highly methyl-esterified form
and subsequently de-esterified in muro by pectin
methylesterases (PMEs). PMEs are regulated by
either differential expression and post-translational
control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against
microbial enzymes. The enzyme folds into a righthanded parallel b-helical structure typical of pectic
enzymes. The inhibitor is almost all helical, with four
Participants:
Daniela Bellincampi, Giulia De Lorenzo, professors;
Claudio Caprari, Benedetta Mattei, Giovanni Salvi, researchers; Manuela Casasoli, Adele Di Matteo, Cinzia
Manfredini, Francesca Sicilia, post-doc fellows; Roberta
Galletti, Vincenzo Lionetti, Daniela Pontiggia, Sara
Spadoni, Francesco Spinelli, PhD students.
Report of activity
Plants are continually exposed to pathogens and, in
most cases, successfully defend themselves. The plant
innate immunity relies on several recognition molecules that cooperate in alerting the plant cell against
the pathogens. We study two protein inhibitors of
pectic enzymes in Arabidopsis and tobacco. These
are PGIPs (Polygalacturonase-Inhibiting Proteins),
which inhibit microbial polygalacturonases (PGs)
and generate elicitor-active oligogalacturonides
(OGs) and PMEI (Pectin Methyl-Esterase
Inhibitors). We aim at enhancing the activity of the
Inhibitors and improving their specificity versus different enzymes. The ability of PGIP to limit fungal
infections is not merely due to its activity as an
inhibitor of PGs, but also to the ability to favour the
formation of OGs, which elicit the oxidative burst
and several defense-responses.
Specific tasks of the project are: (i) characterization
of PGIPs and PMEIs; (ii) production of transgenic
plants with enhanced resistance to pathogens; (iii)
elucidation of the defense-related regulatory effects
of OGs by genomic and proteomic analysis.
Characterization of PGIPs and PMEIs
Botrytis cinerea is a phytopathogenic fungus that causes gray mold in many plant species. During infection,
it secretes several PGs and, among them, BcPG1 is
an important virulence factor. We have characterized
BcPG1 and investigated its interaction with PGIP
45
F. Cervone - Molecular signalling and recognition in plant defense mechanisms
degradation by in muro expression of AnPGII
enhances plant defence responses and affects auxin
sensitivity, possibly by releasing of OGs, which act
antagonistically with auxin in modulating defence
responses and development.
long a-helices aligned antiparallely in a classical upand-down four helical bundle. The two proteins form a
stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative
active site is located. The four helix bundle of the
inhibitor packs roughly perpendicular to the main axis
of the parallel b-helix of PME and three helices of the
bundle interact with the enzyme. The interaction
interface displays a polar character, typical of non-obligate complexes formed by soluble proteins.
Defense responses mediated by
oligogalacturonides
In Arabidopsis, OGs increase resistance to B. cinerea
independently of jasmonate (JA), salicylic acid (SA)
and ethylene (ET) mediated signaling. Microarray
analysis showed that about 50% of the genes regulated by OGs, including genes encoding enzymes
involved in secondary metabolism, show a similar
change of expression during B. cinerea infection. In
particular, expression of Phytoalexin Deficient 3
(PAD3) is strongly up-regulated by both OGs and
infection independently of SA, JA and ET. OG treatments do not enhance resistance to B. cinerea in the
pad3 mutant, nor in ups1, a mutant with severely
impaired PAD3 expression in response to OGs.
Similarly to OGs, the bacterial flagellin peptide elicitor flg22 also enhanced resistance to B. cinerea in a
PAD3-dependent manner, independently of SA, JA
and ET. This idicates that OGs during pathogen
infection contribute to basal resistance against
pathogens through a signaling pathway also activated by typical pathogen-associated molecular pattern
molecules (PAMPs). We suggest to name them HostAssociated Molecular Patterns (HAMPs).
Transgenic plants
To identify PGIPs that efficiently inhibit BcPG1 of B.
cinerea, we expressed the enzyme in P. pastoris and tested its activity in the presence of PGIPs from P. vulgaris
and Arabidopsis. PvPGIP2 is a very efficient inhibitor
of BcPG1 and, when overexpressed in transgenic
tobacco and Arabidopsis, it restricts B. cinerea colonization. In Arabidopsis PGIPs with comparable activity
against BcPG1 are encoded by two genes, AtPGIP1
and AtPGIP2. Arabidopsis plants expressing an antisense AtPGIP1 gene have reduced AtPGIP1 inhibitory activity and are more susceptible to B. cinerea infection. We have also constitutively expressed the genes
AtPMEI-1 and AtPMEI-2 in Arabidopsis and targeted
the proteins into the apoplast. The overexpression of
the inhibitors resulted in a decrease of PME activity in
transgenic plants. While the content of uronic acids in
transformed plants was not significantly different from
that of WT, the degree of pectin methylesterification
was increased of about 16%. Transformed plants were
more resistant to B. cinerea. The reduced symptoms
caused by the fungus on transgenic plants were related
to its impaired ability to grow on methylesterified
pectins.
Transgenic plants expressing the endopolygalacturonase II of Aspergillus niger (AnPGII) were more
resistant to B. cinerea and to the bacterial pathogen
Pseudomonas syringae pv. tabaci. Leaves of both tobacco
and Arabidopsis plants expressing AnPGII accumulated high levels of hydrogen peroxide, peroxidase and
glucanase activity and showed enhanced defence gene
expression. PGs are virulence factors and, on the other
hand, may activate defence responses by releasing
OGs that act antagonistically with auxin in different
bioassays. Interestingly, sensitivity of transgenic
plants to auxin is reduced in different bioassays, whereas resistance to fungal infection is suppressed by
exogenous auxin. We conclude that homogalacturonan
Di Matteo A, Giovane A, Raiola A, Camardella L,
Bonivento D, De Lorenzo G, Cervone F, Bellincampi
D, Tsernoglou D. Structural basis for the interaction
between pectin methylesterase and a specific
inhibitor protein. Plant Cell, 2005; 17(3):849-58.
Epub 2005 Feb 18.
Sicilia F, Fernandez-Recio J, Caprari C, De Lorenzo
G, Tsernoglou D, Cervone F, Federici L. The polygalacturonase-inhibiting protein PGIP2 of Phaseolus
vulgaris has evolved a mixed mode of inhibition of
endopolygalacturonase PG1 of Botrytis cinerea. Plant
Physiol, 2005; 139(3):1380-8. Epub 2005 Oct 21.
Federici L, Di Matteo A, Fernandez-Recio J,
Tsernoglou D, Cervone F. Polygalacturonase inhibiting proteins: players in plant innate immunity?
Trends Plant Sci, 2006; 11(2):65-70. Epub 2006 Jan 9.
46
Molecular and functional approaches to investigate the
physiopathological role of the chemokines and their receptors in
the central nervous system
Principal investigator: Fabrizio Eusebi
Professor of Human Physiology
Dipartimento di Fisiologia Umana e Farmacologia
Tel.: +39 06 49910857, Fax: +39 06 49910851
[email protected]
During the second year of the project, we focused
our attention on two main subjects, going deeper in
the results already obtained the previous year: (i) the
study of
the role of
the chemokine
fractalkine/CX3CL1 on cell movement and migration, performed both in primary cultures of
ippocampal and cerebellar rat neurons (6); (ii) the
study of
the role of
the chemokine
fractalkine/CX3CL1 on the modulation of synaptic
transmission in hippocampal slices (5).
Participants:
Francesca Grassi, Sergio Fucile, Cristina Limatola,
Eleonora Palma, Davide Ragozzino, professors; Silvia
Diangelantonio, Clotilde Lauro, Flavia Trettel, post-doc
fellows; Cristina Bertollini, Myriam Catalano, Raffaela
Cipriani, Francesca Maiolino, Fabrizia Sobrero, PhD
students; Giusi Chece, technician.
Report of activity
Chemokines and their receptors are widely expresses in CNS under normal and pathological conditions.
In the last few years, several activities have been
described for chemokines in the brain: they regulate
neural cell migration and synaptic transmission and
have trophic effects on neurons. The knowledge of
these biological activities in the CNS is relevant to
understand the neuropathological events associated
with several brain diseases, when the levels of
chemokines and chemokine receptors are up-regulated. Aim of this research project is to investigate the
molecular mechanisms underlying the functional
effects of chemokines on the synaptic transmission
and on the development of the CNS. The effects of
chemokines on synaptic transmission will be examined in rodent brain slices, focusing on the changes
induced on short-term neuromodulatory activities
and long-term plasticity. The function of
chemokines in cell migration and survival after toxic
insults, as well as the molecular mechanisms
involved, will be investigated in primary cultures of
cerebellar, hippocampal and cortical neurons
obtained from rat and mouse brain. This part of the
project will be extended to analyse the ability of
chemokines to modulate the homing, proliferation
and differentiation of neuronal stem cells derived
from adult and embryonic mouse brains. This study
has an obvious impact on the understanding of neurogenesis and neurorepair, yielding possible clues to
therapeutical strategies.
Role of the chemokine fractalkine/CX3CL1 on
cell movement and migration
We performed experiments both on ippocampal and
cerebellar neurons. The chemokine CX3CL1 and its
specific receptor CX3CR1 are abundantly expressed
in the CNS, where they mediate microglia-neuron
interaction during physiological and pathological
conditions. Several evidences describe that toxic
insults induce CX3CL1 expression and release from
neuronal cells with microglial recruitment. CX3CL1
released also plays a direct neuroprotective role,
reducing the neuronal damage caused by toxic
insults, and reducing IFN-g and LPS-induced
microglial activation.
Fractalkine/CX3CL1 and its specific receptor
CX3CR1 are constitutively expressed in several
regions of the CNS, and are reported to mediate
neuron-microglial interaction, synaptic transmission
and neuronal protection from toxic insults. CX3CL1
is released both by neuronal and astrocytic cells,
while CX3CR1 is mainly expressed by microglial
cells and neurons. Microglial cells efficiently migrate
in response to CX3CL1, while no evidence is reported to date on CX3CL1-induced neuronal migration.
For this reason we have investigated in vitro the
effects of CX3CL1 on basal migration of neurons
and of the microglial and astrocytic populations, all
these cells being obtained from the hippocampus and
the cerebellum of newborn rats. We report that
CX3CL1 stimulates microglial cell migration but
47
F. Eusebi - Molecular and functional approaches to study chemokines and their receptors in the CNS
efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is
PTX-sensitive and PI3-K-dependent on hippocampal neurons, while it is PTX-sensitive, and PI3-Kand ERK-dependent on cerebellar granules.
Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin,
with mechanisms dependent on PTX-sensitive G
proteins, and on the ERK and PI3-K pathways. Both
the reduction of migration and the increase of neuron adhesion require the activation of the a1 and b6
integrin subunits with the exception of cerebellar
neuron migration, which is only dependent on the a1
subunit.
More
importantly,
in
neurons,
CX3CL1/CXCL12 co-treatment abolished the effect
mediated by a single chemokine on chemotaxis and
adhesion. In conclusion, our findings indicate that
CX3CL1 reduces neuronal migration by increasing
cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.
observed in the absence of afferent fibre stimulation
or AMPA-receptor activation, respectively, indicating
the requirement of sustained receptor activity for its
development. Findings obtained from hippocampal
slices, cultured hippocampal neurons and transfected
HEK cells indicate that a Ca2+-, cAMP- and phosphatase-dependent process is likely to modulate
CX3CL1 effects because i) CX3CL1-induced depression was antagonized by many drugs including intracellular BAPTA, 8Br-cAMP, phosphatase inhibitors,
and pertussis toxin (PTX); ii)
CX3CL1 inhibited forskolin- induced cAMP formation sensitive to PTX; iii) CX3CL1 inhibited
forskolin-induced Ser845 GluR1 phosphorylation
which was sensitive to PTX and dependent on Ca2+
and phosphatases activity. Together these findings
indicate that CX3CL1 negatively modulates AMPA
receptor function at active glutamatergic synapses
through cell signalling pathways by influencing the
balance between kinase and phosphatase activity.
Role of the chemokine fractalkine/CX3CL1 on
the modulation of synaptic transmission
We examined the effects of the chemokine fractalkine
(CX3CL1) on excitatory postsynaptic currents
(EPSCs) evoked by electrical stimulation of Schaffer
collaterals in patch clamped CA1 pyramidal neurons
from rat hippocampal slices. Acute applications of
CX3CL1 caused a sustained reduction of EPSC
amplitudes, with partial recovery after washout.
EPSC depression upon CX3CL1 treatment is postsynaptic in nature, since (i) paired pulse ratio was maintained, (ii) amplitude distribution of spontaneous
excitatory postsynaptic currents shifted to lower values, and (iii) whole cell current responses to ?-amino3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA) were reversibly inhibited. EPSC depression
by CX3CL1 is bona fide mediated by CX3CL1 receptor (CX3CR1), because CX3CL1 was unable to influence EPSC amplitude in CA1 pyramidal neurons
from CX3CR1-KO mice. CX3CL1-induced depression of both EPSC and AMPA-current was not
Ragozzino D, Palma E, Di Angelantonio S, Amici
M, Mascia A, Arcella A, Giangaspero F, Cantore G,
Di Gennaro G, Manfredi M, Esposito V, Quarato PP,
Miledi R, Eusebi F. Rundown of GABA type A
receptors is a dysfunction associated with human
drug-resistant mesial temporal lobe epilepsy. Proc
Natl Acad Sci USA, 2005;102(42):15219-23. Epub
2005 Oct 10.
Fucile S, Miledi R, Eusebi F. Effects of cyclothiazide on GluR1/AMPA receptors. Proc Natl Acad
Sci USA, 2006; 103(8):2943-7. Epub 2006 Feb 10.
Palma E, Amici M, Sobrero F, Spinelli G, Di
Angelantonio S, Ragozzino D, Mascia A, Scoppetta
C, Esposito V, Miledi R, Eusebi F. Anomalous levels
of Cl- transporters in the hippocampal subiculum
from temporal lobe epilepsy patients make GABA
excitatory. Proc Natl Acad Sci USA, 2006;
103(22):8465-8. Epub 2006 May 18.
