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Université De La Méditerranée Faculte De Médecine De Marseille École Doctorale Des Sciences De La Vie Et De La Santé Centre De Recherche En Cancérologie De Marseille

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Université De La Méditerranée Faculte De Médecine De Marseille École Doctorale Des Sciences De La Vie Et De La Santé Centre De Recherche En Cancérologie De Marseille UNIVERSITÉ DE LA MÉDITERRANÉE FACULTE DE MÉDECINE DE MARSEILLE ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTÉ CENTRE DE RECHERCHE EN CANCÉROLOGIE DE MARSEILLE T H È S E Pour l’obtention du Diplôme de DOCTEUR de L’UNIVERSITÉ de la MÉDITERRANÉE SPÉCIALITÉ : Oncologie : Pharmacologie et Thérapeutique Présentée et soutenue publiquement par Mme Laetitia STUHL-GOURMAND Le 30 Mars 2010 ROLE DE NACA ET DE SES PARTENAIRES MOLÉCULAIRES DANS LA DIFFÉRENCIATION ÉRYTHROÏDE NORMALE ET PATHOLOGIQUE DES SYNDROMES MYÉLODYSPLASIQUES Directeur de Thèse : Dr Sophie GOMEZ Membres du Jury de Thèse : Pr Daniel Olive Président Pr Catherine Lacombe Rapporteur Dr Dominique Duménil Rapporteur Dr Christian Chabannon Examinateur 1 A mes deux hommes, mes deux amours : mon fils Anthony et mon mari Sébastien 2 REMERCIEMENTS Je remercie les membres du jury d’avoir accepté de juger ce travail: les Pr Catherine Lacombe, Dr Christian Chabannon, Dr Dominique Duménil et le Pr Daniel Olive de présider ce jury. Je remercie Françoise Birg de m’avoir accueillie au centre de Recherche en Cancérologie de Marseille UMR891. Je remercie ma directrice de thèse Dr Sophie Gomez a qui je dois beaucoup. Merci de m’avoir toujours soutenue tout au long de cette thèse, et de m’avoir accompagné avec patience dans l’aboutissement de ce travail à mon rythme de jeune maman. Je remercie le Dr Patrice Dubreuil de m’avoir accueilli dans son équipe, d’avoir toujours été à l’écoute, et de m’avoir soutenu pour la finalisation de mon projet. Je remercie Véronique Gelsi-Boyer, Virginie Trouplin, Daniel Birnbaum et Norvert Vey, sans qui le projet MDS n’aurait pas existé. M’avoir permis d’aborder la pathologie dans mon travail de thèse a été un vrai plaisir. Je remercie tout particulièrement Véro et Virginie pour leur contribution dans ce projet au quotidien. Je remercie Stéphane Audebert, qui m’a initié aux joies de la protéomique ‘kératine free !’’. Je remercie Patrick Lécine pour son vecteur TAP-tag, certes, mais aussi pour sa bonne humeur et son ouverture d’esprit, en sciences bien sûr ! Je remercie également la collaboration de Daniel Isnardon, notre roi du confocal et de l’apotome pour son temps et son investissement en immunofluorescences. Je remercie le Dr Sophie Lopez pour tous ses bons conseils et ses bons protocoles. J’ai apprécié toutes nos discussions, aussi bien en sciences que sur d’autres sujets. Une personne de qualité telle que toi me manquera. Je remercie Eric Lecocq pour sa bonne humeur, son humour (parfois lourd) mais souvent rafraîchissant. Je remercie Sébastien Letard (alias Sébounet) non seulement de bonne compagnie, il regorge toujours de 1001 merveilles dans ces ampoules et tiroirs pour nous sauver. Je remercie Amandine Chaix pour nos bons moments de discussion et son franc parlé, courage toi aussi tu verras la lumière au bout du tunnel ! Je remercie Laurence Borge pour son investissement dans la fin du projet ERGIC. Je remercie également le reste de l’équipe pour juste avoir été là au quotidien Paulo, Coralie mais aussi Nadine, Nathalie, Katia, Gaëlle et ceux qui sont partis Magali, Edwige, Ying, Julie, Sarah, Carole .... 4 3 Je remercie tous les membres de ma famille : mon fils pour sa patience d’attendre sa maman le soir qui venait le chercher en retard chez sa nounou, mon mari de m’avoir toujours soutenu moralement et remotivé au quotidien, mon papa qui a accepté mes choix de carrière et de partir à 600 Km de la maison, ma petite sœur qui a dû apprendre à vivre sans moi et ma belle-maman, mon beau-frère, mes belles sœurs qui m’ont toujours soutenue avec fierté pour l’aboutissement de mes études. 4 5 - SOMMAIRE - ABBREVATIONS..................................................................................................................................8 INTRODUCTION GENERALE.........................................................................................................12 OBJECTIFS DE LA THESE ..............................................................................................................12 I. NACA (ALPHA CHAIN OF NASCENT POLYPEPTIDE ASSOCIATED COMPLEX).........13 1. DESCRIPTION ..............................................................................................................................................13 a. Gène NACA et ses isoformes.................................................................................................................13 b. Structure de la protéine NACA ..............................................................................................................14 2. LES FONCTIONS DE