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Doctorat De L'université De Toulouse HÈSE En vue de l'obtention du DOCTORAT DE L’UNIVERSITÉ DE TOULOUSE Délivré par l'Université Toulouse III - Paul Sabatier Discipline : Biochimie Présentée et soutenue par Violette GAUTIER Le 18 décembre 2012 Développement de méthodes quantitatives sans marquage pour l’étude protéomique des cellules endothéliales JURY Dr. Philippe Marin, IGF/CNRS (Montpellier) – Rapporteur Dr Thierry Rabilloud, iRTSV/LCBM/CNRS (Grenoble) - Rapporteur Dr Sarah Cianferani-Sanglier, IPHC/CNRS (Strasbourg) - Examinatrice Dr. Bernard Monsarrat, IPBS/CNRS (Toulouse) - Directeur de thèse Dr. Anne Gonzalez de Peredo, IPBS/CNRS (Toulouse) – Co-directrice de thèse Pr. Jean-François Arnal, Université Paul Sabatier (Toulouse) - Président Ecole doctorale : Biologie - Santé - Biotechnologies Unité de recherche : Institut de Pharmacologie et de Biologie Structurale (UMR5089, CNRS) Directeurs de Thèse : Bernard Monsarrat, Anne Gonzalez de Peredo Rapporteurs : Philippe Marin, Thierry Rabilloud RESUME La compréhension du fonctionnement des systèmes biologiques, dont les protéines sont les principaux effecteurs, est un défi majeur en biologie. La protéomique est aujourd’hui l’outil incontournable pour l’étude des protéines. Au cours de ma thèse, j’ai donc utilisé différentes approches protéomiques pour répondre à plusieurs questions biologiques autour des cellules endothéliales, concernant l’étude de mécanismes fonctionnels de protéines d’intérêt ainsi que des processus inflammatoires au sein de ces cellules. Ces différentes études ont nécessité la mise en place et l’optimisation de méthodes de quantification sans marquage (« label free ») essentielles à la fois pour la caractérisation de complexes protéiques et pour l’analyse de protéomes entiers. Cette thèse décrit ainsi dans un premier temps l’utilisation de telles approches pour l’analyse de complexes immunopurifiés dans laquelle un enjeu important consiste souvent à discriminer de façon non ambiguë les composants bona fide du complexe par rapport aux contaminants non-spécifiques. J’ai ainsi notamment pu identifier certains partenaires spécifiques d’une nouvelle famille de facteurs de transcription humains, les protéines THAP, qui jouent un rôle clé dans la prolifération des cellules endothéliales. Dans un second temps, les processus activés par les cellules endothéliales en condition inflammatoire ont été étudiés au niveau de sous-protéomes ou à l’échelle de protéomes entiers, faisant appel à des méthodes de protéomique globale associées à des stratégies de quantification sans marquage. Le glycoprotéome des cellules endothéliales a ainsi d’une part été étudié lors la réponse inflammatoire, grâce à la mise en place d’une méthode d’enrichissement du protéome de surface des cellules. D’autre part, une analyse du protéome entier de ces cellules et de ses modulations lors de la stimulation par des cytokines pro-inflammatoires a également été réalisée. De façon à obtenir une couverture du protéome la plus profonde possible, cette étude a nécessité la mise en place d’une stratégie quantitative impliquant un fractionnement de l’échantillon sur gel 1D. Enfin, une troisième partie s’intéresse plus spécifiquement aux rôles et aux mécanismes d’action de l’interleukine-33 au sein des cellules endothéliales et a requis l’utilisation des méthodes quantitatives précédemment optimisées. ABSTRACT Understanding biological systems, in which proteins are the main effectors, is a major challenge in biology. Proteomics is now an indispensable tool for the study of proteins. During my PhD, I used different proteomic approaches to address several biological questions about endothelial cells for the study of functional mechanisms of proteins of interest as well as inflammatory processes in these cells. These studies involved the development and the optimization of label-free quantitative methods, essential both for the characterization of protein complexes and for the analysis total proteome. This thesis describes first the use of such approaches for the analysis of immunopurified complexes, for which an important issue is often to discriminate unambiguously bona fide components of the complex from non-specific proteins. I could identify specific partners of a new family of human transcription factors, the THAP proteins, which play a key role in endothelial cells proliferation. Then, the processes activated in endothelial cells under inflammatory condition were studied at sub-proteome or entire proteome level, using global proteomics strategies associated with label-free quantification. On the one hand, the glycoproteome has been studied under inflammatory conditions, through the establishment of a method for cell surface proteome enrichment. On the other hand, a whole proteome analysis of these cells was performed after