Proteome-wide Functional Profiling of Serine Hydrolases in the Human Malaria Parasite AEM Rubayet Elahi Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biochemistry Michael W. Klemba, Chair Glenda E. Gillaspy Jinsong Zhu Daniel J. Slade May 7, 2019 Blacksburg, VA Keywords: Plasmodium falciparum, malaria, serine hydrolase, lipase, acylpeptide hydrolase, activity-based protein profiling Copyright © 2019 AEM Rubayet Elahi Proteome-wide Functional Profiling of Serine Hydrolases in the Human Malaria Parasite AEM Rubayet Elahi ABSTRACT The serine hydrolase (SH) enzyme superfamily is one of the largest and most diverse enzyme classes in eukaryotes and prokaryotes. The most virulent human malaria parasite Plasmodium falciparum has over 40 predicted serine hydrolases (SH). Prior investigation on a few of these have suggested their critical role in parasite biology. The majority of the SHs in P. falciparum have not been functionally characterized. Investigation of these uncharacterized SHs will provide new insights into essential features of parasite metabolism and possibly lead to new antimalarial targets. In this study, we have employed activity-based protein profiling (ABPP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to functionally characterize SHs. In our effort to profile plasmodial SHs using ABPP, we have identified a human erythrocyte SH, acylpeptide hydrolase (APEH) in the developing parasites. This finding is the first report of internalization of host hydrolytic enzyme by the parasite. Treatment of parasites with an APEH specific triazole urea inhibitor, AA74-1, caused growth inhibition in parasites with poor potency in the first replication cycle, however, the potency dramatically increased in the second cycle. We show that this unique growth inhibition profile is due to the inability of AA74-1 to inhibit parasite-internalized APEH in vivo. These findings suggest that internalization of active APEH by the parasite is essential for parasite survival. Lipases catalyze the hydrolysis of ester bonds of lipid species such as neutral lipids and phospholipids. Although roles of lipases in propagation, as well as virulence in various organisms, have been acknowledged, in P. falciparum lipases remain understudied. We combined LC-MS/MS with the SH-directed ABPP to identify lipases of SH superfamily in P. falciparum. We have identified 16 plasmodial SHs with putative lipase activity. Bioinformatics analysis of our identified lipases is consistent with our findings. We have screened a panel of various classes of SH inhibitors in a competitive ABPP. A plasmodial putative lipase was potently and specifically inhibited by human monoacylglycerol lipase inhibitor. This inhibition profile suggests it as a monoacylglycerol lipase which plays a role in releasing fatty acids from neutral lipid. This finding shows that how inhibitor screening can aid in building hypotheses on biological roles of an enzyme. Altogether, in this dissertation, we have presented a robust strategy of identifying and functionally characterizing SHs in P. falciparum, which opens the door to the discovery of new biological processes. Proteome-wide Functional Profiling of Serine Hydrolases in the Human Malaria Parasite AEM Rubayet Elahi GENERAL AUDIENCE ABSTRACT Malaria contributed to nearly a half a million deaths in 2017. The vast majority of malaria-related deaths are due to the parasite Plasmodium falciparum. This parasite resides inside human red blood cells (erythrocytes) and grows rapidly during a 48 hour cycle. There are over 40 serine hydrolase (SH) superfamily proteins in the parasite. Biological functions of the majority of SHs in the parasite remains unknown. Study on these SHs will provide new insights into parasite biology, and possibly present new antimalarial drug targets. We used chemical biology techniques to identify and functionally characterize parasite SHs. In one study, we show the parasite intenalized a human erythrocyte SH, acylpeptide hydrolase (APEH). We used an APEH-specific inhibitor to investigate the biological significance of internalized APEH in parasite biology. Treatment of the parasite with the inhibitor resulted in parasite growth inhibition suggesting internalization of APEH is essential for parasite survival. Lipases are enzymes that aid in break down of lipids and have shown to be crucial for growth and pathogenicity in various organisms. Lipases and lipid catabolism remain understudied in the malaria parasite. We used mass spectrometry in our approach to identify 16 lipases in asexual parasites. We have also shown that screening with highly specific inhibitors can help in predicting biological function of a particular enzyme. In summary, in this body of work, we have presented an approach of studying SHs in the malaria parasite, which will provide new insights into parasite biology. To my father and brother vi ACKNOWLEDGEMENTS Foremost, I would like to express my sincere gratitude to my advisor Dr. Michael W. Klemba. I am very fortunate to be able to work with