Cysteine Residues In Proteins Research Articles

Lysines and cysteines: partners in stress?

Modifications of cysteine residues in redox-sensitive proteins are key to redox signaling and stress response in all organisms. A novel type of redox switch was recently discovered that comprises lysine and cysteine residues covalently linked by an nitrogen-oxygen-sulfur (NOS) bridge. Here, we discuss chemical and biological implications of this discovery.

Metabolic activation of 3-aminodibenzofuran mediated by P450 enzymes and sulfotransferases

3-Aminodibenzofuran (3-ADBF) is a potent bladder carcinogen. This study aimed to identify reactive metabolites and the metabolic pathways of 3-ADBF. The in vitro and in vivo studies demonstrated that 3-ADBF was oxidized to the corresponding hydroxylamine by cytochrome P450 enzymes, followed by sulfation of the hydroxyl group mediated by sulfotransferases. The resulting sulfate conjugate was chemically reactive to GSH and cysteine residues of hepatic protein to form the corresponding GSH conjugate and protein adduction. Exposure of 3-ADBF to primary hepatocytes caused protein covalent binding and decreased cell viability. The resultant protein adduction was found to correlate the observed cytotoxicity of 3-ADBF.

Displacement of Native FXYD Protein From Na+/K+-ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity.

The seven mammalian FXYD proteins associate closely with α/β heterodimers of Na+/K+-ATPase. Most of them protect the β1 subunit against glutathionylation, an oxidative modification that destabilizes the heterodimer and inhibits Na+/K+-ATPase activity. A specific cysteine (Cys) residue of FXYD proteins is critical for such protection. One of the FXYD proteins, FXYD3, confers treatment resistance when overexpressed in cancer cells. We developed two FXYD3 peptide derivatives. FXYD3-pep CKCK retained the Cys residue that can undergo glutathionylation and that is critical for protecting the β1 subunit against glutathionylation. FXYD3-pep SKSK had all Cys residues mutated to Serine (Ser). The chemotherapeutic doxorubicin induces oxidative stress, and suppression of FXYD3 with siRNA in pancreatic- and breast cancer cells that strongly express FXYD3 increased doxorubicin-induced cytotoxicity. Exposing cells to FXYD3-pep SKSK decreased co-immunoprecipitation of FXYD3 with the α1 Na+/K+-ATPase subunit. FXYD3-pep SKSK reproduced the increase in doxorubicin-induced cytotoxicity seen after FXYD3 siRNA transfection in pancreatic- and breast cancer cells that overexpressed FXYD3, while FXYD3-pep CKCK boosted the native protein’s protection against doxorubicin. Neither peptide affected doxorubicin’s cytotoxicity on cells with no or low FXYD3 expression. Fluorescently labeled FXYD3-pep SKSK was detected in a perinuclear distribution in the cells overexpressing FXYD3, and plasmalemmal Na+/K+-ATPase turnover could not be implicated in the increased sensitivity to doxorubicin that FXYD3-pep SKSK caused. FXYD peptide derivatives allow rapid elimination or amplification of native FXYD protein function. Here, their effects implicate the Cys residue that is critical for countering β1 subunit glutathionylation in the augmentation of cytotoxicity with siRNA-induced downregulation of FXYD3.

Prodrugs of Persulfide and Sulfide: Is There a Pharmacological Difference between the Two in the Context of Rapid Exchanges among Various Sulfur Species In Vivo?

Sulfide and persulfide are chemically different and one might expect persulfide to be more effective in mediating sulfur signaling because persulfide can directly modify protein cysteine residue. However, rapid scrambling, and interconversions occur among sulfur species. Then there is the question of whether the chemical reactivity differences between sulfide and persulfide would translate into pharmacological differences. Utilizing a delivery system to generate pure hydrogen sulfide (H2 S), hydrogen persulfide (H2 S2 ), and N-acetyl-l-cysteine persulfide (N-CysSSH), we examined the activities of sulfide and persulfide in vitro and in vivo. Persulfide prodrugs exhibited increased activities compared to the H2 S prodrug. In particular, the H2 S2 prodrug offers much-elevated analgesic effects compared to the H2 S prodrug in vivo. Persulfide prodrugs also possess a reduced level of toxicity compared to the H2 S prodrug in vivo, indicating persulfide might represent a better therapeutic paradigm than H2 S.

