S-nitrosothiols and the S-nitrosoproteome of the cardiovascular system

Abstract

Significance: Since their discovery in the early 1990's, S-nitrosylated proteins have been increasingly recognized as important determinants of many biochemical processes. Specifically, S-nitrosothiols in the cardiovascular system exert many actions, including promoting vasodilation, inhibiting platelet aggregation, and regulating Ca(2+) channel function that influences myocyte contractility and electrophysiologic stability.

Recent advances: Contemporary developments in liquid chromatography-mass spectrometry methods, the development of biotin- and His-tag switch assays, and the availability of cyanide dye-labeling for S-nitrosothiol detection in vitro have increased significantly the identification of a number of cardiovascular protein targets of S-nitrosylation in vivo.

Critical issues: Recent analyses using modern S-nitrosothiol detection techniques have revealed the mechanistic significance of S-nitrosylation to the pathophysiology of numerous cardiovascular diseases, including essential hypertension, pulmonary hypertension, ischemic heart disease, stroke, and congestive heart failure, among others.

Future directions: Despite enhanced insight into S-nitrosothiol biochemistry, translating these advances into beneficial pharmacotherapies for patients with cardiovascular diseases remains a primary as-yet unmet goal for investigators within the field.

FIG. 1.

Nitric oxide (NO^•) metabolism in mammalian cells. (A) NO^• synthesized from L-arginine by nitric oxide synthase (NOS) may bind to transition metals (NO-M), undergo oxidation in the presence of molecular oxygen (O₂), or interact with superoxide (^•O₂⁻) to form peroxynitrite (ONOO⁻). In the presence of O₂, NO^• may be converted to nitrosonium (NO⁺), an electrophile that interacts with nucleophilic thiols to form S-nitrosothiols (SNO). Additionally, under conditions of extreme acidity (pH≤3), protonation of NO^• yields the formation of nitrous acid (HNO₂), which is a substrate for the synthesis of the nitrosylating agent, nitrogen trioxide (N₂O₃). S-Nitrosothiol denitrosylation may occur enzymatically or through nonenzymatic exchange with free thiols. Encircled blue labels correspond to reactions that are described in greater detail in (B). M, transition metal (electron acceptor). Adapted from Guikema et al. (50a)

FIG. 2.

Two proposed mechanisms for denitrosylation. (A) The glutathione/S-nitroso-glutathione reductase (GSH/GSNOR) system requires nicotinamide adenine dinucleotide (NADH), reduced glutathione (GSH), and S-nitrosoglutathione (GSNO) for selective denitrosylation of GSNO. Trans-S-nitrosylation from RSNO to GSH forms GSNO. In the presence of NADH, GSNO is converted to glutathione N-hydroxysulfenamide (GSNHOH) by GSNOR. (B) Thioredoxin (Trx) reduces RSNO to induce denitrosylation in a reaction that generates NO^• or HNO₂ and the oxidized form of thioredoxin (TrxSS). Reduction of TrxSS to recycle Trx is catalyzed by thioredoxin reductase (TrxR) in the presence of nicotinamide adenine dinucleotide phosphate (NAPDH). Se, selenium.

FIG. 3.

Trans-S-nitrosylation and S-thiolation reactions. In trans-S-nitrosylation, the nucleophilic thiolate (R′S⁻) of R′SH attacks the nitrogen of an S-nitrosothiol to form R′SNO and RSH, whereas in S-thiolation, the nucleophilic thiolate interacts with the sulfur of an S-nitrosothiol to form a mixed disulfide (RSSR′) and NO⁻.

FIG. 4.

Biotin-switch method for the detection of S-nitrosothiols. Free cysteines (-SH) are blocked (-SX) following exposure of protein samples to methyl-methanethiosulfonate (MMTS). Excess MMTS is removed by running the sample through a microspin column or by protein precipitation with acetone. The sample is then treated with ascorbate and copper sulfate. Ascorbate reduces Cu²⁺→Cu³⁺, which cleaves S-NO bonds, and previously S-nitrosylated (now free) cysteines are labeled with N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide (biotin-HPDP). Biotin-labeled proteins are subjected to Western immunoblotting, target bands are cut from the gel, and then trypsin-digested before liquid chromatography–mass spectrometry (LC-MS) analysis. Alternatively, streptavidin agarose may be used to segregate biotinylated proteins before LC-MS. Adapted from Kettenhofen et al. (71).

FIG. 5.

Cyanide dye-labeling (CyDye)-switch method for the detection of nitrosylated cysteinyl thiols. (A) In the CyDye-switch method, protein samples are subjected to the thiol blocking and reduction steps as outlined in the biotin-switch assay method. Control samples are then labeled with green fluorescence Cy3 dye and experimental (i.e., S-nitrosylated) samples are labeled with red fluorescence Cy5 dye. (B) Using 2D-gel technology, separation of proteins occurs according to gel-specific gradients for molecular weight and pH. Equal protein amounts from both control and experimental conditions are then analyzed by difference gel electrophoresis (DIGE), which exploits differences in Cy3 and Cy5 fluorescence colors to identify cysteinyl thiols modified (i.e., S-nitrosylation or disulfide bond formation) in the experimental conditions. In the example provided, DIGE analysis of mixed protein samples (labeled Merged) reveals the presence of green protein spots that reflect proteins detected with Cy3, but not Cy5 labeling (white arrows). Conversely, proteins labeled by Cy5, but not Cy3 (red circle), reflect cysteinyl thiol modification(s) induced under the experimental conditions. In the merged gel, a color gradient from green to red for protein spots is observed: proteins with greater cysteinyl thiol modification are observed to fluoresce red more strongly. Identification of proteins and specific cysteinyl thiol modification(s) is performed by LC-MS analysis on excised gel spots. Figure 5A is adapted from Kettenhofen et al. (71); Figure 5B is reproduced with permission from Huang et al. (61a)