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Investigations Into the Function of Elp3 in Toxoplasma Gondii

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Investigations Into the Function of Elp3 in Toxoplasma Gondii INVESTIGATIONS INTO THE FUNCTION OF ELP3 IN TOXOPLASMA GONDII Leah R. Padgett Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Pharmacology and Toxicology Indiana University August 2017 Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. _______________________________ Gustavo Arrizabalaga, Ph.D., Chair ________________________________ Travis Jerde, Ph.D. Doctoral Committee ________________________________ Amber Mosley, Ph.D. ________________________________ Richard M. Nass, Ph.D. May 4, 2017 ________________________________ William J. Sullivan, Jr., Ph.D. ii ACKNOWLEDGEMENTS I wish to thank first and foremost my mentor, Dr. Bill Sullivan, who provided me with exceptional training and mentorship. His optimism and passion for science is admirable and infectious. I appreciate him for being open to all of my crazy ideas and guiding me towards the most logical ones to test. I would also like to thank him for supporting my independent research while occasionally reminding me to focus. My thesis committee has provided valuable suggestions regarding my research project and has supported me throughout this journey. I would like to thank each of my thesis committee members: Dr. Travis Jerde, Dr. Amber Mosely, Dr. Richard Nass and Dr. Gustavo Arrizabalaga. I especially would like to thank Dr. Gustavo Arrizabalaga for his enthusiasm for science and hallway pep-talks. Next, I would like to thank my fellow IBMG graduate students, Joe Varberg and Kacie Lafavers. We have experienced the “ups” and the “downs” of this graduate school roller coaster ride together. Specifically, I would like to thank Joe for his brilliant mind and great scientific discussions, and Kacie for her statistical advice. I would also like to thank all of the past and present members of the Sullivan and Arrizabalaga labs for their continuous support and advice throughout the years. I would like to thank Dr. Vicki Jeffers for her training in molecular parasitology techniques, support and friendship. I would also like to thank Dr. Mike Holmes for his insightful scientific conversations and help with the polyribosomal profiling experiments and Dr. Michael Harris for his advice regarding recombinant protein protocols. This work would not be possible without the help of others. First, I would like to thank my collaborators Jenna Lentini and Dragony Fu at the Univeristy of Rochester for performing the in vitro γ-toxin cleavage assay. Next, I would like to thank Joe Varberg for being a great teammate and for his significant contribution to the work presented in Chapter 3 which was adapted from the published journal article “TgATAT-Mediated α-Tubulin Acetylation Is Required for Division of the iii Protozoan Parasite Toxoplasma gondii.” Additionally, I would like to thank Gustavo Arrizabalaga for performing the bioinformatics analyses presented in the published article “Targeting of tail-anchored membrane proteins to subcellular organelles in Toxoplasma gondii” which has been adapted for Chapter 4. I would also like to thank the American Heart Association for providing funding for my research, predoctoral fellowship grant 15PRE25550023. Importantly, I would like to thank my family for their love and encouragement. My parents are my biggest cheerleaders and they have taught me that with hard work and perseverance, anything is possible. I would also like to thank Charles, my best friend and soulmate, for his support and to Cerrah, who reminds me that it is the little things in life that matter most. iv Leah R. Padgett INVESTIGATIONS INTO THE FUNCTION OF ELP3 IN TOXOPLASMA GONDII The parasite Toxoplasma gondii causes life-threatening infection in immunocompromised individuals. Our lab has determined that Toxoplasma Elongator protein-3 (TgElp3) is required for parasite viability. While catalytic domains are conserved, TgElp3 is the only component of the six-subunit Elongator complex present in Toxoplasma; moreover, TgElp3 localizes to the outer mitochondria membrane (OMM). These unusual features suggest that TgElp3 may have unique roles in parasite biology that could be useful in drug targeting. The goals of this thesis were to determine the function of TgElp3 and how the protein traffics to the OMM. In other species, Elp3 mediates lysine acetylation of histones and alpha- tubulin, and its radical S-adenosyl methionine (rSAM) domain is important for the formation of tRNA modifications, which enhance translation efficiency and fidelity. Given its location, histones would not be an expected substrate, and we further determined that tubulin acetylation in Toxoplasma is mediated by a different enzyme, TgATAT. We found that overexpression of TgElp3 at the parasite’s mitochondrion results in a significant replication defect, but overexpression of TgElp3 lacking the transmembrane domain (TMD) or with a mutant rSAM domain is tolerated. We identified one such modification, 5-methoxycarbonylmethyl-2- thiouridine (mcm5S2U) that is likely mediated by TgElp3. These findings signify the importance of TgElp3’s rSAM domain for protein function, and confirms TgElp3 activity at the OMM is essential for Toxoplasma viability as previously reported. To determine how TgElp3 traffics to the OMM, we performed a bioinformatics survey that discovered over 50 additional “tail-anchored” proteins present in Toxoplasma. Mutational analyses found that targeting of these TA proteins to specific parasite organelles was strongly influenced by the TMD sequence, including charge of the flanking C-terminal sequence. Gustavo Arrizabalaga, Ph.D., Chair v TABLE OF CONTENTS LIST OF TABLES ................................................................................................ x LIST OF FIGURES ...............................................................................................xi LIST OF ABBREVIATIONS ............................................................................... xiii CHAPTER 1: Introduction .................................................................................. 1 1.1 Toxoplasma gondii, a protozoan parasite .................................................... 1 1.2 Life cycle of Toxoplasma ............................................................................. 4 1.3 Toxoplasma infection .................................................................................. 7 1.4 Treatment of toxoplasmosis ........................................................................ 8 1.5 Lysine acetylation as a drug target .............................................................. 9 1.6 History of Elp3 ........................................................................................... 10 1.7 Elp3 and human disease ........................................................................... 13 1.8 Identification of an Elp3 homologue in Toxoplasma .................................. 13 1.9 Summary and hypotheses ......................................................................... 15 CHAPTER 2: Characterization of TgElp3 ........................................................ 17 2.1 Introduction ................................................................................................ 17 2.2 Materials and Methods .............................................................................. 18 2.2.1 Plasmid DNA construction .................................................................. 18 2.2.1.1 BioID ............................................................................................. 18 2.2.1.2 Endogenous Replacement ............................................................ 18 2.2.1.3 Destabilization Domain ................................................................. 19 2.2.1.4 Ectopic Overexpression ................................................................ 19 2.2.1.5 Recombinant TgElp3 expression vectors...................................... 20 2.2.2 Parasite culture ................................................................................... 21 2.2.3 Generation of transgenic parasites ..................................................... 21 2.2.4 Affinity purification of biotinylated proteins .......................................... 23 2.2.5 Mass spectrometry analysis ................................................................ 24 2.2.6 Antibodies ........................................................................................... 24 2.2.7 Immunoblotting ................................................................................... 25 2.2.8 Immunofluorescence assays ............................................................... 26 2.2.9 Toxoplasma growth assays ................................................................. 26 vi 2.2.9.1 Plaque Assay ................................................................................ 26 2.2.9.2 Doubling Assay ............................................................................. 27 2.2.10 γ-toxin modification assay tRNA ....................................................... 27 2.2.10.1 RNA isolation .............................................................................. 27 2.2.10.2 Characterization of tRNAs treated with γ-toxin in vitro ................ 28 2.2.11 Recombinant protein expression and purification ............................. 28 2.2.12 Analysis of protein synthesis ............................................................. 30
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