him over the last four years. His unique blend of vision, energy, technical knowledge, and generosity makes him an inspiring role model for my future career. My gratitude extends to my committee members, Drs. Glenda E. Gillaspy, Jinsong Zhu, and Daniel J. Slade for all the thoughtful and inspiring discussions and suggestions. I also like to thank them for their invaluable support and encouragement that helped me to go through this journey. My sincere thanks also go to Dr. Rich Helm and Dr. Keith Ray for their help in mass spectrometry analysis. I would also like to acknowledge Dr. Ken Hsu of the University of Virginia for his invaluable insights in proteomics sample preparation. Thank you, Christie Dapper and all the past members of the lab that I have worked with for the assistance you have given me at some point and time on this project. It was fun working with you all in this lab. In addition, my graduate career would not have been possible without the constant support of my friends. Sajal Dash, Nabil, Sazzad, Sajal Iskander, Archi, Trisha, and Asif, you guys were my family here in Blacksburg. All the fun that we had hanging out together was a major stress relief. I would especially like to thank my loving wife, Fatema Siddiquee, for all the sacrifices you made and the difficulties you had to go through. Thank you for not giving up on me when I was ready to give up on myself. It would not have been possible for me to be here without your love and support. vii Finally, I would like to thank my parents and my siblings. Thank you for believing in me. Whatever I am today, it is because of your love, encouragement, and guidance. I hope my PhD will bring a smile to all of your faces. viii ATTRIBUTIONS Several of my colleagues have aided in research and writing of the chapters of this dissertation. Chapter 2: “Internalization of erythrocyte acylpeptide hydrolase is required for asexual replication of Plasmodium falciparum”, published in mSphere, 2019. Christie Dapper was a Senior Laboratory Specialist in The Klemba lab, Department of Biochemistry, Virginia Tech. Mrs. Dapper is a co-author of the article and contributed to parasite growth inhibition assays presented in figure 2-3A, B, and Table 2-1. Michael W. Klemba, PhD is currently an Associate Professor in Department of Biochemistry, Virginia Tech. Dr. Klemba is a co-author of the article, principal investigator for the grant supporting the work, contributed in experimental design, data analysis, and wrote the manuscript. Chapter 3: “Discovery of putative lipases of the serine hydrolases superfamily in the asexual erythrocyte stage of Plasmodium falciparum”, manuscript in preparation. William Keith Ray, PhD is currently a Senior Research Associate in The Helm Lab, Department of Biochemistry, Virginia Tech. Dr. Ray performed mass spectrometry analysis and data analysis. ix Christie Dapper was a Senior Laboratory Specialist in The Klemba lab, Department of Biochemistry, Virginia Tech. Mrs. Dapper contributed to parasite lysate preparation and experiments presented in figure 3-1B and 3-4C. Seema Dalal, PhD was a Research Scientist in The Klemba lab, Department of Biochemistry, Virginia Tech. Dr. Dalal generated Pf3D7_0709700-YFP parasite line used in the experiments presented in figure 3-4E and figure 3-S2. Rich Helm, PhD is currently an Associate Professor in Department of Biochemistry, Virginia Tech and the director of the Virginia Tech Mass Spectrometry Research Incubator. Dr. Helm contributed to the experimental design and supervised mass spectrometry analysis. Michael W. Klemba, PhD is currently an Associate Professor in Department of Biochemistry, Virginia Tech. Dr. Klemba is the principal investigator for the grant supporting the work, contributed in experimental design, and data analysis. x TABLE OF CONTENTS Abstract ii General audience abstract iv Dedication vi Acknowledgements vii Attributions ix List of figures xiii List of tables xv List of abbreviations xvii Chapter 1 Introduction 1 1.1 The malaria problem 2 1.2 The life cycle of P. falciparum 4 1.3 Plasmodium lipid metabolism 6 1.4 Serine hydrolases of P. falciparum 8 1.5 Activity-based proteomics 12 1.6 Functional profiling of P. falciparum serine hydrolases 15 References 17 Chapter 2 Internalization of erythrocyte acylpeptide hydrolase is 26 required for asexual replication of Plasmodium falciparum 2.1 Abstract 27 2.2 Importance 28 2.3 Introduction 29 xi 2.4 Results 31 2.5 Discussion 43 2.6 Materials and methods 46 Acknowledgements 52 References 53 Supplementary information 60 Supplementary references 65 Chapter 3 Discovery of putative lipases of the serine hydrolases 67 superfamily in the asexual erythrocyte stage of Plasmodium falciparum 3.1 Abstract 68 3.2 Introduction 69 3.3 Results 71 3.4 Discussion 87 3.5 Materials and methods 90 Acknowledgments 97 References 98 Supplementary information 105 Supplementary references 119 Chapter 4 Summary and conclusions 122 4.1 Summary and future directions 123 References 127 xii LIST OF FIGURES Chapter 1 Figure 1-1.