Role of alphaOGG1 in the Maintenance of Mitochondrial Physiology Debora Lia To cite this version: Debora Lia. Role of alphaOGG1 in the Maintenance of Mitochondrial Physiology. Cellular Biology. Université Paris Saclay (COmUE), 2018. English. NNT : 2018SACLS125. tel-01820633 HAL Id: tel-01820633 https://tel.archives-ouvertes.fr/tel-01820633 Submitted on 22 Jun 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. ROLE OF αOGG1 IN THE MAINTENANCE OF MITOCHONDRIAL PHYSIOLOGY Thèse de doctorat de l’Université Paris-Saclay préparée à NNT : 2018SACLS125 : NNT l’Université de Paris-Sud Ecole doctorale n 577 Structure Et Dynamique Des Systemes Vivants (ED-SDSV) Spécialité de doctorat : BIOLOGIE Thèse présentée et soutenue à Fontenay aux Roses, le 16/05/2018, par DEBORA LIA Composition du Jury: Filippo Rosselli President Directeur de Recherche, Institut Gustave Roussy (FANC/BRCA signalling pathway and Cancers) Guy Lenaers Rapporteur Directeur de Recherche, Université d’Angers (PREMMi) Miria Ricchetti Rapporteur Group Leader, Institut Pasteur (Stem Cells and Development) Anne Lombes Examinateur Group Leader, Institut Cochin (Mitochondrie, Bioénergétique, Métabolisme Et Signalisation) Anna Campalans Directeur de thèse Chercheur, CEA (LRIG) “Non è l’assenza di difetti che conta, ma la passione, la generosità, la comprensione e simpatia del prossimo, e l’accettazione di noi stessi con i nostri errori, le nostre debolezze, le nostre tare e virtù.” – Rita Levi-Montalcini "Success consists of going from failure to failure without loss of enthusiasm." - Winston Churchill A mia Madre, il mio sorriso, e a mio Padre, il mio eroe. - 3 - ACKNOWLEDGEMENTS I would never have been able to finish my dissertation without the guidance of my thesis director, the help from friends, and the support from my family… so the acknowledgments will be a bit in English, a bit in French…and a bit in Italian of course so that everyone could recognize himself in the best way. First and foremost, I wish to thank my thesis director, Dr Anna Campalans. Annina, you have always been supportive since the first days I began working on our amazing project; Thank you for having helped me to become the person I am today, both professionally and personally. I know very well that I would never have arrived here without your help. You made me grow step by step, every day. Above all, you gave me the enthusiasm and the motivation to wake up and be happy with my work. And I think this is the greater gift a Teacher can give to a student. I feel privileged for the possibility I had to work with you. Thanks also for all the times you got angry or desperate with me, I needed that moments, especially that moments. Thank you for let me discovered the magic world of mitochondria. This thesis is the result of a beautiful journey we did together all these 3 years. You are a bit like family for me and I just want you to know that I own you a lot, and I will always do... Thanks to Dr Pablo Radicella, for having allowed me to join your group in the first place. Thank you for all the good advices both professional and not, thank you for your kindness. For the Argentine barbecue. Thanks also to your wife, Maria. My thesis committee guided me through all these 3 years. So, many thanks to Dr Anne Lombes, Dr Erika Brunet, Dr Karl Mann and especially to Dr Miria Ricchetti. Thanks for being part of my thesis committee, for carefully evaluating my work and for your helpful comments. All of you have been an inspiration in many ways. Many thanks also to all the members of my thesis committee. Je remercie tous les membres et ex-membres de l’équipe LRIG pour le climat sympathique dans lequel ils m’ont permis de travailler. Les nombreuses discussions que j’ai pu avoir avec chacun des vous m’ont beaucoup apporté. Vous étes des gens spéciaux que j’ai eu l’honneur de connaître. Surtout merci à Dr Anne Bravard, Dr Jacquiline Bernardino-Sgherri, Dr Stefanie Marsin et Dr Anne Marie de Guilmi pour notre soirée entre filles…Merci Emilie pour ton bonheur. I would like to thank Prashant who, as a good friend, was always willing to help and give his best suggestions…and also some hugs in my worst moments. It would have been a lonely lab without you. Many thanks to Dr Jan Baijer for having taught me everything I know about cell sorting and FACs analysis. Thank you for having followed me in my crazy ideas. Lamya, merci pour m’avoir si facilement suivie dans ma folie la plus totale, pour m’avoir accordé ta confiance et pour tous les beaux moments partagés ensemble. - 5 - Grazie Mary (la mia Abib). Insieme abbiamo imparato moltissimo sulla natura umana e su noi stesse in questi ultimi mesi. Mi hai raccolta che ero incasinata, mi hai dovuta cazziare, mi hai dovuto sopportare ma non sei