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Découverte D'une Nouvelle Famille De Protéine Kinases Bactériennes

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Découverte D'une Nouvelle Famille De Protéine Kinases Bactériennes Découverte d’une nouvelle famille de protéine kinases bactériennes : mécanismes de fonctionnement et rôle cellulaire de YdiB, un archétype chez Baccillus subtilis Hien-Anh Nguyen To cite this version: Hien-Anh Nguyen. Découverte d’une nouvelle famille de protéine kinases bactériennes : mécanismes de fonctionnement et rôle cellulaire de YdiB, un archétype chez Baccillus subtilis. Sciences agricoles. Université de Grenoble, 2012. Français. NNT : 2012GRENV017. tel-00721757 HAL Id: tel-00721757 https://tel.archives-ouvertes.fr/tel-00721757 Submitted on 30 Jul 2012 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. THÈSE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITÉ DE GRENOBLE Spécialité : Chimie-Biologie Arrêté ministériel : 7 août 2006 Présentée par Hien-Anh NGUYEN Thèse dirigée par le Dr. Jean-Michel JAULT préparée au sein de l’Institut de Biologie Structurale J.-P. Ebel, et du CEA de Grenoble dans l'École Doctorale Chimie et Sciences du Vivant Découverte d’une nouvelle famille de protéines kinases bactériennes : Mécanisme de fonctionnement et rôle cellulaire de YdiB, un représentant chez B. subtilis Thèse soutenue publiquement le 23 mai 2012 devant le jury composé de : Mme. Patricia DOUBLET Rapporteur Prof. de Bactériologie Moléculaire, Université Lyon 1, Directrice de recherche, MVRL, CNRS UMR 5240 M. Ivan MIJAKOVIC Rapporteur Prof. de Biologie Systemique, AgroParisTech, Rapporteur Directeur de recherche, Micalis, AgroParisTech-INRA UMR 1319 Mme. Patricia RENESTO Examinateur Directrice de recherche UVHCI, CNRS-EMBL-UJF UMI 3265 M. Harald PUTZER Examinateur Directeur de recherche, IBPC, CNRS UPR 9073 M. Christophe GRANGEASSE Examinateur Directeur de recherche, IBCP, CNRS UMR 5086 M. Jean-Michel JAULT Examinateur Directeur de recherche, IBS, CNRS UMR 5075 Université Joseph Fourier / Université Pierre Mendès France / Université Stendhal / Université de Savoie / Grenoble INP Université Joseph Fourier / Université Pierre Mendès France / Université Stendhal / Université de Savoie / Grenoble INP Remerciements Les travaux présentés dans cette thèse ont été princialement réalisés dans le laboratoire de « Membrane & Pathogènes » à l’Institut de Biologie Structurale Jean-Pierre Ebey de Grenoble, mais également dans le laboratoire de Chimie et Biologie des Métaux au CEA de Grenoble ainsi que dans le laboratoire de « Phosphorylation des Protéines et Pathogénie Bactérienne » à l'Institut de Biologie et Chimie des Protéines de Lyon. Je remercie donc ces trois établissements pour m’avoir accueillie et ainsi permis de réaliser mon doctorat. Ce travail, qui faisait partie du projet « P-loop proteins » dans le cadre de l’ANR blanc 2008, a été financé par une bourse du Programe Doctoral International d’Irtelis de la Direction des Sciences du Vivant du CEA (trois années) et par le CNRS sur budget d’unité (trois mois). Je tiens tout particulièrement à remercier le professeur Eva Pebay-Peyroula, qui était membre du jury d’Irtelis, pour m’avoir offert la chance de réaliser ma thèse dans le domaine de la biochimie alors que j’avais une formation de Génie des Procédés et Technologie Alimentaire, et m'avoir accueillie dès mes premiers pas dans son « Laboratoire des Protéines Membranaires », le précédesseur du « Membrane & Pathogènes ». Je remercie très chaleureusement les professeurs Patricia Renesto, Harald Putzer, Christophe Grangeasse, Patricia Doublet et Ivan Mijakovic pour l'honneur qu'ils me font en participant au jury en charge de mon évaluation ce 23 mai 2012. Je voudrais remercier en particulier Patricia Doublet et Ivan Mijakovic pour avoir accepté de se pencher aussi sérieusement sur mon travail ainsi que pour leurs précieuses suggestions. Je remercie très sincèrement mon directeur de thèse, le Dr. Jean-Michel Jault, tout d’abord pour avoir pris le risque d’encadrer en thèse une ingénieur de technologie alimentaire, qui n’avait qu’une connaissance très vague en biochimie et aucune formation en biologie moléculaire. Je le remercie surtout pour sa patience, sa disponibilité illimitée et son enthousiasme scientifique. Je le remercie également pour sa bonne humeur et ses encouragements quotidiens qui contribuèrent à son excellent encadrement pendant ces années. Je remercie toutes les personnes de l’IBS avec qui j’ai interagi au cours de ma thèse et plus particulièrement les membres de « Conserved GTPase/ATPase Team » de Jean-Michel. Merci à Anne-Emmanuelle, Catherine, et particulièrement