The evolution of S-nitrosylation detection methodology and the role of protein S-nitrosylation in various cancers

Abstract

S-nitrosylation (SNO) modification, a nitric oxide (NO)-mediated post-translational modification (PTM) of proteins, plays an important role in protein microstructure, degradation, activity, and stability. Due to the presence of reducing agents, the SNO modification process mediated by NO derivatives is often reversible and unstable. This reversible transformation between SNO modification and denitrification often influences the structure, activity, and function of proteins. The reversibility of SNO modifications also poses a challenge when verifying changes in the biological functions of proteins. Moreover, SNO modification of key signaling pathway proteins, such as caspase-3, NF-κB, and Bcl-2, can affect tumor proliferation, invasion, and apoptosis. The SNO-modified proteins play important roles in both promoting and inhibiting cancer, which indirectly confirms the duality and complexity of SNO modification functions. This article reviews the biological significance of various SNO-modified proteins in different cancers, providing a theoretical basis for determining whether the related changes of SNO-modified proteins are universal in cancers. Additionally, this review presents a comprehensive and detailed summary of the evolution of detection methods for SNO-modified proteins, providing a possible methodological basis for future research on SNO-modified proteins.

Introduction

Cancer places a major burden on global health and the economy [1, 2]. The incidence rates of various cancers, including colorectal cancer, lung cancer, and breast cancer, remain high worldwide [2]. However, the cancer-related mortality rate in high-income countries is significantly lower than that in other countries [2]. The occurrence, progression, and metastasis of cancer involve multiple factors, and the molecular mechanisms are among the most complex of diseases [3]. Several factors can contribute to the progression of cancer, such as exposure to the risk factors, abnormal expression and degradation of oncoproteins and tumor suppressor proteins, abnormal DNA damage repair, and changes in metabolic enzyme activity [3,4,5]. The implementation of the global genome project has provided first-hand information for tackling cancer [6, 7]. Additionally, the establishment and improvement of databases such as Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) have laid the foundation for the advancement of omics technology [8]. The significance of changes in epidemiology, genome, transcriptome, and proteomics in cancer is gradually being discovered and recognized by the academic community [7, 9]. The main characteristic of cancer is the uncontrolled proliferation of cells caused by abnormal genetic changes, including abnormal signaling pathways regulated by gene mutations [10, 11]. The malignant transformations of many oncogenes promote cell division and differentiation, ultimately leading to cancer [12].

The malignant proliferation and differentiation of cancer cells are closely associated with SNO-modified proteins, such as p53 and PTEN [13,14,15]. Changes in protein conformation and function can alter specific signaling pathways. This has given rise to a new field of research focusing on PTMs of protein [13]. PTMs generally involve the covalent binding of chemical groups, peptide chains, amino acids, and complex molecules to proteins [16]. PTMs can alter the conformational function and sub-localization of proteins, thereby affecting the fate of tumor cells [16]. Different PTMs generally occur on different amino acids; for example, succinylation often involves binding to lysine, while SNO often co-occurs with cysteine (Cys) [17]. Recent research has demonstrated that PTMs can alter protein structure to regulate downstream gene binding or mutation, impacting cancer progression. Additionally, PTMs play vital roles in DNA mismatch repair, metabolic pathways, and immune responses in tumor cells [18]. Moreover, there is also a crosstalk between PTMs, which can further influence the outcomes in tumor cells [18].

There are various types of cancer, involving many human organs, and research on SNO-modified proteins has progressed at different rate depending on the cancer types. Furthermore, alterations in SNO-modified proteins vary across different cancers. Therefore, it is essential not only to explore common changes in SNO-modified proteins across cancers, but also to analyze and summarize these changes based on specific cancer types. This article summarizes the preparation and detection methods for SNO-modified proteins and highlights the limitations of current SNO research methods. Additionally, this review discusses the biological significance of SNO-modified proteins in different cancer fields, providing new ideas for the diagnosis and treatment of related cancers.

Definition and formation principle of SNO modification

Common PTMs of chemical groups involve phosphorylation, acetylation, SNO, ubiquitination, and proteolytic cleavage [19,20,21]. SNO modification is a special type of PTMs based on NO derivatives. SNO involves the covalent binding of a NO⁺ group to the thiol group of Cys and protein or peptide segments, forming an S-nitroso thiol bond [22,23,24]. There are four primary mechanisms by which SNO modifications can occur: (1) SNO modification mainly occurs via a reaction between deprotonated Cys residues and NO derivatives. In the presence of sufficient transition metal ions and oxygen, NO mainly forms two types of derivatives: nitroso ions (NO⁺) and peroxynitrite (OONO⁻) ions [24,25,26]. The OONO⁻ is the main reactive nitrogen species, and is primarily generated from a reaction between NO radicals and superoxide anion (O₂⁻) [26]. The NO radicals are generated during the conversion of l-arginine to l-citrulline, while O₂⁻ is generated when oxygen molecules accept electrons during the oxidation of NADPH (Fig. 1) [26]. The formation of OONO⁻ is heavily dependent on the catalytic activity of nitric oxide synthase [25]. Other NO derivatives, such as nitrogen trioxide (N₂O₃), reactive nitrogen species, and nitrogen dioxide, participate in the formation of nitrosothiol bonds to a lesser extent [27,28,29,30]; (2) NO directly binds to the active thiol groups in proteins, transforming them into nitroso thiol groups and reducing the activity of those sites [31]; (3) The direct exchange of NO between nitroso mercaptan and sulfate for nitroso conversion via an oxidation–reduction independent method [28, 32]; (4) S-nitrosoglutathione reductase (GSNOR) promotes the removal of SNO residues from target proteins. Conversely, GSNOR deficiency can lead to an increase in SNO modification [33, 34]. Given the variety of pathways for SNO-modified protein formation, numerous factors influence the specific formation of SNO modifications [35]. These factors include the physical and chemical properties of NO donors, the subcellular localization and acid–base environment of Cys residues, the expression levels of nitrosotransferase and nitrosoreductase, and the redox state of cells or tissues [36]. Not all Cys residues are modified by SNO due to limiting factors, such as sub-localization of Cys thiol groups, acid–base environment, metal ion reactions, and spatial exposure [37,38,39]. Acidic amino acid residues are widely present in SNO-modified peptide motifs, while alkaline amino acid residues have a relatively small impact on SNO modification. Peptide segments based on the E-X-C (glutamic acid-other amino acid-Cys) sequence are more prone to SNO modification [40]. Cys residues with acid–base motifs, low pKa, and high sulfur atom exposure space, are more prone to SNO modification and SNO modification also affects protein stability and function [41]. Endogenous SNO modification often occurs in protein α-helices, whereas NO donor-mediated SNO modification often occurs in protein β-sheets [42]. In addition to the previously mentioned factors, the efficiency of protein SNO modification is also influenced by the concentration of intracellular reactive oxygen species and NO radicals [26]. The intracellular concentration of NO varies from 0.1 nM to 5 mM, with significant variations depending on subcellular localization and time [26]. Within the concentration range of 100–500 nM, NO can participate in the reversible oxidation of thiol groups, and this concentration range is considered sufficient to initiate reversible SNO modifications [26]. Concentrations of NO exceeding 500 nM lead to irreversible SNO modifications, which are often associated with pathological states such as cancer and inflammation [26]. Additionally, the second-order rate constant for the formation of ONOO⁻ (1.7 × 10¹⁰ M⁻¹ s⁻¹) is significantly higher than that for the dismutation of oxygen molecules (5.0 × 10⁵ M⁻¹ s⁻¹) [26]. Therefore, under conditions of high NO and oxygen concentrations, the efficiency of SNO modification can be significantly increased. Various NO donor drugs such as S-nitrosoglyceathione (GSNO), S-nitroso-N-acetylpenicillamine, and sodium nitroprusside (SNP) have been used to generate large amounts of NO radicals and reactive oxygen species in a short period [43]. Furthermore, the modification of Cys thiol groups by SNO can cause conformational changes in proteins, thereby affecting protein–protein interactions, protein–DNA binding, and signal coupling [44,45,46]. Additionally, SNO modification requires NO derivatives to lose one electron and then covalently bind with a Cys thiol group [47]. As a result, SNO modification is often considered a reversible reaction. However, this process is an irreversible reaction in proteins containing metal ions. Notably, SNO modification can lead to the significant decrease in the binding force between the protein and the metal ions [31]; the release of large amounts of metal ions; and the inactivation of proteins [31]. These limiting factors make it difficult to study the function of SNO modification at the cellular and tissue levels.