48
Molecular and enantioselective recognition by receptors and
proteins studied in the gas phase, in free solution and at solidliquid interfaces
Principal investigator: Francesco Gasparrini
Professor of Organic Chemistry
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze
Biologicamente Attive
Tel.: (+39) 06 49912776, Fax: (+39) 06 49912780
[email protected]
nisms originating between target compounds and
active sites on the CSP structure. Hydrogen bonds
were found to be pivotal for chromatographic retention and enantioselectivity. The competitive adsorption isotherms of two model BZDPs (lorazepam and
temazepam) were measured at different mobile phase
components, through the so-called inverse method.
The adsorption data were fitted with a competitive biLangmuir adsorption isotherm. Enantiomeric separations under non-linear conditions were modeled by
using the equilibrium-dispersive (ED) model of chromatography. Theoretical overloaded band profiles
(obtained by solving the system of partial differential
equations described by the ED model) matched, in a
significantly accurate way, the profile experimentally
measured.
Participants:
Bruno Botta, Claudio Villani, professors; Ilaria
D’Acquarica, Beatrice Galli, Mario Giovannoli, Marco
Pierini, researchers; Alessia Ciogli, Deborah Subissati, postdoc fellows; Laura Nevola, PhD student; Giovanna
Cancelliere, technician.
Report of activity
The 2005-2006 scientific activity of the present
research project, focused on the study of the recognition ability of chiral synthetic and naturally occurring
macrocyclic hosts towards peptidic substrates, has
been organized in the following main topics: (a) synthesis and evaluation of novel chiral stationary phases
(CSPs) for HPLC; (b) deposition of carbon nanotubes
(CNTs) on HPLC silica microparticles; (c) gas-phase
enantioselectivity of chiral amido[4]resorcarenes
receptors.
Deposition of carbon nanotubes (CNTs) on
HPLC silica microparticles
Carbon nanotubes (CNTs) constitute a new class of
materials with potential applications ranging from
nanoelectronics to medicinal chemistry. The welldefined inner and outer surfaces of CNTs act as efficient sensors for studying their recognition properties towards a variety of molecular targets, including
small organic ligands and biologically relevant molecules. The major problem encountered in detecting
and analyzing molecular interactions is related to the
difficult manipulation of individual nanotubes, usually present in bundles and ropes and particularly
insoluble in both organic and water-rich media. A
possible way to overcome this limitation consists in
the deposition of CNTs on a chromatographic solid
matrix (e.g. HPLC silica microparticles) to generate
a packing material on which the relative affinities of
potential ligands can be studied by monitoring their
retention under controlled experimental conditions
(organic and water-based eluents, variable temperature). In the present project, we prepared and characterized hybrid materials by deposition of CNTs on
the external surface of porous silica microparticles.
Synthesis and evaluation of novel chiral
stationary phases (CSPs) for HPLC
In the previous project we prepared a new hybrid
brush-type polymeric HPLC chiral stationary phase
(CSP) by the surface-initiated polymerization of the
chiral, enantiopure diacryloyl derivative of trans-1,2diaminocyclohexane on mesoporous azo-activated silica. The new chiral packing material showed broad
applicability, high enantioselectivity, chemical and
thermal inertness and the availability in both the
enantiomeric forms. At the same time, analytical
columns packed with the new material showed high
overall chromatographic efficiency and high loading
capabilities, leading to complete resolutions by short
time analysis. The chromatographic behaviour of a
series of racemic benzodiazepines (BZDPs) was evaluated under linear and non-linear conditions on the
new hybrid polymeric CSP. Differently substituted
BZDPs were employed as probes to make hypotheses
concerning possible molecular interaction mecha-
49
F. Gasparrini - Molecular and enantioselective recognition by receptor and protein studies
Compared to a pure graphitic porous carbon packing,
our materials showed less pronounced hydrophobicity and were suitable for the analysis of polar compounds with water based-eluents. Given the simplicity of the HPLC screening procedure, we expect our
hybrid materials will find a broad range of applications, including the study of the interaction between
CNTs and small-to-medium libraries of biomolecules in water-rich media.
wherein: i) guest A does not present any additional
functionalities besides the amino acid one [alanine
(Ala), PhGly, and phenylalanine (Phe)]; ii) guest A
presents an additional alcohol functionality [serine
(Ser), Thr, and AThr]; iii) guest A contains several
additional functionalities on its aromatic ring [tyrosine (Tyr), TyrOMe, Trp, and 3,4-dihydroxyphenylalanine (DOPA)]. Each category exhibited a specific enantioselectivity depending on the predominant
[1LHA]+ structures and the orientation of the 2aminobutane reactant in the relevant observed
adducts. The results may contribute to the understanding of the exceptional selectivity and catalytic
properties of enzyme mimics towards unsolvated
biomolecules.
Gas-phase enantioselectivity of chiral
amido[4]resorcarenes receptors
Diastereomeric proton-bound [1 HA]+ complexes
L
between selected amino acids [A = phenylglycine
(PhGly), tryptophan (Trp), tyrosine methyl ester
(TyrOMe), threonine (Thr), and allothreonine
(AThr)] and a chiral amido[4]resorcarene receptor
(1L) displayed a significant enantioselectivity when
undergoing loss of the amino acid guest A by way of
the enantiomers of 2-aminobutane (B) in the gas
phase, by ESI-FT-ICR mass spectrometry. The enantioselectivity of the B-to-A displacement was
ascribed to a combination of thermodynamic and
kinetic factors related to the structure and the stability of the diastereomeric [1LHA]+ complexes and
of the reaction transition states. The results of the
present and previous studied allowed classification of
the [1LHA]+ complexes in three main categories
Gasparrini F, Misiti D, Rompietti R, Villani C. New
hybrid polymeric liquid chromatography chiral stationary phase prepared by surface-initiated polymerization. J Chromatogr A, 2005; 1064(1):25-38.
Botta B, Caporuscio F, D’Acquarica I, Delle
Monache G, Subissati D, Tafi A, Botta M, Filippi A,
Speranza M. Gas-phase enantioselectivity of chiral
amido[4]resorcinarene receptors. Chemistry, 2006;
12(31):8096-105.
50
Functional role of the interaction between transcriptional
regulators, nuclear receptors and leukemia-associated fusion
proteins
Principal Investigator: Clara Nervi
Professor of Histology and Medical Embryology
Dipartimento di Istologia ed Embriologia Medica
Tel.: (+39) 06 49768102, Fax: (+39) 06 4462854
[email protected]
binding to RARa and PML/RARa by co-repressors
and co-activators interactions; 2) Conformational
changes of RARa and PML/RARa induced by corepressors, IDs and co-activators interaction; 3)
Effects of N-CoR and SMRT ID peptides on receptor/co-repressor interaction and transcriptional
function; 4) Effects on proliferation and differentiation of leukemic cell lines expressing PML/RARa+
or other fusion proteins (PLZF/RARa+,
AML1/ETO+; BCR/ABL).
Participants:
Daria Brambilla, Francesco Fazi, Lorena Travaglini, postdoc fellows; Maria Rita Mosini, technician.
Collaborations:
Università di Perugia (Prof. Francesco Grignani); Università di
Roma “Tor Vergata” (Prof. Francesco Lo Coco); Lady Davis
Institute for Medical Research, Montreal, Canada (Prof. Wilson
Miller); Med. Klinik III/Abtl. Haematologie, Johann Wolfgang
Goethe-Universitat, Frankfurt, Germany (Prof. Martin
Ruthardt).
Regulation of ligand binding to RARa and
PML/RARa by Co-Rs and co-activators
interactions
Ligand binding assays showed that RA binding to
either RARa and PML/RARa is increased by the
expression of NCoR or of peptides representative of
the C-terminal interacting domain of NCoR (IDC),
but not of mutated ID-C, ID-N polypeptides or transcriptional co-activators TIF2, NSD1 and ACTR. In
addition, we found that RA binding to RARa and
PML/RARa is inhibited by the expression of cDNAs
encoding polypeptides representing the co-repressors/nuclear receptors interaction region present on
the ligand binding domain of RARa (D-E domains)
that probably sequester endogenous co-repressors.
Saturation and competition binding analysis indicated
that ID-C expression did not change the RA binding
affinity of RARa and PML/RARa, but consistently
increased of about two fold the number of RARa and
PML/ RARa ligand binding sites. Moreover, we found
that specific conformational changes that allow ligand
binding to receptors are induced by different interaction peptides.
Report of activity
The PML/RARa, PLZF/RARa and AML1/ETO
leukemia fusion proteins induce acute myeloid
leukemia due to their aberrant interaction with corepressors, N-CoR and SMRT, that recruit a multiprotein complex containing histone deacetylases on
crucial myeloid differentiation genes. This leads to
gene repression contributing to generate a differentiation block. N-CoR and SMRT interact with the
retinoic acid (RA) nuclear receptor RARa and with
the AML-associated RARa-fusion proteins through
interaction domains (IDs: IDC and IDN), that contain an LXXLL (L: leucine, X: any aminoacid) core
sequence. In the case of AML1/ETO the N-CoR
domain interacting with ETO mapped within the
RD3 domain. Binding of polypeptides representative
of co-repressors IDs and RD3 induces nuclear
receptor and fusion proteins conformational changes
similar to those induced by the co-repressor protein
and competitively displaces co-repressors. These
peptides, therefore might have a therapeutic potential. Major aim of this project is to characterize the
molecular mechanisms that regulate ligand binding
and the release of transcriptional regulators from
nuclear receptors and cancer fusion proteins in order
to develop novel therapeutic approaches in neoplasia.
We mainly focused on the: 1) regulation of ligand
Effects of N-CoR and SMRT ID peptides on
receptor/co-repressor interaction and
transcriptional function
The ligand dependent transactivation activity of the
bRARE3-tk-LUC, a reporter construct containing
the RA responsive element (RARE), transfected into
51
C. Nervi - Interaction between transcriptional regulators, nuclear receptors and leukemia-associated fusion proteins
an APL cell line expressing both RARa and
PML/RARa (NB4 cells), is significantly increased by
the expression of the IDC polypeptide, but not by
the IDN. In contrast, NCoR expression significantly
impaired ligand dependent transcriptional activity,
which is consistent with the presence of a transcriptional repressor domain in this protein. Thus, it
appears that IDC expression can increase both the
ligand binding and the transactivation activity of
nuclear receptors.
ferentiation response in cells expressing N-CoR
fragments. Treatment of NB4IDC and NB4R4IDC
cells with proteasome inhibitors re-establishes
PML/RARa expression, but the fusion protein cannot recruit N-CoR, due to the overexpression of
interaction peptides, and, as a result, cannot block
differentiation. Moreover, the SKNO1 RD3 cells differentiate efficiently despite the fact that
AML1/ETO protein is only slightly degraded.
Our data indicate that fusion proteins activity is a
permanent requirement for the leukemia phenotype
and that it is necessary for the leukemia differentiation block. We also showed that fusion protein function is necessary to block myeloid differentiation,
although it may not be sufficient. Thus, full malignant features in leukemia require fusion protein
activity. This implies that the elimination of fusion
proteins function may revert the leukemia phenotype. Thus, fusion proteins are an important target
for molecular therapy of leukemia.
Effects on proliferation and differentiation of
leukemic cell lines expressing PML/RARa or
AML1/ETO fusion proteins
We investigated whether the in vivo interruption of
the contact between fusion proteins and co-repressors could modify the leukemia phenotype. We took
advantage of two well-studied cell line models
expressing of the fusion proteins PML/RARa (NB4
cells and its RA resistant subclone NB4R4) and the
AML1/ETO (SKNO-1). We show that the expression of IDC and IDN RD3 sequences, representing
the fusion protein interaction domains of the N-CoR
protein with the nuclear receptor RAR·, efficiently
blocks these interactions and restores the differentiation potential of leukemia cells. Expression of the
N-CoR fragments can also convert leukemia cells
from RA-resistant to RA responsive. Indeed, in RAresistant NB4R4-IDC or IDN cells the dominant
negative effect on RAR? ?of ?a mutant PML/RAR?
is abolished. SKNO1 cells, which express
AML1/ETO became RA-responsive following the
expression of the RD3 fragment. These data are in
agreement with our findings suggesting that the
AML1/ETO fusion protein is able to block the
RARa pathway and show that this is the direct consequence of the AML1/ETO protein interaction
with co-repressors. These effects were also obtained
by direct protein transduction.
Overall our results indicate that loss of NCoR/SMRT interactions, rather than fusion protein
degradation is primarily responsible for restored dif-
Fazi F, Travaglini L, Carotti D, Palitti F, Diverio D,
Alcalay M, McNamara S, Miller WH Jr, Lo Coco F,
Pelicci PG, Nervi C. Retinoic acid targets DNAmethyltransferases and histone deacetylases during
APL blast differentiation in vitro and in vivo.
Oncogene, 2005; 24(11):1820-30.
Puccetti E, Zheng X, Brambilla D, Seshire A,
Beissert T, Boehrer S, Nurnberger H, Hoelzer D,
Ottmann OG, Nervi C, Ruthardt M. The integrity of
the charged pocket in the BTB/POZ domain is
essential for the phenotype induced by the leukemiaassociated t(11;17) fusion protein PLZF/RARalpha.
Cancer Res, 2005; 65(14):6080-8.
Racanicchi S, Maccherani C, Liberatore C, Billi M,
Gelmetti V, Panigada M, Rizzo G, Nervi C, Grignani
F. Targeting fusion protein/corepressor contact
restores differentiation response in leukemia cells.
EMBO J, 2005; 24(6):1232-42. Epub 2005 Feb 24.
52
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
[email protected]
Participants:
remodelling of the rat SCG intraganglionic
synapses
Maria Egle De Stefano, professor; Arianna Del Signore,
Lucia Leone, post-doc fellows; Loredana Lombardi, PhD
student.
