<<

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

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: S-: 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 -base and hydrophobic motifs ……………………………… 9

1.2.2 Interaction with 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 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 ………………………. 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- 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 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--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 kinetics for SCoR/SCoR mutants with substrates ………………….. 63

Table 2.2 Additional 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 …………… 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 in the world and always pushed me to be my best. I wouldn’t be here without all they have given me. And my sister, Susan Merchak, for setting an example of where hard work can take you. Nor

viii would I be where I am without the love and support of my wife, Caitlin O’Connor. From the time we met, she pushed me to be a better scientist. We’ve already spent countless hours talking science and I can’t wait to spend countless more.

ix Molecular Mechanism and Metabolic Function of the S-nitroso-coenzyme A

Reductase AKR1A1

Abstract

by

COLIN T. STOMBERSKI

The majority of nitric oxide’s (NO) biological effect across cell types and tissues occurs through protein S-nitrosylation, the oxidative, posttranslational modification of thiols in proteins by NO to form S-nitrosothiols (SNOs). Through S-nitrosylation,

NO controls protein activity, stability, interactions with other proteins, and subcellular localization to alter cellular function. NO also modifies small molecule thiols, primarily and coenzyme A, to serve as SNO-based signaling molecules. S-nitroso- glutathione (GSNO) and S-nitroso-coenzyme A (SNO-CoA) signal through S-nitrosylation of target proteins and are regulated by enzymatic denitrosylases, proteins that metabolize

GSNO and SNO-CoA. SNO-CoA was recently identified as a conserved metabolic signal transducer regulated by the activity of S-nitroso-coenzyme A reductases (SCoRs). The discovery of SNO-CoA generates numerous, fundamental questions regarding how SNOs derive specificity in signaling, how recognize different SNOs, and endogenous roles of SNO-CoA in mammals. This thesis addresses these questions by providing a mechanistic understanding of how aldo-keto reductase 1A1 (AKR1A1), the mammalian

1 SCoR, specifically recognizes SNO-CoA among myriad S-nitrosothiols, identifying targets of SCoR-dependent denitrosylation in mammalian cells and tissues, and exploring metabolic processes regulated by the SCoR/SNO-CoA system of S-nitrosylation and denitrosylation. Here we use a combination of in silico molecular modeling, in vitro biochemical assays, cell culture models, and animal models to address these questions. We find that SCoR binds SNO-CoA through both the CoA moiety and NO group; the latter finding provides the first evidence of direct SNO recognition by enzymes and explains how

SCoR can identify SNO-CoA among the multitude of SNOs and CoA derivatives. SCoR also controls the S-nitrosylation of metabolic proteins and alters mitochondrial metabolism. Finally, SCoR regulates mammalian cholesterol metabolism by modulating the S-nitrosylation and secretion of the hypercholesterolemic risk factor proprotein convertase subtilisin/kexin type 9 (PCSK9) to increase low-density lipoprotein receptor

(LDLR) stability and lower serum cholesterol, providing a fundamental mechanism by which nitric oxide controls cholesterol homeostasis. Remarkably, chemical inhibition of

SCoR in mice lowers serum cholesterol levels. Overall, the work presented herein investigates SCoR from basic understanding of the structure—function relationship of the enzyme to potential translational application of targeting SCoR in hypercholesterolemia.

2 Foundation and Experimental Framework

Coenzyme A (CoA) is a central carrier molecule used in the activation and transfer of acetyl and acyl groups for both energetic and biosynthetic metabolic processes. The function of CoA is two-fold: i) CoA facilitates transfer of acetyl and acyl groups to other nucleophiles via a reactive, nucleophilic thiol group; and ii) CoA provides a carrier backbone that serves as a recognition motif for targeted binding to enzymes.

While these characteristic of CoA allow its derivatives to participate in a number of critical enzymatic reactions across metabolism, they also allow CoA to act as a regulatory molecule. Transfer of acetyl, malonyl, succinyl, glutaryl groups to the -amino group of residue side chains in proteins (i.e. lysine , malonyaltion, etc.) or transfer of acyl groups to cysteine residues in proteins (i.e. S-palmitoylation) are key mechanisms of posttranslational control of metabolic protein function. However, a central, broad-based signaling role for CoA had not previously been considered.

Nitric oxide (NO) reacts with thiols to generate S-nitrosothiols (SNOs) in both proteins and small molecules. At the outset of my thesis work, our laboratory had recently discovered that NO could attach to CoA, forming S-nitroso-coenzyme A (SNO-

CoA), and that SNO-CoA could transfer NO to target proteins to affect protein S- nitrosylation. In essence, we have identified for the first time a novel, CoA-derived metabolic signal that transduces through transfer of NO to cysteine residues. Further, we had also found a class of enzymes that control the level of SNO-CoA in cells and tissues

– the SNO-CoA reductases (SCoRs) – to provide a regulated, NO-based signaling mechanism in metabolism. The principle discovery of the SNO-CoA/SCoR system of protein S-nitrosylation changes how we think about both CoA biology and the

3 role/mechanics of SNO-based signaling in cellular metabolism, and opens an entirely new field of study regarding how this system is affected in cells. New questions include: how is SNO-CoA initially made and distributed to target proteins?; how is its production regulated in time/space by signaling inputs and cellular conditions?; how do enzymes recognizes SNO-CoA among all CoA derivatives in a cell?; what metabolic processes are critically regulated by SNO-CoA?; how does CoA-based NO signaling interface with other CoA-based posttranslational modifications?; and so forth.

The work in this thesis begins to tackle these questions by considering the mammalian SCoR, AKR1A1. In particular, we study how AKR1A1 specially binds

SNO-CoA (as opposed to other small molecule SNOs) and how it may preferentially bind

SNO-CoA among CoA derivates, providing important insights into how this system (and other SNO-based systems) operates at the molecular level. Further, we use AKR1A1- deficient cellular and animal systems to interrogate the role of SNO-CoA-mediated metabolic signaling in physiology. To understand how these findings integrate into the wider field of SNO biology requires a thorough background on mechanisms governing protein S-nitrosylation, as presented below.

4 CHAPTER 1:

Protein S-nitrosylation: Determinants of Specificity and Enzymatic Regulation of S-

nitrosothiol-based Signaling

Colin T. Stomberski1,2, Douglas T. Hess1,3, and Jonathan S. Stamler1,3,4*

From the 1Institute for Transformative Molecular Medicine; 2Departments of

Biochemistry and 3Medicine, Case Western Reserve University, Cleveland, OH 44106;

4Harrington Discovery Institute, University Hospitals Cleveland Medical Center,

Cleveland, OH 44106

*This research was originally published in Antioxidants and Redox Signaling. Final

publication is available from Mary Ann Liebert, Inc., publishers at

https://dx.doi.org/10.1089/ars.2017.7403

© 2018 Mary Ann Liebert, Inc., New Rochelle, NY.

5 1.1 Introduction

Reversible posttranslational protein modifications are essential regulators of cellular function. Oxidative modifications of cysteine (Cys) thiols provide a mechanism for redox-based control of protein activity. Generally, reversible Cys modifications subserve signaling functions whereas irreversible modifications result in loss of regulatory control (1, 2). Reversible Cys modifications include Cys sulfenylation (S-OH),

S-glutathionylation (S-SG), and S-nitrosylation (S-NO). Chief among these modifications with respect to prevalence and demonstrated physiological signaling functions is S- nitrosylation, a covalent modification in which a nitric oxide (NO)-derived group is attached to a Cys thiol to generate an S-nitrosothiol (SNO), providing a ubiquitous mechanism for cellular signaling. NO was first identified as the endothelium-derived relaxing factor and was demonstrated to vasodilate blood vessels through activation of guanylyl cyclase to produce the second messenger cGMP (3–5). However, it is now understood that NO exerts the majority of its biological influence through protein S- nitrosylation, affecting myriad cellular processes in physiology and pathophysiology, including the regulation of both activation and deactivation of guanylyl cyclase (2, 6–10).

Thus, NO may be viewed as the prototypic redox-based signal.

NO is predominantly generated by reductases in microbes and plants and by three isoforms of NO synthase (NOS) in mammalian cells: neuronal NOS (nNOS; NOS1), inducible NOS (iNOS; NOS2) and endothelial NOS (eNOS; NOS3). In principle, SNOs can initially form via multiple chemical routes that formally entail a one-electron oxidation, including reaction of NO with thiyl radical, transfer of the NO group from metal-NO complexes to Cys thiolate, or reaction of Cys thiolate with nitrosating species generated by

6 NO auto-oxidation, exemplified by N2O3 (11). However, the emerging evidence favors a primary role for in catalyzing de novo S-nitrosylation in situ (12–16), including under both aerobic and anaerobic conditions. Indeed, a multiplex enzymatic machinery governing the of SNOs was recently described in bacteria

(17). This machinery includes: i) a or that generates nitric oxide; ii) a novel class of enzyme (SNO synthases) that synthesizes SNOs from NO; and iii) trans-S-nitrosylases (Fig. 1.1). The latter group of enzymes can then transfer SNOs between donor and acceptor Cys thiols via trans-S-nitrosylation (18), which likely acts as a primary mechanism to propagate SNO-based signals in physiological settings. S- nitrosylation occurs both in proteins, producing S-nitroso-proteins (SNO-proteins), and in low-molecular-weight (LMW) thiols including glutathione and coenzyme A, generating S- nitrosoglutathione (GSNO) and S-nitroso-coenzyme A (SNO-CoA), respectively (19, 20).

Protein and LMW SNOs (LMW-SNO) exist in thermodynamic equilibria, which are governed by removal of SNO-proteins by SNO-protein denitrosylases (namely Trx1/2 and

Trp14) and/or LMW-SNOs by GSNO and SNO-CoA metabolizing activities (Fig. 1.2). In effect, NO-based is represented by equilibria between LMW-SNOs and protein SNOs, and between SNO-proteins linked by transnitrosylation. Enzymatic governance of these equilibria thereby provides a basis for regulation of NO-based signal transduction.

SNO-CoA is a recently identified (20) metabolic signaling molecule that exerts its function by regulating the S-nitrosylation status of target proteins. SNO-CoA levels are controlled by a class of NADPH-dependent enzymes known as SNO-CoA reductases

(SCoRs). To date, one yeast SCoR (NADP-dependent dehydrogenase 6, Adh6) and

7 one mammalian SCoR (aldo-keto reductase 1A1, AKR1A1) have been identified (20). The broad role of CoA in cellular metabolism suggests that SNO-CoA-based regulation of protein S-nitrosylation could impact a wide range of cellular processes; however, mammalian protein targets of SNO-CoA and cellular functions regulated by the SNO-

CoA/SCoR system are not well defined. Further, the identification of a novel group of denitrosylating enzymes (SCoRs) regulating S-nitrosylation in a manner distinct from previously known enzymatic classes (GSNO reductases and thioredoxins) implies mechanisms governing the specificity of denitrosylating reactions and underscores our general lack of understanding of how denitrosylases recognize their cognate substrates, whether SNO-proteins or LMW-SNOs. The goal of this thesis is to address these two topics

– regulation of metabolism by SNO-CoA and denitrosylase targeting specificity – as they relate to the mammalian SNO-CoA reductase, AKR1A1. To do so first requires an understanding of general mechanisms governing SNO specificity and reactivity, and how denitrosylases have previously been used to probe cellular pathways and processes regulated by SNO-based signaling.

1.2 S-Nitrosothiol specificity

It is well established that protein S-nitrosylation exhibits remarkable spatiotemporal specificity in the targeting of protein Cys residues (2, 21, 22). Physiological amounts of NO typically target one or few Cys within a protein and this is sufficient to alter protein function and associated physiology or pathophysiology (23–25). It has emerged as a general rule that S-nitrosylation and alternative S-oxidative modifications, in particular those mediated by reactive species, most often target separate populations of Cys and, whether the same or different Cys are targeted, exert disparate

8 functional effects (15, 26). Thus, proteomic analyses of Cys modifications have revealed that, under physiological conditions, there is little overlap between different redox-based

Cys modifications (26, 27). Functional specificity is well illustrated in the case of the bacterial transcription factor OxyR, in which S-nitrosylation versus oxygen-based oxidative modification of a single, critical Cys activates distinct regulons (15, 28). Also, in the case of mammalian hemoglobin, S-nitrosylation versus oxidative modification of the same, single Cys mediate vasodilation and vasoconstriction respectively (29). However, S- nitrosylation and alternative oxidative modifications may also target distinct Cys to exert coordinated effects as in the case of the ryanodine receptor/Ca2+-release channel of mammalian skeletal muscle (RyR1), where S-nitrosylation of a single critical Cys and O2- based oxidation of a distinct set of Cys work in concert to activate Ca2+ release from the sarcoplasmic reticulum (30–34). There are a variety of mechanisms implicated in targeting

S-nitrosylation of specific protein substrates and Cys residues within target proteins, as discussed below.

1.2.1 Acid-base and hydrophobic motifs

A role for an acid-base motif in determining the specificity of protein S- nitrosylation was first suggested by the analysis of S-nitrosylation of Cys93 of hemoglobin (Hb) (35). In this model, a residue proximal to Cys93 in the oxygenated, R-state of Hb facilitates base-catalyzed S-nitrosylation whereas a proximal residue in the deoxygenated T-state facilitates acid-catalyzed denitrosylation, coupling oxygenation status of Hb to SNO formation and release in the respiratory cycle

(35, 36). Thus, close proximity of charged acidic and basic side chains would regulate S- nitrosylation by influencing both thiol pKa (and thus reactivity) and SNO stability.

9 Subsequent analysis suggested a more general acid-base motif, (K,R,H,D,E)C(D,E), where proximity of the acidic residue (D,E) is crucial (35). Acid-base motifs may also emerge from protein tertiary structure, as demonstrated for adenosyltransferase (MAT)

(37). S-nitrosylation of Cys121 of MAT is facilitated by distal primary sequence residues that are brought into proximity of Cys121 by tertiary protein structure: Arg357 and Arg363, which lower Cys121 pKa (37). Tertiary structure-derived acid-base motifs have also been shown to direct S-nitrosylation in caspase-3 and aquaporin-1 (23).

It is important to note that the mechanisms through which acidic and basic side chains direct protein S-nitrosylation are not restricted to control of target thiol pKa. In the case of MAT, Asp355 is proximate in tertiary structure to the target Cys121, and the acidic side chain of Asp355 promotes donation of an NO group from GSNO (37). Similarly,

Savidge et al. (38) identified a conserved acid-base motif in the Cys domain of

Clostridium difficile exotoxins TcdA and TcdB, in which tertiary structure brings Glu743 and His653 in proximity to catalytic Cys698 of TcdB. Mutation of Glu743 diminishes S- nitrosylation of Cys698, likely by altering the ability of GSNO to bind in a transnitrosylating orientation. A modified acid-base motif (that included hydrophobic elements) to predict GSNO-mediated protein S-nitrosylation was proposed for the bacterial transcription factor OxyR, based on oriented GSNO binding (2, 28). Docking analysis of multiple proteins has confirmed GSNO binding sites that likely mediate protein S- nitrosylation (2, 39). An expanded acid-base motif, representing and bases within 8Å of sites of S-nitrosylation, has also been implicated in protein-protein interactions that may subserve transnitrosylation, as described below (39).

10 Hydrophobic environments, including membranes or those generated by protein structure and/or protein-protein interactions, can facilitate the formation of protein S- nitrosothiols (23). Regions of hydrophobicity within a protein can provide sites of micellar by concentrating NO and O2 and thereby promote formation of nitrosating species, as was demonstrated for SNO-albumin, or may channel NO to target thiols, as was hypothesized for the transfer of NO from to Cys thiol in hemoglobin (23, 40–

42). Conceivably, interactions with transition metal (iron) nitrosyls within (or adjacent to) hydrophobic regions can also mediate protein S-nitrosylation, as in the cases of non-heme dinitrosyl iron complexes (2, 14, 27). Radical nitrosylating species may also be stabilized in hydrophobic compartments, promoting S-nitrosylation (2, 11). Hydrophobicity might play a role in S-nitrosylation of COX-2, Src , and tubulin at solvent-inaccessible Cys

(6, 22, 43–46). The importance of local hydrophobicity is exemplified by RyR1, in which one of fifty free Cys is targeted for S-nitrosylation and that Cys lies within a hydrophobic -binding region (31, 32).

Proteomic analyses of S-nitrosylation sites have expanded understanding of the role of motifs in targeting S-nitrosylation, in particular by pointing to the potential importance of residues more distal in primary sequence to the targeted SNO-Cys and thereby of trans-

S-nitrosylative interactions between target proteins and both SNO-proteins and LMW-

SNOs (Table 1). Probing of dbSNO 2.0 identified enrichment of charged amino acids in the primary sequence and tertiary structure near SNO-Cys (47). Greco et al. (48) found evidence of a primary sequence acid-base motif in SNO-proteins when the sequence was expanded to include residues -6 and +6 from the target Cys, and identified SNO-Cys within hydrophobic regions of proteins. Analysis of the endogenous mouse liver proteome

11 identified a linear acid-base motif around SNO-Cys and showed preferential location at accessible surfaces of α-helices and coils (27). The conformational flexibility of these secondary structures coupled with the presence of charged residues would aid in protein- protein interactions or protein-LMW thiol interactions to facilitate transnitrosylation.

Marino and Gladyshev (39) analyzed 70 known SNO-Cys sites and provided a spatially expanded acid-base motif within 8Å of the target Cys that might facilitate SNO-forming interactions. SNO site analysis of a dataset derived from treatment with S-nitrosylating agents (49) confirmed acid-base motifs while also emphasizing the importance of local hydrophobicity, and correlated acid-base motifs with -helices and hydrophobic motifs with -sheets. It also identified a role for the +2 position in mediating protein-protein interactions, a position independently shown to facilitate the interaction of GAPDH and

SIRT1 for transnitrosylation (50). Lee et al. (51) identified a linear hydrophobic motif and mutagenic analysis eliminated S-nitrosylation in some motif-containing proteins, validating its predictive power. Recently, Jia et al. (52) identified a mixed acid/ base and hydrophobic motif that is recognized by the cognate transnitrosylase S100A9. Specifically, a complex of iNOS and S100A8/A9 targets proteins with the motif (I,L)XCXX(D,E) for transnitrosylation by S100A9. Introduction of the SNO motif into naive proteins conferred targeting by S100A8/9 (52).

In sum, work identifying protein S-nitrosylation motifs demonstrates multiplex determination of the basis of specificity: charged residues or local hydrophobicity may influence Cys reactivity or may facilitate protein-protein and protein-LMW thiol interactions to target Cys for S-nitrosylation/trans-S-nitrosylation, and these determinants can arise from primary, tertiary, or quaternary structure, whereas thiol pKa is not

12 necessarily predictive of S-nitrosylation (27, 39). It is important to note that, in general, the emerging centrality of transnitrosylation implies an important role for protein tertiary structure that determines solvent accessibility/surface exposure of target thiols, which will not be captured by motifs based on primary sequence or secondary structure per se. The multiplex determination of S-nitrosylation thus underlies the specificity of S-nitrosothiol formation that allows it to act as a broad signaling mechanism. Indeed, computational analysis of the annotated protein data-bases using predicted SNO motifs indicates that

~70% of the proteome may be targeted by S-nitrosylation, mainly at conserved Cys residues (53). However, proteomic analysis of endogenous SNO sites, rather than those from samples treated with RSNO donors (as has generally been the case) may provide a more physiological picture of predictive motifs, inasmuch as supra-physiological amounts of RSNO donors may S-nitrosylate Cys that would not be targeted physiologically or may catalyze higher oxidation states that would not occur in vivo.

1.2.2 Interaction with nitric oxide synthases

Compartmentalization of substrates for S-nitrosylation with a NOS, which may entail their interaction with NOS either directly or through scaffolding proteins, provides a mechanism for specific targeting. High local concentrations of NO may promote the formation of nitrosating species (such as N2O3) (54). Intriguingly, each isoform of NOS is

S-nitrosylated, potentially through metal auto-catalysis, and may thereby act as a transnitrosylating partner for scaffolded proteins, either directly or via formation of LMW-

SNOs (55–60). NOS expression is specific at the level of organs, cells, and subcellular compartments (61). In the heart, nNOS is localized to the sarcoplasmic reticulum whereas eNOS is localized to caveolae; consequently, nNOS regulates RyR2 S-nitrosylation and

13 eNOS regulates L-type Ca2+ channel S-nitrosylation to affect protein activity and myocyte contractility (62, 63). Localization of eNOS to the Golgi generates a local NO pool capable of targeting S-nitrosylation to compartmentalized proteins and addition of a nuclear localization signal to eNOS increases nuclear S-nitrosylation (64). The finest grain of compartmentation is represented by the binding to separate regions of an individual target protein by different NOSs that results in the S-nitrosylation of different Cys (unpublished observations).

Numerous NOS-protein interactions have been demonstrated to target protein S- nitrosylation, either through direct interaction with the target protein or through a protein scaffold. nNOS scaffolds with the n-methyl-D-aspartate receptor (NMDAR) through PSD-

95; flux through the NMDAR activates nNOS to target both NMDAR and PSD-

95 for S-nitrosylation (65–68). The scaffold protein CAPON mediates the interaction of nNOS and the G-protein Dexras1 to target Dexras1 S-nitrosylation (69). nNOS can bind directly and S-nitrosylate in pancreatic -cells, coupling insulin-stimulated

NO production to glucokinase activity (70). Following stimulation of the 2-adrenergic receptor, eNOS interacts with and S-nitrosylates -arrestin 2, GRK2, and dynamin to regulate 2-adrenergic receptor trafficking (36, 71–73). iNOS directly binds and activates

COX-2 via S-nitrosylation of a single Cys residue (43). Jia et al. (52) merged the concepts of NOS scaffolding and NO targeting. A complex including iNOS and S100A8/A9 provides both a source of NO and a mechanism for targeted transnitrosylation by S100A9 of multiple proteins with the motif I/L-X-C-X2-D/E. Interestingly, S100A9 expression is increased in mouse liver lacking a key denitrosylase, potentially identifying crosstalk between transnitrosylating and denitrosylating pathways (74). The co-localization of NOSs

14 with target proteins, either through subcellular compartmentalization or protein-protein interactions, is thus an important mechanism in achieving S-nitrosylation specificity and, in some cases, may initiate transnitrosylation cascades to subserve NO signaling.

1.3 S-Nitrosothiol stability and reactivity

Cellular SNO-protein levels are governed by rates of both S-nitrosylation and denitrosylation. SNOs are readily detected in situ under basal conditions and some protein

SNOs are stable in the presence of NOS inhibitors; further, protein S-nitrosylation increases upon inhibition of denitrosylases, indicating that steady-state S-nitrosylation is a function of denitrosylase activity (i.e. enzymatic breakdown) (20, 75–82) (Fig. 1.3).

Formally, RSNO decomposition may occur through a variety of mechanisms. Reducing agents, metal ions, heat, UV light, reactive oxygen species, and nucleophiles can mediate

SNO decomposition through homolytic and heterolytic cleavage of the SNO bond to radical or ionic species (83, 84). Homolytic bond dissociation energies of RSNOs have been estimated between 20-32 kcal/mol, corresponding to spontaneous decomposition rates of seconds to years; since endogenous SNO probably trend towards the upper end of this range, homolysis is unlikely to be physiologically relevant (83, 85, 86). Although free metals, in particular Cu2+, can rapidly decompose RSNOs in vitro, metals are often sequestered in vivo and probably do not contribute appreciably to physiological denitrosylation (18). Thiols are abundant in cells and can react with S-nitrosothiols to initiate NO transfer between thiols (primary reaction channel) or facilitate NO release and formation (in some cases). Both mechanisms are utilized by enzymatic denitrosylases to reduce SNO-proteins (Fig. 1.2 and vide infra), providing the principal physiological means of denitrosylation. This section discusses control of SNO stability and

15 reactivity before considering enzymatic mechanisms for protein denitrosylation, which predominate in situ and are required for physiological signaling.

1.3.1 RSNO bond chemistry

Initial studies on the stability of LMW RSNOs highlighted the importance of the

R-group in RSNO stability. Electron-withdrawing R-groups (SNO-destabilizing) inhibited

Bacillus cereus spore outgrowth compared to electron-donating R-groups (SNO- stabilizing) (1, 87). Primary and secondary RSNOs decomposed rapidly whereas tertiary

RSNOs, such as S-nitroso-N-acetyl-penacillamine (SNAP), were stable almost indefinitely and could be isolated for storage (83). Roy et al. (88) proposed a model in which tertiary

RSNOs prefer a resonance structure with S=N double-bond character, increasing the strength of the S-N bond and reducing NO lability. However, a kinetic (rather than thermodynamic) basis for “stability” of tertiary RSNOs is likely: the bulky R-group of tertiary RSNOs sterically hinders reactivity (85, 89–91). Indeed, primary and tertiary SNOs adopt planar cis or trans conformations, respectively, with the cis conformation slightly favored thermodynamically over trans (90). A large energy barrier to interconversion between conformers is consistent with S=N double bond character (89), but such conformational restraints, which limit bond flexibility (to increase stability) may apply to either primary or tertiary RSNOs. By contrast, non-planar orientations of RSNO are intrinsically less stable. bonding and other interactions, as may occur in proteins, may also influence SNO stability. Thus, the protein R-group might affect protein SNO stability and reactivity through a variety of mechanisms (discussed further below).

