Proteomic analysis of S-nitrosylated proteins in Arabidopsis thaliana undergoing hypersensitive response

Nitric oxide (NO) has a fundamental role in the plant hypersensitive disease resistance response (HR), and S-nitrosylation is emerging as an important mechanism for the transduction of its bioactivity. A key step toward elucidating the mechanisms by which NO functions during the HR is the identification of the proteins that are subjected to this PTM. By using a proteomic approach involving 2-DE and MS we characterized, for the first time, changes in S-nitrosylated proteins in Arabidopsis thaliana undergoing HR. The 16 S-nitrosylated proteins identified are mostly enzymes serving intermediary metabolism, signaling and antioxidant defense. The study of the effects of S-nitrosylation on the activity of the identified proteins and its role during the execution of the disease resistance response will help to understand S-nitrosylation function and significance in plants.     

Computational Prediction of Candidate Proteins for S-Nitrosylation in Arabidopsis thaliana

Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments “chloroplast,” “CUL4-RING ubiquitin ligase complex,” and “membrane” more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.     

Human spermatozoa contain multiple targets for protein S‐nitrosylation: An alternative mechanism of the modulation of sperm function by nitric oxide?

Nitric oxide (NO) enhances human sperm motility and capacitation associated with increased protein phosphorylation. NO activates soluble guanylyl cyclase, but can also modify protein function covalently via S-nitrosylation of cysteine. Remarkably, this mechanism remains unexplored in sperm although they depend on post-translational protein modification to achieve changes in function required for fertilisation. Our objective was to identify targets for S-nitrosylation in human sperm. Spermatozoa were incubated with NO donors and S-nitrosylated proteins were identified using the biotin switch assay and a proteomic approach using MS/MS. 240 S-nitrosylated proteins were detected in sperm incubated with S-nitroso-glutathione. Minimal levels were observed in glutathione or untreated samples. Proteins identified consistently based on multiple peptides included established targets for S-nitrosylation in other cells e.g. tubulin, GST and HSPs but also novel targets including A-kinase anchoring protein (AKAP) types 3 and 4, voltage-dependent anion-selective channel protein 3 and semenogelin 1 and 2. In situ localisation revealed S-nitrosylated targets on the postacrosomal region of the head and throughout the flagellum. Potential targets for S-nitrosylation in human sperm include physiologically significant proteins not previously reported in other cells. Their identification will provide novel insight into the mechanism of action of NO in spermatozoa.     

S-nitrosylation: An emerging post-translational protein modification in plants

Increasing evidences support the assumption that nitric oxide (NO) acts as a physiological mediator in plants. Understanding its pleiotropic effects requires a deep analysis of the molecular mechanisms underlying its mode of action. In the recent years, efforts have been made in the identification of plant proteins modified by NO at the post-translational level, notably by S-nitrosylation. This reversible process involves the formation of a covalent bond between NO and reactive cysteine residues. This research has now born fruits and numerous proteins regulated by S-nitrosylation have been identified and characterized. This review describes the basic principle of S-nitrosylation as well as the Biotin Switch Technique and its recent adaptations allowing the identification of S-nitrosylated proteins in physiological contexts. The impact of S-nitrosylation on the structure/function of selected proteins is further discussed.     

Detection of thiol modification following generation of reactive nitrogen species: analysis of synaptic vesicle proteins

S-nitrosylation is an important means of regulating the activity of proteins. We have developed a method which allows unbiased identification of thiol modified proteins within a complex mixture following NO generation, by taking advantage of the fact that prior nitrosylation will block subsequent modification of cysteine residues with 1-biotinamido-4-[4'-(maleimidomethyl)-cyclohexane-carboxamid o] butane (biotin-BMCC). Thiol modified proteins are reduced in intensity when revealed by blotting and overlay with avidin-horseradish peroxidase. In the case of a purified synaptic vesicle fraction we observe a high degree of enrichment of specific biotinylated proteins relative to homogenate. We find that thiol modification of proteins in the presence of NO donors is widespread, occurring in the majority of proteins that will react with biotin-BMCC. In a further development of this technique we have depleted the biotinylated proteins from solubilised synaptic vesicles using avidin-agarose and analysed the supernatants with a panel of antibodies. This has allowed us to identify SNARE proteins (soluble NSF attachment protein receptors) as potential targets for S-nitrosylation.     