Extracellular proteases, such as matrix metalloproteases (MMPs), plasmin, tissue-type plasminogen
activator (tPA) and urokinase-type plasminogen activator (uPA) are well known to be involved in synaptic remodelling processes, including learning and
memory. These enzymes have proteins of the extracellular matrix (ECM) as their major targets; however, they may act on synaptic molecules that, extending into the ECM from the pre- or post-synaptic side,
reciprocally interact contributing to synapse stabilisation. Neurexins and dystroglycan (DG) are good
candidates to play this role. DG is a transmembrane
protein complex binding intracellularly dystrophin
and extracellularly molecules such as laminin, agrin,
perlecan, biglycan and neurexins, neurone-specific
cell surface proteins that extend from the presynaptic side into the ECM. The interaction of DG with
neurexins may be one of the direct bridges between
pre- and postsynaptic sides. Recently, it has been
shown that DG is one of the targets of gelatinases,
members of the MMP family, which disrupt the link
between the ECM and the cytoskeleton by disintegrating the dystroglycan complex. We hypothesise
that degradation of DG, through disruption of the
molecular bridge between pre- and postsynaptic side,
is involved in the intraganglionic synaptic detachment induced, in SCG, by pre- and post-ganglionic
nerve crush.
MPPs are a Zn2+-containing family of extracellular
peptidases. They are expressed in various organs,
including the brain, with both glial and neuronal
localisation. The proteolitic activity of MMPs is
counterbalanced by the tissue inhibitors of matrix
metalloprotease (TIMPs). All MMPs require enzymatic activation by cleavage of the propeptide,
achieved by the action of other MMPs, or of serine
proteases such as plasmin, which is produced from
plasminogen by the activity of tPA or uPA.
Collaborations:
Istituto Superiore di Sanità, Laboratorio di Biologia Cellulare,
Roma (Dr. Tamara Petrucci); Centro di Farmacologia Cellulare e
Molecolare, CNR, Dipartimento di Farmacologia Medica,
Università di Milano (Dr. Cecilia Gotti); Dipartimento di
Genetica e Biologia Molecolare, Sapienza-Università di Roma
(Prof. Ernesto Di Mauro, Prof. Alberto Oliverio); Facoltà di
Farmacia, Università di Catanzaro “Magna Grecia”, Roccelletta
di Borgia (CZ) (Prof. Carla Perrone Capano).
Report of activity
Aim of this project is the characterisation in rodent
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 postganglionic 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 characterisation of
extracellular proteinases and their substrates
involved in the remodelling of the rat SCG intraganglionic synapses induced by pre- and post-ganglionic nerve crush; (ii) the modulation of gene
expression in rat SCG induced by damage of the preor post-ganglionic nerves; (iii) the role of dystrophin
and SCG target organs in the maintenance of the
structural and functional integrity of ganglionic circuits in mice.
Characterisation of extracellular proteinases
and their substrates involved in the
53
P. Paggi - Neuronal response to experimental interruption of the neural circuit: a molecular and structural study
(Dp427) in the SCG and in the innervation of some
We have shown (Leone et al., 2005) that SCG neurone axotomy activates MMP-2 and two enzymes
involved in one of its activation pathways, the membrane type-1 MMP (MT1-MMP) and TIMP-2. In
parallel, we found an increase in the 30 kDa DG
degradation fragment, suggesting that DG may be a
MMP-2 substrate also in the SCG (Paggi et al., J.
Physiol Paris, 99:119-24, 2006).
of its muscular (heart and iris) and non-muscular
(submandibular gland) peripheral targets in dys-
trophic mdx mice. SCGs of mdx mice have 36% fewer
neurons than those of wild-type animals and neuronal loss occurs around P10. Retrograde labeling of
ganglionic neurons reveals that lost neurons are
among those innervating muscular targets, which
are affected by the lack of Dp427, as shown by Evans
Modulation of gene expression in rat SCG
Large-scale analysis of gene expression has shown
that both pre- and post-ganglionic nerve crush modulate the expression of numerous genes in rat SCG.
Various genes whose expression changed in response
to pre- and/or post-ganglionic nerve crush at different post-injury time points were identified. Affected
genes were classified according to their function, and
clustered on the basis of their temporal expression
profiles. Among altered genes we found ribosomal
proteins; transcriptional factors (brain finger protein); proteins involved in extracellular matrix
organization/remodelling or in the cell/matrix
interactions (collagen, proteoglycans, annexin II);
proteins involved in neurite outgrowth modulation
(thrombin receptor). To validate the microarray data,
the expression of some modulated genes was
analysed by semiquantitative or real time reverse
transcriptase-PCR. Our results show that, although
the up- and down-regulation of gene expression
induced by nerve crush is generally quantitatively
modest, it involves genes whose function is crucial
for neuronal survival and regeneration. Moreover,
cluster analysis allowed us to correlate the changes
in gene expression with the time-course of the
structural and functional alterations observed in the
SCG after injury (Del Signore et al., Gene expression
pathways induced by axotomy and decentralisation
of rat superior cervical ganglion neurons. Eur J
Neurosci, 2006, 23:65-74).
blue staining of muscle cells in heart and iris already
at P10. In addition, although tyrosine hydroxylase
immunolabeling reveals reduced axonal defascicula-
tion and terminal sprouting in all SCG target organs
examined, poor adrenergic innervation is observed
only in the heart and in the iris. These alterations are
detected as early as P5, when neuronal loss has not
yet occurred. Our results indicate that, in mdx mice,
the lack of Dp427 directly impairs the axonal defas-
ciculation and terminal sprouting of sympathetic
neurons, leading to neuronal death when these
intrinsic alterations combine with structural and/or
functional damage to muscular targets (De Stefano et
al., 2006).
De Stefano ME, Leone L, Lombardi L, Paggi P.
Lack of dystrophin leads to the selective loss of
superior cervical ganglion neurons projecting to
muscular targets in genetically dystrophic mdx mice.
Neurobiol Dis, 2005; 20(3):929-42. Epub 2005 Jul 14.
Leone L, De Stefano ME, Del Signore A, Petrucci
TC, Paggi P. Axotomy of sympathetic neurons acti-
vates the metalloproteinase-2 enzymatic pathway. J
Neuropathol Exp Neurol, 2005; 64(11):1007-17.
Del Signore A, De Sanctis V, Di Mauro E, Negri R,
Role of dystrophin and SCG target organs in
the maintenance of the structural and
functional integrity of ganglionic circuits
Perrone Capano C, Paggi P. Gene expression path-
ways induced by axotomy and decentralization of rat
To explore the autonomic imbalance associated with
Duchenne muscular dystrophy we analyzed the
alterations induced by lack of full-length dystrophin
superior cervical ganglion neurons. Eur J Neurosci,
2006; 23(1):65-74.
54
The role of DNA sequence in the organization of telomeric
chromosomal domains
Principal investigator: Maria Savino
Professor of Biological Physical Chemistry
Tel.: (+39) 06 49912238, Fax: (+39) 06 4440812
[email protected]
Whether histones and specific telomeric proteins
compete for telomeric DNA binding (and hence occupy different telomere domains) or whether nucleosomes are directly involved in the formation of the
telomeric complex is a relevant issue in order to
understand telomeric structure and its dynamics. We
addressed this question by electrophoretic mobility
shift assay (EMSA) and DNase I footprinting, using
nucleosome cores formed on different DNA probes
containing human telomeric sequences of various
lengths. We found that TRF1 specifically recognizes
telomeric binding sites on the nucleosome (Figure
1a), even if binding to nucleosome requires a sixfold
higher concentration of protein than is required to
bind naked DNA (Figure 1b). The formation of the
ternary complex is strongly dependent on the orientation of binding sites on the nucleosome surface,
rather than on the location of the binding sites in
respect to the nucleosome dyad. Strikingly, TRF1
binding causes alterations in nucleosome structure
without dissociation of histone subunits (Figure 1c).
These results indicate that nucleosomes contribute
to the establishment of a telomeric capping complex,
whose structure and dynamics can be modulated by
the binding of telomeric factors (Galati et al., 2006).
Then, we examined whether nucleosomal spacing at
telomeres could be determined by DNA sequence.
We assembled nucleosomal arrays in vitro onto 1500
bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence; we then measured internucleosomal distances by means of AFM imaging, a
technique which allows to analyze nucleosome positioning at the level of single molecules (Scipioni et
al., 2006). We found that nucleosomes assembled
onto human telomeric DNA are randomly positioned, whereas nucleosomes assembled onto 601-8
DNA are preferentially positioned every 200 bp
(Pisano et al., 2006). The AFM-derived nucleosome
organization is in satisfactory agreement with that
predicted by theoretical modeling, based on
Participants:
Stefano Cacchione, researcher; Rosella Mechelli, Luigi
Rossetti, post-doc fellows; Alessandra Galati, Sabrina
Pisano, PhD students; Enrico Marchioni, Emanuela
Pascucci, graduate students.
Collaborations:
Dipartimento di Chimica, Sapienza-Università di Roma (Prof.
Pasquale De Santis, Dr. Anita Scipioni); Istituto di Biologia e
Patologia Molecolari, CNR, Roma (Dr. Roberto Caneva);
Dipartimento di Biochimica G. Moruzzi, Università di Bologna
(Dr. Anna Bergia, Prof. Bruno Samorì); Medical Research
Council, Laboratory of Molecular Biology, Cambridge, UK (Dr.
Linda Chapman, Dr. Daniela Rhodes).
Report of Activity
Telomeres are dynamic nucleoprotein structures that
cap the ends of eukaryotic chromosomes. In humans,
the long (TTAGGG)n double-stranded telomeric
DNA repeats are bound specifically by the two related proteins TRF1 and TRF2 and are also organized
in arrays of tightly spaced nucleosomes. Previous
studies carried out in our laboratory have shown that
telomeric nucleosomes have peculiar sequencedependent features, as a consequence of the sequence
periodicity of 6-8 bp, which is out of phase with the
DNA helical repeat. We demonstrated that nucleosomes formed on telomeric sequences are less stable
than bulk nucleosomes, occupy multiple isoenergetic
positions, and are intrinsically mobile. Whereas the
role of TRF1 and TRF2 in telomeric function has
been extensively studied, little is known about the
involvement of telomeric nucleosomes in telomere
structures and how chromatin formation may affect
binding of the TRFs.
In the last two years, we investigated i) whether
TRF1 is able to bind to telomeric binding sites in a
nucleosomal context; ii) the role of telomeric DNA
sequence in nucleosomal spacing.
55
M. Savino - The role of DNA sequence in the organization of telomeric chromosomal domains
ing sites and alters nucleosome structure. J Mol Biol,
2006; 360(2):377-85. Epub 2006 May 19.
sequence-dependent DNA curvature and flexibility.
These results indicate that DNA sequence plays a
main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays.
Pisano S, Pascucci E, Cacchione S, De Santis P,
Savino M. AFM imaging and theoretical modeling
studies of sequence-dependent nucleosome positioning. Biophys Chem, 2006; 124(2):81-9. Epub 2006 Jul 7.
Scipioni A, Pisano S, Bergia A, Savino M, Samori B,
De Santis P. Recognition on the nanoscale of a DNA
sequence by an inorganic crystal surface.
Chembiochem, 2006; 7(11):1645-8.
Galati A, Rossetti L, Pisano S, Chapman L, Rhodes
D, Savino M, Cacchione S. The human telomeric protein TRF1 specifically recognizes nucleosomal bind-
Fig. 1 - Binding of TRF1 to nucleosomes formed on a 154 bp DNA sequence containing 8 telomeric
repeats. (a) Gel mobility-shift assay. (b) Percentage of unbound DNA and nucleosomes reported as a
function of TRF1 concentration. (c) DNAse I footprinting of TRF1/nucleosome ternary complexes.
Binding of TRF1 protects two non-consecutive sites near the dyad axis (asterisks) and induce alterations of the nucleosome structure.
56
AREA
4
Cellular
and
molecular
immunology
Cellular and molecular immunology - Area 4
Defense mechanisms in innate immunity
Principal investigator: Donatella Barra
Professor of Biochemistry
Tel: (+39) 06 4456663, Fax: (+39) 06 4440062
[email protected]
a detailed characterization of the biological activity
and target specificity of some amphibian antimicrobial peptides, as well as insights into the post-translational processing of natural peptides.
In particular, the effect of temporin A (FLPLIGRVLSGIL-NH2) and temporin B (LLPIVGNLLKSLL-NH2) on Leishmania parasites was investigated,
as these protozoa represent a severe threat for
immunocompromised patients worldwide (Mangoni
et al., 2005). Both peptides have leishmanicidal activity at micromolar concentrations with no cytolytic
effect on human erythrocytes. These temporins, isolated from Rana temporaria, represent the shortest
natural peptides with the maximal leishmanicidal
activity and the lowest number of positive charges,
that are able to maintain their activity in serum.
Their lethal mechanism implies the permeabilization
of the cytoplasmic membrane, inducing a rapid collapse of the membrane potential and reducing intracellular ATP levels.
Furthermore, to shed light on the biological significance of the presence of closely related antimicrobial peptides in a single living organism, we used the
homologous antimicrobial peptides temporins A, B,
and L (FVQWFSKFLGRIL-NH2). We found that
temporins A and B are only weakly active toward
Gram-negative bacteria. However, a marked synergism occurs when each is mixed with temporin L
(Rosenfeld et al., 2006). To study the underlying
mechanisms involved in these activities, we used various experimental strategies to investigate: (i) the
effect of the peptides’ interaction on both the viability and membrane permeability of intact bacteria
and spheroplasts; (ii) their interaction with
lipopolysaccharides (LPS) and the effect of LPS on
the oligomeric state of temporins, alone or combining one with another; (iii) their structure in solution
and when bound to LPS. The data reveal that temporin L synergizes with temporins A and B by preventing their oligomerization in LPS. This should
promote their translocation across the outer mem-
Participants:
Maurizio Simmaco, professor; M. Luisa Mangoni,
Giuseppina Mignogna, Rossella Miele, researchers; Marina
Borro, Giovanna Gentile, post-doc fellows; Alessandra
Franco, technician.
