NO is generated through the oxidation of L-arginine by NO synthase (NOS) and is involved in key physiological functions, including vasodilatation, platelet aggregation, immune defense, cell proliferation and mitochondrial respiration (5-7). As NO is a gas with a short half-life in the body, its activity is concentration-dependent, often confining its action to local target sites (7). Identifying NOS-specific inhibitors can contribute to understanding NO-driven signaling pathways and may provide a basis for developing therapies for conditions linked to abnormal NO levels.

In mammals, NOS exists in three major isoforms: inducible (iNOS/NOS2), neural (nNOS/NOS1) and endothelial (eNOS/NOS3), each showing distinct expression patterns and biological functions across various tissues and cell types (8,9). Under resting conditions, iNOS is generally not expressed. Its expression is triggered by stimuli such as inflammatory mediators, endotoxins and hypoxic environments (9). Pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1, interferon-γ and lipopolysaccharide bind to membrane receptors, activating intracellular kinases that phosphorylate target proteins and initiate specific transcription factors. These transcription factors then translocate into the nucleus, bind promoter sequences and induce iNOS gene expression, leading to sustained, high-output NO production (10,11).

By contrast, nNOS and eNOS are constitutively expressed and their enzymatic activities are fine-tuned by phosphorylation, S-nitrosylation, protein-protein interactions and calcium signaling (9). eNOS is primarily located in endothelial cells and is the predominant isoform involved in regulating vascular tone and function (12). NO derived from endothelial cells inhibits platelet aggregation, prevents thrombus formation and regulates the expression of genes associated with atherosclerosis (AS), thereby blocking leukocyte adhesion and migration to the endothelium, which helps prevent AS progression (13,14). nNOS is mainly found in neurons and is regulated by Ca²⁺ and calmodulin, playing essential roles in learning, memory and neurogenesis (15). Under physiological conditions, nNOS rapidly produces small amounts of NO to maintain internal stability and contribute to neuroprotection (16).

NO signaling functions through both classical and non-classical mechanisms to regulate diverse cellular activities (17). In the classical pathway, NO binds to the heme moiety of soluble guanylate cyclase, facilitating the enzymatic conversion of guanosine triphosphate into cGMP (18). Acting as a second messenger, cGMP activates cGMP-dependent protein kinase G, which in turn regulates downstream targets, including cGMP-regulated kinases, phosphodiesterases and ion channels, thereby regulating mitochondrial dynamics and biogenesis.

By contrast, the non-classical signaling pathway involves the covalent post-translational modification of biomolecules by NO and its reactive derivatives. This mode of action, primarily through S-nitrosylation, plays a regulatory role across various tissues and organ systems (19,20). S-nitrosylation has emerged as a critical form of redox-based signaling that enables cells to manage oxidative stress and limit reactive oxygen species levels (21,22). Importantly, S-nitrosylation is a selective and reversible modification encompassing nitrosylation, transnitrosylation and denitrosylation processes (23). Two principal classes of denitrosylating enzymes have been identified: The Trx system and low-molecular-weight SNO-cleaving enzymes, which include S-nitrosoglutathione reductase (GSNOR) and S-nitroso-coenzyme A reductase (24). Enzymes such as GSNOR and thioredoxin reductase (TrxR) catalyze the denitrosylation of SNOs, restoring free sulfhydryl groups. In parallel, SNOs can donate NO groups to the cysteine thiols of target proteins, a process facilitated by transnitrosylases (Fig. 1). Recent findings indicate that NO-driven S-nitrosylation markedly affects essential cellular processes including transcription, DNA repair, cell growth, differentiation and apoptosis (17,25).

