DISRUPTION OF SOLUBLE EPOXIDE HYDROLASE DIMERIZATION AS A NOVEL THERAPEUTIC TARGET FOR STROKE By Jonathan W. Nelson A THESIS/DISSERTATION Presented to the Department of Molecular and Medical Genetics and the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy April 2013 TABLE OF CONTENTS List of Figures ...................................................................................................................... vi List of Tables ...................................................................................................................... vii List of Abbreviations ......................................................................................................... viii Acknowledgements ........................................................................................................... xiii Dissertation Abstract ........................................................................................................ xvi Chapter 1 Targeting Soluble Epoxide Hydrolase Dimerization As a Stroke Therapy ......... 1 Abstract ........................................................................................................................... 1 Stroke and Society ........................................................................................................... 2 Prevention and Treatment of Stroke .............................................................................. 3 Soluble Epoxide Hydrolase and Stroke ........................................................................... 5 Epoxide Hydrolases...................................................................................................... 5 Soluble Epoxide Hydrolase Protein ............................................................................. 6 sEH and Epoxyeicosatrienoic Acids ........................................................................... 11 sEH Single Nucleotide Polymorphisms (SNPs) ........................................................... 15 sEH Knockout (sEHKO) Mice ...................................................................................... 20 Development of sEH Inhibitors .................................................................................. 22 sEH Inhibitors and Ischemia ...................................................................................... 23 i Are We Ready For a sEH Inhibitor Clinical Trial for Stroke? ...................................... 26 Soluble Epoxide Hydrolase Dimerization As a Novel Therapeutic Target .................... 30 Dimerization and Protein Function ........................................................................... 31 Localization and Protein Function ............................................................................. 34 Peroxisomes and sEH .................................................................................................... 36 Overview of Thesis ........................................................................................................ 37 Chapter 2 Dimerization and Soluble Epoxide Hydrolase Activity .................................... 40 Abstract ......................................................................................................................... 40 Introduction................................................................................................................... 41 Experimental Procedures .............................................................................................. 42 Mutagenesis of sEH Salt-Bridge Residues ................................................................. 42 Creation of sEH-Luciferase construct ........................................................................ 43 Transfection of HEK cells ........................................................................................... 43 Luciferase Activity Assay ............................................................................................ 43 Hydrolase Activity Assay ............................................................................................ 44 Determination of Km and Vmax ................................................................................... 44 Protein Rendering ...................................................................................................... 45 Immunoblotting ......................................................................................................... 45 Statistics ..................................................................................................................... 45 ii Results ........................................................................................................................... 46 Generation of sEH dimerization mutants .................................................................. 46 Split Luciferase System as a Reporter Assay for sEH Dimerization ........................... 48 Dimerization Is Required For sEH Hydrolase Enzymatic Activity .............................. 48 The kinetics of sEH dimerization ............................................................................... 50 Discussion ...................................................................................................................... 57 Chapter 3 Disrupting Dimerization Translocates Soluble Epoxide Hydrolase to Peroxisomes ...................................................................................................................... 63 Abstract ......................................................................................................................... 63 Introduction................................................................................................................... 64 Experimental Procedures .............................................................................................. 66 GFP-sEH and RFP-SKL Plasmids .................................................................................. 66 Primary Cortical Mouse Neuronal Culture ................................................................ 68 Plasmid Purification and Transfection ....................................................................... 68 HEK Cells and Western Blot ....................................................................................... 69 Image Acquisition ...................................................................................................... 69 Image Presentation ................................................................................................... 70 sEH Modeling ............................................................................................................. 71 Statistics ..................................................................................................................... 71 iii Results ........................................................................................................................... 71 Modeling sEH subcellular localization through GFP fusion ....................................... 71 Analysis of sEH subcellular localization ..................................................................... 74 Disrupting sEH dimerization preferentially localizes sEH to peroxisomes ................ 78 Discussion ...................................................................................................................... 83 Chapter 4 Peroxisomal translocation of soluble epoxide hydrolase protects against ischemic stroke injury ....................................................................................................... 87 Abstract ......................................................................................................................... 87 Introduction................................................................................................................... 88 Experimental Procedure ................................................................................................ 90 Expression and purification of TAT-fusion protein .................................................... 90 Western blot and hydrolase assay ............................................................................ 90 Animals and treatment groups .................................................................................. 91 TAT-sEH injection and middle cerebral artery occlusion .......................................... 91 Infarct size analysis .................................................................................................... 92 Immunohistochemistry ............................................................................................. 93 Statistics ..................................................................................................................... 93 Results ........................................................................................................................... 93 Characterization of translocation deficient human soluble epoxide hydrolase ....... 93 iv Translocation deficient human soluble epoxide hydrolase increases infarct damage after stroke ................................................................................................................ 94 Discussion ...................................................................................................................... 97 Chapter 5 Conclusions and Future Directions .............................................................. 103 Conclusions.................................................................................................................