INVESTIGATION OF CANDIDA ALBICANS PRENYLTRANSFERASES: SUBSTRATE RECOGNITION AND ENZYME INHIBITION by Elaina A. Zverina A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2012 Doctoral Committee: Professor Carol A. Fierke, Chair Professor Anna K. Mapp Associate Professor Bruce A. Palfey Professor Ronald W. Woodard © Elaina A. Zverina 2012 DEDICATION To my parents and grandparents ii ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor, Prof. Carol Fierke, for her support, guidance, and encouragement throughout my time in graduate school. I would also like to thank my committee, Prof. Anna Mapp, Prof. Bruce Palfey, and Prof. Ron Woodard, for their helpful comments and suggestions. I would like to thank the Pharmacological Sciences Training Program for funding and training. I sincerely thank all my labmates, both past and present, who have made been great colleagues over the years. I would especially like to thank Drs. Corissa Lamphear, Terry Watt, and James Hougland for their help and discussions about my project. I would like to thank Noah Wolfson, Elia Wright, Xin Liu, Carol Ann Pitcairn, and Andrea Stoddard for all of their help along the way. I would like to thank the Chemical Biology Program for providing an excellent academic environment, and all the members of my class. I would like to thank Prof. Kenneth Merz and Dr. Mark Benson at the University of Florida for collaboration on prenyltransferase inhibitors. I would like to thank all the people who have encouraged me to continue my education, especially Drs. Dave Ehmann, Tom Keating, Adam Shapiro, and Grant Walkup. I would like to thank my family for their unconditional love, support and encouragement. I would like to express my deepest love and gratitude to my parents Alex and Irina. iii TABLE OF CONTENTS DEDICATION ............................................................................................................................ ii ACKNOWLEDGEMENTS ........................................................................................................iii LIST OF FIGURES ................................................................................................................... vi LIST OF TABLES ..................................................................................................................... ix ABSTRACT ................................................................................................................................ x CHAPTER ONE ............................................................................................................... 1 INTRODUCTION ...................................................................................................................... 1 Post-translational modifications .............................................................................................. 1 Protein lipidation ..................................................................................................................... 2 Protein prenylation and processing ......................................................................................... 5 Prenylation and disease implications ...................................................................................... 7 Mammalian FTase and GGTase-I ........................................................................................... 8 Prenyltransferase substrate identification ............................................................................. 11 Computational work .............................................................................................................. 13 Candida albicans prenyltransferases .................................................................................... 15 Objectives of this work ......................................................................................................... 19 REFERENCES ......................................................................................................................... 21 CHAPTER TWO ............................................................................................................ 27 INVESTIGATION OF SUBSTRATE RECOGNITION BY CANDIDA ALBICANS PRENYLTRANSFERASES USING A PEPTIDE LIBRARY APPROACH .......................... 27 INTRODUCTION .................................................................................................................... 27 EXPERIMENTAL PROCEDURES ......................................................................................... 34 Cloning of C. albicans FTase and GGTase-I genes ............................................................. 34 C. albicans FTase and GGTase-I expression and purification ............................................. 36 Peptide libraries .................................................................................................................... 38 Multiple turnover FTase and GGTase-I Activity Assay ....................................................... 42 Single turnover assay ............................................................................................................ 43 Statistical analysis of amino acid sequence in FTase and GGTase-I peptide substrates ...... 44 RESULTS ................................................................................................................................. 46 Peptide substrates of FTase and GGTase-I ........................................................................... 46 Sequence analysis of MTO and STO C. albicans FTase and GGTase-I substrates ............. 51 Statistical analysis of individual amino acid distribution in C. albicans FTase and GGTase-I substrates ............................................................................................................................... 57 Predictions for Cxxx sequences found in C. albicans genome ............................................. 61 DISCUSSION ........................................................................................................................... 65 Comparison of mammalian and C. albicans prenyltransferase substrate pools ................... 65 Comparison of mammalian and C. albicans prenyltransferase substrate recognition .......... 67 REFERENCES ......................................................................................................................... 71 iv CHAPTER THREE ........................................................................................................ 73 MOLECULAR BASIS OF Ca1a2X RECOGNITION IN MAMMALIAN AND CANDIDA ALBICANS PRENYLTRANSFERASES ................................................................................. 73 INTRODUCTION .................................................................................................................... 73 EXPERIMENTAL PROCEDURES ......................................................................................... 80 Multiple Turnover Prenylation Activity Assay ..................................................................... 80 Data analysis ......................................................................................................................... 81 RESULTS ................................................................................................................................. 81 C. albicans FTase reactivity with GCVa2X where X=A, S, Q, M, L and F ......................... 81 C. albicans GGTase-I reactivity with GCVa2X where X=A, S, Q, M, L and F ................... 87 Comparison of mammalian and C. albicans GGTase-I reactivity with GCVa2L ................. 90 Recognition of the X group by mammalian and C. albicans prenyltransferases .................. 92 DISCUSSION ........................................................................................................................... 94 Mammalian and C. albicans FTase recognition of –a2X ...................................................... 94 Mammalian and C. albicans GGTase-I recognition of –a2X ................................................ 98 Recognition of X group by mammalian and C. albicans prenyltransferases ..................... 101 REFERENCES ....................................................................................................................... 105 CHAPTER FOUR ......................................................................................................... 107 INVESTIGATION OF SMALL MOLECULE INHIBITORS OF MAMMALIAN AND CANDIDA ALBICANS PRENYLTRANSFERASES ............................................................. 107 INTRODUCTION .................................................................................................................. 107 EXPERIMENTAL PROCEDURES ....................................................................................... 111 Selection of small molecule prenyltransferase inhibitors ................................................... 111 Prenyltransferase inhibition assays ..................................................................................... 112 Mode of inhibition of C. albicans FTase by compound 36745 .......................................... 113 RESULTS ............................................................................................................................... 114 FTase and GGTase-I inhibition........................................................................................... 114 Mode of inhibition of