In 1992, Stamler et al (26) first identified SNO, a product formed when NO reacts with bovine serum albumin. Protein S-nitrosylation, a covalent modification involving the attachment of NO to reactive cysteine residues, has since been recognized as a key mechanism by which NO mediates its biological functions (27). As NO itself is not an oxidant and does not efficiently interact with protein thiols directly (17), most S-nitrosylation events proceed through an intermediate oxidation process. Initially, NO reacts with O₂ to form NO₂, which then combines with another NO molecule to produce N₂O₃, a more reactive intermediate that reacts with protein thiols to yield nitrosothiols (28). Additionally, NO₂ can oxidize thiols to generate thiol radicals, which subsequently react with NO to form nitrosothiols (17). Other pathways also exist. During the auto-nitrosylation of hemoglobin or in the formation of GSNO by cytochrome c, NO is oxidized by a transition metal such as Fe³⁺ or Cu²⁺ within a metalloenzyme, forming a nitrosonium ion (18). This positively charged intermediate then reacts with a nearby thiol group close to the catalytic center, producing a stable nitrosothiol (18) (Fig. 2).

S-nitrosylation does not occur randomly across all cysteine residues; it is selective and occurs only on specific thiols within proteins (29). The target cysteine typically needs to be spatially close to the NO source for direct recognition by NOS. It is often located in a hydrophobic environment shaped by the tertiary protein structure or membranes, which avoids steric hindrance and increases reaction probability (29,30). Moreover, certain motifs, such as I/L-X-C-X2-D/E, are preferentially targeted by NOS. These motifs often lie within α-helices that present a broad surface area, making them more accessible for S-nitrosylation (31). Proteomic analyses have revealed that the likelihood of S-nitrosylation is determined not only by the surrounding amino acid sequence but also by the pKa of the cysteine thiol, the local pH, the electrophilic nature of nitrosothiols, NOS enzymatic activity and the concentration of external NO (22,27,32-34).

Intracellular SNO levels are shaped not only by the cellular redox environment but also by the action of specific nitrosylases (35,36). Among the most abundant endogenous SNOs, GSNO acts as a key NO donor for protein modification in vivo (37). GSNO is metabolized by GSNOR, a member of the alcohol dehydrogenase family, which catalyzes its breakdown into oxidized glutathione and ammonia (38). In addition to GSNOR, the Trx enzyme family, primarily located in the cytoplasm, forms a central part of the denitrosylation system (39). Unlike the GSNOR system, which specifically targets GSNO, TrxR enables denitrosylation across a broader range of nitrosylated proteins. Changes in the expression or function of reductases such as GSNOR and TrxR can disturb S-nitrosylation dynamics and contribute to disease development (4,40). S-nitrosylation is also regulated through transnitrosylation, a process in which the NO group is transferred from low molecular weight nitrosothiols such as GSNO to cysteine thiols on target proteins (41). GSNO can transfer its S-nitroso group to various substrates, affecting cellular physiology or contributing to disease progression through protein S-nitrosylation (17,41). Together, nitrosylation, denitrosylation and transnitrosylation work in concert to maintain SNO homeostasis in vivo, playing essential roles in normal physiology. Disruptions in these processes can lead to imbalances in SNO levels, resulting in functional disturbances.

Mitochondria, central to cell growth and cycle regulation, function not only as the primary source of cellular ATP but also participate in signal transduction, differentiation and apoptosis. Murray et al (43) identified several proteins modified by S-nitrosylation in rat heart tissue following co-incubation with the NO donor GSNO. These proteins were mainly involved in mitochondrial quality control, energy metabolism and mitochondria-mediated apoptosis. This suggests that S-nitrosylation-mediated control of mitochondrial biogenesis and dynamics may offer a potential strategy for addressing diseases linked to mitochondrial dysfunction (52,53). In plant mitochondria, protein S-nitrosylation has been shown to participate in multiple metabolic pathways, including photorespiration (54), the tricarboxylic acid cycle (55-67), oxidative phosphorylation (54,58), protein processing and turnover (59) and other core metabolic processes (60). Beyond metabolic regulation, S-nitrosylation also influences mitochondrial autophagy. Parkin, a ubiquitin E3 ligase of the RING-between-RING (RBR) family, is a key player in mitochondrial quality control. Ozawa et al (61) reported that S-nitrosylation levels of Parkin increased markedly in GSNO-treated HeLa cells, which increased Parkin's E3 ligase activity and promoted mitochondrial degradation. In addition to its role in regulating mitochondrial pathways, Tang et al (38) demonstrated that mitochondrial GSNOR is critical for maintaining mitochondrial homeostasis. Through the denitrosylation of ANT1, GSNOR contributes to preserving mitochondrial integrity and improving mitochondrial performance.