Recently, Paige et al. (92) demonstrated that a subset of proteins S-nitrosylated by

GSNO in cell lysates were relatively resistant to denitrosylation by GSH, pointing to

16 changes in protein conformation that altered target Cys-SNO accessibility or SNO bond chemistry. Similarly, treatment of spinal cord explants with GSNO resulted in the formation of SNO-proteins and individual SNO-proteins were denitrosylated at substantially different rates following subsequent addition of GSH (93). The crystal structure of S-nitroso-hemoglobin (SNO-Hb) (94) has provided important structural insights into SNO stability. Within the R-state of Hb, Cys93-SNO is buried in a hydrophobic pocket inaccessible to solvent thereby promoting stability; subsequent de- oxygenation of hemoglobin and transition to the T-state exposes SNO to the aqueous environment, facilitating liberation of the NO group for vascular bioactivity (95, 96). More generally, S-nitrosylation of proteins may alter charge distribution surrounding the target

Cys, which may modulate protein-protein or protein-LMW thiol interactions to affect SNO stability and reactivity (39, 97).

S-nitrosothiols may accept electrons and participate in various interactions with the protein backbone (2, 94). The protein R-group may thus influence the nature of the SNO moiety, including the S-N bond itself. Despite demonstrated planarity of the SNO group in crystal structures of small molecule RSNOs (which suggests S-N double bond character), the S-N bond in most RSNOs (1.75-1.80Å) is longer than exemplary S=N double bonds

(89, 98, 99), consistent with the propensity for transnitrosylative reactions (Fig. 1.4).

Various interactions with atoms of the SNO group may alter transnitrosylation kinetics.

Metal or Lewis acid coordination with sulfur destabilizes RSNOs and promotes decomposition; coordination with or oxygen stabilizes RSNOs (100, 101).

Timerghazin et al. (102) proposed a resonance model of the RSNO bond that predicts three structures: the putative RSNO resonance structure with a S-N single-bond and a

17 zwitterionic structure with a S=N double-bond, and a novel ionic structure in which the

RSNO is represented as a non-bonded ion pair. This model provides a framework for understanding SNO-thiol reactions, chiefly the propensity for trans-S-nitrosylating or S- thiolating reactions to occur: interactions favoring electrophilicity at the nitrogen atom would promote transnitrosylation reactions, likely through disulfide (SNO2) formation (103); conversely, interactions favoring sulfur atom electrophilicity would preference S-thiolation (disulfide forming) reactions (Fig. 1.4). Proteins therefore could exert a high degree of control over SNO-thiol reactions through the interaction of charged residues with the SNO bond or by modulating the local chemical environment (104). In this sense, the reactive fate of the SNO-Cys is dictated by the local environment, with protein structure and characteristics of the aqueous milieu determining whether the SNO- protein—thiol reaction will be trans-S-nitrosylative or S-thiolative. Empirically and thermodynamically the general case strongly favors trans-S-nitrosylation, as discussed below.

1.3.2 Protein SNO—thiol reaction bias

The identification of an increasing number of Cys-to-Cys transnitrosylases has established a critical role for transnitrosylation in NO-based signaling (6, 29, 50, 52, 105).

Enzymatically targeted S-nitrosylation may rationalize empirical evidence that transnitrosylation reactions of RSNO predominate under physiological conditions.

Mechanistically, activation barriers for thiolate attack at the N atom (in SNO) are reportedly lower than barriers for attack at the S atom, and transnitrosylation products are predicted to be more thermodynamically favorable (106). Reaction of thiolate with the S atom of RSNO generates the nitroxyl anion (NO-) leaving group, which is a spin-forbidden

18 reaction; conditions conducive to HNO generation, which would be required to make this energetically favorable (107–110), may be disfavored by physiologically relevant proteins.

By contrast, the model reaction of GSNO with GSH in vitro supposes both N- and S- directed thiolate attack to rationalize myriad observed products; however, the control exerted by protein R-groups on SNO-generating thiol reactions is absent in this model, and low-yield S-directed reactions are observed because of high reactant concentrations (111,

112). Reaction of GSNO with an engineered protein nanopore primarily resulted in transnitrosylation reactions at physiological pH (113). In addition, SNAP does not participate in S-thiolation reactions, likely due to R-group steric hindrance (113).

1.3.3 SNO sites do not overlap S-oxidation sites

Examination of the endogenous SNO-proteome has provided information regarding the overlap between S-nitrosylation and other S-oxidative modifications at a given Cys.

Doulias et al. (27) identified 328 SNO-Cys, none of which overlapped with Cys that participate in disulfide-bond formation or were S-glutathionylated. Gould et al. (26) also found minimal overlap between SNO-Cys and annotated disulfide bonds, consistent with predictions based on structural analysis that SNO-Cys were highly unlikely to undergo disulfide formation. Although S-nitrosylation might in principle promote S- glutathionylation, eNOS-deficient mice exhibited little change in the S-glutathionylation proteome (26, 114). The case of RyR1 is illustrative. It possesses ~100 Cys residues, many of which are subject to oxidation in vivo. However, only one is modified by S-nitrosylation and that Cys is not included among those oxidized to either disulfide or sulfenic acid (34).

Vicinality may provide a unique context in which S-thiolation reactions are preferred due to high localized thiol molarity (84), and indeed disulfide bond formation within some

19 proteins can be catalyzed by S-nitrosylation (84, 115, 116). Nonetheless, it should be noted that examples of NO-catalyzed disulfide formation by endogenous NO (i.e. physiological amounts as opposed to high concentrations of NO donors) have, to the best of our knowledge, not been reported. Moreover, current methods of detection are unable to distinguish between disulfide bonds and nitroxyl (SNO2), and thus some putative S-nitrosylation-catalyzed disulfides could instead represent SNO2s. Taken together, current data suggest that physiological SNOs exist primarily as a distinct population, and a confluence of protein-mediated interactions and protein-driven influence on S-nitrosothiol (versus thiol) reactivity, exemplified in protein transnitrosylation reactions, achieves specificity for SNO-based modifications (among the different oxidative

Cys modifications) and determines S-nitrosothiol reactivity. As an illustrative example of multiple determination, the interaction between the anion exchange protein AE1 and SNO- hemoglobin, which results normally in transnitrosylation of AE1 (N-directed attack), leads instead to disulfide formation (S-directed attack) following disruption of the native orientation between SNO in hemoglobin and thiol in AE1 (29).

1.4 Enzymatic denitrosylation

Because protein S-nitrosylation is a ubiquitous post-translational modification that operates across cell types and phylogeny to convey the influence of NO, the requirement for enzymatic machinery governing SNO-protein denitrosylation is evident. Presently, two major classes of denitrosylating enzymes comprising seven proteins have been identified:

SNO-protein denitrosylases, typified by the thioredoxin (Trx) system, and LMW-SNO denitrosylases, including the GSNO reductase and SNO-CoA reductase systems (Table 2).

20 Additional denitrosylase activities have been reported (19), which remain to be validated under physiological conditions.

1.4.1 The thioredoxin system

The Trx system was initially identified as a major component of cellular redox homeostasis through its role as a disulfide reductase (117). The Trx system consists of thioredoxin-1/2 (Trx), (TrxR) and NADPH. Using a dithiol active- site (C-X-X-C), Trx1 reduces target protein disulfide bonds with concomitant oxidation of

Cys32 and Cys35 of Trx1. Subsequent reduction of Trx1 by TrxR is coupled to NADPH consumption, regenerating reduced Trx1. In addition to its role in disulfide reduction, the

Trx system plays a major role in SNO-protein denitrosylation (118–120). Trx-dependent denitrosylation likely proceeds through mixed disulfide formation via attack of the more nucleophilic active site Cys (Cys32 in Trx1) on the sulfur of the SNO bond, release of nitroxyl anion (as HNO), resolution of the mixed disulfide by the second active site Cys to form oxidized Trx, and reduction by TrxR (120) (Fig. 1.5A). Support for this mechanism was found in studies where Trx modified by mutation of the resolving Cys was employed to “trap” target SNO-proteins (121, 122). This analysis showed enhanced interaction of

Trx with target proteins under conditions of elevated NO or SNO, whereas treatment with

H2O2 (meant to oxidize proteins and form disulfide bonds) resulted in comparatively little protein trapping (121). These results indicate that SNO-proteins are a critical cellular substrate for Trx. A proposed, alternative initial step in Trx-mediated denitrosylation is transnitrosylation from the target protein to the active site cysteine of Trx and subsequent release of nitroxyl anion (or HNO) (118, 119). Trx primarily exists in its reduced form due to TrxR activity, which may facilitate its role as a denitrosylase. Trx has also been reported

21 to metabolize GSNO, but inefficiently, and a physiological role for this activity has not been demonstrated (118, 123). The thioredoxin-interacting protein (TXNIP) inhibits Trx denitrosylating activity; endogenous NO relieves this inhibition by repressing TXNIP, providing a mechanism to dynamically regulate Trx-mediated protein denitrosylation in response to NO (81).

Numerous substrates for Trx-mediated denitrosylation have been identified (Table

2). Constitutive and Fas-induced SNO-caspase-3 denitrosylation in the cytosol and mitochondria was ascribed to cytosolic Trx1 and mitochondrial Trx2, respectively (78,

118, 120). Trx also denitrosylates NF-κB following cytokine stimulation, further illustrating the importance of stimulus-coupled denitrosylation in activation of immune signaling (124). Trx likely mediates denitrosylation of insulin signaling components in adipocytes, a finding of particular importance since iNOS is induced in obesity (125, 126).

Proteomic analyses have identified numerous proteins with a wide variety of cellular functions as targets of Trx-mediated denitrosylation, aiding in the elucidation of new roles for endogenous Trx-mediated denitrosylation (80, 121, 127–129). Further, analysis of targets of denitrosylation by Trx identified the motifs C-X5-K and C-X6-K within targeted

SNO-proteins (130).

In addition to its role as a denitrosylase, Trx1 has been identified as a trans-S- nitrosylase (131). Trx1 in the oxidized state is S-nitrosylated at Cys73, which mediates the

S-nitrosylation of caspase-3 and other targets, and uncouples Trx1 transnitrosylase and denitrosylase activities (58, 132, 133). The stability of SNO-Trx is regulated by both Trx- and GSH-mediated denitrosylation (119). Interestingly, charged residues proximal to

Cys73 are required for Trx transnitrosylase activity, consistent with identified motif

22 elements facilitating transnitrosylation reactions (132). Additional Trx1 transnitrosylation motifs have been proposed that involve proximal residues (133). Of note, quantitative allows for the distinction between targets of Trx-mediated

S-nitrosylation and denitrosylation, and may aid in delineating their roles (129).

Thioredoxin-related protein of 14 kDa (Trp14) was recently identified as having denitrosylating activity towards GSNO and SNO-proteins, including caspase-3 and cathepsin B, and this activity was dependent on both TrxR1 and NADPH (134). Future work will be necessary to identify a role for Trp14 denitrosylating activity in vivo.

Similarly, protein disulfide (PDI) has been reported to catalyze the denitrosylation of GSNO and SNO-PDI (135), though an in vivo role for this activity has not been established.

1.4.2 LMW-SNO denitrosylases

Two of the most abundant LMW cellular thiols are glutathione (GSH) and coenzyme A (CoA). GSH is present in cells up to 10mM and is concentrated in airway lining fluid, where endogenous GSNO was first discovered (136); coenzyme A levels significantly vary by cellular compartment, with concentrations near 2.5mM in mitochondria and 0.05mM in the cytosol (137). Both GSH and CoA can interact with and denitrosylate target proteins via transnitrosylation, generating GSNO and SNO-CoA, respectively (Fig. 1.2). Metabolism of GSNO and SNO-CoA by cognate denitrosylases

(GSNO reductase (GSNOR); SNO-CoA reductase (SCoR)), prevents further transnitrosylating activity of these RSNOs, which are in equilibrium with SNO-proteins.

To date, GSNOR and carbonyl reductase 1 (CBR1) have been identified as mammalian

GSNO reductases (138–140), and GSNOR is conserved from bacteria to man; alcohol

23 dehydrogenase 6 (Adh6) and aldo-keto reductase 1A1 (AKR1A1) have been identified as

SCoRs in yeast and mammals, respectively (20). S-nitroso-cysteine (141) and S-nitroso- homocysteine (142, 143) have also been described as LMW-SNOs that may exist in equilibrium with SNO-proteins to regulate protein S-nitrosylation, although dedicated enzymatic mechanisms addressing these LMW-SNOs have yet to be identified and their physiological roles remain to be established.

1.4.3 The GSNO reductase system

Purification of NADH-dependent GSNO-metabolizing activity identified class III (ADH5 in ) as a phylogenetically conserved GSNO reductase (138, 139). ADH5 is found in most tissues, with highest activity in liver (144).

GSNO, the only SNO substrate for ADH5, represents the most efficient substrate for

ADH5, and the enzyme was re-designated as GSNOR (138, 139, 144). GSNO has been detected in airways, but not in cells (136, 139), pointing to efficient metabolism by

GSNOR. Reduction of GSNO proceeds via hydride transfer from the NADH to the nitrogen of the SNO bond with subsequent solvent-derived protonation of the SNO oxygen atom to generate an S-(N-hydroxy) intermediate that resolves to glutathione sulfinamide (138, 145) (Fig. 1.5B). Further, in the presence of GSNOR, treatment of SNO- proteins with GSH in vitro diminishes SNO levels, and addition of NADH causes a sharp decline in the levels of both SNO-proteins and LMW-SNOs (GSNO) (80). Genetic deficiency of GSNOR increased GSNO and SNO-protein levels in situ, indicating GSNO as a physiological target of GSNOR in vivo. These findings demonstrate that LMW-SNOs and SNO-proteins are in a cellular equilibrium that is governed by GSNOR (25, 139, 144,

146, 147).

24 Regulation of GSNOR expression and activity is multifaceted and likely context- dependent. VEGF induces GSNOR mRNA expression in lungs, which is reversed by NOS inhibition (148). Treatment of hepatocytes with Cys-SNO increased GSNOR activity by induction of GSNOR mRNA, which might be transcriptionally-mediated by Sp1 (82, 149).

IL-13 increased GSNOR expression in bronchoalveolar lavage cells from asthmatic patients and in A549 human lung carcinoma cells (150). GSNOR is also regulated by miR-

342-3p, which downregulates GSNOR expression (151). S-nitrosylation of GSNOR might regulate enzymatic activity allosterically: increased S-nitrosylation of GSNOR has been observed in mouse lungs and brain, corresponding with an increase in GSNOR activity

(152, 153). In contrast, treatment of purified Arabidopsis, human, and yeast GSNOR with

Cys-SNO inhibited GSNOR activity through S-nitrosylation of allosteric Cys, and this inhibition was reversed by mutating target Cys to alanine (154). The site(s) of endogenous

GSNOR S-nitrosylation has yet to be identified and the effect of in situ S-nitrosylation on

GSNOR enzymatic activity remains uncertain.

CBR1 is a member of a class of NADPH-dependent enzymes that mediate carbonyl reduction of both physiological substrates and xenobiotics (155). CBR1 is a cytosolic protein that metabolizes endogenous substrates including prostaglandins, steroids and aldehydes, and plays a role in detoxification of aldehydes during oxidative stress (140,

155). The crystal structure of CBR1 confirmed the existence of a GSH , and

CBR1 mediated as much as 30% of NADPH-dependent GSNO reduction in lung cells

(140). Importantly, the NADPH:NADP+ ratio is usually >1, whereas the ratio of

NADH:NAD+ is <1 (140). Thus, NADPH-dependent GSNO reduction by CBR1 and the remaining NADPH-dependent GSNOR reductase(s) should play an important role in

25 steady-state denitrosylation. Furthermore, GSNO might regulate the activity of CBR1

(156, 157). GSNO was shown to reduce CBR1 activity, possibly by catalyzing S- glutathionylation at C227 (156). Treatment with GSNO also promoted formation of a disulfide between vicinal thiols C226 and C227 in CBR1, which altered substrate efficiency (157). Whether these modifications occur in vivo with physiological levels of

GSNO remains to be seen.

1.4.4 GSNOR in physiology and pathophysiology

The physiological role of GSNOR-dependent S-nitrosylation/denitrosylation is best characterized in the cardiovascular system. GSNOR-deficient (GSNOR-KO) mice have low systemic vascular resistance, consistent with systemic vasodilation (158). Although blood pressure may be maintained by increased cardiac output under some conditions,

GSNOR-KO mice are highly susceptible to hypotension, including that induced by anesthesia (144, 158). Thus, GSNO is an EDRF in the classic use of the term.

Part of the cardiovascular influence of GSNOR is exerted through the control by

GSNOR of SNO-Hb levels within red blood cells. GSNOR-KO mice have elevated levels of SNO-Hb within red blood cells (RBCs), which may contribute to cardioprotection (29,

159). These findings are consistent with the demonstration by Zhang et al. (160) of an essential role for SNO-Hb (SNO-Cys93) in hypoxic vasodilation, a critically important mechanism to couple oxygen delivery to local tissue oxygen demand. Mutant Cys93Ala mice are ischemic at baseline and highly susceptible to cardiac injury and mortality (161).

In addition to enhanced levels of SNO-Hb, GSNOR-KO mice are protected from cardiac injury by virtue of increased capillary density (162). Increased S-nitrosylation of HIF-1 in GSNOR-KO mice stabilizes HIF-1 under normoxia, preventing pVHL-mediated

26 degradation and driving cardiac angiogenesis through VEGF induction (162). Thus,

GSNOR may protect from myocardial ischemia through a variety of mechanisms.

GSNOR also influences cardiac (and pulmonary) function by regulating S- nitrosylation of multiple elements contributing to trafficking of the 2-AR (36). GSNOR-

KO mice demonstrate increased 2-AR expression in heart and lungs, and enhanced S- nitrosylation of two key proteins involved in receptor desensitization, internalization and degradation: GPCR kinase 2 (GRK2) and -arrestin 2 (-arr2) (72, 73). GRK2 phosphorylates the 2-AR upon receptor activation, leading to receptor desensitization by recruitment of -arr2. Stimulus-coupled, eNOS-mediated S-nitrosylation of GRK2 inhibits receptor , preventing receptor internalization and desensitization (72). - arrestin 2 also undergoes a cycle of S-nitrosylation and denitrosylation dependent on eNOS, which promotes receptor internalization and recycling (73). eNOS-derived S- nitrosylation of dynamin, a protein involved in regulation of endocytic budding for 2-AR internalization, is also coupled to 2-AR activation; however, it is unclear whether GSNOR mediates its denitrosylation (71).

The role of GSNOR in the heart includes regulation of calcium-handling machinery. GSNOR-KO mice exhibit an impaired inotropic response to isoproterenol that is due, in part, to impaired stimulus-coupled denitrosylation of RyR2 leading to pathological calcium leak (158). By contrast, cardiomyocytes from GSNOR-transgenic

(GSNOR-TG) mice increase contractility in response to isoproterenol without increasing calcium flux (and accordingly, GSNOR-TG mice are resistant to pathological hypertrophy)

(163). S-nitrosylation was shown to affect the activity of multiple calcium-handling proteins, including phospholamban and cardiac troponin C (163). Stimulus-coupled S-

27 nitrosylation was required for phospholamban multimerization to relieve inhibition of

SERCA2a, whereas S-nitrosylation of cardiac troponin C reduced its sensitivity to calcium.

In addition, diminished cardiac contractility in sepsis is worsened in GSNOR-KO mice and improved in GSNOR-TG mice (164). Improvements in GSNOR-TG mice versus GSNOR-

KO mice were attributed to restoration of calcium sensitivity of calcium handling proteins.

Taken together, these data indicate that GSNOR regulates cardiac 2-AR receptor function, calcium flux, and myofilament calcium sensitivity (Fig. 1.6).

Accumulating evidence suggests that aberrant GSNOR-dependent denitrosylation may be relevant to human disease. GSNOR-KO mice display multi-organ dysfunction and mortality in models of sepsis, with particularly notable histological derangement in the liver and thymus (144). These effects as well as enhanced SNO-protein levels, as seen in humans, were reversed by iNOS inhibition (144). Deletion of GSNOR also leads to spontaneous hepatocellular carcinoma (HCC) and carcinogen-induced HCC by downregulation of O6-alkylguanine-DNA (AGT), a DNA repair enzyme

(165). Carcinogen challenge by diethylnitrosamine stimulates iNOS-dependent S- nitrosylation and degradation of AGT, causing increased mutagenesis (165–168). A further role for GSNOR in HCC was demonstrated by Rizza et al. (169), who reported that downregulation of GSNOR increases S-nitrosylation and degradation of mitochondrial chaperone TRAP1, leading to an increase in succinate dehydrogenase (complex II) activity.

Inhibition of succinate dehydrogenase in this setting caused necrosis and of tumor cells. Interestingly, GSNOR is downregulated in HCC patients and is located in a chromosomal region frequently deleted in patients with HCC, strongly implicating loss of

28 GSNOR in human HCC (165, 169). GSNOR is also down-regulated in breast cancers (170) and , the latter associated with activating Ras S-nitrosylation (171).

GSNOR activity may also play a significant role in airway disease. In a murine model of asthma, wild-type mice treated with ovalbumin displayed increased airway

GSNOR, reduced SNO levels, and increased airway hyper-responsiveness compared to

GSNOR-KO mice (172). Inhibition of iNOS restored airway sensitivity to methacholine, confirming the role of SNO in mediating asthma protection. Inhibitors of GSNOR recapitulate the protective phenotype demonstrated in GSNOR-KO mice (173, 174).

Increased GSNOR activity and diminished SNO levels are observed in bronchoalveolar lavage samples from human asthma patients, strongly linking GSNOR to asthma in humans

(150, 175). Single polymorphisms in GSNOR predict asthmatic phenotype in patients as well as response to 2-AR agonists (176, 177). Mechanistically, GSNO may prevent tachyphylaxis to 2-AR agonists by preventing receptor desensitization (72, 172).

Additionally, GSNOR plays a role in the maturation of CF transmembrane conductance regulator protein (CFTR) (178). GSNOR regulates S-nitrosylation of Stip1, a co-chaperone in CFTR folding and maturation, leading to Stip1 stabilization and reduced

CFTR maturation. Bronchial epithelial cells displaying a cystic fibrosis phenotype as a result of the common F508del-CFTR mutant display increased GSNOR activity and reduced CFTR maturation, and genetic or pharmacological inhibition of GSNOR rescues

CFTR maturation by increasing Stip1 S-nitrosylation and degradation (178). Further, restoration of GSNO by inhibition of GSNOR may alleviate cigarette smoke-induced dysfunction of CFTR (179).

29 GSNOR has also been implicated broadly in cell maturation and fate. GSNOR-KO mice display lymphopenia, with reduced peripheral T cell and B cell counts (147).

Inhibition of iNOS reverses the lymphopenia by reducing apoptosis in the thymus. GSNOR has also been shown to play a role in stem cell maturation. Lima et al. (162) observed increased numbers of bone marrow-derived hematopoietic stem cells in GSNOR-KO mice, indicating a potential role for S-nitrosylation in stem cell biology (162). Mesenchymal stem cells (MSCs) derived from GSNOR-KO mice showed reduced capacity for vasculogenesis associated with downregulation of the VEGF receptor PDGFR (180). NOS inhibitors restored vasculogenesis in GSNOR-KO MSCs, whereas NO donors reduced vasculogenesis in wild-type or human MSCs. Additionally, MSCs from GSNOR-KO mice have reduced adipogenesis with a coordinate increase in osteoblastogenesis (181). In this model, increased S-nitrosylation of peroxisome proliferator-activated receptor gamma

(PPAR) prevents transcriptional activation of PPAR target . This biases MSC differentiation and may help explain the observed reduction in body fat mass in GSNOR-

KO mice.

GSNOR has been implicated in tissue recovery from diverse insults. GSNOR inhibition protected zebrafish from acetaminophen-induced liver toxicity through activation of Nrf2, a key stress response protein (182). Protection against liver injury was also observed in GSNOR-KO mice while pharmacological inhibition of GSNOR aided in liver recovery. Notably, cardiac regeneration following myocardial infarction (MI) is enhanced in GSNOR-KO mice (183). Cardiac injury in GSNOR-KO mice was associated with increased proliferation of differentiated cardiomyocytes, expansion of cardiac stem

30 cells, and neovascularization by endothelial cells (183). GSNOR may therefore provide a novel target for regenerative therapy.

Thus, in sum, genetic or pharmacological suppression of GSNOR (and other denitrosylases) provides a means to study the specific roles of endogenous SNO-proteins, and has provided important insights into the role of GSNO and SNO-proteins in both physiology and pathophysiology across multiple tissue types and diseases. Inhibition of endogenous denitrosylation supports physiologically relevant conclusions not accessible by other, e.g. pharmacological, means of SNO enhancement.

