MOLECULAR MECHANISM AND METABOLIC FUNCTION OF THE S- NITROSO-COENZYME A REDUCTASE AKR1A1 by COLIN T. STOMBERSKI Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Jonathan S. Stamler Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May, 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of COLIN T. STOMBERSKI candidate for the degree of Doctor of Philosophy*. Committee Chair Focco van den Akker Committee Members Jonathan Stamler George Dubyak Mukesh Jain Hung-Ying Kao 03-22-2019 *We also certify that written approval has been obtained for any proprietary material contained therein TABLE OF CONTENTS Table of Contents ………………………………………………………………………… i List of Tables ……………………………………………………………………………. v List of Figures ………………………………………………………………………….. vi List of Abbreviations …………………………………………………………………… ix Acknowledgements …………………………………………………………………….. xi Abstract ………………………………………………………………………………….. 1 Foundation and Experimental Framework ……………………………………………….. 3 Chapter 1: Protein S-nitrosylation: Determinants of specificity and enzymatic regulation of S-nitrosothiol-based signaling …………………………………………….. 5 1.1 Introduction …………………………………………………………………. 6 1.2 S-nitrosothiol specificity …………………………………………………….. 8 1.2.1 Acid-base and hydrophobic motifs ……………………………… 9 1.2.2 Interaction with nitric oxide synthases ………………………….. 13 1.3 S-nitrosothiol stability and reactivity ……………………………………….. 15 1.3.1 RSNO bond chemistry ………………………………………….. 16 1.3.2 Protein SNO—thiol reaction bias ………………………………. 18 1.3.3 SNO sites do not overlap S-oxidation sites …………………….. 19 1.4 Enzymatic denitrosylation ………………………………………………….. 20 1.4.1 The thioredoxin system ………………………………………… 21 1.4.2 LMW-SNO reductases …………………………………………. 23 1.4.3 The GSNO reductase system …………………………………… 24 1.4.4 GSNOR in physiology and pathophysiology …………………… 26 i 1.4.5 The SNO-CoA reductase system ……………………………….. 31 1.5 Specificity in denitrosylation ……………………………………………….. 34 1.5.1 Subcellular localization ………………………………………… 34 1.5.2 Interaction of denitrosylases with substrates …………………… 35 1.6 Summary …………………………………………………………………… 37 Chapter 2: Molecular recognition of S-nitrosothiol substrate by its cognate protein denitrosylase …………………………………………………………………… 48 2.1 Abstract …………………………………………………………………….. 49 2.2 Introduction ………………………………………………………………… 50 2.3 Results ……………………………………………………………………… 52 2.3.1 Molecular modeling of SCoR-based specificity …………………. 52 2.3.2 Essential role of SCoRK127A in SNO-CoA reductase activity ……. 52 2.3.3 SCoR binds the CoA backbone and recognizes the SNO moiety in SNO-CoA ……………………………………….. 54 2.3.4 Identification of targets of SNO-CoA/SCoR-mediated S-nitrosylation/denitrosylation …………………………………… 56 2.3.5 SCoR regulates mitochondrial metabolism ………………………. 59 2.4 Discussion ………………………………………………………………….. 60 2.5 Figures and Tables …………………………………………………………. 63 2.6 Experimental Procedures …………………………………………………… 82 2.6.1 Animals …………………………………………………………… 82 2.6.2 Molecular Modeling ……………………………………………… 82 2.6.3 Generation and expression of recombinant wild-type and ii mutant SCoR ……………………………………………………… 82 2.6.4 Kinetic analysis of recombinant SCoR …………………………… 83 2.6.5 CoA and SNO-CoA bead pull-down assays ……………………… 84 2.6.6 SCoR-dependent SNO-CoA reductase activity in mouse kidney lysate and analysis of protein S-nitrosylation …………………….. 84 2.6.7 Identification of SNO-proteins by iTRAQ-coupled LC-MS/MS … 86 2.6.8 Generation of SCoR mammalian expression plasmid ……………. 88 2.6.9 Western blot analysis……………………………………………… 89 2.6.10 Assay of SCoR activity in cell lysate …………………………… 89 2.6.11 Generation of SCoR-deficient HEK293 by CRISPR/Cas9 …….. 89 2.6.12 Stable overexpression of SCoR ………………………………… 90 2.6.13 Analysis of SNO-proteins in SCoR-deficient and SCoR-overexpressing HEK293 lines …………………………… 90 2.6.14 Metabolic analysis using Seahorse XFe24 Analyzer …………… 91 Chapter 3: S-nitroso-coenzyme A reductase regulates low-density lipoprotein metabolism by modulating circulating proprotein convertase subtilisin/kexin 9 ……… 93 3.1 Abstract …………………………………………………………………….. 94 3.2 Introduction ………………………………………………………………… 95 3.3 Results ……………………………………………………………………… 98 3.3.1 SCoR-/- mice are hypocholesterolemic …………………………… 98 3.3.2 SCoR regulates hepatic LDLR and LDLR is required for SCoR-dependent hypocholesterolemia ………………………….. 98 3.3.3 SCoR regulates circulating PCSK9 ………………………………. 99 iii 3.3.4 SCoR regulates PCSK9 secretion ……………………………….. 100 3.3.5 Chemical inhibition of SCoR reduces circulating PCSK9 and total serum cholesterol ………………………………………….. 101 3.4 Discussion …………………………………………………………………. 103 3.5 Figures …………………………………………………………………….. 105 3.6 Experimental Procedures ………………………………………………….. 118 3.6.1 Mice …………………………………………………………….. 