Fig. 1

The main mechanism diagram for the formation of SNO modification

Nitrosothiols are well-known for their ability to undergo chemical reactions with various reducing agents, such as ascorbic acid, glutathione (GSH), and transition metal ions [48]. Due to the reversibility and instability of SNO residues, their half-life ranges from just a few seconds to a few minutes [48]. This reversible nature of SNO bonds has led researchers to recognize SNO as a potential PTM. The reversibility of SNO allows nitroso residues to be reduced back to thiol groups through a process called denitrification [49]. Free thiols in GSH not only scavenge intracellular reactive oxygen species but also serve as a storage reservoir for NO [50]. GSH reaches a stable state by binding to intracellular NO to form GSNO, making GSNO an indirect indicator for evaluating intracellular NO [50]. When endogenous cellular GSH (5–10 mM) is present, SNO-modified protein levels rapidly decrease. However, when GSH is depleted, SNO-modified proteins become more stable, leading to elevated levels of SNO modifications [51]. GSH also receives NO from SNO-modified proteins, yielding GSNO, which is then metabolized by GSNOR. Due to this dual mechanism, even a small amount of GSH is sufficient to maintain thiol groups in their reduced state [51, 52]. Although GSH can denitrify most SNO-modified proteins, some stable SNO proteins, such as SNO-caspase-3, can only be denitrified by the thioredoxin (Trx) system [52]. The Trx system has been identified as a key catalyst for SNO denitrification [53, 54]. The current consensus is that Trx-SNO releases HNO/NO to form thioredoxin disulfide (TrxS2). The conversion of Trx-SNO to the disulfide state is widely observed in both nitrosylation and denitrification processes. The disulfide form, TrxS2, is subsequently reduced back to Trx-(SH)2 through the acceptance of electrons from NADPH [52]. Within the Trx system, Trx1 and Trx2 are the most crucial factors for achieving effective denitrification. Like GSH, Trx1 is a universal regulator of reversible SNO modifications. It uses different domains for transnitrosation (involving SNO-Cys73) and denitrification (involving Cys32 and Cys35) [55, 56]. Peroxiredoxin 1 and cyclophilin A are targets for Trx1 transnitrosation, although not all SNO-Trx1 target cysteines can be denitrified [55]. Trx1 may promote SNO modifications through mechanisms involving inactive thiols Cys69 or Cys73, which are absent in Trx2. Inhibition of Trx1-TrxR1 or Trx2-TrxR2 results in enhanced SNO-modified protein levels, indicating that the primary function of the Trx system is protein denitrification [56]. However, the Trx system can completely denitrify the stable SNO-modified protein population in the presence of GSH [57]. Therefore, by first using the Trx system and then sequentially treating SNO-modified proteins with GSH, complete denitrification of S-nitrosothiol can be achieved, suggesting that the substrate selectivity of GSH and Trx systems for SNO-modified proteins is different [57].

Only about 2% of Cys thiol groups are present in proteins of humans and rodents, as identified by the post-genome project [37, 58]. Although SNO modification is less common compared to other modifications such as phosphorylation [59], it still widely occurs in various proteins, including microtubules, apoptotic proteins, metabolic proteins, redox proteins, vesicles, enzymes, and signal pathway proteins; and induces widespread changes in cellular function [60,61,62]. SNO modification of the Cys residue in enzymes may inhibit the efficiency of enzyme–substrate binding, thereby inhibiting enzymes activity [45]; for example, the activation of tyrosine kinase and T cell activation in immune response [63]. However, in pathological states; such as cancer, cardiovascular injury, and neurological diseases; abnormal SNO modifications regulate cell growth and metabolic pathways, leading to abnormal cellular outcomes [64]. Due to various complex factors—such as the instability of SNO residues, denitrification, variations in microenvironmental pH, and the chemical reaction kinetics of SNO modifications—it remains highly challenging to study SNO modifications comprehensively. Currently, the primary approach involves investigating the function of individual proteins after purification [47].

Evolution of preparation and detection methods of SNO-modified proteins

SNO bonds can be easily converted into thiol bonds by UV light or reducing agents, which presents a significant challenge for directly identifying SNO modification. Despite the advancements in biophysical technology and physical methods for characterizing SNO active sites, including X-ray and nuclear magnetic resonance, the biotin switch technique (BST) remains the primary method for indirectly identifying SNO-modified proteins [65]. For Cys residues to react with NO and form SNO bonds, they must typically be exposed on the protein surface. Therefore, the purification and enrichment of endogenous SNO-modified proteins pose significant challenges, thereby motivating scientists to develop improved methods [66].

At present, there are five main methods for detecting SNO-modified proteins. The first and common method is to use BST to specifically label, enrich, and purify the SNO bonds in proteins, and then use western blot to identify the SNO modification [67]. The second method involves using a mass spectrometer to identify the specific sites of SNO modification and enhances detection sensitivity by detecting peptide segments [67, 68]. The third method, known as gel fluorescence, employs fluorescent labels that bind to specific groups, such as NO radicals or thiol groups, to enable the selective recognition of SNO-modified proteins [67, 69]. The fourth method utilizes the instability of SNO bonds to release NO, and then indirectly recognizes SNO-modified proteins by detecting the formed NO derivatives through chemical colorimetry [67]. Unfortunately, gel fluorescence and chemical colorimetry methods have been rarely used in recent years. The fifth method directly uses physical techniques or computer prediction models to detect SNO bonds. Accurate identification is challenging due to the low abundance, reversibility, and instability of SNO modifications [67].