S-nitrosylation not only affects mitochondrial function but also modifies key transcription factors and their associated regulatory proteins, including NF-κB, p53, hypoxia-inducible factor (HIF) and nuclear respiratory factor 2 (52,62-64). NF-κB is a major transcriptional regulator involved in immune and inflammatory responses, development, cell proliferation and apoptosis. It activates transcription by binding specific DNA sequences within promoter regions. S-nitrosylation of the NF-κB p50 subunit at Cys62 and the p65 subunit at Cys38 impairs this DNA-binding ability, thereby reducing transcriptional activity (65,66). Matthews et al (62) showed that in A549 human lung cancer cells, treatment with the NO donor GSNO led to S-nitrosylation of NF-κB, which suppressed its transcriptional activity and increased the cells' sensitivity to tumor necrosis factor-α-induced apoptosis. Additionally, under excitotoxic conditions triggered by glutamate, eNOS caused S-nitrosylation of the NF-κB p65 subunit. This modification decreased NF-κB activity and offered neuroprotection, improving neuronal survival (67).

S-nitrosylation also influences p53. Specifically, modification at Cys124 within its DNA-binding domain increases the regulation of downstream targets such as superoxide dismutase and catalytic subunits, helping reduce oxidative stress (50). Beyond NF-κB and p53, S-nitrosylation of HIF-1α stabilizes the protein, increasing its protective function in hypoxic environments (68).

S-nitrosylation, as part of redox-based signaling, influences protein function by affecting enzyme activity, subcellular localization and protein-protein interactions. Under conditions of proliferative stress, hematopoietic stem cells show increased levels of NO and SNO, which are associated with the accumulation of misfolded or aggregated proteins. Yi et al (69) identified a mechanistic connection between GSNOR-regulated protein S-nitrosylation and the CHOP-mediated unfolded protein response, which plays a role in maintaining hematopoietic stem cell activity. Their findings suggest that adjusting S-nitrosylation levels could support the expansion of functional hematopoietic stem cells without compromising viability. Lin et al (70) reported that S-nitrosylated recombinant cathepsin B (CTSB) regulates its mRNA reprogramming through the ADD1/MATR3/ADAR1 signaling pathway, affecting the steady-state expression of CTSB. S-nitrosylation may either promote or suppress protein activity depending on the cellular context, timing and location (71). This highlights the need to evaluate how SNO-modified proteins influence downstream signaling processes under specific conditions.

In the complex development of cardiovascular diseases, protein S-nitrosylation is gaining recognition for its distinct role in regulating inflammation, apoptosis, angiogenesis, myocardial contractility and electrophysiological signaling transduction (Table II). Yoon et al (75) showed that increased nNOS activity in hearts with diastolic dysfunction (DD) raised NO levels in vivo, leading to S-nitrosylation of histone deacetylase 2 and worsening clinical symptoms in patients with DD. This study identified protein S-nitrosylation as a contributing factor in DD pathogenesis and proposed it as a potential therapeutic target for treatment-resistant heart failure.