1.4.5 The SNO-CoA reductase system

Coenzyme A (CoA), one of the most abundant LMW cellular thiols, plays a central role in cellular metabolism as a carrier molecular for myriad metabolic intermediates in catabolic energy production and anabolic of , sterols, ketone bodies, and amino acids (184, 185). S-nitroso-coenzyme A (SNO-CoA) had been synthesized in vitro and it was hypothesized that S-nitrosylation of CoA might adversely affect cellular function by limiting the availability of CoA (186–188). However,

NO is not available physiologically in sufficient amounts to modify the CoA pool by mass action. The existence of endogenous SNO-CoA and the establishment of its role in regulating protein S-nitrosylation was provided by the identification of yeast Adh6 and mammalian AKR1A1 as dedicated SNO-CoA reductases (20).

Yeast Adh6 is a -containing enzyme with obligate specificity for NADPH as a cofactor (189), originally identified as an NADPH-dependent, medium-chain cinnamyl alcohol dehydrogenase with broad activity against alcohol substrates (190, 191).

Crystallization of Adh6 revealed an active site structure capable of accepting a wide variety

31 of large or hydrophobic substrates (189, 191), but its physiological substrate(s) and function remained unclear. More recently, Adh6 was shown to account for ~80% of

NADPH-dependent SNO-CoA reductase activity in yeast lysates (20). Adh6 likely catalyzes SNO-CoA reduction via hydride transfer from NADPH and protonation from the aqueous environment to produce CoA-sulfinamide, similar to GSNOR (Fig. 1.5B).

Enzymatic efficiency (Km ~ 180M) towards SNO-CoA is roughly equivalent to efficiency for other known substrates (20, 190).

Knock out of Adh6 in yeast led to a SNO-CoA-dependent increase in both endogenous protein S-nitrosylation and S-nitrosylation induced by treatment with exogenous S-nitrosylating agents (20). Cytosolic thiolase (yeast: Erg10; mammal:

ACAT2) showed increased S-nitrosylation in Adh6-mutant yeast, and Erg10 was S- nitrosylated by SNO-CoA but not other LMW-SNOs, which inhibited thiolase activity, leading to a decrease in the downstream metabolite mevalonate, a key intermediate in sterol biosynthesis. Interestingly, both CoA and acetyl-CoA levels were altered in Adh6-mutant yeast, implicating SNO-CoA in control of additional metabolic processes.

In mammalian tissues, AKR1A1 (henceforth SCoR) was shown to mediate

NADPH-dependent SNO-CoA reduction (20). SCoR is a member of the aldo-keto reductase (AKR) superfamily of enzymes that catalyze a variety of oxidative or reductive reactions (192). AKRs are characterized by a conserved (/)8 barrel and exists as monomers, dimers, or tetramers (192). SCoR is cytosolic and monomeric, and has a role in the reduction of a variety of endogenous and exogenous substrates, including DL- glyceraldehyde and D-glucuronate as well as SNO-CoA (155, 192). However, SNO-CoA is the preferred endogenous substrate for SCoR (with a Km of ~20µM, compared to 1.7mM

32 and 4.2mM for DL-glyceraldehyde and D-glucuronate, respectively) and the only known substrate for human SCoR. AKRs are characterized by an active site tetrad consisting of , aspartate, histidine, and lysine, and catalytic tyrosine residues are conserved throughout AKRs (192, 193). Reduction of SNO-CoA (as for aldehydes) proceeds via hydride transfer with subsequent protonation of the oxygen atom by the active site tyrosine; aspartate and lysine might lower tyrosine pKa through a hydrogen bonding network while histidine serves to orient the substrate (193). Reduction of SNO-CoA by SCoR produces

CoA-sulfinamide, similar to yeast Adh6 (20) (Fig. 1.5B).

SCoR is expressed in most or all mammalian tissues with the highest activity in kidney and liver, and SCoR is responsible for the majority of NADPH-dependent SNO-

CoA reduction (20, 194, 195). Prior to its identification as a SNO-CoA reductase, the only physiological role described for SCoR involved the reduction of D-glucuronic acid in ascorbate synthesis (196, 197). However, for humans and many other mammals, ascorbate intake is required due to genetic mutations preventing endogenous ascorbate synthesis

(198). An additional role for SCoR in the metabolism of the anthracycline anticancer agents daunorubicin and doxorubicin has also been proposed (199); however, this is activity involves reduction of an exogenous agent. Thus, SNO-CoA reduction is likely the predominant endogenous role of SCoR in humans and mammals generally. Although

SCoR-deficient mice display increased S-nitrosylation of SNO-proteins, the physiological roles of SNO-CoA-dependent S-nitrosylation remain largely unexplored. First evidence for a role of SCoR/SNO-CoA in mammalian metabolism was provided by the finding that

SCoR-deficient mouse kidneys are protected from acute ischemia/reperfusion (IR) injury

(200). SCoR-regulated inhibitory S-nitrosylation of M2 led to shunting of

33 glycolytic intermediates into the pentose phosphate pathway, generating both reducing equivalents to protect against IR-induced oxidative stress and the for regenerative biosynthesis (200). Additional studies will further delineate the role of SCoR- mediated S-nitrosylation on metabolic function in other tissues.

1.5 Specificity in denitrosylation

The steady-state level of protein S-nitrosylation is governed by multiple factors: 1) production of NO and cellular nitrosating equivalents; 2) transnitrosylating reactions with

LMW-SNOs and other SNO-proteins; 3) SNO removal by denitrosylases. An illustrative example of the multi-factorial governance of protein S-nitrosylation is provided by

GAPDH. The function of GAPDH is regulated by S-nitrosylation, but GAPDH is also a prototypical transnitrosylase, and the denitrosylation of GAPDH is subserved by Trx,

GSNOR and SCoR (20, 50, 147, 201). Our emphasis on enzymatic denitrosylation highlights its critical role in modulating the dynamic equilibria that govern transnitrosylation of target proteins by LMW-SNOs and by SNO-proteins, and thereby regulate SNO-based signaling under basal conditions and in response to altered levels of

NO production.

1.5.1 Subcellular localization

Subcellular compartmentalization of denitrosylases was first demonstrated for the thioredoxin system. Trx1/TrxR1 and Trx2/TrxR2 localize to the cytosol and mitochondria, respectively, and thus regulate denitrosylation within these compartments (120). Fas- induced denitrosylation of mitochondrial caspase-3 is mediated by mitochondrial-specific

Trx2, and specific inhibition of Trx2 diminishes Fas-dependent signaling in lymphocytes

(120).

34 Early analyses of GSNOR demonstrated localization in both the cytosol and the nucleus, and subcellular localization has been shown to play a role in directing GSNOR activity (202, 203). Localization of GSNOR to myoendothelial junctions (gap junctions between smooth muscle cells and endothelial cells) facilitates stimulus-coupled denitrosylation of connexin-43 (204). S-nitrosylation of connexin-43 is mediated by co- localized eNOS, and stimulation with phenylephrine activates GSNOR-dependent denitrosylation of connexin-43 (204). GSNOR was also shown to localize to the sarcoplasmic reticulum (SR) in the heart where it interacted directly with nNOS and acted to facilitate stimulus-coupled denitrosylation of RyR2 (63, 158). These examples illustrate co-localization of the enzymatic machinery for stimulus-coupled S- nitrosylation/denitrosylation, also likely the case for -arrestin2, which interacts with eNOS and undergoes a cycle of dynamic S-nitrosylation and GSNOR-dependent denitrosylation following 2-AR activation (73). GSNOR localization may also be regulated in physiological contexts and dysregulated in pathology. GSNOR is typically localized in puncta distributed throughout the cytosol, but associates with mitotic spindles during cell division in normal lung cells; in contrast, GSNOR redistributes in a perinuclear fashion in lung carcinoma cells (205).

1.5.2 Interaction of denitrosylases with substrates

In addition to subcellular localization of denitrosylases, specificity in denitrosylation can result from protein-protein or protein-LMW thiol interactions of denitrosylases. The requirement for protein-protein interaction is inherent for Trx because the catalytic mechanism of Trx most likely involves formation of a mixed disulfide between Trx and target substrate (122). Thus, structural motifs predicting protein-protein

35 interactions with Trx that mediate denitrosylation may correspond with motifs that predict

S-nitrosylation (80, 121, 128).

Protein denitrosylation by transnitrosylation of GSH or CoA requires binding of

SNO-protein and LMW partner in a reactive orientation. As described above, thiolase S- nitrosylation is mediated selectively by SNO-CoA; SNO-CoA inhibits thiolase activity whereas GSNO does not (20). Thus, it is likely that SNO-CoA has preferential access to the regulatory Cys in Erg10 (thiolase). In addition, direct interaction of GSNO or SNO-

CoA reductases with target proteins may aid in denitrosylation by rapidly metabolizing newly formed GSNO or SNO-CoA. To date, limited examples of direct interaction of

LMW denitrosylases with target proteins exist. GSNOR was demonstrated to directly bind

Stip1, leading to denitrosylation and stabilization of this co-chaperone involved in CFTR maturation (178). GAPDH is a target of SCoR-mediated denitrosylation and is also predicted to exist in a macromolecular protein complex with SCoR (20, 206). Such protein complexes, and other proteins that co-immunoprecipitate with GSNOR and SCoR, may represent facile targets of denitrosylation by these enzymes.

It is also worth considering that, in general, localization of targets of denitrosylation to particular signaling pathways or functional mechanisms may be identified with specific denitrosylases. analysis of Trx1 targets from substrate trapping experiments in monocytes (THP-1 or RAW264.7 cells) identified targets across multiple cellular processes, including translation, and division, stress response and apoptosis

(121). Similar analysis of Trx1 targets in A549 lung carcinoma cells identified targets important for cell cycle, inflammatory signaling, transcriptional regulation, and RNA processing (128). Analysis of sites of protein S-nitrosylation in hearts from GSNOR-

36 deficient mice found clustering within , kinase binding proteins, and (25). Further, the majority of SNO-CoA-dependent targets of S-nitrosylation identified in Adh6-null yeast were clustered among metabolic enzymes (20).

1.6 Summary

Precisely targeted protein S-nitrosylation has emerged as a key mediator of NO- based redox signaling across classes of protein, cell type and phylogeny, and nitrosylases and denitrosylases play a central role in conferring specificity upon S-nitrosylation-based cellular signaling. From this perspective, denitrosylases govern the dynamic equilibria among SNOs, created by S-nitrosylases, which mediate the cellular functions of NO. Thus, the identification of protein denitrosylases and the genetic manipulation of these enzymes has greatly facilitated our understanding of the in situ role of protein S-nitrosylation in both physiology and pathophysiology.

37 1.5 TABLES AND FIGURES

Table 1.1

S-nitrosylation motif elements from SNO-proteome analyses

Source Motif SNO sites / Motif Element Proposed Identified Ref. Total SNO sites Motif Function Linear Motif* Curated list of NO-regulated 18/27 Charged residues Alter thiol reactivity (K,R,H,D,E)C(D,E) (35) proteins

Curated list of 69/72 Charged residues (at 8Å) Facilitate interactions -- (39) SNO-proteins 45/72 Solvent-exposure --

~216/309 Acidic residues Facilitate interactions DC CXXE Endogenous ~57/142 -helices Facilitate interactions (27) mouse liver 37/309 GC couplet -- GC ? Solvent-exposure --

16/18 Charged residues Alter thiol reactivity (E,D)XXXC Cys-SNO- or (E,D)XXC PAPANO-treated (48) CX(K,H,R) HASMCs 3/18 Hydrophobicity --

SNO-Cys in Charged residues Transnitrosylation (I,L)XCXX(D,E) peripheral blood 5/19 (52) Hydrophobicity (by S100A8/A9) monocytes

200/586 Charged residues -- (D,E)XX(X)C CX(K,R,H) (-helix) CX(E) (-helix)

261/586 Hydrophobicity Facilitate interactions CX(V,I,L) (-sheet) SNAP-treated Alter thiol reactivity CXXXI (49) MS-1 cells IXXXC VXXC CXA

20/586 CXG 60/586 CXT GSNO-treated 80/138 Hydrophobicity -- (A,V,L,I)(A,V,L,I)XXXC (51) proteins ?/133 Charged residues -- KXXXXXC CXXXXXD KXC Cys-SNO or LPS

treatment of BV- (207) ?/133 Hydrophobicity -- AXC 2 cells CXXXXXI

?/133 MXXC 1079/1250 (human) dbSNO 2.0 Charged residues -- Numerous motifs (47) 2315/2647 (mouse)

*Identified linear motifs do not necessarily account for all SNO-sites identified as exemplifying a motif element.

Abbreviations: Cys-SNO, S-nitroso-cysteine; GSNO, S-nitroso-glutathione; HASMC, human aortic smooth muscle cells; LPS, bacterial lipopolysaccharide; NO, nitric oxide; PAPANO, propylamine propylamine NONOate; SNAP, S-nitroso-N- acetyl-D,L-penicillamine; SNO-protein, S-nitroso-protein.

38 Table 1.2

Known targets of enzymatic denitrosylation

Denitrosylase Target

High Molecular Weight Denitrosylases

Caspase-3 (120), Caspase-8 (208), Caspase-9 (120), PTP1B (120), NF-B (124), NSF (209), iNOS (121), nNOS (57), MEK1 (121), STAT3 (121), PRMT1 (121), PA28 (121) Trx1 GAPDH (201), (210), Annexin-1 (80), 14-3-3 (80), ~50 proteins (80), >400 proteins (121), >500 proteins (128)

Trx2 Caspase-3 (120)

Trp14 Caspase-3 (134), Cathepsin B (134)

Low Molecular Weight Denitrosylases

GSNO Reductases

GRK2 (72), -arrestin 2 (73), RyR1 (211), RyR2 (158), PLN (163), NCX (163), Actin (163), TPM (144), cTnC (144), cTnI (144), LTCC (144), SERCA2a (144), MYBPC3 (144), HIF- GSNOR 1 (162), PPAR (181), Cx43 (204), AGT (165), Apaf-1 (170), c-Jun (170), HSF1 (170), CNA (170), CUGBP1 (170), NF-M (170), Ras (171), TRAP1 (169), Stip1 (178), GAPDH (147), Caspase-6 (147), VDAC1 (147), ~85 unique proteins (147), ~76 unique proteins (25) CBR1 No identified targets

SNO-CoA Reductases

AKR1A1 GAPDH (20)

Adh6 Erg10 (20), ~15 proteins (20)

Abbreviations: AGT, O6-alkylguanine-DNA alkyltransferase; Apaf-1, apoptotic protease-activating factor 1; c-Jun, transcription factor AP-1; CNA,  subunit of calcineurin; cTnC, cardiac troponin C; cTnI, cardiac troponin I; CUGBP1, CUG triplet repeat, RNA binding protein 1; Cx43, connexin-43; Erg10, ergosterol biosynthesis protein 10; GAPDH, glyceraldehyde

3-phosphate dehydrogenase; GRK2, G protein-coupled receptor kinase 2; HIF-1, hypoxia-inducible factor 1-alpha; HSF1, heat shock factor 1; iNOS, inducible nitric oxide synthase; LTCC, L-type calcium channel; MEK1, mitogen-activated kinase 1; MYBPC3, cardiac myosin binding protein C; NCX, sodium-calcium exchanger; NF-B, nuclear factor-B;

NF-M, neurofilament 160; nNOS, neuronal nitric oxide synthase; NSF, N-ethylmaleimide-sensitive factor; PA28, proteasome activator complex subunit 2; PLN, phospholamban; PPAR, peroxisome proliferator-activated receptor gamma;

PRMT1, protein N-methyltransferase 1; PTP1B, protein tyrosine phosphatase 1B; RyR1, ryanodine receptor 1;

SERCA2a, sarcoplasmic/endoplasmic reticulum calcium ATPase 2; STAT3, signal transducer and activator of transcription

3; TRAP1, tumor necrosis factor type 1 receptor-associated protein; TPM, tropomyosin; Trp14, thioredoxin related protein

14; Trx1, thioredoxin 1; Trx2, thioredoxin 2; VDAC1, voltage-dependent anion-selective channel protein.

39 Figure 1.1

Figure 1.1. S-nitrosothiol formation occurs via complexed enzymatic machinery.

Schematic representation of the three enzyme complex that mediates the majority of SNO synthesis in vivo. In mammals, nitric oxide is mainly produced by one of three nitric oxide synthases, or NOSs. SNO synthases, likely via transition metals, convert newly synthesized

NO to S-nitrosothiols by fulfilling the one electron requirement for SNO formation.

Nascent SNOs are passed from SNO synthases to transnitrosylases, whose function is to distribute SNOs to target proteins throughout the cell via group transfer chemistry to propagate SNO-based signals.

40 Figure 1.2

Figure 1.2. Coupled, dynamic equilibria that govern protein S-nitrosylation are regulated by enzymatic denitrosylases. (A) SNO-proteins are in equilibrium with LMW-

41 SNOs and can further participate in protein-to-protein transfer of the NO group (trans-S- nitrosylation) to subserve NO-based signaling. (B) Transnitrosylation by both identified

LMW-SNOs (G=glutathione; CoA=Coenzyme A; Cys=Cysteine) and SNO-proteins will result in distinct sets of SNO-proteins that mediate specific SNO signaling cascades. (C)

Distinct enzymatic denitrosylases regulate the coupled equilibria that confer specificity to

SNO-based signaling. These include GSNO reductases and SNO-CoA reductases, which regulate protein S-nitrosylation by GSNO and SNO-CoA respectively. These LMW-SNOs are in equilibrium with cognate SNO-proteins. In contrast, thioredoxins directly denitrosylate SNO-proteins.

42 Figure 1.3

Figure 1.3. Steady-state protein S-nitrosylation reflects denitrosylase activity. (A) In cultured HEK cells, suppression of Trx-mediated denitrosylation with the TrxR inhibitor auranofin results in greatly enhanced steady-state levels of SNO-proteins (as detected by the SNO-RAC method (76)). (B) Following induction of iNOS by systemic administration of bacterial lipopolysaccharide (LPS), steady-state levels of hepatic SNO-protein are greatly increased in the genetic absence of GSNOR (as assessed by photolysis- chemiluminescence; modified from Liu et al. (144)).

43 Figure 1.4

Figure 1.4. Differential RSNO reactivity. By default (middle scheme), a partial negative charge exists on the S atom of the SNO bond; a partial positive charge exists on the N atom.

This intrinsically favors transnitrosylative reactions. Positive charge coordination with the

S atom or negative charge coordination with the N or O atom would further increase electrophilicity of the N atom, enhancing the propensity for transnitrosylation (top). In contrast, negative charge coordination with the S atom or positive charge coordination with the N or O atom would increase electrophilicity of the S atom, preferencing S-thiolation reactions (bottom).

44 Figure 1.5

Figure 1.5. Enzymatic mechanisms of protein denitrosylation. (A) Denitrosylation by thioredoxin requires a direct interaction with target SNO-proteins. Nucleophilic attack by an active Cys leads to mixed disulfide formation between Trx and target proteins with liberation of nitric oxide as a nitroxyl anion (likely as HNO). The mixed disulfide is resolved by the second active site Cys of Trx, generating oxidized Trx and reduced target protein thiol. Trx is subsequently reduced by TrxR and NADPH to regenerate denitrosylating activity. (B) A common reaction scheme exists for the reduction of GSNO and SNO-CoA by their cognate denitrosylases (GSNO reductase and SNO-CoA reductase). Hydride transfer from NAD(P)H to the N atom and protonation of the O atom lead to an S-(N-hydroxy) intermediate that rearranges to sulfinamide.

45 Figure 1.6

Figure 1.6. Stimulus-coupled S-nitrosylation and denitrosylation: cardiomyocytes as an exemplary case. (A) -induced activation of the 2-adrenergic receptor (2-AR) in cardiomyocytes stimulates eNOS activity. eNOS-dependent S-nitrosylation of GRK2 suppresses GRK2 activity whereas S-nitrosylation of -Arr2 facilitates -Arr2 activity, thereby regulating receptor desensitization and internalization. GSNOR negatively regulates the S-nitrosylation status of both GRK2 and -Arr2. (B) nNOS and GSNOR interact directly at the sarcoplasmic reticulum (SR) in cardiomyocytes. 2-AR activation stimulates nNOS-dependent S-nitrosylation of components of calcium handling (PLN,

46 Serca2a, RyR2) and of cardiac myofilaments, which is regulated by GSNOR-dependent denitrosylation. In A and B, SNO depicted in RED indicates inhibition of normal function by S-nitrosylation; SNO depicted in GREEN indicates activation of normal function by S- nitrosylation.

47 CHAPTER 2:

Molecular recognition of S-nitrosothiol substrate by its cognate protein

denitrosylase

Colin T. Stomberski1,2, Hua-Lin Zhou1, Liwen Wang4, Focco van den Akker2, and

Jonathan S. Stamler1,3,5*

From the 1Institute for Transformative Molecular Medicine; 2Departments of

Biochemistry and 3Medicine; and 4Center for Proteomics and Bioinformatics, Department

of , Case Western Reserve University, Cleveland, OH 44106; 5Harrington

Discovery Institute, University Hospitals Cleveland Medical Center, Cleveland, OH

44106

*This research was originally published in the Journal of Biological Chemistry. Colin T.

Stomberski, Hua-Lin Zhou, Liwen Wang, Focco van den Akker, and Jonathan S. Stamler.

Molecular recognition of S-nitrosothiol substrate by its cognate protein denitrosylase. J.

Biol. Chem. 2019; 294(5):1568-1578. © Jonathan S. Stamler

48 2.1 ABSTRACT

Protein S-nitrosylation mediates a large part of nitric oxide’s influence on cellular function by providing a fundamental mechanism to control protein function across different species and cell types. At a steady state, cellular S-nitrosylation reflects dynamic equilibria between S-nitrosothiols (SNOs) in proteins and small molecules (low-molecular-weight

SNOs) whose levels are regulated by dedicated S-nitrosylases and denitrosylases. S- nitroso-coenzyme A (SNO-CoA) and its cognate denitrosylases, SNO-CoA reductases

(SCoRs), are newly identified determinants of protein S-nitrosylation in both yeast and mammals. Since SNO-CoA is a minority species among potentially thousands of cellular

SNOs, SCoRs must preferentially recognize this SNO substrate. However, little is known about the molecular mechanism by which cellular SNOs are recognized by their cognate enzymes. Using mammalian cells, molecular modeling, substrate-capture assays, and mutagenic analyses, we identified a single conserved surface Lys (Lys-127) residue as well as active-site interactions of the SNO group that mediate recognition of SNO-CoA by

SCoR. Comparing SCoRK127A versus SCoRWT HEK293 cells, we identified a SNO-CoA– dependent nitrosoproteome, including numerous metabolic protein substrates. Finally, we discovered that the SNO-CoA/SCoR system has a role in mitochondrial metabolism.

Collectively, our findings provide molecular insights into the basis of specificity in SNO-

CoA–mediated metabolic signaling and suggest a role for SCoR-regulated S-nitrosylation in multiple metabolic processes.

49 2.2 INTRODUCTION

S-nitrosylation mediated control of proteins, a fundamental mechanism for cellular regulation and signaling, operates across phylogeny and cell types. By current estimates,

70% of the proteome is subject to this modification (2, 53). Recent evidence indicates that

S-nitrosylation is enzymatically regulated by protein S-nitrosylases (6) and denitrosylases

(19). S-nitrosylases operate as part of a multi-protein machinery (with NOSs and SNO synthases) for S-nitrosylation (5), analogous to the E1/E2/E3 ubiquitinylation machinery, and it is predicted that hundreds of nitrosylases mediate cellular NO signaling (17). By contrast, denitrosylases are likely fewer in number and fall into two categories: 1) direct protein denitrosylases, exemplified by thioredoxin related proteins (122); and 2) low- molecular weight (LMW) SNO reductases, including S-nitrosoglutathione (GSNO) reductases (139, 140) and S-nitroso-coenzyme A (SNO-CoA) reductases (20). The latter group of enzymes carry out NAD(P)H-dependent reduction of GSNO and SNO-CoA, thereby regulating coupled equilibria between SNO-proteins and LMW-SNOs to control

SNO-protein levels (212).

Two GSNORs and two SCoRs have been identified to date (212). In mammals, the primary SCoR is AKR1A1 (20), the founding member of the aldo-keto reductase superfamily of proteins (213). AKR1A1 has a preference for negatively charged carbonyl- containing substrates (192) including D-glucuronate, an intermediate in ascorbate synthesis in rodents (196). However, interestingly, the physiological substrates in humans are unknown and thus the primary functions of mammalian SCoR remain to be discovered.

We recently demonstrated that SNO-CoA is in fact the likely preferred physiological substrate of SCoRs across phylogeny and that mammalian SCoR thereby regulates S-

50 nitrosylation of proteins (20). Moreover, we have shown that SCoRs confer metabolic growth advantages in yeast (20) and reprogram cellular metabolism in mice to alleviate tissue injury (200). Thus, the SNO-CoA/SCoR system may play a newly discovered role in cellular metabolism.

The emerging paradigm wherein specificity in S-nitrosylation signaling derives, at least partly, from differential reactivities of LMW-SNOs, predicts a molecular basis for

SNO recognition by LMW denitrosylases. However, structure-function relationships for

LMW denitrosylases remain largely unexplored. Thus, there is a general lack of understanding of how denitrosylases recognize their substrates within the cellular milieu.

In particular, it remains unclear if these interactions depend on the R groups in RSNOs

(e.g. glutathione or coenzyme A) and/or whether proteins may recognize the SNO moiety.