scappata via. Grazie per esserci amica mia. Grazie Eleonora (il mio Orgoglio). Non sono sicura che io sarei stata in grado di iniziare questo dottorato se non ti avessi incontrata. Grazie per avermi iniziato ai vini alsaziani ed ai cetrioli. Grazie per le sessioni di psicoterapia. Con te Parigi è diventata veramente casa mia… alla fine l’Universo è stato ed è davvero positivo. Grazie Paola (il nostro Unicorno). Grazie per esserci anche se a distanza. Grazie Elisa (la mia Topa). Le parole non mi bastano…Grazie per avermi fatto ritrovare il senno tutte le volte che lo perdevo. Grazie per il tuo calore, grazie per le nostre risate. Grazie per avermi accolta. Grazie a Stefano, Gladys e Giacomo. Thank you Haser, my dear friend, I know I’ve got a friend in you and I hope you know that you will never find my door close. Grazie Parigi per tutti i tuoi Serbi, i tuoi Shannon, i tuoi Tournesol e le tue L’adada. Grazie ai nuovi amici (Salvatore (Babe), todos los amigos de el BoBo, Ghazi & Dasha, Elma, Ivana, Stafania (la mia Donnaccia)...) che me li hanno fatti scoprire insieme ad una città che mi ha accolta. Grazie Martina (la bella Pazza) sono felice di averti incontrata. Grazie Parigi, perché tra le tue rues non mi perdo. Grazie agli amici di sempre, il mio porto sicuro: Emanuele, Nicola, Marco e Carola. Grazie per tutte le serate Scottish degli ultimi 12 anni. Grazie Ema (Cuore) per tutte le volte che mi hai detto: “Forza che me devi di’ mo?”. Grazie Nicola (Nata) per tutti i tuoi consigli saggi che io non seguo mai. Ed in fine ma non per importanza, vorrei ringraziare la mia famiglia. La mia incredibile, unica famiglia. Durante i miei tre anni di dottorato il vostro appoggio è stato incondizionato e non avete idea di quanto mi sia stato necessario. Grazie Nonna per la scheda telefonica fatta apposta per chiamare la nipote lontana. Grazie Zia Roberta e Zia Rossana per la vostra missione di salvataggio. Zia Rita per i vasetti di sottoli. Grazie alle mie sorelle/cugine per le pizze “all’andata e al ritorno”. Grazie Mamma e grazie Papà. Voi siete la mia forza. Ho talmente tanti motivi per ringraziarvi che faccio fatica a dirne anche uno solo…grazie per tutte le volte che vi ho visti in prima fila ad aspettarmi all’aeroporto tornando a casa, per i pacchi stile tetris da giù, per avermi tollerata nei miei momenti critici, con i miei bronci e con i miei pianti isterici pieni di parolacce. Grazie per le chiamate su Skype ed i nostri saluti pieni di smorfie, risate, boccacce…grazie per le foto di Ginetta, per le lenzuola sempre pulite in camera mia. Grazie per le melanzane alla parmigiana. Grazie per tutte le piccole grandi attenzioni. Grazie per il Vostro Amore. - 7 - ACKNOWLEDGEMENTS 5 INDEX 9 RESUME 11 ABBREVIATIONS 21 Chapter I. INTRODUCTION 23 I Structure and functions of mitochondria 25 a Mitochondria, an overview 25 b The electron transport chain and mtROS production 26 c Structure of mtDNA nucleoids and their replication and transcription 29 d Impact of mtDNA alterations on medicine 36 II Quality controls of mitochondria 39 a ROS scavenging 39 b Mitochondrial Unfolded Proteins Response 40 c The maintenance of a healthy mitochondrial population: the fission/fusion 42 mechanisms i Mitochondrial inner membrane topology 45 d mtDNA maintenance: how to repair or tolerate DNA damage in 46 mammalian mitochondria i Mismatch repair pathway (MMR) 49 ii Homologous recombination and non-homologous end-joining 50 iii Translesion synthesis 50 iv mtDNA degradation 52 e Mitophagy programs 53 f The final response: apoptosis 54 Concluding remarks on mitochondrial dynamics, mitophagy and 57 apoptosis III Base excision repair in mitochondria 61 a Modified base excision and strand incision 64 b Gap filling by DNA polymerase and strand sealing by DNA ligase 66 IV The 8-oxoguanine lesion 67 a G:C to T:A transversion and the GO repair system 68 b The DNA glycosylase OGG1 protein 70 c The absence of OGG1 activity in the cell… what do we know so far? 74 Chapter II. PURPOSE OF THE WORK 79 Purpose of the work 81 Chapter III. RESULTS 83 Results – Section 1 85 a Characterization of the mitochondrial localization of αOGG1 85 b Role of the localization sequences at the N and C ter of αOGG1 in the sub- 89 cellular distribution of the enzyme Results – Section 2 95 a Evaluation of the mitochondrial physiology in OGG1 deficient cells 95 b Mitochondrial dysfunction of OGG1 deficient cells can be complemented 98 by expressing a functional αOGG1 in mitochondria c Influence of oxidative stress on mitochondrial localization of αOGG1 104 d Menadione induces the accumulation of 8oxoG in mtDNA 106 Preliminary results 110 - 9 - a Quantification of mtDNA and mtRNA 110 b Evaluation of apoptosis in U2OS cells in the absence of OGG1 113 Chapter V.