6th International Conference

on Ribonucleases

19-23 June, 2002

Bath, UK

Major Sponsors for the Conference

Alfacell Corporation, Bloomfield, USA European Commission, Brussels, Belgium

Sponsors of Selected Speakers

Cancer Research UK (Sponsored Speaker- Ronald Raines, Madison, USA)

Company of Biologists, UK

(Sponsored Speakers- James Riordan, Boston, USA and Robert Silverman, Cleveland, USA)

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Local Organising Committee

K. Ravi Acharya (Bath, UK)- Coordinator Vasanta Subramanian (Bath, UK)

Robert Shapiro (Boston, USA)

International Scientific Committee

Jaap Beintema (Groningen, The Netherlands) Claudi Cuchillo (Barcelona, Spain) Giuseppe D’Alessio (Naples, Italy) Jan Hofsteenge (Basel, Switzerland)

Masachika Irie (Tokyo, Japan) Allen Nicholson (Detroit, USA)

Ronald Raines (Wisconsin, USA) James Riordan (Boston, USA)

Jan Steyaert (Brussels, Belgium) Richard Youle (Bethesda, USA)

Conference WEB site Designers Jawahar Swaminathan, Bath, UK

Matthew Baker, Bath, UK

Conference Logo Artist Shalini Iyer, Bath, UK

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PROGRAMME

Wednesday 19th June, 2002 3.00 - 8.00pm Registration and Coffee, 8W2.31 6.30 - 7.30pm Dinner (Choices Restaurant) Venue: Lecture Theatre 8W1.1 7.30pm Welcome Ravi Acharya (University of Bath, UK- Conference Organiser) 7.45pm Keynote Address (L1) James Riordan (Harvard Medical School, Boston, USA) Searching for success in the land of the ribonucleases Venue: Claverton Rooms 8.45 - 11.00pm Welcome Reception Lysandra String Quartet Meg Moss- Violin, Michelle Ibison- Violin,

Bob Baker- Viola, Jo Bell- Cello

Thursday 20th June, 2002 7.30 – 9.00am Breakfast (Choices Restaurant) Session 1: Structure and Mechanism of Ribonucleases (L2-L5) Venue: Lecture Theatre 8W1.1 Chair Persons: Giuseppe D’ Alessio and James Riordan 8.30 – 10.15am (Each speaker 20 minutes)

Miquel Coll (CSIC, Barcelona, Spain) Human pancreatic ribonuclease: from monomers to dimers

Claudi Cuchillo (Universitat Autònoma de Barcelona, Bellaterra, Spain) The influence of p2 and other phosphate-binding subsites on the specificity of cleavage by ribonucleases

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Stanley Crooke (Isis Pharmaceuticals Inc, California, USA) Structure and function of human RNase Hs

Xinhua Ji (National Cancer Institute, Frederick, USA) Active site architecture of RNase III: molecular basis for dsRNA cleavage

10.15 – 10.30am Coffee 10.30 – 12.30pm (Each speaker 20 minutes) (L6-L10)

John Julias (National Cancer Institute, Frederick, USA) Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo

Lelio Mazzarella (CNR, Napoli, Italy) Tension in seminal ribonuclease as detected by the structural organization of several forms of the enzyme

Stefan Sarafianos (Rutgers University, New Jersey, USA) Structural basis of RNA/DNA polypurine tract recognition by RNase H of HIV-1 reverse transcriptase

Jan Steyaert (Free University of Brussels, Belgium) A decade of protein engineering: RNase T1 undressed

Alexander Wlodawer (National Cancer Institute, Frederick, USA) Crystallographic and functional studies of a modified form of EDN

12.30 – 2.00pm Lunch (Choices Restaurant) Session 2: Ribonuclease Stability and Folding (L11-L14) Chair Persons: Ronald Raines and Alexander Wlodawer 2.00 - 3.45pm (Each Speaker 20 minutes)

Robert Hartley (NIH/NIDDK, Bethesda, USA) The fold of barstar and the composition of its hydrophobic core

Alexander Makarov (Engelhardt Institute of Molecular Biology, Moscow, Russia) Stability of barnase and binase complexed with barstar mutants

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Mahesh Narayan (Cornell University, Ithaca, USA) The oxidative folding of RNase A: an overview

Nick Pace (Texas A&M University, USA) How to make your favourite ribonuclease more stable

3.45 – 4.00pm Coffee 4.00 – 6.30pm (Main Speaker 20 minutes) (L15-L22)

Jayant Udgaonkar (National Centre for Biological Sciences, Bangalore, India) Incremental loss of structure during the unfolding of barstar

(Short Presentations 10 minutes each)

Marta Bruix (CSIC, Madrid, Spain) An NMR overview of the electrostatics and dynamics at the active site of α-Sarcin

Gayatri Chavali (University of Bath, UK) Crystal structure of angiogenin in complex with its antibody (mAb 26-2F)

Daniel Holloway (University of Bath, UK) Guest-host crosstalk in an angiogenin/RNase A chimeric protein

Doug Laurents (CSIC, Madrid, Spain) Electrostatic interactions in RNase Sa and a charge reversed mutant

Jozef Sevcik (Slovak Academy of Sciences, Bratislava, Slovak Republic) Crystal structure of RNase Sa3- a microbial ribonuclease with cytotoxic activity

Salvatore Sorrentino (University of Naples, Napoli, Italy) The remarkable activity of HP-RNase on double stranded RNA Jawahar Swaminathan (University of Bath, UK) Conformational requirements for the design of specific inhibitors of EDN: crystal structures of EDN at atomic resolution

6.30 – 7.30pm Dinner (Choices Restaurant)

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7.30 – 9.00pm Poster session I (Poster numbers P1-P30) Venue: University Hall

Friday 21st June, 2002 7.30 – 9.00am Breakfast (Choices Restaurant) Session 3: Emerging Ribonucleases, Evolution and Genomics (L23-L26) Venue: Lecture Theatre 8W1.1 Chair Person: Jaap Beintema 8.30 – 10.15am (Each speaker 20 minutes)

Pamela Green (Delaware Biotechnology Institute, Newark, USA) Genetics and genomics of eukaryotic RNases Haruhiko Masaki (University of Tokyo, Japan) A unique group of new ribonucleases targeting specific tRNases Helene Rosenberg (NIAID, NIH, Bethesda, USA) The eosinophil ribonucleases and anti-viral host defense: the evolving story Jianzhi Zhang (University of Michigan, Ann Arbor, USA) Adaptive evolution of a duplicated pancreatic ribonuclease gene in a leaf-eating monkey

10.15 – 10.30am Coffee Session 4: Applications of Ribonucleases (L27-L31) Chair Person: Gerald Gleich 10.30 – 12.30pm (Each speaker 20 minutes)

Wojciech Ardelt (Alfacell Corporation, Bloomfield, USA) A novel cytotoxic ribonuclease from amphibian oocytes

John Burke (University of Vermont, USA) Cellular and antiviral applications of hairpin ribozymes

Ronald Raines (University of Wisconsin, Madison, USA) Ribonuclease A as a chemotherapeutic

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Susanna Rybak (National Cancer Institute, Frederick, USA) RN321: Onconase based targeted therapeutic

Robert Shapiro (Harvard Medical School, Boston, USA) Recent developments in anti-angiogenin therapy

12.30 – 2.00pm Lunch (Choices Restaurant) Session 5: Functions of Ribonucleases in Health and Disease (L32-L35) Chair Persons: Helene Rosenberg and Allen Nicholson 2.00 - 3.45pm (Each Speaker 20 minutes)

Gerald Gleich (University of Utah School of Medicine, USA) Eosinophil granule proteins: Current understanding

Bret Hassel (Univ. of Maryland School of Medicine, Baltimore, USA) Biological functions of RNase-L

Guo-fu Hu (Harvard Medical School, Boston, USA) Nuclear function of angiogenin

Claudia De Lorenzo (Università di Napoli, Italy) ImmunoRNases as anti-tumour agents

3.45 – 4.00pm Coffee 4.00 – 6.30pm (Each Speaker 20 minutes) (L36-L42)

Martin Michaelis (Johann Wolfgang Goethe-Universität, Frankfurt, Germany) Antiviral effects of onconase (R) (Ranpirnase)

Robert Silverman (The Lerner Research Institute, Cleveland, USA) An interferon regulated antiviral enzyme, RNase-L, is a candidate suppressor of hereditary prostate cancer (HPC1)

Vasanta Subramanian (University of Bath, UK) Regulation and expression of angiogenins

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(Short Presentations 10 minutes each)

Josette Badet (INSERM, Paris, France) Expression of angiogenin in the human monocyte/ macrophage lineage.

Kimberly Dickson (University of Wisconsin, Madison, USA) Mechanism of ribonuclease cytotoxicity

Tomas Eckschlager (J.W.Goethe-University Medical Center, Frankfurt, Germany) Effect of BS RNase on chemo sensitive and chemo resistant neuroblastoma cell lines

Susanna Navarro (Universitat Autònoma de Barcelona, Bellaterra, Spain) Studies on the ribonuclease activity profile and ECP expression during HL60 cell differentiation

6.30 – 7.30pm Dinner (Choices Restaurant) 7.30 – 9.00pm Poster session II (Poster numbers P31-P60) Venue: University Hall

Saturday 22nd June, 2002 7.30 – 9.00am Breakfast (Choices Restaurant) Session 6: RNA Degradation and Processing (L43-L46) Venue: Lecture Theatre 8W1.1 Chair Persons: Robert Hartley and Robert Silverman 8.30 – 10.15am (Each speaker 20 minutes)

Agamemnon Carpousis (CNRS, Toulouse, France) RNA processing and decay in E.coli: discovery and characterization of the degradosome Sherif Abou Elela (Université de Sherbrooke, Québec, Canada) Substrate selection by eukaryotic RNase III: one enzyme and two modes of action

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Siew Loon Ooi (Johns Hopkins University School of Medicine, Baltimore, USA) RNA lariat debranching enzyme

Phillip Mitchell (University of Edinburgh, UK) Function of the exosome complex in nonsense-mediated decay

10.15 – 10.30am Coffee 10.30 – 12.30pm (Each speaker 20 minutes) (L47-L52)

Allen Nicholson (Wayne State University, Detroit, USA) Mechanistic analysis of double stranded RNA processing by RNase III

Gadi Schuster (Technion-Israel Institute of Technology, Haifa, Israel) Poly adenylation and degradation of RNA in chloroplast and prokaryotes, similarities and differences

Robert Simons (University of California, Los Angeles, USA) Ribonuclease function during the bacterial cold shock response

David Stern (Cornell University, Ithaca, USA) RNA degradation in plant chloroplasts: the role of CSP41, a unique endoribonuclease

(Short Presentations 10 minutes each)

Sergei Borukhov (State University of New York Health Center at Brooklyn, New York, USA) Structure-function analysis of prokaryotic transcript cleavage factors GreA and GreB Niklas Henriksson (Uppsala University, Sweden) Modulating poly(A)-specific ribonuclease (PARN) activity in vitro by RNA binding proteins

12.45 - 2.00pm Lunch (Choices Restaurant) 2.00 - 4.00pm Trip to American Museum (Bath) 6.00pm Visit to the City of Bath 7.00pm Civic Reception (Roman Baths)

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8.00 - 11.00pm Conference Banquet at the City Guildhall Banquet speech: Tina Shogen (Alfacell Corporation, Bloomfield, USA) Vote of thanks: Vasanta Subramanian (University of Bath, UK, Conference Co-organiser)

Banquet music: ‘Hywel Davies Trio’ Hywel Davies -Piano, Ralf Dorrell –Bass, Trevor Davies -Drums

Sunday 23nd June, 2002

7.30 – 9.00am Breakfast (Choices Restaurant) 9.00am Departure

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Poster Titles

Session I (P1 – P30)

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(P1) STABILIZATION OF PROTEINS BY INTRODUCTION OF NON-NATURAL MODULES Ulrich Arnold , 1,2, Matthew P. Hinderaker3, and Ronald T. Raines 2, 3 1 Department of Biochemistry and Biotechnology, Martin-Luther University, Halle, Germany, 2 Department of Biochemistry, 3 Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, U.S.A. (P2) SOLUTION STRUCTURE OF MONOMERIC BOVINE SEMINAL RIBONUCLEASE BY NUCLEAR MAGNETIC RESONANCE Francesca Avitabilea, Orlando Crescenzia, Alfonso Carotenutob, Anna M. D’Ursib, Teodorico Tancredic and Delia Piconea aDipartimento di Chimica, Università Federico II di Napoli, Via Cintia, 80126, Napoli, Italy. bDipartimento di Scienze Farmaceutiche, Università di Salerno, 84084, Fisciano, Italy. cIstituto Chimica Biomolecolare del CNR, Pozzuoli, Napoli, Italy. (P3) COORDINATED DIVALENT METAL IONS IN THE ACTIVE SITE OF POLY(A)-SPECIFIC RIBONUCLEASE. Yan-Guo Ren, Nikolaos A.A. Balatsos, Leif A. Kirsebom and Anders Virtanen. Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, SE-751 24, Uppsala, Sweden. (P4) ADAPTIVE EVOLUTION IN MEMBERS OF THE RIBONUCLEASE A SUPERFAMILY Jaap J. Beintema, Jean-Yves F. Dubois, Björn M. Ursing Dept. of Biochemistry, University of Groningen, The Netherlands (P5) SOLUTION ISOLATION AND CHARACTERIZATION OF AN ENGINEERED DOMAIN-SWAPPED HUMAN PANCREATIC RIBONUCLEASE Rodríguez, M., Benito, A., Ribó, M. and Vilanova, M. Laboratori d’Enginyeria de Proteïnes, Dept. de Biologia, Fac. de Ciències, Universitat de Girona, Campus de Montilivi s/n, 17071 Girona. Spain. (P6) REGULATION OF RIBONUCLEASE REGB ACTIVITY BY RIBOSOMAL PROTEIN S1 Marco Bisaglia1, Soumaya Laalami2, Jean-Yves Lallemand1, Marc Uzan2 and François Bontems1. 1Equipe ICSN-RMN, Ecole Polytechnique, 91128 Palaiseau, France 2Institut Jacques Monod, 2 Place Jussieu, 75251 Paris cedex 05, France

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(P7) CHARACTERIZATION OF CYTOTOXIC HUMAN PANCREATIC RIBONUCLEASES Bosch, M.1, Benito, A.1, Ribó, M.1, Beaumelle, B.2 and Vilanova, M1. 1 Laboratori d’Enginyeria de Proteïnes, Dept. de Biologia, Fac. de Ciències, Universitat de Girona, Campus de Montilivi s/n, 17071 Girona. Spain. 2 UMR 5539 CNRS, Case 107, Bt 24, Université Montpellier II, 34095 Montpellier Cedex 05. France. (P8) ESSENTIAL STATIONS IN THE INTRACELLULAR PATHWAY OF CYTOTOXIC BOVINE SEMINAL RIBONUCLEASE Aurora Bracale1, Castaldi, F.1, Spalletti-Cernia, D.2, Nitsch, L.2 and D’Alessio, G.1 1 Dipartimento di Chimica Biologica, Università di Napoli “Federico II”, Italy. 2 Dipartimento di Biologia e Patologia Cellulare e Molecolare “L. Califano”, Università di Napoli “Federico II”, Italy. (P9) STUDIES OF THE STRUCTURAL BASIS OF EOSINOPHIL CATIONIC PROTEIN CYTOTOXICITY Esther Carreras1, Ester Boix1, Helene F. Rosenberg,2, Claudi M. Cuchillo1 and M. Victòria Nogués1

1Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain 2Laboratory of Host Defenses, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA (P10) KINETIC ANALYSIS OF CONFORMATIONAL CHANGES IN THE ESCHERICHIA COLI RIBONUCLEASE III PRE-STEADY STATE MONITORED BY CHANGES IN INTRINSIC PROTEIN FLUORESCENCE Adam G. Cassano, Frank E. Campbell, and Michael E. Harris

Department of Molecular Biology and Microbiology and Center for RNA Molecular Biology (P11) CHARACTERISATION OF THE BACILLUS SUBTILIS 5S RRNA MATURASE, RNase M5: ARE RNASE M5 AND TYPE I TOPOISOMERASE CLEAVAGE REACTIONS ANALAGOUS? Jordi Rourera, Frédérique Allemand, Dominique Brechemier-Baey, Harald Putzer and Ciarán Condon. Institut de Biologie Physico-Chimique UPR 9073,13 rue Pierre et Marie Curie, 75005 Paris, France. (P12) CORE MANIPULATIONS, METAL BINDING: RNase T1 AS A TEST-CASE. De Vos, S., Deswarte, J., Backmann, J., Langhorst, U., Steyaert, J., and Loris, R. Department of Applied Biological Sciences, Institute for Molecular Biology and Biotechnology, Free University of Brussels (VUB), Belgium.

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(P13) TRANSCRIPTIONAL ACTIVATION OF THE MOUSE EOSINOPHIL-ASSOCIATED RIBONUCLEASE 2 (EAR2) GENE BY INTRONIC ENHANCER ELEMENTS: A COMMON REGULATORY FEATURE OF HUMAN AND MOUSE EOSINOPHIL-ASSOCIATED RIBONUCLEASE GENES. Kimberly D. Dyer, Joanne M. Moreau and Helene F Rosenberg Laboratory of Host Defenses, National Institutes of Health, U.S.A. (P14) SOLUTION PROPERTIES OF BOVINE SEMINAL RIBONUCLEASE Carmine Ercole1, Francesca Avitabile1, Orlando Crescenzi1, Giuseppe D'Alessio2, Teodorico Tancredi3 and Delia Picone1. 1 Dipartimento di Chimica, Università Federico II di Napoli, Via Cintia, 80126, Napoli, Italy.2 Dipartimento di Chimica Biologica, Università Federico II di Napoli, Via Mezzocannone, 80134, Napoli, Italy.3 Istituto Chimica Biomolecolare del CNR, Pozzuoli, Napoli, Italy. (P15) STRUCTURAL ASPECTS OF ANTISENSE OLIGONUCLEOTIDE-BASED ARTIFICIAL RIBONUCLEASES Martin M. Fabani1, Marina A. Zenkova2, Natalia G. Beloglazova2, Vladimir V. Sil’nikov2, Valentin V. Vlassov2, Kenneth T. Douglas1, Elena V. Bichenkova1*. 1 School of Pharmacy and Pharmaceutical Sciences, University of Manchester, U.K. 2 Institute of Bioorganic Chemistry, Novosibirsk, Russia. (P16) PROCESSIVE PROPERTIES OF EUKARYOTIC RIBONUCLEASES H1: SITES OF RNase H CLEAVAGE ARE AFFECTED BY THE 3'-TERMINUS OF THE DNA. Sergei A. Gaidamakov and Robert J. Crouch. Laboratory of Molecular Genetic NICHD NIH (P17) THE PRESENCE OF LEU-145 IS ESSENTIAL TO MAINTAIN THE ACTIVE SITE ELECTROSTATIC ENVIRONMENT OF RIBONUCLEASE α-SARCIN.

Flor García-Mayoral1, Masip, M.2, García-Ortega, L.2, Pérez-Cañadillas, J.M.1, Martínez del Pozo, A.2, Gavilanes, J.G.2, Rico, M.1 and Bruix, M1. 1Departamento de Espectroscopía y Estructura Molecular, Instituto de Química Física “Rocasolano”, CSIC, Madrid, Spain. 2Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain (P18) THE RELATIVE PROPORTIONS OF RNase A AGGREGATES VARY, THOSE OF DIMERS EVEN INVERTING, AS A FUNCTION OF EXPERIMENTAL CONDITIONS Giovanni Gotte, Francesca Vottariello, and Massimo Libonati (P19) ATTEMPTS TO CHANGE THE SPECIFICITY OF RNase T1 BY RANDOM MUTAGENESIS R. Czaja, M. Hänsler, H. Hoier, K. Höschler, B. Hubner, M. Struhalla, P. Orth, W. Saenger & Ulrich Hahn University of Leipzig, Faculty for Biosciences, Institute for Biochemistry, FRG

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(P20) HIGH-YIELDING PROCEDURES FOR THE PRODUCTION OF PROTEINS BELONGING TO THE PANCREATIC RIBONUCLEASE SUPERFAMILY Michelle C. Hares1, Daniel E. Holloway1, Lori D. Horb1, M. Thomas E. Raven1, Robert Shapiro2, Vasanta Subramanian1 and K. Ravi Acharya1 1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom. 2Center for Biochemical and Biophysical Sciences and Medicine, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, U.S.A. (P21) CRYSTAL STRUCTURE OF TYPE 2 RIBONUCLEASE H FROM PYROCOCCUS HORIKOSHII Tomonori Hata, Yoshimitsu Kakuta, Yoshiaki Kouzuma, and Makoto Kimura Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan (P22) MICROBIAL RIBONUCLEASES AS ANTI-PROLIFERATIVE AGENTS Olga Ilinskaya1, Florian Dreyer2; Ester Vock3 1Department of Microbiology, Kazan State University, Kazan, Russia. 2Rudolf-Buchheim-Institute of Pharmacology, University Giessen, Germany. 3Department of Toxicology, University Wuerzburg, Germany. (P23) CATALYTIC MECHANISM OF AN RNA RESTRICTION ENZYME, COLICIN E5 Sakura Inoue1, Tetsuhiro Ogawa1, Shunsuke Yajima2, Makoto Hidaka1, Haruhiko Masaki1

1Department of Biotechnology, The University of Tokyo, Japan 2Department of Bioscience, Tokyo University of Agriculture, Japan (P24) MAPPING OF ANGIOGENIN CONTACTS WITH SMALL-MOLECULE INHIBITORS BY KINETIC AND DOCKING STUDIES Jeremy L. Jenkins1 and Robert Shapiro1,2 1Center for Biochemical and Biophysical Sciences and Medicine and 2Department of Pathology, Harvard Medical School, MA, USA (P25) EXPRESSION AND CRYSTALLIZATION OF A WOUND-INDUCIBLE RIBONUCLEASE NW FROM NICOTIANA GLUTINOSA Shin Kawano, Yoshimitsu Kakuta, and Makoto Kimura Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan (P26) THE RESIDUE IMMEDIATELY UPSTREAM OF THE RNase P CLEAVAGE SITE IS A POSITIVE DETERMINANT Leif A. Kirsebom, Mathias Brännvall and B. M. Fredrik Pettersson Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Sweden.

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(P27) RIBONUCLEASE A IN TRIFLUOROETHANOL – A STUDY ON UNFOLDING BY PROTEOLYSIS AND STOPPED-FLOW FLUORESCENCE SPECTROSCOPY Jens Köditz, Yvonne Markert, Ralph Golbik* and Renate Ulbrich-Hofmann Martin-Luther University Halle-Wittenberg, Department of Biochemistry & Biotechnology, Institute of Biotechnology and Institute of Biochemistry*, Kurt-Mothes-Str. 3, D-06120 Halle (P28) COMPARATIVE ANALYSIS OF RNase III CLEAVAGE MECHANISM Bruno Lamontagne, Ghada Ghazal, and Sherif Abou Elela Département de Microbiologie et Infectiologie, Université de Sherbrooke, Canada (P29) ENZYMATIC ACTIVITY, DIVERSIFICATION AND IMMUNITY PROTEIN BINDING IN RNase COLICINS. Dan Walker, Lorna, E, Lancaster and Colin Kleanthous. School of Biological Sciences, University of East Anglia, Norwich, U.K. (P30) S. CEREVISIAE RNase III (RNT1) AFFECTS TELOMERE HOMEOSTASIS Stéphanie Larose, Raymund Wellinger and Sherif Abou Elela Département de Microbiologie et d'Infectiologie, Université de Sherbrooke, Canada

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Poster Titles Session II

(P31 – P58)

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(P31) REGULATION OF RIBONUCLEASE EXPRESSION BY ESTRADIOL IN RANA CATESBEIANA (BULLFROG) Pin-Chi Tang1, Huey-Chung Huang2, Sui-Chi Wang1, Jen-Chong Jeng1and You-Di Liao1, 1Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan; 2Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei 100, Taiwan (P32) BIOLOGICAL ACTIONS OF RNase A OLIGOMERIC AGGREGATES Giovanni Gotte1, Josef Soucek2, Massimo Libonati1, Tomas Slavik3, and Josef Matousek3 1Dipartimento di Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Università di Verona, Verona, Italy. 2Institute of Hematology and Blood Transfusion, Prague, Czech Republic. 3Institute of Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic. (P33) NUCLEOPHILE ACTIVATION IN RIBONUCLEASES : A COMBINED X-RAY CRYSTALLOGRAPHIC/AB INITIO QUANTUM CHEMICAL APPROACH

Stefan Loverix1, Pierre Mignon2, Paul Geerlings2 and Jan Steyaert1 1Dienst Ultrastruktuur, VIB (Vlaams Interuniversitair Instituut Biotechnologie), Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium; and 2Eenheid Algemene Chemie (ALGC), Faculteit Wetenschappen, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium. (P34) FUNCTIONAL CHARACTERIZATION OF YEAST AND ARABIDOPSIS RNases FROM THE T2 FAMILY Gustavo C. MacIntosh1, Nicole D. LeBrasseur2, Tracey Millard2 and Pamela J. Green1 1Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA 2MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA (P35) OPTIMUM MODIFICATION OF CATIONIZATION FOR THE POTENT CYTOTOXIC RIBONUCLEASE Takashi Maeda, Junichiro Futami, Midori Kitazoe, Emiko Nukui, Hiroko Tada, Masaharu Seno, Megumi Kosaka, and Hidenori Yamada. Department of Bioscience and Biotechnology, Okayama University, Japan. (P36) DEGRADOSOME DISRUPTION AFFECTS DEGRADATION OF THE rpsO mRNA OF Escherichia coli P. E. Marujo, J. LeDerout and P. Régnier Institut de Biologie Physico-Chimique, Paris, France.

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(P37) PEG CHAINS INCREASE ASPERMATOGENIC AND ANTITUMOR ACTIVITY OF RNase A AND BS-RNase ENZYMES WITHOUT EMBRYOTOXICITY OF RNaseA Josef Matoušek1*, Tomáš Slavík1, Pavla Poučková2, Josef Souček3, Jiří Škvor4 1Institute of Physiology and Genetics, Academy of Sciences of the Czech Republic, Liběchov, Czech Republic. 2Institute of Biophysics, Medical Faculty of the Charles University, Prague, Czech Republic. 3Institute of Hematology and Blood Transfusion, Prague, Czech Republic. 4Seva-Imuno Praha, Prague, Czech Republic (P38) POPULATION SHIFT VERSUS INDUCED FIT: THE INSTRUCTIVE CASE OF BOVINE SEMINAL RIBONUCLEASE SWAPPING DIMER Antonello Merlino1, Luigi Vitagliano2, Filomena Sica1, Adriana Zagari2 and Lelio Mazzarella1,2

1Dipartimento di Chimica, Università degli Studi di Napoli ‘Federico II’, Via Cinthia, Napoli. 2Centro di Studio di Biocristallografia, CNR, Via Mezzocannone 6, Napoli. (P39) MOLECULAR DYNAMICS SIMULATIONS OF 3D DOMAIN SWAPPING DIMERS OF BOVINE PANCREATIC RIBONUCLEASE Antonello Merlino1, Luigi Vitagliano2, Marc Antoine Ceruso3 and Lelio Mazzarella1,2 1Dipartimento di Chimica, Università degli studi di Napoli ‘Federico II’, Via Cynthia, Napoli, Italy. 2Centro di Studio di Biocristallografia, CNR, Via Mezzocannone 6, Napoli, Italy. 3Department of Physiology and Biophysics, Mt Sinai School of Medicine, One Gustave L. Levy Place, New York, USA (P40)THE EFFECT OF NET CHARGE ON THE ACTIVITY, STABILITY, AND CYTOTOXICITY OF RIBONUCLEASE Sa Vladimir A. Mitkevich1, Olga N. Ilinskaya2, Kevin L. Shaw3, Gerald R. Grimsley4, Florian Dreyer5, Gennady I. Yakovlev1, Alexander A. Makarov1, C. Nick Pace4 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia2Department of Microbiology, Kazan State University, 420008 Kazan, Russia3Department of Biology, Grove City College, Grove City, Pennsylvania 16127, USA.4Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843, USA.5Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany (P41) CRYSTAL STRUCTURE OF EOSINOPHIL CATIONIC PROTEIN IN COMPLEX WITH 2’,5’-ADP AT 2 Å RESOLUTION REVEALS THE DETAILS OF THE RIBONUCLEOLYTIC ACTIVE SITE C. Gopi Mohan,1 Ester Boix,1,2 Hazel R.Evans,1 Zoran Nikolovski,2 M. Victòria Nogués,2 Claudi M. Cuchillo2 and K. Ravi Acharya1 1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K. 2Department de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. (P42) THE CONTRIBUTION OF BASIC AMINO ACID RESIDUES IN THE P2 SUBSITE TO RIBONUCLEASE A ACTIVITY Mohammed Moussaoui, M. Victòria Nogués and Claudi M. Cuchillo Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona. 08193-Bellaterra. Spain.

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(P43) MOLECULAR CHARACTERIZATION OF A MOUSE CDNA ENCODING DICER, A RIBONUCLEASE III ORTHOLOGUE INVOLVED IN RNA INTERFERENCE Rhonda H. Nicholson and Allen W. Nicholson, Department of Biological Sciences, Wayne State University, Detroit, Michigan, USA. (P44) STRUCTURAL BASIS FOR A CHANGE IN SUBSTRATE SPECIFICITY OF THE RNase MC1 MUTANT FROM BITTER GOURD SEEDS Tomoyuki Numata, Yoshimitsu Kakuta, Makoto Kimura Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan (P45) SUBSTRATE RECOGNITION MECHAMISM OF AN RNA RESTRICTION ENZYME, COLICIN E5 Tetsuhiro Ogawa1, Shunsuke Yajima2, Makoto Hidaka1 and Haruhiko Masaki1. 1 Department of Biotechnology, The University of Tokyo, Japan. 2 Department of Bioscience, Tokyo University of Agriculture, Japan. (P46) MINI OPEN READING FRAMES (MINI-ORF) AT UPSTREAM OF ORF OF TWO RNases FROM BASIDOMYCETES, Lentinus edodes AND Irpex lacteus. Kazuko Ohgi1, Tatsuya Wada1, Wasanori Iwama1, Tsutomu Tsuji1, Masachika Irie1, Tadashi Itagaki 2, Hiroko Kobayashi 2, Norio Inokuchi2

1Department of Microbiology, Hoshi College of Pharmacy, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. 2Department of Microbiology, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan. (P47) POLYMER CONJUGATED BOVINE PANCREATIC RIBONUCLEASE (RNase A) FOR CANCER THERAPY P. Poučková1, M. Zadinová1, D. Hloušková1, J. Strohalm2, D. Plocová2, M. Špunda1, T. Olejár2, M. Zitko1, J. Matoušek3, J. Souček4

1Institute of Biophysics, Medical Faculty, Charles University, Prague 2, Czech Republic 2Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague 6, Czech Republic 3Institute of Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic 4Institute of Hematology and Blood Transfusion. Prague 2, Czech Republic (P48) DELETION OF THE NH2-TERMINAL β-HAIRPIN OF THE RIBOTOXIN α-SARCIN RENDERS A NON-TOXIC BUT ACTIVE RIBONUCLEASE García-Ortega‡, L., Masip‡, M., Mancheño‡, J:M, Oñaderra‡,M., Lizarbe‡, M.A., García-Mayoral§, M.F., Bruix§, M., Martínez del Pozo‡, A., and Gavilanes‡, J.G. ‡Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, Madrid, Spain §Instituto de Química Física “Rocasolano”, Consejo Superior de Investigaciones Científicas, Madrid, Spain

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(P49) CONTRIBUTION OF THE HYDROPHOBIC RESIDUES OF THE α3β2β5β6 CHAIN FOLDING INITIATION SITE TO THE CONFORMATIONAL STABILITY OF RIBONUCLEASE A Font, J., Ribó, M., Benito, A., Torrent, J. and Vilanova, M. Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, E-17071 Girona, Spain. (P50) ENDORIBONUCLEASE RegB FROM BACTERIOPHAGE T4 : DESIGN OF A NEW POSITIVE SELECTION PLASMID, PRELIMINARY FUNCTIONAL AND STRUCTURAL STUDIES OF THE ENZYME Saida F1., Lallemand J-Y1 and Bontems F.1 1 Laboratoire ICSN-RMN, Ecole polytechnique, Palaiseau, France. (P51) ESSENTIAL DYNAMICS AND HYDROGEN BOND ANALYSIS ON EOSINOPHIL RELATED RNases Sanjeev B.S.1 and Vishveshwara S. 1 1Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India. 560 012. (P52) ANALYSIS OF THE CLEAVAGE SITE SEQUENCE IN HYPER-EDITED dsRNA A. Deirdre J. Scadden and Chris W. J. Smith Department of Biochemistry, University of Cambridge, UK (P53) STRUCTURAL INVESTIGATION OF MONOMERIC AND DIMERIC FORMS OF BOVINE SEMINAL RIBONUCLEASE Filomena Sica1,2, Anna Di Fiore1, Rita Berisio2, Renata Piccoli3, Giuseppe D’Alessio3, Adriana Zagari2,3 & Lelio Mazzarella1,2

1Dipartimento di Chimica, Università degli Studi “Federico II”, Napoli, Italy 2Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy 3Dipartimento di Chimica Biologica, Università degli Studi “Federico II”, Napoli, Italy (P54) SYNTHESIS AND PROPERTIES OF POLYMER CONJUGATED BOVINE SEMINAL AND BOVINE PANCREATIC RIBONUCLEASES J. Souček1, Strohalm2, D. Plocova2, P. Pouckova3, D. Hlouskova3, M. Zadinova3, J. Matousek4, K. Ulbrich3

1Institute of Hematology and Blood Transfusion, U nemocnice l, Prague 2, Czech Republic, 2Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 3 Institute of Biophysics, School of Medicine, Charles University, Prague, Czech Republic, 4 Institute of Physiology and Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic (P55) STUDIES ON THE SUBSTRATE SPECIFICITY OF ONCONASE Suhasini A. N., Sunil Kumar. S., Ravi Sirdeshmukh Centre for Cellular and Molecular Biology, Hyderabad 500,007, India

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(P56) RNase Sa POINT MUTATIONS: STRUCTURAL RESPONSE AND CONFORMATIONAL STABILITY Lubica Urbanikova1, Jozef Sevcik1 , C. Nick Pace2 1 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic 2 Texas A&M University, College Station, Texas, USA (P57) CONTRIBUTION OF ACTIVE SITE RESIDUES TO THE ENZYME ACTIVITY AND THERMOSTABILITY OF RIBONUCLEASE SA Gennady I. Yakovlev1, Vladimir A. Mitkevich1, Kevin L. Shaw2, Saul Trevino3, Stephanie Newsom3, C. Nick Pace3, Alexander A. Makarov1 (P58) THERMAL STABILIZATION OF A LABILE MUTANT ENZYME OF RIBONUCLEASE A BY ANTIBODIES Hina Younus1,2, Jens Köditz2, Mohammed Saleemuddin1, Renate Ulbrich-Hofmann2 1Interdisciplinary Biotechnology Unit, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India. 2Department of Biochemistry/Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany. (P59) AN EFFICIENT SEQUENCE SPECIFIC ARTIFICIAL RIBONUCLEASES - OLIGONUCLEOTIDE CONJUGATED TO IMIDAZOLE CONSTRUCTS Marina Zenkova, Nataliya Beloglazova, Vladimir Sil'nikov, Valentin Vlassov. Novosibirsk Institute of Bioorganic Chemistry, 8, Lavrentiev ave., 630090, Novosibirsk, Russia (P60) OLIGONUCLEOTIDE-PEPTIDE CONJUGATES DISPLAYING SPECIFICITY OF RNase T1 Marina Zenkova, Nadejda Mironova, Dmitryi Pyshnyi, Eugenia Ivanova, Valentin Vlassov. Institute of Bioorganic Chemistry 8, Lavrentiev av., Novosibirsk-90, 630090, Russia

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Presentation Abstracts

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(L1) SEARCHING FOR SUCCESS IN THE LAND OF RIBONUCLEASES James F. Riordan Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Cambridge, MA, USA The occasion of the Sixth International Meeting on Ribonucleases is a good opportunity to reflect on the progress that has occurred in the field since the inaugural meeting in Moscow fourteen years ago. Many who are present here today did not attend that first meeting, and that includes myself. At the time, I was still engaged in a long- term relationship with enzymes that preferred to cleave peptide bonds rather than phosphodiesters. My introduction to ribonucleases came at the hands of angiogenin, an enzyme so catalytically challenged that its hydrolytic activity eluded detection for many, many months. Would success in the land of ribonucleases be equally elusive? This, of course depends on one’s definition of success: merely a favorable or satisfactory outcome or result, or the attainment of wealth, position or honors. What are the ribonucleolytic success stories and are they comparable to achievements with my earlier love, the peptidases? We have been reminded repeatedly of the four RNaseA-related Nobel prizes but we can also cite the two for ribozymes. We could even stretch things to include the three for reverse transcriptases and one more for RNA polymerase. So the land of RNA and ribonucleases has done quite well. Only cholesterol can claim more prizes. The peptidases, on the other hand, are still waiting for their first. In contrast, peptidases figure prominently on the list of most frequently prescribed drugs. Some 30 of the top 100 best sellers are enzyme inhibitors, and of these 11 are directed against peptidases. None of the top 100 is either directed against or involves a ribonuclease unless cGMP phosphodiesterase gets honorary membership in the club. However, we can look ahead with keen anticipation to a number of bona fide candidates, some well along, and ribonucleases should soon achieve the same success in the market as they have in Stockholm.

