Unveiling Ubiquitination as a New Regulatory Mechanism of the ESCRT-III Protein CHMP1B Xènia Crespo-Yañez To cite this version: Xènia Crespo-Yañez. Unveiling Ubiquitination as a New Regulatory Mechanism of the ESCRT-III Pro- tein CHMP1B. Cellular Biology. Université Grenoble Alpes, 2017. English. NNT : 2017GREAV035. tel-01684692 HAL Id: tel-01684692 https://tel.archives-ouvertes.fr/tel-01684692 Submitted on 15 Jan 2018 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 LA COMMUNAUTE UNIVERSITE GRENOBLE ALPES Spécialité : Biologie Cellulaire Arrêté ministériel : 25 mai 2016 Présentée par Xènia CRESPO-YAÑEZ Thèse dirigée par Marie-Odile FAUVARQUE préparée au sein du Laboratoire Biologie à Grande Echelle, Institut de Biosciences et Biotechnologies de Grenoble, CEA Grenoble dans l'École Doctorale de Chimie et Sciences du Vivant Découverte de l'ubiquitination en tant que nouveau mécanisme de régulation de la protéine ESCRT-III CHMP1B Thèse soutenue publiquement le 13 octobre 2017 devant le jury composé de : Mme Christel BROU Directrice de recherche, Institut Pasteur, Examinatrice M. Pierre COSSON Professeur, Université de Genève, Rapporteur M. Jean-René HUYNH Directeur de recherche, Collège de France, Rapporteur Mme Marie-Odile FAUVARQUE Directrice de recherche, CEA, Directrice de thèse M. Winfried WEISSENHORN Professeur, Université Grenoble Alpes, Président et Examinateur Acknowledgments The present work was carried out at the CEA of Grenoble in the laboratory of Large Scale Biology into the BIG Institute directed by Jérôme Garin. I thank the Université Grenoble Alpes for funding my first three years of PhD and giving me the opportunity to teach at the Bachelor and Master levels, and the Fondation pour la Recherche Médicale for funding me for one extra year. First of all, I want to thank the members of my jury for devoting part of their time to the evaluation of my work and the thesis defense. Thanks to Pr. Pierre Cosson and Dr Jean- René Huynh for agreeing to be reporters of my thesis work. Thanks also to Dr Christel Brou and Pr. Winfried Weissenhorn for agreeing to examine my thesis work. I would like to start by thanking all the member of the Gen&Chem team with whom I have share this 4 years. Specially Marie-Odile, my director. First of all, thank you for always believe and trust me. Your faith gave me strength and confidence and sense of duty when I needed it the most. Thank you for your guidance, for your enthusiasm, for our discussion and for introducing me our little flying friends. Thank you for making me feel not so alone at the beginning. And over-all thank you for giving me the opportunity of living this amazing four years within your team. Thanks to Carmen for choosing to work with me in this project, teach me about structural biology and being brave enough to take in charge the enzymatic and interaction part. Thank you very much for our discussions, our confidences and laughs. For sharing with me the moments of morriña for Barcelona and the Mediterranean and our culture. Moltes gràcies Carmen de tot cor. Dominique, in Spanish we will call you “La terremoto”, you are simply the spark of the team. Thank you for your patience with my terrible French accent and my mood, for your always good advices, support and reprimands, for your joy of life and your energy. I wish you the best in your next adventure!! Emmanuelle I thank you for being so motivated during the study of the interaction. It was a tuff part and you and Carmen made the perfect team. But overall I thank you for your complicity and support, for always be there to help me if I needed it and for all the good moments that we have shared inside and outside the lab. Emmanuel I thank you for our discussions, for your help with the fly genetics, for your always very good questions in the meetings or presentations and for being always so calm and so diplomatic no matter how many women are screaming around you!! I have to thank my first partner, Alice, who took the patience to teach me to work with cell cultures and with who I shared my first year “elbow by elbow”. Thank you for your kindness, your permanent smile and your patience. I thank Gilles Courtois for my first year here under his supervision. Thank you for our discussions and for never forget that Barça is the best football team in the word!! I Perrine I thank you for the time we shared. You were the smile of every lunch and coffee break and I have missed your joy and sweetness in the lab. Anne-Claire I thank you also for the first