Collaborations:
Institute of Molecular Biology, Austrian Academy of Sciences,
Salzburg, Austria (Prof. Günther Kreil); Dipartimento di Scienze
Mediche Internistiche, Università di Cagliari, Monserrato (Dr.
Andrea Rinaldi); Centro de Investigaciones Biologicas (CSIC)
Madrid, Spain (Prof. Luis Rivas); Department of Biological
Chemistry, Weizmann Institute of Science, Rehovot, Israel (Prof.
Yechiel Shai).
Report of activity
Peptides are known to play important functions both
in innate and adaptive immunity. Antimicrobial peptides are produced at all levels of the evolutionary
scale, either constitutively or induced in response to
inflammatory stimuli, and represent the first line of
host defence against microbial invasion. The accepted mechanism by which antimicrobial peptides exert
their function is the extensive permeabilization of
bacterial membranes. However, many examples are
known of peptides that penetrate cell wall, without
causing lysis, and act inside the cell by interfering
with host vital processes.
The purpose of this project was the search for novel
antimicrobial peptides from amphibian skin secretions and the study of their mechanisms of action, to
assist in the development of novel peptide-based
drugs against several types of pathologies of microbial or viral origin. Another issue of this research
was the study of structure-function relationships of
a small protein isolated from skin secretions of
Bombina species, Bv8, bearing peculiar pharmacological properties.
The main results achieved during these two years are
59
D. Barra - Defense mechanisms in innate immunity
genomic DNA on the basis of partial protein
sequencing. Expression of this cDNA in Xenopus laevis oocytes allowed detection of isomerase activity.
Proteins related to the frog skin enzyme are present
in several vertebrate species, including humans, as
based on sequence comparison of homologues from
different species. We consider it to be likely that
enzymes related to the frog isomerase are widely distributed among vertebrate species. This finding raises the intriguing possibility that peptides containing
a D-amino acid are also present in these species.
These may be unknown peptides that have not been
isolated. In addition, the presence of D-isomers may
have been overlooked in hormones, antibacterial or
modulatory peptides of the immune system, or neuropeptides that have earlier been characterized.
Indeed, in the past D-amino acids could not be
detected easily with the most common sequencing
techniques or by mass spectrometry. In case of dermorphin, the first animal peptide that was found to
contain a D-amino acid, this residue was discovered
only because the synthetic all-L-isomer was biologically inactive.
brane into the cytoplasmic membrane. This is the
first study that explains how a combination of native
antimicrobial peptides from the same species can
overcome bacterial resistance imposed by the LPS
leaflet.
On Bv8, attempts were made to identify the structural determinants required for receptor binding and
hyperalgesic activity, as a first step in the design of
antagonist molecules. In view of the strict conservation of the amino-terminal end in the AVIT family,
we prepared Bv8 derivatives lacking one or two
amino acid residues from the N-terminus. The binding affinity, the in vitro biological activity, and the
hyperalgesic potency in vivo of these truncated
forms were analyzed, as well as their activity as agonists or antagonists. N-terminal Ala and Ala-Val
deletion does not preclude the ability of the modified
Bv8 to bind the receptors, however influences its
ability to activate the signalling cascade. Truncated
Bv8 forms competitively bind the receptors, antagonizing the Bv8-induced hyperalgesia and providing
the beginnings of the biological basis for designing
therapies to block nociceptive information before it
reaches the brain.
Finally, the presence of D-amino acids in bioactive
peptides from amphibian skin, with either pharmacological (opioid peptides) or antimicrobial activities
(bombinins H) has attracted our attention with the
aim of elucidate the mechanism of conversion of an
L-amino acid encoded by the corresponding DNA
codon into its D-enantiomer in the peptide precursor
during processing. From skin secretions of
Bombinae, we have isolated an enzyme that catalyzes
the isomerization of an L-Ile in position 2 of a model
peptide to D-allo-Ile, possibly by means of a deprotonation/protonation mechanism at the a-carbon of
the second residue (Jilek et al., 2005). From the
enzyme specificity, it appears that in vivo formation of
the D-amino acid is a late reaction in the processing
of the precursors. The amino acid sequence of this
isomerase could be deduced from cloned cDNA and
Mangoni ML, Saugar JM, Dellisanti M, Barra D,
Simmaco M, Rivas L. Temporins, small antimicrobial
peptides with leishmanicidal activity. J Biol Chem,
2005; 280(2):984-90.
Jilek A, Mollay C, Tippelt C, Grassi J, Mignogna G,
Mullegger J, Sander V, Fehrer C, Barra D, Kreil G.
Biosynthesis of a D-amino acid in peptide linkage by
an enzyme from frog skin secretions. Proc Natl Acad
Sci USA, 2005; 102(12):4235-9.
Rosenfeld Y, Barra D, Simmaco M, Shai Y, Mangoni
ML. A synergism between temporins toward Gramnegative bacteria overcomes resistance imposed by
the lipopolysaccharide protective layer. J Biol Chem,
2006; 281(39):28565-74.
60
Interaction of Hepatitis C virus with the immune system:
cryoglobulinemia and lymphomagenesis
Principal investigator: Massimo Fiorilli
Professor of Allergology and Clinical Immunology
Dipartimento di Medicina Clinica
Tel.: (+39) 06 49972031, Fax: (+39) 06 4463877
[email protected]
CD5), “early memory” (medium-sized, IgMhigh
IgDlow CD38 CD27 CD21low CD95 CD5), and
“late memory” (large-sized, IgMlow IgDlow-neg
CD38 CD27low CD21low-neg CD5 CD95) (Figure
1). The B cells expanded in cryoglobulinemia
patients have a “memory” phenotype; this fact,
together with the evidence for intraclonal variation,
suggests that antigenic stimulation by hepatitis C
virus causes the unconstrained expansion of activated VH1-69 B-cells. In some cases, these cells replace
the entire pool of circulating B cells, although the
absolute B cell number remains within normal limits.
Absolute monoclonal VH1-69 B lymphocytosis was
seen in three patients with cryoglobulinemia and
splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here
indicate that the hepatitis C virus-driven clonal
expansion of memory B cells producing a VH1-69
natural Ab escapes control mechanisms and subverts
B cell homeostasis. Genetic alterations may provide a
further growth advantage leading to an overt lymphoproliferative disorder.
The genetic mechanisms of HCV-driven lymphomagenesis are unknown, besides for a suspected role for
t(14;18) involving the Bcl-2 gene. We collected 16
patients with HCV-associated B-cell NHL including
both low grade and high grade histotypes.
Comparative Genomic Hybridization (CGH) in
metaphase and/or microarrays identified genomic
gains and deletions in 11/14 cases tested (78%). 3q
gain and 2q loss were the two most frequent changes
in 3 and 5 cases, respectively. These changes were
mutually exclusive and occurred both as isolated
event or within complex imbalances. Fluorescence in
situ hybridization (FISH) helped to detect an additional cryptic 3q gain and to perform a genomic
restriction of both 3q gain and 2q loss. 3q gain was
detected in 4/6 low grade splenic marginal zone
lymphomas (MZL) and not in other histotypes.
Furthermore, we found one patient with type II
mixed cryoglobulinemia, out of 19 studied, who had
Participants:
Milvia Casato, researcher; Maria Bracci, Deborah
Mancaniello, Marcella Visentini, PhD students; Maurizio
Carbonari, technician.
Collaborations:
Dipartimento di Ematologia, Università di Perugia (Prof. Cristina
Mecucci); Dipartimento di Ematologia, Sapienza-Università di
Roma (Dr. A. Pulsoni); Istituto Dermopatico dell’Immacolata,
Roma (Dr. Giandomenico Russo).
Report of activity
Chronic hepatitis C virus (HCV) infection causes
monoclonal B-cell lymphoproliferative disorders
which include type II mixed cryoglobulinemia (cryoII) and B cell non-Hodgkin’s lymphomas (NHL)
(Fiorilli et al., Rev Clin Exp Hematol 7: 406-423,
2003). The cellular hallmark of cryo-II is the nonneoplastic expansion of a B cell clone producing a
‘natural’ polyreactive IgM autoantibody with
rheumatoid factor activity. In over 80% of the cases
the monoclonal IgM express the same crossidiotype
(CRI) named WA, encoded by the VH1-69 variable
gene. About 15% of patients with cryo-II develop,
over time, a, most commonly derived from WAexpressing B cells. The main goal of our project was
to elucidate i) the characteristics of B-cells clonally
expanded in cryo II, and ii) the genetic anoalies of
HCV-driven lymphomas.
We used anti-idiotype antibodies to track the stages
of differentiation and clonal expansion of VH1-69expressing B-cells in patients with type II mixed
cryoglobulinemia associated with HCV infection.
Owing to their property of producing natural Ab,
these cells are reminiscent of murine B-1 and marginal zone B cells. By immunophenotyping and cell
size analysis, we could define three discrete stages of
differentiation of VH1-69 B-cells: naive (small,
IgMhigh IgDhigh CD38 CD27 CD21high CD95
61
M. Fiorilli - Interaction of Hepatitis C virus with the immune system: cryoglobulinemia and lymphomagenesis
80% of circulating B-cells carrying trisomy 3; nevertheless, this patient had no signs of malignancy
three years after this finding. Together, these observations indicate that trisomy 3q is a marker of clonality rather than of malignancy in HCV-infected
patients. Loss of 2q22.3 was detected in four of five
patients with aggressive MZL or large B-cell lymphoma (LBCL) arising from MZL, as well as in one
case of small lymphocytic lymphoma. The loss of
2q22.3 may highlight a novel pathway of progression from MZL to LBCL driven by HCV. (Matteucci
et al., Distinctive genetic abnormalities in low- and
high-grade lymphomas driven by hepatitis C virus
infection. Leukemia, in press).
Carbonari M, Caprini E, Tedesco T, Mazzetta F,
Tocco V, Casato M, Russo G, Fiorilli M. Hepatitis C
virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type
II cryoglobulinemia: a model of infection-driven
lymphomagenesis. J Immunol, 2005; 174(10):6532-9.
Fig. 1 - A model of differentiation, clonal expansion and transformation of VH1-69+ B cells in HCVassociated type II cryoglobulinemia.
62
Identification of CD28 signals leading to chemokine and survival
gene expression through NF-kB activation
Principal investigator: Enza Piccolella
Professor of Immunology
Tel.: (+39) 06 49917587, Fax: (+39) 06 49917594
[email protected]
Bcl-2 homologues A1/Bfl-1 and Bcl-xL. The most
important costimulatory molecule able to activate
the expression of NF-kB-regulated anti-apoptotic
genes and protect T cells from apoptosis is CD28.
Moreover, NF-kB may counteract apoptosis is some
cell lines by down-regulating Bax expression.
However, the ability of NF-kB to influence expression of both Bcl-2 and Bax is cell-type-specific and
the mechanisms by which NF-kB may regulate Bax
expression are still unknown.
Bax promoter presents a DNA consensus sequence for
binding of the tumour suppressor p53 and p73 proteins. Moreover, the survival of lymphocytes has been
recently connected to the activation of NF-kB and
subsequent inhibition of p73 functions. This is the only
evidence that an interplay between NF-kB and p73
exists and may regulate the fate of lymphocytes.
However, how NF-kB interferes with p73-dependent
activation-induced death in T cells and how p73
induces apoptosis in T cells remain still unknown.
To elucidate these mechanisms we analyzed the
effects of CD28-mediated survival signals on the
expression of pro-apoptotic genes. Bax gene transcription was induced as early as 2 h post irradiation
and CD28 stimulation strongly inhibited bax (Figure
1a, lanes 4 and 5 vs. lanes 2 and 3). Similar results
were obtained at the protein level (Figure 1c, upper
panel). Primary T cells expressed basal proteins levels of p73, which did not change following irradiation (Figure 1c, middle panel). Moreover, data
obtained from radioactive RT-PCR evidenced an
early (2 h after IR) and transient induction of p73a
transcription that was completely inhibited following
CD28 stimulation (Figure 1b, upper panel). By contrast, p53 levels were strongly induced after IR and
were not affected by CD28 (Figure 1c, middle panel).
The analysis of bax gene promoter occupancy by
chromatin immunoprecipitation (ChIP) experiments
revealed that p73, but not p53, was recruited on the
promoter in the early phase of IR-induced transcriptional activation (Figure 1d, upper panel, lanes 1-3).
Participants:
Loretta Tuosto, researcher; Roberta Cianfrocca, PhD
student.
Collaborations:
Istituto Veneto di Medicina Molecolare (VIMM), Padova (Dr.
Antonella Viola); Dipartimento di Dermatologia, Università
“Tor Vergata”, Rome (Dr. Antonio Costanzo); Fondazione
“Andrea Cesalpino”, Policlinico Umberto I, Sapienza-Università di
Roma (Prof. Massimo Levrero)
Report of activity
The nature of the receptors engaged and signals
generated may determine the fate of an antigen-activated T cell. Antigen-dependent activation of T cell
proliferation requires additional signals beyond
those provided by the T cell receptor (TCR). These
signals are delivered by the co-stimulatory receptor
CD28 on T cells. The presentation of antigen to the
TCR (signal 1), in the absence of CD28 (signal 2),
does not lead to an efficient immune response, but
instead may contribute to the activation of apoptosis.
Several studies have shown that the Bcl-2 family of
proteins regulates the death of the majority of activated T cells responding to foreign antigen in vivo.
The ratio of death inducing (Bax) and deathinhibitory members (Bcl-2, Bcl-xL) determines
whether a cell will respond to an apoptotic signal,
mediating the disruption of the mitochondrial membrane and the release of protease activators. The
downregulation of Bcl-2 occurs in vivo in activated
T cells and probably tips the balance in favour of
pro-apoptotic Bcl-2 family members.