BST

Due to the instability of protein SNO groups and the possibility of transnitrosylation and denitrification between protein molecules, it is difficult to detect protein SNO bonds directly. These factors have driven the development and widespread application of BST [22, 28]. The BST process, which was invented by Dr. Snyder [48], can be roughly divided into three steps. The first step involves the use of specific blocking agents (e.g., MMTS and NEM) to comprehensively and stably block the free thiol groups in proteins, causing them to lose their ability to bind with other molecules. The second step involves the use of ascorbic acid to specifically reduce the Cys SNO bond to a thiol bond. The reduction efficiency of ascorbic acid is a critical factor in this step. The third step involves the use of biotin compounds such as HPDP to bind with newly generated Cys thiol bonds, enabling the specific recognition of SNO-modified proteins [22, 33, 48, 70, 71]. The practice of BST has led to the identification of numerous PTMs of proteins, often resulting in false positive and false negative results at SNO sites. These unreliable identification results significantly impact the exploration of the function and biological significance of SNO-modified proteins. To enhance the accuracy of SNO modification identification, many studies have optimized BST by adjusting the concentration and incubation time of MMTS sealants and ascorbic acid [72,73,74,75]. Chao et al. used the BST method and agarose beads to enrich and purify SNO-GNAI2, and proved that reducing the SNO of GNAI2 could effectively alleviate atherosclerosis caused by diabetes [73]. Similar methods have also been applied to detect CSF1R [74], FAK1 [75], VCAM-1 [76], p53 [77], DJ-I [78] and DNMT3B [79]. However, the limitations of BST method still need to be addressed.

Despite adjustments in the reaction time and dose of N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamide (HPDP), issues related to low efficiency and high false-positive rates in the BST method remain unresolved. To enhance the recognition and identification efficiency of SNO-modified proteins, Chen et al. improved the BST method in two ways. First, MMTS was replaced with iodoacetamide, avoiding the Cys residue-blocking failure caused by the high-temperature instability of MMTS reagents [80]. Second, ascorbic acid’s reduction efficiency is unstable because of oxidation in light and air, S-alkylation reagents were used to irreversibly and specifically bind SNO bonds, form S-alkylated biotin proteins, and enhance the recognition efficiency of SNO-modified proteins [80]. Due to the enrichment of detergents such as SDS, peptide signals can be suppressed in mass spectrometry analysis. This phenomenon can decrease the recognition rate and reproducibility of SNO sites. Han and Chen used urea instead of SDS to fully open the three-dimensional structure of the protein, enhancing MMTS’s blocking effect on deep SNO sites [81]. Comparing the effects of experimental schemes with or without SDS on the detection of SNO-modified proteins in HeLa cells, high concentrations of urea significantly improved the performance of mass spectrometry analysis [81]. Martinez-Ruiz and Lamas developed a method for the qualitative detection of SNO-modified proteins using anti-biotin antibodies. By setting up a control group without HPDP, they confirmed the blocking effect of MMTS on free thiol bonds [47]. While these methods improved detection efficiency, they also introduced drawbacks such as complex experimental steps and sample waste [47]. Wang et al. discovered the significance of copper and cuprous ion cycling in the SNO bonds of ascorbic acid-reducing proteins [82]. Cuprous ions decomposed SNO bonds to produce free thiol bonds and copper ions, while ascorbic acid reduced copper ions, completing the metal ion cycle and accelerating SNO bond reduction [82]. In addition, Hao et al. added trypsin after traditional BST labeling to hydrolyze SNO-modified proteins into peptide segments [83]. Because protein structure was much more complex than peptide segments, the process of protein enrichment was likely to cause intermolecular disulfide bond cleavage, leading to incomplete enrichment [83]. Enriching peptide segments could enhance the probability of contact between SNO active sites and agarose beads, thereby enhancing enrichment efficiency [83]. However, the method failed to clarify the primary sequence characteristics of SNO-modified proteins and could not detect specific protein changes by using Western blot [83]. Another strategy for enhancing specific binding with reduced SNO-Cys is to irreversibly bind peptides containing His tags to newly generated thiol bonds [84]. Camerini et al. believed that His labeling simplified the purification process of SNO-modified proteins and identified complex biological samples. The irreversible binding mode greatly reduced the accidental shedding of proteins during the enrichment process [84].

The detection of SNO proteins under physiological conditions is challenging due to the instability, short lifespan, and low abundance of SNO modifications. Benhar utilized the inherent mechanism of Trx in reducing SNO-modified proteins to develop a method for labeling purified recombinant Trx mutants carrying streptavidin tags with SNO-modified proteins [85]. This method was highly sensitive and identified many low-abundance SNO-modified proteins for the first time. However, it also had limitations, including (1) some proteins might not bind to Trx; (2) Trx might react with other oxidative proteins to form disulfides, reducing the specificity of this method [85]. In addition, Ben-Lulu et al. detected the effects of NO donors (S-nitroso-Cys) and pro-inflammatory factors on the SNO-modified protein by using a Trx capture method [86]. After cross comparison with publicly available databases, approximately 150 proteins and 300 possible SNO active sites were successfully identified [86]. Thus, the method of Trx capturing nitroso thiols had strong accuracy in identifying SNO-modified proteins. Despite purification and enrichment via the BST, SNO-modified proteins may still form intermolecular disulfide bonds with other proteins. The proteins, which covalently bond with SNO-modified proteins, are likely to be identified as target proteins for SNO in subsequent mass spectrometry or western blot, leading to false positive results. To address this issue, Huang and Chen developed an irreversible biotinylation program (IBP) based on non-disulfide biotinylation reagents [87]. They converted the reversible biotinylation process of HPDP into irreversible biotinylation by adding DTT to break all disulfide bonds between molecules before enrichment in agarose beads [87]. This method solved the concern of interference of intermolecular disulfide bonds on SNO-modified protein identification [87]. Similarly, Yi et al. [88] and Gao et al. [89] used the IBP method to successfully detect SNO-modified proteins in hematopoietic stem cells and cancers. In addition, Chen et al. also tackled the issue of disulfide bond cleavage during mass spectrometry identification of SNO-modified proteins by covalently binding SNO-Cys and using strong reducing agents like DTT and TCEP to denature and thoroughly digest the protein [42]. This approach improved the purification efficiency of biotin labeling and the sensitivity of mass spectrometry analysis [42].

To further enhance the extraction efficiency of SNO protein, scientists have not only optimized the labeling step in the BST method but also gradually applied novel enrichment methods such as phenylmercury resin and solid-phase resin. Doulias et al. replaced ascorbic acid and biotin HPDP with phenylmercury resin which specifically bond to Cys SNO bonds without affecting other protein modifications such as sulfite and S-alkylation [90]. Therefore, phenylmercury resin could directly covalently bind with protein SNO to complete the enrichment process. This method had lower cost, as well as higher efficiency and specificity compared to traditional BST methods [90]. Additionally, gold nanoparticles, which easily bond to thiol bonds, were suggested as an alternative to biotin HPDP for enriching SNO-modified proteins [91]. Although this method partially improved the sensitivity of enriching SNO-modified proteins, free thiol bonds still interfered with the enrichment process of gold nanoparticles toward SNO-modified proteins [91]. Ibanez-Vea et al. developed a Cys-phosphonate labeling (CysPAT) method to label reduced SNO bonds instead of using biotin HPDP [92]. This method involved titanium dioxide chromatography instead of agarose beads and showed higher accuracy and lower missed detection rates for SNO-modified proteins in mouse macrophages [92]. Gu and Robinson introduced oxidative Cys selective cPILOT (OxcyscPILOT) to detect SNO-modified protein levels. This method involved blocking free thiol bonds, enriching SNO sites with solid-phase resin, and purifying peptide segments with isotope or isobaric labeling [93]. OxcyscPILOT improved detection efficiency and allowed simultaneous measurement of SNO-modified protein and total protein levels across multiple samples [93]. For further advancements, Guo et al. proposed a resin-assisted enrichment and reduction method for SNO-modified proteins [94]. This method directly combined the free thiol bonds generated by ascorbic acid reduction with the resin for enrichment and included isobaric labeling for quantitative analysis. This approach reduced endogenous biotin interference and simplified the process compared to traditional BST, though it did not fully resolve the issue of false positives from free thiol bonds [94]. Forrester et al. enhanced the BST preparation method by using SNO-resin-assisted capture (SNO-RAC) and demonstrated high sensitivity in identifying SNO-modified proteins [95]. SNO-RAC required simpler experimental steps and resulted in higher detection efficiency with less protein loss in subsequent mass spectrometry [70, 95]. Shin et al. used the SNO-RAC method to demonstrate that blocking SNO-GAPDH could help treat traumatic brain injury [96]. Thompson et al. further improved SNO-RAC by using thiopropyl sepharose (TPS) instead of biotin HPDP for protein hydrolysis on the resin. This simplification obviously improved the accuracy of SNO site recognition and identification, particularly when SNO site combined with various quantitative mass spectrometry methods, such as stable isotope labeling by amino acids in cell culture (SILAC), tandem mass tags (TMT), and label-free quantification [97].