Here we use molecular modeling and mutagenic analyses to determine the molecular mechanisms by which SCoRs bind and metabolize SNO-CoA. We then utilize mutant

SCoR that is unable to bind SNO-CoA to identify novel targets of SNO-CoA-mediated S- nitrosylation in cellular systems, and to assess the role of SCoR in mitochondrial metabolism.

51 2.3 RESULTS

2.3.1 Molecular modeling of SCoR-based specificity

To understand how SCoR recognizes SNO-CoA, we performed molecular modeling of SNO-CoA and the SCoR (AKR1A1) active site to identify amino acids potentially mediating the SCoR—SNO-CoA interaction. Docking of SNO-CoA to the

SCoR active site produced two possible binding modes (Fig. 2.1, A and B) with the SNO group oriented towards NADPH and the catalytic Tyr50 (193). Multiple charged or aromatic residues surrounding the SCoR active site (Fig. 2.1, A and B, labeled in red) were predicted to either hydrogen bond with SNO-CoA or facilitate interaction of SCoR with

SNO-CoA via van der Waals forces. Active site binding modes for D-glyceraldehyde and glucuronate were also obtained by docking calculations (Fig. 2.1, C and D). In accordance with previous work demonstrating a marked increase in Km for glucuronate reduction upon mutation of Arg312 (214), our model of glucuronate binding the SCoR active site shows a strong interaction between the glucuronate group and Arg312, providing a level of confidence in our modeling results.

2.3.2 Essential role of SCoRK127A in SNO-CoA reductase activity

To test the role of the putative SNO-CoA-binding residues (Fig. 2.1, red labeled amino acids) in mediating the SCoR—SNO-CoA interaction, we generated and purified recombinant (see section 2.5, Methods) wild-type (SCoRWT) and mutant SCoRs (Fig. 2.2) involving multiple putative substrate-interacting residues, and assessed the impact of these mutations on in vitro reduction of SNO-CoA and DL-glyceraldehyde. Mutations varyingly altered the catalytic efficiency (Kcat/Km) of SCoR for both SNO-CoA and DL- glyceraldehyde (Fig. 2.3A and Table 2.2). Notably, SCoRK127A resulted in an ~90%

52 reduction in SCoR catalytic efficiency for SNO-CoA with no effect on that of DL- glyceraldehyde (Fig. 2.3A). This selective reduction in catalytic efficiency was mediated primarily by an ~7-fold increase in Km (with only a modest reduction in Kcat) (Fig. 2.3C

K127A and Table 2.1). SCoR did not alter Km or Kcat for DL-glyceraldehyde (Fig. 2.3B and

Table 2.1) and only modestly reduced Kcat for glucuronate (Fig. 2.3D and Table 2.1).

Diminished SNO-CoA turnover with unaltered DL-glyceraldehyde reduction by

SCoRK127A was confirmed by following NADPH consumption over 10 minutes (Fig. 2.4).

Importantly, mutation of the active site Tyr50 abolished SCoR SNO-CoA reductase activity (Fig. 2.4), consistent with the previously identified role of Tyr50 in the catalytic mechanism of SCoR (193) and indicating that SNO-CoA reduction by SCoR likely follows the canonical aldo-keto reductase catalytic mechanism, namely hydride transfer from

NADPH and protonation by an active site Tyr (193, 199).

Based on the crystal structure of SCoR, Lys127 lies distant from the active site

(compared to other tested residues) and modeling does not predict an interaction of Lys127 with either DL-glyceraldehyde or glucuronate (Fig. 2.1). Furthermore, mutation of Lys97

(not shown in Figure 2.1), which resides on the same surface as the SCoR active site

(though further away than Lys127), does not alter SNO-CoA or DL-glyceraldehyde reduction by SCoR (Fig. 2.3A and Table 2.2). Importantly, Lys127 is highly conserved among mammalian SCoRs (Fig. 2.3E), suggesting a key role for SCoR-regulated SNO-

CoA signaling in mammals. These results demonstrate that among the tested substrates

(including many previously studied substrates) (193, 215, 216)), SNO-CoA is the kinetically preferred substrate for SCoR (Table 2.1), and that the conserved Lys127 of

SCoR mediates a specific enzyme—substrate interaction to facilitate SNO-CoA reduction.

53 2.3.3 SCoR recognizes the CoA backbone and SNO moiety in SNO-CoA

We next sought to determine how Lys127 interacts with SNO-CoA to facilitate reduction by SCoR. One binding mode of SNO-CoA (Fig. 2.1A) predicted a hydrogen bond between Lys127 and the 3'-phosphate of the 3'-phospho-ADP component of CoA; a second binding mode had the diphosphate linker of SNO-CoA interacting with Lys127

(Fig. 2.1B). We hypothesized that if binding mode 1 is correct, SCoRWT would not effectively metabolize dephospho-SNO-CoA (SNO-CoA lacking the 3'-phosphate).

WT However, reaction of dephospho-SNO-CoA with SCoR produced no change in Km and only a modest reduction in Kcat (Fig. 2.5A and Table 2.1), indicating that this predicted hydrogen bond is not a primary driver of the interaction between SCoR and SNO-CoA

(though it may affect turnover rate). Thus, it is more likely that K127 interacts with the diphosphate linker in the CoA moiety of SNO-CoA, as predicted from binding mode 2 in

Fig. 2.1B where K127 is ~3.0Å from the CoA diphosphate and makes many favorable interactions with the CoA backbone.

The structure of the AKR superfamily is defined by a TIM barrel with interspersing loops, and variations in these loop regions provide specificity to the enzymes (192, 214).

Lys127 resides on a large loop between -sheet 4 and -helix 4 (of the TIM barrel), and provides a solvent-exposed positive charge (lost upon mutation to Ala) that could promote interaction of SNO-CoA with SCoR (Fig. 2.6, A and B). To test the requirement of a positively charged residue at this position, we substituted Lys127 with Arg (Fig. 2.6C) and assessed the ability of SCoRK127R to metabolize SNO-CoA. Reaction of SNO-CoA with

K127R SCoR produced no change in Km and only a modest decrease in Kcat (Fig. 2.5B and

Table 2.1). We further explored the requirement of a positive charge at residue 127 to

54 facilitate the SNO-CoA—SCoR interaction by assessing the ability of SCoRWT,

SCoRK127A, and SCoRK127R to bind bead-bound CoA and SNO-CoA. Both SCoRWT and

SCoRK127R, but not SCoRK127A, efficiently bound CoA and SNO-CoA beads (Fig. 2.5, C and D). Together, these results demonstrate that a positively charged residue at position

127 is sufficient to drive the majority of the SCoR—SNO-CoA interaction, and that this interaction is mediated through the CoA moiety in SNO-CoA (likely through interaction with the diphosphate of the 3'-phospho-ADP component of CoA, as seen in Fig. 2.1B).

Similar to the reaction of SCoRWT with dephospho-SNO-CoA, reaction of SCoRK127R with

SNO-CoA modestly lowered Kcat (Table 2.1). Thus, the role of Lys127 in SNO-CoA metabolism is likely two-fold: i) direct substrate recognition via the CoA diphosphate moiety and ii) specific interaction between Lys127 (as opposed to other positively charged residues) and the 3-phosphate of CoA to facilitate optimal substrate turnover.

By contrast, two mutations (SCoRK23A and SCoRW220A) increased SCoR catalytic efficiency for SNO-CoA by increasing Kcat without altering Km (Fig. 2.3A and Table 2.2).

These mutant enzymes also bound SNO-CoA beads as effectively as SCoRWT (Fig. 2.7, A and B), consistent with unaltered Kms.

Notably, we also observed that SCoRWT bound SNO-CoA more effectively than

CoA (Fig. 2.5, C and E), implying that SCoR interacts with the SNO moiety. Since the

SNO group in SNO-CoA is part of cysteamine (Fig. 2.5F, top), we reasoned that SCoR might also metabolize SNO-cysteamine. Indeed, SCoR reduces SNO-cysteamine (Fig.

2.5F, bottom and Table 2.1), albeit with a higher Km and lower Kcat compared to SNO-

WT K127A CoA. Interestingly, the Km of SCoR for SNO-cysteamine is similar to that of SCoR

55 for SNO-CoA (Table 2.1). These data point to the importance of the CoA moiety for effective SNO-CoA binding and turnover by SCoR.

As a further test of SCoR substrate recognition, we explored competitive inhibition of SNO-CoA reductase activity by CoA and acetyl-CoA (an alternative CoA derivative), finding both to be weak inhibitors (Fig. 2.5G). Using 100M SNO-CoA, CoA and acetyl-

CoA exhibited IC50’s of 1.85mM and 10.4mM, respectively (Fig. 2.5G). Taken together, our results indicate that SCoR interacts with both the CoA and SNO moieties in SNO-CoA.

Thus, SCoR preferentially acts on SNO-CoA and is unlikely to be inhibited by CoA or its derivatives at cytosolic concentrations, allowing SCoR to serve its physiological function as a SNO-CoA reductase.

2.3.4 Identification of targets of SNO-CoA/SCoR-mediated S- nitrosylation/denitrosylation

Previous work (20) has identified numerous substrates of SCoR in yeast, whereas fewer mammalian substrates of SCoR have been identified (27). AKR1A1 constitutes ~90-

95% of NADPH-dependent SNO-CoA reductase activity in murine kidney lysate (Fig.

2.8A), and addition of recombinant wild-type SCoR rescued NADPH-dependent SNO-

CoA reductase activity in SCoR-null kidney lysate to a greater degree than recombinant

SCoRK127A (Fig. 2.8A).

Utilizing the difference between SCoRWT and SCoRK127A activity, we sought to identify targets of SNO-CoA-mediated S-nitrosylation. SNO-CoA treatment of kidney lysate greatly enhanced whole cell protein S-nitrosylation, as visualized by Imperial staining following SNO-RAC (76) (Fig. 2.8B, lanes 2 and 4), and this increase was abrogated by the co-addition of NADPH to wild-type, and to a lesser degree SCoR-null,

56 lysate (Fig. 2.8B, lanes 3 and 5). Addition of recombinant SCoRWT, but not recombinant

SCoRK127A, to SCoR-deficient kidney lysate greatly reduced SNO-CoA-induced S- nitrosylation (Fig. 2.8B, lanes 6 and 7). SNO-proteins regulated by SCoR-dependent metabolism of SNO-CoA (lanes 6 and 7; Fig. 2.8B) were -digested and identified by isobaric tags for relative and absolute quantification (iTRAQ) coupled liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) (Appendix 2.1), providing a genetically-validated list of mammalian targets of SNO-CoA-mediated S- nitrosylation.

To identify endogenous targets of SCoR-dependent denitrosylation we developed a tractable cellular model in HEK293 cells. We deleted endogenous SCoR utilizing the

CRISPR/Cas9 system and stably overexpressed SCoRWT, SCoRY50A (catalytically dead), or SCoRK127A in SCoR-deficient cells (Fig. 2.9A). Both SCoRWT and SCoRK127A (but not

SCoRY50A) rescued SCoR-mediated DL-glyceraldehyde reduction (Fig. 2.9B, top); by contrast, only SCoRWT rescued SCoR-mediated SNO-CoA reduction (Fig. 2.9B, bottom).

Following transient overexpression of iNOS (inducible nitric oxide synthase), SNO- proteins were enriched by SNO-RAC and identified by iTRAQ coupled LC-MS/MS, and the nitrosoproteomes from SCoRWT and SCoRK127A stable cells were compared (Appendix

2.2). Notably, 50 of the 123 SNO-proteins (~40%) identified as substrates of SCoR in

HEK293 cells were also identified as targets of SNO-CoA in mouse kidney lysates

(Appendix 2.3). Appendix 2.3 dataset revealed overrepresentation of proteins involved in glycolysis, the tricarboxylic acid cycle, protein folding, and other cellular metabolic processes (Appendix 2.3).

57 We validated a top subset of putative targets of SCoR-dependent denitrosylation, including -like modifier-activating enzyme 1 (Ube1a/b), alpha- (ENO1), A chain (LDHA), and fatty acid synthase (FASN). Analysis by

SNO-RAC coupled to western blotting demonstrated that SCoRWT, but not SCoRK127A or

SCoRY50A, lowered the levels of endogenous, iNOS-derived SNO-Ube1a/b, SNO-FASN,

SNO-ENO1, and SNO-LDHA (Fig. 2.9C), identifying these proteins as targets of SNO-

CoA-mediated (and SCoR-regulated) S-nitrosylation.

We also assessed whether reduced SNO-CoA binding by SCoRK127A would manifest in changes in S-nitrosylation by endogenous nitric oxide or with low concentrations of NO derived from the long half-life nitric oxide donor DETA-NONOate.

Cells expressing SCoRK127A displayed enhanced basal S-nitrosylation of two CoA- dependent enzymes, SNO-FASN and SNO-ATP-citrate (ACLY), and SNO-FASN and SNO-ACLY levels increased upon treatment with DETA-NONOate in SCoRK127A, but not SCoRWT, expressing cells (Fig. 2.9D). Additionally, SNO-Ube1a/b and SNO-pyruvate kinase M2 (PKM2; a known SCoR target (200) were basally elevated in SCoRK127A cells

(Fig. 2.9D).

Further, we evaluated SCoR activating mutations (K23A and W220A) to determine whether SCoR function is limited by enzyme turnover. Overexpression of SCoRK23A and

SCoRW220A increased SNO-CoA reductase activity in cell lysates (Fig. 2.10, A and B), consistent with in vitro enzyme kinetics (Fig. 2.3A and Table 2.1). However, neither

SCoRK23A nor SCoRW220A had added effect on SNO-FASN, SNO-ACLY or SNO-GAPDH levels under basal conditions or in cells treated with DETA-NONOate (Fig. 2.10C),

58 indicating that SCoR-mediated denitrosylation at baseline and under conditions of nitrosative stress is likely not limited by enzyme turnover.

Taken together, our results suggest that SNO-CoA exists in equilibrium with myriad SNO-proteins across cellular processes to regulate protein S-nitrosylation, with

SCoR controlling these equilibria via reduction of SNO-CoA.

2.3.5 SCoR regulates mitochondrial metabolism

We were interested to know if SCoR-mediated denitrosylation of proteins

(Appendix 2.3) alters mitochondrial metabolism. Utilizing the Seahorse analyzer platform, we measured oxygen consumption rate (OCR, a measure of mitochondrial respiration) and extracellular acidification rate (ECAR, largely a measure of cellular glycolysis) in SCoR-

KO cells stably expressing SCoRWT or SCoRK127A. Cells expressing SCoRK127A displayed enhanced basal OCR (Fig. 2.11, A and C). Treatment with DETA-NONOate reduced OCR in both SCoRK127A and SCoRWT cells, potentially due to SCoR-independent inhibitory S- nitrosylation of respiratory chain components (217, 218). Both SCoRK127A and SCoRWT expressing cells increased OCR in response to cellular stress by oligomycin (ATP synthesis inhibitor) and FCCP (uncoupling agent). Basal ECAR was also slightly enhanced in

SCoRK127A expressing cells (Fig. 2.11, B and D). DETA-NONOate treatment increased

ECAR in both SCoRWT and SCoRK127A expressing cells, likely in response to reduced

OCR. While SCoRWT and SCoRK127A cells increased ECAR in response to oligomycin and

FCCP, DETA-NONOate treated cells were unable to increase ECAR. Taken together, these results suggest that increased S-nitrosylation by endogenous SNO-CoA increases mitochondrial respiration, and to a lesser degree glycolysis, whereas the effects of exogenous NO (to dramatically reduce mitochondrial respiration) are SCoR independent.

59 2.4 DISCUSSION

Our structure/function studies on AKR1A1 provide the first molecular insights into recognition of a SNO substrate and strong support for the proposition that the enzyme’s primary role is to regulate cellular S-nitrosylation. AKR1A1 is the founding member of the aldo-keto reductase family, and is highly conserved in mammals, yet a primary carbonyl substrate has not been identified in man (192). Our results (and previous studies (20, 193,

215, 216)) indicate that SNO-CoA is in fact a preferred endogenous AKR1A1 substrate and that AKR1A1 is the primary mammalian SNO-CoA reductase (SCoR). SNO-CoA recognition by SCoR involves interactions with both the CoA backbone (Lys 127) and the

SNO moiety (active site residues). The latter finding, i.e. recognition of the SNO group, has important consequences for the field as it represents a basis for specificity that has been heretofore missing from conceptual understanding of NO-based signaling. Knowledge that enzyme—RSNO interactions will likely entail molecular recognition of both R and SNO groups could also influence inhibitor design where SCoR inhibition proves beneficial.

Molecular recognition of the SNO group by SCoR is important for SCoR function in situ, as evidenced by preferential binding of SCoR to SNO-CoA vs. CoA. Consequently,

SCoR also catalyzes the reduction of SNO-cysteamine (Fig. 2.5F). But whereas the interaction of the SNO moiety with SCoR provides a mechanism to outcompete high levels of cellular CoA and CoA derivatives, interaction of the CoA moiety with Lys127 will favor binding of SNO-CoA over SNO-cysteamine. Cytosolic concentrations of CoA are also likely to be much higher than cysteamine. Thus, reduction of SNO-cysteamine may be a fortuitous activity rather than a physiological function of the enzyme.

60 How exactly SCoR recognizes the SNO moiety is still unclear. The active site of

SCoR is comprised of four critical residues: proton-donating Tyr50, Asp45 and Lys80, which influence both Tyr50 pKa and nucleotide binding (193), and His113, which is important for substrate orientation (193). Notably, the active site of SCoR is positively charged (Fig. 2.6), a characteristic that should markedly alter the electronic state of the

SNO group through charge coordination with individual S—N—O atoms (104, 212). More specifically, the positively—charged residues near the active site may draw electrons to the N or O (102), promoting interaction with His113 to thereby orient SNO-CoA (and

SNO-cysteamine) for efficient catalysis. This substrate orientation mechanism would be absent in CoA (or Ac-CoA). Interestingly, mutation of Arg312 (a residue lining the active site) to Ala greatly reduced SNO-CoA reductase activity through a primary effect on Kcat.

This decrease in Kcat contrasts with the primarily effect of Lys127 mutation on Km and may indicate importance of Arg312 for SNO recognition and orientation. Collectively, these data suggest a catalytic sequence involving initial attraction of SNO-CoA to SCoR via CoA interaction with Lys127 and subsequent polarization and orientation of the SNO moiety by Arg312 and His113. The reaction would be completed via hydride transfer from

NADPH to the N atom of the S—N—O and protonation of the O atom by Tyr50, similar to the conserved reaction mechanism for carbonyls (199).

The list of SNO-proteins regulated by SCoR includes overrepresentation in several metabolic processes, including glycolysis, TCA cycle and amino acid metabolism

(Appendix 2.3). This is supported by demonstration of altered cellular metabolic parameters, in particular mitochondrial parameters, in cells expressing mutant SCoR with impaired substrate (SNO-CoA) binding. Collectively, these data strengthen the case for an

61 important physiological role for the SCoR/SNO-CoA system in NO-based cellular signaling and metabolism. Future work will determine the role of individual SNO-proteins in mediating metabolic effects and whether SCoR regulates whole-body metabolism in mammalian physiology and pathophysiology.

Interestingly, Lys127 is a putative target of CoA-based acetylation and (219); either modification would neutralize the positive charge at that residue and thereby likely inhibit SCoR activity (similar to the K127A mutation). Consequently,

Lys127 may represent a for crosstalk between CoA-based post-translational modifications that could work in concert to regulate energy demands of the cell.

62 2.5 FIGURES AND TABLES

Table 2.1

Table 2.1. Enzyme kinetics for SCoR/SCoR mutants with substrates.

a a a Km Vmax Kcat Substrate Enzyme (μM) (μM/min) (min-1)

WT 58 ± 4.4 19.2 ± 0.39 959

SNO-CoA K127A 410 ± 27 15.0 ± 0.41 750

K127R 52 ± 4.6 14.6 ± 0.32 730

Dephospho-SNO- WT 49 ± 6.7 14.7 ± 0.53 733 CoA

SNO-Cysteamine WT 476 ± 72 2.45 ± 0.13 123

WT 2554 ± 260 6.14 ± 0.24 306 DL-glyceraldehyde K127A 2469 ± 256 6.24 ± 0.26 312

WT 4555 ± 726 12.08 ± 0.78 604 Glucuronate K127A 4728 ± 907 10.05 ± 0.26 502 a Km and Vmax were determined from Michaelis-Menten curves using GraphPad Prism 7.

Kcat was calculated by dividing Vmax by the enzyme concentration in each assay. Enzyme assays were performed in triplicate.

63 Table 2.2

Table 2.2. Additional enzyme kinetics for SCoR and SCoR mutants.

a a a Km Vmax Kcat Substrate Enzyme (μM) (μM/min) (min-1)

WT 58 ± 4.4 19.2 ± 0.39 959

W22A 64 ± 9.5 12.9 ± 0.54 645

K23A 64 ± 10.2 26.2 ± 1.17 1308 SNO-CoA W220A 46 ± 8.8 22.4 ± 1.08 1122

R312A 132 ± 11.7 8.22 ± 0.21 410

K97A 65 ± 8.3 19.8 ± 0.71 987

WT 2554 ± 260 6.14 ± 0.24 306

W22A 361 ± 241 0.11 ± 0.16 6

K23A 22201 ± 3035 13.0 ± 1.20 650 DL-glyceraldehyde W220A 3333 ± 460 7.69 ± 0.39 384

R312A 4311 ± 319 3.97 ± 0.13 198

K97A 3580 ± 469 8.01 ± 0.45 401

a Km and Vmax were determined from Michaelis-Menten curves using GraphPad Prism 7.

Kcat was calculated by dividing Vmax by the enzyme concentration in each assay. Enzyme assays were performed in triplicate.

64 Figure 2.1

Figure 2.1. Molecular modeling of SNO-CoA within the SCoR active site. (A-D)

Models of SNO-CoA [in binding mode 1 (A) and binding mode 2 (B)], DL-glyceraldehyde

(C), and glucuronate (D) bound to the SCoR active site. Putative SCoR SNO-CoA interacting residues are labeled in red. NADPH carbons are colored green. Modeling was performed in Schrödinger Maestro and images were generated in PyMol.

65 Figure 2.2

Figure 2.2. Purification of Recombinant SCoR. Recombinant His-tagged SCoR was produced in bacteria and purified by nickel affinity chromatography. 1 g of purified protein was separated by SDS-PAGE and visualized by Imperial staining. Lanes are labeled with the corresponding SCoR variant. X is a SCoR mutant that was not further analyzed.

66 Figure 2.3

Figure 2.3. The conserved Lys127 in SCoR facilitates SNO-CoA reductase activity.

WT (A) Catalytic efficiency (Kcat/Km) of SCoR mutants expressed relative to SCoR . Km and

WT Kcat were determined from two independent purifications of SCoR and mutant enzymes, and used to generate an average catalytic efficiency for each enzyme and substrate. Scatter plot represents mean ± SD. (B-D) SCoRK127A inhibits SCoR-mediated SNO-CoA

67 reduction. Michaelis-Menten curves for SCoRWT and SCoRK127A using DL-glyceraldehyde

(B), SNO-CoA (C), and glucuronate (D) as substrates. Enzyme assays were performed in triplicate. Error bars are not shown for data points where standard deviation is very small.

(E) Lys127 is conserved across mammalian species. Amino acid sequence alignment for select mammalian species. Sequences from Uniprot were aligned using ClustalW. Lys127 is highlighted with a black box. The residue number of the final amino acid is listed on the right. * indicates complete residue conservation; : indicates conservation of amino acids with strongly similar properties; . indicates conservation of amino acids with weakly similar properties.

68 Figure 2.4

Figure 2.4. SCoRK127A mutation lowers SNO-CoA reductase activity. NADPH consumption over 10 minutes by SCoRWT (WT), SCoRK127A (K127A), or SCoRY50A

(Y50A) using 100 M NADPH (A), 3 mM DL-glyceraldehyde + 100 M NADPH (B), or

50 M SNO-CoA + 100 M NADPH (C), as substrates. Assays were performed in triplicate and raw absorbance at 340 nm was plotted over time in GraphPad Prism 7. Error bars are not plotted where standard deviation was very small.

69 Figure 2.5

Figure 2.5. SCoR recognizes the CoA backbone and SNO moiety of SNO-CoA. (A)

Dephosphorylated SNO-CoA alters Vmax but not Km. Michaelis-Menten curves for

SCoRWT and SCoRK127A with SNO-CoA or dephospho-SNO-CoA (De-P-SNO-CoA) as substrates. Assays were performed in triplicate. Error bars are not shown for data points where standard deviation is very small. SCoRWT and SCoRK127A with SNO-CoA are from

K127R Fig. 2C and are shown here for comparison. (B) SCoR alters Vmax but not Km.