NACA..........................................................................................................................16 a. NACA dans le cytosol............................................................................................................................16 NACA dans l’adressage des polypeptides naissants............................................................................................16 NACA régulateur négatif de l’apoptose ..............................................................................................................17 NACA interacteur des taxilines...........................................................................................................................20 b. NACA dans le noyau .............................................................................................................................20 NACA co-facteur de transcription.......................................................................................................................20 c. NACA régule la prolifération et la différenciation des cellules T CD8+...............................................22 d. NACA impliquée dans l’hématopoïèse..................................................................................................23 3. REFLEXIONS SUR LA PROTEINE NACA .......................................................................................................23 II. L’ERYTHROPOIESE...................................................................................................................25 1. DEFINITION ET LIGNAGES ............................................................................................................................25 2. MECANISMES MOLECULAIRES ASSOCIES A L ’ERYTHROPOÏESE ....................................................................26 a. Présentation des différents acteurs moléculaires au cours de la différenciation.....................................26 b. Les voies de prolifération et différenciation...........................................................................................28 La voie SCF/KIT.................................................................................................................................................28 La voie EPO/EPO-R............................................................................................................................................29 e. L’apoptose lors de l’érythropoïèse.........................................................................................................36 III. MES TRAVAUX DE RECHERCHE ..........................................................................................37 ARTICLE 1 : NACA IS A POSITIVE REGULATOR OF HUMAN ERYTHROID -CELL DIFFERENTIATION ........................38 Introduction....................................................................................................................................................39 Article scientifique.........................................................................................................................................40 Commentaires ................................................................................................................................................52 ARTICLE 2 : ERGIC-53 A NOVEL INTERACTOR OF NACA, PARTICIPATES TO EPO-R TRAFFICKING ..................54 Introduction....................................................................................................................................................55 Présentation de la technique de Tandem Affinity Purification ............................................................................55 Le transport et la maturation des protéines..........................................................................................................58 Article scientifique.........................................................................................................................................65 6 Résultats supplémentaires ..............................................................................................................................95 Mesure d’EPO-R à la surface cellulaire de cellules stimulées en EPO................................................................95 Détermination de l’interaction entre EPO-R/ERGIC-53 et NACA .....................................................................96 Commentaires ................................................................................................................................................98 ARTICLE 3 : NACA RESCUE ERYTHROID DIFFERENTIATION OF EARLY STAGE MYELODYSPLASTIC SYNDROME - DERIVING PRECURSORS AND PREDICT RESPONSE TO EPO TREATMENT . ............................................................100 Introduction..................................................................................................................................................101 Définition
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