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(L2) HUMAN PANCREATIC RIBONUCLEASE: FROM MONOMERS TO DIMERS Albert Canals1, Joan Pous1, Antoni Benito2, Marc Ribó2, Maria Vilanova2, Miquel Coll1 1Institut de Biologia Molecular de Barcelona, CID-CSIC, Jordi Girona 18–26, 08034 Barcelona, Spain. 2Laboratori d´Enginyeria de Proteïnes. Dept. de Biologia. Universitat de Girona, Campus de Montilvi s/n, 17071 Girona, Spain. Some variants of the human pancreatic ribonuclease (RNase 1) have been crystallised lately, after many years of unfruitful attempts. The substitution -or elimination- of part of the N-terminus appears to be critical for getting high-quality crystals, and has provided the first crystallographic approach to the structure of RNase 1. The final models confirm an overall resemblance to RNase A, with the essential residues of the active-site cleft occupying equivalent positions. However, these structures also reveal subtle disagreements in the loop regions that might lead to different interactions with the ribonuclease inhibitor (RI) (1). Far beyond this comparison of monomeric homologues, the crystal structure, at 2 Å resolution, of one of these variants reveals a novel domain-swapped dimer based on the change of N-terminal domains between the two subunits (2). It confirms a tendency to dimerize that has been long reported for some ribonucleases, but also introduces the idea that subtle changes in key positions may derive in the formation of completely new dimeric structures. As a result, proteins capable of domain swapping may quickly evolve toward an oligomeric form, provided that the new structure acquires an evolutionary advantage. (1) Pous, J., Canals, A., et al and Coll, M. (2000). J. Mol. Biol. 303, 49-59. (2) Canals, A., Pous, J., et al and Coll, M. (2001). Structure 9, 967-976.

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(L3) THE INFLUENCE OF p2 AND OTHER PHOSPHATE-BINDING SUBSITES ON THE SPECIFICITY OF CLEAVAGE BY RIBONUCLEASES Claudi M. Cuchillo, Mohammed Moussaoui and M. Victòria Nogués. Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autónoma de Barcelona. 08193-Bellaterra. Spain. The cleavage of polycytidylic acid (poly(C)) by bovine pancreatic ribonuclease A (RNase A) shows a clear endonucleolytic pattern that has been explained as the consequence of additional interactions by the polymeric substrate in non-catalytic binding sites. The role of these non-catalytic sites on substrate binding and catalysis has been analysed by several approaches. In addition to the active site p1, other specific phosphate binding sites have been identified (pn binding sites). The best characterized are p2, and p0 which are adjacent to the active site (p1) on the 3’- and 5’-side, respectively. It has been demonstrated that electrostatic interactions between phosphate groups in the substrate and basic amino acid residues of the enzyme contribute to the correct alignment of the substrate. Abolition of the electrostatic binding in the p2 region results in a more exonucleolytic cleavage pattern. The cleavage patterns of oligocytidylic acids of different lengths by both wild type and variants of RNase A with mutations at the pn sites are easier to analyse in order to clarify the contribution of the non-catalytic sites to the exo- or endonucleoytic cleavage of the substrate (1). Substrate and products were separated by reversed-phase HPLC and the cleavage preferences were established by extrapolating the results to zero time. The wild type enzyme shows no special preference for either an endonucleolytic or an exonucleolytic cleavage whereas the mutant lacking p2 shows a clear exonucleolytic pattern, the scission taking place specifically on the phosphodiester bond adjacent to the 3’-end of the substrate. Molecular modelling analysis indicated which specific interactions are involved in each cleavage. No drastic changes on the specificity pattern were observed in the mutant lacking p0. In addition to the RNase A studies, the contribution of the p2 site to the endonucleolytic activity has also been confirmed with the cleavage of the polymeric substrate by eosinophil cationic protein (ECP, RNase 3) (2). The structural analysis of this RNase shows the absence of a p2 site and, consequently, a preferential exonucleolytic cleavage pattern is observed. (1) Cuchillo, C.M., Moussaoui, M., Barman, T., Travers, F., and Nogués, M. V. (2002). Protein Science 11, 117-128. (2) Boix, E., Nikolovski, Z., Moiseyev, G. P., Rosenberg, H. F., Cuchillo, C. M., and Nogués, M. V. (1999). J. Biol. Chem. 274, 15605-15614. Research funded by grants 2000SGR 00064 from Generalitat de Catalunya and BMC2000-0138-C02-01 from DGES, Ministerio de Educación y Cultura, Spain.

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(L4) STRUCTURE AND FUNCTION OF HUMAN RNase Hs Stanley T. Crooke1, Walt Lima and Hongjiang Wu2 1 Chairman and CEO, Isis Pharmaceuticals, Carlsbad, CA, USA 2 Antisense Core Research, Isis Pharmaceuticals, Carlsbad, CA, USA We have cloned and expressed two (RNase H1 and RNase H2) of the members of the human RNase H2 family. We have shown that although human RNase H1 is homologous to the E. coli enzyme, it displays structural and enzymatic differences. New data that bear on the structure, enzymology and biological functions presented.

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(L5) ACTIVE SITE ARCHITECTURE OF RIBONUCLEASE III: MOLECULAR BASIS FOR DOUBLE-STRANDED RNA CLEAVAGE Xinhua Ji, Jaroslaw Blaszczyk, Joseph E. Tropea, Mikhail Bubunenko, Karen M. Routzahn, David S. Waugh, and Donald L. Court National Cancer Institute, NIH, USA. Ribonuclease III (RNase III) from Aquifex aeolicus belongs to the family of divalent-cation-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III enzymes are conserved in all studied bacteria and eukaryotes and known to transform precursor RNAs into mature RNAs and to produce small single-stranded RNAs that regulate stage-specific development and mediate RNA interference. Functionally dimeric RNase III proteins from bacteria consist of an endonuclease domain followed by a double-stranded RNA-binding domain (dsRBD). The three-dimensional structures of various dsRBDs have been previously elucidated. Here we present the crystal structures of the endonuclease domain of A. aeolicus RNase III in its ligand-free form and in complex with Mn2+ (1). Our structures reveal a novel all-helical protein fold. The functional dimer is formed via mainly hydrophobic interactions, including a “ball-and-socket” junction that ensures accurate alignment of the two subunits. The protein fold and dimerization create a valley that can accommodate a dsRNA substrate. Six negatively charged side-chains at each end of this valley form a compound active center. Metal ion binding has significant impact on the formation of two RNA-cutting sites within each compound active center. On the basis of structural, genetic, biological, and biochemical data, we have constructed a hypothetical model of RNase III in complex with dsRNA, which provides the first glimpse at RNase III in action 33 years after the first RNase III enzyme was discovered (2). (1) Blaszczyk, J., Tropea, J.E., Routzahn, K.M., Waugh, D.S., Bubunenko, M., Court, D.L., and Ji, X. (2001). Structure 9, 1225-1236. (2) Zamore, P.D. (2001). Molecular Cell 8, 1158-1160.

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(L6) MUTATIONS IN THE RNase H DOMAIN OF HIV-1 REVERSE TRANSCRIPTASE AFFECT THE INITIATION OF DNA SYNTHESIS AND THE SPECIFICITY OF RNase H CLEAVAGE IN VIVO John G. Julias1, Mary Jane McWilliams1, Stefan G. Sarafianos2, Edward Arnold2, and Stephen H. Hughes1 1HIV Drug Resistance Program, National Cancer Institute at Frederick, P.O. Box B, Fredrick, MD 21702-1201 and 2Center for Advanced Biotechnology and Medicine (CABM) and Rutgers University Chemistry Department, 679 Hoes Lane, Piscataway, NJ 08854-5638. Retroviral reverse transcriptases (RTs) contain a DNA polymerase activity that can copy an RNA or a DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the ppt primer used for plus strand DNA synthesis and to remove the tRNA primer used for minus strand DNA synthesis. The crystal structure of HIV-1 RT in complex with an RNA: DNA duplex allowed the identification of the RNase H primer grip, a structural element proposed to play a role in determining the cleavage specificity of RNase H. The RNase H primer grip makes numerous contacts with the DNA strand of the RNA: DNA duplex. We generated mutations in an HIV-1 based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 RT during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus strand and minus strand primers; these ends can be joined to form 2-LTR circles. Analysis of 2-LTR circle junctions showed that mutations in the RNase H domain affected the specificity of RNase H cleavage. Research funded by NCI and NIGMS.

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(L7) TENSION IN SEMINAL RIBONUCLEASE AS DETECTED BY THE X-RAY ANALYSIS OF VARIOUS FORM OF THE ENZYME Lelio Mazzarella Dipartimento di Chimica, Università degli Studi “Federico II”, Napoli, Italy Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy Seminal ribonuclease (BS-RNase) is a homodimer, more than 80% homologous to the pancreatic enzyme, with two chains linked by two consecutive disulphide bridges. The X-ray analysis revealed that the two subunits in the quaternary assembly of this protein presented the interchange of the N-termini (1). This structural feature, firstly uncovered for this molecule, has been proved not to be uncommon in the assembly of oligomeric proteins and is now generally referred to as domain swapping (2). The biological implications of this phenomenon has been widely discussed. From a structural point of view the dimers are stabilised by the additional interface between subunits provided by the swapping, and this interface is responsible for the maintenance of the dimeric structure. Due to the presence of the consecutive interchain disulphides, however, the seminal enzyme in the native form presents the unique feature of being an equilibrium mixture of two isomeric dimers, the swapped and the unswapped form, although the latter is less abundant. A selective reduction of the interchain disulphides of the swapped dimer (MxM) produces a non-covalent dimer held together by the swapping, whereas the unswapped dimer (M=M) produces a stable monomeric derivative. We are currently engaged in the refinement of the structure of M=M, of the swapped non-covalent dimer, and that of the monomeric species. The crystallographic analysis of the various forms of the seminal enzyme has produced an interesting set of structural data, from which it emerges the role of the intersubunits constraints in defining the details of the quaternary structure of the swapped covalent dimer MxM. 1. D'Alessio, G., Di Donato, A., Mazzarella, L. and Piccoli, R. (1997) in Ribonucleases: structure and function (Riordan, J. F., and D'Alessio, G., Eds.) pp 383-423, Academic press, New York. 2. Schlunegger, M. P., Bennett, M.J. and Eisenberg D. (1997). Adv. Protein Chem. 50, 61-122.

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(L8) STRUCTURAL BASIS OF RNA/DNA POLYPURINE TRACT RECOGNITION BY RNase H of HIV-1 REVERSE TRANCRIPTASE Stefan G. Sarafianos1, Kalyan Das1, Chris Tantillo1, Arthur D. Clark1, J. Ding1, Jeanette Whitcomb2, Paul L. Boyer3, Stephen H. Hughes3, and Edward Arnold1 1Center for Advanced Biotechnology and Medicine (CABM) and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA 2ViroLogic, Inc., 270 E.Grand Avenue, S. San Fransisco, CA 94080 3ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Building 539, Frederick, MD 21702 HIV-1 reverse transcriptase (RT) converts single-stranded viral genomic RNA into double-stranded DNA. First strand DNA synthesis is primed by a host tRNA and creates an RNA:DNA duplex. The genomic RNA in this duplex is cleaved by the RNase H of RT, with the exception of a purine-rich segment (polypurine tract or PPT) that is used as primer for second strand synthesis. We have solved and refined at 3.0 Å resolution the structure of a complex of wild-type RT with an RNA:DNA hybrid whose sequence includes PPT, and Fab fragment of a monoclonal antibody. This structure was compared to the structure of a related complex in which the nucleic acid component was a 19:18 DNA:DNA. Although the overall structures are similar, there are clear differences in elements that could influence RNase H activity. A consistent network of amino acids interacts with the DNA primer strand near the RNase H active site. We propose that these amino acids form an element of the RT structure (the “RNase H primer grip”) that determines the trajectory of the template-primer in relation to the RNase H active site. The RNase H primer grip, in interacting with the DNA primer strand, may effectively control the access of the RNA template substrate to the RNase H catalytic site in a nucleic acid sequence-dependent and structure-dependent manner. Hence, a significant narrowing of the minor groove at the A-T stretch of PPT is likely to contribute to its poor cleavage by misplacing the template strand at the RNase H active site. But this model predicts that groove width alone will not determine RNase H cleavage specificity of HIV RT since the RNase H primer grip will also assist in orienting the template-primer substrate relative to the active site. In addition to interactions of RT with phosphates of the template-primer we observe specific contacts of RT with the 2’-OHs of the RNA strand throughout the length of the template, including at the RNase H catalytic site. The contacts of 2’-OHs with conserved amino acid residues that position the RNA:DNA for catalytic cleavage may also contribute to the ability of the RNase H of RT to specifically recognize RNA:DNA hybrids. The most striking feature of the structure is a surprising “unzipping” of six base pairs in the adenine stretch of PPT, including two unpaired bases that compensate each other, a G-T mismatch, and a base-shift. The structural aberration extends to the RNase H active site and may also be related to the inability of RNase H to cleave within this part of PPT. [Supported in part by NIAID, NCI, NIGMS, and DHHS, under contract with ABL].

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(L9) A DECADE OF PROTEIN ENGINEERING: RNase T1 UNDRESSED Steyaert, J. Dienst Ultrastructuur, Vlaams Interuniversitair Instituut Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium RNase T1 enhances the rate cleavage of the dinucleotide GpC cleavage about 1011 fold by binding to the transition state of the reaction with a dissociation constant of 3x1015M. This lecture focuses on the nature of this transition state and reviews the remarkable affinity of RNase T1 for this highest energy intermediate. The ultimate goal is to generate a comparative picture of all intermolecular interactions of the RNase-RNA complex in the ground state versus the transition state. The introduction of delicate changes in the RNA substrate (chemical synthesis) or at the active site of RNase T1 (site-directed mutagenesis) and the subsequent biochemical and structural analysis of the complex allowed to construct an increasingly detailed picture of the assembly of protein side chains used to control the recognition between the enzyme and the substrate undergoing transphosphorylation.

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(L10) CRYSTALLOGRAPHIC AND FUNCTIONAL STUDIES OF A MODIFIED FORM OF EDN Alexander Wlodawer1, Changsoo Chang1, Dianne L. Newton2, and Susanna M. Rybak3

1Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA 2SAIC Frederick, National Cancer Institute, Frederick, MD 21702, USA 3Developmental Therapeutics Program, National Cancer Institute, Frederick, MD 21702, USA The crystal structure of a post-translationally modified form of eosinophil-derived neurotoxin with four extra residues on its N terminus [(-4)EDN] has been solved and refined at atomic resolution (1 Å). Two of the extra residues can be placed unambiguously, while the density corresponding to two others is poor. The modified N terminus appears to influence the position of the catalytically-important His129, possibly explaining the diminished catalytic activity of this variant. However, (-4)EDN has also been shown to be cytotoxic to a Kaposi’s sarcoma tumor cell line and other endothelial cell lines. Analysis of the structure and function suggests that the reason for cytotoxicity is most likely due to cellular recognition by the N-terminal extension since the intrinsic activity of the enzyme is not sufficient for cytotoxicity and the N-terminal extension does not affect the conformation of EDN.

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(L11) THE FOLD OF BARSTAR AND THE COMPOSITION OF ITS HYDROPHOBIC CORE. Robert W. Hartley National Institutes of Health, Bethesda, Maryland, USA Barstar, the specific inhibitor of the ribonuclease barnase, is a small protein with a relatively large and compact hydrophobic core. It is composed of the sidechains of 22 of its 89 residues and is almost completely surrounded by elements of secondary structure, a three-sranded parallel β-sheet and four α-helices. To what extent does the arrangement of the secondary structure depend on the precise composition of this core? To explore this question, total synthesis of the barstar gene has been carried out with part or all of the hydrophobic core residues randomized to Leu, Ile, Val, Met or Phe. When tested in E. coli expression systems, the fraction of such genes producing functional barstar is very small. Selection for success from a pool of some 108 independently randomized genes by phage display, however, turned up many which could be further selected for and tested by a two-plasmid system which depended on the ability of functional barstar to protect the bacteria from the lethal effect of barnase synthesis. In vitro assay could detect barstar activity in all those that tested positive in vivo. While the distribution of residues in the successful cores was by no means random, there are no positions where substitution for the wild-type residue is not permitted. Several functional barstars were found for which none of the 22 core residues were wild-type. In addition to being able to fit together and just fill the alloted space, the primary requirement for the core residues is that they be spaced properly along the chain so that their sidechains point into the core as defined by the arrangement of secondary structures. An alignment of barstar with nine homologous proteins confirms that the hydrophobicity of the residues that align with barstar's core is strongly conserved, although in no case does the same amino acid occur in all nine.

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(L12) STABILITY OF BARNASE AND BINASE COMPLEXED WITH BARSTAR MUTANTS Alexander A. Makarov1, Vladimir A. Mitkevich1, Alexey A. Schulga2, Yaroslav S. Ermolyuk2, Vladimir M. Lobachov1, Gennady I. Yakovlev1, Robert W. Hartley3, C. Nick Pace4, Mikhail P. Kirpichnikov2

1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871 Moscow, Russia 3National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA 4Department of Medical Biochemistry and Genetics, Texas A&M University, College Station 77843-1114, USA Barstar inhibits both barnase and binase by binding tightly at the active site. We studied the relationship between the free energy of barnase and binase complex formation with barstar mutants and the melting temperature of the complex. Asp 35, Asp 39, and Glu 76 are all important residues in inhibitor binding. To weaken the binding, we studied single barstar mutants in which these residues were replaced with alanines. Scanning microcalorimetry and circular dichroism analyses show that when the binding is weakened, the melting of the complex consists of two transitions: the first is ribonuclease denaturation, and the second is denaturation of the free barstar mutant. Linear correlation between melting temperatures of both ribonucleases complexed with barstar and its mutants and free energies of complex formation was found. Analysis of these relationships has shown that a part of energy of complex formation is spent for structural alteration of ribonuclease within the complex without any contribution to an increase in its thermal stability. This work was supported by NIH FIRCA grant TW0105.

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(L13) THE OXIDATIVE FOLDING OF RNase A: AN OVERVIEW Mahesh Narayan, Ervin Welker, Harold A. Scheraga Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, N.Y. 14853-1301, U.S.A Oxidative folding studies of bovine pancreatic Ribonuclease A have been ongoing in this laboratory. Our previous studies1 revealed that this protein folds through a pre-equilibrium involving ensembles of unstructured species (nS) followed by the rate-determining step in which two des species, des[40-95] and des[65-72], with native-like structure (but lacking bonds 40-95 and 65-72, respectively) are formed from the 3S ensemble. The final step is the oxidation of the fourth and final disulfide bond in each des species, to form N.

Our recent low-temperature studies2 of the oxidative folding mechanism have revealed the presence of the two other native-like des species, des[26-84] and des[58-110], which are kinetic traps that cannot oxidize directly to N. They appear to reshuffle back to their unstructured precursors (3S) and then form N via des[40-95] and des[65-72].

The key factor in determining the kinetic fate of these des species is the relative accessibility of both their thiol groups and disulfide bonds3,4. Productive intermediates tend to be disulfide-secure, meaning that their structural fluctuations preferentially expose their thiol groups, while keeping their disulfide bonds buried. By contrast, dead-end species tend to be disulfide-insecure, in that their structural fluctuations expose their disulfide bonds in concert with their thiol groups.

Based on these oxidative folding studies of RNase A, four generic types of oxidative folding pathways may be identified5,6. These four pathways are distinguished by three factors: whether any disulfide intermediates are folded, whether the folded species are metastable, and whether the metastable species oxidize preferentially or reshuffle.

We combine these results with those of earlier studies to suggest a general three-stage model of oxidative folding of RNase A and other single-domain proteins with multiple disulfide bonds. (1) Rothwarf, D. M. & Scheraga, H. A. (1993) Biochemistry 32, 2680-2689. (2) Welker, E., Narayan, M., Volles, M. J. & Scheraga, H. A. (1999) FEBS Lett. 460, 477-479. (3) Wedemeyer, W.J., Welker, E., Narayan, M. and Scheraga, H. A. (2000) Biochemistry 39, 4207-4216. (4) Welker, E., Narayan, M., Wedemeyer, W.J. & Scheraga, H.A. (2001) Proc. Natl. Acad. Sci. USA, 98, 2312-2316. (5) Narayan, M., Welker, E., Wedemeyer, W. J. & Scheraga, H. A. (2000) Acc. Chem. Res. 33, 805-812. 6) Welker, E., Wedemeyer, W.J., Narayan, M. and H.A. Scheraga (2001) Biochemistry, 40, 9059-9064. This research was supported by the National Institutes of Health Grant GM-24893.

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(L14) HOW TO MAKE YOUR FAVORITE RIBONUCLEASE MORE STABLE Nick Pace Medical Biochemistry Dept., Texas A&M Univ., USA This is an interesting time for the protein-folding field. We are gaining a better understanding of the forces stabilizing proteins and this is improving our progress in predicting structure from sequence and in designing more stable proteins. One good method for stabilizing your RNase is to improve electrostatic interactions on the native protein surface. Another is to replace buried charged groups that do not form intramolecular hydrogen bonds with nonpolar side chains. I will illustrate both of these approaches using examples from microbial RNases. By incorporating seven changes in amino acid sequence, we have been able to increase the melting temperature of RNase Sa to over 120°C using a program developed by Steve Mayo’s group to guide us in selecting stabilizing mutations. These results will be discussed. Finally, as a general principle, I will try and convince you that proteins gain more stability from burying polar groups than they do from burying nonpolar groups.

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(L15) INCREMENTAL LOSS OF STRUCTURE DURING THE UNFOLDING OF BARSTAR Jayant B. Udgaonkar1, K. Sridevi1, G. Lakshmikanth2 and G. Krishnamoorthy2

1National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, 560065, India 2Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India Fluorescence Resonance Energy Transfer (FRET) measurements have been utilized to determine how intramolecular distances in the small protein barstar, change upon unfolding. Equilibrium as well as kinetic studies have been carried out, using the core tryptophan, Trp53, as a fluorescence donor, and a thionitrobenzoic acid moiety (TNB) attached to a cysteine residue acts as an acceptor. Intramolecular distance distributions in the denaturant-induced equilibrium unfolding transition were monitored by time-resolved-FRET methods, in which fluorescence decay kinetics are analyzed by the maximum entropy method (MEM). It is seen that the distance between the donor and acceptor increases incrementally with an increase in denaturant concentration. Native protein is seen to expand progressively through a continuum of native-like forms that achieve the dimensions of a molten globule, whose heterogeneity increases with increase in denaturant concentration, and which appear to be separated from the unfolded ensemble by a free energy barrier. To complement the equilibrium unfolding studies, kinetic unfolding studies, in which unfolding was monitored by changes in donor (Trp53) fluorescence intensity due to FRET, were carried out for four different single cysteine containing mutants of barstar with cysteine residues at positions 25, 40, 62, 82. The FRET efficiency between Trp53 and the solvent-exposed residues (25 and 62) is observed to decrease by 15-20% in a faster kinetic phase compared to the rate of core solvation as monitored by changes in Trp53 fluorescence in the absence of FRET. The FRET efficiency between Trp53 and the buried residues (40 and 82) however, changes at a rate similar to that of core solvation. The FRET experiments clearly indicate that during the unfolding of barstar, an overall expansion of the protein occurs independently of and faster than the core solvation.

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(L16) AN NMR OVERVIEW OF THE ELECTROSTATICS AND DYNAMICS AT THE ACTIVE SITE OF α-SARCIN Marta Bruix, García-Mayoral, F., Pérez-Cañadillas, J.M. and Rico, M. Departamento de Espectroscopía y Estructura Molecular, Instituto de Química Física “Rocasolano”, CSIC, Madrid, Spain. The cytotoxic ribonuclease α-sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a universally conserved rRNA loop. To shed light on its highly specific activity, we have determined its solution structure (1) and its electrostatics properties including pKa values for all titrable groups in the pH range of 3-8 (2). Here, we complement those studies by using NMR methods to determine α-sarcin’s backbone dynamics and to measure the pKa values of variants containing altered residues at the active site.

Relaxation data reveals that α-sarcin behaves as an axial symmetric rotor which tumbles with a correlation time τm of 7.54 ± 0.02 ns. The analysis of the internal dynamics shows that α-sarcin is composed of a rigid hydrophobic core together with a few exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the µs to ms time scale are less abundant and in some cases can be assigned to specific processes. Residues His 50, Glu 96 His 137, Arg 121 and Tyr 48 are part of the active site, though only the first three are directly involved in the catalytic mechanism. In contrast to other parts of the protein involved in recognition, this region shows very restricted internal motions and there is no evidence of slow time scale movements on the µs to ms time scale.

The pKa values of the active site mutants H50Q, E96Q, H137Q and H50/137Q reveal that a complex network of interactions sustains a delicate electrostatic balance in the native α-sarcin structure. E96 and H137 are not very sensitive to charge replacements (variations of about ± 0.3 with respect to wild type pKa values). On the contrary, the pKa of H50 suffers important changes, increasing (>0.5 pKa units) or decreasing (-1.1 pKa units) relative to the native value depending on whether a positive or negative charge substitution is made at the active site.

All electrostatic and dynamic data will be interpreted on the basis of the three-dimensional structure and in relation to the particular role of these residues in the processes of recognition and catalysis. These studies complete the biophysical picture of the protein providing important keys to understand its highly specific mode of action. 1. Pérez-Cañadillas, J. M., Santoro, J., Campos-Olivas, R., Lacadena, J., Martínez del Pozo, A., Gavilanes, J. G., Rico, M, and Bruix, M. (2000). J. Mol. Biol. 299, 1061-1073. 2. Pérez-Cañadillas, J. M., Campos-Olivas, R., Lacadena, J., Martínez del Pozo, A., Gavilanes, J. G., Santoro, J., Rico, M. and Bruix, M. (1998). Biochemistry 37, 15865-15876.

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(L17) CRYSTAL STRUCTURE OF ANGIOGENIN IN COMPLEX WITH ITS ANTIBODY (mAb 26-2F) Gayatri B. Chavali1, Anastassios C. Papageorgiou1, 3 Karen A. Olson2, Robert Sapiro2, James Fett2 and K. Ravi Acharya.1

1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK 2Centre for Biochemical & Biological scince & Medicine, Dept of Pathology, Harvard Medical School, Boston, MA, USA 3Present address: Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, BioCity, Turku 20521, Finland

Angiogenin (Ang) is a mediator of angiogenesis associated with tumours and other pathological conditions. The protein possesses a unique ribonucleolytic activity, which is necessary for its angiogenic capability (1). The three dimensional structure of human Ang is similar to that of bovine pancreatic RNase A (2) where structural differences, such as the presence of a cell-binding region, impart to Ang its characteristic activity. The involvement of Ang in tumour progression elicits interest in the development of inhibitors targeted against its function.

One of such inhibitors of Ang is the murine monoclonal antibody, mAb 26-2F. The mAb is an IgG1k with an IC50 binding affinity of 1.6nM, which neutralises the ribonucleolytic, angiogenic, and mitogenic activities of human Ang (3). It also possesses anti-tumour and anti-metastatic activity against human xenografts in mice (4). Towards the goal of engineering neutralising humanised versions of murine mAb 26-2F, we have determined the structure of the complex between human Ang and its Fab fragment.

Structural analysis shows that only a small proportion of residues constituting two discontinuous segments are involved in binding, which agrees with epitope mapping studies (3). Critical contact regions and framework residues that support the conformational integrity of the complementarity determining regions (CDR) have been identified from the structure. The interactions of various CDR residues with the antigen and in addition, the position of both the CDRs as well as the framework residues can be derived from the study. This information is important for carrying out site-directed mutagenesis of the framework residues from murine to their human counterparts while retaining all the residues that are involved in antigen binding. These details facilitate the design of efficient humanised mAb 26-2F counterparts for therapeutic use. Furthermore, the cell-binding region of Ang, which is structurally distinct from RNase A, was observed to undergo significant conformational change upon binding to the antibody. The complex structure may also help in the design of high affinity peptide-based antagonists of Ang. 1. Shapiro, R. et al. (1986). Characteristic ribonucleolytic activity of human angiogenin. Biochemistry 25, 3527-3532. 2. Acharya, K.R. et al. (1994). Crystal structure of human angiogenin reveals the structural basis for its functional divergence from ribonuclease. Proc. Natl. Acad. Sci. USA 91, 2915-2919. 3. Fett, J.W. et al. (1994). A monoclonal antibody to human angiogenin. Inhibition of ribonucleolytic and angiogenic activities and localization of the antigenic epitope. Biochemistry 33, 5421-5427.

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4. Olson, K.A. et al. (2002). Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody. Int. J. Cancer 98, 923-929. This work was supported by the Cancer Research UK and the Medical Research Council, UK (Grants to K. R. A.). We thank our collaborators Drs. James Fett, Karen Olson and Robert Shapiro at CBBSM, Harvard Medical School, USA.

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(L18) GUEST-HOST CROSSTALK IN AN ANGIOGENIN/RNase A CHIMERIC PROTEIN Daniel E. Holloway1, Robert Shapiro2, Michelle C. Hares1, Demetres D. Leonidas1,3, and K. Ravi Acharya1 1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom. 2Center for Biochemical and Biophysical Sciences and Medicine, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, U.S.A. 3Present address: Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece.

Bovine pancreatic ribonuclease (RNase A) and human angiogenin (Ang) share 33% sequence identity but have significantly different functions. RNase A is a digestive enzyme with high catalytic efficiency toward general RNA substrates, whereas Ang is a potent inducer of angiogenesis and is only weakly ribonucleolytic. We are interested in the structural basis for this difference.

Chimeric proteins are powerful tools for investigating functional differences between related proteins (1). One such chimera ("ARH-I"), in which angiogenin residues 58-70 were replaced by residues 59-73 of RNase A, was prepared in order to evaluate the dissimilarity between the two proteins in this region (2). The protein has intermediate ribonucleolytic potency and no angiogenic activity.

We present a 2.1 Å resolution crystal structure of ARH-I that reveals the molecular basis for these characteristics. The RNase A-derived (guest) segment adopts a structure largely similar to that in RNase A, and successfully converts this region from a cell-binding site to a purine-binding site. At the same time, its presence causes complex changes in the angiogenin-derived (host) portion that account for much of the increased RNase activity of ARH-I.

To our knowledge, ARH-I is the first chimeric protein for which it has been possible to explain complex functional characteristics in terms of guest-host interactions. Such interactions probably occur more generally in protein chimeras, emphasizing the importance of direct structural information for understanding the functional behaviour of such molecules.

1. Cunningham, B.C., Jhurani, P., Ng, P. and Wells, J.A. (1989). Science 243, 1330-1336. 2. Harper, J.W. and Vallee, B.L. (1989). Biochemistry 28, 1875-1884. Research funded by Cancer Research UK Grant SP2354/102 (to K.R.A.), Medical Research Council UK Grant 9540039 (to K.R.A.), and the National Institutes of Health Grant CA-88738 (to R.S.).

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(L19) ELECTROSTATIC INTERACTIONS IN RIBONUCLEASE Sa AND A CHARGE REVERSE VARIANT Douglas, V, Laurents 1,2, Huyguens-Despointes, B.M.P.1, Bruix, M.2, Thurkill, R.1, Schell, D.1, Newsom, S.1, Shaw, K. L.1, Treviño, S.1, Rico, M.2, Scholtz, J. M.1, and Pace, C.N.1 1Department of Medical Biochemistry and Genetics, Texas A&M University School of Medicine, College Station TX 77843, U.S.A. 2Departamento de Espectroscopía y Estructura Molecular, Instituto de Química Física “Rocasolano”, CSIC, Madrid, Spain We have been using NMR to investigate the solution structure and dynamics of RNase Sa (1). Here, we report the determination of the pKas of titratable groups in RNase Sa by NMR. Asps 33 and 84, which are buried and form intramolecular hydrogen bonds show depressed pKas, whereas Asp 79, which is buried and lacks intramolecular hydrogen bonds, has an elevated pKa. RNase Sa´s seven tyrosine residues and N-terminal ammonium group all show elevated pKas and the ionization of buried and hydrogen bonded tyrosines is likely coupled to alkaline unfolding. The chief goal of this research is to compare the pKas of wild type RNase Sa with those of a charge reverse variant (5K) containing five carboxylate to lysine substitutions. By NMR, the structure of the 5K variant was observed to be highly similar to the wild-type structure. The pKas of the groups dropped in the variant, as expected form its increased positive net charge. The N-terminal ammonium group and Glu 14 show large pKa decreases relative to their wild type values which could be explained by changes in short range charge-charge interactions. Other groups have smaller pKa changes. A very good correlation was obtained between the experimental ∆pKa values and ∆pKa values calculated using a simple coulombic model that only considers charge-charge interactions. This is evidence that the charge substitutions are responsible for most of the pKa shifts. The best correlation was obtained using a surprisingly high value of 45 for the protein’s dielectric constant. 1. Laurents, D.V., Pérez-Cañadillas, J.M., Santoro, J., Rico, M., Schell, D., Pace, C. N. & Bruix, M., (2001) Proteins Struct. Funct. Genet. 44, 200-211.

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(L20) CRYSTAL STRUCTURE OF RNase Sa3 - A MICROBIAL RIBONUCLEASE WITH CYTOTOXIC ACTIVITY Jozef Sevcik1, Lubica Urbanikova1, Peter A. Leland2, Ronald T. Raines2

1Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic 2Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA Ribonuclease Sa3 is produced by Streptomyces aureofaciens. Crystallization of the recombinant enzyme under different conditions using the hanging drop vapour diffusion method yielded two crystal forms. X-ray diffraction data from crystal form I were collected at 100 K to 2.0 Å, and data from crystal form II were collected at room temperature to 1.7 Å resolution on the EMBL beamlines X31 and BW7A, respectively, at the DORIS storage ring, DESY, Hamburg. Crystal form I is trigonal (P3121) and crystal form II is tetragonal (P41212) with unit cell parameters of a = b = 64.7, c = 69.6 Å, β= 120° and a = b = 34.0, c = 147.2 Å, respectively. The asymmetric units of both crystal forms contain one molecule of the enzyme, which corresponds to a VM = 3.8 Å3 Da-1 with solvent content of 68 % and VM = 1.9 Å3 Da–1 with solvent content of 37 %, respectively (1). Both structures were solved by molecular replacement method using the program AMoRe with RNase Sa as a search model and refined with the program REFMAC5. Refinement of crystal form I and II converged with crystallographic R - factors of 15.5% and 18.6%, respectively. The two structures are almost identical, but a few differences are caused by different crystal packing and/or data collection conditions. There is a high degree of disorder of N - terminal residues and the Trp79 side chain in crystal form II. In contrast, these residues are apparent in the electron density of crystal form I, in which the N - terminal tails of two neighbouring molecules are intertwined with each other. A most interesting property of RNase Sa3 is its cytotoxic activity against the human erythroleukemia cell line K-562. That activity is comparable to that of Onconase® and bovine seminal ribonuclease, which are pancreatic ribonucleases. These two enzymes evade ribonuclease inhibitor, which is one of the conditions for a ribonuclease to be cytotoxic. No microbial ribonuclease is inhibited by porcine ribonuclease inhibitor. Nonetheless, only a few are known to have antitumour activity (e.g., α-sarcin and restrictocin, which are in the eukaryotic subfamily of T1 microbial ribonucleases). RNase Sa3 is the only cytotoxin in the prokaryotic subfamily of T1 microbial ribonucleases. The mechanism by which RNase Sa3 enters mammalian cells, which is a prerequisite for its cytotoxicity, is unknown. Biochemical study and determination of the tertiary structure of RNase Sa3 is likely to shed light on this mechanism. 1. Hlinkova, V., Urbanikova, L., Krajcikova, D., Sevcik, J. (2001). Acta Cryst. D57, 737-739.

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(L21) THE REMARKABLE ACTIVITY OF HP-RNase ON DOUBLE-STRANDED RNA Sorrentino, S.1, Naddeo, M.1, Lombardi, M.1, Russo, A.2, D’Alessio, G.1 1Department of Biological Chemistry, University of Naples “Federico II”, Napoli, Italy 2Department of Life Sciences, Second University of Naples, Caserta, Italy

Under physiological salt conditions double-stranded RNA is resistant to the action of bovine pancreatic ribonuclease (RNase A). The reason is that the sterical requirements for the “in line” mechanism of the enzymatic degradation of RNA cannot be met, given the secondary structure of dsRNA. However, several mammalian pancreatic-type (pt) RNases are able to degrade (more or less efficiently) dsRNA under conditions in which the action of RNase A is essentially undetectable. This ability has been related to the density of positive charges on the RNase protein. The mechanism hypothesized is that degradation of dsRNA by these RNases occurs as a consequence of a previous “destabilization” of the nucleic acid secondary structure. This was in turn related to the number and location of basic charges on the enzyme protein. A comparative analysis of the aminoacid sequences of some ptRNases very active on dsRNA, indicated a possible correlation between location of basic residues on the ribonuclease surface and activity against dsRNA (1).

Human pancreatic-type (HP-RNase), whose activity on dsRNA is very potent (about 500 times higher than that of RNase A), shows a peculiar surface electrostatic potential. It has six basic amino acids at positions where RNase A has instead neutral residues. In particular, in the human enzyme, it was found by modeling that R4 and K102 are located near the binding site for RNA. Furthermore, at position 38, close to the positive side chain of R39, a negative charge is present in RNase A, whereas in HP-RNase there is no negative charge for the substitution of a Gly for Asp. To verify the role of these residues in the mechanism of dsRNA degradation by HP-RNase, we prepared by site-directed mutagenesis and protein expression in E. coli, as described (2), four variants of HP-RNase: R4A, G38D, K102A, and the triple mutant R4A/G38D/K102A (ADA). Their kinetic parameters were determined with poly(A):poly(U) as double helical substrate, using recombinant HP-RNase and bovine RNase A as controls. The enzymatic assays have shown that all three single mutations decrease the activity of HP-RNase by a factor of 2.5, and the activity of the triple mutant ADA was 27 times lower than that of the wild-type enzyme. On the contrary, toward yeast RNA or synthetic single-stranded polyribonucleotides, the four variants showed activity values similar to those of wtHP-RNase. When tested with synthetic poly(dA-dT):poly(dA-dT) in thermal melting assays, the three single variants were found to have a lower helix-destabilizing capability, while the triple mutant ADA was totally inactive. These results indicate that HP-RNase degrades dsRNA more efficiently than other ptRNases because of its stronger local positive electrostatic potential developed by some basic residues, which cooperatively contribute to the binding and destabilization of the double-helical RNA molecule. 1. Libonati, M., Sorrentino, S. (2001) Methods Enzymol. 341, 234-248. 2. Russo, A., Antignani, A., D’Alessio, G. (2000) Biochemistry 39, 3585-3591.

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(L22) CONFORMATIONAL REQUIREMENTS FOR THE DESIGN OF SPECIFIC INHIBITORS OF EDN: CRYSTAL STRUCTURES OF EDN AT ATOMIC RESOLUTION. G. Jawahar Swaminathan*, Daniel E. Holloway, Matthew D. Baker and K. Ravi Acharya Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K. *Present address: EBI, Hinxton, Cambridge, U.K.

