DECIPHERING THE CROSSTALK: CHARACTERIZATION OF THE REGULATION OF THE ER STRESS RESPONSE BY THE DNA REPAIR ENZYME AAG Clara Forrer Charlier Faculty of Health and Medical Sciences Department of Biochemistry and Physiology This thesis is submitted for the degree of Doctor of Philosophy June 2018 DECIPHERING THE CROSSTALK: CHARACTERIZATION OF THE REGULATION OF THE ER STRESS RESPONSE BY THE DNA REPAIR ENZYME AAG- Clara Forrer Charlier – June 2018 SUMMARY The genome is a very dynamic store of genetic information and constantly threatened by endogenous and exogenous damaging agents. To maintain fidelity of the information stored, several robust and overlapping repair pathways, such as the Base Excision Repair (BER) pathway, have evolved. The main BER glycosylase responsible for repairing alkylation DNA damage is the alkyladenine DNA glycosylase (AAG). Repair initiated by AAG can lead to accumulation of cytotoxic intermediates. Here, we report the involvement of AAG in the elicitation of the unfolded protein response (UPR), a mechanism triggered to restore proteostasis in the cell whose dysfunction is implicated in diseases like diabetes, Alzheimer’s disease and cancer. Firstly, we determined that not only human ARPE-19 cells were capable of eliciting the UPR, but that an alkylating agent, methyl methanesulfonate (MMS), also triggers the response, and that in the absence of AAG the response is greatly diminished. Our luciferase reporter assay indicates that the response is activated on multiple branches (IRE1 and ATF6) on both AAG-proficient and deficient cells. Although no transcriptional induction of UPR markers was detected by RT-qPCR at 6 hours post MMS treatment, preliminary western-blot data at 6 and 24h, show activation of key UPR markers (p-eIF2α, BiP and XBP-1) upon MMS treatment in wild-type cells and little or no activation on AAG -/-. To investigate the impact of AAG modulation on the cellular proteome we also conducted a proteomic analysis, identifying 5480 protein groups in wild-type ARPE-19, 5377 in the AAG -/- A2C2 and 5264 in AAG -/- B6C3. After a high-stringency analysis, we identified 13 proteins present only in wild-type cells, indicating promising targets for further investigation into the role of AAG in the UPR. We also identified 44 overrepresented GO-slim terms across all cell lines and 23 overrepresented pathways, mostly related to cellular metabolism processes. Whereas more experiments are required to characterize the nature of AAG’s contribution to the UPR, we demonstrate the existence of crosstalk between the DNA repair response and the ER stress response pathways, that is potentially relevant in a clinical setting. ii ACKNOWLEDGEMENTS There is always a little trepidation whenever one needs to write acknowledgements. It was such a long and intense journey, meeting so many wonderful people that is hard not to feel that you are missing someone important in this section. Firstly, I would like to thank my mother, without whom I wouldn’t be here, both literally and figuratively. Thanks for being such an incredibly wise person, thanks for your support, patience and love. Thanks for stimulating my scientific curiosity and for believing in me. You are my first and best friend, always. And thanks for the mitochondria! I like to thank Lisi, for also believing in me and letting me be a part of her fantastic group. You are not only a fantastic supervisor, but a fantastic human being as well. Every time I replied to somebody saying you were my supervisor I invariably heard back “she is lovely!” or “you are very lucky!”. I know I am. You rock! You are funny, supportive and the owner of a very sharp mind. I have grown as much as a person as a scientist after I met you. Don’t ever change! Have I mentioned you are also a great cook and have the most contagious laughter I ever heard? Thanks for having me! Thanks to Bernadette Moore for all the pep talk, writing tips and for introducing me to the scary world of proteomics. Although my first brush with it was a bit rocky, I am always keen to learn something completely new. Thanks for constantly worrying about my sanity as well! Thanks to Axel Nohturfft for helping (a lot!) with the proteomic data analysis, the plasmids, the cells and for being the husband of the best supervisor ever. iii Thanks to Dr Ruan Elliott and Eleanor Healing for developing the AAG activity assay and applying the AAG activity assay on the bazillions of overexpressors, wild-types and knockout clones I had. Thanks to Dr Paul Skipp and Dr Erika Parkinson for having me on the Proteomics Lab at the University of Southampton. Thanks for enduring all my silly questions with patience and grace (i.e. not laughing at them) and for letting me use the sexiest and most expensive piece of equipment I ever touched. Thanks to