Michaelis-Menten curves for SCoRWT, SCoRK127A and SCoRK127R with SNO-CoA as substrate. Assays were performed in triplicate. Error bars are not shown for data points where standard deviation is very small. SCoRWT and SCoRK127A with SNO-CoA are from

70 Fig. 2C and are shown here for comparison. (C) SCoRK127A reduces binding to SNO-CoA and CoA. Representative western blot for SCoR following incubation of purified recombinant enzymes with SNO-CoA- or CoA-beads. (D) Quantification (n=5) of

SCoRWT, SCoRK127A and SCoRK127R binding to SNO-CoA-beads from Fig. 3C. Bands were quantified using ImageJ. Scatter plot and bars represent mean ± SD. P-value < 0.01 by one- way ANOVA with Tukey’s correction for multiple comparisons. (E) SCoR preferentially binds SNO-CoA. Quantification (n=5) of SCoRWT binding to SNO-CoA- or CoA-beads from Fig. 2.5C. Bands were quantified using Image J. Scatter plot and bars represent mean

± SD. P-value < 0.05 by Student’s t-test. (F) SCoR metabolizes SNO-cysteamine. (Upper)

Linear representation of the core components of SNO-CoA. (Lower) Michaelis-Menten curves for wild-type SCoR and the substrates SNO-CoA and SNO-cysteamine. Assays were performed in triplicate. Error bars are not shown for data points where standard deviation is very small. SCoRWT with SNO-CoA is from Fig. 2.3 and is shown here for comparison. (G) CoA and Acetyl-CoA (Ac-CoA) poorly inhibit SNO-CoA reduction by

SCoR. Increasing concentrations of CoA or Ac-CoA were added to a reaction mix of 100

M SNO-CoA/NADPH and 20 nM SCoRWT. Assays were performed in triplicate. Error bars are not shown for data points where standard deviation is very small. IC50s were calculated in GraphPad Prism 7.

71 Figure 2.6

Figure 2.6. Lys127 provides a positively charged residue near the SCoR active site.

Surface representation of electrostatic charge distribution for SCoRWT (A), SCoRK127A (B), and SCoRK127R (C) demonstrates a strong positive charge near the SCoR active site that is

72 lost upon mutation of Lys127 to Ala but preserved by mutation of Lys127 to Arg. The electrostatic surface was generated in PyMol using the SCoR crystal structure (PDB:

3H4G; H2O and fidarestat removed) and the APBS plug-in with a lower boundary of -2 and an upper boundary of +2. The location of residue 127 is highlighted by a green box.

73 Figure 2.7

Figure 2.7. SCoRK23A and SCoRW220A do not alter binding to SNO-CoA. (A)

Representative western blot for SCoR following incubation of purified recombinant enzymes with SNO-CoA beads. (B) Quantification (n=3) of SCoRWT, SCoRK23A and

SCoRW220A binding to SNO-CoA beads. Bands were quantified using ImageJ. Scatter plot and bars represent mean ± SD. P-value > 0.05 (n.s.) by one-way ANOVA with Tukey’s correction for multiple comparisons.

74 Figure 2.8

Figure 2.8. SCoR regulates SNO-CoA-dependent protein S-nitrosylation in tissue lysates. (A) Recombinant wild-type human SCoR rescues SNO-CoA reductase activity.

SNO-CoA reductase specific activity was measured in kidney lysate from wild type (WT)

SCoR mice or SCoR knockout (SCoR-/-; KO) mice (n=3). Activity was reconstituted in

SCoR-/- lysates by the addition of 150 nM recombinant human SCoRWT or SCoRK127A.

75 Scatter plot and bars represent mean ± SD. P-value < 0.05 by Student’s t-test. (B)

Representative (n=3) Imperial-stained SDS-PAGE gel showing SNO-proteins enriched by

SNO-RAC. Mouse kidney lysates were treated with 50 M SNO-CoA, 100 M NADPH, and recombinant SCoRWT or SCoRK127A (as indicated) for 2 minutes. The S-nitrosylation reaction was stopped by the addition of acetone and SNO-RAC was performed. SCoR and

GAPDH were assessed by western blot analysis. Imperial-stained gels were quantified using ImageJ. Scatter plate and bars represent mean ± SD. P-value <0.05 by Student’s t- test for experimental lanes 6 and 7. mSCoR = endogenous mouse SCoR; hSCoR = recombinant human SCoR.

76 Figure 2.9

Figure 2.9. SCoR regulates endogenous protein S-nitrosylation. (A) Stable overexpression of SCoR in SCoR-knock out (KO) HEK293 cells. Representative western blot analysis of SCoR expression in SCoRWT cells, SCoR-KO cells, or KO cells stably expressing wild-type (+WT), SCoRY50A (+Y50A), or SCoRK127A (+K127A). (B) Stable overexpression of SCoRWT rescues SCoR-dependent SNO-CoA reductase activity in KO

HEK293 cells. Fold change in specific activity of DL-glyceraldehyde and SNO-CoA reducing activity in lysate from indicated cell lines. Data represent the average fold change in specific activity from 3 independent passages. Scatter plots represent mean ± SD. P- value <0.05 by Student’s t-test. (C) SCoR regulates SNO-CoA-dependent protein S- nitrosylation. Western blot analysis of the S-nitrosylation status of putative targets of

77 SCoR-mediated denitrosylation from Dataset S2. iNOS was overexpressed in the indicated cell lines for 24 hours prior to cell harvest. SNO-proteins were enriched by SNO-RAC, separated by SDS-PAGE, and analyzed by western blot. (D) Western blot analysis of the

S-nitrosylation status of putative targets of SCoR-mediated denitrosylation. SCoR-KO

HEK293 cells stably expressing SCoRWT or SCoRK127A were untreated or treated for 20 hours with indicated concentrations of DETA-NONOate (DETA-NO) prior to harvest for

SNO-protein enrichment by SNO-RAC.

78 Figure 2.10

Figure 2.10. SCoR activating mutations do not reduce S-nitrosylation. (A) SCoR-KO

HEK293 cells were transfected with empty vector, SCoRWT, SCoRK23A, or SCoRW220A and harvested after 24 hours. SNO-CoA reductase activity was determined by enzyme assay for indicated cells using 100M SNO-CoA, 100M NADPH, and 25L of cell lysate and normalized to protein content to generate specific activity. Scatter plot and bars represent mean ± SD (n=3) of technical replicates to demonstrate intra-assay consistency. (B)

Western blot analysis demonstrating SCoR expression in the lysates assayed in A. (C)

SCoR-KO HEK293 cells were transiently transfected with SCoRWT, SCoRK23A, or

SCoRW220A for 6 hours and then treated with media  DETA-NONOate (DETA-NO) for

20 hours prior to harvest for assessment of protein S-nitrosylation (+Asc) by SNO-RAC (-

Asc = control).

79 Figure 2.11

Figure 2.11. SCoR regulates cellular energy metabolism. (A and B) SCoR-KO HEK293 cells stably expressing SCoRWT or SCoRK127A were assessed for oxygen consumption rate

(OCR, panel A) and extracellular acidification rate (ECAR, panel B) using the Seahorse xFe24 analyzer. Cells were grown overnight and then treated with or without 125 M

DETA-NONOate (DETA-NO) for 20 hours before analysis. Oligomycin (2 M) and FCCP

(3 M) were co-injected following the 3rd measurement, as indicated by the arrow. Data represent mean  SEM of three independent replicates (with 5 data points each) per experimental group. Error bars are not shown for data points where standard error of the

80 mean is very small. (C and D) Panels C and D summarize data in panels A and B, respectively, before addition of oligomycin and FCCP. Data represent mean  SD. P-value calculated by one-way ANOVA with Tukey’s correction for multiple comparisons.

81 2.6 EXPERIMENTAL PROCEDURES

2.6.1 Animals

Animal studies were approved by the Institutional Care and Use Committee

(IACUC) at Case Western Reserve University. All housing and procedures complied with the Guide for the Care and Use of Laboratory Animals (220) and the American Veterinary

Medical Associations guidelines regarding euthanasia (221).

2.6.2 Molecular Modeling

Static protein/flexible ligand modeling of the interaction of SNO-CoA, DL- glyceraldehyde, and glucuronate were performed using Maestro 9.9 software. The SCoR crystal structure (PDB: 3H4G) was prepared for docking by removing H2O and fidarestat from the PDB file. In Maestro, original were removed and replaced, bond orders were assigned, and the structure was minimized. A grid was prepared around the active site centered at X = -2.0999, Y = -24.4072, Z = -6.5084. CoA, DL-glyceraldehyde, and glucuronate structures were obtained from PubChem. The SNO-CoA structure was generated by editing the CoA structure in Maestro. All ligands were prepared for docking in Maestro using the ligand preparation function. Ligands were docked to the active site grid using XP Glide Docking with post-docking minimization.

2.6.3 Generation and expression of recombinant wild-type and mutant SCoR

The human SCoR coding sequence was previously cloned into a pET21b vector

(20). Human SCoR mutants were generated by site-directed mutagenesis of pET21b-SCoR using the Agilent QuikChange XL II system per manufacturer’s instructions. Mutants were verified by sequencing. Primers for specific mutations are listed in Supporting Information

(Table S2). pET21b-SCoR and mutants were transformed into Rosetta2(DE3)pLysS E. coli

82 (EMD Millipore) and expression was induced by the addition of 100 M isopropyl--D-

1-thiogalactopyranoside (Sigma) at A600nm = 0.4. Bacteria were grown for 4 hours at 25C and recombinant His-tagged SCoR was purified as previously described (20). Purification was assessed by SDS-PAGE followed by Imperial Protein Stain (Thermo Fisher).

2.6.4 Kinetic analysis of recombinant SCoR

Kinetic parameters of recombinant wild-type and mutant SCoR were determined in

50 mM phosphate buffer, pH 7.0 containing 100 M EDTA (Sigma) and 100 M DTPA

(Sigma). Triplicate reactions were performed with 20 nM recombinant SCoR, 100 M

NADPH (Sigma), and varying concentrations of SNO-CoA, dephospho-SNO-CoA, SNO- cysteamine, DL-glyceraldehyde (Sigma) or D-glucuronate (Sigma). SNO-CoA, dephospho-SNO-CoA, and SNO-cysteamine were prepared by reacting equal volumes of

0.1 M CoA (Sigma), dephospho-CoA (Sigma), or cysteamine (Sigma) in 1 M HCl and 0.1

M NaNO2 (Fluka Chemicals) in MilliQ containing 100 M EDTA and 100 M

DTPA. Initial rates were calculated using absorbance decrease at 340 nm and an extinction coefficient of 7.06 mM-1cm-1 (combined for SNO-CoA and NADPH). Kinetic parameters

(Km and Vmax) were determined using GraphPad Prism 7, and Kcat was derived from Vmax and enzyme concentration. For NADPH consumption curves, reactions (50 M SNO-CoA or 3 mM DL-glyceraldehyde with 100 M NADPH and 20 nM enzyme) were allowed to proceed for 10 minutes while measuring absorbance at 340 nm. CoA and Ac-CoA inhibition assays were performed in triplicate as above but with a static concentration of

100 M SNO-CoA and varying concentrations of CoA and Ac-CoA (Sigma) dissolved in

50mM phosphate buffer, pH 7.0 containing 100 M EDTA and 100 M DTPA. IC50 was determined using GraphPad Prism 7.

83 2.6.5 CoA and SNO-CoA bead pull-down assays

CoA beads (50% slurry) were prepared by suspending CoA agarose powder

(Sigma) in water overnight at 4°C. To generate SNO-CoA beads, CoA beads were washed twice with 30 volumes of 10 mM HCl and supernatant was aspirated. Pelleted CoA beads were resuspended in 0.5 mL 10 mM HCl and 0.5 mL 10 mM NaNO2 was added to the suspension to generate SNO-CoA. Immediately following NaNO2 addition, 10 mL washing buffer (150 mM NaCl (Fisher), 1 mM EDTA, 1 mM DPTA, 0.1 mM neocuproine

(Sigma) and 50 mM borate buffer pH 8.2) was added into tube to dilute SNO-CoA beads and the SNO-CoA beads were washed three times with washing buffer. For binding experiments, 1 g recombinant SCoRWT, SCoRK127A, or SCoRK127R was diluted in 1 mL binding buffer (250 mM NaCl, 1 mM EDTA, 1 mM DPTA, 0.1 mM neocuproine and 50 mM borate buffer pH 8.2). 10 L (10 ng) of protein solution was saved for input analysis.

The remaining 990 L of protein solution was incubated with 30 l 50% SNO-CoA or

CoA beads for 2 hours at 4°C in the dark. After incubation, the beads were washed six times with binding buffer, and bound proteins were eluted with 50 l 1X SDS loading dye

(Bio-Rad) containing 5% 2-mercaptoethanol (Sigma).

2.6.6 SCoR-dependent SNO-CoA reductase activity in mouse kidney lysate and analysis of protein S-nitrosylation

The generation of SCoR-/- mice was reported previously (20). Twelve-week old male SCoR+/+ (WT) and SCoR-/- mice were euthanized and tissue samples were collected and immediately frozen in liquid nitrogen. Kidney tissue was Dounce homogenized in 50 mM phosphate buffer, pH 7.0 containing 150 mM NaCl, 100 M EDTA, 100 M DPTA, and protease inhibitor cocktail (Roche). Tissue extracts were cleared by centrifugation (2X;

84 20,000 g, 30 minutes). Protein concentration was determined by bicinchoninic acid assay

(Pierce). Assays for specific activity in kidney lysates were performed in 50 mM phosphate buffer, pH 7.0 containing 50 M SNO-CoA, 100 M NADPH, 100 M EDTA, and 100

M DPTA. Reactions were initiated by the addition of WT or SCoR-/- lysate (500 g protein) with or without 150 nM recombinant SCoRWT or SCoRK127A enzyme added to

SCoR-/- lysate. Initial rate was calculated from the change in absorbance at 340 nm and an extinction coefficient of 7.06 mM-1cm-1 (combined for SNO-CoA and NADPH). For

SNO-protein analysis by SNO-RAC (18), similar reactions were performed using 500

g/mL protein with and without the addition of 100 M NADPH, 50 M SNO-CoA, and

150 nM recombinant enzyme, and allowed to proceed for 2 minutes. Reactions were stopped by the addition of 3 volumes ice cold acetone and proteins were incubated for 20 minutes at -20C. Precipitated proteins were pelleted by centrifugation (4000g at 4C for

5 minutes). Following removal of supernatant, proteins were resuspended in HEN buffer, pH 8.0 (100 mM HEPES, 1 mM DTPA, 0.1 mM neocuproine) containing 2.5% SDS and

0.2% S-methylmethanethiosulfonate (MMTS; Sigma) and incubated at 50C for 20 minutes with frequent vortexing. Proteins were again precipitated with 3 volumes of ice cold acetone and incubated at -20C for 20 minutes. Precipitated proteins were pelleted by centrifugation and resuspended in 1 mL HEN buffer containing 1% SDS. The process of precipitation and resuspension in 1 mL HEN buffer with 1% SDS was repeated. Proteins were then incubated with thiopropyl-sepharose beads (GE Healthcare) for 4 hours in the dark with 30 mM ascorbate (Sigma). Beads were subsequently washed with HEN/1% SDS buffer and 10-fold diluted HEN/1% SDS buffer. Proteins were eluted from the beads in 1X

85 SDS loading dye containing 10% 2-mercaptoethanol. Eluate was separated by SDS-PAGE and SNO-proteins visualized with Imperial protein stain per manufacturer’s instructions.

2.6.7 Identification of SNO-proteins by iTRAQ-coupled LC-MS/MS

SNO-proteins enriched by SNO-RAC (previously described) were separated by

SDS-PAGE and stained with Imperial protein stain. Each column of gel bands was sliced and collected in two 1.5 mL tubes. The gel slices were washed with 500 L of 50% acetonitrile (ACN) / 50% 100 mM ammonium bicarbonate for more than 5 hours with vortexing. After removal of washing buffer, 400 L of 100% ACN was added to gel pieces and vortexed for 10 min. After removal of ACN, gel pieces were dried in a speed vacuum dryer for 10 minutes. 200 L of 10 mM dithiothreitol (DTT) was added to dry gel pieces and vortexed for 45 minutes. 200 L of 55 mM iodoacetamide (IAA) was added to the gel pieces after removal of DTT buffer and incubated for 45 minutes in the dark. After removal of IAA buffer, gel pieces were washed with 400 L of 1x iTRAQ dissolution solution then

400 L ACN, and this cycle was repeated once. Gel pieces were dried for 10 minutes in a speed vacuum dryer. 500 ng trypsin enzyme in 150 L 1X iTRAQ buffer was added to dried gel pieces on ice for 30 minutes, and then incubated overnight at 37C. Following incubation, supernatant from digested protein solution was transferred to a 1.5 mL tube using gel-loading tips. 200 L extraction buffer of 60% ACN / 5% formic acid was added to gel pieces, vortexed for 30 minutes, and sonicated for 15 minutes. Supernatant containing extracts was transferred to 1.5 mL tubes, and extraction was repeated two more times. The digested protein solution was dried completely. To label with iTRAQ reagents, 30 L of iTRAQ dissolution buffer (10x) were added to each sample tube (pH>7.0). iTRAQ reagent (114, 115, 116, 117) was brought to room temperature and

86 70 L of ethanol was added to each reagent. One iTRAQ labeling reagent was added to each sample tube. The labeling reaction was allowed to proceed for more than 5 hours at room temperature with vortexing. After labeling, the samples were mixed together and dried completely.

Prior to mass spectrometry, samples were cleaned-up as follows. 160 L of 5%

ACN containing 0.5% trifluoroacetic acid (TFA) was added to the dried mixed-label sample. C18 ZipTips were wetted 5 times with 20 L of 50% ACN and equilibrated with

100 L of 5% ACN containing 0.5% TFA. Samples were then loaded to the tip by drawing and expelling 50 cycles to ensure complete binding. The tips were washed 10 times with

20 L of 5% ACN containing 0.5% TFA. Peptides were eluted 3 times from tips with 20

l of 60% ACN containing 0.1% formic acid, combined, and dried completely.

Digested peptides were separated by UPLC (, Milford, MA) with a Nano-

ACQUITY UPLC BEH300 C18. Separated peptides were continuously injected into an

Orbitrap Elite hybrid mass spectrometer (Thermo Finnigan) by a nanospray emitter (10

µm, New Objective). A linear gradient was used in chromatography by using mobile phase A (0.1% formic acid in water) and B (100% ACN) at a flow rate of 0.3 µL/min.

Chromatography started with 1% mobile phase B and gradually increased to 40% at 130 minutes, then increased to 90% within 2 minutes and stayed at 90% for 10 minutes to clean the column. All mass spectrometry data were acquired in a positive ion mode. A full MS scan (m/z 300-1800) at resolution of 120,000 was conducted; ten MS2 scans

(m/z 100-1600) were activated from five most intense peptide peaks of full MS scans.

CID and HCD cleavage modes were performed alternatively on same peptides selected from full MS scans. MS2 resolution of HCD is 15,000.

87 Bioinformatic software MassMatrix was used to search MS data against a database composed of sequences of mouse or human proteins (depending on origin of sample) from

Uniprot and their reversed sequences were used as a decoy database. Modifications such as oxidation of methionine and labeling of cysteine (IA modifications) were selected as variable modifications in searching. For iTRAQ label searches, MS tagging of N terminus,

Lys and/or Tyr were selected as variable modifications to test labeling efficiency and as fixed modifications for quantitative iTRAQ analysis. Trypsin was selected as an in-silico enzyme to cleave proteins after Lys and Arg. Precursor ion searching was within 10 ppm mass accuracy, and ions were within 0.8Da for CID cleavage mode and 0.02Da for HCD cleavage mode. 95% confidence interval was required for protein identification.

2.6.8 Generation of SCoR mammalian expression plasmid

The SCoR coding sequence was amplified from pet21b-SCoR with the following primers:

F, 5-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGCGGCTTCCTGTGTTCTA-3; and R, 5-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAGTACGGGTCATTAAAGGG-3; or

R, 5-GGGGACCACTTTGTACAAGAAAGCTGGGTTGTACGGGTCATTAAAGGGGTA-3 (to remove stop codon).

The SCoR coding sequence was cloned into Gateway vector pDONR221 (Thermo

Fisher) per manufacturer’s instructions and verified by sequencing. Mutant pDONR221-

SCoR vectors were generated by site-directed mutagenesis using primers and procedures as described previously. Sequence-verified wild-type and mutant SCoR constructs were shuttled to pcDNA-DEST40 (Thermo Fisher) per manufacturer’s instructions and verified by sequencing.

88 2.6.9 Western blot analysis

Western blot analyses were performed using standard methods. Antibodies used were: SCoR (Santa Cruz Biotechnology, sc-100500); GAPDH (Abcam, ab181602); FASN

(, 3180S); Ube1a/b (Cell Signaling, 4891S); ENO1 (Cell Signaling,

13410S); LDHA (Cell Signaling 3582S); ACLY (Cell Signaling, 13390); PKM2 (Santa

Cruz Biotechnology, sc-365648); NOS2 (Santa Cruz Biotechnology, sc-8310).

2.6.10 Assay of SCoR activity in cell lysate

HEK293 cells were purchased from the American Type Culture Collection (ATCC) and cultured at 37C, 5% CO2 in growth media [DMEM (Gibco) supplemented with 10% fetal bovine serum (Sigma), and 1X antibiotic-antimycotic (Gibco)]. Empty vector, wild- type or mutant SCoR cDNA constructs were transfected into SCoR-WT or SCoR-KO

HEK293 cells using Lipofectamine 2000 (Thermo Fisher) per manufacturer’s instructions.

After 24 hours, cells were washed 2x with cold PBS and harvested in PBS with a cell scraper. Cells were pelleted at 1500 RPM for 3 minutes and supernatant was aspirated.

Cells were resuspended in 50 mM phosphate buffer, pH 7.0 containing 100 M EDTA,

100 M DTPA, and protease inhibitor cocktail (Roche). Cells were lysed by sonication and cell debris pelleted by centrifugation (20000g at 4C for 15 minutes). The resulting supernatant was used for activity assays as described previously using 100 M SNO-CoA or 3 mM DL-glyceraldehyde, 100 M NADPH, and 25 L of cell lysate. SCoR expression was verified by western blot analysis.

2.6.11 Generation of SCoR-deficient HEK293 by CRISPR/Cas9

HEK293 cells were transfected with the Dharmacon Edit-R system in order to generate SCoR-deficient HEK293 cells, per manufacturer’s instructions. Briefly, HEK293

89 cells were transfected with a mixture of Cas9 plasmid, TracrRNA, and crRNA (targeting human SCoR; Dharmacon, CR-005087-05) using Dharmafect Duo (Dharmacon). After 24 hours, cells were trypsinized and subcultured in growth media supplemented with 3.3

g/mL puromycin hydrochloride (Gibco). Cells were selected for 2 weeks with frequent media changes, and then single colonies were manually selected and individually plated for expansion. Loss of SCoR expression was verified by western blot analysis.

2.6.12 Stable overexpression of SCoR

Wild-type or mutant SCoR in pcDNA-DEST40 without a stop codon (or empty vector) was linearized using PvuI restriction enzyme (Thermo Fisher). Wild-type or SCoR- deficient HEK293 cells were transfected with linearized constructs using Lipofectamine

2000 (Thermo Fisher) per manufacturer’s instructions. After 48 hours, cells were trypsinized and subcultured in growth media supplemented with 500 g/mL Geneticin

(Gibco). After several passages, SCoR expression was verified by western blotting and

SCoR activity was assessed as described previously.

2.6.13 Analysis of SNO-proteins in SCoR-deficient and -overexpressing HEK293 lines

SCoR-HEK cell lines were maintained in growth media supplemented with 500

g/mL Geneticin. For experiments using iNOS as the endogenous nitric oxide source, cells were transfected with human inducible nitric oxide synthase in pcDNA-3.1 vector using

Lipofectamine 2000 per manufacturer’s instructions. After 24 hours, cells were washed with and harvested in cold PBS containing 100 M EDTA and 100 M DTPA. Cells were pelleted by centrifugation at 1500RPM for 3 minutes. The supernatant was aspirated and cells were frozen in liquid nitrogen. For SNO-RAC analysis of SNO-proteins, cells were thawed on ice and lysed by sonication in HEN buffer containing 1% NP-40 and 0.01%

90 MMTS. Cell debris was pelleted by centrifugation (20000g at 4C for 20 minutes) and the supernatant used for SNO-RAC analysis, as described previously. Proteins from replicates used for SNO-protein identification by mass spectrometry were separated by SDS-PAGE and stained with Imperial protein stain, and gels were processed for mass spectrometry as described above. Alternatively, SNO-proteins were visualized by western blotting using standard methods and antibodies listed above.

For experiments using a nitric oxide donor, cells stably expressing SCoRWT or

SCoRK127A were treated for 20 hours with complete growth media containing indicated concentrations of DETA-NONOate (Cayman Chemicals). DETA-NONOate was freshly prepared in 0.01 M NaOH and diluted in complete growth media to the final experimental concentration before addition to cells. SNO-proteins were enriched and analyzed by western blotting as described in the previous paragraph. Alternatively, SCoR-KO cells were transfected with SCoRWT, SCoRK23A, or SCoRW220A for 6 hours using PolyJet transfection reagent (Signagen) per manufacturer’s instructions. After 6 hours, transfection media was removed and replaced with fresh growth media with or without DETA-

NONOate. After 20 hours, cells were harvested for SNO-protein analysis as described above.

