Clemson University TigerPrints All Dissertations Dissertations 5-2011 REGULATION OF TRYPANOSOMA BRUCEI HEXOKINASE 1 AND 2 ON MULTIPLE LEVELS: TRANSCRIPT ABUNDANCE, PROTEIN EXPRESSION AND ENZYME ACTIVITY Heidi Dodson Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Biochemistry Commons Recommended Citation Dodson, Heidi, "REGULATION OF TRYPANOSOMA BRUCEI HEXOKINASE 1 AND 2 ON MULTIPLE LEVELS: TRANSCRIPT ABUNDANCE, PROTEIN EXPRESSION AND ENZYME ACTIVITY" (2011). All Dissertations. 703. https://tigerprints.clemson.edu/all_dissertations/703 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. REGULATION OF TRYPANOSOMA BRUCEI HEXOKINASE 1 AND 2 ON MULTIPLE LEVELS: TRANSCRIPT ABUNDANCE, PROTEIN EXPRESSION AND ENZYME ACTIVITY A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Biochemistry and Molecular Biology by Heidi Cornelia Dodson May 2011 Accepted by: Dr. James C. Morris, Committee Chair Dr. Kimberly S. Paul Dr. Michael G. Sehorn Dr. Kerry S. Smith ABSTRACT Trypanosoma brucei, a unicellular eukaryotic parasite, is the causative agent of African sleeping sickness in sub-Saharan Africa. The parasite encounters two main environments as it progresses through its life cycle: the tsetse fly and the mammalian bloodstream. Nutrient availability is distinct in the two environments, requiring the parasite to utilize diverse metabolic pathways to efficiently produce ATP for survival. Bloodstream form parasites (BSF), residing in a glucose rich environment, rely solely on glycolysis for energy, while procyclic form (PF) parasites metabolize readily available proline and threonine in addition to glucose. T. brucei expresses two hexokinases, the first enzyme in the glycolytic pathway, that are 98.5% identical at the nucleotide level and 98% similar at the amino acid level. These two enzymes, TbHK1 and TbHK2, are differentially expressed in both BSF and PF parasites. Here, I identify a means of regulation of TbHK1 gene expression and a novel mechanism for regulating TbHK1 enzyme activity. Lastly, I have characterized, the bioflavonoid quercetin, a small molecule regulator TbHK1. Mechanisms involved in regulating TbHK1 and TbHK2 gene expression have not been extensively studied. I have found that TbHK1 uses differential polyadenylation to regulate gene expression in variable environmental conditions. A recent study by Siegel et al. (2010), revealed that TbHK1 contains seven predominant polyadenylation sites, whereas, TbHK2 contains only one. Using a reporter gene system to assess transcript level and protein expression, I have determined that these seven different 3’UTR lengths ii result in differential steady state transcript abundance and expression level, dependent on nutrient availability. Further, I have identified a novel means of regulation of TbHK1 enzyme activity. Enzymatic studies of recombinant TbHK1 reveal that TbHK is inactivated under acidic conditions. However, the addition of glycerol-3-phosphate to the reaction at acidic pH maintained T. brucei hexokinase activity. I propose this regulation may play an important role in the biology of the parasite during differentiation and subsequent acidification of glycosomes, the peroxisomal like organelle that houses glycolytic enzymes. Lastly, the TbHKs were identified as possible drug targets because they are essential to the BSF parasite and only ~30% similar to human glucokinase, the human equivalent of TbHK1. We have found that quercetin, a known trypanocide, is a potent inhibitor of TbHK1. Further, taking advantage of the fluorescent nature of the compound, I found that the compound localizes to the same subcellular compartment that houses TbHK1 and interacts with the protein near the active site as a mixed inhibitor. iii DEDICATION For my family: Daddy and Mama~ Thank you for teaching me the value of kindness and hard work. Jason, Alicia, Abby & Jake~ Thank you for your continuous love and support. iv ACKNOWLEDGMENTS This work would not have been possible without the help and guidance of numerous people. First, I would like to thank Dr. James Morris for the opportunity to work in his lab and for his guidance and encouragement during my graduate career. Also, I would like to thank Dr. Meredith Morris for her insight and direction. In addition, my committee, Dr. Kim Paul, Dr. Michael Sehorn, and Dr. Kerry Smith, have proven immensely helpful throughout the duration of this project. I would also like to thank former and present lab members who have provided useful suggestions for my work and who along the journey also became wonderful friends. These lab members include Dr. Jeremy Chambers, Dr. Todd Lyda, Dr. Marcia Hesser, Ms. April Joice, and Mr. Andrew Sayce. Further, the faculty, graduate students, and staff of the Genetics and Biochemistry Department at Clemson University have contributed in a myriad of ways to the completion of my degree. I would also like to thank the Biology Department at North Greenville University for the foundation that they gave me in the study of science. Finally, without love and encouragement from my precious family and dear friends, this work would not have been possible. Thank you all for walking alongside me in this journey. v TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGMENTS ............................................................................................... v LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ........................................................................................................ ix CHAPTER I. LITERATURE REVIEW .............................................................................. 1 Life cycle of T. brucei .............................................................................. 2 Cell Structure and Biology ....................................................................... 5 Glycolysis in T. brucei ........................................................................... 11 Gene Expression .................................................................................... 17 References .............................................................................................. 24 II. DIFFERENTIAL POLYADENYLATION INFLUENCES GENE EXPRESSION IN THE AFRICAN TRYPANOSOME IN RESPONSE TO ENVIRONMENTAL CUE ............................................................. 38 Abstract .................................................................................................. 38 Introduction ............................................................................................ 40 Materials and Methods ........................................................................... 42 Results .................................................................................................... 44 Discussion .............................................................................................. 53 References .............................................................................................. 58 III. GLYCEROL-3-PHOSPHATE ALTERS TRYPANOSOMA BRUCEI HEXOKINASE ACTIVITY IN RESPONSE TO ENVIRONMENTAL CHANGE ..................................................... 61 vi Table of Contents (Continued) Abstract ................................................................................................. 61 Introduction ............................................................................................ 63 Materials and Methods ........................................................................... 65 Results .................................................................................................... 68 Discussion .............................................................................................. 79 References .............................................................................................. 83 IV. QUERCETIN, A FLUORESCENT BIOFLAVONOID, INHIBITS TRYPANOSOMA BRUCEI HEXOKINASE 1 ................................................................................... 87 Abstract .................................................................................................. 87 Introduction ............................................................................................ 89 Materials and Methods ........................................................................... 91 Results .................................................................................................... 94 Discussion ............................................................................................ 103 References ............................................................................................ 106 V. EXPRESSION OF TRYPANOSOMA BRUCEI HEXOKINASE 1 IS REGULATED BY A METAZOAN-LIKE MICRORNA BINDING ELEMENT