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Structural and Functional Studies of ALIX Ricardo Pires

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Structural and Functional Studies of ALIX Ricardo Pires Structural and functional studies of ALIX Ricardo Pires To cite this version: Ricardo Pires. Structural and functional studies of ALIX. Biomolecules [q-bio.BM]. Université Joseph- Fourier - Grenoble I, 2008. English. tel-00332814 HAL Id: tel-00332814 https://tel.archives-ouvertes.fr/tel-00332814 Submitted on 21 Oct 2008 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. To the memory of my beloved grandmother Mariazinha. Acknowledgements A special thanks to my PhD supervisor, Prof. Winfried Weissenhorn, for the quality of his scientific guidance throughout these four years of work, for every opportunity to learn and to be involved in such interesting scientific projects. I would like to thank the two members of my thesis advisory committee, Dr. Christoph Müller and Dr. Michael Knop for all the discussions, suggestions and advice during the PhD work. I would like to thank Prof. Rémy Sadoul, for his collaboration in this work, for his help as a thesis advisory committee member and especially for his enthusiastic discussions, suggestions and criticisms. Thanks also to his group, in particular to Béatrice Blot and Christine Chatellard-Causse for their help and kindness. I am very grateful to the European Molecular Biology Laboratory for all the scientific opportunities and in particular to Dr. Stephen Cusack and Prof. Rob Ruigrok, leading the new "unité mixte" Unit of Virus Host Cell Interactions (UVHCI) where I performed the second half of my PhD work. I am also grateful to all those who contributed to the present work, in particular Dr. Guy Schoehn for all the electron microscopy work, Dr. Eric Forest and Dr. Luca Signor for the mass spec analysis, Dr. Dmitri Svergun and Dr. Manfred Roessle for the collaboration in SAXS experiments, Dr. Marc Jamin for the MALLS analysis, Prof. Heinrich Göttlinger for testing the Alix dimerization mutants, Dr. Carlo Petosa, Dr. Andrew McCarthy and Dr. Charles Sabin for crystallographic advice, Dr. Juan Sanchez-Weatherby and Dr. Monika Budayova-Spano for the work and advice in optimization of crystals and Dr. Véronique Boyer for the immunofluorescence analysis. I would also like to thank all scientific and non-scientific UVHCI staff for their help and kindness. A special thanks to all my friends and colleagues, who have supported me during this difficult but nevertheless creative period. A special word to Carlos Fernández Tornero for his help and inspiration and to Thibault Crepin for the careful reading of the thesis. Last, but not least, special thanks to my family for their continuous encouragements and full-time support: to my father Euclides, who has been my inspiration throughout life and the person who best understands my fears and my dreams, to my mother Regina, with whom I learnt to be persistent and perfectionist and whose love and support is specially important each and every day, to my brother Alexandre, always silent, but always there, to Milene, for reminding me what life is about! Index Abbreviations......................................................................................................................i Abstract............................................................................................................................. iii Résumé .............................................................................................................................. v Chapter I - INTRODUCTION .............................................................................................. 1 The Endosomal Sorting Pathway.................................................................................... 3 Biogenesis of multivesicular bodies: the ESCRT machinery......................................... 4 Overall mechanism of the ESCRT machinery................................................................ 5 Structure and function of the ESCRT complexes .......................................................... 7 ESCRT-0..................................................................................................................................7 ESCRT-I...................................................................................................................................8 ESCRT-II............................................................................................................................... 10 ESCRT-III.............................................................................................................................. 12 Deubiquitylation in the ESCRT pathway ...................................................................... 15 Membrane disassembly of the ESCRT machinery ...................................................... 17 Reasons for exploring the ESCRT machinery ............................................................. 19 Alix, a multifunctional protein ....................................................................................... 22 Modular organization of Alix ......................................................................................... 22 Alix homologues in Saccharomyces cerevisiae ........................................................... 25 Interactions between Alix and the ESCRT machinery ................................................. 26 Functional links between Alix and MVB trafficking....................................................... 27 Mammalian homologs of Alix........................................................................................ 30 Alix cooperates with ALG-2 to promote apoptosis ....................................................... 32 Alix supports budding of some enveloped viruses....................................................... 34 Dictyostelium reveals a role for Alix in developmental signaling ................................. 35 Alix in the recycling endocytic pathway ........................................................................ 36 Structure of mammalian Alix......................................................................................... 38 Structural details of Alix-CHMP4 interaction ................................................................ 41 Structural basis of Alix interaction with YPXnL late domain ......................................... 43 Aims of the present work............................................................................................... 47 Objectifs........................................................................................................................... 49 Index Chapter II - RESULTS ..................................................................................................... 51 Mapping and preliminary characterization of Alix major domains........................... 53 Oligomerization of Alix................................................................................................... 60 Alix forms monomers and dimers in solution ............................................................... 60 Mapping Alix dimerization interface.............................................................................. 63 Global kinetics of H/D exchange .................................................................................. 63 Local kinetics of H/D exchange .................................................................................... 64 Alix-V mutants impair dimerization and adopt elongated conformations..................... 71 Solution structure of Alix by small angle X-ray scattering (SAXS) .......................... 74 Alix-'PRD folds into a crescent-shaped dimer ............................................................ 74 Alix-V mutant adopts an extended conformation ......................................................... 75 Crystallization and preliminary X-ray crystallographic analysis of Alix.................. 82 Crystallization of Alix-V dimer....................................................................................... 82 Crystallization of Alix-V mutant..................................................................................... 84 Alix interaction with lipid bilayers ................................................................................ 88 Monomeric and dimeric Alix interact with liposomes ................................................... 88 Alix-'PRD and Alix-V dimerize upon lipid bilayer binding ........................................... 91 Alix-'PRD liposome interaction does not depend on the vesicle curvature................ 92 Alix-'PRD deforms lipid membranes in vitro ............................................................... 93 Alix distribution on the lipid bilayer ............................................................................... 97 Binding of monomeric and dimeric Alix-V to EIAV late domain ............................. 101 Characterization of Alix-CHMP4B interaction ........................................................... 102 Alix binds a C-terminal region of CHMP4B ................................................................ 102 CHMP4B forms ring-like polymers in vitro.................................................................
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