Human Eosinophil-derived neurotoxin (EDN) is a small, basic protein that

belongs to the ribonuclease A superfamily. EDN displays antiviral activity and also causes the neurotoxic Gordon phenomenon following an intrathecal injection into rabbits. Although EDN and RNase A have similar molecular topologies, they share only 36% sequence identity, and the ribonucleolytic activity of EDN is between 3- and 30-fold lower than that measured for RNase A. However, this enzymatic activity is essential for the observed cytotoxic, neurotoxic and antiviral activity of EDN. There are subtle differences in substrate specificity between RNase A and EDN, making the task of identifying specific inhibitors of EDN activity more challenging. Additionally EDN also contains a characteristic Trp-X-X-Trp sequence signature between residues 7 and 10, specifying the site of an unusual C-mannosylation of Trp7.

In order to address the substrate specificity of EDN as well as design tight-binding inhibitors for the same, we have determined the crystal structures of recombinant EDN at atomic resolution (1), and with different mononucleotide and polynucleotide inhibitors at resolutions ranging between 0.98 Å and 1.8 Å respectively. The results of these rigorous and systematic studies provide a detailed 3-dimensional map of the conformational freedom of various active subsite residues in EDN, as well as address the importance of certain subsites (P0 and B1) for the design of potent inhibitors. The results obtained from our studies are comparable with the values obtained from enzyme kinetics with these inhibitors. The atomic resolution (0.98 Å) structure of EDN and the minimum conformational requirements that a potent inhibitor should fulfill to be an efficient and specific inhibitor of EDN enzymatic activity will be presented during the meeting. Swaminathan, G.J., Holloway, D.E., Veluraja, K. and Acharya, K.R. (2002). Biochemistry 41, 3341-3352. This work is supported by the Medical Research Council (U.K.) (Programme Grant 9540039) to K.R.A

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(L23) GENETICS AND GENOMICS OF EUKARYOTIC RIBONUCLEASES Pam Green1, Jim Kastenmayer2, Gustavo MacIntosh1, and Nikki LeBrasseur3. 1Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA 2Plant Research Laboratory, Michigan State University, E. Lansing, MI, USA 3Present Address: Journal of Cell Biology, Rockefeller University Press, NY, USA

We have addressed the function of ribonucleases in both plant and yeast systems. Plants lack homologs of Xrn1p, the major yeast cytoplasmic 5’ to 3’ exoribonuclease that functions in mRNA decay. However, Arabidopsis appears to compensate for this at least in part by the presence of a cytoplasmic version of the yeast nuclear 5’ to 3’ exoribonuclease, Rat1p/Xrn2p (1). This Arabidopsis enzyme, called AtXRN4, has been shown to have 5’ to 3’ exoribonuclease activity that is blocked by poly(G) in yeast. Because AtXRN4 is expressed in Arabidopsis at a lower level than expected based on yeast, it may have a more specialized function. We isolated a T-DNA insertion mutant of AtXRN4 and then used microarray analysis followed by RNA gel blots experiments to identify several potential substrates. Similar strategies should reveal substrates for other cytoplasmic ribonucleases in Arabidopsis.

Even less is known about the biological function of extracellular ribonucleases such as those in the ubiquitous T2 superfamily. We have taken a genetic approach to address this question in yeast and Arabidopsis. Yeast cells that carry a mutated version of RNY1, the only member of this family present in their genome, are larger than wild type cells, as well as osmo- and temperature-sensitive for growth. Accordingly, RNY1 expression is increased by heat shock and osmotic stress (2). The Arabidopsis genome contains five genes corresponding to members of the T2 family. Interestingly, plants lacking only one of such genes, RNS1, exhibit characteristics that parallel the yeast mutants. The rns1-2 seedlings are larger than wild type. This phenotype is particularly evident in roots; roots of two-week-old mutants are longer than wild-type roots, while plants overexpressing RNS1 have roots that are shorter than wild type. RNS1 is also induced by several stress conditions, including high salt concentrations and wounding. Our results suggest that RNases from the T2 family have previously unexpected roles during growth and development. 1. Kastenmayer, J., Green, P.J. (2000) Proc. Natl. Acad. Sci. USA 97, 13985-13990. 2. MacIntosh, G.C., Bariola, P.A., Newbigin, E., Green, P.J. (2001) Proc. Natl. Acad. Sci. USA 98, 1018-1023. Research funded by grants from the National Science Fundation (NSF) and the Department of Energy (DOE).

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(L24) A UNIQUE GROUP OF NEW RIBONUCLEASES TARGETING SPECIFIC tRNases Haruhiko Masaki1, Tetsuhiro Ogawa1, Kozo Tomita2, Sakura Inoue1, Shunsuke Yajima3

1Department of Biotechnology and 2Department of Integrated Biosciences, The University of Tokyo, Japan, 3Department of Bioscience, Tokyo University of Agriculture, Japan

Colicins are toxic proteins produced by respective Col plasmids and kill Escherichia coli cells that do not have the plasmids. Colicins first bind to specific surface receptors and then enter the sensitive cells toward final targets. Three types of modes of killing action have been known; (1) colicins E1, K, Ia and B form ion-channels in the cytoplasmic membrane, (2) colicins E2, E7, E8 and E9 degrade DNA, and (3) colicin E3 cleaves a specific site of 16S-rRNA within 70S ribosome to stop protein synthesis. In the latter nuclease-type colicins, producer cells are protected from the colicin action by plasmid-coded specific inhibitors, referred to as immunity proteins. Base on phenotypic analogy to the case of E3, colicins E5 and D have long been believed to stop protein synthesis by inactivating ribosomes. We recently revealed that the actual targets of colicins E5 and D are not ribosomes but tRNAs.

Colicin E5 specifically cleaves tRNAs for Tyr, His, Asn and Asp between the first and second letters of those anticodons. On the other hand, colicin D specifically cleaves all the four isoaccepting tRNAs for Arg at the 3' end of their anticodon loops. Both reactions give a 2', 3'-cyclic phosphate and a 5' OH in the ends of RNA products and these activities are exclusively carried on their C-terminal small RNase domains referred to as CRDs, where respective Imm proteins tightly bind. Colicins E5 and D thus proved to form a new unique group of ribonucleases targeting specific tRNAs as "tRNases". In spite of these similar properties and phenomenal consequences they bring about, there is no sequence homology between colicins E5 and D. Moreover, modes of substrate recognition and catalytic mechanisms seem quite different between the two colicins. In particular, E5-CRD is an “RNA restriction enzyme” that cleaves the GU sequence, and has a unique catalytic mechanism in which no histidine residues are involved. (1) Ogawa, T., et al. (1999) Science, 283, 2097-2100. (2) Tomita, K., et al. (2000) Proc. Natl. Acad. Sci. USA, 97, 8278-8283.

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(L25) EOSINOPHIL RIBONUCLEASES AND ANTI-VIRAL HOST DEFENSE: THE EVOLVING STORY Helene F. Rosenberg1, Joseph B. Domachowske2, Jianzhi Zhang3, Joanne M. Moreau1, Kimberly D. Dyer1, Nora L. Vasquez1

1Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, Maryland, USA 2Department of Pediatrics, SUNY Upstate Medical University, Syracuse, New York, USA 3Department of Ecology and Evolution, University of Michigan, Ann Arbor, Michigan, USA Eosinophils remain among the most enigmatic of cells, as our appreciation of their detrimental activities—e.g….asthma and allergic disease--far outweighs our understanding of their beneficial effects. Among the major secretory effector proteins of eosinophils are two ribonucleases—the eosinophil-derived neurotoxin (EDN/ RNase 2) and eosinophil cationic protein (ECP / RNase 3) in primates and their highly divergent orthologs, the eosinophil-associated ribonucleases (EARs) in rodents. The rapid diversification observed among these ribonucleases suggested that their ultimate target(s) might be similarly efficient at generating sequence diversity while maintaining an unalterable susceptibility to ribonucleolytic cleavage. These thoughts have prompted us to consider a role for the eosinophil ribonucleases in host defense against single-stranded RNA virus pathogens. We will detail our studies of the antiviral activity of the human eosinophil ribonucleases against the human pathogen, respiratory syncytial virus (RSV), and that of the mouse EARs against a related, natural rodent pathogen, pneumonia virus of mice (PVM), and present our results on the differential expression of ribonuclease genes in the setting of primary virus infection in vivo (1). 1. Rosenberg, H. F. and Domachowske, J. B. (2001) Eosinophils, eosinophil ribonucleases and their role in host defense against respiratory virus pathogens. J. Leukoc. Biol. 70, 691-698.

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(L26) ADAPTIVE EVOLUTION OF A DUPLICATED PANCREATIC RIBONUCLEASE GENE IN A LEAF-EATING MONKEY Jianzhi Zhang1, Ya-ping Zhang2, and Helene F. Rosenberg3

1 Departments of Ecology and Evolutionary Biology and Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA; Email:jianzhi@umich.edu 2 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China 3 Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA Although complete genome sequences of over 60 representative species have shown the abundance of duplicated genes in all three domains of life, the roles of gene duplication in organismal adaptation and biodiversity remain poorly understood. Moreover, the evolutionary forces behind the functional divergence of duplicated genes are often unknown, leading to disagreement on the relative importance of positive Darwinian selection versus relaxation of functional constraints in this process. Methodologically, earlier studies relied largely on DNA sequence analysis but lacked functional assays of duplicated genes, thus frequently generated contentious results. Here we use both computational and experimental approaches to address these questions in a study of the pancreatic ribonuclease gene (RNASE1) and its duplicate (RNASE1B) in a leaf-eating colobine monkey, douc langur. We show that RNASE1B has evolved rapidly under positive selection for enhanced ribonucleolytic activity in an altered microenvironment, a response to increased demands for the enzyme in digesting bacterial RNAs. At the same time, the ability to degrade double-stranded RNA, a non-digestive activity characteristic of primate RNASE1, has been lost in RNASE1B, indicating functional specialization and relaxation of purifying selection. Our findings provide direct evidence for the contribution of gene duplication to organismal adaptation and demonstrate the power of combinatory approaches of sequence analysis and functional assays in delineating the molecular basis of adaptive evolution.

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(L27) A NOVEL CYTOTOXIC RIBONUCLEASE FROM AMPHIBIAN OOCYTES Wojciech Ardelt, Eugene Vidunas, Shailendra Saxena, Hung-Suen Lee, Abha Saxena, Alejandro Viera, and Kuslima Shogen Alfacell Corporation, Bloomfield, NJ, USA. Onconase (ranpirnase), the first described amphibian cytotoxic ribonuclease and its natural variant were originally isolated in our laboratory more than decade ago (1,2). They are now well characterized enzymes and Onconase is presently in Phase III clinical trials in the US and in Europe for malignant mesothelioma. We now report on the discovery and characterization of a novel cytotoxic ribonuclease from the same source (Rana pipiens oocytes). We have isolated four natural variants of this enzyme and determined their complete amino acid sequences. The sequences were checked against sequences registered with the Protein Information Resource and were found to be original. All variants are single chain proteins of 114 amino acid residues. The variants are substantially similar to Onconase (36-39% identity) as well to Ribonuclease A. The three catalytic residues are strictly conserved at the homologous positions. Eight cysteine residues are paired. Molecular masses, as calculated from amino acid sequences vary from 12968 to 13077 daltons and the isoelectric points from 9.95 to 10.16. The variants were found N-glycosylated at two identical sites.

Recombinant forms of these variants were obtained by cloning the synthetic genes in the pET-11d or pET-22b plasmid vectors and expressing them in E.coli BL21(DE3) cells.They were characterized along with the natural variants.

All variants were able to degrade highly polymerized RNA as well as other Ribonuclease substrates including dinucleotides. Their pH optima for enzymatic activity varied from 7.2 to 7.8 and were distinctly different from that of Onconase (6.0). Natural variants were over hundred times less active than Onconase towards highly polymerized RNA as substrate. Despite this difference their in vitro cyototoxicity against cancer cell lines was of the same order as that of Onconase.

The recombinant variants were fully active enzymatically and fully cytotoxic to the cancer cell lines tested. It seems, therefore, that glycan moieties found in natural variants are not the prerequisite for cytotoxicity. 1. Ardelt, W., Mikulski, S.M., and Shogen, K. (1991). J.Biol. Chem. 266, 245-251. 2. Ardelt, W., Lee, H-S., Randolph, G., Viera, A., Mikulski, S.M., and Shogen, K. (1996). Proc.4th Int. Meeting on Ribonucleases, Groningen, S3P6.

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(L28) CELLULAR AND ANTIVIRAL APPLICATIONS OF HAIRPIN RIBOZYMES Attila A. Seyhan, Michele T. Shields, Cynthia A. Villarimo, Danielle Vitiello, Zhenxi Zhang and John M. Burke University of Vermont, Department of Microbiology and Molecular Genetics, Burlington, VT, USA 05405 Hairpin ribozymes are endoribonucleases that carry out a reversible RNA cleavage reaction, generating products with 5’- OH and 2’,3’-cyclic phosphate termini. Their sequence specificity is largely due to the formation of approximately ten canonical base-pairs between ribozyme and substrate, and so can be manipulated in the laboratory. To explore the application of ribozymes as experimental therapeutics, we have focused on developing ribozymes to inhibit the replication of Sindbis virus, a member of the alphaviridae that replicates very aggressively in the cytoplasm of mammalian and insect cells. We have systematically identified and addressed several problems in achieving reproducible and efficient ribozyme inhibition of viral replication. These include (1) identification of suitable target sites within the viral RNA genome, (2) expression of small ribozymes within mammalian cells, (3) cytoplasmic localization of ribozyme transcripts, and (4) the site and mechanism of action of antiviral ribozymes.

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(L29) RIBONUCLEASE A AS A CHEMOTHERAPEUTIC Ronald T. Raines Departments of Biochemistry and Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA A mammalian secretory ribonuclease, such as ribonuclease A, can be made to be toxic to mammalian cells (1). This cytotoxicity relies on the ribonuclease maintaining high ribonucleolytic activity and conformational stability while gaining the ability to evade the ribonuclease inhibitor protein that is endogenous to the cytosol of mammalian cells (2). This principle has been demonstrated with multiple variants of both ribonuclease A and human pancreatic ribonuclease. Recent work has revealed the pathway by which secretory ribonucleases gain access to cellular RNA as well as means to enhance the ability of ribonucleases to target specific human cells. Leland, P.A. and Raines, R.T. (2001). Chem. Biol. 8, 405-413. Leland, P.A., Schultz, L.W., Kim, B.-M., and Raines, R.T. (1998). Proc. Natl. Acad. Sci. USA 95, 10407-10412. Research funded by grant CA73808 (NIH).

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(L30) RN321: ONCONASE BASED TARGETED THERAPEUTIC Susanna Rybak,1 Dianne Newton, 2 Kuslima Shogen, 3 Stanley Mikulski, 3 and Edward Sausville 1 1 Developmental Therapeutics Program, National Cancer Institute-Frederick, USA 2 SAIC Frederick, Frederick, USA 3 Alfacell Corporation, Bloomfield, USA RNases linked to antibodies can become target-specific drugs. Onconase linked by a disulfide bond to RFB4, an anti-CD22 antibody, specifically kills non-Hodgkin’s lymphoma cells in vitro and in murine models of human lymphoma. The therapeutic index or difference between killing a target cancer cell and non-target healthy cell is greater than 200,000 fold. These striking results have resulted in approval for a clinical trial by the National Cancer Institute, USA. This targeted RNase is now termed RN321. Funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000.

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(L31) RECENT DEVELOPMENTS IN ANTI-ANGIOGENIN THERAPY Robert Shapiro1,2, Richard Y.T. Kao1, Jeremy L. Jenkins1, Karen A. Olson1,2, and James W. Fett1,2 1Center for Biochemical & Biophysical Sciences & Medicine and 2Department of Pathology, Harvard Medical School, MA, USA Angiogenin (ANG) is an angiogenic protein in the pancreatic RNase superfamily whose expression has been shown to be elevated in many human cancers. Monoclonal antibodies and an antisense oligonucleotide directed against ANG are highly effective at inhibiting the establishment and/or metastatic spread of several distinct types of human tumors in athymic mice. Strikingly, recent metastasis experiments with antisense have revealed a strict correlation between the extent of reduction in ANG expression in the primary tumors and the degree of protection achieved. These findings identify ANG as a potentially important target for anticancer therapy. As ANG antagonists for clinical use, low molecular weight compounds would offer tremendous advantages over the agents (proteins, oligonucleotides) tested extensively in mice thus far. One attractive strategy for developing such inhibitors is to target the ribonucleolytic active site of ANG, which has been demonstrated to be essential for angiogenicity. Earlier efforts in this direction had focused on small nucleotides. Although some of the compounds tested had mid-to-upper nanomolar Ki values with RNase A and other homologues, Ki values with ANG were no better than ~500 µM under physiological conditions. As an alternative, we have now devised a high-throughput screening assay for ANG and used it to conduct a wider search for low molecular weight inhibitors. Screening of 18,310 compounds from the National Cancer Institute (NCI) Diversity Set and ChemBridge DIVERSetTM yielded 15 hits that inhibit the enzymatic activity of ANG with Ki values < 100 µM. Two inhibitors seemed to be particularly amenable to both rational design and combinatorial approaches for improving affinity, suggesting that they might have promise as potential leads. One of these, NCI compound 65828 (8-amino-5-(4'-hydroxy-biphenyl-4-ylazo)-naphthalene-2-sulfonate; Ki = 81 µM), was tested for antitumor activity in athymic mice. Local treatment with modest doses of 65828 significantly delayed the formation of subcutaneous tumors from human prostate (PC-3) and colon adenocarcinoma (HT-29) cancer cells. ANG is the likely target involved because (i) a 65828 analogue with much lower activity against the enzymatic activity of ANG failed to exert any antitumor effect, (ii) the average number of interior blood vessels was smaller in the treatment groups than in the controls, and (iii) 65828 appears to have no direct effect on the tumor cells. Our findings provide considerable support for the targeting of the enzymatic active site of ANG as a strategy for developing new anticancer drugs. This work was supported by the National Institutes of Health, U.S.A. (Grant CA-88738 to R.S.) and the Endowment for Research in Human Biology, Inc.

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(L32) THE EOSINOPHIL GRANULE RNASES: CURRENT UNDERSTANDING G.J. Gleich Dept of Dermatology, University of Utah Medical school, Salt Lake City, Utah, USA Shortly after the discovery of the eosinophil, its presence in peripheral blood was related to parasitic disease and bronchial asthma. By the early part of the last century the eosinophil had been associated with anaphylaxis, and the concept that it might alter the severity of anaphylaxis had been put forth. For many years this hypothesis was aggressively pursued, and it was only after the discoveries that eosinophils are able to kill parasites and that eosinophil granule and proteins are rich in cytotoxic molecules that it was abandoned. Over the past three decades the eosinophil has been extensively investigated and its biochemistry and physiology elucidated. The results of these studies have shown that the eosinophil is armed with a series of cytotoxic granule proteins that are able to kill helminths and to inflict damage on tissues. For example, exposure of larval forms of parasites, such as microfilariae of Onchocerca volvulus and Trichinella spiralis, to eosinophils causes the death of the organisms. In addition, eosinophils and their granule proteins are able to damage normal tissues, and in many diseases the presence of eosinophils is strongly associated with tissue dysfunction. The mechanisms of the eosinophil-mediated damage to tissues have also been thoroughly investigated. These studies have shown that the granule proteins including the eosinophil peroxidase, the major basic proteins and the eosinophil associated ribonucleases are important mediators of damage.

The eosinophil ribonucleases were discovered during biochemical analyses of granule proteins, and both the eosinophil-derived neurotoxin and the eosinophil cationic protein are homologous to pancreatic ribonuclease. Both of these molecules possess ribonuclease activity, but the eosinophil-derived neurotoxin is about 10 to 50 times more active than is the cationic protein. Remarkably both of these proteins cause a neurotoxin reaction in rabbits and guinea pigs referred to as the Gordon phenomenon, and it appears that ribonuclease activity is needed for expression of neurotoxicity. The cDNAs and the genes for these molecules have been cloned and sequenced, and both lie in a cluster on chromosome 14, along with the gene for angiogenin.

In this presentation the role of the eosinophil associated ribonucleases in immunity will be discussed as well as the current status of our information concerning the multiplicity of these RNases in humans and rodents.

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(L33) BIOLOGICAL FUNCTIONS OF RNase-L Bret A. Hassel1,2, Krish Chandrasekaran3, Xiao-Ling Li1, Zara Mehrabyan3 and Carianne Judge2

1Greenebaum Cancer Center, 2Program in Cell and Molecular Biology 3Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD, USA The 2-5A system is an interferon (IFN)-regulated RNA decay pathway comprised of three major biochemical activities. A family of 2-5A synthetases is induced by IFN and, when activated by double-stranded RNA (dsRNA), polymerizes ATP into 2’,5’-linked oligoadenylates (2-5A). 2-5A, in turn, binds the latent endoribonuclease, RNase-L, resulting in its dimerization and activation. An RNase-L inhibitor, RLI, interacts with RNase-L and attenuates its activity. RNase-L mediates the biological functions of the 2-5A pathway including antiviral, growth inhibitory and proapoptotic activities, through the degradation of single stranded viral and cellular RNAs. We have utilized cells derived from RNase-L-deficient mice, and cells transfected with RNase-L expression vectors to explore the roles of RNase-L independent of virus infection. These studies revealed two novel functions for RNase-L in senescence and excitotoxic mitochondrial stress. Specifically, we found that while most cell lines apoptose in response to ectopic RNase-L expression, RNase-L-transfected human diploid fibroblasts (HDFs) remain viable, but undergo senescence more rapidly than their vector-transfected counterparts. Consistent with this observation, RNase-L-transfected HDFs exhibited a reduced in vitro lifespan, a senescent morphology, and increased senescence-associated beta-galactosidase staining. Studies into a potential role for RNase-L in mitochondrial (mt) gene expression stemmed from our finding that mtRNAs were destabilized in response to excitotoxic stress in neurons (1), and the recent report demonstrating RNase-L-dependent regulation of mtRNAs in IFN treated cells (2). Treatment of wild type mouse embryonic fibroblasts (MEFs) with a sodium ionophore to induce mt stress dramatically reduced mtRNAs. In contrast, mtRNAs from RNase-L deficient MEFs were unaffected in these conditions, suggesting that RNase-L mediates mtRNA turnover in response to mt stress. Current work is focused on confirming the physiologic role of RNase-L in excitotoxic stress in neurons, and determining the role of altered mt gene expression in RNase-L-induced apoptosis. Interestingly, ageing associated defects in mitochondrial function are well established, suggesting a mechanistic link between the activities of RNase-L in mtRNA regulation and senescence. (1) Chandrasekaran, K, Liu, LI, Hatanpaa, K, Shetty, U, Mehrabyan, Z, Murray, PD, Fiskum, G, and Rapoport, SI. (2001). Mitochondrion. 1, 141-150. (2_ Le Roy F, Bisbal C, Silhol M, Martinand C, Lebleu B, Salehzada T. (2001). J. Biol Chem. 276, 48473-82. Research funded in part by grant # AI39608 to BAH from NIAID, NIH.

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(L34) NUCLEAR FUNCTION OF ANGIOGENIN Zheng-ping Xu1, James F. Riordan1 and Guo-fu Hu1,2

1Center for Biochemical and Biophysical Sciences and Medicine, and 2Department of Radiology, Harvard Medical School, One Kendall Square, Building 600, Floor 3, Cambridge, MA 02139, USA

Angiogenin, a weak ribonuclease but a potent angiogenic protein, is translocated to the nucleus of endothelial cells where it accumulates in the nucleolus and binds to DNA. Nuclear translocation is dependent on cell density, is related to cell growth and is necessary for angiogenesis. Because angiogenin is concentrated in the nucleolus where ribosomal biogenesis takes place, we investigated the involvement of nuclear angiogenin in rRNA synthesis. Exogenous angiogenin enhances the production of 45S rRNA precursor in endothelial cells. Reduction of endogenous angiogenin by antisense oligonucleotides decreases the transcription of 45S rRNA. In isolated endothelial nuclei, angiogenin stimulates the synthesis of nascent RNA in an α-amanitin independent manner, indicating RNA polymerase I is involved in angiogenin-stimulated transcription. Indeed, Northern Blotting analysis shows that the initiation site sequence of 45S rRNA is among the products stimulated by angiogenin. An angiogenin-binding element (ABE) from the non-transcribed region of rDNA has been identified. It has the sequence of 5’-CTCTCTCTCTCTCTCTCCCTC-3’, and exhibits angiogenin-dependent promoter activity in a promoter-less luciferase reporter system. These results suggest that the nuclear function of angiogenin is related, at least in part, to its capacity to induce rRNA synthesis. Because the growth rate of the cells is essentially the rate of overall protein production that is dependent on the availability of an adequate supply of rRNA, angiogenin-stimulated rRNA transcription might also be necessary for cell growth induced by other angiogenic factors. Research funded by the Endowment for Research in Human Biology, Inc, Boston, MA, USA and the National Institute of Health (Grant # 91086)

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(L35) IMMUNO RNases AS ANTITUMOUR AGENTS Claudia De Lorenzo, Donald B. Palmer*, Sonia Di Gaetano, Angela Arciello, Annarita Tedesco, Mary A. Ritte, Renata Piccoli and Giuseppe D’Alessio Dipartimento di Chimica Biologica, Università di Napoli Federico II, Via Mezzocannone 16, 80134 Napoli, Italy; *Department of Immunology, Division of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, London, UK Aim of the study. Most conventional anticancer treatments are characterized by lack of tumour cell specificity. An ideal anticancer drug should be selectively targeted to tumour cells, non-immunogenic, nor inducing drug-resistance. Immunotoxins (ITs), hybrid reagents in which a monoclonal antibody reactive to tumour cells is fused to a potent cytotoxin, are of great interest as therapeutic agents, in that the antibody moiety drives the toxin to target cells. Nevertheless, ITs may still be aspecifically toxic and immunogenic. A new approach strategy is to prepare immunoRNases (IRs), in which the toxin is replaced by a non-toxic and non immunogenic RNase, and the mAb by an scFv directed towards a tumour specific receptor antigen (1). By this way, only upon specific antigen recognition and internalisation the RNase becomes cytotoxic. The aim of our study is the construction of antibody based new drugs, selective for tumour cells and potentially immunotolerated. To this purpose, human pancreas RNase (HP-RNase) was fused to murine and human scFv specifically directed towards tumour associated antigens. Methodology. A chimeric protein was prepared by fusing the cDNA encoding HP-RNase to the cDNA encoding a murine scFv directed towards the ErbB2 receptor. For the construction of the corresponding fully human immunoRNase, a cDNA encoding a human anti-ErbB2 scFv was isolated from a phage library through a double-selection strategy on live cells and fused to HP-RNase. An additional tumour-directed immunoRNase was prepared by fusing to HP-RNase the cDNA encoding a human scFv anti-CEA (carcinoembryonic antigen). The chimeric proteins, as well as their scFv moieties, were expressed in E. coli, purified and characterised structurally and functionally. Results. We found that the anti-ErbB2 murine-human IR is active as an RNase, specifically binds to the receptor, and induces their death (2). We also showed that the anti-ErbB2 human scFv we isolated, upon internalisation in receptor-positive cancer cells, strongly inhibits their proliferation (3). The chimeric cDNAs encoding the two fully human immunoRNases, i.e. the anti-ErbB2 and anti-CEA IRs, have been obtained and successfully expressed in E.coli. Their enzymatic and biological activities are at present under test. Conclusions. New potential anticancer drugs have been obtained. We demonstrated that a chimeric human-murine immunoRNase, specifically directed towards ErbB2-positive cancer cells, is able to recognise, enter and kill target cells. Furthermore, we isolated a human anti-ErbB2 scFv capable by itself to inhibit target cell proliferation. Two immunoRNases, whose moieties are for the first time both of human origin, and which recognise tumour-specific antigens, may be proven to be valuable tools for cancer therapy.

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(1) Rybak, S.M. and Newton, D.L. (1999) Exp. Cell Res. 253, 325-335. (2) De Lorenzo, C., Nigro, N., Piccoli, R. and D’Alessio, G. (2002) FEBS Lett., 517, in press. (3) De Lorenzo, C., Palmer, D.B., Piccoli, R., Ritter, M.A. and D’Alessio, G. (2002) Clin. Cancer Res., in press. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, Italy, and from Medical Research Council, UK.

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(L36) ANTIVIRAL EFFECTS OF ONCONASE(R) (RANPIRNASE) Martin Michaelis1, Mikulski, S.2, Shogen, K.2, Doerr, H.W.1, Cinatl J. jr.1 1Institut fuer Medizinische Virologie, Klinikum der J.W. Goethe-Universitaet Frankfurt/Main, Germany. 2Alfacell Corporation, Bloomfield, New Jersey, USA Onconase (Ranpirnase) is a ribonuclease that is isolated from the Northern Leopard frog (Rana pipiens). It is currently under investigation in clinical trials for its antitumoural activity (1,2). Besides its antitumoural potential onconase was already shown to inhibit HIV replication in vitro (3,4). Investigations of our group demonstrated, that replication of the RNA viruses coxsackie virus (IC50=5.2µg/ml) and echovirus type 7 (IC50=5.9µg/ml) is also inhibited by onconase in vitro. In contrast to this, replication of DNA viruses human cytomegalovirus and herpes simplex virus type 1 was not influenced in concentrations up to 20µg/ml. Onconase slightly inhibited the reactivation of human immunodeficiency virus type 1 (HIV-1) in U1 cells (containing two copies of HIV-1 proviral DNA per cell) after addition of 12-0-tetradecanoylphorbol-13-acetate (TPA). Extinction was 1.0 for TPA (100nM, 2 days) treated cells compared to 0.7 for TPA (100nM, 2 days) and onconase (1µg/ml, 2 days) treated cells, indicated by HIV-1 p-24 staining. Stimulation of HIV-1 replication using TNF-alpha (25ng/ml) for two days was not influenced by onconase treatment (TNF-alpha= 1.45, TNF-alpha onconase (2 µg/ml) = 1.34). (1) Mikulski, S.M., Grossman, A., Carter, P. et al. (1993) Int. J. Oncol. 3, 57-64. (2) Vogelzang, N.J., Aklilu, M., Stadler, W.M. et al. (2001) Invest. New Drugs 19, 255-260. (3) Youle, R.J., Wu, Y.N., Mikulski, S.M. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 6012-6016. (4) Saxena, S.K., Gravell, M., Wu, Y.N. et al. (1996). J. Biol. Chem. 271, 20783-20788. Research funded by the friendly society ”Hilfe für krebskranke Kinder Frankfurt e.v.” and its foundation ”Frankfurter Stiftung für krebskranke Kinder”.

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(L37) THE INTERFERON REGULATED ANTIVIRAL ENZYME, RNase L, IS A CANDIDATE SUPPRESSOR OF HEREDITARY PROSTATE CANCER (HPC1) Robert H. Silverman Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, USA Prostate cancer is a leading cause of malignancy in U.S. males accounting for 198,000 new diagnoses and 31,500 deaths per year. Investigations into the genetic basis of prostate cancer indicate that the RNase L gene (RNASEL) is a strong candidate for the hereditary prostate cancer gene, HPC1 (1). Two HPC1-affected families were characterized which contained inactivating mutations in RNASEL. Loss of heterozygosity of RNASEL was shown in prostate tumor tissue and lymphoblasts from heterozygous RNASEL+/- individuals had one-half the RNase L activity compared to RNASEL+/+ family members. In addition, we have recently characterized a missense mutation in RNASEL present in prostate cancer patients that reduces enzymatic activity by three-fold. This latter mutation is predictive of prostate cancer risk (2). While previous findings demonstrated that RNase L has antiviral, antiproliferative and apoptotic activities, the linkage to HPC1 provides the first evidence that RNase L is a tumor suppressor. Activation of RNase L requires unusual short 2’,5’-linked oligoadenylates (2-5A) produced from ATP by interferon-inducible 2-5A synthetases in response to double-stranded RNA. Involvement of RNase L in apoptosis is being studied by transfecting cells with 2-5A and monitoring activation of stress-activated protein kinases, caspases and cleavage of death substrates. Our hypothesis is that the tumor suppressor function of RNase L is due to activation of a ribotoxic stress pathway resulting in apoptosis. 1. Carpten, J. et al. (2002) Nature Genetics 30,181-184. 2. Casey, G. et al. (2002) under review. Research funded by a grant from the U.S. National Cancer Institute, National Institutes of Health, CA44059.

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(L38) REGULATION AND EXPRESSION OF ANGIOGENINS Vasanta Subramanian*, Jason Lawrence*, Ben Crabtree*, Robert Shapiro@ and Matthew Rolfe*. *Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom. @CBBSM, Harvard Medical School, One Kendall Square, 600 Building, 3rd Floor, Cambridge, MA 01239, USA. The Angiogenins (Ang) belong to the RNase superfamily and at least 4 members of this family have now been identified and found to be present across phyla. The murine Angiogenins include Ang1, Angrp, Ang3 and Ang4. The first identified member of this family the human ANG1 was isolated from tumour cell conditioned medium and its weak RNase activity was found to be necessary for its angiogenic activity. Of the murine Angs, Angrp is not angiogenic in CAM assays but Ang3 was found to induce neovascularisation. Ang1, Angrp and Ang3 have also been identified in zebrafish. We have isolated and characterised genomic clones for the Ang1 and Angrp genes and carried out a comparative analysis of the proximal regulatory elements of Ang1 and Angrp. Data on the characterisation of the regulatory elements for Ang1 and the developmental expression of Angiogenins in mouse embryogenesis will be discussed. The work is funded by the Medical Research Council UK

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(L39) EXPRESSION OF ANGIOGENIN IN THE HUMAN MONOCYTE/MACROPHAGE LINEAGE Nadine Pavlov 1, Arnaud Poulain 1, Brigitte Bauvois2, Josette Badet 1 1 Unité 427 INSERM “ Human development: growth and differentiation ”, Université René Descartes, Paris, FRANCE. 2 Unité 365 INSERM “ Interferons and Cytokines ”, Institut Curie, Paris, FRANCE Angiogenesis is a key event in tumour growth and metastasis, and is thus a therapeutic target to block solid-tumour expansion (1). Angiogenin is one of the most potent inducers of neovascularisation in experimental models in vivo [(2) for review]. The possible involvement of angiogenin in the development of cancer is suggested by its overexpression in patients with a variety of tumours. Since macrophages are key effector cells in inflammatory and tumoral angiogenesis (3), we examined angiogenin expression in the human monocyte/macrophage lineage by using indirect immunofluorescence microscopy, in situ hybridisation, and a two-antibody sandwich enzyme-labelled immunoassay. Differentiation of myeloblastic HL60 and monoblastic U937 leukemia cell lines and normal blood monocytes along the macrophage pathway was associated with an increase in angiogenin expression. Macrophages can be involved in different steps of the angiogenic cascade by their capacity to secrete the key mediators of angiogenesis. Our data indicate that angiogenin is one of them. 1. Bergers, G., Javaherian, K., Lo, K.-M., Folkman, J., and Hanahan, D. (1999) Science 284, 808-812 2. Badet, J. (2000) in Encyclopaedic Reference of Vascular Biology & Pathology (Bikfalvi, A., ed), pp. 16-29, Springer-Verlag, Berlin Heidelberg 3. Sunderkötter, C., Steinbrink, K., Goebeler, M., Bhardwaj, R., and Sorg, C. (1994) J. Leukocyte Biol. 55, 410-422 Research funded by the Institut National de la Santé et de la Recherche Médicale and by a grant from the Association de la Recherche sur le Cancer (grant ARC-5424). N.P. was supported by fellowships from the Association de la Recherche sur le Cancer, the Ligue Nationale contre le Cancer and Novo Nordisk Pharmaceutique.

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(L40) MECHANISM OF RIBONUCLEASE CYTOTOXICITY Kimberly A. Dickson1, Marcia C. Haigis1, and Ronald T. Raines1,2

1Department of Biochemistry and 2Department of Chemistry University of Wisconsin – Madison, USA Select ribonucleases are toxic to mammalian cells and are exceptionally toxic to tumor cells. The anti-tumor activity demonstrated by cytotoxic ribonucleases such as Onconase (ONC) varies depending on the type of tumor. The goal of this project is to characterize the interactions of ribonucleases at the surface of a variety of human cells and to reveal the pathway of their internalization. Both ONC and RNase A demonstrate non-specific binding to the surface of human tumor cell lines. Binding of RNase A to the surface of K-562 cells is not altered by the presence of colominic acid, heparin sulfate, NaCl, polylysine, or nucleotides (3´-UMP and 5´-AMP). Similarly, treating K-562 cells with EDTA, trypsin, and neuraminidase has no effect on RNase A binding. Preliminary data suggests that non-specific binding to the cell surface is rampant and does not correlate with susceptibility to toxic ribonucleases. In addition, ONC and RNase A exhibit dose-dependent uptake into acidic endosomal vesicles. On-going work is investigating the co-localization of ONC and RNase A with lipid rafts. Supported by CA73808 (NIH).

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(L41) EFFECT OF BS RNase ON CHEMO SENSITIVE AND CHEMO RESISTANT NEUROBLASTOMA CELL LINES. Tomas Eckschlager, Josef Matousek*, Jindrich Činátl**, Jiri Činátl, jr.# Dept.Pediatric Oncology, 2nd Medical Faculty and University Hospital Motol, Prague, * Inst. Animal Physiology and Genetic, Libechov, ** Klinlab s.r.o., Prague, Czech Republic, .# Inst. Medical Virology, J. W. Goethe-University Medical Center, Frankfurt a. Main, Germany

There were confirmed, that bovine seminal RNase /BS RNase/ induces apoptosis in several human malignant tumours in vivo and in vitro (1, 2). One may speculate that BS RNase can be used as an anticancer drug. Therefore the aim of our study was comparing of its efficiency on chemosensitive and chemoresistant neuroblastoma cells in vivo.

Methods: four neuroblastoma cell lines (UKF NB1, UKF NB 2, UKF NB 3, and UKF NB 4) and three chemoresistant (vincristine, cis-platin, doxorubicin) derived from each of the above mentioned cell lines were cultivated for 7 days with different concentrations of BS RNase. Cell lines were derived from high-risk neuroblastoma (N-myc amplification, del 1p36). Vincristine- and doxorubicin-resistant lines express high levels of P-glycoprotein. Tumour cells viability was evaluated by MTT test. Apoptosis was evaluated as percentage of Anexin V binding cells using flow cytometry. Flow cytometry was also used for evaluation of P-glycoprotein and cell cycle kinetics (DNA content).