NF-kB family play a key role in regulating apoptosis
of T lymphocytes. Several studies have evidenced
that NF-kB activation can suppress cell death pathways through the transcriptional activation of antiapoptotic genes of the Bcl-2 family. In particular,
NF-kB positively regulates the transcription of the
63
E. Piccolella - CD28 signals leading to chemokine and survival gene expression through NF-kB activation
5).Altogether these data indicate that the in vivo
recruitment of RelA/NF-kB on the bax gene promoter in CD28 co-stimulated T lymphocytes is paralleled by a decrease in IR-induced p73 recruitment
and transcriptional activity, thus revealing a novel
mechanism by which NF-kB regulates memory T
cell survival (Cianfrocca et al., RelA/NF-kB recruitment on the Bax gene promoter antagonizes p73dependent apoptosis in costimulated T cells.
Submitted for publication).
The down-regulation of p73 expression by siRNA
duplexes was associated with a significant reduction
(around 40%) of IR-induced apoptosis (Figure 2a).
Moreover, CD28 stimulation significantly inhibited
p73 and RNA polymerase II (pol II) recruitment on
the bax gene promoter (Figure 2c, upper and middle
panels, lanes 4 and 5 vs. 2 and 3). The loss of both
p73 and pol II bindings correlated with the recruitment of RelA on the bax gene promoter in CD28
stimulated cells (Figure 2d, upper panel, lane
Fig. 1 - CD28 interferes with IR-induced apoptosis by both
inducing bcl-xL and inhibiting bax gene expressions. Human
CD4+ T cells were exposed to 5 Gy of X rays-radiation and cultured in the presence (B7) for different times. (a) RT-PCR analysis
of bcl-xL, bax, bim, puma and GAPDH of irradiated T cells. (b)
Radioactive RT-PCR for p73 and GAPDH performed on mRNA
extracted from irradiated T cells. (c) 24 h post-irradiation Bax,
p73, p53, and RelA protein expressions were analyzed by
Western Blotting. (d) Anti-p73 and p53 ChIPs were analyzed by
PCR with bax promoter-specific primers.
Fig. 2 - CD28 promotes the recruitment of RelA to the bax
gene promoter and interfere with p73-induced bax gene transcription. (a,b) Human T cell-blasts were transfected with 5 mg
of control siRNA specific for GFP (siRNA ctr) or 5 mg of p73specific siRNA duplexes (siRNA p73). After 48 h, p73 expression
was evaluated by western blotting (b) and T cells were exposed
to 5 Gy X ray radiation for further 24 h. Apoptosis induction
was measured by Propidium Iodide (PI) staining (a). (c,d)
Human CD4+ T cells were IR and cultured in the presence (B7)
or absence (ctr) of adherent Dap/B7 cells for different times. (c)
Anti-p73, anti-pol II ChIPs were analyzed by PCR with bax promoter-specific primers. (d) Anti-RelA ChIPs were analyzed by
PCR with bax and bcl-xL promoter-specific primers.
64
Receptor-triggered signals leading to activation of NK cell
migration and functions
Principal investigator: Angela Santoni
Professor of Immunology
Tel./Fax: (+39) 06 44340632
[email protected]
are trying to define the cross-talk between the signalling pathways generated by integrin and chemokine
receptors leading to the control of NK cell migration.
Furthermore, we are investigating in depth the signaldriven events regulating cytoskeletal rearrangement
and secretory granule polarization. Finally, we are
attempting to identify the receptor pairs involved in
the control of T cell responses by NK cells.
Participants:
Angela Gismondi, Ricciarda Galandrini, Stefania
Morrone, Gabriella Palmieri, Fabrizio Mainiero, Rossella
Paolini, professors; Marco Cippitelli, Giovanni Bernardini,
researchers; Matteo Barba, Claudia Carlino, Federica
Micucci, Giovanna Peruzzi, Alessandra Porzia, Raffaele
Strippoli, Ilaria Tassi, PhD students; Cristina Cerboni, Rosa
Molfetta, Alessandra Soriani, Helena Stabile, Alessandra
Zingoni, post-doc fellows.
Analysis of the signalling events triggered by
integrins and chemokines and their relevance
for NK cell migration
Collaborations:
Department of Pediatrics, Institute of Molecular Medicine
”Angelo Nocivelli”, University of Brescia, Brescia, Italy (Prof.
Luigi Notarangelo); Department of Microbiology and
Immunology and the Cancer Research Institute, University of
California, San Francisco, CA, USA (Prof. Lewis L. Lanier).
Our attention has been recently focused on the signalling pathways implicated in the control of actin
polymerization, namely WASp/Arp2/3 and
p38MAPK/HSP27 pathways. In collaboration with
Prof. Luigi Notarangelo, University of Brescia, we
have demonstrated that WASp is functionally
involved in integrin-supported chemokine-induced
NK cell migration using cultured NK cells from
Wiskott-Aldrich or X-linked thrombocytopenia
(XLT) patients carrying different mutations of
WASp coding gene. We found that NK cells from
these patients failed to migrate on beta 1 (VCAM-1)
and beta 2 (ICAM-1) integrin ligands in response to
chemokines such as Fractalkine (CX3CL1) or SDF1(CXCL12). We have also information on the molecular mechanisms that control WASp function, in that
triggering of NK cells through chemokine receptors
or integrin engagement rapidly results in Cdc42
activation, and induction of WASp tyrosine phosphorylation. Moreover, we observed that upon integrin ligation or chemokine stimulation, WASp associates with the Src kinase Fyn and the focal adhesion
kinase Pyk-2, a tyrosine kinase that acts as receptorproximal link between integrin and chemokine
receptor signalling and is required for NK cell
migration. These results suggest that upon integrin
or chemokine receptor triggering WASp function
can be regulated by the coordinate action of both
Cdc42 activity and WASp tyrosine phosphorylation.
Report of activity
Natural killer (NK) cells represent one of the main
effectors of the immune surveillance against microbial
infections and tumors, by exhibiting cytotoxic functions and secreting a wide array of cytokines and
chemokines. They also play a major immunoregulatory role and can shape adaptive immune responses by
cognate interaction with dendritic cells and T lymphocytes. NK cells are mostly present in the bloodstream,
spleen and bone marrow, but are also resident in nonlymphoid organs and can rapidly extravasate and infiltrate inflamed and neoplastic tissues. NK cell migration across endothelium and into the tissues is governed by adhesion molecules and chemotactic factors.
The specificity of NK cells mainly depends on the
interplay between inhibitory receptors for MHC class
I molecules, and a wide array of activating receptors
that act in concert to induce efficient elimination of
target cells.
The present study is aimed at identifying the main signal transduction pathways which regulate NK cell
migration, and effector and regulatory functions. We
65
A. Santoni - Receptor-triggered signals leading to activation of NK cell migration and functions
A paper containing these results is in preparation (A.
Gismondi et al.)
In regard to the analysis of p38 MAPK /HSP27
pathway, we have previously identified some of the
downstream p38 effector molecules that are involved
in MIP-1beta (CCL4)-induced NK cell migration,
such as MK2 and its substrate, the heat shock protein
27 (HSP27). This latter is a member of the stressinducible small heat shock protein family that functions as an actin barbed-end capping protein and,
once phosphorylated, affects F-actin generation and
cell migration. There are three phosphorylation sites
on human HSP27 (denoted as Ser-15, Ser-78, and
Ser-82). By using specific antibodies against the Ser78 and Ser-15, our data indicate that NK cell stimulation with FN or CCL4 leads preferentially to phosphorylation of Ser-78. To provide direct demonstration of the functional role of HSP27 in CCL4induced NK cell migration, we produced lentiviral
vectors encoding two HA-tagged HSP27 proteins: a
wild-type HSP27 (HA-HSP27-wt) and a dominant
negative phosphorylation-deficient mutant (HAHSP27-S78, 82A), in which two (Ser-78 and Ser-82)
of the three known phosphorylation sites were
mutated to alanine. Preliminary experiments indicate that an NK cell line transfected with lentivirus
enconding HSP27 mutant exhibits an impaired ability to migrate on FN in response to CXCL12. A
paper containing these results is in preparation (F.
Mainiero et al.).
almost undetectable at day 7. The expression of
ULBP1, ULBP2 and ULBP3 is delayed, with the
majority of donors (48-77%) showing maximal
induction at day 5 after stimulation. NKG2DL
expression was observed mainly on T cells that were
undergone at least one division (as evaluated by
CFSE labeling). We have then explored whether
NKG2DL expression on activated T cells would
result in increased susceptibility to lysis mediated by
autologous NK cells. SEB or PMA plus ionomycinactivated T cells were killed by IL-2 activated NK
cells, and cytotoxicity was dependent on NKG2D
and involved a perforin–dependent mechanism.
We are presently investigating the molecular mechanism(s) involved in the induction of NKG2DL
expression on activated T cells and we are examining whether ATM kinase that is required for
NKG2DL expression in response to genotoxic damage, is involved in the induction of NKG2DL expression on antigen-activated T cells. We found that
ATM is phosphorylated upon TCR engagement, and
that caffeine, a pharmacological inhibitor of
ATM/ATR kinaseses, blocks MICA induction on
activated T cells with a mechanism involving NF-kB.
Experiments are ongoing to demonstrate whether
antigen-dependent T cell proliferation activates the
DNA damage response involving ATM/ATR activation. A manuscript (C. Cerboni et al.) containing
these results has been submitted to Blood and is
under revision.
NK cell regulation of T cell-mediated
responses
Increasing evidence suggest that NK cells can negatively regulate adaptive immune responses, but the
receptor pairs controlling the cognate NK-T cell
interaction are largely unknown. Thus, we have analyzed whether activated T cells could express the ligands for a major NK activating receptor NKG2D,
and become susceptible to NK cell-mediated lysis.
Our results indicate that both CD4+ and CD8+ T
cells express the NKG2D ligands (NKG2DL) MICA,
ULBP1, ULBP2 and ULBP3 upon SEB superantigen
or alloantigen stimulation. MICA is induced in the
majority of the donors tested (90%), and its expression is maximal on day 3, decreases at day 5 and is
Galandrini R, Micucci F, Tassi I, Cifone MG,
Cinque B, Piccoli M, Frati L, Santoni A. Arf6: a new
player in FcgammaRIIIA lymphocyte-mediated
cytotoxicity. Blood, 2005; 106(2):577-83.
Cippitelli M, Fionda C, Di Bona D, Piccoli M, Frati
L, Santoni A. Hyperthermia enhances CD95-ligand
gene expression in T lymphocytes. J Immunol, 2005;
174(1):223-32.
Micucci F, Zingoni A, Piccoli M, Frati L, Santoni A,
Galandrini R. High-efficient lentiviral vector-mediated gene transfer into primary human NK cells. Exp
Hematol, 2006; 34(10):1344-52.
66
Analysis of the role of preTCR-triggered NF-kB in T cell
leukemogenesis: relationship with activated Notch signaling
Principal Investigator: Isabella Screpanti
Professor of General Pathology
Tel. (+39) 06 44700816, Fax (+39) 06 4464129
[email protected]
binding of p50 and p52 and RelB, that suggested the
increased generation of p50p/p50 inhibitory
homodimers and p52/RelB heterodimers, characteristically mediating the alternative pathway of NFkB activation.
Together, these data suggested that Notch3 signaling is able to activate both the canonical and alternative NF-kB pathways and that the relative balance
between the two pathways depends on the presence
or absence of a functional pre-TCR.
Participants:
Maria Pia Felli, Alessandra Vacca, professors; Diana
Bellavia, Antonio F. Campese, researchers; Saula
Checquolo, Claudio Talora, post-doc fellows; Samantha
Cialfi, Giuseppina Di Mario, Rocco Palermo, PhD students;
Massimo Zani, technician.
Collaborations:
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).
Analysis of Notch3 relationship with NF-kB
pathways
Stimuli, that lead to activation, nuclear translocation
and DNA binding of NF-kB heterodimers impact on
IKK complex assembling and activation. In the
canonical NF-kB pathway, the IKKa/IKKb complex
catalyzes phosphorylation, polyubiquitination and
subsequent proteosomic degradation of IKBa. We
demonstrated that IKBa degradation is absent in
thymocytes from N3-IC/pTa-/- mice, which also
display a decrease of IKKa/IKKb complex formation and an increase of IKKa homodimers, characteristic of the alternative pathway.
It has been previously shown that the activation of the
alternative pathway mainly depends on NF-kB-inducing kinase (NIK) that specifically activates IKKa. We
therefore examined the protein expression levels of
NIK and its binding to IKKa in thymocyte extracts
obtained from different mice. Our results showed that
thymocytes from N3-IC tg thymocytes display higher
levels of NIK as well as increased IKKa/NIK complex, when compared to WT and double mutant thymocytes. Thus, unespectedly, in thymocytes from double mutant mice, in which we observed a preferential
activation of an IKKa-dependent alternative pathway,
the protein levels of NIK, as well as its interaction
with IKKa, were decreased.
We thus examined the possibile interaction between
Notch3 and the IKKa kinase and observed that
Notch3 forms a complex with IKKa in the extracts of
Report of activity
We previously observed that thymocytes from Notch3IC (N3-IC) transgenic (tg) mice display overexpression
of pTa/pre-TCR chain and increased NF-kB activity
(Bellavia et al., EMBO J. 19:3337, 2000). Furthermore,
we demonstrated that combined overexpression of
pTa and Notch3 in tg mice, triggers important molecular events, some of which are NF-kB-mediated and
cooperate in sustaining Notch3-induced lymphomagenesis (Felli et al., Oncogene, 24:992, 2005; Talora et al.,
EMBO rep., 4:1067, 2003).