Prodrugs of Persulfide and Sulfide: Is There a Pharmacological Difference between the Two in the Context of Rapid Exchanges among Various Sulfur Species In Vivo ?

Sulfide and persulfide are chemically different and one might expect persulfide to be more effective in mediating sulfur signaling because persulfide can directly modify protein cysteine residue. However, rapid scrambling, and interconversions occur among sulfur species. Then there is the question of whether the chemical reactivity differences between sulfide and persulfide would translate into pharmacological differences. Utilizing a delivery system to generate pure hydrogen sulfide (H2S), hydrogen persulfide (H2S2), and N-acetyl-l-cysteine persulfide (N-CysSSH), we examined the activities of sulfide and persulfide in vitro and in vivo. Persulfide prodrugs exhibited increased activities compared to the H2S prodrug. In particular, the H2S2 prodrug offers much-elevated analgesic effects compared to the H2S prodrug in vivo. Persulfide prodrugs also possess a reduced level of toxicity compared to the H2S prodrug in vivo, indicating persulfide might represent a better therapeutic paradigm than H2S.

Low-Toxicity Sulfonium-Based Probes for Cysteine-Specific Profiling in Live Cells.

Despite being a low-abundance amino acid, cysteine plays an essential role in regulating protein function and serves as a satisfactory target of post-translational modifications and drug developments. To comprehensively assess reactive-cysteine-containing proteins, the development of chemical proteomic probes to label cysteine residues in human cells is an important objective. Cysteine modification using sulfonium-based probes is a novel method to identify reactive cysteine residues in proteins. Herein, we reported a set of "cysteine-reactive sulfonium-based (C-Sul)" probes to label the reactive cysteine sites in cellular proteins. Notably, water-soluble C-Sul probes have a significantly enhanced stability and cellular uptakes, displaying a high specificity toward reactive cysteines and compatibility with quantitative proteomic profiling. In comparison to the conventional iodoacetamide-based probe, C-Sul particularly has no inhibitory effects on cell viability, enabling its application in proteomic profiling of reactive cysteine residues under biorelevant conditions. We propose C-Sul probes as optimal tools of cysteine profiling for further broadly basic research.

Rational Design of Self-Assembling Artificial Proteins Utilizing a Micelle-Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope.

Self-assembling artificial proteins (SAPs) have gained enormous interest in recent years due to their applications in different fields. Synthesis of well-defined monodisperse SAPs is accomplished predominantly through genetic methods. However, the last decade has witnessed the use of a few chemical technologies for this purpose. In particular, micelle-assisted protein labeling technology (MAPLabTech) has made huge progress in this area. The first generation MAPLabTech focused on site-specific labeling of the active-site residue of serine proteases to make SAPs. Further, this methodology was exploited for labeling of N-terminal residue of a globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue of globular proteins. In addition, we disclose the synthesis of redox-sensitive SAPs and their systematic self-assembly and disassembly studies using size-exclusion chromatography. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering.

Synthetic Thiol and Selenol Derived Amino Acids for Expanding the Scope of Chemical Protein Synthesis.

Cells employ post-translational modifications (PTMs) as key mechanisms to expand proteome diversity beyond the inherent limitations of a concise genome. The ability to incorporate post-translationally modified amino acids into protein targets via chemical ligation of peptide fragments has enabled the access to homogeneous proteins bearing discrete PTM patterns and empowered functional elucidation of individual modification sites. Native chemical ligation (NCL) represents a powerful and robust means for convergent assembly of two homogeneous, unprotected peptides bearing an N-terminal cysteine residue and a C-terminal thioester, respectively. The subsequent discovery that protein cysteine residues can be chemoselectively desulfurized to alanine has ignited tremendous interest in preparing unnatural thiol-derived variants of proteogenic amino acids for chemical protein synthesis following the ligation-desulfurization logic. Recently, the 21st amino acid selenocysteine, together with other selenyl derivatives of amino acids, have been shown to facilitate ultrafast ligation with peptidyl selenoesters, while the advancement in deselenization chemistry has provided reliable bio-orthogonality to PTMs and other amino acids. The combination of these ligation techniques and desulfurization/deselenization chemistries has led to streamlined synthesis of multiple structurally-complex, post-translationally modified proteins. In this review, we aim to summarize the latest chemical synthesis of thiolated and selenylated amino-acid building blocks and exemplify their important roles in conquering challenging protein targets with distinct PTM patterns.