Results: BS RNase significantly reduced cell growth and induced apoptosis in chemo sensitive and chemo resistant cell lines and those effects were dose dependent. BS RNase did not induced increasing of P-glycoprotein expression but change cell kinetics (increase percentage of S phase). We suppose that increasing of S phase is caused by retardation of DNA synthesis.

Conclusion: BS RNase significantly reduces neuroblastoma growth in vitro by induction of apoptosis, even if they are chemo resistant. One may speculate that this enzyme may be used as anticancer drug, which is effective in multidrug resistant malignancies. (1) Matousek J.: Comp Biochem Physiol C Toxicol Pharmacol (2001) 129,175-191 (2) Michaelis M, Matousek J, Vogel JU et al: Anticancer Drugs (2000) 11, 369-376 .

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(L42) STUDIES ON THE RIBONUCLEASE ACTIVITY PROFILE AND ECP EXPRESSION DURING THE HL60 CELL DIFFERENTIATION. Susanna Navarro, Zoran Nikolovski, M. Victoria Nogués and Claudi M. Cuchillo. Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autónoma de Barcelona. 08193-Bellaterra. Spain. The superfamily of pancreatic ribonuclease includes several human proteins that are involved in different biological process or in some cases of unknown function. Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are both ribonucleases localised in the eosinophil secondary granules and are implied on the immune response system but with specific effects. The present work is focused on ECP detection. Previous studies have described that messenger RNA encoding ECP has been detected in eosinophils, neutrophils, basophils and macrophages and the ECP expression has been determined in eosinophils and a low amount in monocytes and non-differentiated cell lines. In this context we are analysing the ribonuclease activity profile and ECP expression during the HL60 cell differentiation, a promyelocytic cell line. Ribonuclease activity profile based on molecular mass separation is determined using SDS-PAGE activity staining with polyuridylic acid as substrate and ECP is detected by Western blot using an specific antibody that recognizes specifically the D115-Y122 ECP sequence and detects glycosylated and non glycosylated forms (1). HL-60 cells can be induced to differentiate to morphologically and functionally mature granulocytes by incubation with all-trans-retinoic acid, to monocytes with cholecalciferol ( vitamine D3 ) and to macrophages with phorbol 12 myristate 13-acetate. Electron microscopy, cell staining with the Wright-Giemsa colouring and biochemical probes as esterase activity, Luxol Fast Blue staining and the nitroblue tetrazolium (NBT) test are used to assess the differentiation process. This study will help us to identify the ECP expression and its potential role in the immune system. 1. Boix, E., Carreras, E., Nikolovski, Z., Cuchillo, C. M., and Nogués, M. V. (2001). J. Leukoc. Biol. 69, 1027-1035. Research funded by grants 2000SGR 00064 from Generalitat de Catalunya and BMC2000-0138-C02-01 from DGES, Ministerio de Educación y Cultura, Spain. S. N is a recipient of a predoctoral fellowship grant from Ministerio Ciencia y Tecnología ( Spain ).

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(L43) RNA PROCESSING AND DECAY IN E. coli: DISCOVERY AND CHARACTERIZATION OF THE DEGRADOSOME Anne Leroy1, Nathalie Vanzo1, Sandra Sousa2 and Marc Dreyfus2 and Agamemnon J. Carpousis1 1Laboratoire de Microbiologie et Génétique Moléculaire, (CNRS, UMR 5100), Paul Sabatier Université, 118 rue de Narbonne, 31062 Toulouse, France. 2Laboratoire de Génétique Moléculaire (CNRS, UMR 8541), Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris, France. Messenger RNA instability is an intrinsic property that permits timely changes in gene expression by limiting the lifetime of a transcript. The ribonuclease E (RNase E) of E. coli is a single-strand-specific endoribonuclease involved in the processing and degradation of ribosomal and messenger RNA. A nucleolytic multienzyme complex now known as the RNA degradosome was discovered during the purification and characterization of RNase E (1). The other major components are a 3’-specific exoribonuclease (polynucleotide phosphorylase = PNPase), a DEAD-box RNA helicase (RNA helicase B = RhlB) and enolase. The DEAD-box proteins are ubiquitous enzymes involved in translation initiation, ribosome assembly and mRNA splicing. RNase E is a large multidomain protein with an N-terminal catalytic domain and a C-terminal ‘non-catalytic part’ containing RNA binding sites and a protein ‘scaffold’ that interacts with PNPase, RhlB and enolase (2). Our current research is focused on elucidating the physiological role of the degradosome in RNA metabolism. The rne gene has been replaced with alleles encoding deletions in the non-catalytic part of RNase E. RNase E activity was tested using a PT7-lacZ reporter gene whose message is particularly sensitive to degradation since its translation is uncoupled from transcription. Both hypo- or hyper-active mutants were obtained showing that the non-catalytic region has positive and negative effectors of mRNA degradation. Expression of the mutant proteins in vivo anti-correlates with activity showing that autoregulation compensates for defective function. There is no simple correlation between RNA binding and activity in vivo. Disrupting RhlB and enolase binding resulted in hypoactivity whereas disrupting PNPase binding resulted in hyperactivity. An allele (rne131), expressing the catalytic domain alone, was put under Plac control. In contrast to rne+, low expression of rne131 severely affects growth. Even with autoregulation, all of the mutants lose when grown in competition with wild type. Although the catalytic domain of RNase E is sufficient for viability, our results show that elements in the non-catalytic part, which affect mRNA degradation, are necessary for normal activity in vivo. 1. Carpousis, A.J., Van Houwe, G., Ehretsmann, C.P. and Krisch, H.M. (1994). Cell 76, 889-900. 2. Vanzo, N.F., Li, Y.S., Py, B., Blum, E., Higgins, C.F., Raynal, L.C., Krisch, H.M. and Carpousis, A.J. (1998). Genes & Dev. 12, 2770-2781. This research was funded by the CNRS with additional support from the Cancer Research Association (ARC), the Ministry of Education (MENRT) and the Midi-Pyrénées Region.

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(L44) SUBSTRATE SELECTION BY EUKARYOTIC RNase III: ONE ENZYME AND TWO MODES OF ACTION Sherif Abou Elela , Bruno Lamontagne, and Ghada Ghazal Groupe ARN/RNA Group, Département de Microbiologie et d’infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4 Rnt1p is an RNA processing enzyme involved in the maturation of pre-rRNA, 4 snRNAs, and several snoRNAs. As a member of the dsRNA specific RNase III family it shares the main structural features of the bacterial enzyme, like the dsRBD and the nuclease domain, suggesting similar mechanism of function. However, unlike the bacterial enzyme, Rnt1p recognizes RNA with conserved AGNN terminal tetra-loop structure and cleaves at a fixed distance from the loop. The solution structures of two different substrates of Rnt1p reveal a common fold for the terminal loop with the universal G in syn conformation and extensive base stacking. The structure suggests that Rnt1p recognize the shape of the tetraloop and use it for the initial interaction with its substrates. This is in marked contrast with the recognition mode of most dsRNA binding proteins including RNase III, which are believed to interact primarily with the minor groove of the double helix and recognize the shape of the A-form dsRNA. To examine the ability of Rnt1p to recognize the dsRNA helix we have tested the binding and cleavage of RNA substrates with different stem length and sequences. The result confirm a primary role of the tetraloop in positioning the enzyme for cleavage and demonstrate that AGNN tetraloop with only 9bp is capable of directing RNA cleavage in single stranded region. Surprisingly, changes in the sequence of the RNA helix adjacent to the tetraloop inhibited the binding to Rnt1p suggesting an additional helix dependent interaction with the enzyme. Specific combinations of stem and tetraloop sequences inhibited binding and cleavage. Consequently, we found that substrates with 16bp or longer stems could bind to Rnt1p in AGNN independent matter and could be unspecifically cleaved under special conditions. Substrates with stems shorter than 12bp do not support binding in absence of the conserved AGNN tetraloop suggesting that one turn of the helix is needed for AGNN independent binding. These results reveal two mechanisms of RNA recognition by Rnt1p. The first recognizes the shape of the terminal tetraloop and the other recognizes the minor groove. This duality of substrate recognition may either reflect the evolutionary origin of Rnt1p dsRNA or project two alternative mechanisms of function that allow Rnt1p to differentiate its natural substrate from foreign duplex RNA like viral RNA. Research funded by grant MOP-14305 from the Canadian Institute for Healt Research (CIHR).

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(L45) RNA LARIAT DEBRANCHING ENZYME Siew Loon Ooi and Boeke, J. D. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Nucleic acids are usually linked by 3’-5’ phosphodiester bonds. Interestingly, in both procaryotes and eucaryotes, there exist low levels of nucleic acids containing 2’-5’ phosphodiester bonds. Examples of branched RNAs include intron lariats in eucaryotes, Y-like transplicing intermediates in trypanosomes and group II intron lariats in eucaryotic organelles and prokaryotes. Multicopy single-stranded DNAs (msDNAs) form a set of RNA molecules with a DNA branch found in certain prokaryotes. In msDNAs, the DNA is linked to the RNA via a 2’-5’ phosphodiester bond, creating a branched fork structure. Branched nucleic acids were first discovered by Wallace and Edmonds in the population of nuclear polyadenylated RNAs. These branched nucleic acids are mostly derived from intron lariats, one of the intermediate structures in pre-mRNA splicing. RNA lariat debranching enzyme (DBR) is a 2'-5' phosphodiesterase. It specifically hydrolyzes the 2'-5' phosphodiester linkage of RNA intron lariats between the G residues of 5' splice site and the A residues of the branchpoint. The gene that encodes for RNA lariat debranching enzyme (DBR) is highly conserved, and has been cloned from the yeast S. cerevisiae, S. pombe, C. elegans, A. thaliana, mouse and human. The original mutant allele dbr1-1 was isolated in S. cerevisiae based on its reduced Ty1 transposition frequency. S. cerevisiae dbr1 mutants are viable, have modest growth defects and accumulate intron lariats missing the linear sequences on the 3' side of the branchpoint. S. cerevisiae dbr1 mutants have mild pseudohyphal growth defects and intronic snoRNAs accumulate in the lariat form. The phenotypes of dbr1 mutants are intriguing, but it is not clear whether the enzyme plays a direct role in the processes affected. To understand how Dbr1p is involved in Ty1 transposition and other cellular pathways, we perform a genome wide synthetic lethality screen in S. cerevisiae. Results from this screen will be presented. Research funded by a grant from NIH

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(L46) FUNCTION OF THE EXOSOME COMPLEX IN NONSENSE-MEDIATED DECAY Philip Mitchell and David Tollervey Wellcome Centre for Cell Biology, Institute for Cell and Molecular Biology, King’s Buildings, University of Edinburgh, Edinburgh EH9 3RJ.

Eukaryotic cells have evolved a number of mRNA surveillance mechanisms that degrade aberrant mRNA transcripts, including the nuclear turnover of pre-mRNAs that are defective in splicing or 3’ end formation and the cytoplasmic degradation of mRNAs lacking a termination codon. A key player in mRNA surveillance mechanisms is the exosome, a complex of multiple 3’->5’ exoribonucleases. The exosome is also a major activity in the turnover of canonical mRNA transcripts following initial deadenylation. The cytoplasmic functions of the exosome in mRNA decay are dependent upon Ski7p, a putative GTPase Ski7p, and the Ski complex, consisting of Ski3p, Ski8p and the putative RNA helicase Ski2p.

Nonsense-mediated decay (NMD) is a well-studied surveillance mechanism that degrades transcripts containing premature termination codons, thereby preventing the translation of truncated proteins that may otherwise have deleterious, dominant negative phenotypes. It is also important in the regulated expression of normal transcripts, since it can modulate the stability of alternatively spliced transcripts and those containing regulatory, small upstream ORFs. NMD requires Upf1p, Upf2p and Upf3p, factors that form a surveillance complex which registers the termination event as aberrant and triggers subsequent accelerated degradation.

The generally accepted model for NMD is that the accelerated degradation of nonsense codon containing transcripts is achieved by deadenylation-independent decapping, followed by 5’->3’ exonucleolytic decay. We have recently obtained data demonstrating a distinct 3’->5’ NMD pathway involving deadenylation in yeast that requires the exosome and its cofactor Ski7p. Given the central role of the exosome in normal mRNA turnover in mammalian cell extracts, we anticipate that this exosome-dependent 3’->5’ NMD pathway will be the major degradation mechanism for early nonsense codon containing transcripts in higher eukaryotes. In support of this, an earlier report showed that a β-globin transcript containing an early nonsense codon is highly destabilised but is degraded via a deadenylation-dependent mechanism. We are currently addressing the role of the Upf proteins and a requirement for the Ski7p GTPase domain in the 3’ NMD pathway. This work was supported by the Wellcome Trust.

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(L47) MECHANISTIC ANALYSIS OF DOUBLE-STRANDED RNA PROCESSING BY RIBONUCLEASE III Allen W. Nicholson Department of Biological Sciences, Wayne State University, Detroit, Michigan, USA. Members of the ribonuclease III superfamily of double-strand(ds)-specific endoribonucleases are highly conserved in bacteria and eukaryotes, and participate in diverse functional roles from structural and messenger RNA maturation to small interfering(si) RNA formation in the RNA interference pathway. RNase III family members are distinguished by conserved domain elements, including a dsRNA-binding motif (dsRBM) and a catalytic domain, which often occurs as a tandem repeat. The most studied member is Escherichia coli RNase III. Recent structural data on the E. coli RNase III dsRBM (1) and the catalytic domain of Aquifex aeolicus RNase III (2) provide a new basis with which to understand the molecular mechanisms of substrate recognition, and phosphodiester hydrolysis. We have shown that a truncated form of Escherichia coli RNase III containing only the catalytic domain retains the ability to site-specifically cleave model substrates in vitro (3). Optimal activity is observed in the presence of Mn2+ (~5 mM) and low salt, with higher Mn2+ concentrations conferring inhibition. The truncated enzyme retains strict specificity for dsRNA, is inhibited by ethidium bromide, and is sensitive to specific W-C bp sequences (“antideterminants”) which also inhibit substrate binding by the holoenzyme. An alanine-scanning mutational analysis of the catalytic domain indicates the involvement of several conserved residues in the mechanism of dsRNA cleavage. One of these residues, Glu117 is essential for catalytic activity (4), and has been shown to coordinate a divalent metal ion (2). Another conserved residue, D45, is in the catalytic domain “signature motif.” Mutation of D45 to alanine also blocks catalytic activity in vitro. The locations of these residues in the putative active site; their interactions with metal ion and water molecules, and the inhibitory action of Mn2+ provides new information on the hydrolytic mechanism of RNase III. 1. Kharrat A., Macias M.J., Gibson T.J., Nilges M. and Pastore A. 1995. Structure of the dsRNA-binding domain of E. coli RNase III. EMBO J. 14: 3572-3584. 2. Blaszczyk, J., Tropea, J.E., Bubunenko, M., Routzahn, K.M., Waugh, D.S., Court, D.L. and Ji, X. 2001. Structure 9, 1225-1236. 3. Sun W., Jun, E. and Nicholson, A.W. 2001. Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain. Biochem. 40: 14976-14984. 4. Sun, W. and Nicholson, A.W. 2001. Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical rtequirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant. Biochem. 40: 5102-5110. Research supported by the NIH (GM56457 and GM56772)

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(L48) POLYADENYLATION AND DEGRADATION OF RNA IN CHLOROPLAST AND PROKARYOTES, SIMILARITIES AND DIFFERENCES Gadi Schuster, Shlomit Yehudai-Resheff, Varda Liveanu, Ruth Rott Department of Biology, Technion – Israel Institute of Technology, Haifa 32000, Israel. The molecular mechanism of mRNA degradation in the chloroplast includes a series of sequential events that are endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products and exonucleolytic degradation by PNPase and possibly other exonucleases, yet to be discovered. A similar mechanism exists in E. coli, where polyadenylation is performed mainly by poly(A)-polymerase I but when absent, PNPase is doing the job. In the chloroplast, we found that only PNPase performs both polyadenylation and degradation of the transcript and there is no a poly(A)-polymerase I homologue. Additional difference between these two systems is the absent of the “degradosome”, a high molecular weight complex described in E. coli which includes PNPase, RNase E, RNA-helicase and the enzyme enolase, in the chloroplast. Preliminary results of polyadenylation and degradation of RNA in the cyanobacteria Synechocystis disclosed a chloroplast type system, suggesting that E. coli acquired the poly(A) polymerase I and the degradosome late in its evolutionary process. The first endonucleolytic cleavage, possibly the rate-limiting step of the RNA degradation process, is believed to be carried out by RNase E. Analyzing the RNase E of cyanobacteria, chloroplast of green alga and the nuclear gene of Arabidopsis as compared to E. coli, revealed three conserved sequences (boxes I, II and III) in addition to the SI RNA-binding domain described before. Biochemical analysis of these proteins suggested box I as the catalytic site and box III to be involved in the tri-phosphates 5’ end inhibition of RNase E activity. No homology to the putative catalytic domain was detected in other proteins in the data bank that are not related to RNase E.

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(L49) RIBONUCLEASE FUNCTION DURING THE BACTERIAL COLD SHOCK RESPONSE Robert W. Simons1, Rudolf K. Beran1, Annie Prud’homme-Genereux2 and George A. Mackie2 1Department of Microbiology and Molecular Genetics, University of California at Los Angeles, CA, USA 2Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada When bacterial cells are shifted to cold temperatures, growth stops for several hours, during which time a small number of key cold shock response (CHR) proteins are expressed, following which growth resumes. The CSR is accompanied by dramatic changes in mRNA decay rates. To explore this question we examined the function of the RNA degradosome and its components during the CSR. The degradosome remains functional at low temperatures, and the levels of at least two of its components, ribonuclease E (RNaseE) and polynucleotide phosphorylase (PNPase), are induced. An RNA helicase, CsdA, is also induced, and may also assemble into the degradosome. Interestingly, PNPase is required at low temperatures for proper RNaseE expression and/or function. The likely biological significance of these enzymatic changes during the CSR will be discussed. Research supported by grants from the National Science Foundation (RWS) and the Canadian Institutes of Health Research (GAM)

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(L50) RNA DEGRADATION IN PLANT CHLOROPLASTS: THE ROLE OF CSP41, A UNIQUE ENDORIBONUCLEASE Thomas J. Bollenbach, Dana A. Tatman and David B. Stern Boyce Thompson Institute for Plant Research, Cornell University, Ithaca NY, 14853, USA. CSP41 is a nucleus-encoded 41 kDa chloroplast RNA stem-loop binding protein. In spinach chloroplast extracts, CSP41 is part of a three protein petD RNA binding complex, which presumably functions to inhibit endo- and exonucleolytic attack of the 3’ stem-loop. CSP41 is also an endoribonuclease. Although CSP41 is a single strand specific endonuclease, it has a preference for stem-loop containing substrates. CSP41 activity has an absolute requirement for either Mg2+ or Ca2+. The enzyme generates 3’ hydroxyl terminated products, which are likely polyadenylated and degraded by polynucleotide phosphorylase in vivo. We used an antisense approach to study the role of CSP41 in tobacco. Three stable antisense lines were generated, which compared to wild-type plants contained 5, 1 and 0.1% residual CSP41, respectively. Although none of the antisense lines had a visible phenotype, RNase protection assays revealed at least two-fold more petD RNA relative to wild-type plants, and the level varied inversely with the amount of residual CSP41. The degradation rates of chloroplast RNAs were studied in whole chloroplast extracts. The degradation rate of the full-length rbcL transcript in wild type chloroplast extracts was 8-fold higher than that in the antisense line with the lowest level of CSP41. Furthermore, the degradation rates in the three antisense lines correlated directly with the CSP41 level. Taken together, these data suggest that the CSP41 antisense plants are deficient in their ability to degrade petD and rbcL transcripts. The current model of RNA turnover in the chloroplast predicts rate-limiting endonucleolytic cleavages within the stem-loop structure of the 3’ UTR and within the coding region. It appears that CSP41 may catalyze this rate-limiting step. In a complementary approach, we are attempting overexpression by stably integrating the csp41 gene into the chloroplast genome. Preliminary data suggest that these plants have an abnormal growth phenotype, and their molecular characters are under investigation. (1) Yang, J., Schuster, G., and Stern, D. B. (1996). Plant Cell 8, 1409-1420. (2) Yang, J., and Stern, D. B. (1997). J. Biol. Chem. 272, 12784-12880. Research funded by a grant from the U.S. Dept. of Energy Biosciences Program.

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(L51) STRUCTURE-FUNCTION ANALYSIS OF PROKARYOTIC TRANSCRIPT CLEAVAGE FACTORS GREA AND GREB Oleg Laptenko, Jookyung Lee, and Sergei Borukhov. State University of New York Health Science Center at Brooklyn, Department of Microbiology and Immunology, 450 Clarkson Ave, BSB 3-27, Brooklyn, NY 11203, USA. Two homologous prokaryotic proteins GreA and GreB are involved in endonucleolytic hydrolysis of nascent RNA in ternary complexes (TCs) of RNA polymerase (RNAP). The RNA cleavage is a Mg2+-dependent reaction which typically occurs 2-18 bases upstream from the 3'-terminus, followed by dissociation from the TC of the 3'-proximal fragment carrying the phosphate group. The 5'-proximal fragment remains in TC, and can be extended in the presence of rNTPs. The exact molecular mechanism of Gre action is unknown, however it is believed that Gre factors act by binding to RNAP, interacting with nascent RNA 3'-terminus, and inducing conformational changes near the enzyme's catalytic center, which leads to an activation of its intrinsic RNase activity. Biochemical data indicate that the same RNAP catalytic center is responsible for both RNA synthesis and RNA hydrolysis reactions. The biological role of Gre-stimulated nucleolytic reaction may include enhancement of transcription fidelity, suppression of transcriptional pausing and arrest, and stimulation of RNAP promoter escape. To study the interactions of E. coli RNAP with Gre factors we used two approaches: (i) protein-protein photocross-linking using radiolabeled Gre proteins carrying selected Cys-substituted surface-exposed residues derivatized with photoactive bifunctional reagents; (ii) general Fe-EDTA hydroxyl-radical protein footprinting and localized hydroxyl-radical mapping of Gre-RNAP complexes using Fe2+-ion placed at RNAP catalytic center, and (iii) site-directed mutagenesis of GreA and GreB followed by their in vitro transcription analysis. Based on the results obtained we conclude that both Gre factors bind to RNAP primarily through their C-terminal globular domain (residues 77-158) at a site near the opening of the "secondary channel", a presumed entry site for the substrates, visible on the 3D-crystal structure of T. aquaticus RNAP core. The β' subunit of RNAP is the primary target for Gre binding, although β subunit may also provide additional contacts for C-terminal domain of Gre. The N-terminal coiled-coil domain of Gre factors (residues 1-76), responsible for the induction of nucleolytic activity in RNAP, binds the nascent RNA in TCs through a patch of conserved basic residues. This domain protrudes through RNAP secondary channel interacting with the extended coiled-coil subdomain of β'-subunit (beginning of conserved region β'-F) and with portions of conserved regions β'-E and β'-G. 6 highly conserved amino acid residues of Gre protein comprising the tip of coiled-coil domain are located in close proximity to the catalytic site (conserved region β'-D). We propose that these interactions cause allosteric changes at the active center of RNAP resulting in the activation of the dormant RNase activity of this enzyme. This work was supported by NIH Grant, GM54098-06A1 to S.B.

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(L52) MODULATING POLY(A)-SPECIFIC RIBONUCLEASE (PARN) ACTIVITY IN VITRO BY RNA BINDING PROTEINS Niklas Henriksson , Javier Martinez and Anders Virtanen Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, SWEDEN mRNA degradation is an important step in the regulation of eucaryotic gene expression. The first step in mRNA decay is the removal of the poly(A)-tail at the 3’-end of the mRNA. Poly(A)-specific ribonuclease (PARN), the only so far characterized mammalian poly(A)-specific exoribonuclease, participates in the initial degradation of the mRNA poly(A) tail. In vivo, the mRNA is associated with RNA binding proteins. We have therefore investigated how RNA binding proteins affect the activity of PARN. We have focused on two proteins binding the mRNA poly (A) tail: Poly(A) binding protein (PABP) and the 100 kDa human coactivator (p100). For our studies, we have used recombinant proteins, expressed in prokaryotic (E.coli) and eucaryotic (insect cell) expression systems. The RNA binding capacity was verified using electrophoretic mobility shift experiments. Both PABP and p100 were found to inhibit the effect of PARN in an in vitro deadenylation assay. Therefore, the amount of RNA binding proteins in the cytoplasm might be a way for the cell to control mRNA degradation.

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Poster Abstracts

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(P1) STABILIZATION OF PROTEINS BY INTRODUCTION OF NON-NATURAL MODULES Ulrich Arnold , 1,2, Matthew P. Hinderaker3, and Ronald T. Raines 2, 3 1 Department of Biochemistry and Biotechnology, Martin-Luther University, Halle, Germany, 2 Department of Biochemistry, 3 Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, U.S.A. Conventional site-directed mutagenesis is restricted to the 20 naturally occurring amino acids. The introduction of non-natural modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding pathways, and biological function. By use of the intein-based method of expressed protein ligation (Fig. 1) [1], chemically synthesized peptides, which might contain non-natural modules, can be ligated to expressed proteins. Here, this method was used to introduce a non-natural module into ribonuclease A (RNase A). Initially, semisynthetic wild-type (wt) RNase A was produced by this means and full-length RNase A was produced by intein-mediated protein expression. Both RNase A variants had the same ribonucleolytic activity as did wt-RNase A. Subsequently, an RNase A variant was produced in which R-nipecotic acid – S-nipecotic acid [2] replaces the RNase A β-turn at residues Asn113–cisPro114 (Fig. 1, 2). Beta-turns are a way for the polypeptide chain to reverse its direction as is necessary for the folding of proteins into their native, compact structure. Furthermore, loops of the protein structure are often involved in recognition processes. However, due to their flexibility they also represent points instability. Also the R-nip–S-nip RNase A variant showed ribonucleolytic activity comparable to that of the wt-RNase A, indicating that it is folded properly. The transition temperature as a measure for the conformational stability of this enzyme variant, however, is increased. This stabilization is attributed to a native-like pre-organization of the C-terminal part of the RNase A molecule. A control, in which Asn113–cisPro114 was replaced by the stereoisomer R-nip–R-nip (Fig. 2), failed to fold thereby proving that the incorporation of the non-natural module is not neglected by the protein structure.

Figure 1: Scheme for semisynthesis of RNase A variants by expressed protein ligation.

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Figure 2: Structure of Asn113–cisPro114 in RNase A and the non-natural modules R-nip–S-nip (β-turn mimic) and R-nip–R-nip (control). [1] Muir, TW, Sondhi, D & Cole, PA (1998) Proc. Natl. Acad. Sci. USA 95, 6705-6710. [2] Huck, BR, Fisk, JD & Gellman, SH (2000) Org. Lett. 2, 2607-2610. This work was supported by the Arthur B. Michael Fund postodoctoral fellowship for U.A., by NIH Training Grant GM08506 for M.P.H., and by NIH Grant GM44783 for R.T.R.

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(P2) SOLUTION STRUCTURE OF MONOMERIC BOVINE SEMINAL RIBONUCLEASE BY NUCLEAR MAGNETIC RESONANCE Francesca Avitabilea, Orlando Crescenzia, Alfonso Carotenutob, Anna M. D’Ursib, Teodorico Tancredic and Delia Piconea

aDipartimento di Chimica, Università Federico II di Napoli, Via Cintia, 80126, Napoli, Italy. bDipartimento di Scienze Farmaceutiche, Università di Salerno, 84084, Fisciano, Italy. cIstituto Chimica Biomolecolare del CNR, Pozzuoli, Napoli, Italy. Within the ribonuclease family, bovine seminal ribonuclease (BS-RNase) is the only dimeric protein, constituted by two identical subunits linked to each other through two disulphide bridges. Each subunit is homologous (80% identity) to bovine pancreatic RNase A, with all the residues of the active site present at the same sequence position in both enzymes. However, beside the ribonuclease activity, BS-RNase shows several peculiar biological activities, such as cytotoxicity toward malignant cells, immunosuppression and antispermatogenesis (1). The 3D structure of BS-RNase is very peculiar, since it is the only known dimeric enzyme characterized, in solution, by an equilibrium between two different structures. In the form indicated as MxM the N-terminal arm is swapped between the subunits, whereas in the form indicated as M=M no swapping occurs. The structure of MxM has been solved by X-ray diffraction at 1.9 Å resolution (2), whereas no structural data are yet available for the M=M form. In order to understand the features which induce the N-terminal domain swapping of BS-RNase we have started a structural study of its monomeric derivative (mBS-RNase), which is an obligatory intermediate in the folding process leading to the dimer, by heteronuclear NMR. The monomer can be obtained even from the native protein by selective reduction of the interchain disulphide bridges followed by blocking of the sulphydryl groups. While displaying a ribonuclease activity higher than that of the parent dimeric protein, the monomer does not retain any of the special biological functions. Analysis of heteronuclear 3D spectra suggests that the structure of mBS-RNase is very similar to that of bovine pancreatic ribonuclease (RNase A), the main differences being concentrated in the hinge region (residues 16-22), i.e. in the region primarily involved in the swapping of the N-terminal domains between the two subunits. (1)D'Alessio, G. et al. (1997) In Ribonucleases: Structures and Functions (D'Alessio, G. and Riordan, J. F. , Eds.), Academic Press, New York, N.Y. pp. 383-423. (2) Mazzarella, L. et al. (1993) Acta Crystallogr. D49, 389-402. Research fundend by a grant from MURST (PRIN 2000) and CNR Agenzia 2000.

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(P3) COORDINATED DIVALENT METAL IONS IN THE ACTIVE SITE OF POLY(A)-SPECIFIC RIBONUCLEASE. Yan-Guo Ren, Nikolaos A.A. Balatsos, Leif A. Kirsebom and Anders Virtanen. Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, SE-751 24, Uppsala, Sweden. Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and highly poly(A) specific exonuclease that degrades mRNA poly(A)tails. It interacts with the mRNA 5’ end located cap structure during degradation and this amplifies the processivity. PARN belongs to the RNase D family of nucleases. Here we provide direct evidence for coordination of divalent metal ions in the active site of PARN. The minimal substrate length can be modulated by the presence of different divalent metal ions. Penta- and trinucleotides (A5 and A3) were efficiently degraded to dinucleotides (A2) by PARN in the presence of Mg2+ or Cd2+. However, in the presence of Mn2+, Zn2+ or Co2+, the enzyme degraded effectively A2 to mononucleotides. Therefore, the identity of the divalent metal ion can modulate the length of the RNA substrate to be degraded. Four conserved residues of PARN are essential for activity, i.e. D28, E30, D292 and D382. When each of these residues was substituted by cysteine, the enzyme was inactive in the presence of Mg2+. However, activity could be rescued when Mg2+was replaced by soft metal ions, such as Mn2+, Zn2+, Co2+ and Cd2+. To locate divalent metal ions in the active site of PARN, the metal ion switch approach has been used. Soft metal ions coordinate soft atoms, like sulfur, while the latter is avoided by the hard Mg2+. Given that A3 is the shortest substrate efficiently cleaved, a non-bridging oxygen of the phosphodiester bond of the substrate was replaced with sulfur (A3S), to produce two isomers with RP-and SP-configurations. Although the RP-isomer could be deadenylated in the presence of Mg2+, the kobs was reduced 35 times compared to the A3 substrate. Perforning the reactions with Mn2+, Zn2+, Co2+ or Cd2+ instead of Mg2+, profoundly improved deadenylation. PARN activity could not be rescued when the SP- isomer was used. We propose that the architecture of the active site of PARN resembles the one of the 3’-exonuclease of Klenow polymerase. However, in contrast to the Klenow polymerase where the pro-S oxygen coordinates the divalent ion, our data strongly suggest that the pro-R oxygen of the RP-isomer interacted with divalent ions in the transition state.

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(P4) ADAPTIVE EVOLUTION IN MEMBERS OF THE RIBONUCLEASE A SUPERFAMILY Jaap J. Beintema, Jean-Yves F. Dubois, Björn M. Ursing Dept. of Biochemistry, University of Groningen, The Netherlands The ribonuclease A superfamily (RNase) is the only ‘vertebrate specific’ protein family with enzymatic activity identified in the human genome. Two Nobel prizes Chemistry were awarded to four persons in 1972 and 1984 for studies on the most prominent member of this superfamily: bovine pancreatic RNase. Homologues of this enzyme have been isolated, sequenced and characterized from many sources and several of them have interesting potential medical applications, but their functional biology is still largely unknown. Our studies concentrated on members of the mammalian RNase 1 family. These enzymes are expressed at high levels in the pancreas of ruminants (e.g. ox, Bos taurus), species with ruminant-like digestion and several ones with cecal digestion. But an ubiquitous presence at low levels at other sites in mammals indicates other still unknown physiological functions of the enzyme. Sequence studies of the enzyme showed interesting examples of homoplasy of amino acid sequences of the enzyme in species with cecal digestion (pig, guinea-pig, fruit bat, elephant), in paralogous and orthologous sequences of cetartiodactyls (whales and artiodactyls), and of ruminant and kangaroo sequences. Mammalian angiogenins constitute another family in the RNase A superfamily. These proteins have low RNase activities, but are more closely related to active enzymes isolated from birds and reptiles than to members of other mammalian RNase A families. Angiogenins react strongly with a mammalian RNase inhibitor protein (in contrast to non-mammalian members of the superfamily). X-ray studies show that the interaction sites of mammalian RNase and angiogenins with the inhibitor are strikingly different, which may indicate that these sites evolved independently.

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(P5) SOLUTION ISOLATION AND CHARACTERIZATION OF AN ENGINEERED DOMAIN-SWAPPED HUMAN PANCREATIC RIBONUCLEASE Rodríguez, M., Benito, A., Ribó, M. and Vilanova, M. Laboratori d’Enginyeria de Proteïnes, Dept. de Biologia, Fac. de Ciències, Universitat de Girona, Campus de Montilivi s/n, 17071 Girona. Spain. Protein aggregation in vivo is the origin of different severe diseases. The study of how proteins aggregate in vitro can help in the understanding of how this process proceeds in vivo. Domain swapping has been proposed as a mechanism that explains the formation of oligomeric proteins and aggregates. This mechanism is based on the mutual exchange of an entire domain between each one of the molecules of a dimer. Ribonucleases are good models for the study of the structural determinants leading to a dimeric or oligomeric state because monomer and dimeric domain-swapped molecules occur in the nature. Recently, we have reported the crystal structure, at 2Å resolution, of an engineered human pancreatic ribonuclease, which has revealed a new kind of domain-swapped dimer based on the exchange of N-terminal domains between the two subunits. In this structure the hinge loop (16-22 residues), connecting the swapped domains with the rest of the molecules, becomes organised in a 310 helix structure. The swapping is stabilised at both hinge peptides by hydrogen bonds and stacking interactions between residues of different chains (1). Although evidence of this dimeric structure in solution was also reported, until present its isolation had not been achieved. In this work we describe the conditions that favour the presence of this human dimeric form in solution and their isolation. The equilibrium dissociation constant at 29ºC between dimer and monomer forms of this HP-RNase variant is at the milimolar range. Work is in progress to characterise the stability in solution and the kinetic parameters of this dimeric form. (1) Canals, A. et al. (2001) Structure, 9, 967-976. Work supported by grants BMC2000-0138-CO2-02 from MCyT and SGR00-0064 from CIRIT.

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(P6) REGULATION OF RIBONUCLEASE RegB ACTIVITY BY RIBOSOMAL PROTEIN S1 Marco Bisaglia1, Soumaya Laalami2, Jean-Yves Lallemand1, Marc Uzan2 and François Bontems1. 1Equipe ICSN-RMN, Ecole Polytechnique, 91128 Palaiseau, France 2Institut Jacques Monod, 2 Place Jussieu, 75251 Paris cedex 05, France RegB, a bacteriophage T4 endoribonuclease, is involved in the regulation of the virus life-cycle. It specifically cleaves in the middle of early mRNA intergenic GGAG sequences. Cleavages by RegB result in mRNA inactivation and destabilisation. RegB efficiency is modulated by the S1 ribosomal protein that, probably, promotes the correct RNA conformation for its recognition by the enzyme. We are currently analysing the molecular basis of the role of S1 by combining biochemical and NMR spectroscopic studies. S1 is a modular protein composed of six repetitions of a conserved domain called “S1 motif” found in many RNA-binding protein. Genetically engineered fragments of the S1 protein were tested for their ability to stimulate RegB. We have shown that the C-terminal two-third of the molecule are sufficient to fully stimulate RegB. Mono- bi- and tri-modules derived from the C-terminal domain were used to define the smallest part of S1 molecule involved in RegB activity. Two RNA molecules were used as substrates. The first possesses a well-defined secondary structure (as probed by NMR spectroscopy) whereas the second is unstructured. Interestingly, the cleavage of the first molecule only depends on the presence of two contiguous S1 modules, while the cleavage of the second requires the four C-terminal modules. This suggests that the nature of the substrates governs the RNA-S1 interaction. To understand in detail the S1-RNA interactions, we have started to identify, by NMR spectroscopy, the S1 residues involved in the interaction with either substrate. We also have achieved the structure determination of the last S1 domain, as a first step toward the reconstruction of the whole C-terminal region.