In order to investigate the possible role of pTa in
mediating NF-kB constitutive activity, we monitored
the ability of Notch3 to activate NF-kB in the
absence of pTa, by utilizing previously established
N3-IC/pTa-/- double mutant mice (Bellavia et al.,
PNAS 99:3788, 2002). We observed that NF-kB
DNA binding activity is significantly increased in
N3-IC tg, but, interestingly, it is still higher in N3IC/pTa-/- double mutant, with respect to wild type
(WT) mice, suggesting that pre-TCR is dispensable
for Notch3 induced NF-kB activation. However, we
also observed that while in N3-IC thymocytes most
of DNA-bound NF-kB complexes are p50/p65, in
double mutant thymocytes it was evident a decreased
DNA-binding of p65, accompanied by an increased
67
I. Screpanti - The role of preTCR-triggered NF-kB in T cell leukemogenesis: relationship with Notch signaling
the levels of cyclin D1 completely returned to the
wild type levels, the levels of IL7Ra were further
increased and the levels of Bcl2A1 remained at an
intermediate level in thymocytes from N3-IC/pTa/- double mutant mice.
Experiments of Chromatin ImmunoPrecipitation
demonstrated that the recruitment of p65, responsible for the transcriptional activity of canonical NFkB complex, to the promoters of those different
genes is readily detected in chromatin extracts of
N3-IC tg thymocytes. In contrast, in the absence of
pTa, p65 is never recruited to the different gene promoters and p52 appears to be the main NF-kB transcriptional complex component recruited to the promoter of different genes. Moreover, while RelB is
also recruited on the promoter of IL7Ra and Bcl2A1
genes, justifying their increase, it is not recruited
onto the cyclin D1 promoter. This suggests an
increased formation of inhibitory p52 homodimers
in cyclin D1 promoter, justifying the lack of increase
of cyclin D1 mRNA levels.
Overall, our data suggest that a finely tuned interplay between Notch3 and pre-TCR pathways converges on the regulation of NF-kB activity, leading
to differential NF-kB subunit dimerization that regulates distinct gene clusters involved in either cell
differentiation or proliferation/leukemogenesis.
thymocytes obtained from both N3-IC and N3IC/pTa-/- mice, suggesting that this direct contact
is pre-TCR independent .
Altogether our observations suggest that Notch3 is
able to activate canonical and alternative NF-kB
pathways by regulating the assembling and activation of different IKK complexes. In addition and
more importantly, Notch3 appears to be able to
directly trigger the alternative pathway, independently on NIK.
Gene expression profiles depend on
differential activation of canonical and
alternative NF-kB pathways
It has been recently reported that the two NF-kB
pathways differentially activate a number of genes.
The canonical pathway has been suggested to be
mainly involved in the transcriptional control of
genes involved in lymphoid cell proliferation and
protection from apoptosis, while the alternative pathway plays a central role in the expression of genes
involved in development of lymphoid organs. We
previously reported that enforced expression of
Notch3 is effective in inducing T cell leukemia only
in the presence of a functional pre-TCR; whereas, in
the absence of pTa, Notch3 is able to rescue the
blockage of the pre-TCR check-point by forcing
immature DN thymocytes to progress toward a more
differentiated phenotype, without restoring total
thymocyte yield (Bellavia et al., PNAS 99, 3788,
2002). We thus hypothesized that the Notch3induced differential activation of the two NF-kB
pathways may play a specific role in either thymocyte
proliferation/leukemogenesis or differentiation.
We compared the expression levels of genes known
to be responsive to NF-kB and differentially involved
in these processes in thymocytes obtained from wild
type, N3-IC tg and N3-IC/pTa-/- double mutant
mice. We considered cyclin D1, known for its
growth-promoting function in several cancers, Bcl2A1, an antiapoptotic gene previously described as a
pre-TCR-dependent and Notch3-induced regulator
of thymocyte survival and IL7Ra, known to play a
critical role in early thymocyte development.
All of these genes are upregulated in thymocytes of
N3-IC tg mice, while their expression was variably
influenced by the absence of pre-TCR. Indeed, while
Felli MP, Vacca A, Calce A, Bellavia D, Campese AF,
Grillo R, Di Giovine M, Checquolo S, Talora C,
Palermo R, Di Mario G, Frati L, Gulino A, Screpanti
I. PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.
Oncogene, 2005; 24(6):992-1000.
Talora C, Cialfi S, Oliviero C, Palermo R, Pascucci
M, Frati L, Vacca A, Gulino A, Screpanti I. Cross
talk among Notch3, pre-TCR, and Tal1 in T-cell
development and leukemogenesis. Blood, 2006;
107(8):3313-20. Epub 2005 Dec 20.
Vacca A, Felli MP, Palermo R, Di Mario G, Calce A,
Di Giovine M, Frati L, Gulino A, Screpanti I. Notch3
and pre-TCR interaction unveils distinct NF-kappaB
pathways in T-cell development and leukemia. EMBO
J, 2006; 25(5):1000-8. Epub 2006 Feb 23.
68
Molecular mechanisms regulating the immune response in the
testis: a balance between immune privilege, tissue homeostasis
and autoimmune disorders
Principal investigator: Elio Ziparo
Professor of Embryology
Dipartimento di Istologia ed Embriologia Medica
Tel.: (+39) 06 49766586, Fax: (+39) 0649766340
[email protected]
Recently, some membrane receptors have been considered to be responsible for the immune-privileged
status in various systems. Several results have
implied that the PD-1 molecule, expressed by activated T and B peripheral lymphocytes, might be
involved in the negative regulation of immune
responses. Two ligands for PD-1, PD-L1 and PD-L2,
have been identified almost simultaneously by different groups. PD-L1 is costitutively expressed in nonlymphoid organs and parenchymal tissues, such as
heart and placenta, whereas PD-L2 expression is
restricted to macrophages and DCs. The broad
expression of PD-L1 within non-lymphoid tissues
suggests that this molecule may serve to regulate
self-reactive T or B cell responses in peripheral tissues and/or may serve to regulate inflammatory
responses at these sites.
We investigated the possibility of an involvement of
PD-1/PD-Ligand system in maintaining the
immune privilege in the testis.
The expression of PD-L1 protein in mouse total
testis and seminiferous tubules has been studied. By
Western blot analysis, we could not detect any constitutive expression of PD-L1 neither in total testis
lysates nor in freshly prepared seminiferous tubules.
Since it is established that IFN-g up-regulates PDL1 expression in many cell types (endothelial cells,
myoblasts and keratinocytes), we treated seminiferous tubules with increasing doses of IFN-g for 24
hours. PD-L1 was strongly induced by IFN-g treatment in a dose dependent manner.
Being acquired that Sertoli cells play an important
role in immune protection of germ cells and are
known to show antigen presenting cell characteristics (such as phagocytic activity and interleukin production), we proposed that they could respond to
IFN-g treatment and contribute to the increment of
PD-L1 expression in seminiferous tubules. To test
this hypothesis, we treated Sertoli cell cultures with
various doses of IFN-g for 24 hours and analyzed
PD-L1 expression by flow cytometry. Our results
Participants:
Antonio Filippini, professor; Anna Riccioli, researcher;
Claudia Giampietri, Donatella Starace, post-doc fellows,
Valentina Dal Secco, Alessio Paone, PhD students; Roberta
Galli, graduate student; Fabrizio Padula, Simonetta
Petrungaro, technicians
Collaborations:
Dipartimento di Medicina Sperimentale, Università di L’Aquila
(Prof. Paola De Cesaris).
Report of activity
The classic immunologically privileged districts
include the anterior chamber of the eye, the brain,
the pregnant uterus and the testis. In the testis, during spermatogenesis, a myriad of surface and intracellular proteins that are considered as “non self ” by
the immune system is expressed; yet, these new autoantigens are tolerated by the testis. The immunogenicity of proteins expressed by germ cells is not
diminished, as shown by their ability to elicit strong
autoimmune reactions when injected elsewhere in
the body. It is then the testis itself that confers protection. In spite of its immunologically privileged
status, the testis is not isolated from the immune system and recent reports confirmed that CD8+ memory T cells survey it effectively though their response
is hampered by immunosuppressive mechanisms
present on site. Peripheral tolerance is based on the
observation that self-reactive T cells that migrate
into peripheral tissues appear to be held in check by
immunemodulatory controls. Multiple components
of the peripheral tolerance exist to suppress and
subvert adaptive immunity and promote tolerance,
including low-dose exposure of T cells to their antigen, accompanied by inadequate or inappropriate signals from the antigen-presenting cells; local production of immunosuppressive molecules and cytokines
as TGF-b and activin.
69
E. Ziparo - Molecular mechanisms regulating the immune response in the testis
strong stimulus for T cell proliferation. Of note,
CD4+ T cells never undergone division when co-coltured with Sertoli, even in the presence of ConA
with and without anti-PD-L1 blocking antibody. On
the contrary, CD8+ T cells proliferated in co-cultures with Sertoli cells, although their proliferation
was significantly increased by the addition of the
anti-PD-L1 blocking antibody. These data, confirming the Sertoli cell ability to inhibit T cells proliferation, demonstrate that PD-L1 contributes to the
inhibition of CD8+ T lymphocyte proliferation.
We believe that no single immunemodulatory mechanism accounts for the establishment and maintenance of tolerance towards germ cells within the
testis in vivo. Our results have firstly demonstrated
that Sertoli cells are capable of dampening CD8+ T
cells proliferation by delivering an inhibitory co-signal through the surface PD-L1.
show that PD-L1 protein is induced in a dose and
time dependent manner with a maximum expression
at 72 hours. As shown by Northern blotting, IFN-g
stimulation of Sertoli cells increases PD-L1 mRNA
levels as early as 4 h of IFN-g treatment and
remained at the same level until 24 hours.
Although Sertoli cells are known to express MHC
class I and therefore to provide Signal 1 to CD8+ T
cells, instead MHC class II is absent on these cells.
Since TCR engagement is a requirement for the
transmission of specific inhibitory signals through
PD-L1, we wanted to verify the ability of Sertoli cells
to express MHC-II after IFN-g treatment. We found
that MHC class II was absent on untreated Sertoli
cells but, interestingly, was strongly induced in the
presence of IFN-g simultaneously to PD-L1.
In addition, we verified Sertoli cell ability to provide
positive co-stimulation through CD28 family members. The expression of CD80 (B7-1), CD86 (B7-2)
and CD40 was assayed by flow cytometric analysis.
No positive co-stimulatory molecules were detectable
on Sertoli cells either in control and IFN-g treated
cultures.
To evaluate the functional role of PD-L1 expressed
on Sertoli cells in establishing immune tolerance, we
tested the effect of blockade of PD-L1 on T cell proliferation. Sertoli cells were pre-incubated for 48
hours with IFN-g to induce PD-L1 and MHC class
II expression. Subsequently, cells were cultured, at
various E:T ratio, with freshly isolated CFSE-labeled
syngenic T cells in the presence of neutralizing antiPD-L1 mAb or respective isotype control Ab. On day
3 and 5 of co-culture, proliferation of T cells was
assessed by measurement of CFSE dilution. Both
CD8+ and CD4+ T lymphocytes showed no proliferative response either in the presence or absence of
neutralizing B7-H1 antibody. Therefore, we evaluated inhibiting potential of PD-L1 for T cells activation by adding to the culture Concanavalina A, a
Riccioli A, Dal Secco V, De Cesaris P, Starace D,
Gandini L, Lenzi A, Dondero F, Padula F, Filippini A,
Ziparo E. Presence of membrane and soluble forms
of Fas ligand and of matrilysin (MMP-7) activity in
normal and abnormal human semen. Hum Reprod,
2005; 20(10):2814-20. Epub 2005 Jun 24.
Romano F, Chiarenza C, Palombi F, Filippini A,
Padula F, Ziparo E, De Cesaris P. Platelet-derived
growth factor-BB-induced hypertrophy of peritubular smooth muscle cells is mediated by activation of
p38 MAP-kinase and of Rho-kinase. J Cell Physiol,
2006; 207(1):123-31.
Riccioli A, Starace D, Galli R, Fuso A, Scarpa S,
Palombi F, De Cesaris P, Ziparo E, Filippini A.
Sertoli cells initiate testicular innate immune
responses through TLR activation. J Immunol, 2006;
177(10):7122-30.
70
AREA
5
New
antimicrobial
and antiviral
agents
New antimicrobial and antiviral agents - AREA 5
New perspectives in the design for anti-HIV-1 agents targeted to
reverse transcriptase (RT) and integrase (IN): synthesis and in
vitro evaluation
Principal investigator: Marino Artico
Professor of Medicinal Chemistry
Dipartimento di Studi Farmaceutici
Tel. (+39) 064462731, Fax: (+39) 0649913627
[email protected]
groups (at C5 and benzylic positions) is crucial for
high antiviral action. Prompted by computational
evidences and with the aim to establish further SARs
on amino DABOs, we also prepared a series of novel
N,N-disubstituted 2-amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F2-N,NDABOs). These derivatives showed a high activity
against some relevant (K103N, Y181C, Y188L,
IRLL98) NNRTIs-resistant mutants, and behaved as
slow-binding tight-binding inhibitors of HIV-1 RT.
In fact, besides low association rates (kon) values,
these molecules are endowed with very low dissociation rates (koff) values, and show a characteristic
increase in potency (i.e. lower Ki values) upon preincubation in the presence of the enzyme alone (Mai et
al., 2005).
Participants:
Roberta Costi, Roberto Di Santo, Antonello Mai, Romano
Silvestri, professors; Rino Ragno, researcher; Gabriella De
Martino, Giuseppe La Regina; post-doc fellow; Gaetano
Miele, Francesco Piscitelli, PhDstudents.
Collaborators:
Università di Roma “Tor Vergata” (Profs. Alberto Bergamini,
Chiara Ciaprini, Anna Sinistro); CNR Pavia (Profs. Emanuele
Crespan, Giovanni Maga); Università di Napoli “Federico II”
(Profs. Antonio Lavecchia, Ettore Novellino).
Report of activity
Human immunodeficiency virus (HIV) is the etiological cause of acquired immunodeficiency syndrome
(AIDS), a pandemic disease that affects almost 40
million people. The search here summarized aimed at
continuing our optimization process of the HIV-1
RT and IN inhibitor classes DABO, IAS and ADKs.