2.6.14 Metabolic analysis using Seahorse XFe24 Analyzer

All assays were performed on the Agilent Seahorse XFe24 analyzer using the Cell

Energy Phenotype Test Kit. XF24 microplates were coated with 5 g/mL poly-D-lysine

(Corning) in water for 3 hours. Poly-D-lysine was aspirated and plates washed two times with DPBS (Gibco). SCoR-KO cells stably expressing SCoRWT or SCoRK127A were trypsinized and resuspended in complete growth media to a concentration of 4x105 cells /

91 mL. 100 L of cell suspension was added to 10 wells per plate per cell line (40000 cells per well). 250 L of complete growth media was added to blank wells. Cells were allowed to settle and adhere for 5 hours before 150 L of complete growth media was added to cell wells for overnight growth. After 24 hours, 200 L of media was removed from cell wells and replaced with 200 L fresh growth media with or without DETA-NONOate to a final concentration of 125 M (5 wells per cell line were untreated; 5 wells per cell line were treated). Cells were incubated for another 20 hours before analysis. Cell metabolic analysis was performed following manufacturer’s instructions. Briefly, assay medium was prepared as Agilent phenol red-free base medium supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM (all Agilent reagents). 200 L of media was removed from all wells and wells were washed with 1 mL of assay medium. 450 L of fresh assay media was added to each cell and blank well. Cells were incubated at 37C with room air for 1 hour prior to analysis. XFe24 sensor cartridge was prepared per manufacturer’s instructions. After 1 hour incubation, cells were analyzed using the default Cell Energy

Phenotype program, injecting 2 M oligomycin and 3 M FCCP after the third measurement.

92 CHAPTER 3:

S-nitroso-coenzyme A reductase regulates low-density lipoprotein metabolism by

modulating circulating proprotein convertase subtilisin/kexin 9

Colin T. Stomberski1,2 and Jonathan S. Stamler1,3,4*

From the 1Institute for Transformative Molecular Medicine; 2Departments of

Biochemistry and 3Medicine, Case Western Reserve University, Cleveland, OH 44106;

4Harrington Discovery Institute, University Hospitals Cleveland Medical Center,

Cleveland, OH 44106

93 3.1 ABSTRACT

Protein S-nitrosylation is the covalent modification of cysteine thiols by nitric oxide in proteins to exert multifaceted control of cellular function. We recently described novel enzymatic regulation of protein S-nitrosylation via metabolism of S-nitroso-coenzyme A

(SNO-CoA) by dedicated enzymes, SNO-CoA reductases (SCoRs), and identified a role for yeast SCoR in ergosterol metabolism. However, the role of SCoR/SNO-CoA in regulation of mammalian cholesterol metabolism remains unexplored. Here we show that

SCoR-deficient mice display low-density lipoprotein receptor (LDLR)-dependent hypocholesterolemia due to reduced circulating proprotein convertase subtilisin/kexin 9

(PCSK9). Tissue and cell-based analyses revealed that SCoR regulates PCSK9 S- nitrosylation to reduce PCSK9 secretion, and mutation of Cys301 in PCSK9 prevents the inhibitory effect of S-nitrosylation on PCSK9 secretion. Thus, S-nitrosylation of PCSK9 represents a novel mechanism controlling PCSK9 function and is the first example of direct regulation of PCSK9 by a posttranslational modification. Further, in- dosing of the

SCoR inhibitor imirestat reduced serum cholesterol and circulating PCSK9 in normal mice and in apolipoprotein B100/cholesteryl ester transferase protein transgenic mice, but not

LDLR-deficient mice. These results indicate that targeting the SCoR/SNO-CoA system may provide a novel treatment paradigm for hypercholesterolemia by modulating circulating PCSK9.

94 3.2 INTRODUCTION

Much of the ubiquitous influence of nitric oxide (NO) on cellular function in physiology and pathophysiology occurs via protein S-nitrosylation, the covalent attachment of NO to cysteine (Cys) thiol(s) in proteins (2, 7). Steady-state S-nitrosylation is governed by a set of dynamic equilibria between S-nitroso-thiols (SNOs) in proteins and in small molecule thiols regulated by the concerted actions of nitrosylases and denitrosylases (17, 212). S-nitroso-coenzyme A (SNO-CoA) and its dedicated denitrosylase (SNO-CoA reductase; SCoR) represent a novel mechanism regulating protein S-nitrosylation with the potential for broad ranging effects on cellular metabolism

(20, 200, 222).

Hypercholesterolemia and associated atherosclerosis leading to coronary heart disease, stroke, and peripheral artery disease is a leading cause of morbidity and mortality in the United States and globally (223). A protective role for NO in atherosclerotic plaque formation and progression has been established using multiple animal models (224).

Endothelial nitric oxide synthase (eNOS)-deficiency in apolipoprotein E (ApoE) knockout mice increases atherosclerotic lesion size (225, 226). Chemical inhibition of NO synthesis also increases atherosclerotic lesion size in hypercholesterolemic rabbits (227) and ApoE- deficient mice (228). Conversely, dietary delivery of the nitric oxide donor reduced aortic plaque size (229) and ApoE-deficient mice transgenically overexpressing eNOS have reduced atherosclerotic plaque size (230). Atherosclerosis- induced endothelial dysfunction and lipoproteins themselves reduce eNOS activity (231), generating a loop in which atherosclerotic plaques reduce NO production and promote further atherogenesis. In contrast, the direct role of nitric oxide in regulating circulating

95 cholesterol (lipoprotein) levels is less clear. Long-term feeding of the nitric oxide synthase inhibitor L-N- (L-NNA) to rats increased total serum cholesterol (232); treatment of hypercholesterolemic rabbits with sodium nitroprusside selectively lowered

LDL cholesterol (233). These findings suggest a role for NO in regulating serum cholesterol levels.

The low-density lipoprotein receptor (LDLR) regulates serum cholesterol homeostasis by internalizing LDL (and other lipoproteins) (234), and mutations in LDLR cause familial hypercholesterolemia (235). LDLR internalization is facilitated by the autosomal recessive hypercholesterolemia protein (ARH) and S-nitrosylation of ARH is required for LDLR/LDL endocytosis (236). LDLR stability is controlled, in part, by the negative regulator proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is synthesized and secreted into circulation primarily from hepatocytes. Circulating PCSK9 binds to the LDLR and biases it for lysosomal degradation, leading to decreased cell- surface LDLR, reduced LDL clearance, and hypercholesterolemia. Gain- and loss-of- function mutations in PCSK9 cause familial hypercholesterolemia and hypocholesterolemia, respectively (237). As such, inhibitors of PCSK9 drastically lower serum cholesterol and are associated with reduced cardiovascular events (238, 239). While a number of transcriptional programs regulate PCSK9 production (240–242), direct modulation of PCSK9 function by posttranslational modifications has not been described.

Our previous work (20) described a mechanism by which the yeast SCoR/SNO-

CoA system regulates cholesterol homeostasis via inhibition of cytosolic thiolase (Erg10 in yeast), the initial step in mevalonate (a precursor to cholesterol) biosynthesis. Therefore, we sought to determine whether mammalian SCoR would regulate cholesterol and

96 lipoprotein metabolism in animals. Using a combination of animal models and cell culture models, we demonstrate that mammalian SCoR regulates serum cholesterol in an LDLR- dependent manner by modulating the secretion of PCSK9. Genetic inhibition of SCoR increased PCSK9 S-nitrosylation concomitant with reduced PCSK9 secretion, and mutation of C301 in PCSK9 prevented the SNO-induced reduction in PCSK9 secretion.

Accordingly, long-term in-diet dosing of the SCoR inhibitor imirestat reduced circulating

PCSK9 and total cholesterol, identifying a potential new paradigm in the treatment of hypercholesterolemia.

97 3.3 RESULTS

3.3.1 SCoR-/- mice are hypocholesterolemic

To assess the potential role of mammalian SCoR in cholesterol regulation, we subjected wild-type and SCoR-/- mice to overnight fasting prior to serum collection. Total serum cholesterol was significantly lower in SCoR-/- mice (Fig. 3.1A) whereas serum triglycerides were unchanged (Fig. 3.1B). We also assessed serum cholesterol and triglycerides in unfasted wild-type and SCoR-/- mice and observed reduced total serum cholesterol (Fig. 3.1C) with unchanged serum triglycerides (Fig. 3.1D). Pooled serum from unfasted mice was fractionated into distinct lipoprotein pools, revealing a reduction in both

LDL cholesterol and HDL cholesterol (Fig. 3.1E). Together, these results indicate that

SCoR regulates serum cholesterol in mice.

3.3.2 SCoR regulates hepatic LDLR and LDLR is required for SCoR-dependent hypocholesterolemia

The liver eliminates excess cholesterol from the body as bile or bile salts and is thus central to regulating total circulating cholesterol levels, and hepatic lipoprotein receptors are required for uptake of specific lipoproteins and lipids into the liver. Because serum lipoprotein fractionation showed reductions in both LDL and HDL cholesterol, we assessed hepatic expression of the LDL receptor (receptor-mediated endocytosis of LDL) (243) and scavenger receptor class B type I (SR-BI; receptor-mediated endocytosis and/or lipid uptake from HDL) (244). Surprisingly, while SR-BI protein levels were unchanged (Fig.

3.3) in livers from SCoR-/- mice, LDLR protein levels were elevated (Fig. 3.2, A and B).

Additionally, expression levels and levels of S-nitrosylation of the cholesterol synthesis proteins acetyl-CoA acetyltransferase 2 (ACAT2; cytosolic thiolase) and 3-hydroxy-3-

98 methyl-glutaryl-CoA reductase (HMGCR; rate-limiting step in cholesterol synthesis) were unchanged (Fig. 3.4). These results suggest that enhanced hepatic LDLR expression may be responsible for relative hypocholesterolemia in SCoR-/- mice. We next generated SCoR-

/-/LDLR-/- mice to test the requirement of LDLR for SCoR-dependent hypocholesterolemia.

Deletion of SCoR in the absence of LDLR did not lower total serum cholesterol, providing evidence that LDLR is required for SCoR-mediated reduction in cholesterol (Fig. 3.2C).

Further, elevated LDLR protein levels were not due to altered LDLR transcription (Fig.

3.2B) and SCoR does not regulate the S-nitrosylation statue of LDLR (Fig. 3.4), suggesting an alternate mechanism regulating LDLR stability.

3.3.3 SCoR regulates circulating PCSK9

A key, negative posttranscriptional regulator of LDLR stability is PCSK9, a serum protein produced and secreted primarily from the liver (245). Binding of PCSK9 to LDLR promotes lysosomal degradation of LDLR. As such, PCSK9-/- mice are hypocholesterolemic due to increased hepatic LDLR protein levels (246), displaying reductions in both LDL cholesterol and HDL cholesterol; conversely, adenovirus-mediated expression of PCSK9 in mice leads to LDLR-dependent hypercholesterolemia (247). We assessed serum PCSK9 in wild-type and SCoR-/- mice and found reduced serum PCSK9 in

SCoR-deficient animals (Fig. 3.2D). Interestingly, this reduction in serum PCSK9 was concomitant with increased hepatic PCSK9 protein (Fig. 3.2, A, E and F) without altered

PCSK9 transcription (Fig. 3.2E). Further, S-nitrosylation of PCSK9 is greatly elevated in livers from SCoR-/- mice (Fig. 3.2F), identifying PCSK9 as a target of SCoR/SNO-CoA- mediated S-nitrosylation. The combination of reduced circulating PCSK9 and increased

99 hepatic PCSK9 suggests that SCoR-mediated S-nitrosylation of PCSK9 may regulate its secretion.

3.3.4 SCoR regulates PCSK9 secretion

To test whether SCoR regulates PCSK9 secretion, we generated human hepatoma

HepG2 cell lines stably expressing SCoR-targeting shRNA. Knockdown of SCoR in

HepG2 cells reduced the amount of PCSK9 acutely secreted into cell media (Fig. 3.5, A and B) while increasing the amount of cellular PCSK9 (Fig. 3.5, A and B), further suggesting a secretion defect. Consequently, LDLR expression was elevated in SCoR- replete HepG2 cells (Fig. 3.5, C and D). Additionally, SNO-PCSK9 was elevated in SCoR knockdown HepG2 cells (Fig. 3.5, E and F).

PCSK9 consists of three domains (237): i) a cleaved prodomain that acts as a chaperone for the catalytic domain; ii) a catalytic domain required for self-cleavage and maturation; and iii) a structural Cys-His rich domain (CHRD). While PCSK9 contains 26

Cys residues, 24 of the 26 exist in disulfide bonds in the mature protein (248).

SNO-PCSK9 analysis in mouse livers (Fig. 3.2F) and HepG2 cells (Fig. 3.5, E and F) demonstrated elevated S-nitrosylation in the cleaved peptide containing the catalytic domain and the CHRD; only Cys-301 exists as a free Cys in the cleaved peptide (248). To address whether Cys-301 may mediate the effect of S-nitrosylation on PCSK9 secretion, we transiently expressed PCKS9WT and PCSK9C301A in SCoR-deficient HEK293 cells

(222). Acute treatment with ethyl ester SNO-cysteine (a SNO donor) reduced secretion of

PCSK9WT but not PCSK9C301A (Fig. 3.5, G and H). These results are consistent with a regulatory role for S-nitrosylation of PCSK9 at Cys-301 in modulating PCSK9 secretion, and provide the first evidence for direct posttranslational control of PCSK9 function.

100 3.3.5 Chemical inhibition of SCoR reduces circulating PCSK9 and total serum cholesterol

Biological inhibitors (PCSK9-directed monoclonal antibodies) of PCSK9 have demonstrated efficacy in lowering LDL cholesterol and improving outcomes for patients with familial hypercholesterolemia and in those patients with prior cardiovascular events

(238, 239, 249). Despite much interest, small molecule inhibitors of PCSK9 have proven challenging to develop (237). Imirestat (AL-1576) is a small molecule inhibitor of SCoR that is orally bioavailable and active in mice (250). To test whether chemical inhibition of

SCoR could also regulate serum cholesterol, we in-diet dosed mice with imirestat for 4 weeks. After 4 weeks of imirestat treatment, SCoR activity was reduced in the livers of treated mice (Fig. 3.6A) to a similar degree as seen in SCoR-/- mice (20). Total serum cholesterol was reduced in imirestat-treated mice (Fig. 3.6B) and serum fractionation revealed reductions in both LDL and HDL cholesterol (Fig. 3.6C). Consistent with SCoR-

/- mice, serum PCSK9 was reduced following imirestat treatment (Fig. 3.6D). Expression analysis in livers from imirestat-treated mice demonstrated increases in both LDLR and

PCSK9 protein levels (Fig. 3.6, E and F) with no change in LDLR, PCSK9, or sterol- response element binding protein 2 (SREBP2; transcription factor regulating cholesterol genes) mRNA levels. Further, treatment of LDLR-/- mice with imirestat did not lower cholesterol (Fig. 3.6H), indicating that the LDLR is required for imirestat-mediated reduction in cholesterol.

Lipoprotein profiles differ substantially between mice and humans (251), with mice carrying the majority of circulating cholesterol in HDL while humans carry the majority of circulating cholesterol in LDL. To test the effect of imirestat in a more human-relevant

101 mouse model, we utilized transgenic mice expressing both apoB100 and cholesteryl ester transfer protein (CETP). This mouse has a lipoprotein profile similar to normocholesterolemic humans (252), with higher LDL cholesterol than HDL cholesterol.

In-diet dosing of imirestat substantially reduced total serum cholesterol in the apoB100/CETP transgenic mouse (Fig. 3.6I). Lipoprotein fractionation revealed a drastic reduction in LDL cholesterol with only a modest lowering of HDL cholesterol (Fig. 3.6J), consistent with the effect of imirestat on LDLR protein stability. Together, our data suggest that small molecule inhibition of SCoR may represent a novel mechanism to treat hypercholesterolemia via a reduction in serum PCSK9 and increased hepatic LDLR.

102 3.4 DISCUSSION

The central finding of this work is that S-nitrosylation of PCSK9 controlled by the

SCoR/SNO-CoA system provides a physiological mechanism by which nitric oxide regulates cholesterol homeostasis, and identifies SNO-CoA-mediated S-nitrosylation as a major posttranslational modification affecting PCSK9 function. S-nitrosylation of PCSK9 reduces the secretion of PCSK9, thus stabilizing the LDLR and lowering circulating cholesterol. These results, along with others (200, 222), increasingly support a paradigm in which SNO-CoA has an essential role as a metabolic signaling molecule, and that SCoR- regulated, SNO-CoA-based S-nitrosylation serves as a general mechanism to control metabolic signaling. Further, inhibition of SCoR by imirestat mimicked the effect of genetic SCoR deletion, potentially identifying a novel way of targeting PCSK9 for treatment of hypercholesterolemia by exploiting SNO-CoA-mediated S- nitrosylation/denitrosylation.

The findings presented in this chapter generate many questions regarding exactly how S-nitrosylation alters PCSK9 secretion. Secreted proteins are initially sorted into membrane-bound vesicles that transport cargo from the ER to the Golgi apparatus (253).

ER vesicles are formed by the coat protein complex II (COPII), consisting of the SAR1

GTPase, SEC23/SEC24 heterodimers, and SEC13/SEC31 heterotetramers. While some soluble cargo proteins enter COPII vesicles by passive diffusion, others are actively captured by receptor-mediated selection (254). To this end, SEC24 primarily drives active sorting of ER cargo and genetic deficiency of SEC24A causes hypocholesterolemia by reducing PCSK9 secretion (255). However, SEC24A resides on the cytoplasmic surface of

ER cargo vesicles while soluble cargo (e.g. PCSK9) exist within the ER lumen; thus, these

103 proteins are physically separated and require a transmembrane adaptor protein for PCSK9 cargo sorting in the ER. SURF4 was recently identified as a cargo receptor for PCSK9 and deletion of SURF4 from HEK293 cells prevented PCSK9 secretion (256). One possible mechanism for the effect of SNO on PCSK9 secretion is that S-nitrosylation of PCSK9 prevents SURF4-dependent cargo selection of PCSK9, thus lowering the rate of PCSK9 secretion. Future work will address whether S-nitrosylation of PCSK9 alters its ability to interact with SURF4. Further, SEC23A has been shown to be a SNO-protein in hepatocytes and the S-nitrosylation of SEC23A required eNOS (257). This indicates that cytosolic components of the COPII complex may be regulated by S-nitrosylation and could be subject to the SCoR/SNO-CoA system. Whether the S-nitrosylation status of COPII proteins changes with genetic deletion of SCoR and how S-nitrosylation might generally alter the function of ER cargo sorting (and thus PCSK9 secretion) remains to be explored.

To this end, it will be important to determine whether secretion of other COPII proteins is altered by either genetic or chemical inhibition of SCoR, i.e. is this mechanism specific to

PCSK9 or more general for secreted proteins.

SNO-based regulation of a secreted protein, in particular, brings into focus the temporality of protein regulation by S-nitrosylation. That is to say, PCSK9 exists only transiently within a hepatocyte and can only be subject to SNO modification and regulation of secretion during that time. S-nitrosylation and denitrosylation are often coupled to physiological stimuli that activate NOSs and denitrosylases (19, 77, 120). Therefore, it is likely that stimulus-coupled activation of hepatic NOSs will temporally regulation the S- nitrosylation of PCSK9. What physiological stimuli lead to this regulation are unknown.

PCSK9 expression and serum levels vary diurnally with cholesterol synthesis (258) and

104 fasting reduces PCSK9 expression and serum levels in humans and mice (259, 260). NO production also appears to vary with circadian rhythm (261). If nutritional stimuli can modulate hepatic NOS activity is still unclear. Thus, how PCSK9 and NO production integrate spatiotemporally to regulate PCSK9 function requires additional consideration.

Further, whether SNO-based regulation of PCSK9 is substantial in the presence of SCoR is not known. Indeed, basal S-nitrosylation of PCSK9 is very low in wild-type mice (Fig.

3.2F) and therefore SNO modification of PCSK9 may only become apparent when SCoR activity is reduced or inhibited.

105 3.5 FIGURES

Figure 3.1

Figure 3.1. SCoR-/- mice are hypocholesterolemic. (A and B) Total serum cholesterol

(A) and serum triglycerides (B) from overnight fasted 12-week old male SCoR+/+ (n=20) and SCoR-/- mice (n=21). P-values were calculated by Student’s t-test. Bars represent mean and SD. (C and D) Total serum cholesterol (C) and serum triglycerides (D) from unfasted

24-week old male SCoR+/+ (n=14) and SCoR-/- mice (n=14). P-values were calculated by

106 Student’s t-test. Bars represent mean and SD. (E) Pooled serum from 24-week old unfasted male SCoR+/+ mice (n=7) and SCoR-/- mice (n=7) was separated by fast protein liquid chromatography to obtain individual lipoprotein fractions. Cholesterol was assayed in each fraction. Lipoprotein fractions were labeled according to known standards.

107 Figure 3.2

Figure 3.2. SCoR regulates hepatic LDLR by modulating serum PCSK9. (A) Western blot analysis for hepatic LDLR and PCSK9 from unfasted 24-week old male SCoR+/+ (n=7) and SCoR-/- (n=7) mice. (B) Quantification of LDLR protein levels from panel A (bands were quantified using ImageJ) and quantitative real-time PCR analysis of hepatic LDLR mRNA from the same tissue samples as in panel A. P-value was calculated by Student’s t- test. Bars represent mean and SD. (C) Total serum cholesterol from unfasted 16-week old male SCoR+/+/LDLR-/- (n=12) and SCoR-/-/LDLR-/- mice (n=7). Bars represent mean and

SD. (D) Serum PCSK9 from unfasted 24-week old male SCoR+/+ (n=16) and SCoR-/-

(n=16) mice. Serum PCSK9 was measured by ELISA. P-value was calculated by Student’s t-test. Bars represent mean and SD. (E) Quantification of PCSK9 protein levels from panel

A (bands were quantified using ImageJ) and quantitative real-time PCR analysis of hepatic

108 PCSK9 mRNA from the same tissue samples as in panel A. P-value was calculated by

Student’s t-test. Bars represent mean and SD. (F) Western blot analysis PCSK9 S- nitrosylation status in livers from unfasted 24-week old SCoR+/+ (n=4) and SCoR-/- (n=4) male mice. SNO-PCSK9 was enriched from liver lysates by SNO-RAC, separated by SDS-

PAGE, and analyzed by western blot.

109 Figure 3.3

Figure 3.3. SCoR does not alter hepatic SR-BI expression. Western blot analysis (left) of scavenger receptor BI expression in livers from unfasted 24-week old SCoR+/+ (n=7) and SCoR-/- (n=7) male mice. Bands were quantified (right) using ImageJ.

110 Figure 3.4

Figure 3.4. SCoR does not regulate the S-nitrosylation status of hepatic LDLR,

HMGCR, or ACAT2. Western blot analysis (left) of LDLR, HMGCR, and ACAT2

(thiolase) S-nitrosylation status in livers from unfasted 24-week old SCoR+/+ (n=4) and

SCoR-/- (n=4) male mice. SNO-proteins were enriched from liver lysates by SNO-RAC,

111 separated by SDS-PAGE, and analyzed by western blot. Bands were quantified (right) using ImageJ. SNO levels were normalized to total protein levels. P-values were calculated by Student’s t-test. Bars represent mean and SD.

112 Figure 3.5

Figure 3.5. SCoR regulates PCSK9 and LDLR in cell culture models of PCSK9 secretion. (A) Western blot analysis for cellular and secreted (media) PCSK9 in HepG2 cells stably expressing control or SCoR-targeting shRNA. An equal number of cells were cultured for 24 hours in serum-free Optimem media, washed with PBS, and given fresh

(PCSK9-free) Optimem, and harvested after 3 hours. (B) Quantification (n=6) of mature

(cellular) PCSK9 (left) and secreted (media) PCSK9 (right; normalized to total mature

PCSK9) from panel A. Bands were quantified using ImageJ. P-values were calculated by

113 Student’s t-test. Bars represent mean and SD. (C) Western blot analysis for LDLR PCSK9 in HepG2 cells stably expressing control or SCoR-targeting shRNA. An equal number of cells were cultured for 24 hours in serum-free Optimem media, washed with PBS, and given fresh Optimem, and harvested after 3 hours. (D) Quantification (n=3) of LDLR from panel C. Bands were quantified using ImageJ. P-values were calculated by Student’s t-test.

Bars represent mean and SD. (E) Western blot analysis of PCSK9 S-nitrosylation status in

HepG2 cells stably expressing control or SCoR-targeting shRNA. An equal number of cells were cultured for 24 hours in serum-free Optimem media. SNO-PCSK9 was enriched from cell lysates by SNO-RAC, separated by SDS-PAGE, and analyzed by western blot. (F)

Quantification (n=3) of SNO-PCSK9 (mature band, normalized to total mature PCSK9) from panel E. Bands were quantified using ImageJ. P-values were calculated by Student’s t-test. Bars represent mean and SD. (G) Western blot analysis for cellular and secreted

(media) PCSK9 in SCoR-deficient HEK transiently expressing PCSK9WT or PCSK9C301A.

An equal number of cells were transfected and cultured for 24 hours, washed with PBS, given fresh Optimem media with or without 200M SNO-cysteine ethyl ester (EtCySNO), and harvested after 90 minutes. (H) Quantification (n=3) secreted (media) PCSK9

(normalized to total mature PCSK9) from panel G. Bands were quantified using ImageJ.

