Protein Modification and Catabolic Fates of Lipid
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PROTEIN MODIFICATION AND CATABOLIC FATES OF LIPID PEROXIDATION PRODUCTS by CHUAN SHI Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Dissertation Advisor: Gregory P. Tochtrop, Ph.D. Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________Chuan Shi candidate for the Doctor of Philosophy degree *. Rajesh Viswanathan (signed)_______________________________________________ (chair of the committee) Anthony Pearson ________________________________________________ Michael Zagorski ________________________________________________ Henri Brunengraber ________________________________________________ Gregory Tochtrop ________________________________________________ ________________________________________________ (date) _______________________Dec. 8, 2016 *We also certify that written approval has been obtained for any proprietary material contained therein. This thesis is dedicated to my parents in the deepest appreciation and gratitude for their unconditional love, endless support and continuous encouragement throughout every step in my life TABLE OF CONTENTS Table of Contents ................................................................................................................. i List of Figures ......................................................................................................................v List of Schemes ................................................................................................................. vii List of Tables ................................................................................................................... viii Acknowledgement ............................................................................................................. ix List of Abbreviations ......................................................................................................... xi Abstract ..............................................................................................................................xv Chapter 1. General Introduction ..........................................................................................1 1.1 Oxidative stress ..........................................................................................................2 1.2 Lipid peroxidation ......................................................................................................3 1.3 Fates of LPO products and their relevance on disease states ....................................5 1.3.1 Reactions with proteins and other endogenous nucleophiles .............................5 1.3.2 Metabolic fate ......................................................................................................8 1.4 Research Strategy ....................................................................................................11 1.4.1 Characterization of the enzymes in catabolic pathway of 4-HNE ....................11 1.4.2 Investigation of the relevance of EKODEs in diseases or aging .......................13 1.5 References ................................................................................................................15 Chapter 2. Identification of Relevant Enzymes in the Catabolic Pathways of 4- Hydroxyacids and Lipid Peroxidation Products ...............................................................23 2.1 Introduction ..............................................................................................................24 2.2 Results and Discussions ...........................................................................................28 2.2.1 Kinase activity assay .........................................................................................28 i 2.2.2 Kinase purification ............................................................................................33 2.2.3 Verification of kinase candidates ......................................................................39 2.3 Conclusions ..............................................................................................................43 2.4 Experimental Sections .............................................................................................44 2.4.1 Materials and Methods ......................................................................................44 2.4.2 GHP-CoA kinase activity assay ........................................................................46 2.4.3 Kinase purification ............................................................................................53 2.4.3 Kinase purification ............................................................................................48 2.4.4 Verification of kinase candidate ........................................................................50 2.5 Acknowledgement ....................................................................................................52 2.6 References ................................................................................................................53 Chapter 3. Mechanistic Study of Protein Modification by Epoxyketooctadecenoic Acids (EKODEs) ..........................................................................................................................59 3.1 Introduction ..............................................................................................................60 3.2 Results and Discussions ...........................................................................................63 3.2.1 Mechanistic study of the reactions of EKODE model compounds with analogues of nucleophilic amino acids ......................................................................63 3.2.2 Reactions of EKODEs with cysteine ................................................................68 3.2.3 Mass spectrometric characterization of the modification of human serum albumin (HSA) by trans-EKODE-(E)-IIb .................................................................73 3.3 Conclusions ..............................................................................................................79 3.4 Experimental Sections .............................................................................................80 3.4.1 Materials and Methods ......................................................................................80 ii 3.4.2 Mechanistic study of the reactions of EKODE model compounds with analogues of nucleophilic amino acids ......................................................................81 3.4.3 Reactions of EKODEs with cysteine ................................................................87 3.4.4 Mass spectrometric characterization of the modification of HSA by trans- EKODE-(E)-IIb .........................................................................................................89 3.5 Acknowledgement ....................................................................................................92 3.6 References ................................................................................................................93 Chapter 4. Immunochemical Detection of EKODE-Cysteine Adducts in Oxidative Stress, Aging, and Diseases ..........................................................................................................99 4.1 Introduction ............................................................................................................100 4.2 Results and Discussions .........................................................................................103 4.2.1 Strategies for antigen preparation ....................................................................103 4.2.2 Production and characterization of antibodies against EKODE-cysteine adduct ..................................................................................................................................108 4.2.3 Immunochemical detection of EKODE-cysteine adduct in nervous system...113 4.2.4 Immunochemical detection of EKODE-cysteine adduct in cardiovascular disease and other tissues ...........................................................................................117 4.3 Conclusions ............................................................................................................121 4.4 Experimental Sections ...........................................................................................122 4.4.1 Materials and Methods ....................................................................................122 4.4.2 Synthesis of antigens against EKODE-cysteine adduct .................................123 4.4.3 Production and characterization of anti-EKODE-Cys antibodies ...................126 iii 4.4.4 Immunochemical detection of endogenous EKODE-Cys adducts in biological samples ....................................................................................................................130 4.5 Acknowledgement ..................................................................................................133 4.6 References ..............................................................................................................134 Chapter 5. Future Directions ...........................................................................................140 5.1 Identification of relevant kinase in the catabolic pathways of 4-hydroxyacids .....141 5.2 Protein modification