DEFINING THE INTERACTOME AND FUNCTIONAL REGULATION OF THE O-GLCNACASE ENZYME DURING OXIDATIVE STRESS by Jennifer A. Groves A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland October, 2017 Abstract The essential post-translational modification O-linked β-N-acetylglucosamine (O- GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. O- GlcNAc is dynamically added and removed from proteins by the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), respectively. Dysregulation of O-GlcNAc-cycling is implicated in the etiology of numerous diseases, including cancer, neurodegeneration, and metabolic dysfunction. Underpinning these observations, O-GlcNAc regulates nearly every major cellular process including transcription, translation, protein degradation, protein localization, and the cell cycle. Furthermore, many forms of cellular stress and injury, including oxidative stress, elicit an increase in O-GlcNAcylation on numerous proteins, which has been demonstrated to promote cell survival. However, the mechanisms by which OGT and OGA are regulated during oxidative stress to alter O-GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevated O- GlcNAc levels in U2OS cells but has little impact on the activity of OGT. In contrast, the expression and activity of OGA are enhanced. We hypothesized that this seeming paradox could be explained by proteins that bind to and control the local activity or substrate targeting of OGA, thereby resulting in the observed stress-induced elevations of O- GlcNAc. To identify potential protein partners and regulators, we utilized BioID proximity biotinylation in combination with Stable Isotopic Labeling of Amino Acids in Cell Culture (SILAC). This analysis revealed 90 OGA-interacting partners, many of which exhibited increased binding to OGA upon stress. The associations of OGA with fatty acid synthase (FAS), filamin-A, heat shock cognate 70 kDa protein, and OGT were confirmed by co- immunoprecipitation. The pool of OGA bound to FAS demonstrated a substantial (∼85%) ii reduction in specific activity, suggesting that FAS inhibits OGA. Consistent with this observation, FAS overexpression augmented stress-induced O-GlcNAcylation on a subset of proteins. Although the mechanism by which FAS sequesters OGA remains unknown, these data suggest that FAS fine-tunes the cell's response to stress and injury by remodeling cellular O-GlcNAcylation. Finally, our studies characterized the use of two commercially available and three non-commercially available antibodies for detecting and enriching full- length OGA from lysates of mouse and human origin in order to facilitate future studies probing the regulation of OGA in disease. Dissertation Advisor: Natasha E. Zachara, Ph.D. Dissertation Reader: Steven M. Claypool, Ph.D. iii Preface My goals for graduate school were to strengthen my knowledge of biological life science and medical research at a molecular level, learn a broad range of techniques and skills that would apply to numerous career paths, and to develop meaningful friendships and a long-lasting professional network. My success in each of these areas is a direct result of the help and support of a number of individuals. First and foremost, I am incredibly grateful that Dr. Natasha E. Zachara gave me the opportunity to train in her laboratory. Natasha fostered my development into a confident and independent scientist, never wavered on her support of my non-academic career goals, and went above and beyond to help in times of personal need. I would also like to thank the members of my thesis committee, Dr.’s Ronald Schnaar, Robert Cole, Katherine Wilson, and Steven Claypool, for their insight, suggestions, and support of my professional goals. Furthermore, I would like to thank my formal and informal collaborators, Dr.’s Jennifer Van Eyk, Akhilesh Pandey, Alfredo Quiñones-Hinojosa, Partha Banerjee, Gerald Hart, Michael Wolfgang, and Sagar Shah, as well as Robert O’Meally, Caitlyn Bowman, and Chris Saeui, for their contributions to this work and my professional development. This work was supported by grants from the National Institutes of Health, including a pre-doctoral National Research Service Award from the National Institute on Aging. I would also like to acknowledge all past and current members of the Zachara laboratory, especially Dr.’s Albert Lee, Kamau Fahie, Marissa Martinez, Devin Miller, Thiago Dias, and Russell Reeves, as well as Gokben Yildirir, Cathrine McKen, Roger Henry, Peter Natov, Srona Sengupa, and Austin Maduka. These individuals have become iv part of my family, providing scientific and technical guidance as well as friendship and laughter during the hardships of graduate school. None of this work could have been successful without the efforts of the Biological Chemistry Department Graduate Program and Administrative Office, especially Darlene Sutton, Danelle Daniels, and Natalie Peters. Finally, I would like to thank my family for their support of my career that took me thousands of miles across the country, and for their faith that I will reach any goal I set my mind to. v Table of Contents ABSTRACT ................................................................................................................................................................. II PREFACE .................................................................................................................................................................. IV TABLE OF CONTENTS .......................................................................................................................................... VI LIST OF TABLES .................................................................................................................................................. VIII LIST OF FIGURES ................................................................................................................................................... IX ABBREVIATIONS ..................................................................................................................................................... X AMINO ACID CODES ........................................................................................................................................... XIII CHAPTER 1: : DYNAMIC O-GLCNACYLATION AND ITS ROLES IN THE CELLULAR STRESS RESPONSE AND HOMEOSTASIS .......................................................................................................................... 1 SUMMARY ...................................................................................................................................................................................... 2 INTRODUCTION ............................................................................................................................................................................. 3 BIOSYNTHESIS OF O-GLCNAC .................................................................................................................................................... 5 The hexosamine biosynthetic pathway ......................................................................................................................... 6 O-GlcNAc transferase ............................................................................................................................................................ 7 O-GlcNAcase ............................................................................................................................................................................ 10 O-GLCNAC AND THE CELLULAR STRESS RESPONSE .............................................................................................................. 11 The PI3K/Akt pathway ....................................................................................................................................................... 14 Heat shock protein expression ........................................................................................................................................ 15 Calcium homeostasis ........................................................................................................................................................... 18 Reactive oxygen species ..................................................................................................................................................... 19 Mitochondrial dynamics .................................................................................................................................................... 20 Inflammation .......................................................................................................................................................................... 22 INTERPLAY BETWEEN O-GLCNAC AND OTHER PTMS ........................................................................................................ 23 Phosphorylation .................................................................................................................................................................... 23 Other PTM roles ..................................................................................................................................................................... 26 MOLECULAR MECHANISMS OF DISEASE ..................................................................................................................................