stimulation with pro- inflammatory cytokines. To obtain deep proteome coverage, this study required the implementation of a quantitative strategy involving sample fractionation by 1D gel. Finally, the third section focuses specifically on the roles and mechanisms of action of interleukin-33 in endothelial cells, and required the use of quantitative methods previously optimized. REMERCIEMENTS Ma thèse se termine, j’en profite donc pour remercier toutes les personnes avec lesquelles j’ai travaillé ou que j’ai côtoyées durant ces 4 années, et qui ont participé à rendre ces années très enrichissantes et passionnantes. Pour commencer, je souhaite remercier l’ensemble des membres du jury, le président Jean- François Arnal, les rapporteurs Philippe Marin et Thierry Rabilloud et également Sarah Cianferani- Sanglier pour avoir accepté d’évaluer mon travail de thèse et pour l’intérêt qu’ils y ont porté. Bernard Monsarrat et Odile Schiltz, je vous remercie sincèrement de m’avoir permis de réaliser ma thèse dans votre équipe et de participer à différents projets. Merci aussi de votre soutien. Je tiens ensuite tout particulièrement à remercier Anne Gonzalez. Un grand merci pour ta disponibilité, ton soutien, ta grande implication, tes conseils toujours très avisés tout au long de ma thèse. Ils m’ont permis de garder espoir malgré les difficultés rencontrées parfois, notamment avec les protéines THAP. Je suis ravie d’avoir réalisé ma thèse sous ta direction et celle de Bernard, et au final, d’avoir eu l’opportunité de travailler sur des projets très différents. Ces années ont été très intéressantes et très formatrices. J’en garde un très bon souvenir. Au cours de ma thèse, j’ai eu le plaisir de travailler avec de nombreuses personnes, d’abord au sein de l’équipe. Merci à David pour ta disponibilité et tes solutions à tout, à Emmanuelle pour ton aide et ton soutien dans tout moment, à Florence pour ton initiation à la MRM, ainsi qu’à Nicolas Delcourt. Et un merci tout particulier à Mathilde, merci pour toute ton aide sur différents projets et ta bonne humeur, c’était super de travailler avec toi ! Je remercie également les collaborateurs avec qui mes projets de thèse ont été réalisés et tout d’abord, Jean-Philippe Girard et son équipe, en particulier Raoul Mazars qui m’a co-encadrée avec Anne durant mon Master 2 Recherche, Corinne Cayrol et Emma Lefrançais pour les projets IL- 33. Merci aussi à l’équipe d’Ambra Mari et particulièrement à Sophies Mourgues pour les travaux sur TFIIH. Merci à l’équipe de Jean-Claude Florent et à Michel Azoulet de l’Institut Curie à Paris, pour la synthèse des sondes chimiques pour l’analyse des glycoprotéines. Ça a été un réel plaisir de côtoyer l’ensemble de l’équipe au quotidien pendant ces années. Merci à tous pour vos conseils, les discussions et la bonne ambiance. Un grand merci à Bertrand (j’aurais pas réussi à te battre finalement, « abusé » !!), Karima (« Pouah », surtout à toi !!), Luc (toi par contre, j’ai réussi à te battre ! «Vachement » facile !), Marlène (Surtout à toi aussi « Charlie » !!) et Mathilde (« Qui ?? ») pour tous les très bons moments qu’on a passés, pour les nombreux fous- rire, et les très nombreux goûters, les grands moments de créativité et aussi pour vos conseils et votre aide. Merci également aux autres sportifs, Renaud (Merci pour les multiples installations d’Endnote !!) et Marc, à Alex, Chrystelle, Thomas, Roxana, Carine et Carole pour les discussions et les pauses, à Manue et Marie-Pierre pour votre gentillesse, vos encouragements et pour les cours à la fac, ainsi qu’à Esthelle, Laure, Nicolas, Stéphane, Sandrine, Michel, François. Merci également à Emilie, Fabien, Lucie et en particulier à Nawel pour les très bons moments passés et leur soutien. Enfin un grand merci à toute ma famille et à tous mes amis pour leurs encouragements et leur soutien sans faille ! SOMMAIRE LISTE DES PRINCIPALES ABREVIATIONS ............................................................................................... 1 INTRODUCTION .................................................................................................................................. 3 PARTIE I. L’ANALYSE PROTEOMIQUE NANOLC-MS/MS PAR SPECTROMETRIE DE MASSE ............................................... 5 I. Principe général de la spectrométrie de masse .......................................................................................... 5 II. Stratégie générale de l’analyse protéomique nanoLC-MS/MS .................................................................... 6 II-1. Préparation de l’échantillon peptidique ................................................................................................ 7 II-2. Fractionnement peptidique par chromatographie liquide.....................................................................
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