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(P7) CHARACTERIZATION OF CYTOTOXIC HUMAN PANCREATIC RIBONUCLEASES Bosch, M.1, Benito, A.1, Ribó, M.1, Beaumelle, B.2 and Vilanova, M1. 1 Laboratori d’Enginyeria de Proteïnes, Dept. de Biologia, Fac. de Ciències, Universitat de Girona, Campus de Montilivi s/n, 17071 Girona, Spain. 2 UMR 5539 CNRS, Case 107, Bt 24, Université Montpellier II, 34095 Montpellier Cedex 05, France. It is well known that some members of the ribonuclease superfamily are cytotoxic against different tumoral cell lines. Several studies have focused on understanding the contribution of ribonucleolytic activity, affinity for ribonuclease inhibitor protein (RI), thermostability and cell internalisation to cytotoxicity. However, at present the mechanism of ribonuclease-mediated cytoxicity is not completely understood. From a molecular model of a complex between RI and human pancreatic ribonuclease (HP-RNase), constructed using the crystallographic structures of RI-RNase complex (1) and that of HP-RNase (2), we have designed a set of HP-RNase variants. They have been constructed with the aim of either evading RI or favour cell attachment while preserving ribonucleolytic activity and thermostability. The whole set of variants have been produced in an E. coli expression system and purified to homogeneity. Their thermostability, catalytic parameters and ability to evade RI have been measured. Cytotoxicity against different human tumoral cell lines has been tested. Finally, intracellular routing has been analysed by fluorescence labelling. Preliminary results show that some of the HP-RNase variants evading RI were not cytotoxic while others inhibited by RI presented this property, with IC50 values close to that of onconase. These results suggest that a good internalisation and arrival to the cell cytosol could overcome the inhibitor effect of RI. (1) Kobe, B. & Deisenhofer, J. (1995) Nature, 374, 183-186 (2) Pous, J. et al. (2000) J. Mol. Biol. 303, 49-59 Work supported by grants PB96-1177-CO2-02, BMC2000-0138-CO2-02 from MCyT and SGR00-0064 from CIRIT.

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(P8) ESSENTIAL STATIONS IN THE INTRACELLULAR PATHWAY OF CYTOTOXIC BOVINE SEMINAL RIBONUCLEASE Aurora Bracale1, Castaldi, F.1, Spalletti-Cernia, D.2, Nitsch, L.2 and D’Alessio, G.1 1 Dipartimento di Chimica Biologica, Università di Napoli “Federico II”, Italy. 2 Dipartimento di Biologia e Patologia Cellulare e Molecolare “L. Califano”, Università di Napoli “Federico II”, Italy. Seminal RNase (BS-RNase) is a powerful cytotoxic agent, selective for malignant cells (1). Some aspects of its mechanism of action have been clarified: the protein binds at the extracellular matrix (ECM) and reaches the cytosol, where it degrades rRNA, but only in malignant cells (2). A study aimed at tracing the path of BS-RNase to the cell cytosol has shown that BS-RNase is internalized into endosome-like vesicles both in normal and malignant cells. RNase A, and a monomeric derivative of seminal RNase, both inactive as cytotoxins, do not bind to any cell types, and are not internalized. A dimeric, cytotoxic variant of RNase A does instead bind, and is internalized. Thus the ECM is a selective station for cytotoxic RNases. Drugs that affect endosome functionality (NH4Cl, nigericin) do not protect malignant cells from BS-RNase cytotoxicity, leading to the conclusion that BS-RNase does not translocate from endosomes directly to the cytosol. Colocalization studies have also shown that BS-RNase colocalizes with TGN38, a marker of the trans-Golgi network (TGN). The effects of brefeldin A (BFA), and the addition of a C-terminal KDEL peptide, the consensus sequence for ER localization, have indicated that BS-RNase does not progress through ER (3). An independent approach was based on the preparation of a series of monomeric derivatives of BS-RNase. Only monomers in which Cys-31 and –32 (involved in the intersubunit disulfides in BS-RNase) are linked through mixed disulfide bonds to thiol compounds, (M(SSR)2 monomers), bound effectively to isolated plasma membranes (PM), as did BS-RNase. These monomers were found to be cytotoxic for malignant cells, whereas monomers with S-alkylated Cys-31 and –32 (M(S-alkyl)2 monomers) did not bind to PM and were not cytotoxic. Apparently, the mixed disulfides enabled the monomers to reconstitute into dimeric BS-RNase using cell sulfhydryls for -SH -S-S- interchange reactions. Based on these results, we can propose the following cytotoxic pathway in malignant cells for seminal RNase and M(SSR)2 monomers: ECM → PM → endosomes → TGN → cytosol. (1) Youle, R. J. and D’Alessio, G. (1997) in Ribonucleases: Structures and Functions, Academic Press, New York, N.Y., 491-509. (2) Mastronicola, M. R. Piccoli, R. and D'Alessio, G. (1995) Eur. J. Biochem. 230, 242-249. (3) Bracale, A., Spalletti-Cernia, D., Mastronicola, M., Castaldi, F., Mannucci, R., Nitsch, L. and D’Alessio, G. (2002) Biochem. J. 362, 553-560. Research funded by grants from AIRC, MIUR and CNR, Italy.

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(P9) STUDIES OF THE STRUCTURAL BASIS OF EOSINOPHIL CATIONIC PROTEIN CYTOTOXICITY Esther Carreras1, Ester Boix1, Helene F. Rosenberg,2, Claudi M. Cuchillo1 and M. Victòria Nogués1

1Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain 2Laboratory of Host Defenses, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA Eosinophil cationic protein (ECP) is a member of the superfamily of pancreatic RNases, it is found in the secondary granules of eosinophils. It exhibits several biological activities related to the immune system. In addition to the RNase activity it is toxic to bacteria, helminths and the host epithelial tissues and has antiviral activity against the single stranded RNA virus Respiratory Syncitial Virus (RSV). The molecular mechanism of ECP cytotoxicity is not clear. It has been proposed it may be due to the capacity of the protein to disrupt cell membranes. The cytotoxic activity has been related to the basic amino acids residues located on the surface of the protein and its interactions with the negatively charged molecules on cell membrane. The aim of this work is to study the role of specific basic and aromatic amino acid residues on the cytotoxic properties of the protein. Mutants of rECP were constructed by site directed mutagenesis, expressed in E.coli and purified from inclusion bodies. Recombinant ECP (rECP) and mutants were tested for its toxicity to bacteria, E.coli strain BL21DE3 and S. aureus strain 502 A, mammalian cell lines (HL60s cells and K562 cells) and for its antiviral activity to RSV. From preliminarY results the study was focused on bactericidal activity. The bactericidal activity of rECP variants were assessed and compared to rECP wild type. We have observed that rECP mutants show different degrees of bactericidal activity depending on the location of the basic and aromatic amino acids on the surface of the protein. To analyse the relationship between cytotoxicity and the effect on the membrane disruption we have checked the leakage of liposomes prepared from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[ phospho-rac -(1-glycerol)]. Leakage was determined by fluorescence using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) method. The leakage was assayed for wild type and variants of rECP. The results have indicated a preferential effect on tryptophan residues on the membrane stability and no effect was observed for RNase A and EDN. Supported by Grants BMC2000-0138-C02-01 from DGES, Ministerio de Educación y Cultura and 2000SGR 00064 from DGR, Generalitat de Catalunya (SPAIN).

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(P10) KINETIC ANALYSIS OF CONFORMATIONAL CHANGES IN THE ESCHERICHIA COLI RIBONUCLEASE III PRE-STEADY STATE MONITORED BY CHANGES IN INTRINSIC PROTEIN FLUORESCENCE Adam G. Cassano, Frank E. Campbell, and Michael E. Harris

Department of Molecular Biology and Microbiology and Center for RNA Molecular Biology Case, Western Reserve University 10900 Euclid Ave,. Cleveland, OH, USA The Ribonuclease III (RNase III) family of enzymes is required for critical RNA processing steps involved in numerous biological processes in both prokaryotes and eukaryotes. Despite new structural information, very little is known about the conformational changes that accompany catalysis. To address this issue, we examined the intrinsic protein fluorescence of RNase III from Escherichia coli in the pre-steady state. During a single round of binding and cleavage by the enzyme, we detect two distinct fluorescence transitions, which we interpret as two separate conformational changes. The first fluorescence transition displays a requirement for either Ca2+ or Mg2+ and is relatively pH independent. Additionally, the first transition is dependent on the substrate concentration with a second order rate constant of 1.0 ± 0.1 × 108 M-1s-1 at pH 7.3. These data are consistent with this conformational change corresponding to binding. The second fluorescence transition displays a requirement for Mg2+ only and is sensitive to pH. This transition is not dependent on substrate concentration and displays a first order rate constant of 6.4 ± 0.8 s-1 at pH 7.3. These data suggest this conformational change occurs after substrate binding. Comparison of the second fluorescence transition with a cleavage reaction monitored by 32P-labelled substrate under identical conditions indicates that the conformational change corresponds with the chemical step. Circular dichroism experiments confirm a change in protein structure upon substrate binding in the presence of Ca2+ and suggest that a substrate conformational change occurs as well. These techniques not only provide new insight into the structural dynamics of RNase III catalysis, but also provide an important new tool for the dissection of the molecular basis of RNase III substrate recognition and chemical mechanism. AGC is a Howard Hughes Pre-Doctoral Fellow.

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(P11) CHARACTERISATION OF THE BACILLUS SUBTILIS 5S RRNA MATURASE, RNASE M5: ARE RNASE M5 AND TYPE I TOPOISOMERASE CLEAVAGE REACTIONS ANALAGOUS? Jordi Rourera, Frédérique Allemand, Dominique Brechemier-Baey, Harald Putzer and Ciarán Condon. Institut de Biologie Physico-Chimique UPR 9073,13 rue Pierre et Marie Curie, 75005 Paris, France. Over twenty-five years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin, M. L. & Pace, N. R. (1974) Nature 252, 598-600). We now know that RNase M5 is encoded by a gene of previously unknown function, that is highly conserved among the low G+C Gram-positive bacteria, and have renamed the gene rnmV. The rnmV gene is non-essential in B. subtilis and strains lacking RNase M5 do not make any mature 5S rRNA whatsoever, indicating that this process is not necessary for ribosome function. Large 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the E. coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or other system for 5S rRNA production, with an imperfect division along Gram-negative and Gram-positive lines. Gene array analysis has shown that RNase M5 has few, if any, messenger RNA substrates in B. subtilis and thus its activity appears to be restricted to 5S rRNA maturation. The N-terminal half of RNase M5 constitutes a Toprim domain, found in the the archaebacterial reverse gyrases, type I toposiomerases and DNA primases. We have successfully modelled the Toprim domain of RNase M5 on the equivalent domain of the E. coli primase DnaG. Since topoisomerases cleave double-stranded DNA to permit strand passage while relaxing supercoils, we believe the RNase M5 and type I topoisomerase cleavage mechanisms to be very analagous. We have begun a mutational analysis of highly conserved amino acids in this and in other regions of RNase M5 to map the functional domains of the enzyme to confirm this hypothesis.

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(P12) CORE MANIPULATIONS, METAL BINDING: RNase T1 AS A TEST-CASE. De Vos, S., Deswarte, J., Backmann, J., Langhorst, U., Steyaert, J., and Loris, R. Department of Applied Biological Sciences, Institute for Molecular Biology and Biotechnology, Free University of Brussels (VUB), Belgium. Differential scanning calorimetry, urea denaturation and X-ray crystallography were combined to study the structural and energetic consequences of refilling an engineered cavity in the hydrophobic core of RNase T1 with CH3, SH and OH groups (1). Three valines that cluster together in the major hydrophobic core of T1 were each replaced by Ala, Ser, Thr and Cys. Compared to the wild type protein, all these mutants reduce the conformational and thermodynamic stability of the enzyme considerably. The relative order of stability equals Val > Ala ≈ Thr > Ser at all three positions. The effect of introducing a sulfhydryl group is more variable. Surprisingly, a Val → Cys mutation in a hydrophobic environment can be as or even more destabilizing than Val → Ser. Furthermore, our results reveal that the penalty of introducing an OH group into a hydrophobic cavity is roughly the same as the gain obtained from filling the cavity with a CH3 group. The inverse equivalence of the behavior of hydroxyl and methyl groups seems to be crucial for the proteins’ unique three-dimensional structure. In the crystalline state, RNase T1 binds calcium ions at different lattice-dependent positions. In solution, its conformational stability is remarkably increased in the presence of divalent metal ions as well. Combining urea unfolding studies and X-ray crystallography, we compared the presence of several metal ions at specific sites in the protein to their contribution to the overall stabilizing effect in solution (2). We constructed thermodynamic cycles involving particular metal ions and specific carboxylate functions. The resulting coupling energies indicate that some (but not all) metal ions found at lattice contacts in crystal structures may indeed significantly contribute to stability enhancement in the presence of metal ions in solution. 1. De Vos, S. et al (2001). Biochemistry 40, 10140-10149. 2. Deswarte, J. et al (2001). Eur. J. Biochem. 268, 3993-4000. This work was supported by the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie (IWT), and the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO). R. Loris and J. Deswarte are postdoctoral fellow and research assistant, respectively, at the FWO. S. De Vos received financial support from the IWT.

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(P13) TRANSCRIPTIONAL ACTIVATION OF THE MOUSE EOSINOPHIL-ASSOCIATED RIBONUCLEASE 2 (EAR2) GENE BY INTRONIC ENHANCER ELEMENTS: A COMMON REGULATORY FEATURE OF HUMAN AND MOUSE EOSINOPHIL-ASSOCIATED RIBONUCLEASE GENES. Kimberly D. Dyer, Joanne M. Moreau and Helene F Rosenberg Laboratory of Host Defenses, National Institutes of Health, U.S.A. The eosinophil-associated ribonuclease (Ear) gene cluster of Mus musculus contains at least thirteen intact open reading frames that are highly divergent orthologs of the human eosinophil ribonucleases - eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP). We demonstrate that the presence of the non-coding exon 1 and the intron in tandem with 361 nucleotides of 5’ sequence containing the putative promoter of the Ear2 gene enhances transcription 20 to 50 fold over activity observed with promoter alone in four different mouse cell lines (3T3, M1, LA-4 and RAW264) in the dual luciferase assay. The presence of intronic enhancer elements has been observed in human EDN and ECP [1-3] and three similar purine-rich elements are present in the intron of the Ear2 gene [4]. Disruption of any of the three sites by site directed mutagenesis reduces transcriptional activity of the promoter-exon-intron reporter to less than 70% of the activity of the wild-type construct in three of four cell lines. Gel shift analysis reveals the formation of specific DNA-protein complexes when oligonucleotides containing the respective sites are incubated with LA-4 nuclear extract. We have identified one of the three sites as an NFATc1 site based on the ability of an antibody directed against NFATc1 to disrupt the specific DNA-protein complex in the gel shift assay. The NFATc1 intronic enhancer site is also found in the newly identified genes encoding Ear1, Ear5, Ear7, Ear10, Ear11, Ear12 and Ear13 suggesting conservation of important intronic regulatory elements among members of the human and mouse eosinophil-associated ribonucleases. Our results suggest that the conservation of transcriptional activation elements despite rapid diversification and minimal conservation of nucleotide sequence, argue for the importance of this transcriptional element to the overall scheme of regulated expression among members of this unique gene family. Additionally the isolation of multiple mouse Ear genes (eight to date) suggests that the diversity of this family is probably genomic based and that the multiple open reading frames isolated to date (more than fifteen) represent unique genes. 1. Handen, J.S. and H.F. Rosenberg (1997) J. Biol. Chem .272,. 1665-1669 2. Tiffany, H.L., et al. (1996) J. Biol.Chem. 271, 12387-12393. 3. van Dijk, T.B., et al. (1998) Blood 91,. 2126-2132. 4. McDevitt, A.L., et al. (2000) Gene 267, 23-30. Research funded by the National Institutes of Health as part of the intramural research division.

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(P14) SOLUTION PROPERTIES OF BOVINE SEMINAL RIBONUCLEASE Carmine Ercole1, Francesca Avitabile1, Orlando Crescenzi1, Giuseppe D'Alessio2, Teodorico Tancredi3 and Delia Picone1. 1 Dipartimento di Chimica, Università Federico II di Napoli, Via Cintia, 80126, Napoli, Italy. 2 Dipartimento di Chimica Biologica, Università Federico II di Napoli, Via Mezzocannone, 80134, Napoli, Italy. 3 Istituto Chimica Biomolecolare del CNR, Pozzuoli, Napoli, Italy. Bovine seminal ribonuclease (BS-RNase) is the only dimeric protein of the pancreatic type ribonuclease family. The two identical subunits are linked through disulphide bridges between two adjacent cysteines, located at position 31 and 32. Each subunit is homologous (80% identity) to bovine pancreatic RNase A. In particular, both enzymes exhibit active sites constituted by identical amino acid residues in the same sequence position. Moreover, BS-RNase is the only known dimeric enzyme characterised, in solution, by an equilibrium between two different structures (1): in the form dubbed MxM the N-terminal arms are swapped between the subunits, whereas in the form indicated as M=M no swapping occurs. In the native protein, the equilibrium ratio between MxM and M=M is 70:30.

We expressed in E. coli and purified BS-RNase. After refolding and oxidative dimerization, the sample is mainly in the M=M form; with time, interconversion into the swapped form occurs, until essentially the same MxM to M=M equilibrium ratio as for the native protein is reached. The kinetics of this process was monitored both at 4 °C and at 37 °C.

These investigations are being extended to mutants obtained by substituting residues from the sequence of RNase A into the corresponding positions of BS-RNase. At the outset, we concentrated on the region which is structurally most affected by the swapping, the so-called hinge peptide. A comparison of the exchange behaviour of the chimeric mutants and of the parent wild-type ribonuclease will be also presented to clarify the features which determine the swapping of BS-RNase. 1. Piccoli, R., Tamburrini, M., Piccialli, G., Di Donato, A., Parente, A., and D'Alessio, G. (1992). Proc. Natl. Acad. Sci. U.S.A. 89, 1870-1874. Research funded by a grant from MURST (PRIN 2000) and CNR Agenzia 2000.

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(P15) STRUCTURAL ASPECTS OF ANTISENSE OLIGONUCLEOTIDE-BASED ARTIFICIAL RIBONUCLEASES Martin M. Fabani1, Marina A. Zenkova2, Natalia G. Beloglazova2, Vladimir V. Sil’nikov2, Valentin V. Vlassov2, Kenneth T. Douglas1, Elena V. Bichenkova1*. 1 School of Pharmacy and Pharmaceutical Sciences, University of Manchester, U.K. 2 Institute of Bioorganic Chemistry, Novosibirsk, Russia.

The creation of artificial ribonucleases is one of the most challenging approaches towards RNA targeting [1-2]. Their design has normally been based on imitation of the active site of natural ribonucleases (e.g. RNase A or T1) by chemical conjugation of hydrolytically active groups (imidazole, aliphatic amino groups, etc.) that normally constitute the catalytic domain of natural ribonucleases. The binding site of the artificial nucleases, designed to deliver a hydrolytic construct to the RNA target, normally contains intercalating or polycationic groups able to bind RNA. Alternatively, the binding site can be an oligonucleotide moiety complementary to the target RNA, recognising the desired RNA region(s) via specific Watson-Crick hydrogen-bonding.

Recently we proposed and designed a number of molecular scaffolds expected to provide artificial RNase activity against a chosen region of tRNAPhe. We synthesised some representatives of this new type of artificial ribonuclease containing bis- and tetra- imidazole cleaving constructs conjugated to antisense oligonucleotides via flexible linkers of different structures and lengths. Biochemical assays of the hydrolytic activities of these artificial ribonucleases showed that their ability to cleave tRNAPhe is governed by intrinsic properties of the respective cleaving constructs.

Molecular modelling was used to determine the prefered orientation(s) of the cleaving group(s) in the vicinity of the cleavage site to provide a structural basis to explain the hydrolytic strengths of various conjugates. Bis-imidazole cleaving constructs were found to be conformationally highly flexible, with no preferred specific conformation regardless of their initial modelling position, implying the absence of interactions between cleaving groups and target able to stabilise an ‘active’ conformation. The hydrolytic activity of bis-imidazole structures appears to be a random event, with no particular pre-scaffolded conformation or interactions with the target site. These data are in agreement with the experimentally determined limited hydrolytic activity (≈25%) found for these compounds.

For the Tetra-Imidazole containing compounds molecular modelling showed that preferable orientation(s) of cleaving constructs to form a pre-organized ‘active’ conformation, strongly dependant on the chemical structure of the linker connecting these constructs to the antisense oligonucleotide. The inclusion of deoxyribothymidine into the linker significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a semi-stacking interaction with the neighbouring nucleotide residue. This is consistent with the observed hydrolytic activity found for these ribonucleases. 1. Podyminogin M.A., Vlassov V.V., Giege R. “Synthetic RNA-cleaving molecules

mimicking ribonuclease A active center. Design and cleavage of tRNA transcripts”, Nucleic Acids Research (1993), 21 (25), 5950-5956.

2. Beloglazova N.G., Sil’nikov V.N., Zenkova M.A., Vlassov V.V. “Cleavage of yeast tRNAPhe with complementary oligonucleotide conjugated to a small ribonuclease mimic”, FEBS Letters (2000), 481, 277-280.

Research is funded by Wellcome Trust (CRIG) grant.

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(P16) PROCESSIVE PROPERTIES OF EUKARYOTIC RIBONUCLEASES H1: SITES OF RNase H CLEAVAGE ARE AFFECTED BY THE 3'-TERMINUS OF THE DNA. Sergei A. Gaidamakov and Robert J. Crouch. Laboratory of Molecular Genetic NICHD NIH Ribonucleases H (enzymes that degrade RNA in RNA-DNA hybrids) are ubiquitous and highly conserved. RNA-DNA hybrids are positioned in the enzyme such that the binding region interacts with both RNA and DNA 7-8 nucleotides from the catalytic site with contacts to the DNA strand throughout this distance. For a typical RNA-DNA hybrid of 18 bp, cleavage by E. coli RNase HI can occur as close as 5-6 nucleotides from the 5'-end of the RNA; a result consistent with the structural data. Extending the 3'-end of the DNA by a few (unpaired) bases shifts the site of cleavage one nucleotide closer to the 5'-end of the RNA, indicating single-stranded DNA can interact with the basic protrusion. Surprisingly, addition of any of several other substituents to the 3'-end of the DNA also permits cleavage at the site closer to the 5'-end of the RNA, suggesting DNA is not required for positioning the substrate for this "extra" cleavage. Similar changes in cleavage of RNA-DNA hybrids by mouse and human RNases H1 have been observed even though these eukaryotic RNases H1 possess a separate domain that interacts with duplex RNAs. The functional role of this dsRNA-binding domain (highly conserved 40 amino acids) is unknown. We demonstrate that RNase H1 from eukaryotic organisms (such as human mouse and S. pombe) have processive properties due to dsRNA-binding domain. Eukaryotic enzymes digest poly(rA-dT) in a different manner compared with E. coli RNase HI. Deletion of dsRNA-binding domain from human or S. pombe RNase H1 converts digestion manner to a nonprocessive type, similar to bacterial RNase HI.

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(P17) THE PRESENCE OF LEU-145 IS ESSENTIAL TO MAINTAIN THE ACTIVE SITE ELECTROSTATIC ENVIRONMENT OF RIBONUCLEASE α-SARCIN. Flor García-Mayoral1, Masip, M.2, García-Ortega, L.2, Pérez-Cañadillas, J.M.1, Martínez del Pozo, A.2, Gavilanes, J.G.2, Rico, M.1 and Bruix, M1. 1Departamento de Espectroscopía y Estructura Molecular, Instituto de Química Física “Rocasolano”, CSIC, Madrid, Spain. 2Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain Extracellular fungal RNases constitute a family of structurally related proteins, represented by RNase T1, which includes ribotoxins such as α-sarcin. RNase T1 active site residues involved in catalysis are conserved in all fungal RNases, except for Phe 100, which is not present in the ribotoxins. In these proteins, this Phe is replaced by Leu. One of the most remarkable differences between α-sarcin and RNase T1 active sites is the pKa value of the catalytic histidine. In α-sarcin, His 137 has a low pKa of 5.8, whereas in RNase T1 His 92 has a pKa of 7.4. The existence of a π/cation interaction in RNase T1 between His 92 and Phe 100 was proposed as one of the possible explanations for the stabilization of the protonated form of the imidazole ring. In this work, α-sarcin Leu 145 has been mutated to Phe. This mutant has been purified, characterized from structural and enzymatic point of view and the individual pKa of His 137 measured by NMR methods. The analysis of the far-UV circular dichroism, fluorescence spectra and 1H NMR data confirm that the mutated protein retains the native conformation. The thermal denaturation was also identical to that exhibited by the wild-type protein. The enzymatic properties of the mutant were only slightly modified. Thus, the L145F variant of α-sarcin retained the ability to specifically inactivate the ribosomes, releasing the α-fragment. However, a higher level of non-specific activity was observed for the variant. The L145F variant cleaves ApA releasing the same products as the wild-type protein, although with altered kinetic parameters. A pKa of 7.1 for His 137 was determined analyzing a set of 1D 1 H-NMR spectra recorded at 17 pH values. Thus, substitution of α-sarcin Leu-145 by Phe results in a mutant protein which still retains the wild-type ability to specifically cleave the rRNA in the ribosomes. However, its active site has been sufficiently altered as to improve its ability as non-specific RNase. Additionally, the results presented show how Leu 145 has a major role in preserving the unique electrostatic properties of the α-sarcin active site.

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(P18) THE RELATIVE PROPORTIONS OF RNase A AGGREGATES VARY, THOSE OF DIMERS EVEN INVERTING, AS A FUNCTION OF EXPERIMENTAL CONDITIONS Giovanni Gotte, Francesca Vottariello, and Massimo Libonati The study of protein aggregation, an event or remarkable interest, can also be approached using bovine RNase A as a model. By lyophilizing RNase A from 40% acetic acid solutions, two dimeric aggregates -a “less basic” and a “more basic” dimer, named according to their chromatographic properties- form in the invariable ratio of 1:4. In addition, two trimeric and two tetrameric conformers, as well as higher order oligomers are produced (1). The structures of the two dimers and trimers have been solved, and a plausible model for the two tetramers was proposed. The aggregates form by the 3D domain-swapping of the N-terminal or C-terminal ends of the protein or both. With the aim to contribute to the understanding of the manner in which RNase A aggregation occurs, we studied the aggregation process under many experimental conditions, always avoiding a lyophilization step. Methodology. RNase A (type XII-A) was dissolved at high concentration (200 mg/ml) in various media (ethanol, trifluoroethanol, water, NaCl, various buffers at different pH values etc.) and incubated at temperatures comprised between 23 °C and 70 °C for times ranging from a few minutes to 2 hours. The treatment was ended by diluting the incubation mixtures 1:100 with 0.2 M sodium phosphate buffer, pH 6.7, and transferring them in to an ice-bath. The samples were then gel filtered or ion-exchange chromatographed to separate the (possibly formed) products. The already well characterized oligomers (1) were always used as standards to identify the various aggregated species formed under the different experimental conditions, each one being then quantified by measuring the area of its peak and calculating its percentage relative to the sum of the areas of all eluted peaks. Results. Under all conditions tested RNase A forms aggregates, their amounts and relative proportions varying in dependence on the environmental conditions used. Relatively high quantities of dimers (about 10% and 18% for the less basic and the more basic dimer, respectively), trimers or tetramers form in aqueous ethanol or trifluoroethanol at 60 °C. Under milder conditions (all media at 23 °C, NaCl, water, buffers at acid or alkaline pH values at 60 °C), lower amounts of the two dimers and negligible amounts of higher aggregates form, but –interestingly- the proportions of the two dimeric conformers are inverted, the less basic dimer definitely prevailing now over the more basic one. Conclusions. The results reported, together with the analysis of the thermal unfolding of native RNase A dissolved in the various media tested, suggest that aggregation of the enzyme protein is of course highly dependent on the unfolding conditions. When the unfolding mainly concerns the N-terminal end of the protein, that is relatively richer in hydrophilic residues, the production of the less basic dimer, formed by the 3D domain-swapping of the N-terminal α-helix of each monomer, prevails over that of the more basic dimer. When also the C-terminal end of RNase A, relatively richer in hydrophobic amino acids, unfolds, the more basic dimer, formed by the 3D domain-swapping of the C-terminal β-strand, prevails over the other, possibly because of the induction to

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aggregation exerted by the relatively more abundant hydrophobic residues (2) present in the C-terminal ends of the two monomers. (1) Gotte, G., Bertoldi, M., and Libonati, M. (1999) Eur. J. Biochem. 265, 680-687. (2) Chiti, F., Taddei, N., Baroni, F., Capanni, C., Stefani, M., Ramponi, G., and Dobson, C.M. (2002) Nature Struct. Biol. 9, 137-142. Research funded by a grant from the Italian MURST-PRIN 2000-2001

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(P19) ATTEMPTS TO CHANGE THE SPECIFICITY OF RNase T1 BY RANDOM MUTAGENESIS R. Czaja, M. Hänsler, H. Hoier, K. Höschler, B. Hubner, M. Struhalla, P. Orth, W. Saenger & Ulrich Hahn University of Leipzig, Faculty for Biosciences, Institute for Biochemistry, FRG Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far. Therefore we applied a semirational approach by randomizing the guanine binding site. A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of six amino acid positions within the recognition loop was screened on RNase indicator plates. The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S-N43W-N44H-Y45A-E46D (RNaseT1-8/3) exhibits an altered preference towards purine nucleotides. The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type. Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2´3´-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable [1]. Variant 9/5 with the guanine recognition segment K41E-Y42F-N43R-N44N-Y45W-E46Q has been cocrystallised with the specific inhibitor 2’-GMP. The crystal structure has been refined to a crystallographic R factor of 0.198 at 2.3 Å resolution [2]. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 Å, 2’-GMP is fixed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2’-GMP is located about 4.2 Å apart from its position in wild-type ribonuclease T1 – 2’-GMP complexes, allowing a Ca2+ ion, coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine affinity and turnover rate. The presence of Ca2+ ions was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extend. Further Variants have been constructed an crystallized. 1. B. Hubner, M. Hänsler & U. Hahn: Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment.(1999) Biochemistry, 38, 1371-1376 2. K. Höschler, H. Hoier, B. Hubner, W. Saenger, P. Orth & U. Hahn: Structural analysis of an RNase T1 variant with an altered guanine binding segment.(1999) J. Mol. Biol. 294, 1231-1238 Research funded by the “Deutsche Forschungsgemeinschaft“ and the German “Fonds der Chemischen Industrie“

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(P20) HIGH-YIELDING PROCEDURES FOR THE PRODUCTION OF PROTEINS BELONGING TO THE PANCREATIC RIBONUCLEASE SUPERFAMILY Michelle C. Hares1, Daniel E. Holloway1, Lori D. Horb1, M. Thomas E. Raven1, Robert Shapiro2, Vasanta Subramanian1 and K. Ravi Acharya1 1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom. 2Center for Biochemical and Biophysical Sciences and Medicine, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, U.S.A. We present a strategy potentially of general use for the production of proteins belonging to the pancreatic ribonuclease superfamily. The procedures were originally developed for the murine angiogenin homologues, angiogenin-related protein (mAngrp), angiogenin-3 (mAng-3) and angiogenin-4 (mAng-4) (1), and have since been adapted for the human proteins angiogenin (hAng), eosinophil-derived neurotoxin (EDN), and mutants thereof. Escherichia coli is often the vehicle of choice for recombinant protein production as it is inexpensive and easy to manipulate. However, 10 % of the codons in the mAngrp gene are classed as ‘rare’ in E. coli, posing a significant translational barrier. In addition, ‘leaky’ expression of this gene was found to be extremely deleterious to E. coli growth. Because of these factors, attempts to express mAngrp from various vectors in E. coli resulted in negligible accumulation of recombinant protein. Use of the E. coli strain BL21-CodonPlus(DE3)-RIL (Stratagene), which provides extra copies of the tRNAs encoded by the argU, ileY and leuW genes, obviated the need to engineer the codon usage of the gene. To overcome the toxicity problem, the vector pET-22b(+) (Novagen), which houses the tightly-regulated T7lac promoter, was employed. This combination resulted in high-level production of mAngrp in the form of inclusion bodies. Several ribonuclease refolding procedures described in the literature were ineffective for mAngrp inclusion bodies. However, the arginine-assisted method identified by several other laboratories in the ribonuclease field (2) refolded mAngrp with high efficiency. We went on to develop a two-step purification procedure that removed the need for a tangential flow concentrator, yielding 8 mg of pure, enzymatically-active protein per litre of culture. The yields of mAng-3 and mAng-4 were even higher, in the region of 12 and 30 mg/L, respectively. When existing synthetic, codon-optimized genes encoding hAng, EDN, and mutants thereof were expressed from pET vectors in E. coli BL21(DE3), the inclusion bodies that formed could be refolded and purified by these procedures in similarly high yields. These data suggest that the combination of a tightly-regulated promoter, E. coli BL21-CodonPlus(DE3)-RIL cells, and arginine-assisted refolding of the protein has general application for high-level production of pancreatic ribonuclease superfamily proteins.

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1. Holloway, D. E., Hares, M. C., Shapiro, R., Subramanian, V. and Acharya, K.R. (2001) Protein Expression and Purification 22, 307-317. 2. Buchner, J. and Rudolph, R. (1991) Bio/Technology 9, 157-162. Research funded by Cancer Research UK Grant SP2354/102 (to K.R.A.), Medical Research Council UK Grant 9540039 (to K.R.A.), and the National Institutes of Health Grant CA-88738 (to R.S.).

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(P21) CRYSTAL STRUCTURE OF TYPE 2 RIBONUCLEASE H FROM PYROCOCCUS HORIKOSHII Tomonori Hata, Yoshimitsu Kakuta, Yoshiaki Kouzuma, and Makoto Kimura Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan In a series of structural genomics on a hyperthermophilic archaeon, Pyrococcus horikoshii, we determined the crystal structure of type 2 ribonuclease H (PhRNase HII) at 2.0 Å resolution by molecular replacement with RNase HII from Thermococcus kodakaraensis (1) as a search model. The gene (PH1650) encoding the PhRNase HII was placed under the control of T7 phage promoter on the expression plasmid pET-22b and induced with IPTG. The recombinant PhRNase HII thus produced was purified to homogeneity by ion-exchange column chromatography on a POROS HS/M column. The absorbed protein was eluted by a linear gradient of NaCl concentration from 0 to 0.5 M in 10 mM Na-phosphate buffer, pH 6.5. The protein was further purified by butyl Toyopearl 650 M.in 10 mM Na-phosphate buffer, pH 6.5 containing 2.0 M ammonium sulfate. The purified PhRNase HII was crystallized under 0.1 M Tris HCl buffer (pH 8.5) with 2.0 M ammonium sulfate at 18° C. The crystal belongs to the space group P212121 with its cell dimensions of a=27.4 Å, b=96.5 Å, c=154.6 Å, and α=β=γ = 90°. The crystal structure was determined at 2.0 Å resolution by molecular replacement. The PhRNase HII is composed of two distinct domains. The N-terminal domain belongs to the α / β class of proteins, having seven α-helices and five stranded β-sheet, while the C-terminal domain forms a compact structure consisting of two α-helices. As a whole the PhRNase HII is highly homologous to other archaeal type 2 RNase Hs from Archaeglobus fulgidus, Methanococcus jannaschii and Thermococcus kodakaraensis. Muroya, A., Tsuchiya, D., Ishikawa, M., Haruki, M., Morikawa, M., Kanaya, S., and Morikawa, K. (2001) Protein Sci. 10, 707-714.

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(P22) MICROBIAL RIBONUCLEASES AS ANTI-PROLIFERATIVE AGENTS Olga Ilinskaya1, Florian Dreyer2; Ester Vock3 1Department of Microbiology, Kazan State University, Kazan, Russia. 2Rudolf-Buchheim-Institute of Pharmacology, University Giessen, Germany. 3Department of Toxicology, University Wuerzburg, Germany. The mammalian and microbial ribonucleases are two important families of RNases whereby the latter ones cannot be inhibited by the cytosolic ribonuclease inhibitor. That offers a preferable possibility to use the cytotoxic microbial RNases in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. The present study was performed to examine some important elements of mechanism of cytotoxicity induced by the guanine-specific ribonuclease secreted by Bacillus intermedius (binase). The anti-proliferative action of the binase was studied by the WST cytotoxicity test in different chicken, mouse and human cell lines. The proliferation rate of chicken embryo fibroblasts, either normal or Rous sarcoma virus-transformed, was significantly reduced by binase treatment. Among mouse fibroblasts, v-ras-transformed NIH3T3 cells were sensitive to binase, whereas the growth of non-transformed, v-src- or v-fms-transformed NIH3T3 cells was not affected. A 48h treatment with binase inhibited the Ca2+-dependent K+ current of v-ras-transformed NIH3T3 cells but had no effect on the membrane currents in non-transformed and in v-src- or v-fms-transformed NIH3T3 cells. Ca2+-activated K+ channels are almost ubiquitously distributed in mammalian cells and constitute a major link between second messenger systems and electrical activity of the cell. These alterations under binase treatment suggest that mammalian cells expressing the ras-oncogene are a potential target for the anti-proliferative action of binase. In earlier papers we reported that binase exhibits genotoxic effects by induction of forward AraR mutation and by histidine reverse mutation as well as by prophage-induction activity and by SOS-response in bacterial test-systems (1,2). Now we treated cultured human lung epithelial cells A549 with binase at high concentration to detect the induction of DNA double-strand breaks (DSB) assessed by pulsed-field gel electrophoresis. Induction of DSB by binase was seen only after cell viability was reduced to less than 30% of the control values, indicating that DSB were the consequence of extragenomic damage and viability loss. In contrast, the crosslinker diepoxybutane induced DSB by a genotoxic mode of action in concentrations that did not affect cell survival. The DNA fragments produced by diepoxide were initially large and were converted to smaller fragments (monotonously from 7 Mbp to less than 1 Mbp) within 72 h in the course of cell death (3). In contrast, the molecular size distribution of DNA fragments, generated by binase after 72h treatment, peaked below 100 kbp, implicates activation of DNA-degrading enzymes in the course of cell death and supports the cytotoxic mechanisms of DNA fragmentation.

1. Ilinskaya O, Ivanchenko OB, Karamova NS. (1995) Bacterial ribonuclease: mutagenic effect in microbial test-systems. Mutagenesis 10,165-170

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2. Ilinskaya ON, Karamova NS, Ivanchenko OB, Kipenskaya LV. (1996) SOS-inducing ability of native and mutant microbial ribonucleases Mutation Research 354, 203- 209

3. Vock EH, Lutz WK, Ilinskaya O, Vamvakas S. (1999) Discrimination between genotoxicity and cytotoxicity for the induction of DNA double-strand breaks in cells treated with aldehydes and diepoxides. Mutation Research 441,:85-93

This work was supported by DAAD (Deutscher Akademischer Austauschdienst).