Although the BST method has undergone numerous upgrades and changes, it still faces limitations such as cumbersome steps, sample loss, and a high incidence of false positives. Additionally, the specificity and sensitivity for detecting SNO-modified proteins require further enhancement. These issues present considerable challenges for the precise identification and isolation of individual SNO-modified proteins.

Mass spectrometry

Mass spectrometry is pivotal in analyzing SNO-modified proteins and active sites. Despite its importance, the pretreatment process for mass spectrometry is complex, and endogenous metabolites such as biotin compounds and oxidized Cys can affect the identification results [98]. Therefore, it is particularly important to develop more accurate detection methods for SNO modifications to overcome current limitations. Derakhshan et al. proposed a new method for identifying SNO-modified proteins based on mass spectrometry analysis, called SNO Cys site identification (SNOSID) [98]. After denaturation and elution with DTT, the SNO-modified protein was identified using nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) [98]. However, this method also faced some challenges, such as the accuracy of mass spectrometry instruments, potential recognition errors from low-abundance peptide segments, the requirement for large protein samples, and potential biases in results due to the differential reactivity of SNO sites to ascorbic acid reduction [98].

With advancements in mass spectrometry, quantitative analysis using gel mass spectrometry is valuable in identifying SNO-modified proteins. Common labeling methods include isotope labeling, such as isotope-coded affinity tag (ICAT) and SILAC, and isobaric labeling like iTRAQ and TMT [99]. Zhang et al. enriched SNO peptides by combining fluorescent solid-phase resin with iTRAQ labeling (FluoroTRAQ) and successfully identified dynamic changes in SNO-modified proteins induced by GSNO stimulation [68]. This FluoroTRAQ approach enriched the database and provided new references for quantitative analysis of SNO modifications. FluoroTRAQ also improved the ability to detect low-abundance SNO-modified proteins and effectively reduced the interference of free thiol bonds [68]. Qu et al. utilized iodoTMT switch assay (ISA) to bind specifically and irreversibly with SNO residual and Cys, enhancing the stability of the Cys SNO bond. SNO peptide segments labeled with iodoTMT were enriched using anti-TMT resin and then identified and quantitatively detected for SNO modification sites via liquid chromatography-tandem mass spectrometry [100]. ISA could identify SNO active sites induced by lipopolysaccharide oxidative stress in mice, demonstrating high sensitivity due to its unique irreversible binding [100]. Chung et al. utilized cyclic reactive pyridyldithiol and iodoacetyl-based tan mass tags for a parallel dual labeling strategy. Mass spectrometry analysis showed that SNO active sites below 30% could be recognized by both above markers [101]. Different labeling reagents exhibited different affinities with protein SNO active sites, which explained the significant differences in the types and sites of SNO-modified proteins detected across studies. This parallel strategy maximized the detection of all SNO active sites and enhanced sensitivity [101]. Compared to gel separation methods, the isotope labeling technology could dynamically monitor SNO-modified protein and more accurately quantify SNO-modified protein levels [42]. SILAC, by detecting the content of labeled amino acids absorbed and utilized by cells, could infer the type and relative quantification of SNO-modified proteins, thus avoiding bias from chemical labeling [42]. The types and subcellular localizations of SNO-modified proteins were identified by adding pollution-free and non-toxic SILAC amino acids to the culture medium. Meanwhile, Ong and Mann showed that the SILAC labeling method did not affect the normal physiological function and proliferation rate of cells, and its inherent labeling could be recognized and screened by mass spectrometry. The signal intensity was directly related to protein abundance [102].

Other methods

The indirect labeling method has several disadvantages, including being error-prone, involving complex procedures, and leading to high sample loss. In contrast, the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-based spin-trapping method effectively addresses these issues [103]. Sircar et al. used DMPO as a spin capture agent to combine thiol radicals generated by SNO protein after photolysis with nitrones to form DMPO nitroketone adducts [57]. These adducts are highly stable and can be directly labeled and identified using anti-DMPO antibodies [57, 103]. In addition, Sircar et al. used this method to demonstrate that GSH, as a denitrification agent, participated in the SNO modification of glutaraldehyde-reducing protein 1 and the R1 subunit of ribonucleic acid-reducing enzyme in liver cancer cells [57]. Compared to BST, the DMPO-based spin-trapping method is more convenient and efficient. Moreover, because it does not rely on reducing agents, the DMPO-based spin-trapping method offers better reproducibility [103].

Advancements in computer technology have introduced new approaches to exploring the structure and function of SNO-modified proteins. Chen et al. used X-ray crystallography to identify the specific structure of the active site in PTP1B crystals modified by SNO. This method revealed the mixed form of sulfate anion and nitroso mercaptan, confirming six Cys sites detected by nano spray electrospray ionization mass spectrometry (nESI-MS) [104]. The integration of these technologies provided a detailed view of the three-dimensional structure of SNO sites, although the high cost of the experimental instruments was a notable limitation [104]. Wang et al. used two-dimensional microelectrophoresis and microfluidic technology to separate SNO-modified proteins and developed a biomarker chip for rapid detection of SNO modification [105]. This innovation marked the first successful application of microfluidic technology to the separation and identification of complex biological samples, producing aging-related SNO-modified protein maps in mouse brains. This foundational work had potential applications in developing biomarker chips for widespread oxidative stress-induced SNO-modified proteins [105].

The post-genomic era has provided extensive protein sequence data, fostering the development of computational models for predicting SNO sites. Xu et al. developed a novel iSNO-AAPair prediction model by using all possible coupling effects between residues on the protein chain and the next residue [106]. The prediction results of the iSNO-AAPair model were compared with a validated SNO-modified protein dataset, demonstrating that the model achieved high identification accuracy. However, the model also had limitations. It only considered the independent positions of amino acids, without accounting for the correlations between them [106]. In reality, the arrangement of amino acid positions had inherent connections. Therefore, it could potentially enhance the accuracy of predicting SNO active sites by including the internal correlation effects between amino acids [106]. Li et al. designed a model combining incremental feature selection with maximum correlation and minimum redundancy [107]. This model used physicochemical properties, sequence conservation, and amino acid frequency to train and recognize SNO modifications. It enhanced detection accuracy, reduced the time and cost of traditional sample preparation, and provided valuable data for subsequent biological experiments [107]. Xu et al. also developed an iSNO-PseAAC predictor using hundreds of Cys SNO active sites and nearly a thousand non-Cys SNO active sites as the training set [108]. The success rate of SNO-modified protein validation through independent dataset recognition was as high as 90%, demonstrating the potential of prediction models in the identification of SNO-modified sites [108].

