Investigating Amine Oxidase Domain Containing Genes - amx-1 and amx-2 - in Caenorhabditis elegans A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Reetobrata Basu December 2014 © 2014 Reetobrata Basu. All Rights Reserved. 2 This thesis titled Investigating Amine Oxidase Domain Containing Genes - amx-1 and amx-2 - in Caenorhabditis elegans by REETOBRATA BASU has been approved for the Department of Biological Sciences and the College of Arts and Sciences by Janet S. Duerr Associate Professor of Biological Sciences Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT BASU, REETOBRATA, M.S., December 2014, Biological Sciences Investigating Amine Oxidase Domain Containing Genes - amx-1 and amx-2 - in Caenorhabditis elegans Director of Thesis: Janet S. Duerr Monoamine (MA) neurotransmitters affect multiple behaviors in animals. MA homeostasis is achieved partly by monoamine-oxidase (MAO) enzymes - a drug target for many human neuropsychiatric disorders. In C. elegans the MA pathway is similar to that in humans and the worm shows MA dependent behaviors, affected by MAO inhibitor (MAOI) treatments. We cloned, expressed and purified the C. elegans genes - amx-1 and amx-2 in heterologous systems. Absorption spectra indicated that AMX1 and AMX2 bind the redox cofactor flavin adenine dinucleotide (FAD). Biochemical assays with wild-type (N2) or mutant worm lysates showed significant differences in MAO and histone demethylase (HDM) activities between them. Purified AMX1 and AMX2 had very low in vitro HDM activity independently, but both significantly increased the HDM activity of worm lysates. AMX1 had negligible in vitro MAO activity, whereas AMX2 had high in vitro MAO activity, with substrate and inhibitor specificities. 4 DEDICATION To my mother Manisha Basu 5 ACKNOWLEDGMENTS I would like to express my deepest appreciation for my advisor, Professor Janet Duerr. Without her guidance and persistent help this dissertation would not have been possible. I would also like to thank the members of my committee Professor Robert Colvin, Professor Daewoo Lee and Professor Sarah Wyatt for their time, support and invaluable guidance in various areas in my thesis. For the help in several aspects of my research, I am greatly thankful to Professor Tomohiko Sugiyama and Professor Mark Berryman (Ohio University, Athens, Ohio), Professor Ralf Baumeister and Ruth Jahne (Freiberg University, Germany), and Professor David Katz and Professor Kelly Williams (Emory University, Atlanta, Georgia). I must thank Amrita Basu, Jian Li, and Nilesh Khade for their constant help and support in different parts of my thesis. I would like to thank my parents and my wife for their love and encouragement and for always being my strength. 6 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 4 Acknowledgments............................................................................................................... 5 List of Tables ...................................................................................................................... 8 List of Figures ..................................................................................................................... 9 Chapter 1: Introduction ..................................................................................................... 11 1.1: Overview on Monoamine Metabolism. .................................................................. 11 1.2: Monoamines and Their Roles in Humans. .............................................................. 14 1.2.1: Dopamine .......................................................................................................... 15 1.2.2: Serotonin ........................................................................................................... 17 1.2.3: Norepinephrine (Noradrenaline) and Epinephrine (Adrenaline) ...................... 19 1.2.4: Tyramine and Octopamine ................................................................................ 20 1.3: Monoamine Oxidase in Humans............................................................................. 21 1.4: Monoamine Pathophysiology in Humans and Monoamine Oxidase Inhibitors. .... 25 1.5: Caenorhabditis elegans as a Model Organism. ...................................................... 28 1.6: MA Trafficking in C. elegans. ................................................................................ 30 1.7: The Roles of MAs in C. elegans. ............................................................................ 32 1.8: Amine Oxidase (AO) Domain Containing Genes in C. elegans. ........................... 34 1.9: Histone Demethylases in Vertebrates and C. elegans. ........................................... 37 Chapter 2: Background Experiments and Hypothesis ...................................................... 42 2.1: Monoamine Dependent Behavior Assays. .............................................................. 42 2.1.1: Sensitivity to Exogenous DA and 5HT............................................................. 43 2.1.2: MA-dependent Movement ................................................................................ 46 2.1.3: Pharyngeal Pumping ......................................................................................... 47 2.1.4: Egg-laying Behavior and Embryos in utero ..................................................... 47 2.1.5: Effect of MAOIs ............................................................................................... 48 2.2: amx Transgenic Studies .......................................................................................... 48 2.3: In situ Monoamine Levels. ..................................................................................... 51 7 2.4: Hypothesis. ............................................................................................................. 52 Chapter 3: Materials and Methods .................................................................................... 54 3.1: Bacterial Plasmid Construction .............................................................................. 54 3.1.1: Gateway-amx plasmids ..................................................................................... 55 3.1.2: 2GFP-T-amx plasmids ...................................................................................... 58 3.2: Yeast Plasmid Construction .................................................................................... 60 3.3: Protein Expression and Purification from Bacterial Constructs ............................. 63 3.4: Protein Expression and Purification from Yeast Constructs .................................. 65 3.5: Protein Expression Analysis by Western Blot ........................................................ 70 3.6: Detection of FAD by Absorption Spectra Analysis................................................ 71 3.7: Preparation of Worm Protein Extracts .................................................................... 71 3.8: Biochemical Assay for Monoamine Oxidase Activity ........................................... 72 3.9: Biochemical Assay for Histone Demethylase Activity .......................................... 73 Chapter 4: Results ............................................................................................................. 75 4.1: Cloning of amx cDNAs into Bacterial Expression Plasmids. ................................. 75 4.2: Expression and Purification of AMX Proteins from Bacteria. ............................... 78 4.3: Cloning of amx cDNAs into Yeast Expression Plasmids. ...................................... 81 4.4: Expression and Purification of AMX Proteins from Yeast. ................................... 82 4.5: FAD Absorption Spectrum. .................................................................................... 85 4.6: Biochemical Activity of Wild-type and Mutant Worm Lysates. ............................ 88 4.7: HDM Activity of AMX Proteins ............................................................................ 90 4.8: MAO Activity of AMX Proteins. ........................................................................... 94 Chapter 5: Discussion And Summary ............................................................................. 101 References ....................................................................................................................... 108 Appendix A: Table 2 – Primer Information .................................................................... 128 Appendix B: Table 3 – Expression Conditions Tested In E. Coli .................................. 130 Appendix C: Nucleotide Sequencing Results ................................................................. 131 Appendix D: Protein Domain Analyses Results ............................................................. 135 Appendix E: Supplementary Figures .............................................................................. 137 8 LIST OF TABLES Page Table 1 - Monoamine Dependent Behavior Assays ......................................................45 Table 2 - Primer Information .......................................................................................128