As mentioned earlier, mitochondrial GSNOR is critical for maintaining mitochondrial homeostasis via denitrosylation of ANT1. A decline in mitochondrial GSNOR expression during heart failure increases S-nitrosylation of ANT1 at Cys160. In line with this, overexpression of either mitochondrial GSNOR or a non-nitrosylated mutant, ANT1 Cys160A, markedly improved mitochondrial function, offering a new potential target for heart failure therapy (38). Aortic aneurysm and coarctation, life-threatening conditions that can lead to aortic dissection and sudden death, have also been associated with increased SNO levels. Aortic tissues from patients with coarctation showed elevated S-nitrosylation compared to healthy controls (76). Pan et al (77) identified Plastin 3, modified at Cys566, as a key protein affected by S-nitrosylation in this disease. Additionally, Zhang et al (78) found that the S-nitrosylation level of Septin2 was markedly increased in diseased vascular tissues and peripheral blood mononuclear cells of patients with aortic coarctation. This modification altered the TIAM1/RAC1/NF-κB signaling cascade, contributing to macrophage-driven vascular inflammation and aortic pathology. Two compounds, NSC23766 and R-ketoglutarate, targeting this pathway, were found effective in preventing and treating the condition.

In conclusion, the levels of a number of protein SNOs are upregulated in various cardiovascular diseases. De-nitrosylation can play a role in protecting the heart and alleviating vascular damage. The research on SNO modification in cardiovascular physiology and pathology has made certain progress, suggesting that S-nitrosylation of proteins plays an important role in regulating cardiovascular diseases. These findings suggest that detailed exploration of the molecular role of S-nitrosylation in cardiovascular physiology and disease may guide new drug development and help identify novel therapeutic strategies. In addition, continuously promoting the research on the targets and mechanisms between SNO and cardiovascular diseases and integrating them with clinical treatment, will bring good news to more patients with cardiovascular diseases.

Studies have shown that NO is involved in a wide range of neural processes, including development, neurotransmitter release and synaptic plasticity (7). However, abnormal S-nitrosylation driven by excessive NO can contribute to neuronal damage in various neurodegenerative diseases (79) (Table III). Nakamura et al (29) reported that under normal conditions, nitrosylated NMDA receptors remain inactive, helping maintain physiological NO levels and supporting normal neuronal function. By contrast, under pathological conditions, overactivation of the extrasynaptic NMDA-nNOS pathway leads to excess NO production, driving the formation of abnormal nitrosylated proteins and accelerating disease progression. In Alzheimer's disease (AD), NO targets the Cys644 residue of guanosine triphosphate-bound Drp1, forming SNO-Drp1, which overactivates the protein and triggers mitochondrial fragmentation. This results in insufficient energy for neurotransmission and contributes to synaptic failure and cognitive decline (80). S-nitrosylation of Drp1 also promotes mitochondrial overactivation and fission (81,82). Wang et al (82) found that S-nitrosylation of Drp1 was present in all cases of sporadic AD. Inhibiting this modification prevented Aβ-induced mitochondrial fragmentation, synaptic dysfunction and neuronal death, suggesting that SNO-Drp1 could serve as a potential therapeutic target for preserving neuronal and synaptic integrity in AD. Furthermore, Nakamura et al (83) demonstrated, using in vitro and in vivo AD models and postmortem brain samples from AD patients, that enzymes from various biochemical pathways can undergo a cascade of S-nitrosylation events, ultimately leading to synapse loss.

In PD, both animal models and human tissue studies have shown that S-nitrosylation of the parkin protein disrupts its neuroprotective function by impairing substrate ubiquitination and suppressing its ubiquitin ligase activity (84). Tsang et al (85) further confirmed that nitrosative stress contributes to PD progression by impairing prosurvival proteins such as parkin and X-linked inhibitor of apoptosis protein via distinct pathways, indicating that abnormal S-nitrosylation is a key factor in neurodegeneration. In glioma cells, S-nitrosylation of extracellular signal-regulated kinase 1/2 (ERK1/2) within the MAPK pathway suppresses its phosphorylation, promoting apoptosis (86). On the other hand, S-nitrosylation of caspase-3 in glioma cells inhibits apoptosis and facilitates tumor progression (87). Altogether, aberrant S-nitrosylation plays a critical role in the pathogenesis of AD, PD and gliomas.