After decades of development, the purification and detection methods for SNO-modified proteins have gradually improved, but several significant challenges need to be addressed. The commonly used methods for detecting SNO modifications are listed in Table 1. Firstly, although SNO-modified protein detection methods are continuously evolving, most studies have been based on exogenous NO donors or anti-nitrosylation drugs to induce exogenous SNO modification. The active sites identified under these conditions are mostly hypothetical or potential SNO sites, rather than actual SNO sites present in vivo [109]. Secondly, some SNO detection methods can only qualitatively detect sites but cannot determine the functional active sites. The precise determination of functional sites can provide a solid foundation for the exploration of the biological behavior of SNO-modified proteins in the future [109]. Thirdly, negative controls and identification errors are crucial in omics identification methods, and peptides screened and identified by mass spectrometry may only report the highest abundance and most stable small portion of all proteins or peptides due to instrument accuracy issues [109]. Fourthly, the complex sample preparation process and low repeatability, as well as the high experimental cost, have also led to slow progress in the study of SNO-modified protein function.

Table 1 Summary of advantages and disadvantages of commonly used methods for identifying SNO-modified proteins

Research progress on SNO-modified proteins in cancer

SNO modification plays an important role in protein stability and catalytic activity, and it significantly impacts the survival and death of tumor cells [110, 111]. The functions of SNO-modified proteins in various cancers are listed in Table 2. The SNO modification of signaling pathway proteins can determine the outcome of various cancers, such as lung cancer, colorectal cancer, breast cancer, liver cancer, and ovarian cancer [112,113,114]. In addition, SNO modifications of different proteins have fundamentally different effects on cancer cells, implying that a general increase or decrease in SNO modifications cannot completely determine the outcome of all cancers [113]. Detailed analysis is needed to understand the impact of a single SNO-modified protein on specific cancer types.

Table 2 List of the active sites in SNO modification proteins and the function of SNO proteins in different cancers

Lung cancer

SNO-modified proteins that control apoptotic signaling pathways, such as Bcl-2 and FLICE inhibitory proteins (FLIp), play crucial roles in lung cancer. These SNO-modified proteins influence tumor cell behaviors, including apoptosis, anti-apoptosis, autophagy, and adhesion. Azad et al. found that two Cys residues (Cys158 and Cys229) of Bcl-2 could be stimulated by various exogenous NO donors to promote Bcl-2 SNO modification [115]. SNO modification prevented Bcl-2 from being ubiquitinated and degraded by proteases, enhancing the anti-apoptotic ability of tumor cells. NO scavengers were used to inhibit Bcl-2 SNO modification, resulting in the promotion of its ubiquitination and degradation, thus inhibiting tumor progression [115]. At the same time, Chanvorachote et al. found that SNO modification of Bcl-2 had an anti-apoptotic effect on cisplatin resistance in lung cancer cells. Notably, cisplatin enhanced the degradation and reduced the expression level of Bcl-2 by enhancing the level of reactive oxygen species [116]. However, NO could promote SNO modification of Bcl-2 by reacting with reactive oxygen species, inhibit the degradation of Bcl-2, and increase tumor resistance to cisplatin [116]. Wright et al. compared the relationship between SNO modification and autophagy levels in lung epithelial cells and cancer cells. They found that SNO modification of Bcl-2 enhanced the protein stability of Bcl-2 and promoted the formation of Bcl-2-Beclin-1 polymer, thereby inhibiting autophagy in malignant tumors and ultimately promoting the survival of malignant cells [117]. FLIp can inhibit the death signal of Fas ligands, and reactive oxygen species play important roles in Fas ligand-induced downregulation of FLIp levels and activation of tumor cell apoptosis [118]. Wang et al. found that reactive oxygen species scavengers significantly inhibited the expression level of FLIp, while NO enhanced the SNO modification of FLIp and enhanced protein stability by reacting with reactive oxygen species. Subsequently, upregulation of FLIp expression reduced the death signal transduction of the Fas ligand and promoted tumor cell survival [118].

SNO modification significantly influences the metastatic ability of lung cancer cells by altering the expression levels of crucial proteins. SNO modification can enhance metastasis by increasing the expression of proteins like Caveolin-1 (Cav-1) and ezrin and decreasing the expression of Vascular Endothelial Growth Factor D (VEGFD). Chanvorachote et al. investigated the interaction between NO and Cav-1 in lung cancer. They found that SNO modification of Cav-1 could inhibit protein ubiquitination and degradation efficiency [119]. They also found that SNO modification might help maintain high levels of expression of Cav-1 in lung cancer and promote the improvement of tumor cell metastasis ability [119]. In addition, Zhang et al. analyzed clinical data from lung cancer patients and found a significant correlation between the SNO level of ezrin and the metastasis abilities of cancer cells [120]. SNO inhibitors were shown to reduce ezrin tension, thereby inhibiting tumor cell invasion and metastasis. Zhang et al. identified Cys117 as the sole active site for SNO modification, which could be targeted for precise regulation of malignant behavior in tumor cells [120]. In another study, Zhang et al. discovered that baicalin, a flavonoid, could particularly reduce NO levels, inhibit the SNO modification of ezrin, and decrease ezrin tension in inflammatory environments [121]. This reduction in tension was closely related to the decreased invasive and metastatic abilities of non-small cell lung cancer cells [121]. In addition, He et al. found that low levels of VEGFD expression were closely associated with the malignancy of lung adenocarcinoma [122]. Excessive NO promoted SNO modification at the Cys277 site of VEGFD, leading to reducing VEGFD expression and promoting lung adenocarcinoma progression and angiogenesis. GSNOR could counteract this effect by promoting the denitrosylation of VEGFD, thus reducing lung cancer occurrence and transformation [122].

SNO modification not only has a protective effect on lung cancer cells but also promotes cancer cell apoptosis. SNO modification also affects the spatial conformation of proteins, such as peroxiredoxin-2 (PRDX2) and STAT3; and disrupts their function, leading to cell death. Zhang et al. applied the NO donor GSNO to inhibit lung cancer and found that the SNO level of PRDX2 was significantly increased [123]. The SNO modification at Cys51 and Cys172 sites disrupted the antioxidant function of PRDX2, leading to the accumulation of reactive oxygen species in tumor cells and ultimately activating the AMPK-SIRT1 signaling pathway, thus promoting tumor cell apoptosis [123]. Cao et al. demonstrated that NO donors significantly increased the concentration of NO in mitochondria and enhanced the SNO modification of STAT3. SNO modification competitively inhibited the phosphorylation of STAT3 protein, thereby inducing cell apoptosis [124]. In addition, SNO modifications of p53 and histone deacetylase 6 protein (HDAC6) also played an important role in cancer suppression [77, 125]. Above all, SNO modification can play a dual role in promoting and inhibiting cancer in lung cancer, depending on whether SNO modification occurs in oncogenic or inhibitory proteins.