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(P23) CATALYTIC MECHANISM OF AN RNA RESTRICTION ENZYME, COLICIN E5 Sakura Inoue1, Tetsuhiro Ogawa1, Shunsuke Yajima2, Makoto Hidaka1, Haruhiko Masaki1

1Department of Biotechnology, The University of Tokyo, Japan 2Department of Bioscience, Tokyo University of Agriculture, Japan Colicin E5 kills Escherichia coli cells by specifically cleaving tRNAs for tyrosine, histidine, asparagine, and aspartic acid between the common Q and U of their anticodons (Q is a modified nucleotide of G). The carboxyl-terminal ribonuclease domain of colicin E5, referred to as E5-CRD, recognizes not only tRNAs with either a QU or GU sequence at the anticodon but also the GpUp dinucleotide, leaving a 2', 3’-cyclic phosphate and a 5’-OH. This indicates that the chemical reaction of the transesterification of E5-CRD includes a nucleophilic attack of the ribose 2’-OH group on the phosphate group that forms a transition state with pentacovalent phosphorus, followed by the cleavage of the P-O5' bond forming a 2’, 3’-cyclic phosphate. E5-CRD is a unique ribonuclease in the absence of a histidine residue within the molecule, which has been implicated as an indispensable catalytic residue for traditional ribonucleases. X-ray crystallographic studies of the complex of E5-CRD and dGpdU, an inactive substrate homologue, revealed the precise structure of the active site of E5-CRD. Based on this result, site-directed mutagenesis on the active site has identified some residues essential for the catalytic reaction. We propose a new catalytic mechanism of a ribonuclease that has no histidine residue. 1. T. Ogawa, K. Tomita, T. Ueda, K. Watanabe, T. Uozumi and H. Masaki (1999) Science, 283, 2097-2100

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(P24) MAPPING OF ANGIOGENIN CONTACTS WITH SMALL-MOLECULE INHIBITORS BY KINETIC AND DOCKING STUDIES Jeremy L. Jenkins1 and Robert Shapiro1,2 1Center for Biochemical and Biophysical Sciences and Medicine and 2Department of Pathology, Harvard Medical School, MA, USA Angiogenesis, or new blood vessel formation, provides vital nourishment for growth and metastasis of solid tumors in vivo. Angiogenin (ANG) is an RNase A homolog and a potent inducer of angiogenesis. Importantly, its ribonucleolytic activity is essential for its biological activity, suggesting that the enzymatic site may be a target for development of new anticancer agents. We recently identified two small-molecule inhibitors of ANG in a fluorescence-based high-throughput screen [Lead 1 (KD = 81 µM) and Lead 2 (KD = 41 µM)]; these compounds bind ANG about 10-fold more tightly than any nucleotide inhibitors under physiological conditions. The aims of the current study were to perform structure-based searching for analogs of the two lead compounds with enhanced inhibitory activity and, subsequently, to map their contact residues on ANG. Leads 1 and 2 were docked to ANG with the program AutoDock 3.0 to predict critical structural cores for binding. A similarity search of online chemical databases was then carried out and analogs with similar core structures were assayed for inhibition of Ang-catalyzed cleavage of an octanucleotide substrate. Using this approach, we evaluated over 70 analogs of Leads 1 and 2. Among these, two analogs of each lead were selected for further analysis; the Lead 1 analogs displayed KD values that were 3.2 and 20-fold tighter than their parent compound (KD = 25 and 4 µM), and the Lead 2 analogs showed 2-fold greater inhibition than their parent compound (KD = 20 and 24 µM). Significantly, one analog of Lead 2 displays a higher affinity for ANG than for RNase A; this preference has not been observed for any small-molecule tested previously. Docking was again employed to generate model complexes of ANG with the second-generation leads. On the basis of the docking models, ANG variants were used in kinetic assays to map the contact ANG residues for the parent leads and the 4 analogs. The results indicated several residues in or near the active site were involved in binding the small-molecule inhibitors; in particular, Ala replacement of Arg5, a phosphate-binding residue in the P2 subsite, dramatically reduced binding to all inhibitors and effectively eliminated Lead 1 binding. This was in agreement with the docking models, which predicted ionic interactions between Arg5 and sulfonates from the Lead 1 series or carboxylates from the Lead 2 series. Replacement of Gln117 or Gln12, shown previously to increase nucleotide binding, lowered KD values for the leads as well. These data, in addition to results with His8Ala, Asn68Ala, and ∆119-123 variants, support the computational models for use in structure-based design of improved ANG inhibitors. Research supported by the National Institutes of Health (grant CA88738).

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(P25) EXPRESSION AND CRYSTALLIZATION OF A WOUND-INDUCIBLE RIBONUCLEASE NW FROM NICOTIANA GLUTINOSA Shin Kawano, Yoshimitsu Kakuta, and Makoto Kimura Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan Ribonuclease NW (RNase NW), the wound-inducible RNase in Nicotiana glutinosa leaves, preferentially cleaves guanylic acid (1). We expressed the cDNA encoding RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant RNase NW (rRNaseNW) secreted into medium was purified to homogeneity by a series of column chromatography. The rRNase NW was crystallized in the presence of 5’-GMP. The crystal structure of rRNase NW bound to 5’-GMP was determined at 1.5 Å resolution by molecular replacement with the tomato RNase LE (2) as a search model. The rRNase NW structurally belongs to the (α + β) class of proteins, having eight α-helices and six β-strands and its structure is highly similar to those of other plant RNases, including RNase MC1 from bitter gourd seeds. The guanine ring of 5’-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: he guanine moiety is stabilized in the hydrophobic pocket via a sandwich-like stacking interaction with two aromatic side chains of Tyr17 and Phe89. In addition, the guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79. This result reveals that Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of guanine base by RNase NW, providing insight into the way in which RNase NW preferentially cleaves guanylic acid. (1) Kariu, T., Sano, K., Shimokawa, H., Itoh, R., Yamasaki, N., and Kimura, M. (1998) Biosci. Biotechnol. Biochem. 62, 1144-1151. (2) Tanaka, N., Arai, J., Inokuchi, N., Koyama, T., Ohgi, K., Irie, M., and Nakamura, K. (2000) J. Mol. Biol. 298, 859-873.

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(P26) THE RESIDUE IMMEDIATELY UPSTREAM OF THE RNase P CLEAVAGE SITE IS A POSITIVE DETERMINANT Leif A. Kirsebom, Mathias Brännvall and B. M. Fredrik Pettersson Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Sweden. The tRNA genes are transcribed as precursors with extra nucleotides located both at the 5'- and 3'-termini and in the spacer regions between tRNA genes in multimeric tRNA transcripts. Consequently the tRNA precursors have to be processed to generate functionally tRNA. RNase P is an endoribonuclease responsible for generating the 5' end of matured tRNA molecules. In Escherichia coli, this ribonucleoprotein complex consists of the catalytic RNA subunit and a basic protein, M1 RNA and C5, respectively. The reaction requires divalent metal ions preferentially Mg2+ and correct and efficient cleavage occurs in vitro in the absence of protein. In almost half of the tRNA precursors in E. coli the residue at –1 are base paired with the base at position +73 where the residue at +73 is part of the "RNase P RNA-RCCA" interaction (residue +73 underlined). A possibility is that the residue at –1 interacts with M1 RNA as a result of formation of the +73/294 interaction in the RNase P RNA-complex. This would expose the cleavage site as we previously suggested. Moreover, analysis of available genome sequences reveals that in most bacteria the preferred nucleotide immediately upstream of the RNase P cleavage site (normally referred to the –1 position) is uridine. Here we studied the importance of the residue immediately upstream the cleavage site in the M1 RNA mediated cleavage reaction using various model substrate derivatives carrying base substitutions and modifications at the 2' position in the ribose. The different substrates were studied with respect to cleavage site recognition and kinetics of cleavage. The in vitro studies were correlated with in vivo analysis where we used different nonsense tRNA constructs carrying changes at the –1 position. Based on our findings we suggest that the residue at the –1 position is a positive determinant in the M1 RNA catalyzed reaction. In addition, the data suggest that the 2' OH immediately upstream of the cleavage site is important for Mg2+-coordination.

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(P27) RIBONUCLEASE A IN TRIFLUOROETHANOL – A STUDY ON UNFOLDING BY PROTEOLYSIS AND STOPPED-FLOW FLUORESCENCE SPECTROSCOPY Jens Köditz, Yvonne Markert, Ralph Golbik* and Renate Ulbrich-Hofmann Martin-Luther University Halle-Wittenberg, Department of Biochemistry/Biotechnology, Institute of Biotechnology and Institute of Biochemistry*, Kurt-Mothes-Str. 3, D-06120 Halle ,Germany. Although enzymes are widely used in organic solvents, their conformational changes in these media are poorly understood. Trifluoroethanol (TFE) is a particularly interesting solvent for studying the influence of organic solvents on the structure of proteins, because it promotes the formation of short-range H-bonds inducing helical structures. On the other hand, TFE disrupts the tertiary structure by weakening the hydrophobic interactions in proteins resulting in a TFE-denatured state at high TFE concentration, the so-called helix-coil-helix state. Therefore, the investigation of unfolding and refolding of proteins in TFE allows to analyze the interplay of secondary structure-forming and tertiary structure-destroying forces. In this paper, we report on the unfolding of ribonuclease A (RNase A) in TFE, which was followed by proteolysis and stopped-flow fluorescence spectroscopy. Proteolysis has been proven to be appropriate for detection of local structural changes being undetectable by spectroscopic methods (1). Moreover, this technique allows the determination of unfolding rate constants in the pretransition region, where spectroscopic methods fail (2). The latter, however, is based on the assumption that the proteolysis of the native protein can be neglected, which is the case for thermolysin as protease (3). Since thermolysin is inactive in TFE, this method had to be modified for studying RNase A in TFE. Proteinase K was used as protease in combination with the mutant enzyme A20P-RNase A. This RNase A variant was shown to have the same activity and thermodynamic stability as wild-type RNase A, but is more resistant toward proteinase K digestion in the native state (4). By this means, unfolding rate constants could be determined as a function of TFE concentration (0–40 %, v/v). Moreover, the stabilization of secondary structure in wild-type RNase A at low TFE concentrations (0-20 %, v/v) was detected by the proteolysis with proteinase K. The proteolytic experiments were completed by kinetic measurements of unfolding of wild-type and A20P-RNase A in 40-70 % (v/v) TFE by stopped-flow fluorescence spectroscopy. These results provide detailed information on structural changes of RNase A at different TFE concentrations. (1) Arnold, U. and Ulbrich-Hofmann, R. (2000) J. Protein Chem. 19, 345-352 (2000) (2) Arnold, U. and Ulbrich-Hofmann, R. (2001) Eur. J. Biochem. 268, 93-97 (3) Arnold, U. and Ulbrich-Hofmann, R. (1997) Biochemistry 36, 2166-2172 (4) Markert, Y., Köditz, J., Mansfeld, J., Arnold, U. and Ulbrich-Hofmann, R. (2001) Protein Eng. 14, 101-106 The research was supported by grants from the Max-Buchner foundation and from the Land Sachsen-Anhalt.

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(P28) COMPARATIVE ANALYSIS OF RNASE III CLEAVAGE MECHANISM Bruno Lamontagne, Ghada Ghazal, and Sherif Abou Elela Département de Microbiologie et Infectiologie, Université de Sherbrooke, Canada RNase III family is a class of double-stranded RNA endoribonucleases involved in the processing of many cellular RNAs and was recently implicated in gene repression by RNA interference (RNAi). The basic mechanism of RNA cleavage appears to be conserved among the different members of the RNase III family. However, the substrate requirement and cleavage site selection vary between enzymes from different organisms. Budding yeast RNase III (Rnt1p) introduces two staggered cleavages at each side of the RNA duplex like its bacterial and fission yeast orthologues. However, unlike its orthologues, Rnt1p recognizes a conserved class of AGNN terminal tetraloops and uses it to position its cleavage sites. This surprising observation suggests that the dsRNA specific nuclease recognizes the fold of a single stranded loop and uses a different mechanism of RNA binding than that used by other dsRNA binding proteins that recognize the minor groove of the dsRNA helix (1). To understand the mechanism of RNA recognition by eukaryotic RNase III and trace its evolutionary origin, we examined the substrate requirements and cleavage conditions of three RNases III from E.coli (RNase III), S. pombe (Pac1) and S. cerevisiae (Rnt1p). In vitro transcribed RNA varying in length and tetraloop sequences were used as model substrates. All three enzymes cleaved perfect intermolecular RNA duplex, but with varying efficiencies. RNase III was the most efficient followed by Pac1 and Rnt1p. Rnt1p poorly cleaved its RNA duplex under physiological salt concentrations underscoring the need for the conserved AGNN tetraloop. Next, we tested all three enzymes for cleavage of different RNA with stem-loop structures closely related to Rnt1p natural substrates either with conserved AGNN tetraloop or with GAAA tetraloop, which is not recognized by Rnt1p. As predicted, Rnt1p cleaved well the substrate containing the AGNN tetraloop while the cleavage of the GAAA containing substrate was detected only under special conditions. Bacterial RNase III did not differentiate between the two substrates, producing the same cleavage pattern. Surprisingly, Pac1 recognized the two substrates differently resulting in two different cleavage patterns. In presence of the GAAA tetraloop the cleavage pattern was random as observed with the bacterial RNase III. In contrast, Pac1 specifically cleaved the RNA containing the AGNN tetraloop at a fixed distance from the terminal loop similar to Rnt1p. However, the absolute distance between the cleavage sites and the tetraloop was different from that of Rnt1p, which probably reflect the structural differences of each protein. These results indicate that Pac1 could preferentially recognize the AGNN tetraloop. Pac1 recognition of the terminal tetraloop is consistent with the observation that Drosophila Staufen dsRBD interacts with the terminal tetraloop and suggests that perhaps the eukaryotic dsRBD containing proteins evolved to recognize tetraloop structures in a different matter than that of bacteria. Lebars, I., Lamontagne, B., Yoshizawa, S., Abou Elela, S., and Fourmy, D. (2001) Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing. EMBO J., 20, 7250-7258.

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(P29) ENZYMATIC ACTIVITY, DIVERSIFICATION AND IMMUNITY PROTEIN BINDING IN RNase COLICINS. Dan Walker, Lorna E Lancaster and Colin Kleanthous. School of Biological Sciences, University of East Anglia, Norwich, U.K. Colicin E3 is an antibacterial protein produced by E.coli during times of nutrient or environmental stress as a means of reducing competition from closely related microbial populations. The cytotoxicity of this protein is due to a highly specific RNase activity which catalyses a single, site-specific cleavage of 16S ribosomal rRNA resulting in the arrest of protein synthesis. In common with other E colicins, colicin E3 (a 60-kDa toxin) possesses three domains; a central receptor-binding domain which attaches the toxin to the outer membrane of bacteria, an N-terminal domain responsible for translocating the toxin into cells and a C-terminal, 12-kDa RNase domain (E3 RNase). This cytotoxic domain also binds a specific immunity protein known as Im3 (10-kDa) which inhibits the activity of the RNase and so prevents the suicide of the producing organism.

Our current work involves the identification and study of active site residues of E3 RNase, and in particular the position of these residues relative to the immunity protein binding site. The X-ray crystal structure of the E3 RNase-Im3 complex (1) in conjunction with site directed mutagenesis has revealed that the immunity protein (inhibitor) binds to E3 RNase at a position distant from the active site, a situation far removed from most other enzyme inhibitor complexes (e.g. barnase-barstar). Further to this, the residues of E3 RNase involved in immunity binding are formed from almost contiguous sequence, involving most of the residues of the N-terminal half of E3 RNase (42 residues of the 96 residue domain). Mutations in this region of the protein have little or no effect on the cytotoxic RNase activity of colicin E3. Residues that have so far been identified which either abolish or severely compromise activity are exclusively located in the C-terminal half of E3 RNase suggesting that the dual functionality of this small domain can be assigned to two functionally "independent" regions. These results are discussed in relation to the closely related DNase type colicins and the evolution of colicin-immunity specificity (2). (1) Carr, S., Walker, D., James, R et al (2000) Structure 8 (9) 949-960 (2) Kleanthous, C. and Walker, D. (2001) TIBS 26 (10) 624-631 Research funded by grants from BBSRC and the Wellcome Trust.

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(P30) S. CEREVISIAE RNase III (RNT1) AFFECTS TELOMERE HOMEOSTASIS Stéphanie Larose, Raymund Wellinger and Sherif Abou Elela Département de Microbiologie et d'Infectiologie, Université de Sherbrooke, Canada The bacterial RNase III family includes a growing number of dsRNA specific nucleases involved in RNA processing and degradation. Eukaryotic RNase III were implicated in rRNA and snoRNA processing and more recently in the regulation of mRNA required for development. Interestingly, this group of enzymes were also implicated in the process that leads to RNA interference (RNAi). Clearly, RNase III is central for the metabolism of cellular RNA and thus understanding this enzyme is essential to understand the mechanism of gene expression. We use Saccharomyces cerevisiae RNase III (Rnt1p) as model to identify new RNA targets and understand the cellular function the eukaryotic RNase III. Using biochemical and genetic techniques we demonstrate the implication of Rnt1p in telomere maintenance. The telomere maintenance is a critical step in the genetic life span of an organism. Telomere length is regulated by a homeostasis between degradation and lengthening processes. The lengthening process is catalyzed by a ribonucleoprotein particle called the telomerase. We have found that deletion of Rnt1p result in over expression of the RNA component of the telomerase and leads to longer telomeric repeats. We are currently delineating the mechanism by which Rnt1p could alter the telomere homeostasis.

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(P31) REGULATION OF RIBONUCLEASE EXPRESSION BY ESTRADIOL IN RANA CATESBEIANA (BULLFROG) Pin-Chi Tang1, Huey-Chung Huang2, Sui-Chi Wang1, Jen-Chong Jeng1and You-Di Liao1,

1Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan; 2Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei 100, Taiwan Multiple ribonucleases in a living organism are widely found, but the function and regulation of individual ribonucleases are still not clear. In the present study, we found that one oocytic ribonuclease, RC-RNase, was developmentally expressed in the liver and stored in the oocyte of bullfrog, while another ribonuclease, RC-RNase L1, is constitutively expressed and retained in the liver at all stages. In females, the expression of RC-RNase increased with the degree of maturity and the concentration of plasma estradiol during oogenesis. In males, the RC-RNase gene was activated in the liver and the newly synthesized protein was secreted into plasma if estradiol was administrated. To investigate the mechanism of estrogen-mediated activation of ribonuclease expression, we cloned the RC-RNase promoter and analyzed the putative transcription factor binding sites, e.g., TATA box, ERE, AP1 and CAAT box. Using luciferase as a reporter gene, we found that an estrogen response element (ERE) in the promoter of RC-RNase was essential for both basic transcription and estradiol-mediated gene activation in estrogen receptor-positive MCF7 cells. These results support the notion that RC-RNase is synthesized in the liver upon stimulation by estradiol during oogenesis, then secreted into the bloodstream and stored in oocytes for embryonic development.

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(P32) BIOLOGICAL ACTIONS OF RNase A OLIGOMERIC AGGREGATES Giovanni Gotte1, Josef Soucek2, Massimo Libonati1, Tomas Slavik3, and Josef Matousek3 1Dipartimento di Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Università di Verona, Verona, Italy. 2Institute of Hematology and Blood Transfusion, Prague, Czech Republic. 3Institute of Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic. Bovine seminal RNase (BS-RNase), the only known ribonuclease dimeric in nature, has important and peculiar biological actions. Aim of this work is the study of possible biological activities that RNase A could acquire when oligomerized in form of dimers, trimers, and tetramers, obtained by lyophilizing the enzyme from 40% acetic acid solutions. Methodology. Oligomers of RNase A (type XII-A, Sigma) were obtained by lyophilizing solutions of the enzyme in 40% ac. acid. Ion-exchange chromatography of the lyophilized material, dissolved in 0.2 M NaP buffer, pH 6.7, was performed with a Pharmacia Source 15S HR 10/16 column and a 0.085-0.180 M NaP (pH 6.7) gradient. Every oligomeric species (dimer, trimer or tetramer) consist of two conformers, a “less basic” and a “more basic” dimer (named according to their elution properties). They were often directly tested in the biological assays; all treatments (dyalisis, concentration etc.) were in fact reduced to a minimum because they cause a more or less remarkable dissociation of the oligomers, whose stability decreases in the order dimers-trimers-tetramers. Aspermatogenic activity was studied histologically. After ICR mice injections (100 µg/ml of each oligomer into the left test) both testes were removed, and their weight indexes calculated. The width of spermatogenic layers and the diameter of the seminiferous tubules were measured microscopically. Embryotoxicity was determined by using two cell embryos flushed from the oviducts of superovulated mice. Embryos were cultured for 72 h in CZB medium in the presence of 100 µg/ml of each RNase A aggregate. Experiments with native, monomeric RNase A, BS-RNase and/or onconase were carried out in parallel. Antitumor activity was tested on the two human tumor cell lines ML-2 and HL-60, cultured in microtiter plates in RPMI 1640 medium supplemented with 10% fetal calf serum. RNase A aggregates, monomeric RNase A, BS-RNase or onconase were assayed in triplicate, and cells cultured for 2 days. 4 hours before ending the experiment, cells were pulse-labelled with 3H-thimidine, and harvested. Mean values obtained were expressed as cpm, and inhibition of DNA synthesis was expressed as percentage of control. Results. Aspermatogenic activity: in comparison with BS-RNase, dimers and trimers of RNase A show a lower aspermatogenic activity, but no embryotoxicity. Therefore, if injected in cancerous animal these oligomers could result to have smaller side effects. The results so far obtained with the two RNase A tetramers are uncertain because of the relatively high instability of these aggregates. We must also point out that all results could be affected by the general low stability of all aggregated species: in other words,

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the effects of the aggregates could actually be more pronounced. Antitumor action: all oligomeric species if assayed directly, i.e., after their elution and without any treatment, exert highly significant antiproliferative effects. While the two dimeric conformers show on both ML-2 and HL-60 cells a slightly lower activity than BS-RNase and onconase, the activities of RNase A trimers, BS-RNase and onconase appear to be similar to each other. Instead, the two tetramers show a definitely higher antiproliferative action than BS-RNase or onconase. Moreover, the more basic conformers of all species are more active than the less basic ones, and the cytotoxic effect of all RNase A oligomers is more pronounced on HL-60 than on ML-2 cells, on the contrary of what was found with BS-RNase and onconase. Research funded by grants from the Grant Agency of the Czech Republic No. 523/01/0114, the Grant Agency of Ministry of Health No. 0023736001, and Italian MURST-PRIN 2000 and 2001.

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(P33) NUCLEOPHILE ACTIVATION IN RIBONUCLEASES : A COMBINED X-RAY CRYSTALLOGRAPHIC/AB INITIO QUANTUM CHEMICAL APPROACH Stefan Loverix1, Pierre Mignon2, Paul Geerlings2 and Jan Steyaert1

1Dienst Ultrastruktuur, VIB (Vlaams Interuniversitair Instituut Biotechnologie), Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium; and 2Eenheid Algemene Chemie (ALGC), Faculteit Wetenschappen, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium. Ribonucleases catayze the nucleophilic displacement of the 5’-leaving nucleoside by the entering 2’-oxyen atom in RNA. High resolution crystal structures of RNases in complex with 3’-mononucleotides demonstrate the accommodation of the nucleophilic 2’-OH group of these substrates in a binding pocket, consisting of the catalytic base (glutamate or histidine) and a positively charged hydrogen bond donor (lysine or histidine). In RNase T1, the representative of a superfamily of microbial RNases, the catalytic base Glu58 exerts its catalytic function in cooperation with the positively charged His40. Initially, we solved the X-ray structures of His40Ala, Glu58Ala and the corresponding double mutant of RNase T1, all complexed with the substrate 3’-GMP. Subsequently, ab initio quantum chemical calculations were performed on these Michaelis complexes, showing negative charge built up (= increased nucleophilicity) on the 2’-oxygen atom upon substrate binding. High level calculations demonstrate improved hydrogen bonding between the 2’-OH group and Glu58 in the presence of His40, via donation of a hydrogen bond to the 2’-oxygen. Similar findings were obtained for pancreatic RNase A, the representative of a superfamily of mammalian RNases. In RNase A, Lys41 has been shown to donate a single hydrogen bond to the rate limiting transition state of the reaction. This catalytically important residue has long been thought to interact with one of the nonbridging phosphoryl oxygens. However, our results indicate that the presence of Lys41 increases the nucleophilicity of the 2’-oxygen atom by strengthening the hydrogen bond between 2’-OH and the catalytic base His12. We found that such a catalytic dyad, consisting of the catalytic base and a cationic hydrogen bond donor, is also present in angiogenin, a member of the RNase A superfamily, and RNase MC1, a member of the RNase T2 family. As such, a catalytic dyad responsible for nucleophile activation seems to be a general, hitherto unrecognized feature of eukaryotic ribonucleases. Research funded by a grant from the ‘Fond voor Wetenschappelijk Onderzoek-Vlaanderen’ and the ‘Vrije Universiteit Brussel’. We would like to acknowledge Dr. Vishveshwara S. for providing us with the coordinates of angiogenin modelled with CpA and Dr. Kimura M. for the coordinates of RNase MC1 complexed with 3’-UMP.

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(P34) FUNCTIONAL CHARACTERIZATION OF YEAST AND ARABIDOPSIS RNases FROM THE T2 FAMILY Gustavo C. MacIntosh1, Nicole D. LeBrasseur2, Tracey Millard2 and Pamela J. Green1 1Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA 2MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA Extracellular and vacuolar ribonucleases (secretory RNases) have been well-studied at the enzymatic and structural levels. However, little is known regarding their biological functions. One family of secretory RNases, the RNase T2 family, is particularly widespread, with members in various kingdoms. Expression patterns and cellular localization suggest that these enzymes could be involved in nutrient recycling. A defined biological role has only been described for the S-RNases, a plant T2 RNase subfamily. S-RNases produce a cytotoxic response during the self-incompatibility process characteristic of several plant families. S-like RNases, another plant subfamily, are found in self-compatible as well as –incompatible plant species, indicating that they are not involved in self-incompatibility. Recent studies have shown that S-like RNases are induced as a response to stress conditions not related to nutrient or Pi limitations. To understand the biological roles of members of the T2 family, we constructed an insertional mutant of RNY1, the only member of this family in yeast. RNY1 encodes an extracellular RNase whose expression is regulated by a number of stress conditions, including heat shock and osmotic stress. Inactivation of Rny1 leads to the formation of unusually large cells that are osmo- and temperature-sensitive and flocculate in culture. These phenotypes can be complemented by RNY1, as well as by both structurally related and unrelated secretory RNases. Complementation is dependent on RNase activity, and the addition of RNase A to the culture media is sufficient to revert the flocculation phenotype. Our studies in yeast are complemented by those of a plant S-like RNase from Arabidopsis thaliana. This extracellular enzyme, RNS1, is induced by Pi-starvation, indicating a role in Pi remobilization (1). However, RNS1 is also induced by wounding, both locally and in distal, undamaged leaves, suggesting additional functions for RNS1, possibly involving defense responses. Arabidopsis mutants that lack RNS1 activity show an increase in growth that is particularly evident in roots. This phenotype and the regulation patterns of RNS1 suggest that T2 RNases in yeast and plants may have similar functions. When coupled with a recent report demonstrating an effect of RNA on membrane permeability (2), our work suggests a previously unrecognized role for Rny1 and possibly other secretory RNases in regulating membrane permeability or stability, a hypothesis that presents a new perspective for understanding their functions. 1. Bariola, P. A., Howard, C. J., Taylor, C. B., Verburg, M. T., Jaglan, V. D., Green, P. J. (1994) Plant J. 6, 673-685. 2. Vlassov, A., Khvorova, A., Yarus, M. (2001) Proc. Natl. Acad. Sci. USA 98: 7706-7711.

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Research funded by grants from the National Science Fundation (NSF) and the Department of Energy (DOE).

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(P35) OPTIMUM MODIFICATION OF CATIONIZATION FOR THE POTENT CYTOTOXIC RIBONUCLEASE Takashi Maeda, Junichiro Futami, Midori Kitazoe, Emiko Nukui, Hiroko Tada, Masaharu Seno, Megumi Kosaka, and Hidenori Yamada. Department of Bioscience and Biotechnology, Okayama University, Japan. Cationization of a protein is considered to be a powerful method for internalization of a functional protein into cells. Although the mechanism is unclear yet, cationized proteins appear to adsorb to cell surface by electrostatic interantions. Since ribonucleases have a latent cytotoxic potential, cationized RNases could be useful cancer chemotherapeutics. In this regard, We have modified the carboxyl groups of non-toxic bovine RNase A and human RNase 1 with ethylenediamine in amide bonds and found that thus cationized RNases are highly cytotoxic in spite of their remarkable decrease in RNase activity (1). Further, we investigated the effect of the degree of the modification of carboxyl groups with ethylenediamine on the cytotoxicity of RNase A. The results indicate that cationization of a protein in optimum level is important to keep the protein function in cytosol as much as possible. Now, we study the optimum modification for highest cytotoxicity of cationized RNases. 1. Junichiro Futami, Takashi Maeda, Midori Kitazoe, Emiko Nukui, Hiroko Tada, Masaharu Seno, Megumi Kosaka, and Hidenori Yamada. (2001). Biochemistry. 40, 7518-7524.

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(P36) DEGRADOSOME DISRUPTION AFFECTS DEGRADATION OF THE rpsO mRNA OF Escherichia coli P. E. Marujo, J. LeDerout and P. Régnier Institut de Biologie Physico-Chimique, Paris, France. The Escherichia coli RNA degradosome is a multiprotein complex involved in degradation of RNA. This complexe is mainly composed of endoribonuclease E (RNase E), polynucleotide phosphorylase (PNPase), the DEAD-box RNA helicase (RhlB) and a glycolytic enzyme, enolase. Presently it is admitted that the N-terminal half of RNase E is responsable for its endonucleolytic activity and that its carboxyl-terminal half contains the binding sites for PNPase, RhlB and enolase. RNase E is a ribonuclease that controls the stability of a great number of messengers and that it is also implicated in the maturation of stable RNAs. Some RNase E mutants were isolated that code for truncated RNase E polypeptides lacking the carboxyl-terminal half. One of these mutants encodes a truncated polypetide of 616 amino acid residues (allele rne 131) which doesn’t allow association of the degradosomal proteins. In our laboratory we analysed the decay of the rpsO transcript which codes for the protein ribosomal S15. This message can be degraded by two mechanisms: one dependent of RNase E and another one dependent from the exonucleases 3’-5’. When RNase E is present (half-lives in wild-type and rne1 strain are 1,3min and 9min, respectively), initiates rpsO mRNA degradation by removing the 3’ stabilizing stem and loop structure that protects the core of the message from the attack of the exoribonucleases 3’-5’. The initial cleavage occurs at the M2 site just upstream of the hairpin of the transcription terminator. In a RNase E deficient strain rpsO message is degraded by a mechanism dependent on 3’-5’ exonucleases and polyadenylation. We analysed the decay of rpsO message in the rne 131 strain described above to investigate whether and how degradosome disruption affects the degradation of the rpsO mRNA. Kinetics of decay showed that the rpsO transcript was stabilized more or less 8 fold in a rne 131 strain (half-lives in wild-type and rne 131 strain is 1,3min and 10min, respectively). Unexpectively, primer extension analysis and accumulation of decay intermediates show that rpsO mRNA is processed at the M2 site in this strain thus suggesting that its stabilization does not only reflect inhibition of the rate limiting cleavage. Stabilization in the rne 131 strain of mRNA degraded by RNase E and poly(A) dependent pathways led us to conclude that both mechanisms of decay are affected upon degradosome disruption. Interestingly, these data also imply that degradosome could coordinate the activity of RNase E and poly(A) dependent degradation of the rpsO transcript. All of the results that will be shown suggest that the RNase E and poly(A) degradation mechanisms are coordinated in the cell and that RNase E inside the degradosome controls the coordination between the two mechanisms.

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(P37) PEG CHAINS INCREASE ASPERMATOGENIC AND ANTITUMOR ACTIVITY OF RNase A AND BS-RNase ENZYMES WITHOUT EMBRYOTOXICITY OF RNaseA Josef Matoušek1*, Tomáš Slavík1, Pavla Poučková2, Josef Souček3, Jiří Škvor4 1Institute of Physiology and Genetics, Academy of Sciences of the Czech Republic, Liběchov, Czech Republic. 2Institute of Biophysics, Medical Faculty of the Charles University, Prague, Czech Republic. 3Institute of Hematology and Blood Transfusion, Prague, Czech Republic. 4Seva-Imuno Praha, Prague, Czech Republic RNase A and BS-RNase are monomeric and dimeric bovine enzymes respectively with antitumor, aspermatogenic and embryotoxic activity While these biologic activities of the RNase A are only minor, the activity of BS-RNase in these phenomena is mostly significant. The aim of study is to compare the biologic activities of free and PEG conjugated of these RNases. Aspermatogenic activity was studied histologically. Both testes were extirpated after ICR mice injections, and their index weights, after their body and testes weights, calculated, the width of spermatogenic layers and diameter of seminiferous tubules of testes were microscopically measured. Embryotoxicity was investigated on two cell embryos obtained from superovulatetd mice which were flushed from their oviducts. Embryos were cultured in CZB medium with tested ribonuclease for 72 hrs. Developmental stages of embryos were evaluated under stereomicroscope. Antitumor effect was searched on athymic nude mice CD-1. Human melanoma was obtained from a surgical specimen (cut in small pieces 3x3 mm) and transplanted subcutaneously. The free ribonucleases were administered in doses 250µg/20 g three times in a week for three weeks. The PEG conjugated RNase A and BS-RNase were applied in the same way in doses 50 µg per injection (about 18 µg of each ribonuclease in the conjugate). Tumor dimensions were measured using a slide calliper. Antibodies against free and conjugated RNases were studied by non-competitive ELISA test. For the direct and indirect immunofluorescence in vitro the IgG against BS-RNase and RNase A were used from the immunized rabbits. The obtained results prove that the use of PEG polymers is associated with the increase of aspermatogenic effect of both RNases, without decreasing the body weight of the experimental mice. Antigenically, the RNase A is much weaker compared to BS-RNase. RNase A is not able to be adsorbed in vitro on the surface of melanoma cells that BS-RNase can do it. The conjugation with PEG substantially increased antitumor activity of both RNases injected into athymic mice with melanoma tumors. The significant increasing of antitumor activity of monomeric RNase A, without embryotoxicity, is the most important results of the influence of PEG conjugation to this RNase. Research funded by a grant from the Grant Agency of the Czech Republic no. 523/01/0114.

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(P38) POPULATION SHIFT VERSUS INDUCED FIT: THE INSTRUCTIVE CASE OF BOVINE SEMINAL RIBONUCLEASE SWAPPING DIMER Antonello Merlino1, Luigi Vitagliano2, Filomena Sica1, Adriana Zagari2 and Lelio Mazzarella1,2

1Dipartimento di Chimica, Università degli Studi di Napoli ‘Federico II’, Via Cinthia, Napoli. 2Centro di Studio di Biocristallografia, CNR, Via Mezzocannone 6, Napoli. Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like superfamily. This enzyme exists as two conformational isomers with distinctive structural and biological properties. The structure of the major component is characterized by the swapping of the N-terminal segment (MXM BS-RNase)1. Here we report the crystal structures of the ligand-free MxM BS-RNase (2.5 Å resolution) and its complex with deoxycitidylyl-3’,5’-adenosine (2.1 Å resolution) derived from isomorphous crystals. Interestingly, the comparison between this novel ligand-free form and the previously published sulphate-bound structure reveals significant differences. In particular, the ligand-free MxM BS-RNase is closer to the structure of MxM BS-RNase complexes2 than to the sulphate-bound form. These results reveal that MxM BS-RNase presents a remarkable flexibility despite the structural constraints of the disulphide bridges and the swapping of the N-terminal helices. The findings that MxM BS-RNase may adopt different quaternary structures has important implications for the enzyme ligand binding process. Indeed, a population shift rather than a substrate induced conformational transition occurs upon substrate binding by MxM BS-RNase. 1. Mazzarella, L., Capasso, S., Demasi, D. et al. 1993. Acta Cryst. D 49:389-402. 2. Vitagliano, L., Adinolfi, S., Riccio, A. et al. 1998. Protein Sci. 7:1691-1699.

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(P39) MOLECULAR DYNAMICS SIMULATIONS OF 3D DOMAIN SWAPPING DIMERS OF BOVINE PANCREATIC RIBONUCLEASE Antonello Merlino1, Luigi Vitagliano2, Marc Antoine Ceruso3 and Lelio Mazzarella1,2

1Dipartimento di Chimica, Università degli studi di Napoli ‘Federico II’, Via Cynthia, Napoli, Italy. 2Centro di Studio di Biocristallografia, CNR, Via Mezzocannone 6, Napoli, Italy. 3Department of Physiology and Biophysics, Mt Sinai School of Medicine, One Gustave L. Levy Place, New York, USA Under lyophilizing conditions, bovine pancreatic ribonuclease (RNase A) forms two different 3D domain swapping dimers. Crystallographic investigations have revealed that these dimers display very different quaternary structures1,2. The structure of the most abundant dimer (Dimer I) is characterized by the swapping of the C-terminal-strands1. On the other hand, in the minor dimer (Dimer II), which presents a more compact structure, a swapping of the N-terminal helices occurs2. Since important catalytic residues of RNase A are located in N-and C- terminals, both dimers present composite active sites. We have recently investigated the dynamical properties of RNase A by using X-ray crystallography and molecular dynamics. Here we describe the local as well as the global motions of both RNase A 3D domain swapping dimers as derived by essential dynamics performed on 3ns molecular dynamics simulations in water. The molecular dynamics simulation of DimerII suggests that the dynamical properties of the composite active sites are virtually identical to those of RNase A despite the swapping of N-teminal fragments. The molecular dynamics analysis also shows that the two subunits of DimerII retain the breathing motion previously reported for the monomeric RNase A. Furthermore, the coupling between the breathing motion of the two subunits may provide a structural explanation for the modulated activity exhibited by this dimer. The analysis of the flexibility of the most abundant dimer indicates that the different quaternary structures of the two dimers induce quite different dynamical behaviors. 1. Liu, Y., Gotte, G., Libonati, M. and Eisenberg, D. 2001. Nat. Struct. Biol. 8: 211-214. 2. Liu, Y., Hart, P.J., Schlunegger, M.P. and Eisenberg D. 1998. PNAS. 95: 3437- 3442.