Dr J. A. Pêgas Henriques, for the all the support back in Brazil (bureaucratic and otherwise) and for putting me in touch with Lisi. Thanks to Dr Izabel Vilella for encouraging me to embark in the adventure that was my PhD here in England. It wouldn’t have happened if not for you. Thanks for all the fantastic people I met on this journey, specially my colleagues from 04AY04: Larissa Milano, for the help on the lab, for listening to me moan since my Masters and for being the only other fellow coffee drinker in the office. And for helping me survive a whole week eating only salmon on all meals at Egmond aan Zee. Thanks Natalie Mayhead for all the fun, signing, booze, science, Christmases and nerdy stuff and for putting up with my weirdness and grumpiness. Thanks even for the occasional hug (ewww!). Thanks Abdullah Aljohani for being such a great colleague and for sharing my love for good food. Thanks for all the recipes and all the laughter we had together. Thank you and Fahad Alhumaydhi for the warm reception I got when I first arrived, wide-eyed and really scared. Go Team AAG! Thanks to our extended lab family: the lovely and smart Amy Barber (although she never gave me the pictures of our last lab Christmas party), the stylish Matshediso Zachariah (one day I’ll drop by to camp with you and see the lions you promised), the graceful Diana Bordin (now gazing at the northern lights) and grumpy Leoncikas Vytautas (former colleague and Singstar Addict). I am eternally iv happy for being part of your lives and I am going to miss you a lot. Maybe I’ll even cry a little (but just a little). Hidden somewhere, probably, so I don’t ruin my reputation. Love you all! Thanks for the CNPq/Science Without Borders for giving this priceless opportunity (and paying me on time). It was a childhood dream come true. Finally, thanks for all the people that helped make this happen in some way, being with technical advice, food, alcohol, laughter or for lending me an ear when I needed. Sorry if I forgot to mention you here! v CONTENTS 1 INTRODUCTION .................................................................................... 25 DNA DAMAGE AND REPAIR ....................................................................... 25 1.1.1 Lesions repaired by BER ............................................................ 27 1.1.2 Alkyladenine DNA Glycosylase/Methylpurine DNA Glycosylase (AAG/MPG) ............................................................ 41 ER STRESS AND UNFOLDED PROTEIN RESPONSE ............................................. 52 1.2.1 IRE1 ........................................................................................... 54 1.2.2 PRKR-Like Endoplasmic Reticulum Kinase - PERK ..................... 58 1.2.3 Activating Transcription Factor 6 .............................................. 63 1.2.4 Endoplasmic Reticulum-Associated Degradation - ERAD ......... 65 PROBLEM STATEMENT AND OBJECTIVES ........................................................ 69 2 EVALUATION OF THE PHENOTYPES RESULTING FROM AAG EXPRESSION IN DIFFERENT SUBCELLULAR COMPARTMENTS ................. 71 INTRODUCTION ........................................................................................ 71 AIM ....................................................................................................... 74 OBJECTIVES ............................................................................................. 74 MATERIALS AND METHODS ........................................................................ 74 2.4.1 Cloning strategies ...................................................................... 74 2.4.2 Cell culture ................................................................................. 78 2.4.3 Isogenic cell line generation ...................................................... 79 2.4.4 Cell line characterization ........................................................... 79 RESULTS ................................................................................................. 84 2.5.1 Generation and characterization of the AAG overexpressing isogenic cell lines ....................................................................... 84 2.5.2 AAG overexpression sensitizes cells to alkylating damage ....... 86 2.5.3 AAG knockdown seems to decrease cell fitness ........................ 87 DISCUSSION ............................................................................................ 89 3 AAG AND THE UPR: CELL LINE DEVELOPMENT AND CHARACTERIZATION ............................................................................. 92 vi INTRODUCTION ........................................................................................ 92 AIMS ....................................................................................................