Johanna pour avoir entamné le sujet de YdiB. Merci à Maria et Bastien pour votre grande contribution et à Anne-Emmanuelle pour la continuation. Merci également à Eric, Carmen, Sandrine, Jean, Benjamin, Jonathan, Argel, Pankaj, Shahid, Isabelle, Lina, Céline, Stéphanie, Fréderique, Charlotte et Emilie pour leur presence, leurs sourires, leurs aides et supports quotidiens. Catherine, je te remercie pour ta gentillesse et ta sensibilité. C’ést toujours toi qui est venue vers moi pour m’aider lorsque tu sentais que j’en avais besoin, même si je ne le disais pas. Merci de m’avoir considérée comme une petite soeur et merci pour toutes nos discussions partagées autour de la musique. NGUYEN Hien-Anh Remerciements Un grand merci à Christophe Grangeasse et également à son équipe, pour m’avoir accueillie et m’avoir consacré une grande attention durant chacun de mes séjours à l’IBCP. Son support scientifique, en tant que d’une part notre direct collaborateur et d’autre part membre du comité de ma thèse, a été indispensable à la réalisation de ce travail, mais également à ma construction en tant que scientifique. C’était dans son laboratoire, avec les aides précieuses de Sébastien Guiral, Emmanuelle Bechet et Aurore Fleurie que les premières démarches sur YdiB en tant que kinase ont été réalisées. Toute ma reconnaissance va également à Anne Marie Di Guilmi du groupe « Pneumococcus » de l’IBS pour sa disponibilité en tant que membre de comité de thèse et particulièrement pour ses précieux conseils concernant des techniques d’immunofluorescentes, tout comme un généreux support de materiel du Pr. Errington Jeffrey à l’Université de Oxford. Je tiens à remercier également sincèrement Patrice Catty, Martine Cuillel et tous les membres de l’équipe Biologie des Métaux du CBM au CEA Grenoble avec qui j’ai intéragi au cours de mes séances de développement des radioactives membranes. Un merci particulier à Alain Grunberg, Georges Eminet, Chantal Robesson, Blandine Caidou, Odile Rossignol et tous les membres de l’équipes administrative et informatique que je ne peux pas citer intégralement ici, pour leur accueil, leur travail et leur responsabilité qui ont beaucoup facilité la vie scientifique des étudiants étrangers, moi incluse. Enfin, mon séjour en France durant ces années de thèse n’aurait pas été aussi enrichissant si je n’avais pas eu des amis proches à mes côtés. Merci à Phương, Minh, anh Minh, anh Nhật, cô Hương, chị Ngọc, chị Thùy, Trân, Quỳnh, Christophe, Laurent, Nina, Brice, Fréderic. Merci à Marie-Laure, Alain, Muriel, Guillaume, Karine, Damien, Cécile, Gérard et les cinq petits chérubins, avec qui je me sens bien adoptée. A mes parents, pour leur amour sans conditions et sans limites A Benjamin, avec qui je me retrouve et me renouvelle NGUYEN Hien-Anh Avant-propos « The only thing that interferes with my learning is my education » (Albert Einstein) « For actually the earth had no roads to begin with, but when many men pass one way, a road is made » (Lu Hsun, My old home, 1921) «… Nobody said it was easy No one ever said it would be so hard I'm going back to the start… » (Coldplay music band, The Scientist) NGUYEN Hien-Anh Avant-propos After all…, tomorrow is another day… (Margaret Mitchell, Gone with the wind, 1936) NGUYEN Hien-Anh Abstract & Key words Abstract : Genome sequencing data has revealed genes encoding uncharacterized protein family UPF0079 which are exclusively found in bacteria; broadly distributed in this kingdom and possess an ATP-binding motif in their sequences. Biochemical characterization and physiological role elucidation of UPF0079 will undoubtedly increase our fundamental biology knowledge, and also remain a prerequisite towards the development of new antimicrobial compounds. Our investigation on YdiB, an archetype of this family in Bacillus subtilis revealed both autophosphorylating and protein phosphotransferase activities. The dual-specificity Ser/Thr and Tyr kinase activity of YdiB seems to require oligomerization is upregulated by basic molecule activators such as natural polyamines or poly-L-lysine. The 10 most conserved residues were studied to gain insights into molecular mechanism of the kinase YdiB. To characterize the function of phosphorylation events linked to YdiB, starting with the B. subtilis ydiA-B-C-D-E operon we showed that YdiB and YdiC function as cognate protein kinase/phosphatase towards two ribosome-related protein substrates YdiD and YdiE. Some co- localization between YdiB and ribosomes were observed. Furthermore, YdiB is capable of phosphorylating both the ribosomal 50S and 30S subunits as well as two ribosome-binding GTPases EngA
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