P-values were calculated by Student’s t-test. Bars represent mean and SD.

114 Figure 3.6

Figure 3.6. Chemical inhibition of SCoR lowers serum cholesterol via reduced serum

PCSK9. (A) SNO-CoA reductase specific activity was measured in liver lysate from male

115 control mice or mice fed a diet containing imirestat for 4 weeks (n=3). P-value was calculated by Student’s t-test. Bars represent mean and SD. (B) Total serum cholesterol from 6-hour fasted 20-week old male control mice (n=10) or mice fed a diet containing imirestat for 4 weeks (n=10). P-value was calculated by Student’s t-test. Bars represent mean and SD. (C) Pooled serum from 6-hour fasted 20-week old male control mice (n=7) or mice fed a diet containing imirestat for 4 weeks (n=7) was separated by fast protein liquid chromatography to obtain individual lipoprotein fractions. Cholesterol was assayed in each fraction. Lipoprotein fractions were labeled according to known standards. (D)

Serum PCSK9 from 6-hour fasted 20-week old male control mice (n=10) or mice fed a diet containing imirestat for 4 weeks (n=10). Serum PCSK9 was measured by ELISA. P-value was calculated by Student’s t-test. Bars represent mean and SD. (E) Western blot analysis for hepatic LDLR and PCSK9 from 6-hour fasted 20-week old control mice (n=7) or mice fed a diet containing imirestat for 4 weeks (n=7). (F) Quantification of LDLR and PCSK9 protein levels from panel E. Bands were quantified using ImageJ. P-values were calculated by Student’s t-test. Bars represent mean and SD. (G) Quantitative real-time PCR analysis of hepatic SREBP2, LDLR, and PCSK9 from 6-hour fasted 20-week old control mice (n=7) or mice fed a diet containing imirestat for 4 weeks (n=7). Bars represent mean and SD. (H)

Total serum cholesterol from 6-hour fasted 16-week old male LDLR-/- mice (n=10) or

LDLR-/- mice fed a diet containing imirestat for 4 weeks (n=9). Bars represent mean and

SD. (I) Total serum cholesterol from 6-hour fasted 16-week old male CETP/ApoB100 transgenic mice (n=3) or CETP/ApoB100 transgenic mice fed a diet containing imirestat for 8 weeks (n=4). Bars represent mean and SD. (J) Pooled serum from 6-hour fasted 16- week old male CETP/ApoB100 transgenic mice (n=3) or CETP/ApoB100 transgenic mice

116 fed a diet containing imirestat for 8 weeks (n=4) was separated by fast protein liquid chromatography to obtain individual lipoprotein fractions. Cholesterol was assayed in each fraction. Lipoprotein fractions were labeled according to known standards.

117 3.6 EXPERIMENTAL PROCEDURES

3.6.1 Mice

Mouse studies were approved by the Institutional Care and Use Committee

(IACUC) at Case Western Reserve University. All housing and procedures complied with the Guide for the Care and Use of Laboratory Animals and the American Veterinary

Medical Associations guidelines regarding euthanasia. Generation of SCoR-/- mice was described previously (200). C57BL/6J and LDLR-/- mice were purchased from Jackson

Laboratory. To generate SCoR and LDLR double knockout mice, SCoR-/- male mice were initially bred to LDLR-/- female mice. Genotyping of SCoR+/+ and SCoR-/- mice was performed using the PCR protocol from Deltagen with these primers: common forward:

5′-GCAGAGATTCAACAAGTCTCCCCTC-3′; mutant reverse: 5′-

GGGCCAGCTCATTCCTCCCACTCAT-3′; common reverse: 5′-

AGCTAAGGCTCCGAGCAGTGCTAAC-3′. Genotyping of SCoR-/-/LDLR-/- mice was performed following the PCR protocol from Jackson Labs using the following primers: common forward: 5′-TATGCATCCCCAGTCTTTGG-3′; wild-type reverse: 5′-

CTACCCAACCAGCCCCTTAC-3′; mutant reverse: 5′-

ATAGATTCGCCCTTGTGTCC-3′. CETP-ApoB100 transgenic mice were purchased from Taconic and genotyped with the following primers: CETP forward: 5′-

CGAGCCAGCTACCCAGATA-3′; CETP reverse: 5′-

GTCAGCTGTGTGTTGATCTGG-3′; ApoB100 forward: 5′-

AAGCCACACTCCAACGCATA-3′; ApoB100 reverse: 5′-

TCAAGTTGGAAACAGTCTTTGG-3′. All mice were maintained on a 12-hour light/dark cycle. Beginning at 8 weeks of age, SCoR-/- mice were fed AIN-93M mature rodent diet

118 (Research Diets) supplemented with 1% ascorbic acid; SCoR+/+ mice were fed AIN-93M.

For imirestat treatment, mice were fed control diet (AIN-93M) or experimental diet (AIN-

93M supplemented with 0.0125% imirestat) for indicated lengths of time. For serum and tissue collection, mice were anesthetized with and euthanized by terminal exsanguination and removal of the liver. Livers were snap frozen in liquid nitrogen and stored at -80C until analysis. Blood was transferred to serum separator tubes (BD

Microtainer, 365967) and allowed to coagulate for 20 minutes at room temperature. Serum was separated by centrifugation at 2000g for 15 minutes at 4C and stored at -80C until analysis.

3.6.2 Serum Chemistries

Total serum cholesterol and serum triglycerides were measured by standard enzymatic methods. For 12-week old SCoR+/+ and SCoR-/- mice, serum cholesterol and serum triglycerides were measured at University Hospitals Cleveland Medical Center

Clinical Laboratory. Lipoprotein fractionation was performed by the Vanderbilt

University Medical Center Lipid Lab. Briefly, lipoprotein fractions were separated from

0.1 mL of serum by gel filtration column chromatography. Approximately 70 fraction

(0.25 mL) are collected and the amount of cholesterol in each fraction is determined using microtiter plate, enzyme-based assays. Cholesterol profiles are constructed and calibration of the column with purified lipoprotein fractions permits quantitation of cholesterol in various lipoprotein classes. Serum PCSK9 was measured by solid phase sandwich ELISA using the PCSK9 Quantikine ELISA kit (R&D Systems, MPC900) following manufacturer’s instructions.

3.6.3 Western Blotting

119 Western blotting analysis was performed by standard procedures on proteins extracted from tissues or cells. Antibodies used: SCoR/AKR1A1 (Santa Cruz

Biotechnology, sc-100500); GAPDH (Proteintech, 10494-1-AP); LDLR (Abcam, ab52818); mouse PCSK9 (R&D Systems, AF3985); ACAT2 (Cell Signaling, 11814);

SR-BI (Proteintech, 21277-1-AP); HMGCR (Abcam, ab174830); and human PCSK9

(Cell Signaling, 85813). All blots were quantified using ImageJ (NIH).

3.6.4 Mouse Tissue Analysis by Western blotting and quantitative reverse transcription PCR

For mouse tissue protein analysis, frozen liver tissue was lysed by mechanical homogenization in RIPA buffer (5-10 L lysis buffer / 1 mg tissue; Alfa Aesar, J62524) containing protease inhibitors (Roche, 04693159001). Lysate was clarified by centrifugation (20000g, 4C, 30 minutes, X2) and protein concentration determined by the bicinchoninic acid (BCA) method. Proteins were analyzed by SDS-PAGE and immunoblotting.

For SNO-protein analysis in mouse livers, SNO-RAC was performed as described previously (76). Frozen liver tissue was lysed by mechanical homogenization in HEN buffer (100 mM HEPES, 1 mM EDTA, 0.1 mM neocuproine, pH 8.0) with 150 mM NaCl,

1% Nonidet P-40 (NP-40), 0.1% S-methylmethanethiosulfonate (MMTS) and protease inhibitors (Roche). Lysate was clarified by centrifugation (20000g, 4C, 30 minutes, X2) and protein determined by BCA method. 4 mg of protein was added to HEN buffer containing 2.5% SDS and 0.2% MMTS and incubated at 50C for 20 minutes with frequent vortexing. Proteins were precipitated with ice cold acetone and re-dissolved in HEN buffer with 1% SDS (HENS). Protein precipitation and resuspension was repeated once and

120 protein concentration estimated by BCA assay. 1-2 mg of protein was incubated with or without 30 mM ascorbate and 50 L of thiopropyl-sepharose (50% bead slurry) for 3-4 hours with rotation in the dark. Protein-bound beads were washed 4 times with 1 mL of

HENS buffer and 2 times with 1 mL of HENS buffer diluted 1/10 with MilliQ grade water.

SNO-proteins were eluted in 2X loading dye containing 10% -mercaptoethanol, separated by SDS-PAGE, and analyzed by immunoblotting.

For mouse mRNA analysis, mRNA was isolated from frozen liver tissue using

RNeasy mini prep kit (Qiagen, 74104) following manufacturer’s instructions. RNA was quantified using a NanoDrop microvolume spectrophotometer (Thermo). 1 g of RNA was converted to cDNA using High-Capacity RNA-to-cDNA kit (Applied Biosystems,

4387406). Quantitative PCR was performed on Applied Biosystems OneStepPlus system using TaqMan Universal Master Mix II (without UNG). Briefly, 10 L of TaqMan

Universal Master Mix II, 1 L of TaqMan Assay probe (listed below for each gene), 1 L of cDNA product, and 8 L of DNase/RNase free water was combined for each reaction.

Reactions were performed in triplicate for each sample using the following program: 10 minutes at 95C followed by 40 cycles of 15 seconds at 95C and 1 minute at 60C. Data were analyzed using the comparative CT method. TaqMan Assay probes (Thermo) are as follows: Ldlr (Mm01177349_m1); Pcsk9 (Mm0126310_m1); Srebf2 (Mm01306292_m1);

Gapdh (Mm99999915_g1).

3.6.5 Cell Lines and Culture

HepG2 and 293T/17 cells were obtained from ATCC and cultured at 37C, 5% CO2 in growth media [DMEM (Gibco) supplemented with 10% fetal bovine serum (Sigma), and 1X antibiotic-antimycotic (Gibco), and 1X GlutaMax (Gibco)]. SCoR knockout

121 HEK293 were described previously (Chapter 2) and were cultured at 37C, 5% CO2 in growth media.

3.6.6 Generation of HepG2 stably expressing SCoR-targeting shRNA

HepG2 cells stably expressing SCoR-targeting shRNA were produced as follows.

293T/17 cells were plated on 6-well plates and transfected with either mammalian non- targeting Mission shRNA (Sigma, SHC002) or SCoR-targeting Mission shRNA (Sigma,

TRCN0000231969) in combination with Mission Lentiviral packaging mix (Sigma,

SHP001) using PolyJet transfection reagent (SignaGen, SL100688) per manufacturers’ instructions. After 24 hours, media was aspirated from cells and 2 mL of fresh growth media was applied. After 24 hours, 2 mL of media containing lentivirus was collected and stored at 4C overnight. Viral media production was repeated. The resulting 4 mL of viral media was centrifuged to remove any cells and 3.5 mL of media retained. 1.5 mL of growth media was added to bring the final volume to 5 mL of viral media. 4 L of 10 mg/mL polybrene (Sigma, TR-1003) was added to media to a final concentration of 8 g/mL. The viral supernatant was applied to HepG2 cells and allowed to incubate for 6 hours. After 6 hours, 10 mL of growth media was added to the viral media and cells were incubated for

72 hours. After 72 hours, HepG2 cells were split into growth media supplemented with 2

g/mL puromycin (Gibco) and cultured consecutively to select for shRNA expressing cells.

3.6.7 Cell-based PCSK9 Secretion Assays and SNO-protein Analysis

PCSK9 secretion assays in HepG2 cells were performed as follows. Equal numbers of HepG2shCtrl and HepG2shSCoR were plated onto dishes coated with 5 g/cm2 rat tail I (Corning) and cultured for 24 hours. After 24 hours, growth media containing 2

122 g/mL puromycin was removed from cells and replaced with plain Opti-MEM media

(Gibco) supplemented with 1.5 g/mL puromycin. Cells were cultured for another 24 hours and then washed twice with warm PBS. Fresh Opti-MEM (with 1.5 g/mL puromycin) was added to cells and cells were incubated for 3 hours at 37C, 5% CO2 to monitor PCSK9 secretion. After 3 hours, media was collected and centrifuged to pellet any cellular material.

Cells were wash 3 times with cold PBS and harvested in RIPA buffer containing protease inhibitors (Roche). Cells were lysed by brief sonication, cell debris pelleted by centrifugation (12000g, 4C, 10 minutes), and protein concentration determined by BCA assay. Proteins were separated by SDS-PAGE and analyzed by immunoblotting. For media

PCSK9 analysis, equal volumes of media were separated by SDS-PAGE and analyzed by immunoblotting.

For SNO-protein analysis in HepG2shCtrl and HepG2shSCoR cells, equal numbers of

HepG2shCtrl and HepG2shSCoR were plated onto dishes coated with 5 g/mL rat tail collagen

I (Corning) and cultured for 24 hours. After 24 hours, growth media containing 2 g/mL puromycin was removed from cells and replaced with plain Opti-MEM media (Gibco) supplemented with 1.5 g/mL puromycin. Cells were cultured for another 24 hours and harvested in HEN buffer with 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.1% S- methylmethanethiosulfonate (MMTS) and protease inhibitors (Roche). Cells were lysed by brief sonication and cell debris pelleted by centrifugation (12000g, 4C, 10 minutes). The entire cell lysate was brought to 2 mL with HEN buffer and 2.5% SDS and 0.2% MMTS was added. Lysate incubated at 50C for 20 minutes with frequent vortexing. Proteins were precipitated with ice cold acetone and re-dissolved in HEN buffer with 1% SDS (HENS).

Protein precipitation and resuspension was repeated once and protein concentration

123 estimated by BCA assay. 1 mg of protein was incubated with or without 30 mM ascorbate and 50 L of thiopropyl-sepharose (50% bead slurry) for 3-4 hours with rotation in the dark. Protein-bound beads were washed 4 times with 1 mL of HENS buffer and 2 times with 1 mL of HENS buffer diluted 1/10 with MilliQ grade water. SNO-proteins were eluted in 2X loading dye containing 10% -mercaptoethanol, separated by SDS-PAGE, and analyzed by immunoblotting.

PCSK9 secretion assays in SCoR knockout HEK293 cells were performed as follows. The PCSK9 coding sequence in pEntr223.1 (Dharmacon) was shuttled to pcDNA-

DEST40 vector (Invitrogen) using LR Clonase II (Invitrogen) per manufacturer’s instructions and verified by sequencing. PCSK9C301A was generated by site-directed mutagenesis of pcDNA-DEST40-PCSK9WT using the Agilent QuikChange XL II system per manufacturer’s instructions. The sequencing primers were: 5-

GCCAGGCGCTGGGCGGCGGCGTTGAG-3 and 5-

CTCAACGCCGCCGCCCAGCGCCTGGC. Mutation was verified by sequencing. Equal numbers of SCoR knockout HEK293 cells were plated onto dishes coated with 5 g/cm2 poly-D-lysine (Corning) and cultured for 24 hours in growth media. Cells were transfected with PCSK9WT or PCSK9C301A using PolyJet transfection reagent (SignaGen) per manufacturer’s instructions. After 24 hours, cells were washed twice with warm PBS and an equal volume of plain Opti-MEM. 1M Ethyl ester SNO-cysteine (EtCySNO) was freshly prepared by mixing equal volumes of 2M sodium with 2M acidified ethyl ester cysteine to generate 1M EtCySNO. EtCySNO was immediately added to designated cells. After 90 minutes, media was collected media was collected and centrifuged to pellet any cellular material. Cells were wash 3 times with cold PBS and harvested in HEN buffer

124 with 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.1% S-methylmethanethiosulfonate

(MMTS) and protease inhibitors (Roche). Cells were lysed by brief sonication, cell debris pelleted by centrifugation (12000g, 4C, 10 minutes), and protein concentration determined by BCA assay. Proteins were separated by SDS-PAGE and analyzed by immunoblotting. For media PCSK9 analysis, equal volumes of media were separated by

SDS-PAGE and analyzed by immunoblotting.

125 CHAPTER 4: GENERAL DISCUSSION AND FUTURE DIRECTIONS

The discovery (20) of phylogenetically conserved SNO-CoA reductase activity has opened new avenues of investigation into the role of S-nitrosylation in cellular function, in particular metabolic signaling and regulation. However, work presented here (and elsewhere (20, 200)) only begins to scratch the surface of nitric oxide’s ability to signal through SNO-CoA. The targets of SNO-CoA-mediated S-nitrosylation/denitrosylation identified in Chapter 2 (Appendix 2.1-2.3) suggest a role for SNO-CoA-based signaling across multiple metabolic processes. Indeed, SCoR regulates distinct aspects of cellular metabolism including mitochondrial function (Chapter 2) and mammalian cholesterol metabolism (Chapter 3). Altered mitochondrial function upon loss of SCoR function suggests that energy metabolism is regulated by the SCoR/SNO-CoA system, and preliminary data (not shown) gathered during this thesis suggests that SCoR regulates whole body glucose and lipid metabolism during times of fasting and during obesity, opening many avenues of studies into the broad ranging effects of SNO-CoA in mammalian metabolism.

SCoR is central to SNO-CoA-based signaling – denitrosylases determine steady- state levels of S-nitrosylation, much like other negative, enzymatic regulators of posttranslational modifications (e.g. phosphatases, deacetylases, etc.) determine steady- state levels of their cognate posttranslational modifications. Yet how SCoR expression and enzymatic activity is regulated to set S-nitrosylation levels is not understood.

Transcriptional control of GSNOR expression is context-dependent and, to a certain extent, responds to NO input (discussed in section 1.4.3). How SCoR expression is regulated in physiology and pathophysiology and whether its expression responds to NO levels remains

126 to be explored. Preliminary data from our laboratory in the murine macrophage cell line

RAW264.7 (data not shown) suggests that SCoR expression is induced at the mRNA level upon macrophage activation by bacterial lipopolysaccharide (LPS). LPS strongly induces the expression of inducible nitric oxide synthase (iNOS), producing large quantities of NO.

Accordingly, treatment of macrophages with a NO donor (in the absence of LPS activation) also induces SCoR expression. This suggests NO-responsive transcriptional control of

SCoR expression and identifies a potential role for SCoR in inflammatory disease states

(asthma and diabetes, among others) where iNOS is induced. The transcriptional pathways regulating SCoR expression both in physiology and pathophysiological states remain to be identified.

As with GSNOR (discussed in section 1.4.3), it is likely that SCoR activity and stability are posttranscriptionally regulated. The first evidence for direct modulation of

SCoR’s SNO-CoA reductase activity was found in murine kidneys following ischemia/reperfusion injury (200). Following ischemia/reperfusion injury, SCoR activity was significantly reduced without altered SCoR protein levels (200), implying some mechanism (likely a posttranslational modification) to control SCoR activity. The modifications of SCoR that alter its activity remain to be discovered. The structure— function studies presented in Chapter 2, however, provide at least one potential mechanism to regulate SCoR’s SNO-CoA reductase activity. Lys-127 is the primary residue in SCoR driving binding to SNO-CoA. While other positively charged amino acids (such as Arg) can substitute for Lys and preserve the SCoR—SNO-CoA interaction, Lys-127 is highly conserved across mammalian species, suggesting Lys is the preferred positively charged amino acid at that residue. A key difference between Lys and Arg is the ability of Lys to

127 be reversibly, posttranslationally modified by CoA derivatives, including acetyl-CoA, malonyl-CoA, and succinyl-CoA. Modification of Lys-127 by any CoA derivative is likely to inhibit binding of SNO-CoA to SCoR, thus inhibiting SCoR’s SNO-CoA reductase activity. The conservation of Lys-127 therefore allows regulatory cross-talk between CoA- based posttranslational modifications. Future work defining the physiological and pathophysiological settings in which Lys-127 is modified, the enzymes responsible for the addition and removal of CoA-based modifications to/from Lys-127, and how SNO-based signals interface with other CoA-derived signaling molecules to regulate SCoR activity will provide depth in understanding how the SCoR/SNO-CoA system operates to control cellular metabolism. Further, GSNOR activity may be regulated by S-nitrosylation of

GSNOR itself (discussed in section 1.4.3). However, whether SCoR is S-nitrosylated endogenously remains to be seen. SCoR’s Cys-46 is next to a Asp-45, a critical residue required for SCoR binding to NAD(P)H and therefore its enzymatic activity (193, 262).

Likewise, Cys-133 is proximal to the SNO-CoA binding residue Lys-127. S-nitrosylation of Cys residues often leads to protein conformational change; consequently, S-nitrosylation of these Cys residues might alter the location of residues critically important for SCoR’s

SNO-CoA reductase activity.

More generally, how SCoR exists spatially with SNO-CoA and targets of CoA- mediated denitrosylation is still unclear. As discussed in Chapter 1, protein denitrosylation by CoA (forming SNO-CoA by transnitrosylation) requires the binding of CoA to target

SNO-proteins in a reactive orientation. Upon denitrosylation by SNO-CoA, it’s likely that either the protein undergoes a conformational change that prevents re-nitrosylation by

SNO-CoA or a transient, localized equilibrium is established between SNO-CoA and the

128 target protein. In the latter case, interaction of SCoR with protein targets of CoA-mediated denitrosylation may aid in denitrosylation by rapidly metabolizing SNO-CoA, thus shifting the equilibrium away protein S-nitrosylation. This was illustrated recently by pyruvate kinase M2, which both exists in complex with SCoR and is a target of SCoR-dependent denitrosylation (200). Systematic identification of proteins complexed with SCoR overlaid with targets of SCoR-dependent denitrosylation in relevant cells and tissues will identify both facile targets of SCoR and biological processes regulated by the SCoR/SNO-CoA system. Utilization of catalytically dead SCoR will define whether SCoR activity alters its ability to bind target proteins. Further, examination of binding partners to SCoRK127A will determine if this surface Lys residue also mediates target protein binding (in addition to

SNO-CoA binding); if so, it would provide an additional layer of regulation at this residue.

Given the potential for transnitrosylation cascades in propagating SNO-based signaling, it is unlikely that SCoR interactors and denitrosylation targets will completely overlap. Thus, proteins that do satisfy both criteria may represent either early regulatory nodes in transnitrosylation cascades or critical downstream effector proteins in SNO-CoA-based signaling. In this way, SCoR could regulate protein S-nitrosylation both generally and specifically, as needed. Additionally, the source of NO (i.e. the different isoforms of NOS) will further dictate how SCoR interacts with SNO-signaling cascades, as NOS isoform expression varies between tissues, with physiological and pathological states, and over the lifespan of the organism. Thus, a detailed understanding of the SCoR/SNO-CoA system will require integration of NO source, SCoR binding partners, SCoR targets of denitrosylation, and SCoR regulation to fully appreciate the influence of SNO-CoA-based signaling on mammalian metabolism.

129 APPENDIX:

Appendix 2.1: Putative targets of SNO-CoA-mediated S-nitrosylation in mouse kidney lysates

Proteins identified in two biological replicates with an average fold change >1.2 and RSD <45% between replicates.