Goal of this research was the discovery of new compounds endowed with high activity against wild type
(WT) HIV-1 and drug resistant mutant strains, and
reduced cytotoxicity.
Indolyl aryl sulfones. N-(2-hydroxyethyl)carboxyamide and
N-(2-hydroxyethyl)carboxy
hydrazide
derivatives.
Predictive 3-D QSAR models using the GRID and
GOLPE programs combination were obtained using a
receptor based alignment by means of docking IASs
into the non-nucleoside binding site (NNBS) of RT.
The derived 3-D QSAR models showed conventional
correlation (r2) and cross-validated (q2) coefficients
values ranging from 0.79 to 0.93 and from 0.59 to 0.84,
respectively. All described models were validated by an
external test set compiled from previously reported
pyrryl aryl sulfones. The most predictive 3-D QSAR
model was then used to predict the activity of novel
untested IASs. The synthesis of six designed derivatives (prediction set) allowed to disclose new IASs
endowed with high anti-HIV-1 activities.
RT Inhibitors
5-Alkyl-2-alkylamino-6-(2,6-difluorophenylalkyl)-3,4dihydropyrimidin-4(3H)-ones
(F2-NH-DABOs).
Pursuing our dihydro-alkoxy-benzyl-oxopyrimidine
(DABO) family optimization effort, we developed
some 2-alkylamino-6-[1-(2,6-difluorophenyl)alkyl]3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F2-NHDABOs) characterized by a further anchor point
(hydrogen bond with C2–NH function) into the nonnucleoside binding site (NNBS), and highly active
against both wild type and the Y181C HIV-1 strains.
Against the Y181C mutant strain, a properly sized
C2–NH side chain and the presence of two methyl
Indolyl aryl sulfones. Novel derivatives of the indole-2carboxamide. An updated 3-D QSAR model was
obtained by increasing the training set composition
from 70 to 101 molecules. With the aim to both validate the new 3-D QSAR model and design new antiHIV-1 compounds, we engineered a prediction set of
eight IASs by simply modifying the substituents at
73
M. Artico - New perspectives in the design for anti-HIV-1 targeted agents
the 2-hydrazide position. The combination of the
structure-based alignment obtained by docking and
the 3-D QSAR model quantitative estimation of the
pEC50 values, proved to be highly effective and
allowed the disclosure of new potent IAS derivatives.
Against the HIV-1 WT strain, some new compounds
were exceptionally potent in the sub-nanomolar
range of concentration in both MT-4 and C8166
cells. These compounds also showed excellent
inhibitory activity against the HIV-1 WT (IIIB) and
the primary isolates HIV-112 and HIV-1-AB1 in
limphocytes and against the HIV-1 WT (IIIBBa-L)
in macrophages. These compounds were also found
more active than efavirenz against the viral RT carrying the K103N mutation (Ragno et al., 2006).
IN Inhibitors
Quinolinonyl diketo acids. Recently, compounds containing a distinct dioxobutanoic acid moiety, named aryl
diketo acids (ADKs) have been identified as potent and
specific inhibitors of HIV-1 multiplication targeting
the integration process. We designed some ADKs
based on quinolinone skeleton. Among the tested
derivatives the bifunctional compounds were potent IN
inhibitors for both steps of the reaction (3’-processing
and strand transfer) and exhibited both high antiviral
activity against HIV-1 infected cells and low cytotoxicity. Molecular modeling studies provide a plausible
mechanism of action, which is consistent with ligand
SARs and enzyme photo-crosslinking experiments.
Further we described an improved and standardized
assay aimed at evaluating IN inhibitors taking
advantage of the transcriptional activity of E-DNA
produced by HIV-derived vectors in the absence of
replication competent virus. In this context, the use
of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral vector infectivity thus making it a safe and cost-effective
assay for evaluating novel IN inhibitors
TIBO analogs. The synthesis and biological evaluation of sulfone analogo of TIBO have been undertaken. The most interesting results were obtained for
truncated derivatives within this series. The most
potent compound in cell-based assays showed EC50
= 0.07 mM, CC50 > 200 mM, SI > 2857 and was 2
times less potent than TIBO prototype R82913, but
more selective. 3-D QSAR studies and docking simulations were developed on TIBO sulfone derivatives
in order to rationalize their anti-HIV-1 potencies
and, in perspective, to predict the activity of novel
untested sulfone derivatives.
Mai A, Artico M, Ragno R, Sbardella G, Massa S,
Musiu C, Mura M, Marturana F, Cadeddu A, Maga G,
La Colla P. 5-Alkyl-2-alkylamino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-one s, a new series
of potent, broad-spectrum non-nucleoside reverse
transcriptase inhibitors belonging to the DABO family.
Bioorg Med Chem, 2005; 13(6):2065-77.
Arylthio-isopropyl-pyridinylmethylpyrrolemethanol.
New arylthio-isopropyl-pyridinylmethyl-pyrrole
methanol (AThP) derivatives related to capravirine
(S-1153) were synthesized and tested for their ability to block the replication cycle of HIV-1 in infected cells. The newly synthesized AThPs were found
active in the range 0.007-53 mM. Even if these compounds were generally less potent than S-1153, nevertheless, their SIs were in some cases comparable to
that of the reference drug. In fact, the cytotoxicity
of AThPs was generally lower than that of S-1153.
A selected number of AThPs showed activity
against the Tyr181Ile, Lys103Asn, and Leu100Ile
mutated rRT. Docking calculations were also performed to investigate the binding mode of AThPs
into the non-nucleoside binding site of HIV-1 RT
and to rationalize some structure-activity relationships and resistance data (Di Santo et al., 2006).
Ragno R, Coluccia A, La Regina G, De Martino G,
Piscitelli F, Lavecchia A, Novellino E, Bergamini A,
Ciaprini C, Sinistro A, Maga G, Crespan E, Artico M,
Silvestri R. Design, molecular modeling, synthesis, and
anti-HIV-1 activity of new indolyl aryl sulfones. Novel
derivatives of the indole-2-carboxamide. J Med Chem,
2006; 49(11):3172-84.
Di Santo R, Costi R, Roux A, Artico M, Lavecchia A,
Marinelli L, Novellino E, Palmisano L, Andreotti M,
Amici R, Galluzzo CM, Nencioni L, Palamara AT,
Pommier Y, Marchand C. Novel bifunctional quinolonyl
diketo acid derivatives as HIV-1 integrase inhibitors:
design, synthesis, biological activities, and mechanism
of action. J Med Chem, 2006; 49(6):1939-45.
74
New antimicrobial and antiviral agents - AREA 5
Synthesis and biological evaluation of aroyl-pyrrolyl-hydroxyamides as new histone deacetylase inhibitors
Principal investigator: Antonello Mai
Professor on Medicinal Chemistry
Dipartimento di Studi Farmaceutici
Tel.: (+39) 06 49913392, Fax: (+39) 06 491491
[email protected]
HDAC inhibitory activity) was 2-fold less potent
than SAHA used as reference (Figure 1). Finally,
both 1c and 1f failed in sensitizing primary tumor
cells, resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), to TRAIL-induced
apoptosis.
Participants:
Enrico Morera, Giorgio Ortar, Mario PaglialungaParadisi, professors; Rino Ragno, researcher; Ilaria Cerbara,
Riccardo Pezzi, Dante Rotili, Sergio Valenti, PhD students;
Silvia Simeoni, graduate student.
Collaborations:
Aryloxopropenyl-Cinnamyl-N-hydroxyamides.
To
explore the effect on the anti-HDAC activity of the
replacement of pyrrole with benzene in the (aryloxopropenylpyrrolyl) hydroxyamide series, novel aryloxopropenyl-cinnamyl-hydroxamates 2 were prepared and tested both against maize HD1-B and –A
enzymes, and in K562 cells as hyperacetylating
agents. Such compounds turned out to be highly
more potent (up to three magnitude orders) than the
pyrrole counterparts, but lacking in selectivity
towards the class II HDACs. Low concentrations of
2 are able to induce proliferation block and/or cell
death in a variety of tumor cell lines. In preliminary
in vivo studies, cutis from normal mice or from mice
exposed to DMBA-TPA treatment was analyzed by
immuno-histochemistry or stained with hematoxylin
and eosin, respectively. The tested compounds
strongly induce histone acetylation in normal animals and are able to reduce the tumor size in treated
animals. Moreover, the tested compounds completely
block the further number increase of chemicallyinduced papillomas. Compounds 2 have been patented by DAC srl in 2005 (PCT/EP2005/054949).
Università di Siena (Prof. Silvio Massa); University of Innsbruck
(Prof. Gerald Brosch); University of Innsbruck (Prof. Peter
Loidl, Dr. Florian Jesacher); Sapienza-Università di Roma
(Prof. Anna Tramontano); Università Cattolica del Sacro CuoreRoma (Prof. Roberto Scatena, Dr. Patrizia Bottoni); II
Università degli Studi di Napoli (Dr. Lucia Altucci); Dipartimento
Oncologia Sperimentale, IEO, Milano (Prof. Pier Giuseppe
Pelicci, Dr. Saverio Minucci).
Report of activity
Design, synthesis and biological validation of
unselective and class I-, class II-, and class IIIselective HDACi
Aryloxopropenyl-Pyrrolyl-Hydroxy-Amides. Class IISelective HDACi. Chemical manipulations performed
on aroyl-pyrrolyl-hydroxy-amides (APHAs) led to
(aryloxopropenyl) pyrrolyl hydroxamates 1a-w.
From inhibition studies against maize HDACs some
benzene meta-substituted compounds emerged as
highly class II-selective HDACi, the most selective
being the 3-chloro- and 3-fluoro-substituted compounds 1c (SI = 71.4) and 1f (SI = 176.4). The
replacement of benzene with 1-naphthyl ring afforded 1s, highly active against the class II homologue
HD1-A (IC50 = 10 nM) but less class II-selective
than 1c,f. When tested against human HDAC1 and
HDAC4, 1f showed no HDAC1inhibitory activity,
while was able to inhibit HDAC4. Moreover, in
human U937 acute myeloid leukemia cells 1f did not
produce any effect on apoptosis, granulocytic differentiation, and cell cycle, whereas 1s (with class I
Uracil-based Hydroxyamides (UBHAs). Starting from
the pharmacophore model for HDACi design, a novel
series of hydroxamates bearing a uracil moiety as
connecting unit (CU) was prepared and tested.
Almost all compounds exhibited HDAC inhibiting
activity at low nanomolar concentrations, the Nhydroxy-6-(3,4-dihydro-4-oxo-6-benzyl- and -6phenyl-2-pyrimidinylthio) hexanamides 3d and 3l
(Figure 2) being more potent than SAHA in enzy-
75
A. Mai - Synthesis and biological evaluation of aroyl-pyrrolyl-hydroxy-amides as new histone deacetylase inhibitors
Fig. 1 - Effects of 1f and 1s on human leukaemia U937 cells.
Fig. 2 - Novel uracil-based HDACi.
matic assays. Such compounds caused also hyperacetylation in NIH3T3 cell core histones, and were
endowed with interesting antiproliferative and
cytodifferentiating effects in human leukemia HL-60
cells.
against human SIRT1 and SIRT2 enzymes. In yeast
in vivo assay, these two small molecules were as
potent as sirtinol. Compounds lacking the 2-hydroxy
group at the naphthalene moiety or bearing several
modifications at the benzene 2’-position of the aniline portion (carbethoxy, carboxy, and cyano), were
1.3 to 13 times less potent than sirtinol, whereas the
2’-carboxamido analogue was totally inactive. Both
(R)- and (S)-sirtinol enantiomers had similar
inhibitory effects on the yeast and human enzymes
demonstrating no enantioselective inhibition.
Sirtinol analogues as class III (sirtuin) HDACi. The sirtuin family of enzymes are named after their founding member, the Sir2 protein of Saccharomyces cerevisiae. In yeast, Sir2 is critical for transcriptional
silencing at three specific loci: the telomeres, ribosomal DNA, and the silent mating type loci. Sir2-like
proteins also deacetylate non-histone proteins,
including the tumor suppressor p53. The human Sir2
ortholog, SIRT1, has been implicated in a variety of
important disease-related processes including silencing of p53, inflammatory responses, cell defense and
survival, and fatty acid metabolism, and finding molecules that modulate these enzymes is considered a
possible route for disease treatment. 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-[(2hydroxy-1-naphthalenylmethylene)amino]-N-(1phenylethyl) benzamide (ie, meta- and para-sirtinol)
were from 2- to 10-fold more potent than sirtinol
Mai A, Massa S, Rotili D, Cerbara I, Valente S,
Pezzi R, Simeoni S, Ragno R. Histone deacetylation
in epigenetics: an attractive target for anticancer
therapy. Med Res Rev, 2005; 25(3):261-309.
Inoue S, Mai A, Dyer MJ, Cohen GM. Inhibition of
histone deacetylase class I but not class II is critical
for the sensitization of leukemic cells to tumor
necrosis factor-related apoptosis-inducing ligandinduced apoptosis. Cancer Res, 2006; 66(13):6785-92.
Mai A, Massa S, Rotili D, Simeoni S, Ragno R,
Botta G, Nebbioso A, Miceli M, Altucci L, Brosch G.
Synthesis and biological properties of novel, uracilcontaining histone deacetylase inhibitors. J Med
Chem, 2006; 49(20):6046-56.
