Interactions and Functions of the Ubiquitin Specific Protease 7 in Human Cells by Anna-Marie Georges A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Molecular Genetics University of Toronto © Copyright by Anna-Marie Georges 2019 i Interactions and Functions of the Ubiquitin Specific Protease 7 in Human Cells Anna-Marie Georges Doctor of Philosophy Molecular Genetics University of Toronto 2019 ABSTRACT The ubiquitin-specific protease 7 (USP7) is a deubiquitylating enzyme that regulates many important cellular processes and is highly studied for its contributions to cancer. Our lab has previously discovered a binding pocket in the USP7 N-terminal TRAF domain that mediates interactions with target proteins. In this thesis I demonstrate how I contributed to the identification of a second binding pocket located in the Ubl2 ubiquitin-like structure of the USP7 C-terminal domain. Furthermore, to gain a more comprehensive understanding of the interactions and functions of USP7, I used a proteomics approach to profile USP7 interactions in cancer cells. This confirmed reported associations of USP7 with USP11, PPM1G phosphatase and TRIP12 and identified novel interactions with FBXO38 and two DEAD/DEAH-box RNA helicases, DDX24 and DHX40. I show that USP11, PPM1G, DDX24, TRIP12 and FBXO38 bind USP7 through its TRAF binding pocket, while DHX40 interacts through the Ubl2 pocket. Motifs in USP11 and DDX24 that are critical for USP7 binding were also identified. Modulation of USP7 expression levels and inhibition of USP7 catalytic activity showed that USP7 consistently stabilizes DDX24, DHX40, TRIP12 and FBXO38 dependent on its catalytic activity, while USP11 and PPM1G levels were not consistently affected. Together these results better define the mechanisms of USP7 interactions and identify FBXO38, DDX24, and DHX40 as new USP7 ii targets. Furthermore, a BioID approach was used to profile the interactions and putative functions of FBXO38, revealing an interaction with KIF20B, a Kinesin-6 protein required for cytokinesis. I show that depletion of either FBXO38 or USP7 result in dramatic decreases in total KIF20B levels and its localization to the midbody, which were manifested in cytokinetic defects. Furthermore, cytokinetic defects associated with USP7 silencing were rescued by restoring FBXO38 or KIF20B. Therefore, I have identified novel roles for USP7 and FBXO38 in the regulation of cytokinesis. iii ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest gratitude to my advisor Dr. Lori Frappier, for giving me the opportunity to work in such an incredible and supportive lab. You have been the most compassionate, patient, encouraging, approachable and supportive supervisor any grad student could ask for. I will always remember our fun conference trips (especially in Croatia and Montenegro!) and our mutual love of dancing at the MoGen retreats and Christmas parties! Through your mentorship, I have become a better writer, critical thinker and project manager, which will no doubt be instrumental in the success of my future scientific career. Thank you to my committee member Drs Anne-Claude Gingras and Sachdev Sidhu for their encouragement and support and for providing me with invaluable insight throughout my graduate career. Thank you for constantly challenging and motivating me to become a better researcher and scientist. I would also like to thank Dr. Vivian Saridakis for her collaboration and guidance for some of the more biochemical aspects of this project. I want to extend a huge thank you to the Frappier lab family, both past and present. A special shout out to my lab bestie and lunch buddy, Kathy Shire. Thank you for all your technical help with experiments (especially cloning!) and patiently listening to all my frustrations and failures, and sharing my excitement during the successes! I will miss our extensive discussions on Doctor Who (and the many other TV shows we shared in common), and I will especially miss your cakes, even the vegan ones. Thank you to Dr. Natasha Malik for being such an amazing mentor for my first few years, and helping with getting my project started. Jaime, Umama, Carlos, Ashley and Sam, I am so grateful to have met you all and I wish you all the best in your future research endeavors. I would like give a special thanks to my MoGen crew, who were a huge support during my entire graduate career and have become my life-long friends. I feel incredibly lucky to have met and bonded so strongly with such an amazing group of people. I love you all and look forward to the many more memories to come as we progress through the next chapter of our lives! A very special thank you to my incredibly supportive parents, who constantly remind me of how proud they are for my pursuit of graduate school and a career in science. Thank you for your unconditional love and support and for shaping me into the incredibly hard working and disciplined person I am today. iv Finally, this thesis is dedicated to my best friend and life partner, Jonathan, who has been my biggest supporter and cheerleader during my entire graduate career and all other aspects of my life. Thank you for your undying love, emotional support, patience and constantly motivating me to grow professionally and to keep my head held high during the many rough patches of grad school. I couldn’t have done this without you! v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iv TABLE OF CONTENTS ............................................................................................................ vi LIST OF FIGURES ..................................................................................................................... ix LIST OF TABLES ........................................................................................................................ x LIST OF ABBREVIATIONS ..................................................................................................... xi CHAPTER 1 Introduction ........................................................................................................... 1 1.1 THE UBIQUITIN SYSTEM .............................................................................................. 2 1.1.1 Ubiquitin and Ubiquitin conjugation ...................................................................... 2 1.1.2 Polyubiquitylation ................................................................................................... 2 1.1.3 Monoubiquitylation ................................................................................................. 4 1.1.4 E3 ligases ................................................................................................................ 5 1.1.5 Deubiquitylating enzymes ...................................................................................... 7 1.2 UBIQUITIN-SPECIFIC PROTEASE 7 ............................................................................. 9 1.2.1 Deubiquitylation by USP7 .................................................................................... 10 1.2.2 USP7 protein interacting domains and mechanisms of USP7 interactions .......... 12 1.2.3 Roles of USP7 in tumor suppressor regulation ..................................................... 14 1.2.3.1 USP7 plays a key role in regulating the p53 pathway ........................... 14 1.2.3.2 Additional roles of USP7 in tumor suppressor and oncoprotein regulation ............................................................................................... 15 1.2.4 Additional Cellular Functions of USP7 ................................................................ 17 1.2.4.1 Roles of USP7 in cell cycle regulation ................................................... 17 1.2.4.2 USP7 in DNA replication, chromatin remodeling and transcription ..... 18 1.2.4.3 Functions of USP7 in DNA repair .......................................................... 19 1.2.5 The development of USP7 inhibitors for anti-cancer therapies ............................ 20 1.3 CYTOKINESIS ................................................................................................................ 21 1.3.1 The role of the kinesin family member 20B in cytokinesis and abscission .......... 23 1.4 THESIS OUTLINE AND RATIONALE ......................................................................... 24 CHAPTER 2 Identification and characterization of USP7 targets in cancer cells .............. 25 2.1 INTRODUCTION ............................................................................................................ 26 2.2 MATERIALS AND METHODS ...................................................................................... 28 vi 2.2.1 Cell Lines .............................................................................................................. 28 2.2.2 Plasmids and siRNA ............................................................................................. 28 2.2.3 Affinity Purification coupled to Mass Spectrometry (AP-MS) ............................ 29 2.2.4 Transfections and USP7 inhibitor treatment ......................................................... 30 2.2.5 Western blotting .................................................................................................... 31 2.2.6