months of my thesis that we spend together, for having the great idea of launching this project and collecting the initial material, for all your advices about how to do biochemistry, for your motivation towards science and for having done such a clear and clean lab books! I also have to thanks Laurence Aubry for very useful and constructive discussions about endocytosis, a new and complex field for me and other members of my thesis committee, Pr. Rémi Sadoul and Pr. Winfried Weissenhorn for their powerful advices. And of course, I do thank all the other members of the Gen & Chem team with who I have shared these years for being such a wonderful team: Magda, Caro, Cathy, Agnes, Ilham, Amandine, Anna and to my best squash partner and super effective lab secretary Julie. I won’t forget Nolwenn Miguet who kindly welcomed me at IBS. I would also like to thanks Irma Thesleff, group leader at the Institute of Biotechnology in Helsinki, who gave me the first opportunity to work in academic research. In Helsinki I felt as a biologist for the first time in my life. Those two years have opened many doors for me. I also thanks all the team of Emili Salò, especially Maria Almuedo, for the 6 months I spent in their lab in Barcelona learning about the curious Planarian model system and that warmness, companionship, friendship and fun is not in conflict with good scientific production. And I thank all the friends that I have meet in Grenoble. They are my friends but also my second family. Always there. I will never forget all that we have shared together. The good moments, and the hard ones. I already miss you all so much: Vanesa, Serena, Valeria, Angel, Martino, Mateo, Leonardo, Vic, Robert, Nathalie, Isabel, Jaione, Tomas, Eugenia, Maddi, Jorge, Kike and specially Raquel with who I have shared the last months, both writing the thesis and both taking care of each other’s mental health ;) It will never be the same, but I hope that we can keep laughing about it for many many years wherever we are. I thank the city of Grenoble and the French Alps. This corner of the world has taken and expanded my heart and strengthened my body. And I finish by thanking my family. Especially my Mum, she is my strength, my peace, my confident and counselor, my soul mate. Mamá, without your hard work, at the job and at my education, and without your trust, I would have never get to arrive here. Gracias familia porque a pesar de los años y la distancia siempre siento cerca vuestra presencia, vuestro apoyo, vuestra confianza y vuestro amor. Y gracias por dejar de llamarme la loca de las moscas! Os amo. II Synopsis en français J’ai effectué ma thèse dans le groupe du Dr. Marie-Odile Fauvarque qui met en œuvre des stratégies de génétique moléculaire sur des modèles de cellules humaines en culture et chez la mouche drosophile pour l'identification et l'étude de la fonction des protéines dans la signalisation intracellulaire. Dans ce contexte, mes travaux visaient à produire des connaissances fondamentales sur le système ubiquitine dans le contrôle du trafic endocytaire, en particulier de récepteurs membranaires impliqués dans la réponse inflammatoire (TNFR, ILR) ou la différenciation et la croissance cellulaire (EGFR). Je me suis notamment intéressée au rôle du complexe formé par l’interaction entre une protéine de la voie endocytaire, CHMP1B, et la protéase d’ubiquitine UBPY (synonyme USP8). CHMP1B est un membre de la famille ESCRT-III qui, via des processus de changements de conformation et de polymérisation à la membrane, contrôle la biogenèse des vésicules intraluménales (ILVs) au niveau des endosomes tardifs pour former les corps multivésiculaires (MVBs). Ces derniers fusionnent avec les lysosomes, assurant ainsi la protéolyse des récepteurs internalisés et l‘arrêt de la signalisation intracellulaire. Alternativement, les récepteurs peuvent être renvoyés à la membrane plasmique à partir des endosomes précoces ou tardifs via des vésicules de recyclage. Le trafic intracellulaire et le tri des récepteurs dans ces différents compartiments subcellulaires jouent un rôle majeur dans l’activation, la durée et la terminaison des signaux intracellulaires. Or, la liaison covalente d’une ou plusieurs ubiquitine (un polypeptide très conservé de 76 aminoacides) au niveau des récepteurs est un signal majeur déclenchant leur internalisation. En hydrolysant cette ubiquitine, UBPY peut stopper l’internalisation des récepteurs au niveau de la membrane plasmique, ou bien, favoriser leur entrée dans le MVB.