Characterizing Sumoylation in the Malaria Parasite

CHARACTERIZING SUMOYLATION IN THE MALARIA PARASITE, PLASMODIUM FALCIPARUM by Katherine Hays Reiter A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July, 2015 © 2015 Katherine Reiter All Rights Reserved ABSTRACT Parasite resistance to first-line anti-malarial drugs has accelerated the need for new drugs with novel targets for effective malaria treatment. Many of the current anti- malarial drugs work in part by overwhelming the parasite stress response. In eukaryotes, the oxidative stress response machinery includes antioxidants, as well as SUMOylation, a post-translational modification that involves the covalent attachment of small ubiquitin- related modifiers (SUMOs) to proteins. In this thesis, we hypothesized that SUMOylation plays an essential role in parasite stress survival, and that parasite-specific SUMOylation inhibitors could prove effective in combination with current anti-malarial drugs. We demonstrated that SUMOylation levels peak during the asexual trophozoite stage and are modulated with stress treatment, suggesting SUMO plays a role during the parasite stress response. We further analyzed the susceptibility of the SUMO pathway for targeted inhibitor design through structural and biochemical characterization of the human and Plasmodium falciparum (Pf) SUMO conjugation enzymes. We demonstrated that human and P. falciparum have distinct SUMO E1-E2 enzyme interactions, resulting from divergent interfaces. To further assess the essential biological functions of SUMOylation in the parasite, we engineered parasite strains for the conditional degradation of SUMO conjugation machinery. These strains allow for the inducible and reversible control of E1 and E2 enzyme degradation, providing a tool to study the temporal functions of SUMO throughout the parasite life cycle. In its entirety, this thesis provides evidence that SUMOylation is involved in the parasite response to stress, and provides a biochemical and structural foundation for the development of parasite specific inhibitors of the SUMO pathway as a novel and synergistic therapeutic for malaria. ii Thesis Advisor: Dr. Michael J. Matunis Thesis Readers: Dr. Cynthia Wolberger, Dr. Sean Prigge, and Dr. Jürgen Bosch Alternate Thesis Readers: Dr. Valeria Culotta and Dr. Photini Sinnis iii ACKNOWLEDGEMENTS Obtaining a Ph.D. relies on a diverse team of individuals who provide essential support and encouragement throughout your graduate school years. First, I would like to thank my thesis advisor, Dr. Michael Matunis, for allowing me to work on a somewhat non-traditional project in his lab. Although the lab focused neither on x-ray crystallography nor parasitology, you encouraged me to tackle projects by embracing collaboration, a skill that will undoubtedly be useful in my future endeavors. While you may be unaware, there are also some ‘classic Mike’ words-of-wisdom phrases that get passed around the lab, and will stick in my mind. For example, ‘If you don’t have time to do it right the first time, then when will you have time to repeat it,’ is surely a motto that is worth remembering. You have also helped to develop and improve my presentation skills tremendously, and I will always remember to fill up the white space. It’s been fun working with you in the lab and ‘thanks for the opportunity!’ I would also like to thank the members of my thesis committee: Dr. Jürgen Bosch, Dr. Valeria Culotta, Dr. Sean Prigge, and Dr. Cynthia Wolberger for valuable advice and discussions that helped shape this project and improve my scientific thinking. Every graduate student will witness, perhaps several rounds, of turnover in the lab, but I am grateful to say that all of the current and past members of the Matunis lab have had a tremendous impact in shaping me into the graduate student I am today. Thank you to past ‘sisters’ for providing a comfortable family environment and current members for helpful discussions. I am also grateful to members of the Prigge, Sullivan, Sinnis and Jacobs-Lorena labs in the MMI department for training me in Plasmodium culturing, and demonstrating patience when I visited them regularly for questions. iv Navigating the often complex nature of a Ph.D. also requires a behind the scenes support staff. Thank you to Sharon Warner who was truly a mother to students – we could always count on you! Thank you Shannon, Brandon, and the administration staff for keeping this department running smoothly; and thanks to Karen Griffen, who not only kept our labware fully stocked, but always knew the right times to provide moral support in passing. I also want to acknowledge all of the labs in the BMB and MMI departments for always being willing