In the clinic, neurodegenerative diseases can be prevented or treated by controlling NO production through drug delivery or the modulation of NOS activity (88). In addition, by inhibiting the excessive release of α-synuclein, which oligomerizes, aggregates and deposits in the cytoplasm, Parkin SNO can be reduced and cells can be protected from extracellular α-synuclein oligomer-induced toxicity, thus treating diseases (89). Therefore, gaining a deeper understanding of how dysregulated S-nitrosylation pathways contribute to neurological disorders will improve insight into their molecular basis and support the search for therapeutic targets.

S-nitrosylation plays dual roles in cancer, acting as both a tumor suppressor and promoter and is involved across multiple stages of tumor progression. Abnormal S-nitrosylation patterns have been identified in various cancers, including pancreatic, lung, liver and prostate, where they influence tumor behavior in both positive and negative directions (Table IV). In pancreatic ductal adenocarcinoma (PDAC), dysregulated NO production by iNOS and eNOS contributes to tumor development and correlates with poor prognosis, although the detailed mechanisms remain unclear (90,91). Tan et al (92) used site-specific proteomics to identify 585 S-nitrosylation sites on 434 proteins in PDAC tissues and PANC-1 cell lines, reporting markedly more S-nitrosylated proteins in tumor samples than in adjacent normal tissue. Deng et al (93) demonstrated that tumor necrosis factor-related apoptosis-inducing ligand triggered apoptosis in human thyroid carcinoma FRO cells by increasing NO levels, which led to S-nitrosylation of GAPDH and its accumulation in the nucleus. In another study, increased S-nitrosylation of the parkin protein, driven by iNOS and GSNOR deficiency, was found to contribute to hepatocellular carcinoma formation in mice (94). Similarly, Wei et al (95) identified S-nitrosylation of O6-alkylguanine-DNA alkyltransferase (AGT) at Cys145 in both in vivo and in vitro models. This modification promoted AGT degradation through the proteasome and led to the accumulation of mutagenic alkylamines, accelerating liver tumor progression.

In the context of non-small cell carcinoma (NSCLC), Zhang et al (96) found that inflammatory conditions caused S-nitrosylation of the cytoskeletal regulator ezrin, promoting mechanical signaling between the cytoskeleton and membrane and increasing cell invasiveness and metastatic capacity. This provides a new perspective on NSCLC progression and potential therapeutic approaches. S-nitrosylation has also been shown to influence the function of epigenetic regulators. DNA methyltransferases (DNMTs), which control DNA methylation patterns in mammals, are key epigenetic enzymes. Okuda et al (97) discovered that S-nitrosylation of DNMT suppressed its enzymatic activity and promoted tumor formation. They also identified DBIC, a compound that blocks S-nitrosylation of DNMT3B at low concentrations (IC₅₀ ≤100 nM), without interfering with its catalytic function. DBIC markedly reduced tumor cell proliferation and malignant transformation. Taken together, these findings show that disruptions in S-nitrosylation contribute to cancer development.

Although SNO modification accounts for a relatively low proportion of overall protein PTMs, it still plays an important role in tumor development and inhibition (98). Increasing the level of SNO modification in certain proteins by developing NO donors, inhibiting GSNOR and denitrification and enhancing the targeting of SNO modification and protein active sites with nanomaterials may provide a new approach for tumor cell therapy. Conversely, reducing the SNO levels of certain cancer-promoting proteins can promote their ubiquitination and degradation and reverse inhibit tumor growth. This can be achieved by using NO scavengers or NOS inhibitors to reduce the activity of certain pro-cancer proteins and using exogenous GSNOR to enhance cell denitrification and inhibit tumor growth. Investigating the role of S-nitrosylation in tumorigenesis can help identify potential targets for early intervention. For patients already diagnosed with cancer, targeting S-nitrosylation-modified proteins may provide new therapeutic options and help delay the onset of drug resistance.