76
AREA
6
Biology
of
malaria
Biology of malaria - AREA 6
Interactions between semiochemicals and olfactory-mediated
behaviour of Afrotropical malaria vectors for the development of
new monitoring and control tools
Principal investigator: Mario Coluzzi
Professor of Parasitology
Dipartimento di Scienze di Sanità Pubblica
Tel.: (+39) 06 4455780, Fax: (+39) 06 49914653
[email protected]
the comparative investigation of olfactory-mediated
behaviour is particularly relevant because, as other
insects, mosquitoes use olfactory cues to perform
important behaviours related to several traits of
their natural history, including, among others, finding blood sources and oviposition sites. The behavioural response of anopheline mosquitoes to the
odour of alternative hosts (human vs. monkey)
arranged in a choice set-up using odour-baited entry
traps (OBETs) was assessed in West African endemic malaria zones by field experiments where procedures analogous to those adopted in olfactometer
laboratory tests were followed. All major human
malaria vectors including An. gambiae s.l., An. funestus, and An. nili clearly expressed a preference for
human odour, with >90% of mosquitoes caught in
the human-baited trap, in accordance with the
hypothesis that the strongly anthropophilic feeding
preferences of An. gambiae did not evolve from an
ancestral association with non-human primates.
Similar studies carried out in three villages of
Madagascar indicated that An. funestus ‘preferred’
human to calf odour and molecular identification of
An. gambiae s.l. samples revealed the exclusive presence of An. arabiensis in samples from two villages
and a marked predominance of An. gambiae s.s. in the
third one, in accordance to what known of the distribution of the two species on the island.
Hosts providing blood for ovarian development are
found by responsive females through a complex
sequence of responses mediated by the chemical properties of the host, including its output of carbon dioxide, sweat and skin volatiles. Electrophysiological studies on the antennae of An. gambiae s.s. showed selective
sensitivity to some sweat compounds, with a threshold
(10±6 g) comparable to that of known olfactory stimulants and the synthetic mosquito repellent DEET.
Wind-tunnel bioassays have provided evidence that
human-specific compounds affect the behaviour of
highly anthropophilic An. gambiae s.l., seemingly
inhibiting the ‘upwind flight’ response to known long-
Participants:
Vincenzo Petrarca, Lucilla Seganti, professors; Maria Pia
Conte, Alessandra della Torre, researchers; Carlo
Costantini, post-doc fellow; Sabina Bietolini, Fabio
Candura, Beniamino Caputo, Marco Pombi, Federica
Santolamazza, PhD students
Collaborations:
Centre National de Recherche et Formation sur le Paludisme,
Ouagadougou, Burkina Faso (Prof. N’Falé Sagnon, Dr. W. M.
Guelbeogo); Institut Pasteur, Dakar, Senegal (Dr. Mawlouth
Diallo); Institut Pasteur, Antananarivo, Madagascar (Dr JeanBernard Duchemin, Dr Vincent Robert); Chemical Ecology
Unit, IACR Rothamsted, UK (Prof. John Pickett); Natural
Resources Institute, University of Greenwich, UK (Dr. Gabriella
Gibson, Dr. Steve Torr)
Report of activity
Sibling species belonging to the Anopheles gambiae
sensu lato (s.l.) complex and to the An. funestus complex are the most important vectors of human
malaria in sub-Saharan Africa, which alone accounts
for about 90% of the malaria problem in the world.
These species exploit a remarkable diversity of larval habitats and hosts: some breed in freshwater and
others in brackish or mineral water; analogously,
some are highly anthropophilic, i.e. they take their
blood meals almost exclusively off humans, or are
otherwise almost totally zoophilic. Such differences
in behavioural and ecological traits largely account
for their contrasting role in malaria transmission.
The nominal species, An. gambiae sensu stricto (s.s.) is
the most synanthropic both at the larval and adult
stage, hence its higher vectorial capacity and major
role in malaria transmission. Knowledge of the
behavioural mechanisms and genes involved in the
quest of these mosquitoes towards appropriate hosts
or breeding sites is instrumental to the development
of new vector control tools. Within this framework,
79
M. Coluzzi - Semiochemicals and behaviour of Afrotropical malaria vectors for new monitoring and control tools
range attractants, and may serve either to ‘mask’ the
attractants present or, more probably, to ‘arrest’
upwind flight when mosquitoes arrive at a host under
natural conditions.
Habitat dispersion field surveys, associated with
cytogenetical and molecular laboratory investigations, of sympatric larval An. gambiae s.l. (An. arabiensis, An. gambiae s.s. molecular form M and form S),
present in an arid savanna area of Burkina Faso
revealed the existence of marked ecological niche
partitioning phenomena among the two molecular
forms of An. gambiae s.s. In particular, the M-form
prevailed (70±3 %) in habitats of permanent or semipermanent nature originating by the emergence of
the water table (filtration sites), whereas the S-form
prevailed (58±2 %) in ephemeral puddles and pools
due to accumulation of rain water over impermeable
soil (residual sites). As expected from the differential
distribution of the An. gambiae s.s. M-form larvae,
gravid females scattered their egg load over a higher
number of sites when the residual (1.7±0.04) rather
than the filtration (1.3±0.04) flora was present as a
substrate. This behaviour may represent an adaptive
strategy to spread the risk of losing the offspring in
unpredictable ephemeral habitats, and provide evidence that semiochemicals released by the bacterial
flora of larval habitats do indeed provide olfactory
cues of an adaptive nature.
The correct determination of the vector age (particularly in the case of the An. gambiae complex of sibling species, the major Afrotropical malaria vector)
provides valuable information in evaluation of the
effectiveness of vector control strategies and in
malaria epidemiology. We focused our efforts to validate the efficacy of gas chromatography-mass spectrometry on cuticular hydrocarbon (CHC) composition of single specimens field collected in endemic
countries or reared in seminatural conditions. Forty-
eight cuticular hydrocarbons (CHCs) were characterized and identified as 14 n-alkanes, 16 monomethyl
alkanes, 13 dimethyl alkanes, 5 alkenes, with mainchain lengths ranging from C17 to C47, and the
results are consistent with those from other
Culicidae species. Qualitative differences were not
observed between laboratory pools of three females
and males, between different age-groups (0-16 days)
and between single field specimens, whereas quantitative differences in CHC profiles were observed.
Differences between sexes were more marked in individuals aged 0-2 days than in older ones. Both sexes
undergo strong CHC profile changes with age, and
individuals aged 0-2 days differ remarkably from the
older ones. The possibility of exploiting these
changes for estimating the age of mosquito was
explored and allowed to assign more than 85% of
females and 75% of males to the correct age-group.
These results show that the method is promising,
and may improve the rationale in the design of alternative control strategies based on basic studies on
vector biology.
Ayala FJ, Coluzzi M. Chromosome speciation:
humans, Drosophila, and mosquitoes. Proc Natl Acad
Sci USA. 2005, 102(S1):6535-42.
Slotman M, Della Torre A, Powell JR. Female
sterility in hybrids between Anopheles gambiae and A.
arabiensis, and the causes of Haldane’s rule. Evolution
Int J Org Evolution. 2005, 59:1016-26.
Slotman MA, Della Torre A, Calzetta M, Powell
JR. Differential introgression of chromsomal
regions between Anopheles gambiae and An. arabiensis.
Am J Trop Med Hyg. 2005, 73:326-35.
80
Biology of malaria - AREA 6
Immunogenetic analysis of the 5q31-q33 chromosomal region in
West African ethnic groups differing in the susceptibility and
immune response to Plasmodium falciparum malaria
Principal investigator: David Modiano
Professor of Parasitology
Dipartimento di Scienze di Sanità Pubblica
Tel.: (+39) 06 4455780, 06 49914933, Fax: (+39) 06 49914653
[email protected]
the balance between Th1 and Th2-type T cell
responses. In conclusion, IRF-1 is involved in multiple stages of the immune response and as a consequence is likely to affect susceptibility to infectious
diseases. A role for this transcriptional regulator in
both malaria mortality and parasite density has been
suggested by studies of Plasmodium berghei infection
conducted in IRF-1 knockout mice but such a role
has not been investigated in humans so far.
Interestingly, the IRF-1 locus lies in the 5q31 human
genome region, which contains a cluster of genes
encoding immunological molecules such as cytokines
and growth factors, and has been therefore thought
to play a role in response to parasitic diseases. This
has been investigated by linkage analysis studies that
demonstrated the involvement of this region in the
control of Plasmodium falciparum blood parasite
densities as well as of intensity of Schistosoma mansoni infection, but the P. falciparum infection level
locus (Pfil) has not been identified yet.
In order to gain insights into molecular mechanisms
of protective immunity to malaria infection, we
investigated whether genetic variation at the IRF-1
locus affects resistance to malaria infection in
humans and therefore if there is any evidence for
IRF-1 of being the Pfil locus. We addressed these
questions by studying IRF-1 diversity and performing a gene candidate association study in West
African ethnic groups showing striking differences
in their susceptibility and immune response to
malaria. The sample comprised 190 unrelated individuals aged > 10 years, 85 belonging to the Fulani
(mean age ± SE; 29.8 ± 2.0 years) and 105 to the
Mossi (39.2 ± 1.7 years) ethnic groups, who were
recruited during a cross-sectional epidemiological
survey conducted by Modiano and colleagues in
rural areas of Burkina Faso. We genotyped 14 Single
Nucleotide Polymorphisms (SNPs) in a region
extending from 5 kb upstream to 5 kb downstream of
the IRF-1 gene. The locus shows a high degree of
genetic diversity. All SNPs are polymorphic in both
Participants:
Mario Coluzzi, professor; Giacomo Paganotti, Federica
Verra, post-doc fellows; Valentina Mangano, Germana
Bancone, PhD students
Collaborations:
The Wellcome Trust Center for Human Genetics, Oxford, UK (Prof.
Dominic Kwiatkowski); Department of Immunology, Wenner-Gren
Institute, University of Stockholm, Sweden (Prof. Marita TroyeBlomberg); Centre National de Recherche et Formation sur le
Paludisme, Ouagadougou, Burkina Faso (Dr. Bienvenu
Sodiomon Sirima)
Report of activity
Interferon Regulatory Factor 1 (IRF-1) is a transcription factor that regulates the expression of a
number of genes whose products play crucial roles
in innate as well as adaptive immunity. IFN-gamma
is the strongest IRF-1 inducer known and IRF-1
promotes transcription of several IFN-gamma stimulated genes, acting as an important mediator of this
cytokine activities. The expression of IRF-1 is also
induced by TNF-alpha and antagonized by IL-4.
IRF-1 activity is essential for recognition of microorganisms and antigen presentation, as it regulates
the expression of Toll-like Receptor 9, MHC class I
and class II genes, and also of LMP2 and MECL1,
encoding two proteasome subunits. IRF-1 is
required for maturation of natural killer cells by regulating the transcription of IL-15, and it has a pivotal role in monocyte/macrophage differentiation. In
macrophages it triggers the expression of iNOS , on
which relies one of the principal mechanisms of
cytotoxicity against pathogens, and Bf , part of the
alternative complement cascade. Moreover, IRF-1
levels seem to be critical for T cell development in
the thymus as well as for B cell growth. IRF-1 triggers IL-2p40 expression and represses IL-4 transcription, and it is therefore central in modulating
81
D. Modiano - Immunogenetic analysis in ethnic groups differing in susceptibility to falciparum malaria
populations, with high minor allele frequency and the
average heterozygosity of the region is consequently also very high (HM = 0.49 and HF = 0.48). This
observation may eventually suggest the action of
balancing selection on the locus. Two SNPs showed
different frequencies among Fulani and Mossi
(rs10213701: Minor Allele Frequency (MAF)
MAFM= 0.42; MAFF=0.27, P=0.004; rs2549005:
MAFM=0.49 MAFF=0.34, P=0.006).
In association analysis, using the presence of P. falciparum parasite in the blood as phenotype, we
observed significant associations in both Fulani and
Mossi, but with different sets of SNPs across the
IRF-1 locus. One IRF-1 promoter polymorphism,
rs2706384, showing similar MAF in Mossi (0.41)
and Fulani (0.51) was associated with protection
against P. falciparum infection in both populations.
These similar MAFs and the fact that the parasite
rate amongst Fulani is lower than in the Mossi
across all the three rs2706384 genotypes does not
suggest the involvement of this SNP in the Fulani specific resistance to malaria. This polymorphism
might instead be a general marker of a protective
factor involved in parasite clearance in populations
leaving in malaria endemic areas. The overall association pattern suggests either a heterozygous advantage effect or of a recessive disadvantage effect. We
tested both models performing a Mantel-Haenszel
analysis stratified by ethnicity in the overall sample.
The heterozygous advantage model is the one that
best fits our data as it is significant in both ethnic
groups separately (PM =0.017 and PF =0.003), and
in the sample set as a whole has a higher significance
level (P= 0.0004). The protective effect of the
rs2706384 heterozygous genotype on parasite density was also evaluated. After log transformation of
parasite counts we performed a three way analysis of
variance adjusted for ethnicity and age in the overall
sample. Consistently with what observed with
respect to parasite rates, heterozygous CA individuals showed lower geometric means of parasite density (4.5±1.5 parasites/Ìl) than CC (13.0±1.2;
P=0.011) and AA (7.3±1.9; P=0.081) homozygous
individuals.
This study provides the first evidence of a specific
locus within the 5q31 region that is associated with
the control of malaria infection, and raises the possibility that IRF-1 could be the Pfil locus. Linkage disequilibrium mapping of the surrounding genomic
region is ongoing to clearly identify causative mutation/s, and targeted functional experiments are
being carried on for determination of molecular
mechanisms affected by genetic variation. The role of
IRF-1 as a genetic determinant of human response
to malaria is worthy of investigation in larger studies of resistance to severe malaria.
Paganotti GM, Palladino C, Modiano D, Sirima BS,
Raberg L, Diarra A, Konate A, Coluzzi M, Walliker
D, Babiker HA. Genetic complexity and gametocyte
production of Plasmodium falciparum in Fulani and
Mossi communities in Burkina Faso. Parasitology.
2006;132(5): 607-14.
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96
97
Finito di stampare nel mese di maggio 2007
presso il
Centro Stampa d’Ateneo
Università degli Studi di Roma La Sapienza
P.le Aldo Moro, 5 - 00185
www.editricesapienza.it

Report of activity 2005-2006

Transcript

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