to answer my questions or share reagents. I truly appreciate the open door policy that we have in our departments. I have also developed lifelong friendships over the past 6 years, and certainly they have helped to make graduate school a bit more fun. My partner, Andrew Bober, continues to remind me to step outside my comfort zone, even when we’re 80 ft above the ground. Finally, I would like to acknowledge my family for their years of continued support and direction. You are always in my thoughts, and the ‘amazing adventures of the Reiter girls’ continues to unfold. v This thesis is dedicated in memory of John Richard Reiter A little frog Riding on a banana leaf, Trembling. Kikaku (1660-1707) vi TABLE OF CONTENTS Title Page i Abstract ii Acknowledgements iv Table of Contents vii List of Tables x List of Figures xi Abbreviations xiii Chapter 1: Introduction 1 Main text 2 Figures 15 Chapter 2: Identification of Biochemically Distinct Properties of the SUMO Conjugation Pathway in Plasmodium falciparum 18 Abstract 19 Introduction 20 Materials and Methods 24 Results 33 Discussion 42 Figures 48 Tables 60 vii Chapter 3: Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems 61 Abstract 62 Introduction 63 Materials and Methods 67 Results 71 Discussion 79 Figures 82 Tables 89 Chapter 4: Development of Parasite Strains for the Conditional Degradation of SUMO Conjugation Pathway Components 90 Abstract 91 Introduction 92 Materials and Methods 96 Results 100 Discussion 103 Figures 104 Appendix: Structural Methods for the Co-crystallization of Pf Uba2Ufd-Ubc9 111 Figures 117 viii Overview and Future Directions 119 References 126 Curriculum vitae 145 ix LIST OF TABLES Table 1-1. Data collection, phasing and refinement statistics Table 2-1. Data collection, phasing and refinement statistics x LIST OF FIGURES Figure 1-1. Life cycle of Plasmodium falciparum Figure 1-2. SUMO conjugation and deconjugation Figure 1-3. Oxidative stress derived from hemoglobin consumption in P. falciparum Figure 2-1. The sumoylation pathway is conserved in P. falciparum Figure 2-2. Sumoylation is differentially regulated during P. falciparum red blood cell stages Figure 2-3. Human and P. falciparum SUMO E1 and E2 enzyme interactions are distinct Figure 2-4. Hs Ubc9 and Pf Ubc9 interact specifically with human and P. falciparum E1- activating enzymes, respectively Figure 2-5. Crystallographic structure of Pf Ubc9 Figure 2-6. Human and P. falciparum Uba2Ufd and Ubc9 interfaces are divergent Figure 2-7. Ubc9 chimeras restore E1 and E2 interaction Figure 2-8. Ubc9 chimeras affect human and P. falciparum E1 enzyme interactions Figure 3-1. SUMO levels are modulated by oxidative stress Figure 3-2. Structural conservation of human Uba2Ufd-Ubc9 complex Figure 3-3. P. falciparum has a divergent Uba2Ufd-Ubc9 interface Figure 3-4. Single mutations throughout the Pf Uba2Ufd-Ubc9 interface are sufficient to disrupt binding Figure 3-5. Mutations in Pf Ubc9 α1-helix and β1-β2 loop disrupt RanGAP1 conjugation Figure 3-6. Select residues mediate Plasmodium specific Uba2Ufd-Ubc9 binding and conjugation activity. Figure 4-1. Conditional degradation pathway overview xi Figure 4-2. Sequence alignment of P. falciparum SCF components Figure 4-3. Validation of Tir1 attB integration Figure 4-4. Validation of endogenous ecDHFR tag integration xii ABBREVIATIONS AND NOMENCLATURE ATP adenosine triphosphate CO2 carbon dioxide DEAE diethylaminoethyl DNA deoxyribonucleic acid E1 ubiquitin or SUMO activating enzyme E2 ubiquitin or SUMO conjugating enzyme E3 ubiquitin or SUMO ligase EDTA ethylenediaminetetraacetic acid FKBP FK506 binding protein GFP green fluorescent protein GG di-glycine GST glutathione S-transferase HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid kDa kiloDalton M molar mL milliliter NEDD8 neural precursor cell expressed, developmentally down-regulated 8 NEM N-ethylmaleimide PBS phosphate-buffered saline PIAS protein inhibitor of activated STAT PMSF phenylmethanesulfonylfluoride PTM post-translational modification xiii RING really interesting new gene S. cerevisiae Saccharomyces cerevisiae S. pombe Saccharomyces pombe SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SENP sentrin-specific protease SIM SUMO interaction motif STUbl SUMO-targeted ubiquitin ligase SUMO small ubiquitin-related modifier TNX-100 triton X-100 Tris tris(hydroxymethyl)aminomethane Ub ubiquitin µg microgram µL microliter µm micrometer µM micromolar YFP yellow fluorescent protein xiv CHAPTER 1 INTRODUCTION 1 The global burden of malaria Globally, malaria remains a public health challenge and a leading cause of death in malaria endemic countries. Over 3.3 billion people are

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