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(P40)THE EFFECT OF NET CHARGE ON THE ACTIVITY, STABILITY, AND CYTOTOXICITY OF RIBONUCLEASE Sa Vladimir A. Mitkevich1, Olga N. Ilinskaya2, Kevin L. Shaw3, Gerald R. Grimsley4, Florian Dreyer5, Gennady I. Yakovlev1, Alexander A. Makarov1, C. Nick Pace4 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia 2Department of Microbiology, Kazan State University, 420008 Kazan, Russia 3Department of Biology, Grove City College, Grove City, Pennsylvania 16127, USA 4Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843, USA 5Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) from Streptomyces aureofaciens is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K+D25K, E74K) with a pI=10.2. The addition of five lysines reverses the net charge from –7 to +3 at pH 7. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the activity and stability have been measured as a function of pH. We find that the pH of maximum activity and the pH of maximum stability do not vary with the pI of the enzyme. RNase Sa and its charge reversal variants were also tested for cytotoxicity. Cytotoxicity assays were conducted with normal and v-ras-transformed NIH3T3 mouse fibroblasts. RNase Sa was not cytotoxic. The positively charged 5K variant, despite its lower catalytic activity as compared to wild-type, was cytotoxic. Based on these data, we concluded that a positive charge on the molecule is an important determinant of ribonuclease cytotoxicity. The cytotoxic 5K variant preferentially killed v-ras-NIH3T3 fibroblasts, suggesting that mammalian cells expressing the ras-oncogene are a potential target for the antiproliferative action of ribonuclease-based drugs. This work was supported by NIH FIRCA grant TWO1058.

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(P41) CRYSTAL STRUCTURE OF EOSINOPHIL CATIONIC PROTEIN IN COMPLEX WITH 2’,5’-ADP AT 2 Å RESOLUTION REVEALS THE DETAILS OF THE RIBONUCLEOLYTIC ACTIVE SITE C. Gopi Mohan,1 Ester Boix,1,2 Hazel R.Evans,1 Zoran Nikolovski,2 M. Victòria Nogués,2 Claudi M. Cuchillo2 and K. Ravi Acharya1 1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K. 2Department de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. Eosinophil cationic protein (ECP) is located in the eosinophil granule matrix (1). It shows marked toxicity against helminth parasites, bacteria, single stranded RNA viruses as well as host epithelial cells. Secretion of human ECP is related to the eosinophil-associated allergic, asthmatic and inflammatory diseases. ECP belongs to the pancreatic ribonuclease superfamily of proteins and the crystal structure of ECP in the unliganded form (determined previously) showed a conserved RNase A fold (2,3). We have now determined a high resolution (2.0 Å) crystal structure of ECP in complex with adenosine-2’,5’-diphosphate (2’,5’-ADP) which has revealed the details of the ribonucleolytic active site. Residues Gln-14, His-15 and Lys-38 make hydrogen bond interactions with the phosphate at the P1 site, while His-128 interacts with the purine ring, at the B2 site. A new phosphate binding site P-1, has been identified which involves Arg-34 residue. This study is the first detailed analysis of the nucleotide recognition site in ECP and provides a starting point for the understanding of its substrate specificity and low catalytic efficiency when compared with the eosinophil-derived neurotoxin (EDN), a close homolog. 1. Ackerman, S. J. (1993) in Eosinophils: Biological and Clinical Aspects (Makino, S., and Fukuda, T., Eds.) pp-33-74, CRC 2. Boix, E., Leonidas, D. D., Nikolovski, Z., Nogués, M. V., Cuchillo, C. M., and Acharya, K. R. (1999) Biochemistry 38, 16794-16801 3. Mallorquí-Fernández, G., Pous, J., Peracaula, R., Aymami, J., Maeda, T., Tada, H., Yamada, H., Seno, M., de Llorens, R., Gomis-Rüth, F.X., and Coll, M. (2000) J. Mol. Biol. 300, 1297-1307 This work was supported by MRC (U.K.) Program Grant 9540039 and Grants BMC2000-0138-C02-01 from Ministerio de Educación y Cultura and 2000SGR 00064 from CIRIT, Generalitat de Catalunya (Spain), to M.V.N. and C.M.C.

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(P42) THE CONTRIBUTION OF BASIC AMINO ACID RESIDUES IN THE P2 SUBSITE TO RIBONUCLEASE A ACTIVITY Mohammed Moussaoui, M. Victòria Nogués and Claudi M. Cuchillo Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona. 08193-Bellaterra. Spain. Bovine pancreatic ribonuclease A (RNase A) is an endonuclease which catalyses the depolymerization of RNA. The endonuclease activity of RNase A is based on the cooperative structure between the multisubsite binding structure of RNase A and the phosphate group of the substrate. Cleavage of oligocytidylic acids by RNase A showed that the RNase activity can be either endo- or exonuclease depending on the integrity of its subsites. This work focuses on the analysis of the effect of either the degree of integrity of the p2 subsite and the nature of the 3’-phosphate terminal group of the substrate on the catalytic process. The analysis was carried out by comparing, by reversed-phase HPLC, the pattern of cleavage of the oligonucleotides (Cp)4C>p and (Cp)4Cp by the native and p2 modified enzymes. The p2 modified forms (substitution of Lys-7 and Arg-10 by either His or Gln, K7H/R10H-RNase A and K7Q/R10Q-RNase A) were obtained by site directed mutagenesis in order to gradually eliminate the electrostatic interaction between the phosphate groups of the substrate and the amino acids residues of the p2 subsite of the enzyme. Both forms of the substrate are used to analyse the effect of the terminal 2’,3’- cyclic phosphate and 3’-phosphate forms on the catalysis by the different forms of the enzyme. The results indicate: 1) the endonuclease activity of RNase A decreases to become an exonuclease activity in proportion to the degree of interaction between the phosphate group of the oligonucleotide and the basic amino acids residues at positions 7 and 10 of the enzyme. 2) The presence of a 3’-phosphate terminal group in the substrate ((Cp)4Cp) instead of a 2’,3’cyclic phosphate ((Cp)4C>p) allows a better interaction of the substrate in the presence of less basic amino acids in the p2 subsite. 3) An endonuclease activity is found with the substrate (Cp)4Cp when the amino acid residues of the modified enzyme are less basic than those in the native form. Research funded by grants 2000SGR 00064 from Generalitat de Catalunya and BMC2000-0138-C02-01 from DGES, Ministerio de Educación y Cultura, Spain.

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(P43) MOLECULAR CHARACTERIZATION OF A MOUSE CDNA ENCODING DICER, A RIBONUCLEASE III ORTHOLOGUE INVOLVED IN RNA INTERFERENCE Rhonda H. Nicholson and Allen W. Nicholson, Department of Biological Sciences, Wayne State University, Detroit, Michigan, USA. Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse cellular RNA maturation and degradation pathways. A recently identified eukaryotic RNase III family member, named “Dicer”, functions in the RNA interference (RNAi) pathway by producing 21-23 bp dsRNAs which target the selective destruction of homologous RNAs. RNAi is operative in animals, plants and fungi, where it is proposed to inhibit viral reproduction and retroposon movement, as well as participate in developmental pathways. RNAi functions in mammalian cells, including mouse oocytes and embryos. This article reports the cDNA sequence characterization and expression analysis of the mouse Dicer orthologue. Based on the cDNA sequence, the Dicer polypeptide is 1906 amino acids and has a predicted molecular mass of 215 kDa. Mouse Dicer contains a DExH/DEAH helicase motif; a PAZ domain; a tandem repeat of RNase III catalytic domain sequences; and a dsRNA-binding motif. The Dicer gene maps to a single locus on the distal portion of mouse chromosome 12. The Dicer transcript is expressed from the embryonic through adult stages of development. The Dicer transcript is also present in a wide variety of adult mouse organs. The highly conserved set of functional domains and the occurrence of a single-copy gene strongly indicate that the encoded protein is the RNase III orthologue responsible for dsRNA processing in the RNAi pathway. Nicholson, R.H. and Nicholson, A.W. 2002. Molecular characterization of a mouse cDNA encoding Dicer, a ribonuclease III orthologue involved in RNA interference. Mamm. Genome 13: 67-73 Research supported by the NIH (GM56457)

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(P44) STRUCTURAL BASIS FOR A CHANGE IN SUBSTRATE SPECIFICITY OF THE RNASE MC1 MUTANT FROM BITTER GOURD SEEDS Tomoyuki Numata, Yoshimitsu Kakuta, Makoto Kimura Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan Ribonuclease MC1 (RNase MC1), isolated from the bitter gourd seeds, is a uridine-specific RNase and belongs to the RNase T2 family. Mutations of Asn71 by Thr (N71T) and Ser (N71S) altered the substrate specificity from uridine-specific to guanosine-specific as shown by the transphosphorylation of diribonucleoside monophosphates (1). To elucidate the change of substrate specificity of these mutant enzymes, the crystal structure of the mutant N71T in complex with 5’-GMP was analyzed. The mutant N71T was overproduced by secreting to growth medium of the methylotrophic yeast Pichia pastoris and purified to homogeneity by a series of chromatography. The N71T liganded with 5’-GMP was crystallized against a reservoir containing 0.2 M ammonium acetate, 0.1 M tri-sodium citrate, pH 5.6, 27.5% polyethyleneglycol 8000. The X-ray diffraction analysis showed that the crystals belonged to a space group of P212121 with unit cell dimensions of a = 38.4 , b = 65.8 , c = 75.9Å , and α = β = γ= 90° . The crystal structure of the mutant N71T in complex with 5’-GMP was determined at 1.5 Å resolution by molecular replacement with the wild type structure (2) as a search model. The structure shows that the side chains of Gln9 and Thr71 interact with O6 and N7, respectively, of the guanine base by hydrogen bonding. In addition, the guanine base is stacked with the hydrophobic side chain of Leu73 and Phe80. From these results, it was revealed that substitution of Asn to Thr leads to an expansion of the base-binding site, thereby making it possible to insert larger guanine base into the B2 site. 1. Numata, T., Suzuki, A., Yao, M., Tanaka, I., and Kimura, M. (2001) Biochemistry 40, 524-530 2. Nakagawa, A., Tanaka, I., Sakai, R., Nakashima, T., Funatsu, G., and Kimura, M. (1999) Biochim. Biophys. Acta 1433, 253-260

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(P45) SUBSTRATE RECOGNITION MECHAMISM OF AN RNA RESTRICTION ENZYME, COLICIN E5 Tetsuhiro Ogawa1, Shunsuke Yajima2, Makoto Hidaka1 and Haruhiko Masaki1.

1 Department of Biotechnology, The University of Tokyo, Japan. 2 Department of Bioscience, Tokyo University of Agriculture, Japan. We recently showed that colicin E5 kills Escherichia coli cells by cleaving anticodons of specific tRNAs, such as tRNATyr, tRNAHis, tRNAAsn and tRNAAsp. Since these tRNAs contain a unique sequence UQU (or UGU in precursor tRNAs) in their anticodon-loops, we assumed that colicin E5 directly recognizes this UGU sequence. Then we purified the C-terminal active domain of colicin E5 (E5-CRD: C-terminal Ribonuclease Domain) and incubated with various “minihelices”, which mimicked the anticodon-stem/loop structure of tRNATyr with variations in the UGU sequence. The mutation of the 5’ uridine of the UGU to cytosine had no effect on the cleavage by E5-CRD. In contrast, mutations of the central guanine or the 3’ uridine made the minihelices completely resistant, suggesting that E5-CRD recognized at least the GU sequence. Furthermore, E5-CRD proved to cleave dinucleotide GpUp, so that other residues were not necessary for the minimum recognition. However, residues or phosphate groups adjacent to 5’ or 3’ to the GpUp also influenced to various extents on kinetic parameters of the enzymatic reaction. Thus some subsite structure of E5-CRD most likely determines the preference of E5-CRD to substrate tRNAs. Ogawa, T., Tomita, K., Ueda, T., Watanabe, K., Uozumi, T. and Masaki, H. (1999). Science 283, 2097-2100. Research funded by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.

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(P46) MINI OPEN READING FRAMES (MINI-ORF) AT UPSTREAM OF ORF OF TWO RNASES FROM BASIDOMYCETES, Lentinus edodes AND Irpex lacteus. Kazuko Ohgi1, Tatsuya Wada1, Wasanori Iwama1, Tsutomu Tsuji1, Masachika Irie1, Tadashi Itagaki 2, Hiroko Kobayashi 2, Norio Inokuchi2

1Department of Microbiology, Hoshi College of Pharmacy, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. 2Department of Microbiology, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan. Irpex lacteus excreted more 45Kda RNase from the cultured cells at early stage of cultivation, then RNase Irp1 (30KDa) and 2 (24KDa). These three RNases have the same N-terminal sequence. I. lacteus RNases were a kind of heat-shock protein. CDNA of RNase Irp consisted of three domains, this is, N-terminal core enzyme domain-S/T rich domain-1.5KDa C-terminal domain. RNase Le45 from L. edodes has very similar domain structure, although it was not yet proved to be a heat-shock protein, because of the presence of many nucleolytic enzymes disturbed the conclusive experiments. In order to investigate the mechanism of this heat-shock, we investigated the nucleotide sequence of cDNA encoding RNase Irp. And we observed the mini open reading frames (mini-ORF) upstream of RNase Irp ORF. The similar analysis of the cDNA of RNase Le 45K showed the presence of mini-ORF upstream of the ORF of the RNase. In fungal world, such mini-ORF are thought to be related many metabolic regulation including phosphate starvation and heat-shock (1). Similar to this finding, in RNase Le2 (2) having no such domain structure, we also detected the mini-ORF. 1. Fujiwara M., Horiuchi H., Ohta A., Tkagi M.. (1997) Biochem. Biophys. Res. Commun., 236, 75-78 2. Kobayashi H., Inokuchi N., Koyama T., Irie M.,. (1998) Biosci. Biotechnol. Biochem., 62 1604-1608

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(P47) POLYMER CONJUGATED BOVINE PANCREATIC RIBONUCLEASE (RNase A) FOR CANCER THERAPY P. Poučková1, M. Zadinová1, D. Hloušková1, J. Strohalm2, D. Plocová2, M. Špunda1, T. Olejár2, M. Zitko1, J. Matoušek3, J. Souček4

1Institute of Biophysics, Medical Faculty, Charles University, Prague 2, Czech Republic 2Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague 6, Czech Republic 3Institute of Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic 4Institute of Hematology and Blood Transfusion. Prague 2, Czech Republic Even in a similar way regarding the preparation of polymer conjugates with BS-RNase, the hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for RNase A modification to prevent its degradation in bloodstream or its fast elimination. Two HPMA-polymers (classic and star-like) were synthesized and their antitumor effect was tested on various human tumors transplanted into the nude mice. Athymic female nude miceCD-1 (18-20 g), housed in sterile conditions and fed with sterile diet, were used for therapeutic experiments. Human melanoma was obtained from a surgical specimen and pieces (3x3 mm) were transplanted subcutaneously on the right flak of the mice. Neuroblastoma and ovarian tumor cells sustained in a culture were injected at the concentration of 107/mouse in the same way. Both the RNase A conjugates injected intravenously or intraperitoneally into the nude mice bearing melanoma, neuroblastoma or ovarian tumor, caused significant reduction of transplanted tumors following ten daily doses of 5 and 1 mg/kg, respectively. Wild RNase A injected in doses 10 mg/kg exerted only negligible antitumor activity. Histological examination confirmed very potent cytotoxic effect of RNase A conjugates on ovarian tumor. Despite the antitumor activity observed in vivo, the in vitro cytotoxic activity of RNase A conjugates was negligible and did not differ from that caused by the wild RNase A. The in vitro experiments with 125I–labeled preparations demonstrated that polymer conjugates were internalized by tumor cells very poorly in contrast to the dose-dependent internalization of the wild BS-RNase. Surprisingly, mice injected with EL-4 leukemic cells, which were preincubated for 2 hours with RNase A cojugates, exerted significantly prolonged survival compared with the control mice. For the first time the presented results demonstrate that polymer conjugated RNase A may be regarded as a potential anticancer drug. This work was supported by Grant Agency of the Czech Republic No. 307/96/K226 and No. 523/01/011 and Grant Agency of Czech Ministry of Health No. 0023736001

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(P48) DELETION OF THE NH2-TERMINAL β-HAIRPIN OF THE RIBOTOXIN α-SARCIN RENDERS A NON-TOXIC BUT ACTIVE RIBONUCLEASE García-Ortega‡, L., Masip‡, M., Mancheño‡, J:M, Oñaderra‡,M., Lizarbe‡, M.A., García-Mayoral§, M.F., Bruix§, M., Martínez del Pozo‡, A., and Gavilanes‡, J.G. ‡Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, Madrid, Spain §Instituto de Química Física “Rocasolano”, Consejo Superior de Investigaciones Científicas, Madrid, Spain Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by cleaving only one single phosphodiester bond of the 28S rRNA. α-Sarcin is the best characterized member of this family(1). This protein is a potent cytotoxin which promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with non-toxic ribonucleases of the RNAse T1 family. The significant structural differences between both groups of proteins are related to both the presence of a long amino-terminal β-hairpin in ribotoxins and the different length of their unstructured loops (2). In order to study the role of this protuberant amino-terminal β-hairpin, a deletion mutant ∆(7-22) of α-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation than the wild-type protein according to a complete spectroscopic characterization based on CD, fluorescence and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA but lacks the specific ability to specifically cleave rRNA in intact ribosomes. Indeed, It displays a decreased interaction with lipid vesicles and shows a behavior compatible with the absence of one vesicle-interacting region. The results obtained indicate that α-sarcin interacts with the ribosome at two regions, the well-known sarcin-ricin loop of the rRNA and a different region recognized by the β-hairpin of the protein. In addition, this protein portion is involved in interaction with cell membranes. In agreement with this, the deletion mutant exhibits a very low cytotoxicity on human rhyabdomysarcoma cells. 1. Martínez-Ruiz, A., García-Ortega, L., Kao, R., Lacadena, J., Oñaderra, M., Mancheño, J.M., Davies, J., Martínez del Pozo, A. and Gavilanes, J.G. (2001) Methods Enzymol. 341, 335-351. 2. Pérez-Cañadillas, J.M., Santoro, J., Campos-Olivas, R., Lacadena, J., Martínez del Pozo, A., Gavilanes, J.G., Rico, M., and Bruix, M. (2000) J. Mol. Biol. 299, 1061-1073. This work was supported by grants BMC2000-0551 from the Ministerio de Ciencia y Tecnología (Spain) and PB98-0677 from the Ministerio de Educación y Cultura (MEC) (Spain).

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(P49) CONTRIBUTION OF THE HYDROPHOBIC RESIDUES OF THE α3β2β5β6 CHAIN FOLDING INITIATION SITE TO THE CONFORMATIONAL STABILITY OF RIBONUCLEASE A Font, J., Ribó, M., Benito, A., Torrent, J. and Vilanova, M. Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, E-17071 Girona, Spain. It is nowadays accepted that protein folding does not occur by random conformational search but proceeds via local folded co-operative growth and is initiated by short-range hydrophobic interactions that result in the formation of a single or multiple chain folding initiation sites (CFIS) within the polypeptide chain. The C-terminal hydrophobic hairpin-like structure consisting of residues 106-118 was proposed by Scheraga and co-workers as one of the main CFIS for the folding of Ribonuclease A (RNase A). The unfolding process of wild-type RNase A and 14 single variants of this 106-118 region, has been monitored by fluorescence, fourth-derivative UV-spectroscopy and by FTIR-spectroscopy (1). Comparison with model systems suggested that the differences in ∆GU among the variants were attributable to both hydrophobic interactions (expressed as the energy of burial of hydrophobic surface) and the packing density. By a direct comparison of pressure- and heat-induced unfolding thermodynamics, it was shown that two particular residues (I106 and especially V108) were, among those analyzed, the most important sites for RNase A stability. Recently, Rico and co-workers (2) have redefined the C-terminal CFIS postulated by Scheraga. This hydrophobic region of exceptional stability, includes the four stranded region: Lys62-Ala 64, Cys 72- Ser 75, Ile 106-Cys 110 and Val 116-His 119, together with the face of helix III containing Val 54 and Val 57. In order to complete the work initiated by our group and to probe the strategic importance of all the hydrophobic residues within the redefined CFIS to the stability of the protein, temperature induced unfolding of seven new variants (V54A, V54G, V57A, V57G, V63A, V63G, A64G) has been monitored by UV absorbance. Wild-type RNase A and its variants have been characterized by mesuring the kinetic parameters in front of low and high molecular mass substrates and registration of circular dichroism spectra for the folded and unfolded states. Our results show that the amino acid changes introduced do not modify either the catalytic parametres or the overall protein conformation. However, thermodynamic analysis reveals that substitutions at positions 54 and 57 are the most destabilizing ones. Taken together our results shed light on which CFIS positions are the more critical for RNAse A stability and eventually to the folding-unfolding process. 1. Torrent, J., Connelly, J.P., Coll, M.G. et al (1999) Biochemistry 38, 15952-15961. 2. Neira, J.L., Sevilla, P., Menéndez, M. et al (1999) J. Mol. Biol. 285, 627-643. Work supported by grants PB96-1177-CO2-02 and BMC2000-0138-CO2-02 from de MCyT and SGR00-0064 from the CIRIT.

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(P50) ENDORIBONUCLEASE RegB FROM BACTERIOPHAGE T4 : DESIGN OF A NEW POSITIVE SELECTION PLASMID, PRELIMINARY FUNCTIONAL AND STRUCTURAL STUDIES OF THE ENZYME Saida F1., Lallemand J-Y1 and Bontems F.1

1 Laboratoire ICSN-RMN, Ecole polytechnique, Palaiseau, France. The regB gene, from bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The regulation mechanism involves the destabilization and the inactivation of phage T4 mRNAs (1). RegB cleaves with high specificity the consensus sequence GGAG when located in intergenic regions (2). Most of the sequences cleaved by RegB are within the ribosome-binding site that is crucial for efficient initiation of the translation (3). Here, we present evidences for the striking high toxicity of endoribonuclease RegB that prevents manipulation of this gene in E. coli with standard genetic methods. We propose a protocol for low expression of this protein using a runaway replication plasmid specially engineered for T7 RNA polymerase induction. We, also, describe the design of a new positive selection plasmid based on regB cytotoxicity. This plasmid, called pTOXR, combines yeast and bacterial genetic characteristics in order to bypass regB lethality. First functional studies of RegB by random and site directed mutagenesis and preliminary structural NMR analysis of a point mutant of RegB (R52L) are under investigation. 1. Sanson B., and Uzan M. (1993). J. Mol. Biol. 233, 429-446. 2. Ruckman J., Ringquist S., Brody E. and Gold L. (1994). J. Biol. Chem. 269, 26655- 26662. 3. Sanson B. and Uzan M. (1995). FEMS Microbiol. Rev., 17, 141-150.

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(P51) ESSENTIAL DYNAMICS AND HYDROGEN BOND ANALYSIS ON EOSINOPHIL RELATED RNASES Sanjeev B.S.1 and Vishveshwara S. 1

1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India. Ribonucleases (RNases), which catalyze RNA, are structurally similar and carry out diverse functions. It is interesting to identify the structural features that are common to the family and highlight the differences. Molecular Dynamics (MD) simulations followed by Essential Dynamics (ED) analysis is one approach that can elucidate dynamics in different regions of the protein in atomic detail. Eosinophil Cationic Protein (ECP) is a member of RNase A super family and is a toxin found mainly in eosinophil. The rate of evolution of ECP is the fastest among all the mammalian proteins. We have carried out equilibrium dynamics simulations on ECP. The simulation was followed by Essential Dynamics (ED) analysis. The important features obtained from ECP simulation and ED will be discussed. The analysis performed on the top modes obtained from ED will also be presented. Specifically, the studies on hydrogen bonds in general, as well as in relation to correlated motion will be presented. The simulation brings out some of the alternate conformations of sidechains, such as for the active site histidine residue, His128, moving from the inactive conformation to the active conformation. The preliminary results from ECP studies indicate that the sidechain-sidechain cum sidechain-mainchain clusters of hydrogen bonds are involved in the concerted motion of the protein. Two conformational subspaces connected with a long transition period are observed. The subspaces are differentiated chiefly with sidechain torsion angles of Trp35 and Phe76. The transition between the conformational subspaces is accompanied by increased radius of gyration. Hydrogen bond analysis would be compared with that obtained from a similar study of Eosinophil Derived Neurotoxin. Further such analyses are also performed on other members of the RNase A superfamily of proteins. Research funded from the Department of Science and Technology (DST), India. B.S.S. thanks Council for Scientific and Industrial Research for fellowship.

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(P52) ANALYSIS OF THE CLEAVAGE SITE SEQUENCE IN HYPER-EDITED dsRNA A. Deirdre J. Scadden and Chris W. J. Smith Department of Biochemistry, University of Cambridge, UK Extended dsRNA duplexes can be hyper-edited by adenosine deaminases that act on RNA (ADARs). Long uninterrupted dsRNA is relatively uncommon in cells, and is frequently associated with infection by DNA or RNA viruses. Moreover, extensive adenosine to inosine editing has been reported for various viruses. A number of cellular antiviral defence strategies are stimulated by dsRNA. An additional mechanism to remove dsRNA from cells may involve hyper-editing of dsRNA by ADARs, followed by targeted cleavage. We have recently described a cytoplasmic endonuclease activity that specifically cleaves hyper-edited dsRNA (Scadden and Smith, 2001). Cleavage occurs at specific sites consisting of alternating I•U and U•I base pairs. In contrast, unmodified dsRNA and even deaminated dsRNAs that contain four consecutive I•U base pairs are not cleaved. Moreover, dsRNAs in which alternating I•U and U•I base pairs are replaced by isomorphic G•U and U•G base-pairs are not cleaved. Thus the cleavage of deaminated dsRNA appears to require an RNA structure that is unique to hyper-edited RNA, providing a molecular target for the disposal of hyper-edited viral RNA. We have now analysed the cleavage of a number of RNA duplexes containing various combinations of G•U, U•G, I•U and U•I pairs in order to determine what is absolutely required for cleavage. In addition, RNA-DNA duplexes have been used to analyse the effects of deoxy groups on cleavage. Systematic analysis of RNA duplexes containing inosine residues has enabled insight into the groups important for cleavage. Scadden, A.D.J. and Smith, C.W.J. (2001) Specific cleavage of hyper-edited dsRNAs. EMBO J., 20, 4243-4252. This work was supported by the Wellcome Trust and Newnham College, University of Cambridge.

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(P53) STRUCTURAL INVESTIGATION OF MONOMERIC AND DIMERIC FORMS OF BOVINE SEMINAL RIBONUCLEASE Filomena Sica1,2, Anna Di Fiore1, Rita Berisio2, Renata Piccoli3, Giuseppe D’Alessio3, Adriana Zagari2,3 & Lelio Mazzarella1,2

1Dipartimento di Chimica, Università degli Studi “Federico II”, Napoli, Italy 2Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy 3Dipartimento di Chimica Biologica, Università degli Studi “Federico II”, Napoli, Italy Native bovine seminal ribonuclease is an equilibrium mixture of two homodimeric isomers, MxM and M=M (1). The former, whose crystal structure has been solved at 1.9 Å resolution (2), is characterised by the swapping of N-terminal helices (residues 1-15) between subunits, phenomenon absent in M=M. The two different folds of N-tails are directed by the hinge peptide (residues 16-22) which connects the exchanging domain to the rest of the protein. The enzyme chains are covalently joined by two disulphide bridges, whose reduction followed by carboxymethylation with iodoacetamide produces a monomeric derivative (MCAM) and a non-covalent swapped dimer (NCD) which are both active (1). Here, we present the details of the crystallisation and the structural determination of MCAM, M=M and NCD. In the case of the monomeric derivative, we have obtained crystals of the free and liganded enzyme at an alkaline pH and we have determined their structures at a resolution of 1.45 and 1.65 Å, respectively. The model of liganded MCAM provides interesting information on the substrate binding of a pancreatic like ribonuclease whose catalytic histidine residues are deprotonated. The unswapped isomer has been crystallised in the unliganded form and in this structure, determined using data to 2.2 Å resolution, each subunit adopts a fold similar to MCAM. The best crystals of the non-covalent dimer have been grown in the presence of deoxycytidyl-3’,5’-deoxyadenosine and the structure of the complex has been determined at 2.0 Å resolution. In the three unswapped structures, the hinge loop is disordered, whereas in NCD it adopts a well defined conformation. 1. D'Alessio, G., Di Donato, A., Mazzarella, L., and Piccoli, R. (1997) in Ribonucleases: structure and function (Riordan, J. F., and D'Alessio, G., Eds.) pp 383-423, Academic press, New York. 2. Mazzarella, L., Capasso, S., Demasi, D., Di Lorenzo, G., Mattia, C. A., and Zagari, A. (1993). Acta Crystallogr. Sect D 49, 389-402.

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(P54) SYNTHESIS AND PROPERTIES OF POLYMER CONJUGATED BOVINE SEMINAL AND BOVINE PANCREATIC RIBONUCLEASES J. Souček1, Strohalm2, D. Plocova2, P. Pouckova3, D. Hlouskova3, M. Zadinova3, J. Matousek4, K. Ulbrich3

1Institute of Hematology and Blood Transfusion, U nemocnice l, Prague 2, Czech Republic, 2Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 3 Institute of Biophysics, School of Medicine, Charles University, Prague, Czech Republic, 4 Institute of Physiology and Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Modification of biologically active proteins (enzymes, cytokines) with poly(ethylen glycol) results in longer persistence in the bloodstream, in a decrease in their immunogenicity and in improvement in resistance to proteolytic degradation. Poly[N(2-hydrxypropyl)methacryl amide] (PHPMA) was used for the modification of bovine seminal ribonuclease (BS-RNase), the enzyme exhibiting anticancer activity in vitro and, if administered intratumorally, also in vivo. Here the synthesis of PHPMA conjugated with monomeric bovine pancreatic ribonuclease (RNase A) is described and its properties are compared with those of BS-RNase polymers. BS RNase was isolated and purified from bull seminal vesicle fluid. RNase A was purchased from Sigma comp. Hydrophylic poly [N-(2-hydroxypropyl)methacrylamide] (PHPMA) was also used for RNase A modification. Athymic female nude mice CD-1 weighing 18-20g were used for therapeutic experiments on human melanoma. Human melanoma obtained from a surgical specimen was transplanted subcutaneously into the nude mice. Treatment of the mice started when the tumor area reached 5 x 5 mm. The tumor dimensions were measured twice a week and tumor volumes from these data were calculated. We have confirmed that both the wild dimeric BS-RNase and unimeric RNase A were entirely ineffective in vivo after i.p. or i.v. administration. On the contrary, both the structures of PHPMA-BS-RNase and PHPMA-RNase A conjugates proved to be highly cytotoxic after intravenous or intraperitoneal administration. Surprisingly the intratesticular injection of 100 µg (BS-RNase or RNase A equivalent) of all conjugates showed a very low aspermatogenic activity and, in contrast to the broad antitesticular effect of wild BS-RNase, was insignificant. Similar effect was also observed on embryotoxicity where no RNase conjugate exerted embryotoxic activity on bovine embryos at the concentration of 100 µg/ml compared with a high embryotoxic activity of wild BS-RNase. Presented data demonstrate significant antitumor effect of PHPMA – polymer conjugated BS RNase and/or RNase A on human melanoma transplanted on the nude mice. This work was supported by Grant Agency of the Czech Republic grant No. 307/96/K226 and No. 523/01/0114

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(P55) STUDIES ON THE SUBSTRATE SPECIFICITY OF ONCONASE Suhasini A. N., Sunil Kumar. S., Ravi Sirdeshmukh Centre for Cellular and Molecular Biology, Hyderabad 500007, India Onconase – a protein from frog (Rana pipiens) eggs is a member of the pancreatic RNase superfamily and known to have anti-cancer and anti-viral activity. These properties of onconasemay be related to its selective action on cellular or viral RNAs; it is found to degrade cellular tRNA in preference to rRNA, when injected into the cells1. In an attempt to understand the molecular specificity of onconase further, we have compared the action, in vitro, of onconase and other pancreatic type RNases on cellular RNAs such as rRNA, tRNA, mRNA and studied specific cleavages produced by the enzyme in these substrates. As reported earlier, in rabbit reticulocyte lysates, onconase was found to degrade tRNA more efficiently than other RNAs. Using reticulocyte lysates, we compared the action of onconase, bovine pancreatic RNase and human pancreatic RNase on polysomal and externally added free ribosomal RNA, over a wide range of enzyme concentrations. As expected, all these RNases degraded free RNA more readily than polysomal RNA of reticulocytes. But this difference was uniquely large for onconase as compared to the other two RNases. Using purified E.coli tRNAlys and yeast tRNAphe, as moel substrates, we observed discrete cleavage patterns . Similarly, onconase action on in vitro transcribed mRNAs also exhibited distinct patterns and suggested limited cleavages. Together these observations support the occurance of site-specific cleavages in these substrates. Using 3’ end labelled tRNAlys and tRNAphe we mapped the specific cleavage sites in these substartes. The most prominent cleavages occured in the TψC and the variable loops of the tRNAs. Onconase specificity and cleavge site determination for the tRNAlys3 – the tRNA used in HIV replication is being studied and these studies would be reported. We conclude that onconase recognizes specific cleavage sites in its substrates. In tRNAs, these sites may be readily accessible, whereas in other RNAs like rRNAs, they may be shielded by associated proteins and far less accessible. Thus onconase is a unique pancreatic RNase homologue exhibiting higher order specificity which may be an important feature for its anticancer or antiviral activity. Saxena, S.K., Sirdeshmukh R Ardelt, W., Mikulski, S.M. Shogen, K. and Youle, R (2002) J.Biol.Chem, 277, 15142-15146, . We are greatful to the Council of Scientific and industrial Research, Govt. of India, for Research Fellowship to ANS. We thank technical support of Mr Y Ramadasu. Dr MRS Srinivas participated in the initial experiments. Onconase was a kind gift from Dr Richard Youles’s lab at NINDS, NIH, Bethesda, MD, USA.

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(P56) RNase Sa POINT MUTATIONS: STRUCTURAL RESPONSE AND CONFORMATIONAL STABILITY Lubica Urbanikova1, Jozef Sevcik1 , C. Nick Pace2 1 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic 2 Texas A&M University, College Station, Texas, USA RNase Sa, a ribonuclease from Streptomyces aureofaciens, has been thoroughly studied biochemically and structurally. This small enzyme has also been used to study the contribution of hydrogen bonding by polar groups and hydrophobic bonding by nonpolar groups to the conformational stability of proteins (1,2). The conformational stability of all of the mutants was determined by analyzing thermal denaturation curves. The observed decreases in stability were (in kcal/mol): 0.5 for S24A, 2.3 for N39S, 2.3 for Y51F, 0.3 for I71V, 1.5 for Y80F, 0.3 for Y86F and 0.3 for T95A. Structures of all of these mutants were refined against high resolution synchrotron data: S24A (1.1 Å resolution, R = 12.1 %), N39S (1.6 Å, 17.6 %), Y51F (1.5 Å, 12.7 %), I71V (1.5 Å, 11.9 %), Y80F (1.2 Å, 13.2 %), Y86F (1.7 Å, 13.1 %), T95A (1.3 Å, 12.2 %). The only mutation that caused structural changes which significantly exceeded the limits of error was the N39S mutant. Substantial conformational changes in this mutant resulted in a large decrease in the conformational stability and the loss of enzymatic activity. The cavity caused by removing of the methylene group in I71V mutant is partly filled with the side chains of neighboring residues and does not contain any water molecule. Serine, threonine and tyrosines which were mutated are located at the surface of the molecule and are partially accessible to solvent. Removing the hydroxyl group caused changes in solvation of the molecule. In all these mutant structures, the intramolecular hydrogen bonds at the site of mutation were lost and new intermolecular (to water molecules) were formed. In summary, 1 – 2 hydrogen bonds were lost in each mutant. Our results show that intramolecular hydrogen bonds at exposed sites on the surface of a protein can make favourable contribution to the conformational stability of the protein. None of the mutated amino acids are directly involved in the catalytic function of the enzyme, therefore changes in enzymatic activity were not expected. However, decreases in enzymatic activity were observed for most of the mutants. Surprisingly, a fourfold increase in activity was observed for the I71V mutant. Conformational changes caused by mutations, influence of structural flexibility, changes in enzymatic activity, and decrease in stability caused by the loss hydrogen bonds will be discussed. 1. Hebert, E.J., Giletto, A., Sevcik, J., Urbanikova, L., Wilson, K.S., Dauter, Z., Pace, C.N. (1998). Biochemistry 37, 16192-16200. 2. Pace, C.N., Horn, G., Hebert, E.J., Bechert, J., Shaw, K., Urbanikova, L., Scholtz, J.M., Sevcik, J. (2001). J. Mol. Biol. 312, 393-404.

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(P57) CONTRIBUTION OF ACTIVE SITE RESIDUES TO THE ENZYME ACTIVITY AND THERMOSTABILITY OF RIBONUCLEASE SA Gennady I. Yakovlev1, Vladimir A. Mitkevich1, Kevin L. Shaw2, Saul Trevino3, Stephanie Newsom3, C. Nick Pace3, Alexander A. Makarov1 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia. 2Department of Biology, Grove City College, Grove City, Pennsylvania 16127, USA. 3Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843, USA It has been suggested that residues at enzyme active sites might make unfavorable contributions to the enzyme stability (1). We have studied this in RNase Sa by replacing active site residues and measuring the effect on the thermal stability of the mutant enzyme. The Gln38Ala replacement in the RNase Sa substrate-binding site results in a one order of magnitude lowering of the enzyme activity, and an increase in the protein melting temperature, Tm, of 3.5°C. Similarly, the Glu74Lys substitution causes a sixfold decrease in enzyme activity and a 3.1°C increase in Tm. On the other hand, substitutions of Glu54 and Arg65 in the catalytic site, which substantially decrease the enzyme activity, result in a decrease of 4-6°C in Tm. Similarly, the replacement of Glu41 in the enzyme active center, which, according to our data, has no catalytic function causes a decrease in Tm. Finally, the His85Gln substitution inactivates the enzyme but has no effect on Tm. Thus, the results show that the hypothesis, coupling the contribution of individual residues to the protein stability and function, is not an absolute one. Residues in an active center may be involved in catalysis, substrate binding, and/or structural stabilization of the active center. It is evident that the residues participating in the substrate binding are not fully complementary to the environment. Owing to this, the replacement of such residues, causing the elimination of their function, will result in increased stability of the protein. In our case, Gln38 and Glu74 are such residues, the replacement of which increases thermal stability of RNase Sa. Shoichet, B.K., Baase, W.A., Kuroki, R. and Matthews, B.W. (1995) Proc. Natl. Acad. Sci. USA 92, 452-456. This work was supported by the NIH FIRCA grant TWO1058.