Differential metabolism of arsenicals regulates Fps1-mediated arsenite transport.

Arsenic is an environmental toxin that exists mainly as pentavalent arsenate and trivalent arsenite. Both forms activate the yeast SAPK Hog1 but with different consequences. We describe a mechanism by which cells distinguish between these arsenicals through one-step metabolism to differentially regulate the bidirectional glycerol channel Fps1, an adventitious port for arsenite. Cells exposed to arsenate reduce it to thiol-reactive arsenite, which modifies a set of cysteine residues in target proteins, whereas cells exposed to arsenite metabolize it to methylarsenite, which modifies an additional set of cysteine residues. Hog1 becomes arsenylated, which prevents it from closing Fps1. However, this block is overcome in cells exposed to arsenite through methylarsenylation of Acr3, an arsenite efflux pump that we found also regulates Fps1 directly. This adaptation allows cells to restrict arsenite entry through Fps1 and also allows its exit when produced from arsenate exposure. These results have broad implications for understanding how SAPKs activated by diverse stressors can drive stress-specific outputs.

Protein Lipidation by Palmitate Controls Macrophage Function.

Macrophages are present in all tissues within our body, where they promote tissue homeostasis by responding to microenvironmental triggers, not only through clearance of pathogens and apoptotic cells but also via trophic, regulatory, and repair functions. To accomplish these divergent functions, tremendous dynamic fine-tuning of their physiology is needed. Emerging evidence indicates that S-palmitoylation, a reversible post-translational modification that involves the linkage of the saturated fatty acid palmitate to protein cysteine residues, directs many aspects of macrophage physiology in health and disease. By controlling protein activity, stability, trafficking, and protein–protein interactions, studies identified a key role of S-palmitoylation in endocytosis, inflammatory signaling, chemotaxis, and lysosomal function. Here, we provide an in-depth overview of the impact of S-palmitoylation on these cellular processes in macrophages in health and disease. Findings discussed in this review highlight the therapeutic potential of modulators of S-palmitoylation in immunopathologies, ranging from infectious and chronic inflammatory disorders to metabolic conditions.

Computational and structural investigation of Palmitoyl-Protein Thioesterase 1 (PPT1) protein causing Neuronal Ceroid Lipofuscinoses (NCL).

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of progressive neurodegenerative disorders, associated with 14 Ceroid Lipofuscinosis Neuronal genes (CLN1-14). The mutations in the Palmitoyl-Protein Thioesterase 1 (PPT1) protein serve as one of the major reasons for the causative of NCL. The PPT1 involves degrading and modifying cysteine residues in proteins or peptides by removing thioester-linked fatty acyl groups like palmitate prefers acyl chains of 14-18 carbons in length. In this study, we have analyzed the impact of PPT1 mutations on the deleteriousness, stability, conservative nature of amino acid, and impact of mutations on the protein structure. We have also used molecular dynamics simulations using GROMACS to perceive the alteration in the dynamic behavior of the PPT1 at the residual level. In this study, we have retrieved 23 PPT1 mutations from the UniProt database, and these were subjected to a series of analyses using varied computer algorithms. From these analyses, out of 23 mutations, 16 mutations were identified as deleterious. Among 16, eight mutations were identified to destabilize the protein structure, and finally, two mutations (W38C and L222P) were found to be positioned in the highly conserved region. The structural impact study observed that the mutant proline could disrupt the alpha helix formed by the leucine at position 222. Finally, from the molecular dynamics simulations, we observed that due to the mutations (W38C and L222P), the protein had experienced higher deviation, fluctuation, and lower compactness. These structural changes elucidate that these mutations can impact the structure and function of the PPT1 protein.