Colorectal cancer

Research into the role of SNO modification in colon cancer reveals significant effects on tumor cell proliferation, despite the relatively low incidence of SNO modifications. Key signaling pathway proteins such as NF-κB, caspase-3, Fas, and Cav-1 are significantly impacted by SNO modifications, which often inhibit tumor growth and promote apoptosis. Williams et al. investigated colorectal cancer cells treated with NO donors (specifically, NO nonsteroidal anti-inflammatory drugs) and identified potential SNO-modified proteins such as p53, β-catenin, and NF-κB [126]. Both p65 (Cys38) and p50 (Cys62) subunits of NF-κB were found to be possible SNO modification sites. NO donors likely regulated the key signaling proteins’ functions and stability via SNO modification, thereby inhibiting tumor growth and promoting apoptosis [126]. Kashfi found that the cytotoxic effects of NO donors were both time-dependent and concentration-dependent. SNO modifications of NF-κB, p65, caspase-3, and β-catenin also correlated with the duration and concentration of NO donors [127]. Interestingly, the activity of caspase-3, which was typically negatively correlated with SNO modification, increased with SNO modification in this study. The inconsistent findings may be because the SNO modification ratios were different across the studies [127]. Low SNO modification ratios might not significantly impact caspase-3 function, or the SNO modification groups of caspase-3 could transfer to other anti-apoptotic proteins, reducing caspase-3 stability and promoting apoptosis [127]. These research findings provided new ideas for enriching treatment strategies in colorectal cancer. In addition, Leon-Bollotte et al. found that the Cys199 and Cys304 sites in Fas could be modified by SNO under the action of NO donors [128]. Replacing Cys with valine at these sites revealed that Cys304 mutation inhibited the transfer of Fas to lipid rafts, thereby blocking the function of death-related complexes and ultimately inhibiting apoptosis. This effect was not observed in the Cys199 mutant cells, highlighting the critical role of Cys304 in inducing apoptosis in colorectal cancer [128]. In addition, SNO modifications of cIAP1, tumor necrosis factor receptor-associated protein 1 (TRAP1), GAPDH, p65, β-catenin, and caspase-3 also play important roles in cancer suppression [129,130,131,132]. Although there may be multiple SNO sites within the same protein, not all SNO modifications at each site can affect protein function, and the rate of SNO modification can also affect the balance of cellular function.

SNO modification demonstrates a dual role in colon cancer, both inhibiting and promoting cancer development. This duality complicates the understanding of SNO modification’s overall impact on tumor biology. Key signaling pathway proteins such as focal adhesion kinase-1 (FAK1), DNA methyltransferase 3B (DNMT3B), latent TGF-β binding proteins 1 (LTBP1), and hypoxia-inducible factor 1-alpha (HIF1α) are affected by SNO modification, influencing protein activity and ultimately disrupting cell viability. Rizza et al. found that the lack of GSNOR increased the phosphorylation levels of FAK1 protein through SNO modification at Cys459 and Cys658 sites [75]. GSNOR-deficient cancer cells showed significant tumor inhibition and pro-apoptotic trends when cells were treated with FAK1 inhibitors. The increased SNO modification enhanced the apoptosis evasion of cancer cells, promoting their invasion and metastasis ability [75]. Okuda et al. found that SNO modification at Cys651 site inhibited the activity of DNMT3B. This inhibition led to decrease methylated cytosine in CpG islands, causing an abnormal increase in the transcription of pro-cancer genes like cyclin D2 [79]. NO inhibitors could prevent the formation of SNO-DNMT3B, restore DNMT3B enzyme activity and inhibit colorectal cancer proliferation and transformation [79]. In addition, Zhao et al. reported that SNO modification of Cys833 and Cys839 increased ubiquitination and promoted the degradation of LTBP1. This reduction in LTBP1 activity led to a decrease in TGF-β secretion, consequently promoting the early development of colorectal cancer by blocking TGF-β’s tumor-inhibiting effects [133]. Although Cav-1 had a promoting effect on lung tumor metastasis [119], it still could inhibit tumor growth [134]. Sanhueza et al. posited that Cav-1, which acted as both a nitric oxide synthase inhibitor and tumor suppressor, could inhibit HIF1α by preventing the SNO modification of critical cysteine residues (Cys800 and Cys520) in HIF1α [134]. This inhibition reduced HIF1α’s transcriptional activity and protein stability. Under hypoxic conditions, when HIF1α activity was significantly inhibited, tumor angiogenesis pathways might also be suppressed in colorectal cancer. Consequently, increased expression of Cav-1 could inhibit tumor growth by suppressing HIF1α activity pathways [134]. Conversely, decreased Cav-1 expression led to SNO modification of HIF1α and enhanced activity of HIF1α, ultimately promoting cancer progression. In addition, SNO modifications of E2–estrogen receptor β (ESR2), topoisomerase II α protein (TOP2A), and PTEN also played necessary roles in cancer promotion [135,136,137]. Although there are relatively few studies on the promotion of colorectal cancer by SNO modification, the cancer promoting effect of SNO modification has been widely reported in other cancer types. The variability may be related to differences in protein phenotypes between various tumors.

Breast cancer

SNO-modified proteins (p120 catenin and vascular cell adhesion molecule-1 VCAM-1) exert significant effects on the proliferation, invasion, and metastasis of breast cancer by regulating metabolism, cell adhesion factors, and signaling pathways. Zamorano et al. found that recombinant Galectin-8 promoted the expression of nitric oxide synthase, and triggered the SNO modification of p120 catenin [138]. This modification reduced the binding force between adhesion molecules, destabilized the adhesion protein complex, and decreased adhesion between cancer cells and endothelial cells. Consequently, the migration and metastasis abilities of cancer cells were enhanced [138]. Koning et al. investigated the effect of SNO modification on VCAM-1, a crucial protein in the cell adhesion process. SNO inhibitors could reduce both the SNO modification and protein expression levels of VCAM-1, and inhibit the metastasis of tumor cells [76]. Thus, reducing the SNO modification of VCAM-1 might serve as a potential treatment for metastatic breast cancer [76]. Besides, when the denitrification activity of GSNOR was inhibited in cancer cells, Cañas et al. detected a significant increase in the SNO modification levels of APAF1, c-Jun, HSF1, and calcineurin phosphatase (CaN) by using biomarkers and antibody array incubation methods [139]. In addition, the inhibition of GSNOR activity enhanced the anti-apoptosis ability of breast cancer cells and promoted the survival of tumor cells. This indicated that SNO modification might promote tumor growth by regulating the activity of apoptosis-related proteins in breast cancer [139]. In addition, SNO modifications of tyrosine kinase c-Src (c-Src), Ras, EGFR, and FLIp also play crucial roles in cancer promotion [140,141,142,143].