Average Mouse Uniprot Human Uniprot Gene Name fold change ID ID (K127A/WT)

3.15 Q91V64 Q96CN7 ISOC1 CGI-111

2.62 P12658 P05937 CALB1 CAB27

2.19 Q8QZS1 Q6NVY1 HIBCH

2.17 P09411 P00558 PGK1 PGKA MIG10 OK/SW-cl.110

1.93 P85094 Q96AB3 ISOC2

1.93 P47754 P47755 CAPZA2

1.89 Q60759 Q92947 GCDH

1.87 P68368 P68366 TUBA4A TUBA1

1.87 O88844 O75874 IDH1 PICD

1.87 P68369 Q71U36 TUBA1A TUBA3

1.86 P05213 P68363 TUBA1B

1.80 P18760 P23528 CFL1 CFL

1.78 Q9WVL0 O43708 GSTZ1 MAAI

1.78 Q99K48 Q15233 NONO NRB54

1.78 P02088 P68871 HBB

1.78 Q99KP3 Q9Y2S2 CRYL1 CRY

1.75 Q99L20 P09211 GSTP1 FAEES3 GST3

1.73 Q9R0P3 P10768 ESD

1.73 P16331 P00439 PAH

1.71 Q9D8N0 P26641 EEF1G EF1G PRO1608

1.70 P56399 P45974 USP5 ISOT

1.68 Q61316 P34932 HSPA4 APG2

1.67 P49945 P02792 FTL

1.67 P10630 Q14240 EIF4A2 DDX2B EIF4F

1.65 Q9DCS2 Q96S19 METTL26 JFP2 C16orf13

1.64 P35700 Q06830 PRDX1 PAGA PAGB TDPX2

1.62 Q8BTY1 Q16773 KYAT1 CCBL1

1.62 Q8BWT1 P42765 ACAA2

1.62 Q9JII6 P14550 AKR1A1 ALDR1 ALR

1.61 P54822 P30566 ADSL AMPS

130 1.61 P70290 Q00013 MPP1 DXS552E EMP55

1.60 P00329 P07327 ADH1A ADH1

1.60 Q922D8 P11586 MTHFD1 MTHFC MTHFD

1.59 P12382 P17858 PFKL

1.59 Q9D6R2 P50213 IDH3A

1.59 P60710 P60709 ACTB

1.59 P16460 P00966 ASS1 ASS

1.59 P09671 P04179 SOD2

1.59 P05064 P04075 ALDOA ALDA

1.58 P50247 P23526 AHCY SAHH

1.58 Q5U5V2 A2RU49 HYKK AGPHD1

1.57 P54071 P48735 IDH2

1.57 P34914 P34913 EPHX2

1.57 Q80W22 Q86YJ6 THNSL2

1.57 P16858 P04406 GAPDH GAPD CDABP0047 OK/SW-cl.12

1.56 Q3TNA1 O75191 XYLB

1.56 Q9CZU6 O75390 CS

1.56 Q9Z0S1 O95861 BPNT1

1.56 Q91Y97 P05062 ALDOB ALDB

1.56 Q9EQ20 Q02252 ALDH6A1 MMSDH

1.55 P05202 P00505 GOT2

1.55 P10648 P09210 GSTA2 GST2

1.55 P68033 P68032 ACTC1 ACTC

1.55 Q61699 Q92598 HSPH1 HSP105 HSP110 KIAA0201

1.54 Q6ZQ38 Q86VP6 CAND1 KIAA0829 TIP120 TIP120A

1.54 Q9ERD7 Q13509 TUBB3 TUBB4

1.54 Q9WUM4 Q9ULV4 CORO1C CRN2 CRNN4

1.54 Q78JT3 P46952 HAAO

1.53 P47857 P08237 PFKM PFKX

1.53 Q91WR5 P52895 AKR1C2 DDH2

1.53 Q00612 P11413 G6PD

1.53 Q9Z1Q5 O00299 CLIC1 G6 NCC27

1.53 O70338 P13489 RNH1 PRI RNH

1.52 O55222 Q13418 ILK ILK1 ILK2

1.52 Q8BFR5 P49411 TUFM

1.52 P11499 P08238 HSP90AB1 HSP90B HSPC2 HSPCB

1.52 Q8K183 O00764 PDXK C21orf124 C21orf97 PKH PNK PRED79

1.52 P28474 P11766 ADH5 ADHX FDH

1.51 Q9JHK4 Q92696 RABGGTA

131 1.51 Q07076 P20073 ANXA7 ANX7 SNX OK/SW-cl.95

1.51 O35855 O15382 BCAT2 BCATM BCT2 ECA40

1.51 Q8R3P0 P45381 ASPA ACY2 ASP

1.51 P57780 O43707 ACTN4

1.50 P68372 P68371 TUBB4B TUBB2C

1.50 Q9WVM8 Q8N5Z0 AADAT KAT2

1.50 Q9D6Y7 Q9UJ68 MSRA

1.49 P38060 P35914 HMGCL

1.49 Q7TMM9 Q13885 TUBB2A TUBB2

1.49 Q9CPY7 P28838 LAP3 LAPEP PEPS

1.49 Q61598 P50395 GDI2 RABGDIB

1.48 P99024 P07437 TUBB TUBB5 OK/SW-cl.56

1.48 Q8BKZ9 O00330 PDHX PDX1

1.48 Q9D964 P50440 GATM AGAT

1.48 Q9D0I9 P54136 RARS

1.48 Q93092 P37837 TALDO1 TAL TALDO TALDOR

1.48 P14152 P40925 MDH1 MDHA

1.47 P61922 P80404 ABAT GABAT

1.47 Q9QXD6 P09467 FBP1 FBP

1.47 Q9DBL1 P45954 ACADSB

1.47 Q9D2R0 Q86V21 AACS ACSF1

1.47 P70441 O14745 SLC9A3R1 NHERF NHERF1

1.47 Q91WT7 Akr1c14 mCG_1727

1.47 O09174 Q9UHK6 AMACR

1.47 Q9JLJ2 P49189 ALDH9A1 ALDH4 ALDH7 ALDH9

1.47 Q8BGA8 Q6NUN0 ACSM5 MACS3

1.46 Q9JK81 Q9HB07 C12orf10

1.46 P63101 P63104 YWHAZ

1.46 P58252 P13639 EEF2 EF2

1.46 Q62433 Q92597 NDRG1 CAP43 DRG1 RTP

1.45 P17427 O94973 AP2A2 ADTAB CLAPA2 HIP9 HYPJ KIAA0899

1.45 P52825 P23786 CPT2 CPT1

1.45 Q8VCR7 Q96IU4 ABHD14B CIB

1.44 Q9CQV8 P31946 YWHAB

1.44 Q8VC30 Q3LXA3 TKFC DAK

1.44 Q9D7B6 Q9UKU7 ACAD8 ARC42 IBD

1.44 D3Z7P3 O94925 GLS GLS1 KIAA0838

1.44 P11930 A8MXV4 NUDT19

1.44 Q61171 P32119 PRDX2 NKEFB TDPX1

132 1.44 P99029 P30044 PRDX5 ACR1 SBBI10

1.44 P97429 P09525 ANXA4 ANX4

1.44 Q8VDM4 Q13200 PSMD2 TRAP2

1.44 Q9QXG4 Q9NR19 ACSS2 ACAS2

1.43 P22599 P01009 SERPINA1 AAT PI PRO0684 PRO2209

1.43 P97494 P48506 GCLC GLCL GLCLC

1.43 Q8BH00 Q9H2A2 ALDH8A1 ALDH12

1.43 Q3UPL0 O94979 SEC31A KIAA0905 SEC31L1 HSPC275 HSPC334

1.43 P70168 Q14974 KPNB1 NTF97

1.43 Q9QZE5 Q9Y678 COPG1 COPG

1.43 Q9DBL7 Q13057 COASY PSEC0106

1.42 P17563 Q13228 SELENBP1 SBP

1.42 Q99LX0 Q99497 PARK7

1.42 Q9DBM2 Q08426 EHHADH ECHD

1.42 Q99K67 Q9UDR5 AASS

1.41 P07759 P01011 SERPINA3 AACT GIG24 GIG25

1.41 P26443 P00367 GLUD1 GLUD

1.41 P97823 O75608 LYPLA1 APT1 LPL1

1.41 Q9EPL9 O15254 ACOX3 BRCOX PRCOX

1.41 P55264 P55263 ADK

1.41 P07901 P07900 HSP90AA1 HSP90A HSPC1 HSPCA

1.40 Q60676 P53041 PPP5C PPP5

1.40 Q60866 Q96BW5 PTER

1.40 Q9Z2I9 Q9P2R7 SUCLA2

1.40 Q99JY9 P61158 ACTR3 ARP3

1.40 Q9DCY0 Keg1

1.40 Q9CQN1 Q12931 TRAP1 HSP75

1.40 Q99MR8 Q96RQ3 MCCC1 MCCA

1.40 Q80X81 Acat3

1.40 Q64105 P35270 SPR

1.39 Q3ULJ0 Q8N335 GPD1L KIAA0089

1.39 Q9Z1Q9 P26640 VARS G7A VARS2

1.39 Q68FH4 Q01415 GALK2 GK2

1.39 P11983 P17987 TCP1 CCT1 CCTA

1.39 Q3THS6 P31153 MAT2A AMS2 MATA2

1.39 Q9D1A2 Q96KP4 CNDP2 CN2 CPGL HEL-S-13 PEPA

1.39 Q7TNE1 Q9HAC7 SUGCT C7orf10 DERP13

1.39 P52480 P14618 PKM OIP3 PK2 PK3 PKM2

1.39 Q05920 P11498 PC

133 1.39 Q3TC72 Q96GK7 FAHD2A CGI-105

1.39 O09172 P48507 GCLM GLCLR

1.39 Q9JHU4 Q14204 DYNC1H1 DHC1 DNCH1 DNCL DNECL DYHC KIAA0325

1.39 Q91YH6 P15313 ATP6V1B1 ATP6B1 VATB VPP3

1.39 Q3UNX5 Q53FZ2 ACSM3 SAH

1.38 Q02053 P22314 UBA1 A1S9T UBE1

1.38 P05201 P17174 GOT1

1.38 P62814 P21281 ATP6V1B2 ATP6B2 VPP3

1.38 P80315 P50991 CCT4 CCTD SRB

1.38 P16125 P07195 LDHB

1.37 Q9WTP6 P54819 AK2 ADK2

1.37 P10126 P68104 EEF1A1 EEF1A EF1A LENG7

1.37 Q8CC88 A3KMH1 VWA8 KIAA0564

1.37 Q9D826 Q9P0Z9 PIPOX LPIPOX PSO

1.37 P45952 P11310 ACADM

1.36 Q91ZA3 P05165 PCCA

1.36 Q64516 P32189 GK

1.36 Q91V92 P53396 ACLY

1.36 Q8VDN2 P05023 ATP1A1

1.36 P47934 P43155 CRAT CAT1

1.35 Q99K51 P13797 PLS3

1.35 P63038 P10809 HSPD1 HSP60

1.35 P80313 Q99832 CCT7 CCTH NIP7-1

1.35 Q91X52 Q7Z4W1 DCXR SDR20C1

1.35 Q8R1B4 Q99613 EIF3C EIF3S8

1.35 P20108 P30048 PRDX3 AOP1

1.35 P47791 P00390 GSR GLUR GRD1

1.34 Q6P1B1 Q9NQW7 XPNPEP1 XPNPEPL XPNPEPL1

1.34 Q8CG76 O43488 AKR7A2 AFAR AFAR1 AKR7

1.34 Q68FD5 Q00610 CLTC CLH17 CLTCL2 KIAA0034

1.34 Q9D051 P11177 PDHB PHE1B

1.34 Q62468 P09327 VIL1 VIL

1.34 P40936 O95050 INMT

1.34 Q8BP47 O43776 NARS

1.34 P23492 Q12923 PTPN13 PNP1 PTP1E PTPL1

1.34 Q8BH86 Q7Z3D6 DGLUCY C14orf159 UNQ2439/PRO5000

1.34 Q8QZT1 P24752 ACAT1 ACAT MAT

1.34 Q6PB66 P42704 LRPPRC LRP130

1.33 Q9D0K2 P55809 OXCT1 OXCT SCOT

134 1.33 Q9CZD3 P41250 GARS

1.33 Q3TW96 Q3KQV9 UAP1L1

1.33 P42125 P42126 ECI1 DCI

1.33 Q9R0H0 Q15067 ACOX1 ACOX

1.33 P14824 P08133 ANXA6 ANX6

1.33 Q8BVE3 Q9UI12 ATP6V1H CGI-11

1.32 Q91XE0 Q6IB77 GLYAT ACGNAT CAT GAT

1.32 P15532 P15531 NME1 NDPKA NM23

1.32 Q64442 Q00796 SORD

1.32 Q61233 P13796 LCP1 PLS2

1.32 Q91WT9 P35520 CBS

1.32 Q9DB29 Q2TAA2 IAH1

1.31 P13707 P21695 GPD1

1.31 Q8VDD5 P35579 MYH9

1.31 P62192 P62191 PSMC1

1.31 Q00896 P01009 SERPINA1 AAT PI PRO0684 PRO2209

1.31 Q9CZN7 P34897 SHMT2

1.31 P17751 P60174 TPI1 TPI

1.31 Q99L13 P31937 HIBADH

1.30 Q3ULD5 Q9HCC0 MCCC2 MCCB

1.30 P47199 Q08257 CRYZ

1.30 Q9DBE0 Q9Y600 CSAD CSD

1.30 P97351 P61247 RPS3A FTE1 MFTL

1.30 P17742 P62937 PPIA CYPA

1.30 Q3UZZ6 Sult1d1 St1d1

1.29 P51174 P28330 ACADL

1.29 Q9EQH3 Q96QK1 VPS35 MEM3 TCCCTA00141

1.29 O35488 O14975 SLC27A2 ACSVL1 FACVL1 FATP2 VLACS

1.29 Q9JHI5 P26440 IVD

1.29 Q8K010 O14841 OPLAH

1.29 P80318 P49368 CCT3 CCTG TRIC5

1.29 P06801 P48163 ME1

1.28 Q9WUA2 Q9NSD9 FARSB FARSLB FRSB HSPC173

1.28 P35505 P16930 FAH

1.28 P53395 P11182 DBT BCATE2

1.28 Q07417 P16219 ACADS

1.28 Q99LB7 Q9UL12 SARDH DMGDHL1

1.28 Q99NB1 Q9NUB1 ACSS1 ACAS2L KIAA1846

1.28 Q922B2 P14868 DARS PIG40

135 1.28 P11352 P07203 GPX1

1.28 Q8R0Y6 O75891 ALDH1L1 FTHFD

1.28 Q8R2V9 P53992 SEC24C KIAA0079

1.28 Q8CIE6 P53621 COPA

1.28 Q8BMF4 P10515 DLAT DLTA

1.28 P17182 P06733 ENO1 ENO1L1 MBPB1 MPB1

1.27 O55125 Q9BPW8 NIPSNAP1

1.27 Q01853 P55072 VCP

1.27 P00920 P00918 CA2

1.27 P63017 P11142 HSPA8 HSC70 HSP73 HSPA10

1.27 Q9Z2I8 Q96I99 SUCLG2

1.27 A3KMP2 Q5R3I4 TTC38

1.26 P56389 P32320 CDA CDD

1.26 P06151 P00338 LDHA PIG19

1.26 P51855 P48637 GSS

1.26 P80314 P78371 CCT2 99D8.1 CCTB

1.26 P61979 P61978 HNRNPK HNRPK

1.26 Q8BIH0 Q9H0E3 SAP130

1.26 Q8VCN5 P32929 CTH

1.26 P45376 P15121 AKR1B1 ALDR1

1.25 Q9Z2V4 P35558 PCK1 PEPCK1

1.25 Q8CHT0 P30038 ALDH4A1 ALDH4 P5CDH

1.25 P35486 P08559 PDHA1 PHE1A

1.25 Q03265 P25705 ATP5A1 ATP5A ATP5AL2 ATPM

1.25 P42932 P50990 CCT8 C21orf112 CCTQ KIAA0002

1.25 P10649 P09488 GSTM1 GST1

1.24 Q7TNG8 Q86WU2 LDHD

1.24 Q9CWS0 O94760 DDAH1 DDAH

1.24 Q8BVI4 P09417 QDPR DHPR SDR33C1

1.24 P10639 P10599 TXN TRDX TRX TRX1

1.24 Q9QXD1 Q99424 ACOX2

1.24 P21981 P21980 TGM2

1.23 Q3UEG6 Q9BYV1 AGXT2 AGT2

1.23 Q8R016 Q13867 BLMH

1.23 Q8BWF0 P51649 ALDH5A1 SSADH

1.22 P50516 P38606 ATP6V1A ATP6A1 ATP6V1A1 VPP2

1.22 Q9QYB1 Q9Y696 CLIC4

1.22 Q60597 Q02218 OGDH

1.22 Q9DCG6 P30039 PBLD MAWBP

136 1.22 O09173 Q93099 HGD HGO

1.21 Q9JMH6 Q16881 TXNRD1 GRIM12 KDRF

1.21 Q9DCM0 O95571 ETHE1 HSCO

1.21 Q99MZ7 Q9BY49 PECR SDR29C1 PRO1004

1.20 Q11136 P12955 PEPD PRD

1.20 P47738 P05091 ALDH2 ALDM

1.20 Q9D0S9 Q9BX68 HINT2

1.20 P50544 P49748 ACADVL VLCAD

1.20 Q80SW1 O43865 AHCYL1 DCAL IRBIT XPVKONA

1.20 Q9DCD0 P52209 PGD PGDH

137 Appendix 2.2: Putative targets of SCoR-dependent denitrosylation in HEK293

Proteins identified in two biological replicates with an average fold change >1.2 and RSD <55% between replicates.

Average Human Uniprot ID Gene Name fold change (K127A/WT) 2.375 P62937 PPIA CYPA

2.133 P61956 SUMO2 SMT3B SMT3H2

2.117 P00338 LDHA PIG19

2.0985 P15531 NME1 NDPKA NM23

2.021 P22314 UBA1 A1S9T UBE1

1.967 P53396 ACLY

1.9655 P07195 LDHB

1.943 Q00839 HNRNPU C1orf199 HNRPU SAFA U21.1

1.9385 P09936 UCHL1

1.929 Q14974 KPNB1 NTF97

1.9245 P40926 MDH2

1.893 P09874 PARP1 ADPRT PPOL

1.891 P62917 RPL8

1.8855 P12277 CKB CKBB

1.8725 Q01813 PFKP PFKF

1.8525 P35228 NOS2 NOS2A

1.8265 P30041 PRDX6 AOP2 KIAA0106

1.808 Q00610 CLTC CLH17 CLTCL2 KIAA0034

1.802 P34932 HSPA4 APG2

1.7805 P60709 ACTB

1.7775 P14866 HNRNPL HNRPL P/OKcl.14

1.774 Q00325 SLC25A3 PHC OK/SW-cl.48

1.771 P49321 NASP

1.7695 P00558 PGK1 PGKA MIG10 OK/SW-cl.110

1.766 P41252 IARS

1.7635 P13010 XRCC5 G22P2

1.7595 Q7KZF4 SND1 TDRD11

1.7595 P08670 VIM

1.758 P22234 PAICS ADE2 AIRC PAIS

1.754 P36578 RPL4 RPL1

1.7485 P23526 AHCY SAHH

1.744 Q13263 TRIM28 KAP1 RNF96 TIF1B

1.7355 P68363 TUBA1B

1.735 Q5VWX1 KHDRBS2 SLM1

138 1.7295 P19338 NCL

1.7295 P62140 PPP1CB

1.7085 P31939 ATIC PURH OK/SW-cl.86

1.7085 P45974 USP5 ISOT

1.6975 O43707 ACTN4

1.694 P08238 HSP90AB1 HSP90B HSPC2 HSPCB

1.6905 P55072 VCP

1.688 Q92945 KHSRP FUBP2

1.685 P50395 GDI2 RABGDIB

1.6795 B5ME19 EIF3CL

1.6745 P12236 SLC25A6 ANT3 CDABP0051

1.67 Q15393 SF3B3 KIAA0017 SAP130

1.665 P13639 EEF2 EF2

1.6625 Q16881 TXNRD1 GRIM12 KDRF

1.66 P30101 PDIA3 ERP57 ERP60 GRP58

1.659 P26639 TARS

1.654 P78371 CCT2 99D8.1 CCTB

1.6485 P06733 ENO1 ENO1L1 MBPB1 MPB1

1.647 P40227 CCT6A CCT6 CCTZ

1.6465 Q06830 PRDX1 PAGA PAGB TDPX2

1.6435 Q99832 CCT7 CCTH NIP7-1

1.6435 Q9NZI8 IGF2BP1 CRDBP VICKZ1 ZBP1

1.643 P68104 EEF1A1 EEF1A EF1A LENG7

1.6425 P26640 VARS G7A VARS2

1.6425 P08865 RPSA LAMBR LAMR1

1.64 P63244 RACK1 GNB2L1 HLC7 PIG21

1.6395 P25705 ATP5A1 ATP5A ATP5AL2 ATPM

1.6395 O14787 TNPO2

1.631 Q08211 DHX9 DDX9 LKP NDH2

1.63 P23396 RPS3 OK/SW-cl.26

1.623 P17987 TCP1 CCT1 CCTA

1.617 Q86VP6 CAND1 KIAA0829 TIP120 TIP120A

1.617 P07900 HSP90AA1 HSP90A HSPC1 HSPCA

1.616 P14618 PKM OIP3 PK2 PK3 PKM2

1.6085 P68371 TUBB4B TUBB2C

1.6025 Q15758 SLC1A5 ASCT2 M7V1 RDR RDRC

1.5995 P0DMV8 HSPA1A HSP72 HSPA1 HSX70

1.599 P10809 HSPD1 HSP60

1.5975 Q13509 TUBB3 TUBB4

139 1.595 P52272 HNRNPM HNRPM NAGR1

1.594 P31943 HNRNPH1 HNRPH HNRPH1

1.593 P11142 HSPA8 HSC70 HSP73 HSPA10

1.5905 P29401 TKT

1.587 P11940 PABPC1 PAB1 PABP1 PABPC2

1.586 Q14157 UBAP2L KIAA0144 NICE4

1.579 P61978 HNRNPK HNRPK

1.575 P50991 CCT4 CCTD SRB

1.572 P06744 GPI

1.569 P13637 ATP1A3

1.5665 Q9P258 RCC2 KIAA1470 TD60

1.5635 P49327 FASN FAS

1.56 P13796 LCP1 PLS2

1.551 P21333 FLNA FLN FLN1

1.549 Q9BQG0 MYBBP1A P160

1.549 P11586 MTHFD1 MTHFC MTHFD

1.5465 O75390 CS

1.546 P38646 HSPA9 GRP75 HSPA9B mt-HSP70

1.544 P30048 PRDX3 AOP1

1.542 P50990 CCT8 C21orf112 CCTQ KIAA0002

1.541 O43175 PHGDH PGDH3

1.527 P22102 GART PGFT PRGS

1.5265 Q8NC51 SERBP1 PAIRBP1 CGI-55

1.5055 P34897 SHMT2

1.49 P23246 SFPQ PSF

1.4845 Q15084 PDIA6 ERP5 P5 TXNDC7

1.4695 O15067 PFAS KIAA0361

1.4685 P31153 MAT2A AMS2 MATA2

1.4685 P17858 PFKL

1.462 O14654 IRS4

1.4585 Q14166 TTLL12 KIAA0153

1.4495 P39023 RPL3 OK/SW-cl.32

1.447 P54886 ALDH18A1 GSAS P5CS PYCS

1.439 Q13162 PRDX4

1.4345 P49368 CCT3 CCTG TRIC5

1.416 Q15233 NONO NRB54

1.401 P14625 HSP90B1 GRP94 TRA1

1.3635 P27708 CAD

1.362 P00390 GSR GLUR GRD1

140 1.3395 O75369 FLNB FLN1L FLN3 TABP TAP

1.3325 P26641 EEF1G EF1G PRO1608

1.3015 P06576 ATP5B ATPMB ATPSB

1.2895 P13489 RNH1 PRI RNH

1.2855 Q15437 SEC23B

1.283 Q9NY93 DDX56 DDX21 NOH61

1.2795 P26599 PTBP1 PTB

1.272 P17844 DDX5 G17P1 HELR HLR1

1.2405 Q92841 DDX17

1.226 P33993 MCM7 CDC47 MCM2

1.224 P52292 KPNA2 RCH1 SRP1

1.221 P36957 DLST DLTS

141 Appendix 2.3: Overlapping Proteins in Appendix 2.1 and 2.2

Human Uniprot ID Gene Name

P62937 PPIA CYPA

P00338 LDHA PIG19

P15531 NME1 NDPKA NM23

P22314 UBA1 A1S9T UBE1

P53396 ACLY

P07195 LDHB

Q14974 KPNB1 NTF97

Q00610 CLTC CLH17 CLTCL2 KIAA0034

P34932 HSPA4 APG2

P60709 ACTB

P00558 PGK1 PGKA MIG10 OK/SW-cl.110

P23526 AHCY SAHH

P68363 TUBA1B

P45974 USP5 ISOT

O43707 ACTN4

P08238 HSP90AB1 HSP90B HSPC2 HSPCB

P55072 VCP

P50395 GDI2 RABGDIB

P13639 EEF2 EF2

Q16881 TXNRD1 GRIM12 KDRF

P78371 CCT2 99D8.1 CCTB

P06733 ENO1 ENO1L1 MBPB1 MPB1

Q06830 PRDX1 PAGA PAGB TDPX2

Q99832 CCT7 CCTH NIP7-1

P68104 EEF1A1 EEF1A EF1A LENG7

P26640 VARS G7A VARS2

P25705 ATP5A1 ATP5A ATP5AL2 ATPM

P17987 TCP1 CCT1 CCTA

Q86VP6 CAND1 KIAA0829 TIP120 TIP120A

P07900 HSP90AA1 HSP90A HSPC1 HSPCA

P14618 PKM OIP3 PK2 PK3 PKM2

P68371 TUBB4B TUBB2C

P10809 HSPD1 HSP60

Q13509 TUBB3 TUBB4

P11142 HSPA8 HSC70 HSP73 HSPA10

P61978 HNRNPK HNRPK

142 P50991 CCT4 CCTD SRB

P13796 LCP1 PLS2

P11586 MTHFD1 MTHFC MTHFD

O75390 CS

P30048 PRDX3 AOP1

P50990 CCT8 C21orf112 CCTQ KIAA0002

P34897 SHMT2

P31153 MAT2A AMS2 MATA2

P17858 PFKL

P49368 CCT3 CCTG TRIC5

Q15233 NONO NRB54

P00390 GSR GLUR GRD1

P26641 EEF1G EF1G PRO1608

P13489 RNH1 PRI RNH

143 REFERENCES

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stress. Nat. Struct. Biol. 5, 247–249

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Protein S-nitrosylation: purview and parameters. Nat. Rev. Mol. Cell Biol. 6, 150–

166

3. Palmer, R. M., Ferrige, A. G., and Moncada, S. (1987) Nitric oxide release

accounts for the biological activity of endothelium-derived relaxing factor. Nature.

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