Evaluation of methods to analyze redox state in immune cells

Reactive oxygen species (ROS) is produced in immune cells during immune responses and is necessary for host defense and inflammation. Furthermore, ROS acts as signals for gene expression and is required for T cell proliferation and activation. While low levels of ROS play important roles in cell activation, high levels of ROS induce significant damage to cells. To monitor redox state in living cells we generated transgenic mice expressing a green fluorescent protein (roGFP) whose fluorescence varies with redox state (J Invest Dermatol. 34, 1701-1709, 2014). Since the redox state may change during in vitro analysis it should be necessary to fix the cells. Here we evaluate the fixation methods to analyze redox state in vitro. We compared aldehyde and organic solvent-based fixation methods. To fix redox state of roGFP protein N-ethylmaleimide which react thiol and modify cysteine residues in protein was used. Splenocytes were isolated from roGFP mice and treated with hydrogen peroxide (oxidized state) or DTT (reduced state) to induce the maximum oxidation and reduction status. Oxidized or reduced cells fixed with various fixation methods were accessed by flow cytometry. Organic solvents lead to a severe loss of fluorescence of roGFP protein. On the other hand, fixation with aldehyde and N-ethylmaleimide was useful to maintain fluorescence and redox status of roGFP. This system should be a powerful and convenient tool for analyzing redox state in various types of immune cells in vitro.

CgGH insertion functional domain analysis in transgenic G1 and G2 and G3 mutiara catfish (Clarias gariepinus) broodstock

Catfish is one of the most important freshwater fish farming commodities in Indonesia. Higher catfish production can be achieved by cultivating transgenic catfish carrying the growth hormone (GH) gene of African catfish (Clarias gariepinus GH, CgGH). This research focuses on analysis of the presence of the CgGH gene in transgenic G1, G2, and G3 mutiara catfish broodstock, as an indication of stable CgGH inheritance. CgGH gene was isolated using the RNeasy mini kit and RT-PCR. RT-PCR revealed amplicons measuring approximately 600 bp in transgenic G0, G1, G2, and G3 mutiara catfish. The CgGH consensus sequence similarities ranged from 93.76% to 97.06%, with four functional domain sites (somatotropin-1, somatotropin-2, four α-helix, N-glycosylation, four cysteine residues) of fish GH proteins. The functional domains of fish GH proteins are conserved in G1, G2, and G3 and indicate stable exogenous GH inheritance to produce transgenic catfish strains in each generation.

Regulation of Cell Polarity by Posttranslational Protein Palmitoylation.

Cell polarity is a common feature of many living cells, especially epithelial cells, and plays important roles in development, tissue homeostasis, and diseases. Therefore, the signaling pathways involved in establishing and maintaining cell polarity are tightly controlled. Protein S-palmitoylation has been recently recognized as an important posttranslational modification involved in cell polarity, via dynamic covalent attachment of fatty acyl groups to the cysteine residues of cell polarity proteins. Here, we describe the methods to study the function and regulation of S-palmitoylation of cell polarity proteins.

Allicin, an Antioxidant and Neuroprotective Agent, Ameliorates Cognitive Impairment.

Allicin (diallylthiosulfinate) is a defense molecule produced by cellular contents of garlic (Allium sativum L.). On tissue damage, the non-proteinogenic amino acid alliin (S-allylcysteine sulfoxide) is converted to allicin in an enzyme-mediated process catalysed by alliinase. Allicin is hydrophobic in nature, can efficiently cross the cellular membranes and behaves as a reactive sulfur species (RSS) inside the cells. It is physiologically active molecule with the ability to oxidise the thiol groups of glutathione and between cysteine residues in proteins. Allicin has shown anticancer, antimicrobial, antioxidant properties and also serves as an efficient therapeutic agent against cardiovascular diseases. In this context, the present review describes allicin as an antioxidant, and neuroprotective molecule that can ameliorate the cognitive abilities in case of neurodegenerative and neuropsychological disorders. As an antioxidant, allicin fights the reactive oxygen species (ROS) by downregulation of NOX (NADPH oxidizing) enzymes, it can directly interact to reduce the cellular levels of different types of ROS produced by a variety of peroxidases. Most of the neuroprotective actions of allicin are mediated via redox-dependent pathways. Allicin inhibits neuroinflammation by suppressing the ROS production, inhibition of TLR4/MyD88/NF-κB, P38 and JNK pathways. As an inhibitor of cholinesterase and (AChE) and butyrylcholinesterase (BuChE) it can be applied to manage the Alzheimer’s disease, helps to maintain the balance of neurotransmitters in case of autism spectrum disorder (ASD) and attention deficit hyperactive syndrome (ADHD). In case of acute traumatic spinal cord injury (SCI) allicin protects neuron damage by regulating inflammation, apoptosis and promoting the expression levels of Nrf2 (nuclear factor erythroid 2-related factor 2). Metal induced neurodegeneration can also be attenuated and cognitive abilities of patients suffering from neurological diseases can be ameliorates by allicin administration.