Diabetes mellitus is closely linked to disrupted S-nitrosylation, which affects all stages of insulin activity, from its synthesis and secretion to signaling and degradation in target tissues (99). This regulation plays an important role in insulin-related metabolic disorders. In type 2 diabetes, insulin resistance (IR) is influenced by S-nitrosylation through three key mechanisms: i) S-nitrosylation of insulin receptor-β, insulin receptor substrate 1 and AKT1 either suppresses their activity or accelerates their degradation, thereby impairing insulin signaling. ii) S-nitrosylation of PPARγ and PDE3B reduces their activity, weakening adipocyte function and contributing to adipocyte IR. iii) S-nitrosylation of SIRT1 increases acetylation of NF-κB p65, boosting NF-κB's transcriptional activity, which triggers pro-inflammatory gene expression and worsens IR.

More recently, attention has turned to the role of S-nitrosylation in cellular metabolism, particularly in relation to type 2 diabetes and insulin resistance. Zhou et al (100) demonstrated that insulin signaling is directly tied to protein S-nitrosylation, where NO covalently binds to cysteine residues to form SNOs, serving as functional effectors of insulin action. This finding highlights the relevance of S-nitrosylation in metabolic regulation and supports its growing importance in diabetes research.

Zhou et al (99) identified a new enzyme, S-nitro-coenzyme A-assisted nitrotransferase (SCAN), which uses SNO-CoA as a cofactor to S-nitrosylate various target proteins. SCAN has been shown to regulate insulin signaling under normal conditions, while its dysregulation may contribute to the development of diabetes. This discovery advances the understanding of islet signaling pathways and suggests new possibilities for treating diseases such as obesity and diabetes. In addition, a number of individuals with diabetes develop neuropathy. Li et al (101) found that in the hippocampus of diabetic rats, defective autophagy caused by S-nitrosylation of the ATG4B protein plays a significant role in CNS neuropathy. This finding reveals a novel mechanism involved in diabetes-related CNS complications.

Carvalho-filho et al (102) indicate that aspirin treatment not only reduces iNOS protein levels but also S-nitrosylation of IRβ, IRS-1 and Akt. These changes are associated with improved insulin resistance and signaling, suggesting a novel mechanism of insulin sensitization evoked by aspirin treatment. These findings also suggest that inhibition of iNOS might be a major mediator of the insulin-sensitizing effects of IKKβ inhibition. In conclusion, targeting S-nitrosylation presents an opportunity for novel or refined drug development that could provide a novel and more precise approach for the management of metabolic diseases.

Fig. 4 systematically summarized the aforementioned 24 studies related to 'S-nitrosylation modification and disease occurrence and development'. Among them, 16 studies have shown that NO-mediated S-nitrosylation modification of target proteins has a promoting effect on the occurrence of diseases. For example, in a study where the protein S-nitrosylation of HDAC2 plays a key role in the development of DD, NO reduction and protein denitrosylation markedly improved the mitral early wave/mitral annulus movement ratio and the effect on movement time in DD mice, but it did not markedly affect the ejection fraction (75). The results indicated that the denitrosylation of HDAC2 induced by antioxidants could alleviate DD. Furthermore, in another study to determine whether the level of SNO-Prx2 in the brains of Patients with PD has pathophysiological significance, the researchers found that the ratio of SNO-Prx2 to total Prx2 was comparable to the ratio of cell death in PD cell-based Models (103). The results indicated that there were pathophysiological-related amounts of SNO-Prx2 in the human brain of patients with PD and the production of SNO-Prx2, interfering with the normal antioxidant system, might lead to neuronal cell death. Conversely, eight studies have shown that S-nitrosylation modification of target proteins has an inhibitory effect on the occurrence of diseases. For instance, Microcystins-LR induces apoptosis of human colon cancer cells (SW480) through the SNO-GAPDH-Siah1 signaling pathway (with significant differences between the treatment group and the control group) (104). This study provides further understanding of the in vitro molecular mechanism of Microcystins-LR-induced colorectal toxicity. Finally, it is worth noting that in metabolic system diseases, all six studies revealed that S-nitrosylation modification of target proteins can aggravate insulin resistance. Therefore, targeted inhibition of S-nitrosylated proteins may be an effective strategy for treating metabolic system diseases.