118 3.6.2 Serum Chemistries ……………………………………………… 119 3.6.3 Western Blotting ………………………………………………… 119 3.6.4 Mouse Tissue Analysis by Western blotting and quantitative reverse transcription PCR ……………………………………… 120 3.6.5 Cell Lines and Culture …………………………………………… 121 3.6.6 Generation of HepG2 stably expression SCoR-targeting shRNA .. 122 3.6.7 Cell-based PCSK9 Secretion Assays and SNO-protein Analysis .. 122 Chapter 4: General Discussion and Future Directions ………………………………… 126 Appendix ……………………………………………………………………………… 130 Appendix 2.1 Putative targets of SNO-CoA-mediated S-nitrosylation in mouse kidney lysates …………………………………………………………. 130 Appendix 2.2 Putative targets of SCoR-dependent denitrosylation in HEK293 . 138 Appendix 2.3 Overlapping Proteins in Appendices 2.1 and 2.2 ………………. 142 References ……………………………………………………………………………. 144 iv LIST OF TABLES Table 1.1 S-nitrosylation motif elements from SNO-proteome analyses ……………….. 38 Table 1.2 Known targets of enzymatic denitrosylation ………………………………… 39 Table 2.1 Enzyme kinetics for SCoR/SCoR mutants with substrates ………………….. 63 Table 2.2 Additional enzyme kinetics for SCoR and SCoR mutants …………………… 64 v LIST OF FIGURES Figure 1.1 S-nitrosothiol formation occurs via complexed enzymatic machinery ……… 40 Figure 1.2 Coupled, dynamic equilibria that govern protein S-nitrosylation are regulated by enzymatic denitrosylases ………………………………………………… 41 Figure 1.3 Steady-state protein S-nitrosylation reflects denitrosylase activity ………… 43 Figure 1.4 Differential RSNO reactivity ………………………………………………... 44 Figure 1.5 Enzymatic mechanisms of protein denitrosylation …………………………. 45 Figure 1.6 Stimulus-coupled S-nitrosylation and denitrosylation: cardiomyocytes as an exemplary case …………………………………………………………………… 46 Figure 2.1 Molecular modeling of SNO-CoA within the SCoR active site …………… 65 Figure 2.2 Purification of Recombinant SCoR …………………………………………. 66 Figure 2.3 The conserved Lys127 in SCoR facilitates SNO-CoA reductase activity ….. 67 Figure 2.4 SCoRK127A mutation lowers SNO-CoA reductase activity …………………. 69 Figure 2.5 SCoR recognizes the CoA backbone and SNO moiety of SNO-CoA ……… 70 Figure 2.6 Lys127 provides a positively charged residue near the SCoR active site …… 72 Figure 2.7 SCoRK23A and SCoRW220A do not alter binding to SNO-CoA ……………… 74 Figure 2.8. SCoR regulates SNO-CoA-dependent protein S-nitrosylation in tissue lysates ……………………………………………………………………………. 75 Figure 2.9 SCoR regulates endogenous protein S-nitrosylation ……………………….. 77 Figure 2.10 SCoR activating mutations do not reduce S-nitrosylation ………………… 79 Figure 2.11 SCoR regulates cellular energy metabolism ………………………………. 80 Figure 3.1 SCoR-/- mice are hypocholesterolemic …………………………………….. 106 Figure 3.2 SCoR regulates hepatic LDLR by modulating serum PCSK9 ……………. 108 vi Figure 3.3 SCoR does not alter hepatic SR-BI expression ……………………………... 110 Figure 3.4 SCoR does not regulate the S-nitrosylation status of hepatic LDLR, HMGCR, or ACAT2 ………………………………………………………………….. 111 Figure 3.5 SCoR regulates PCSK9 and LDLR in cell culture models of PCSK9 secretion ………………………………………………………………………………. 113 Figure 3.6 Chemical inhibition of SCoR lowers serum cholesterol via reduced serum PCSK9 …………………………………………………………………………. 115 vii ACKNOWLEDGEMENTS I would like to first thank my thesis advisor, Dr. Jonathan S. Stamler, for the opportunity to pursue my degree in his laboratory and for providing strong mentorship throughout the years. The lessons I learned on how to be a scientist will stay with me throughout my career. Jonathan knew when to challenge me, how to push me to become a better scientist, and when to press me to focus; but he also gave me the freedom to chase scientific ideas, and ultimately we made (in my view) some great discoveries together. I am endlessly grateful for the support from all members of the Stamler Lab. Without these great scientists, my thesis work would not be where it is today. In particular, I want to thank Dr. Hua-lin Zhou and Dr. Divya Seth for their help and support over the years, from scientific discussions to experimental troubleshooting; Dr. Puneet Anand for helping me at the outset of my time in the lab; Zhaoxia Qian for her invaluable work in maintaining our mouse colony and providing any mouse cross that I needed; and Precious McLaughlin for all her technical support. I would also like to acknowledge Dr. Focco van den Akker in the Department of Biochemistry at Case Western Reserve University. Our collaborations with Focco provided insights into both projects presented in this thesis. We can only hope that these productive collaborations continue. Finally, I am ever indebted to my loving and supportive family. My parents, Tom and Jean Stomberski, provided me with all the opportunities