Pillars and Gaps of S-Nitrosylation-Dependent Epigenetic Regulation in Physiology and Cancer.

Nitric oxide (NO) is a diffusible signaling molecule produced by three isoforms of nitric oxide synthase, which release NO during the metabolism of the amino acid arginine. NO participates in pathophysiological responses of many different tissues, inducing concentration-dependent effect. Indeed, while low NO levels generally have protective effects, higher NO concentrations induce cytotoxic/cytostatic actions. In recent years, evidences have been accumulated unveiling S-nitrosylation as a major NO-dependent post-translational mechanism ruling gene expression. S-nitrosylation is a reversible, highly regulated phenomenon in which NO reacts with one or few specific cysteine residues of target proteins generating S-nitrosothiols. By inducing this chemical modification, NO might exert epigenetic regulation through direct effects on both DNA and histones as well as through indirect actions affecting the functions of transcription factors and transcriptional co-regulators. In this light, S-nitrosylation may also impact on cancer cell gene expression programs. Indeed, it affects different cell pathways and functions ranging from the impairment of DNA damage repair to the modulation of the activity of signal transduction molecules, oncogenes, tumor suppressors, and chromatin remodelers. Nitrosylation is therefore a versatile tool by which NO might control gene expression programs in health and disease.

Hypothiocyanous acid oxidizes collapsin response mediator protein 2 in brain pericytes

AbstractBackgroundPericytes cover brain endothelium to enable maintenance of the blood‐brain barrier (BBB). Decreasing pericyte numbers are reported in Alzheimer’s disease and could lead to BBB dysfunction, triggering vascular inflammation and the recruitment of immune cells. Neutrophils contain high contents of myeloperoxidase, a heme enzyme that produces several oxidising species, including hypothiocyanous acid (HOSCN). HOSCN specifically targets cysteine residues in proteins and low molecular weight thiols, and is more stable than other oxidants in biological systems. A microtubule stabilising protein, collapsin response mediator protein 2 (CRMP2), is oxidised by hydrogen peroxide (H2O2) in some cell types, purportedly through a peroxiredoxin (Prx) relay mechanism. Here, we have tested whether HOSCN can oxidise CRMP2 and lead to disruption of microtubules (MT) in pericytes.MethodMurine pericytes were harvested and cultured, then treated with sub‐lethal concentrations of HOSCN and H2O2. Western blotting was used to observe CRMP2 and Prx oxidation, along with CRMP2 phosphorylation at sites important for MT binding. Fluorescent staining, live‐cell imaging and in vitro scratch assays were used to view cytoskeleton alterations and pericyte migration. Immunoprecipitation and proximity ligation assays were used to investigate physical associations between CRMP2 and Prxs.ResultTreatment of pericytes with HOSCN led to maximal CRMP2 oxidation after 60 min, whereas H2O2 reaches this in 5 min. Oxidation was closely associated with pericyte shrinkage, less growing microtubules and lower pericyte cell migration. CRMP2 oxidation occurred at the same time as Prx2 oxidation, and there was a physical association between these proteins, but it is not possible to conclude a redox relay occurs.ConclusionWe hypothesized that the longer‐lived oxidant HOSCN could oxidize CRMP2 and impair pericyte function at sub‐lethal doses. Indeed, HOSCN led to less dynamic MTs and subsequent lower migration of pericytes, likely lowering their ability to cover empty area on the inflamed vasculature to protect endothelial function. This effect is reversible upon HOSCN removal, nevertheless HOSCN could be a cause of BBB disruption during neuroinflammation. The mechanisms of CRMP2 oxidation are currently being investigated. Strategies that protect this MT regulatory protein from oxidation could have therapeutic benefit in neurological disease.