The pro-cancer effect of SNO-modified proteins has been widely studied in breast cancer, but some SNO-modified proteins (e.g., ERK, JAK2, and TGF-β) can inhibit the progression of breast cancer. Feng et al. used NO donor SNP to stimulate breast cancer cells and found that SNP had a significant cancer cell inhibitory effect. SNP could promote SNO modification at the ERK Cys183 site by significantly increasing intracellular NO concentration [144]. SNO-modified ERK also decreased its own phosphorylation level, thereby promoting apoptosis of cancer cells. In addition, NO scavenger hemoglobin (HB) reversed the apoptosis-promoting state of SNO-modified ERK, further highlighting the significance of SNO-ERK in promoting apoptosis of breast cancer [144]. Bouaouiche et al. demonstrated that NO donor glyceryl trinitrate (GTN) inhibited the invasion and metastasis of breast cancer by promoting SNO modification levels [145]. Specifically, the activity of JAK2 was significantly inhibited because of SNO modification. The reduction in JAK2 activity led to the inhibition of the JAK2-STAT3 signaling pathway, ultimately suppressing tumor cell growth and metastasis [145]. Letson et al. found that the concentration of NO was decreased significantly, while the activity of TGF-β gradually increased in precancerous breast cancer lesions. Treatment with a nitric oxide synthase inhibitor further increased TGF-β expression levels in breast cancer cells [146]. The SNO active sites of TGF-β1, such as Cys355, Cys356, and Cys389, were critical for its dimerization. Replacement of these cysteine residues with alanine resulted in a stronger dimerization binding force of TGF-β1, whereas SNO modification weakened the dimerization binding force and inhibited TGF-β1 activity [146]. Therefore, SNO-modified TGF-β inhibited its own activity and ultimately inhibited the malignant transformation of tumors. In summary, an increase in SNO modification levels in oncogenic signaling proteins typically inhibits their activation, diminishes their oncogenic effects, and reduces breast tumor proliferation and metastasis.

Other cancer

Compared to other types of cancer, SNO modification has been extensively studied in lung cancer, colorectal cancer, and breast cancer. However, SNO-modified proteins also play an important role in solid tumors such as liver cancer, melanoma, thyroid cancer, glioma, ovarian cancer, head and neck cancer, and prostate cancer. In liver cancer, SNO-modified O⁶ alkylguanine DNA alkyltransferase (AGT) and TRAP1 promote the proliferation of tumor cells. Tang et al. explored the pathogenic mechanism of SNO modification in GSNOR-deficient mice by using nitric oxide synthase inhibitors [147]. GSNOR deficiency caused SNO modification of AGT and inhibited the repair process of damaged DNA, thereby also promoting the occurrence of liver cancer. The application of nitric oxide synthase inhibitors alleviated SNO stress in GSNOR-deficient mice and reduced liver cancer pathogenicity [147]. Wei et al. reported that NO donors (GSNO) could increase SNO-AGT and reduce AGT activity in liver cancer. The absence of GSNOR exacerbated this effect, further decreased AGT activity and enhanced the malignant transformation potential of liver cells [148]. Thus, targeting the inhibition of AGT SNO modification could enhance cancer treatment efficacy [148]. In addition, Rizza et al. studied the impact of GSNOR knockdown on TRAP1 and succinate dehydrogenase (SDH) in liver cancer cells. GSNOR deficiency promoted SNO modification at the Cys501 site of TRAP1 and accelerated TRAP1 degradation [149]. Due to the negative feedback regulation between TRAP1 and SDH, the decrease in TRAP1 levels led to an upregulation of SDH expression, thereby promoting tumor progression. Consequently, SDH inhibitors might become new therapeutic targets for GSNOR-deficient liver cancer [149]. Although there have been some studies on promotion of liver cancer proliferation by SNO-modified proteins, they have not been extensively explored to clinically inhibit liver cancer.

SNO modification has been found to exert pro-cancer effects in melanoma by inhibiting key apoptotic proteins and promoting cell survival. Godoy et al. found that melanoma cells treated with cisplatin had high concentrations of NO, and the increase in NO levels also promoted SNO modification of caspase-3 and prolyl hydroxylase-2 (PHD2). The activity of caspase-3, a key protein in the apoptosis pathway, was found to be significantly reduced due to SNO modification at its active site, leading to the inhibition of apoptosis [150]. Similarly, SNO modification also inhibited the activity of PHD2, which is responsible for degrading the pro-cancer HIF1α. This inhibition increased the stability of HIF1α, thereby promoting the survival of malignant cells [150]. Vavvas et al. found that SNO modification competitively inhibited the ubiquitination of Bcl-2 and reduced its degradation efficiency. This led to an increased expression of Bcl-2 in melanoma cells and promoted tumor cell survival [151]. Conversely, nitric oxide synthase inhibitors reduced the SNO modification of Bcl-2, accelerated its degradation and promoted cell apoptosis through the activation of the cysteine protease family [151]. In addition, Ferraz et al. studied the intrinsic mechanism of nanomaterials containing S-nitroso mercaptosuccinic acid in inhibiting melanoma and found that S-nitroso mercaptosuccinic acid nanomaterials significantly enhanced the level of SNO-modified proteins, enhanced intracellular oxidative stress levels, and ultimately promoted cell apoptosis by activating caspase-3 [152]. However, Ferraz et al. did not specify whether caspase-3 was modified by SNO during the process. Therefore, further research is needed to determine the significance of SNO modification of caspase-3 in cell apoptosis [152]. However, there is relatively little research on the anti-cancer effect of SNO modification in melanoma.

SNO modification exhibits diverse effects on glioma tumor cells. Jin et al. used NO donors (SNP and GSNO) to treat glioma cells and explored the role of SNO modification of ERK1/2 in cell apoptosis. Cys183 of ERK1/2 was an active site for SNO modification [153]. There was a competitive inhibitory relationship between SNO and phosphorylation modification of ERK1/2. With the increase of SNO modification, the phosphorylation level of ERK1/2 was further decreased, which led to inhibition of tumor cell proliferation [153]. Guequén et al. demonstrated that pro-inflammatory factors could activate nitric oxide synthase and enhance the SNO modification of VE-cadherin and p120 [154]. SNO modification increased endothelial cell permeability and promoted glioma angiogenesis and tumor cell metastasis. Nitric oxide synthase inhibitors mitigated endothelial cell permeability by reducing protein SNO modification, thereby inhibiting tumor progression [154]. Despite SNO modification displaying both promotive and inhibitory effects on cancer in gliomas, its impact on relevant proteins has been investigated across other cancer types. Consequently, modulating the SNO modification levels of proteins common to various tumors can potentially offer a strategy for treating multiple cancers simultaneously.

In thyroid cancer, pancreatic cancer, and head and neck cancer, the effects of SNO-modified proteins are significantly different. SNO-GAPDH induces apoptosis in thyroid cancer, while SNO-STAT3 reduces proliferation and metastasis in pancreatic cancer. In addition, SNO modification of mitogen-activated protein kinase phosphatase-1 (MKP-1) inhibits the therapeutic efficacy of radiotherapy in head and neck cancer. Du et al. discovered that increased expression of nitric oxide synthase led to SNO modification of GAPDH and promoted its translocation to the nucleus [155]. SNO-GAPDH induced TNF-related apoptosis-inducing ligands (TRAIL) and enhanced apoptosis in thyroid cancer. Conversely, inhibitors of nitric oxide synthase decreased SNO modification and nuclear translocation of GAPDH, and weakened the apoptotic effect of TRAIL [155]. Tan et al. identified over 400 SNO-modified proteins and sites in pancreatic cancer by using mass spectrometry. Additionally, SNO modification was found to involve several signaling pathways implicated in pancreatic cancer development, including the Jak-STAT3 and PI3K-Akt pathways [156]. Thus, SNO-modified proteins exhibited a very wide range of functions in pancreatic cancer. In addition, the use of nitric oxide synthase inhibitors significantly reduced the level of STAT3 SNO modification while enhancing the phosphorylation level, and ultimately promoted the proliferation and invasion of cancer cells [156]. In head and neck cancer, Guan et al. elucidated the mechanism of MKP-1 and radiotherapy sensitivity through extensive study of SNO modifications. Cys258 served as the SNO active site of MKP-1 [157]. The NO donor GSNO was found to enhance the SNO modification of MKP-1, stabilize its protein structure, and prolong its half-life. Additionally, GSNO enhanced the activity of MKP-1 phosphatase. Consequently, SNO-modified MKP-1 enhanced the anti-apoptotic effect of radiotherapy in head and neck cancer, suggesting that inhibiting SNO modification might enhance radiosensitivity in tumors [157]. These findings underscore the dual role of SNO modification across various cancer types.