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(P58) THERMAL STABILIZATION OF A LABILE MUTANT ENZYME OF RIBONUCLEASE A BY ANTIBODIES Hina Younus1,2, Jens Köditz2, Mohammed Saleemuddin1, Renate Ulbrich-Hofmann2 1Interdisciplinary Biotechnology Unit, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India. 2Department of Biochemistry/Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany. The region between the α-Helix 2 and the β-strand 1 was identified as being first exposed during thermal unfolding of ribonuclease A (RNase) (1). Recently, the stabilizing potential of antibodies recognizing the labile region (32−43) of RNase has been investigated (2). With the aim of selecting a better model enzyme for studying the enzyme stabilization by antibodies, L35S-RNase was choosen. This mutant enzyme was constructed by site directed mutagenesis, expressed in E. coli as inclusion bodies, renatured and purified. The specific activity of L35S-RNase was 42.9% as compared to the wild type RNase. Its thermal stability was remarkably decreased due to the point mutation within the labile region. The difference in Tm values amounted to 8.7oC. Antibodies raised against the synthetic dodecapeptide SRNLTKDRAKPV corresponding to the labile region 32−43 on RNase (antipeptide IgG) and those against the entire RNase (antiRNase IgG) bound to the mutant enzyme with the same affinity as to the wild type (2). L35S-RNase immobilized on Sepharose-4B precoupled either with the antipeptide IgG or antiRNase IgG proved to be more resistant to thermal inactivation at 65oC than the soluble enzyme. The stability achieved was highest when L35S-RNase was immobilized onto antipeptide IgG-Sepharose. This preparation retained almost 100% activity after incubation at 65oC for 2 hours while the enzyme bound to antiRNase IgG-Sepharose retained about 60%. In comparison, the soluble enzyme retained less than 20% activity. If the antiRNase IgG-Sepharose was pretreated with the dodecapeptide 32−43 before the binding of L35S-RNase, the stability was decreased, indicating that antiRNase IgG contains a remarkable fraction of antibodies recognizing the labile region of the enzyme and mainly contributing to the stabilizing effect. The studies confirm that antibodies recognizing specific labile regions of enzymes are highly effective in stabilizing these enzymes. 1. Arnold, U., Rücknagel, K.P., Schierhorn, A., Ulbrich-Hofmann, R.(1996). Eur. J. Biochem. 237, 862-869. 2. Younus, H., Owais, M., Rao, D.N., Saleemuddin, M.(2001). Biochim. Biophys. Acta 1548, 114−120. Research funded by a grant from Deutscher Akademischer Austauschdienst (DAAD) to H.Y and from Max-Buchner foundation to J.K are gratefully acknowledged.

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(P59) AN EFFICIENT SEQUENCE SPECIFIC ARTIFICIAL RIBONUCLEASES - OLIGONUCLEOTIDE CONJUGATED TO IMIDAZOLE CONSTRUCTS Marina Zenkova, Nataliya Beloglazova, Vladimir Sil'nikov, Valentin Vlassov Novosibirsk Institute of Bioorganic Chemistry, 8, Lavrentiev ave., 630090, Novosibirsk, Russia Development of oligonucleotide conjugates capable of cleaving RNA at complementary sequences provides new tools for investigation of RNA structure and functions and opens new possibilities for design of antisense therapeutics. New RNA cleaving dendrimer-based constructs containing two or four imidazole groups have been designed by using the precursor approach. The parent 16-mer oligonucleotide built up with methoxyoxalamido modifiers upon standard phosphoramidite oligonucleotide synthesis was post-synthetically functionalized with histamine. The groups were placed at the 5’-end of antisense oligonucleotide via several types of linkers differing in their lengths and structures. The conjugates were active in the reaction of site-directed cleavage of tRNAPhe under physiological conditions. The main cleavage site was the phosphodiester bond C63−A64, where the location of imidazole groups of the conjugates was expected. A complete cleavage of the target RNA was achieved after 2 hours of RNA incubation at 37°C in the presence of conjugates containing four imidazole groups taken at a concentration of 0.01 mM. The cleavage activity of the conjugates correlates with the linker structure and number of Im groups in the reaction domain. For all the conjugates tested, the conjugates bearing four Im residues display a higher activity than the conjugates containing two imidazoles. It was found that the conjugates with RNA-cleaving domain attached to oligonucleotide via cyclohexcyl-containing linkers displayed a higher cleavage rate than those with a nucleotide-containing linker or non-nucleotide unit. This work was supported by the grants from Wellcome Trust 063630 and RFBR 02-04-48664, 00-I5-97969, and Program on Gene-targeted biologically active compounds.

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(P60) OLIGONUCLEOTIDE–PEPTIDE CONJUGATES DISPLAYING SPECIFICITY OF RNASE T1 Marina Zenkova, Nadejda Mironova, Dmitryi Pyshnyi, Eugenia Ivanova, Valentin Vlassov. Institute of Bioorganic Chemistry 8, Lavrentiev av., Novosibirsk-90, 630090, Russia Oligonucleotides bearing different reactive groups and other functional entities have become a commonplace tool in many diagnostic and therapeutic applications. Here we report the design of oligonucleotide-peptide conjugates capable of RNA cleaving and displaying specificity of RNase T1 We investigated cleavage of several RNAs - in vitro transcript of human tRNALys

3, in vitro transcript of yeast tRNAPhe, yeast tRNAPhe and fragment of HIV1 RNA, comprising PBS – with oligodeoxyribonucleotides conjugated to peptide with alternating basic and hydrophobic aminoacids. Three conjugates under the study were: pep-TCAA (1), pep-CCAAACA (2) and pep-CCC-TGG-ACC-CTC-AGA-T-3’ (3). Conjugate (3) targeted anticodon stem, variable loop and ТΨС-steam (bases 38-53) of tRNALys

3 delivered peptide to С56-А57 phosphodiester bond in the ТΨС-loop. The sequences of conjugates (1) and (2) were random and had no complementary sites in all used RNAs. Our study show that these conjugates are capable to cleave phosphodiester bonds in RNA with high efficacy. The main cleavage sites occurred in GpX-sequences for conjugate (1) and (2). We found that cleavage sites for conjugates (1) and (2) are located in the similar elements of tRNA secondary structure: D-loop, variable loop, junction, anticodon loop. Some weak cuts were observed in the steams in the absence of magnesium, reflecting the tRNA structure breezing. For conjugate (3) both sequence-specific and GpX ribonuclease activities were observed. Main cleavage sites for conjugate (3) were phosphodiester bond at C56-A57 and GpX-sequences within the single-stranded region of tRNALys

3 the same as for conjugates (1) and (2). Probing of tRNALys

3 in the presence of .conjugate (3) using RNAse T1 revealed loosening of D-loop structure combined with the unfolding of anticodon and ТΨС-stems. In that way D-loop start to be accessible for cleavage. We found that random oligonucleotide- peptide conjugates (1) and (2) are capable to cleave RNA catalytically with the reaction turn over more than 200, whereas tight binding of the conjugate (3) to RNALys

3 lead to essential reduction of cleavage efficiency. This work was supported by the grants from WellcomeTrust 063630 and RFBR 02-04-48664, 00-I5-97969, and Program on Gene-targeted biologically active compounds.

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List of Participants

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Acharya K Ravi Ardelt Wojciech University of Bath Alfacell Corporation Dept. of Biology and Biological Sciences Director, Biochemistry Claverton Down 225 Belleville Ave Bath BA2 7AY Bloomfield, NJ 07003 UK Tel: +44 1225386238 USA Tel: +1 9737488082 E-mail: k.r.acharya@bath.ac.uk E-mail: bw_ardelt@hotmail.com

Arnold Ulrich Avitabile Francesca Martin-Luther-University Complesso Universitario di Monte S.Angelo Dept. Biochemistry/Biotechnology Dipartimento di Chimica Kurt-Mothes Str. 3 Via Cintia 45 06114 Halle I-80126 Napoli Germany Tel: +49 3455524865 Italy Tel: +39 081674415 E-mail: arnold@biochemtech.uni-halle.de E-mail: francesca@chemistry.unina.it

B.S. Sanjeev. Badet Josette Indian Institute of Science Université René Descartes Molecular Biophysics Unit Faculté de Pharmacie Bangalore 560012 4 avenue de l'Observatoire India Tel: +91 803092611 75006 Paris E-mail: sanjeev@mbu.iisc.ernet.in France Tel: +33 153739604 E-mail: badet@pharmacie.univ-paris5.fr

Baker Matthew Balatsos Nikos University of Bath Uppsala University Dept. Biology and Biochemistry Dept of Cell and Molecular Biology Claverton Down BMC Box 596, Se-751 24 Bath BA2 7AY Uppsala UK Tel: +44 1225384015 Sweden Tel: +46 184714586 E-mail: bssmdb@bath.ac.uk E-mail: nikos.balatsos@icm.uu.se

Beintema Jaap Benito Antoni Biochemisch Laboratorium Universitat de Girona Nijenborgh 4 Lab. d'Enginyeria de Proteïnes 9747 AG Groningen Campus de Montilivi s/n The Netherlands Tel: +31 503132140 17071 Girona E-mail: beintema@chem.rug.nl Spain Tel: +34 972418388 E-mail: antoni.benito@udg.es

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Bichenkova Elena Bisaglia Marco University of Manchester ICSN-RMN School of Pharmacy & Pharm. Sci. Ecole Polytechnique Coupland 3 91128 Palaiseau Manchester M13 9PL France Tel: +33 169334853 UK Tel: +44 1612752401 E-mail: marco.bisaglia@polytechnique.fr E-mail: elena.bichenkova@man.ac.uk Boix Ester Bontems Francois Universitat Autonoma de Barcelona Groupe ICSN-RMN Dpt. Bioquimica i Biologia Molecular Ecole Polytechnique Fac. Ciencies F91128 Palaiseau 08193 Bellaterra France Tel: +33 169334832 Spain Tel: +34 935811707 E-mail: francois.bontems@polytechnique.fr E-mail: ester.boix@uab.es

Borukhov Sergei Bosch I Grau Montserrat State University of New York Universidad Girona Dept. Microbiology and Immunology C/ Rocapastora, n° 2 Health Science Centre at Brooklyn BESALU 17850 450 Clarkson Avenue BSB 3-27 Girona New York 11203 Tel: +1 7182703752 Spain Tel: +34 972590384 E-mail: serbor@aol.com E-mail: montse@fc.udg.es

Bracale Aurora Bruix Marta Universita' "Federico II" di Napoli Inst. Quim. Fis. Rocasolano Via Mezzocannone, 16 CSIC 80134- Napoli Serrano 119 Italy Tel: +39 0812534604 28006 Madrid E-mail: abracale@unina.it Spain Tel: +34 915619400 x 1122 E-mail: mbruix@iqfr.csic.es

Burke John Callaghan Anastasia The University of Vermont University of Cambridge Microbiology & Molecular Genetics Dept. of Biochemistry 306 Stafford Hall 80 Tennis Court Road 95 Carrigan Drive, Burlington, Vermont 05405 Cambridge CB2 1GA USA Tel: +1 8026568503 UK Tel: +44 1223766020 E-mail: john.burke@uvm.edu E-mail: anastasia@cryst.bioc.cam.ac.uk

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Carpousis Agamemnon Carreras-Margalef Esther CNRS-UPS Universitat Autonoma de Barcelona Lab. Microbiology & Molecular Genetics Dept. de Bioquímica i Biologia Molecular UMR 5100, 118, route de Narbonne Facultat Ciencies 31062 Toulouse Cedex 04 08193 Bellaterra, Barcelona France Tel: +33 561335894 Spain Tel: +34 935811707 E-mail: carpousi@ibcg.biotoul.fr E-mail: ecarreras@einstein.uab.es

Cassano Adam Chavali Gayatri Case Western Reserve University University of Bath Department of Molec. Biol. and Micro. Dept. Biology and Biochemistry 10900 Euclid Ave Claverton Down Cleveland, OH 44106 Bath BA2 7AY USA Tel: +1 2163681877 UK Tel: +44 1225383091 E-mail: agc@po.cwru.edu E-mail: bssgbc@bath.ac.uk Coll Miguel Condon Ciaran Institut de Biologia Molecular - CSIC Institut de Biologie Physico-Chimique Jordi Girona 18 13 rue Pierre et Marie Curie 08034 Barcelona 75005 Paris Spain Tel: +34 934006149 France Tel: +33 158415123 E-mail: mcoll@ibmb.csic.es E-mail: condon@ibpc.fr

Crabtree Ben Crooke Stanley University of Bath Isis Pharmaceuticals, Inc. Department of Biology and Biochemistry 2292 Faraday Ave Claverton Down Carlsbad, CA 92008 Bath BA2 7AY USA Tel: +1 7606032311 UK Tel: +44 1225 385250 E-mail: scrooke@isisph.com E-mail: bssbac@bath.ac.uk

Crooke Rosanne Cuchillo Claudi Isis Pharmaceuticals Inc. Universitat Autònoma de Barcelona 2292 Faraday Ave Dept de Bioquímica i Biologia Molecular Carlsbad, CA 92008 Facultat de Ciències USA Tel: +1 7606032326 08193-Bellaterra E-mail: rcrooke@isisph.com Spain Tel: +34 935812565 E-mail: claudi.cuchillo@uab.es

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D'Alessio Giuseppe De Lorenzo Claudia University of Naples "Federico II" University of Naples "Federico II Dept. of Biological Chemistry Department of Biological Chemistry Via Mezzocannone, 16 Via Mezzocannone, 16 80134-Napoli 80134 Napoli Italy Tel: +39 0812534731 Italy Tel: +39 0812534510 E-mail: dalessio@unina.it E-mail: cladelor@hotmail.com

De Vos Stefan Depledge Nigel Vrije Universiteit Brussel University of Bath IMOL-Ultrastructure Dept. of Biology and Biochemistry Paardenstraat 65 Claverton Down 1640 Sint-Genesius-Rode Bath BA2 7AY Belgium Tel: +32 23590268 UK Tel: +44 1225385250 E-mail: stedevos@vub.ac.be E-mail: bssnd@bath.ac.uk

Deshpande Ashlesha Dickson Kimberley University of Bath University of Wisconsin - Madison Dept. Biology and Biochemistry Department of Biochemistry Claverton Down 433 Babcock Dr. Bath BA2 7AY Madison, WI 53706 UK Tel: +44 1225383091 USA Tel: +1 6082629874 x 3357 E-mail: bssad@bath.ac.uk E-mail: kdickson@biochem.wisc.edu Dyer Kimberly Eckschlager Tomas LHD/NIAID/National Institutes of Health University Hospital Motal Bldg 10 Room 11N104 V Uvalu 84 10 Center Drive MSC 1886 150 06 Praha Bethesda, MD 20892-1886 Czech Republic Tel: +42 0606364730 USA Tel: +1 3014022429 E-mail: eckschlagertomas@yahoo.com E-mail: kdyer@niaid.nih.gov

Elela Sherif Abou Ercole Carmine Universite de Sherbrooke Complesso Universitario di M.S.Angelo Département de Microbiologie et d'Infectiologie Dipartimento di Chimica Faculté de Médecine Via Cintia 3001, 12e avenue Nord, Sherbrooke, Quebec 80126-Napoli Canada J1H 5N4 Tel: +1 8195645275 Italy Tel: +39 081674410 E-mail: sabou@courrier.usherb.ca E-mail: ercoleca@hotmail.com

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Evans Hazel Fabani Martin University of Bath University of Manchester Dept. Biology and Biochemistry Pharmacy & Pharmaceutical Sciences Claverton Down Oxford Road Bath BA2 7AY Manchester M13 9PL UK Tel: +44 1225383091 UK Tel: +44 1612752401 E-mail: bsphre@bath.ac.uk E-mail: martin.fabani@man.ac.uk

Gaidamakov Sergei Garcia-Mayoral Maria Flor LMG NICHD NIH Química Física Rocasolano,CSIC Building 6B Room2B231 MSC2790 Serrano, 121 Bethesda, MD 20892-2790 28006 Madrid USA Tel: +1 3018169619 Spain E-mail: gaidamas@mail.nih.gov Spain Tel: +34 915619400 E-mail: fgarcia@iqfr.csic.es

Garcia-Ortega Lucia Gleich Gerald Universidad Complutense de Madrid University of Utah Medical School Departamento de Bioquimica Department of Dermatology, Facultad de Quimicas 30 North 1900 East 28040 Madrid Salt Lake City Utah 84132-2409 Spain Tel: USA Tel: +1 8015818963 E-mail: lucia@solea.quim.ucm.es E-mail: gerald.gleich@hsc.utah.edu

Goonewardene Tyronne Gotte Giovanne University of Oxford DSVN UK Tel: +44 1865 741 360 Sezione di CHIMICA BIOLOGICA E-mail: t.goonewardene@icrf.icnet.uk Strada Le Grazie 8 37134 VERONA Italy Tel: +39 0458027694 E-mail: giovanni.gotte@univr.it Green Pamela Hahn Ulrich Delaware Biotechnology Institute Universität Leipzig 15 Innovation Way Fakultät für Biowissenschaften Newark Institut für Biochemie DE 19711 Talstrasse 33, D-04103 Leipzig USA Tel: +1 302 831 6160 Germany Tel: +49 3419736991 E-mail: green@dbi.udel.edu E-mail: uli.hahn@uni-leipzig.de

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Hares Michelle Hartley Robert University of Bath NIH/NIDDK/LCDB Dept. Biology and Biochemistry 50 Center Drive Claverton Down Rm. 3126, MS8028 Bath BA2 7AY Bethesda, MD 20814 UK Tel: +44 1225383091 USA Tel: +1 3014966870 E-mail: m.c.hares@bath.ac.uk E-mail: hartleyr@helix.nih.gov

Hassel Brett Hata Tomonori University of Maryland Kyushu University School of Medicine Laboratory of Biochemistry 655 West Baltimore Street, 9th flr BRB Faculty of Agriculture Baltimore, MD 21201 Fukuoka 812-8581 USA Tel: +1 4103282344 Japan Tel: +81 926422854 E-mail: bhassel@som.umaryland.edu E-mail: hatatomo@agr.kyushu-u.ac.jp

Henriksson Niklas Holloway Daniel Biomedical Center, Uppsala University of Bath Department of Cell and Molecular Biology Dept. Biology and Biochemistry Box 596 Claverton Down SE-751 24 UPPSALA Bath BA2 7AY Sweden Tel: +46 184714697 UK Tel: +44 1225383091 E-mail: niklas.henriksson@icm.uu.se E-mail: bssdeh@bath.ac.uk

Horb Lori Hu Guofu University of Bath Harvard Medical School Dept. of Biology and Biochemistry Center, Biochem. Biophys.Sci Claverton Down One Kendall Sq, Building 600, Bath BA2 7AY Cambridge, MA 02139 UK Tel: +44 1225383091 USA Tel: +1 16176216109 E-mail: bsslh@bath.ac.uk E-mail: guofu_hu@hms.harvard.edu

Ilinskaya Olga Inokuchi Norio University of Kazan Nihon University Microbiology Department College of pharmacy Kremlevskaya str.,18 7-7-1,Narashinodai 420008 Kazan Funabashi,Chiba,274-8555 Russia Tel: +7 8432315191 Japan Tel: +81 4474654543 E-mail: olga.ilinskaya@mail.ru E-mail: inokun@pha.nihon-u.ac.jp

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Inoue Sakura Iyer Shalini Tokyo University University of Bath 5-16-1-1114 Dept. Biology and Biochemistry Omorikita Claverton Down Ota-ku, Tokyo 143-0016 Bath BA2 7AY Japan Tel: +81 358413078 UK Tel: +44 1225383091 E-mail: sakura@mcb.bt.a.u-tokyo.ac.jp E-mail: bspsi@bath.ac.uk

Jenkins Jeremy Ji Xinhua Harvard Medical School National Cancer Institute Center for Biochem/Biophys. Sci. & Medicine 1050 Boyles Street, Building 539 One Kendall Sq, Building 600, Third Floor Frederick, MD 21702 Cambridge, MA 02139 USA Tel: +1 3018465035 USA Tel: +1 6176216116 E-mail: jix@ncifcrf.gov E-mail: jeremy_jenkins@hms.harvard.edu

Juarez-Morales Jose-Luis Julius John University of Bath NCI-Frederick Department of Biology and Biochemistry 7th and Military Rd Claverton Down Bld 539 Room 116 Bath BA2 7AY Frederick, MD 21702 UK Tel: +44 1225 826826 X 5903 USA Tel: +1 3018461611 E-mail: bspjlj@bath.ac.uk E-mail: jjulias@mail.ncifcrf.gov

Kawano Shin Kimura Makoto Kyushu University Kyushu University Department of Laboratory of Biochemistry Bioscience and Biotechnology Faculty of Agriculture Hakozaki 6-10-1 Fukuoka 812-8581 Fukuoka 812-8581 Japan Tel: +81 926422853 Japan Tel: +81 926422854 E-mail: mkimura@agr.kyushu-u.ac.jp E-mail:

Kirsebom Leif Koboyashi Hiroko Uppsala University Nihon University Department of Cell and Molecular Biology Department of Microbiology Box 596, BMC Narashinodai 7-7-1 SE-751 24 Uppsala Funabashishi,Chiba 274-8555 Sweden Tel: +46 184714068 Japan Tel: +81 474654584 E-mail: leif.kirsebom@icm.uu.se E-mail: hirokok@pha.nihon-u.ac.jp

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Köditz Jens Kouzuma Yoshiaka Martin-Luther-University Halle-Wittenberg Kyushu University Inst. f. Biotechnology Hakozaki, 1-13-30-201 Kurt-Mothes-Str. 3 Higashi-ku 06120 Halle Fukuoka, 812-0053 Germany Tel: +49 3455524869 Japan Tel: +81 926424215 E-mail: koeditz@biochemtech.uni-halle.de E-mail: kouzuma@agr.kyushu-u.ac.jp Lamontagne Bruno Lancaster Lorna Universite de Sherbrooke University of East Anglia 256 rue Montréal 61 Stafford Street Sherbrooke Norwich Quebec NR2 3BD Canada J1H 1E3 Tel: +1 8195633886 UK Tel: +44 1603592695 E-mail: bruno.lamontagne@hermes.usherb.ca E-mail: l.lancaster@uea.ac.uk

Larose Stephanie Laurents Douglas Universite de Sherbrooke Instituto de Quimica Fisica "Rocasolano" Département de Microbiologie et d'Infectiologie CSIC Faculté de médecine Serrano 119 Sherbrooke, Canada, J1H 5N4 28006 Madrid 3001, 12e avenue Nord, Sherbrooke, Quebec Spain Tel: +34 915619400 Canada J1H 5N4 Tel: +1 81982068681-5789 E-mail: doug@malika.iem.csic.es E-mail: steph_larose@hotmail.com

Lawrence Jason Lewis Roger University of Bath University of Nevada Dept. of Biology and Biochemistry Coll. Agriculture, Biotech.and Nat. Resources Claverton Down Reno, NV 89557 Bath BA2 7AY USA Tel: +1 7757841095 UK Tel: +44 1225385250 E-mail: lewis@cabnr.unr.edu E-mail: bssjml@bath.ac.uk

Liao You-Di Libonati Massimo Academia Sinica The University of Verona Institute of Biomedical Sciences Dept Neurological Sciences Taipei Fac. Medicine TAIWAN, 115 Strada Le Grazie 8 Tel: +886 227899167 37134 Verona, Italy Tel: +39 0458027166 E-mail: ydliao@ibms.sinica.edu.tw E-mail: massimo.libonati@univr.it

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Lima Walt Loverix Stefan Isis Pharmaceuticals Inc. Vrije Universiteit Brussel 2292 Faraday Ave Dienst Algemene Chemie Carlsbad, CA 92008 Pleinlaan, 2 USA Tel: +1 6196032387 1050 Brussels E-mail: wlima@isisph.com Belgium Tel: +32 26293516 E-mail: sloverix@vub.ac.be

MacIntosh Gustavo Maeda Takashi University of Delaware Okayama University Delaware Biotechnology Institute Department of Bioscience and Biotechnology 15 Innovation Way Tsushima-Naka Newark, DE 19711 Okayama, 700-8530 USA Tel: +1 3028314644 Japan Tel: +81 862518217 E-mail: gustavo@dbi.udel.edu E-mail: maedata@biotech.okayama- u.ac.jp Maizel Jnr Jacob Makarov Alexander NIH/NCI-Frederick Engelhardt Institute of Molecular Biology Building 469, Room 151 Russian Academy of Sciences Frederick MD 21702-1201 Vavilov str. 32 USA Tel: +1 13018465532 Moscow 119991 E-mail: jmaizel@ncifcrf.gov RussiaTel: +7 0951354095 E-mail aamakarov@genome .eimb.relarn.ru

Martinez del Pozo Alvaro Marujo Paulo Universidad Complutense Fondation Edmond de Rothschild Dept. Bioquimica y Biologia Molecular I Institut de Biologie Physico-Chimique Facultad de Quimica 13, Rue Pierre et Marie Curie E-28040 Madrid 75005 Paris Spain Tel: +34 913944259 France Tel: +33 158415149 E-mail: alvaro@bbm1.ucm.es E-mail: marujo@ibpc.fr

Masaki Haruhiko Matousek Josef Tokyo University Acad Sciences of the Czech Republic Yayoi Institute of Animal Physiology & Genetics Bunkyo-ku 27721 Libechov Tokyo 113-8657 Czech Republic Tel: +42 0206697024 Japan Tel: +81 358413080 E-mail: matousek@iapg.cas.cz E-mail: hmasaki@mcb.bt.a.u-tokyo.ac.jp

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Mazzarella Lelio Merlino Antonello Comp. Universatario di Monte Sant'Angelo Università degli Studi di Napoli 'Federico II Dept. Chemistry Dipartimento di chimica Via Cynthia Via Cyntia 80126 Napoli 80126, Napoli Italy Tel: +39 081674279 Italy Tel: +39 081674281 E-mail: mazzarella@chemistry.unina.it E-mail: merlino@chemistry.unina.it

Michaelis Martin Mignon Pierre Klinikum der Johann Wolfgang Vrije Universiteit Institut fuer Medizinische Virologie Dienst Algemen Paul Ehrlich-Str. 40 Pleinlaan, 2 60596 Frankfurt am Main 1050 Brussels Germany Tel: +49 6963017162 Belgium Tel: +32 26293516 E-mail: michaelis@em.uni-frankfurt.de E-mail: pmignon@vub.ac.be

Mitchell Philip Mitkevich Vladimir Wellcome Centre for Cell Biology Russian Academy of Sciences University of Edinburgh Institute of Molecular Biology Institute of Cell and Molecular Biology Vavilov Str. 32 Edinburgh EH9 3JR Moscow 119991 UK Tel: +44 1316507081 Russia Tel: +7 0951359824 E-mail: pmitch@holyrood.ed.ac.uk E-mail: snayper@mailru.com Mohan C Gopi Motoyoshi Naomi University of Bath Nihon University College of Pharmacy Dept. Biology and Biochemsitry 7-7-1, Narashinodai Claverton Down Funabashi-shi Bath BA2 7AY Chiba, 274-8555 UK Tel: +44 1225384015 Japan Tel: +81 474654584 E-mail: bsscgm@bath.ac.uk E-mail: n-moto@pha.nihon-u.ac.jp

Moussaoui Mohammed Narayan Mahesh Universitat Autonoma de Barcelona Cornell University Dpt. Bioquimica i Biologia molecular Baker Laboratory of Chemistry and Fac. Ciencies Chemical Biology Cerdanyola del valles (Barcelona) 08193 Ithaca, 14853-1301 Spain Tel: +34 935811707 USA Tel: +1 6072554860 E-mail: mohammed.moussaoui@uab.es E-mail: mn43@cornell.edu

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Navarro-Cantero Susanna Nicholson Allen Universitat Autonoma de Barcelona Wayne State University Dept. de Bioquimica i Biologia Molecular Department of Biological Sciences Campus de Bellaterra 5047 Gullen Mall 08193 Barcelona Detroit, MI 48202 Spain Tel: +34 935811707 USA Tel: +1 3135772862 E-mail: sncantero@wanadoo.es E-mail: anichol@biology biosci.wayne.edu Nicholson Rhonda Nogues M. Victoria Wayne State University Universitat Autònoma de Barcelona Department of Biological Sciences Dept de Bioquímica i Biologia Molecular 5047 Gullen Mall Facultat de Ciències Detroit, MI 48202 08193-Bellaterra USA Tel: +1 3135778002 Spain Tel: +34 935811256 E-mail: rnichol@genetics.wayne.edu E-mail: victoria.nogues@uab.es

Numata Tomoyuki Ogawa Tetsuhiro Kyushu University Tokyo University Laboratory of Biochemistry Yayoi Faculty of Agriculture Bunkyo-ku Fukuoka 812-8581 Tokyo 113-8657 Japan Tel: +81 926422854 Japan Tel: +81 358413078 E-mail: rinsei@agr.kyushu-u.ac.jp E-mail: tetsu@mcb.bt.a.u-tokyo.ac.jp

Ohgi Kazuko Ooi Siew-Loon Hoshi College of Pharmacy Johns Hopkins University Dept. Microbiology School of Medicine 2-4-41 Ebara, Shinagawa-ku Hunterian 617, 725 N. Wolfe Street Tokyo 142-8501 Baltimore, MD 21205 Japan Tel: +81 354985754 USA Tel: +1 4106141469 E-mail: ohgi@hoshi.ac.jp E-mail: sooi@jhmi.edu Pace Nick Piccoli Renata Texas A&M Univ University of Naples "Federico II" Medical Biochemistry Dept. Dipartimento di Chimica Biologica College Station Via Mezzocannone 16 TX 77843-1114 80134 Naples USA Tel: +1 9798451788 Italy Tel: +39 0812534733 E-mail: nickpace@tamu.edu E-mail: piccoli@unina.it

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Picone Delia Pouckova Pavla University of Naples "Federico II" Charles University Complesso Universitario di Monte S. Angelo Institute of Biophysics via Cintia Salmovska 1 80126 Naples Prague 2, 120 00 Italy Tel: +39 081674406 Czech Republic Tel: +42 0224965704 E-mail: picone@chemistry.unina.it E-mail: pavla.pouckova@lfl.cuni.cz

Pouckova Petra Raines Ronald RCD Ltd University of Wisconsin-Madison Americka 632 Department of Biochemistry Dobrichovice 252 29 433 Babcock Drive Czech Republic Madison, WI 53706-1544 Czech Republic Tel: +42 029913163 USA Tel: +1 6082628588 E-mail: petrapouckova@hotmail.com E-mail: raines@biochem.wisc.edu

Ramanathan Natesh Raven Tom University of Bath University of Bath Dept. Biology and Biological Science Dept. Biology and Biochemistry Claverton Down Claverton Down Bath BA2 7AY Bath BA2 7AY UK Tel: +44 1225383091 UK Tel: +44 1225383019 E-mail: bssrn@bath.ac.uk E-mail: bssmter@bath.ac.uk

Ribo Marc Rico Manuel Universitat de Girona Instituto de Quimica Fisica "Rocasolano" Laboratori d'Enginyeria de Proteïnes CSIC Fac. de Ciències Serrano 119 Campus de Montilivi s/n, 17071 Girona 28006 Madrid Spain Tel: +34 972418388 Spain Tel: +34 915619400 E-mail: marc.ribo@udg.es E-mail: mrico@iqfr.csic.es

Riordan James Rosenberg Helene Harvard Medical School National Institutes of Health Center for Biochem. Biophys.Sci. & Medicine Laboratory of Host Defences One Kendall Sq, Building 600, Third Floor Building 10, room 11N104 Cambridge, MA 02139 NIAID, 9000 Rockville Pike, USA Tel: +1 6176216132 Bethesda MD E-mail: James_Riordan@hms.harvard.edu 20892, USA Tel: +1 3014021545 E-mail: hr2k@nih.gov

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Rybak Susanna Saida Fakhri NCI-Frederick Laboratoire ICSN-RMN Bldg. 320, Rm. 9 DCSO, Ecole Polytechnique Frederick, MD 21702 Route de Saclay USA Tel: +1 3018465215 91128 Palaiseau Cedex E-mail: rybak@ncifcrf.gov France Tel: +33 169334855 E-mail: fakhri.saida@polytechnique.fr

Sarafianos Stefan Saxena Shailendra Rutgers University Alfacell Corporation 68 Oswestry Way 225 Belleville Avenue Somerset NJ 08873 Bloomfield USA Tel: +1 7322354482 New Jersey 07003 E-mail: stefan@cabm.rutgers.edu USA Tel: +1 9737488082 E-mail: ssaxena2@aol.com

Scadden Dierdre Schuster Gadi University of Cambridge Technion Department of Biochemistry Haifa 32000 80 Tennis Court Road Israel Cambridge CB2 1GA Israel Tel: +97 248293171 UK Tel: +44 1223333665 E-mail: gadis@tx.technion.ac.il E-mail: adjs@mole.bio.cam.ac.uk

Sevcik Jozef Shapiro Robert Slovak Academy of Sciences Harvard Medical School Institute of Molecular Biology Center for Biochem. Biophys.Sci. & Dubravska cesta 21 Medicine 84251 Bratislava One Kendall Sq, Building 600,3rd Floor Slovak Republic Tel: +42 1259307435 Cambridge, MA 02139 E-mail: umbisevc@savba.savba.sk USA Tel: +1 6176216132

E-mail: Robert_Shapiro@hms.harvard.edu

Shogen Kuslima Sica Filomena Alfacell Corporation University of Naples "Federico II" 225 Belleville Avenue University of Naples "Federico II", Bloomfield Via Cynthia New Jersey 07003 80126 Napoli USA Tel: +1 9737488082 Italy Tel: +39 081674479 E-mail: info@alfacell.com E-mail: sica@chemistry.unina.it

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Silverman Robert Simons Robert Lerner Research Institute University of California Department of Cancer Biology, NB40 Dept. Microbiology & Mol. Genetics The Cleveland Clinic Foundation 1602 Molecular Science 9500 Euclid Avenue, Cleveland, Ohio 44195 Los Angeles, CA 90095 USA Tel: +1 2164459650 USA Tel: +1 3108258890 E-mail: silverr@ccf.org E-mail: bobs@microbio.ucla.edu Sorrentino Salvatore Stern David University of Naples "Federico II" Cornell University Via Mezzocannone, 16 Boyce Thompson Institute 80134 Napoli Tower Rd Italy Tel: +39 0812546534 Ithaca, NY 14853 E-mail: ssorrent@unina.it USA Tel: +1 6072541306 E-mail: ds28@cornell.edu

Steyaert Jan Subramanian Vasanta Free University of Brussels University of Bath Paardenstraat 65 Dept. of Biology and Biochemistry B-1640 Sint-Genesius-Rode Claverton Down Belgium Tel: +32 23590248 Bath BA2 7AY E-mail: jsteyaer@vub.ac.be UK Tel: +44 1225 386315 E-mail: bssvss@bath.ac.uk

Suhasini Avvaru Swaminathan Jawahar Center for Cellular and Molecular Biology European Bioinformatics Institute Hyderabad 500007 Hinxton, India Tel: +91 407192562 Cambridge E-mail: suhasini@ccmb.res.in UK Tel: +44 1223492524 E-mail: jawahar@ebi.ac.uk

Turton Kathryn Udgaonkar Jayant University of Bath Tata Institute of Fundamental Research Department of Biology & Biochemistry National Centre for Biological Sciences Claverton Down GKVK Campus Bath BA2 7AY Bangalore 560064 UK Tel: +44 1225383091 India Tel: +91 803636421 E-mail: bspkm@bath.ac.uk E-mail: jayant@ncbs.res.in

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Urbanikova Lubica Van Hoof Ambro Slovak Academy of Sciences University of Arizona Institute of Molecular Biology MCB/HHMI Dubravska cesta 21 Tucson, AZ 842 51 Bratislava USA Tel: +1 5206214576 Slovak Republic Tel: +42 1259307435 E-mail: ambro@u.arizona.edu E-mail: umbiurbi@savba.savba.sk

Vilanova Maria Vitagliano Luigi University of Girona Instituto di Biostrutture e Bioimmagini Laboratori d'Enginyeria de Proteïnes CNR Dept.de Biologia, Fac. de Ciències Via Mezzocannone 4 Campus de Montilivi s/n, 80134 Napoli 17071 Girona, Spain Tel: +34 972418173 Italy Tel: +39 0812536614 E-mail: dbmvb@fc.udg.es E-mail: luigiv@chemistry.unina.it Wlodawer Alexander Wu Hongjiang National Cancer Insitute Isis Pharmaceuticals Inc. Macromolecular Crystallography Laboratory 2292 Faraday Ave Bldg. 536, Rm. 5 Carlsbad, CA 92008 Frederick, MD 21702 USA Tel: +1 7606032348 USA Tel: +1 3018465036 E-mail: hwu@isisph.com E-mail: wlodawer@ncifcrf.gov

Yakovlev Gennady Younus Hina Englehardt Institute of Molecular Biology Martin Luther University Vavilov Str 32 Dept of Biochemistry/Biotechnology Moscow 119991 Halle-Wittenberg Russia Tel: +7 0951350468 06099 Halle E-mail: yakovlev@imb.ac.ru Germany Tel: +49 3455524905 E-mail: hinayounus@rediffmail.com

Zenkova Marina Zhang Jianzhi Novosibirsk Institute of Bio-organic Chemistry University of Michigan 8, Lavrentiev ave Departments of Ecology (and others) 630090 Novosibirsk 3003 Natural Science Building Russia Tel: +7 3832333761 830 North University Ave, Ann Arbor MI E-mail: marzen@niboch.nsc.ru 48109, USA Tel: +1 7347630527 E-mail: jianzhi@umich.edu

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