Drug-drug interactions induced by Linderane based on mechanism-based inactivation of CYP2C9 and the molecular mechanisms

Linderane (LDR) is a main furan-containing sesquiterpenoid of the common herbal medicine Lindera aggregata (Sims) Kosterm. Our early study indicated that LDR led to mechanism-based inactivation (MBI) of CYP2C9 in vitro, implying possible drug-drug interactions (DDIs) in clinic. In the present study, influence of LDR on the pharmacokinetics of the corresponding hydroxylated metabolites of CYP2C9 substrates in rats was investigated. Pharmacokinetic studies revealed that pretreatment with LDR at 20 mg/kg for 15 days inhibited the metabolism of both tolbutamide and warfarin catalyzed by CYP2C9. As for 4-hydroxytolbutamide, the Cmax was decreased, the t1/2z was prolonged, and the Vz/F was increased, all with significant difference. As for 7-hydroxywarfarin, the AUC0-t/AUC0-∞ and CLz/F were significantly decreased and increased, respectively. Furthermore, the underlying molecular mechanisms based on MBI of CYP2C9 by LDR were revealed. Two reactive metabolites of LDR, furanoepoxide and γ-ketoenal intermediates were identified in CYP2C9 recombinant enzyme incubation systems. Correspondingly, covalent modifications of lysine and cysteine residues of CYP2C9 protein were discovered in the CYP2C9 incubation system treated with LDR. The formation of protein adducts exhibited obvious time- and dose-dependence, which is consistent with the trend of enzyme inhibition caused by LDR in vitro. In addition to the apoprotein of CYP2C9, the heme content was significantly reduced after co-incubation with LDR. These data revealed that modification of both apoprotein and heme of CYP2C9 by reactive metabolites of LDR led to MBI of CYP2C9, therefore resulting in the inhibition of biotransformation of CYP2C9 substrates to their corresponding metabolites in vivo.

A sticky situation: regulation and function of protein palmitoylation with a spotlight on the axon and axon initial segment.

In neurons, the axon and axon initial segment (AIS) are critical structures for action potential initiation and propagation. Their formation and function rely on tight compartmentalisation, a process where specific proteins are trafficked to and retained at distinct subcellular locations. One mechanism which regulates protein trafficking and association with lipid membranes is the modification of protein cysteine residues with the 16-carbon palmitic acid, known as S-acylation or palmitoylation. Palmitoylation, akin to phosphorylation, is reversible, with palmitate cycling being mediated by substrate-specific enzymes. Palmitoylation is well-known to be highly prevalent among neuronal proteins and is well studied in the context of the synapse. Comparatively, how palmitoylation regulates trafficking and clustering of axonal and AIS proteins remains less understood. This review provides an overview of the current understanding of the biochemical regulation of palmitoylation, its involvement in various neurological diseases, and the most up-to-date perspective on axonal palmitoylation. Through a palmitoylation analysis of the AIS proteome, we also report that an overwhelming proportion of AIS proteins are likely palmitoylated. Overall, our review and analysis confirm a central role for palmitoylation in the formation and function of the axon and AIS and provide a resource for further exploration of palmitoylation-dependent protein targeting to and function at the AIS.

Endogenous S-nitrosocysteine proteomic inventories identify a core of proteins in heart metabolic pathways

Protein cysteine residues are essential for protein folding, participate in enzymatic catalysis, and coordinate the binding of metal ions to proteins. Enzymatically catalyzed and redox-dependent post-translational modifications of cysteine residues are also critical for signal transduction and regulation of protein function and localization. S-nitrosylation, the addition of a nitric oxide equivalent to a cysteine residue, is a redox-dependent modification. In this study, we curated and analyzed four different studies that employed various chemoselective platforms coupled to mass spectrometry to precisely identify S-nitrosocysteine residues in mouse heart proteins. Collectively 1974 S-nitrosocysteine residues in 761 proteins were identified and 33.4% were identified in two or more studies. A core of 75 S-nitrosocysteine residues in 44 proteins were identified in all four studies. Bioinformatic analysis of each study indicated a significant enrichment of mitochondrial proteins participating in metabolism. Regulatory proteins in glycolysis, TCA cycle, oxidative phosphorylation and ATP production, long chain fatty acid β-oxidation, and ketone and amino acid metabolism constitute the major functional pathways impacted by protein S-nitrosylation. In the cardiovascular system, nitric oxide signaling regulates vasodilation and cardiac muscle contractility. The meta-analysis of the proteomic data supports the hypothesis that nitric oxide signaling via protein S-nitrosylation is also a regulator of cardiomyocyte metabolism that coordinates fuel utilization to maximize ATP production. As such, protein cysteine S-nitrosylation represents a third functional dimension of nitric oxide signaling in the cardiovascular system to ensure optimal cardiac function.