Functional changes of SNO-modified proteins implicated in inhibiting ovarian cancer, such as p65, Akt, STAT3, and EGFR, have also been observed in other cancer types [158]. Tang et al. discovered that nitrosamine treatment promoted SNO modification at the Cys336 site of Apo2L/TRAIL receptor DR4. SNO modification enhanced the stability of the Apo2L/TRAIL receptor DR4 protein and activated caspase-8, thereby stimulating the apoptosis pathway in ovarian cancer [159]. However, SNO modifications of caspase-3 play essential roles in cancer promotion [160]. In addition, Gao et al. employed nitric oxide synthase overexpression to elevate NO concentration in ovarian cancer cells and increase SNO modification of glycolytic proteins. The Cys351 site of phosphofructose kinase (PFKM) underwent SNO modification, which enhanced the activity and stability of the PFKM tetramer, thereby reducing downstream metabolite feedback inhibition and accelerating glycolysis [89]. The heightened sugar metabolism further drove tumor growth and metastasis. Notably, the substitution of the Cys351 site of PFKM with alanine significantly decreased the proliferation rate of ovarian cancer cells overexpressing nitric oxide synthase, highlighting the critical role of the Cys351 site in PFKM SNO modification [89].

The impact of SNO modification on the function of prostate cancer proteins is profound. NF-κB (p50), Snail, and YY1 can be modified with SNO to reduce drug resistance and promote cell death in prostate cancer. Similarly, SNO-modified androgen receptor (AR) becomes inactive and inhibits the growth and metastasis of prostate cancer. Conversely, SNO modification of integrin α6 (ITG α6) enhances tumor migration and invasion ability, which highlights the complex role of SNO modification in prostate cancer. Bonavida et al. investigated the effects of NO donors on prostate cancer treatment and revealed that high NO concentrations could mediate SNO modification of NF-κB (p50), Snail, and YY1. This modification weakened the nuclear translocation ability of NF-κB and led to reduced expression of target genes Snail and YY1 [161]. Additionally, SNO modification inhibited the DNA binding ability of Snail and YY1, suppressed tumor epithelial-mesenchymal transition and reduced drug resistance in prostate cancer cells [161]. In addition, Isaac et al. identified multiple Cys residue sites in ITG α6 susceptible to SNO modification, with Cys86 modification significantly impacting prostate cancer adhesion ability. SNO modification reduced ITG α6 binding to laminin-β1, diminished prostate cancer adhesion and enhanced tumor cell migration and metastasis [162]. Qin et al. proposed that increased NO concentration led to SNO modification of the Cys601 site in AR in prostate cancer. Although SNO modification did not affect AR distribution, it weakened DNA binding ability, resulting in androgen receptor inactivation and further inhibition of prostate cancer metastasis [163]. In addition, Firdaus et al. induced SNO modifications at the Cys224, Cys278, and Cys830 sites in the colony-stimulating factor 1 receptor (CSF1R) by using GSNO, which enhanced the inhibitory effect on prostate cancer cells by reversing the uncoupling effect of nitric oxide synthase and modulating the proportion of immune cells in the tumor microenvironment, including macrophages and effector T cells [74]. This finding has laid the foundation for the development of potential therapeutic strategies, given the effects of GSNO on SNO modifications of various proteins in cancer cells [74].

In various cancers, SNO modification has been found to have two opposite effects: promoting and inhibiting cancer. The main reasons include: (1) SNO modification can appear in both cancer-promoting and inhibiting proteins and affect protein conformation and function, leading to different tumor outcomes [115, 128]. (2) The sites and amounts of SNO modification can also affect changes in protein function. Even if non-functional sites are modified by SNO, they still do not affect protein function. When the SNO modification rate is low, changes in protein function are not enough to reverse the outcome of tumor cells [127, 140]. (3) The reversibility of SNO modification and the role of denitrification can both affect the stability of SNO modification [139, 147].

Because SNO modification has a dual effect in promoting and inhibiting cancer, two primary therapeutic approaches are currently used to suppress tumors: NO donors and antioxidants. The GSH/Trx systems play central roles in SNO bond cleavage metabolism while also synergistically reducing oxidative stress. These systems are often upregulated in cancer cells to enhance survival [164, 165]. NO donors typically increase intracellular NO concentration within a short time frame, rapidly depleting GSH and Trx reserves and disrupting their activity, which in turn elevates the levels of SNO-modified proteins [126, 164, 166, 167]. To prevent cells from experiencing prolonged nitrosative stress, SNO-based defense signals must be promptly turned off. Most SNO-modified proteins are denitrified by GSH, the most abundant source of intracellular thiols [168]. Antioxidants often accelerate the reduction of SNO-modified proteins, alter their conformations, and help maintain protein homeostasis [115, 164]. Trx1-mediated transnitrosation can act on caspase-3, thereby inhibiting caspase-3 activity and promoting cell survival [169]. However, the roles of NO donors and antioxidants in cancer are not straightforward. For instance, SNO modification of the pro-apoptotic protein Bcl-2 can reduce its activity, thereby protecting tumor cell survival [117]. Conversely, NO donors significantly increase SNO modification of proteins such as p53, β-catenin, and NF-κB, reducing their activities and consequently inhibiting tumor growth [127].

Conclusion and future perspectives

Given the potential of SNO modification to inhibit tumor development, further research into its therapeutic applications is warranted. Increasing the level of SNO modification in certain proteins could offer a novel approach to tumor cell therapy. This can be achieved through various methods, such as (1) The development of NO donors increases local NO concentration and enhances SNO modification levels. (2) Inhibition of GSNOR and denitrification function promotes the elevation of SNO modification. (3) Utilization of nanomaterials enhances the targeting of SNO modification and protein active sites, thereby improving the efficiency of SNO modification. 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 nitric oxide synthase inhibitors to reduce the activity of certain pro-cancer proteins and using exogenous GSNOR to enhance cell denitrification and inhibit tumor growth.

Although SNO modification accounts for a relatively low proportion of overall protein PTMs, it still plays an important role in tumor development and inhibition. SNO modification affects the structure, expression level, intracellular localization, and protein–protein interactions of various cancer-related proteins. Due to the poor stability of SNO modification, it is very difficult to directly recognize and explore related functions of SNO modification. Existing identification and purification methods often involve complex procedures and result in substantial sample loss, and hinder progress in validating the biological functions of SNO-modified proteins. Despite these obstacles, there is still considerable potential for further investigation into the role of SNO-modified proteins in cancer development and inhibition.