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190323111.Pdf Characterization of polymers of nucleotide biosynthetic enzymes By Sajitha Anthony April, 2017 A dissertation presented to the faculty of Drexel University College of Medicine in partial fulfillment for the requirements for the degree of Doctor of Philosophy in Molecular and Cellular Biology and Genetics i ii ACKNOWLEDGEMENTS First and foremost, I would like to thank my mentor Dr. Jeffrey Peterson for all of his support and guidance throughout the five years that I have been in his lab. He has truly inspired me with his tremendous enthusiasm for science and his constant encouragement. He has changed, for the better, the way I approach and see science. I could not have asked for a better mentor. Secondly, I would like to thank my committee members for all of their great ideas and their never-ending support. I thank them for answering all of my questions so patiently and always listening to what I had to say. After every meeting I’ve had with them, I always felt encouraged and confident. I would also like to thank my collaborators over at the University of Washington—Justin Kollman, Anika Burrell, and Matthew Johnson. It has been such a pleasure working with them. The journey of discovery that we took together has been very exciting. This was my first collaboration, and they have made it such a memorable and positive experience. iii I would like to thank my lab members, in particular Alex for being an awesome cubemate, colleague, and friend. He has been the sounding board for so many of my project’s ideas over the years and has given such knowledgeable input. I would also like to thank Jackie for being a good colleague and friend. You have advised me on various things and shared so much of your knowledge with me. Also, I thank both of them for sitting in on so many of my practice talks! I would like to thank my mother, who has always supported me throughout my life. She has always emphasized the importance of education and hard work. She has pushed me to achieve my goals and has always advised me to never settle. Without her undying support and encouragement throughout the years, I would not be where I am today. And last, but certainly not least, I would like to thank my dear husband Christian. It has been a busy year with us having to adjust to his recent move here, our marriage and my thesis writing all at once. Through it all, he has stood by me always ready to offer encouragement and help. His technological knowhow has been a tremendous help to me. He has guided me through formatting issues (and numerous recoveries!) of my thesis. I am truly grateful for him. Here’s to the future! iv TABLE OF CONTENTS LIST OF TABLES .............................................................................................. xi LIST OF ILLUSTRATIONS ............................................................................. xii ABSTRACT ....................................................................................................... xv Chapter 1: Introduction ...................................................................................... 1 Nucleotide biosynthesizing enzymes form macromolecular structures within cells ....................................................................................................... 2 .......................................................................................................................... 3 The many roles of nucleotides in cellular metabolism ................................... 3 The importance of nucleotides in dividing and quiescent cells .................. 3 Nucleotides as intermediates in cellular communication ........................... 5 The role of nucleotides in the nervous system ............................................ 6 Nucleotide sugars as activators of metabolites for lipid synthesis ............ 6 Nucleotides as cofactors in reactions (S-Adenosyl-L-methionine) ............. 7 Nucleotides as electron donors .................................................................... 8 v Nucleotides as energy carriers (ATP, GTP) ................................................ 8 The process of purine synthesis and catabolism in the cell ........................... 9 De novo nucleotide synthesis ....................................................................... 9 The salvage pathway .................................................................................. 11 Regulation of synthesis .............................................................................. 13 Catabolism of purine nucleotides .............................................................. 14 Purine nucleotide salvage deficiencies and neurological pathologies ...... 15 Purine nucleotide salvage deficiencies and mitochondrial depletion syndromes ................................................................................................... 17 Deficiencies of the purine de novo synthesis pathway which result in immunodeficiency ....................................................................................... 17 Pyrimidine synthesis and catabolism ........................................................... 19 De novo pyrimidine synthesis .................................................................... 19 The pyrimidine salvage pathway ............................................................... 20 Regulation of pyrimidine synthesis ........................................................... 21 Catabolism of pyrimidine nucleotides ....................................................... 22 Pyrimidine nucleotide salvage deficiencies ............................................... 23 De novo pyrimidine nucleotide synthesis deficiencies .............................. 24 De novo pyrimidine nucleotide synthesis and immunodeficiency ............ 25 Inosine monophosphate (IMPDH) and de novo nucleotide synthesis ......... 26 vi The IMPDH reaction .................................................................................. 26 The structure of IMPDH ............................................................................ 27 The role of the Bateman domain and nucleotides in the regulation of IMPDH activity .......................................................................................... 29 Activation of IMPDH by monovalent cations ............................................ 31 Isoforms of IMPDH..................................................................................... 32 Moonlighting functions of IMPDH ................................................................ 33 Therapeutic targeting of IMPDH activity .................................................... 35 MPA ............................................................................................................ 35 Ribavirin ..................................................................................................... 37 Other inhibitors of IMPDH ........................................................................ 38 IMPDH as filamentous structures ................................................................ 39 Physical characteristics of IMPDH filaments ........................................... 39 History of the discovery of IMPDH filaments ........................................... 40 Regulation of IMPDH filament assembly .................................................. 43 The effect of IMPDH filament assembly on catalytic activity .................. 48 IMPDH can co-assemble with CTPS into filaments ................................. 48 CTPS in pyrimidine synthesis ....................................................................... 50 Structural characteristic of CTPS ............................................................. 51 vii Isoforms and expression ............................................................................. 52 The regulation of CTPS activity .................................................................... 54 Allosteric regulation ................................................................................... 54 Regulation of CTP by post translational modifications ............................ 55 The targeting of CTPS activity as a therapeutic .......................................... 56 Cyclopentenylcytosine Triphosphate (CPEC) and c3Urd .......................... 57 Gemcitabine ................................................................................................ 58 Azaserine .................................................................................................... 60 DON ............................................................................................................ 62 CTPS as filamentous structures ................................................................... 66 The discovery of CTPS filaments ............................................................... 66 Regulation of CTPS filament assembly ..................................................... 68 The biological role of CTPS filament assembly ......................................... 77 Chapter 2: Identification of CTPS1 associating proteins within the filaments ........................................................................................................................... 84 Introduction ................................................................................................... 85 Materials and methods .................................................................................. 89 Cell culture and transfection ......................................................................... 89 Plasmid ......................................................................................................
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