Unbiased identification of cysteine S-nitrosylation sites on proteins

Covalent addition of nitric oxide (NO) to Cys-sulfur in proteins, or S-nitrosylation, plays pervasive roles in the physiological and pathophysiological modulation of mammalian protein functions. Knowledge of the specific protein Cys residues that undergo NO addition in different biological settings is fundamental to understanding NO-mediated signal transduction. Here, we describe in detail an MS-based proteomic protocol for facile, high-throughput and unbiased discovery of SNO-Cys residues in proteins from complex biological samples. The approach, termed SNOSID (SNO-Cys site identification), can be used to identify endogenous and chemically induced S-nitrosylation sites in proteins from tissues or cells. Identified SNO-Cys sites may provide insights into novel mechanisms and proteins that mediate NO bioactivities in health and disease. SNOSID builds on the biotin-switch method for covalent addition of disulfide-linked biotin at S-nitrosylation sites on proteins. Biotinylated proteins are then subjected to trypsinolysis and the resulting biotin-tagged peptides are affinity-captured on streptavidin-agarose. After selective elution with beta-mercaptoethanol, the peptides are sequenced using nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS). Validation that identified peptide ions as originating from authentic NO-Cys-containing precursor proteins can be provided by establishing that these peptide ions are absent from control samples where S-NO bonds were subjected to prior photolysis, using a UV transilluminator. The protocol requires approximately 2 days for sample processing, including the incubation time for proteolysis. An additional 1-2 days is needed for sample analysis by nLC-MS/MS and data analysis/interpretation.     

Nitrosative stress inhibits the aminophospholipid translocase resulting in phosphatidylserine externalization and macrophage engulfment: implications for the resolution of inflammation

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-L-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.     

Bacillus anthracis-derived nitric oxide induces protein S-nitrosylation contributing to macrophage death

Bacillus anthracis, a causative agent of anthrax, is able to germinate and survive within macrophages. A recent study suggested that B. anthracis-derived nitric oxide (bNO) is a key aspect of bacterial defense that protects bacterial DNA from oxidative burst in the macrophages. However, the virulent effect of bNO in host cells has not been investigated. Here, we report that bNO contributes macrophage killing by S-nitrosylation of bioenergetic-relating proteins within mitochondria. Toxigenic Sterne induces expression of the bnos gene and produces bNO during early stage of infection. Nitroso-proteomic analysis coupled with a biotin-switch technique demonstrated that toxigenic infection induces protein S-nitrosylation in B. anthracis-susceptible RAW264.7. For each target enzyme tested (complex I, complex III and complex IV), infection by B. anthracis Sterne caused enzyme inhibition. Nω-nitro-l-arginine methyl ester, a NO synthase inhibitor, reduced S-nitrosylation and partially restored cell viability evaluated by intracellular ATP levels in macrophages. Our data suggest that bNO leads to energy depletion driven by impaired mitochondrial bioenergetic machinery that ultimately contributes to macrophage death. This novel mechanism of anthrax pathogenesis may offer specific approach to the development of therapeutics.     

S-nitrosylation of IRP2 regulates its stability via the ubiquitin-proteasome pathway

Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.     

New insights into protein S-nitrosylation. Mitochondria as a model system

The biological effects of nitric oxide (NO) are in significant part mediated through S-nitrosylation of cysteine thiol. Work on model thiol substrates has raised the idea that molecular oxygen (O(2)) is required for S-nitrosylation by NO; however, the relevance of this mechanism at the low physiological pO(2) of tissues is unclear. Here we have used a proteomic approach to study S-nitrosylation reactions in situ. We identify endogenously S-nitrosylated proteins in subcellular organelles, including dihydrolipoamide dehydrogenase and catalase, and show that these, as well as hydroxymethylglutaryl-CoA synthase and sarcosine dehydrogenase (SarDH), are S-nitrosylated by NO under strictly anaerobic conditions. S-Nitrosylation of SarDH by NO is best rationalized by a novel mechanism involving the covalently bound flavin of the enzyme. We also identify a set of mitochondrial proteins that can be S-nitrosylated through multiple reaction channels, including anaerobic/oxidative, NO/O(2), and GSNO-mediated transnitrosation. Finally, we demonstrate that steady state levels of S-nitrosylation are higher in mitochondrial extracts than the intact organelles, suggesting the importance of denitrosylation reactions. Collectively, our results provide new insight into the determinants of S-nitrosothiol levels in subcellular compartments.     

Basal and stimulated protein S-nitrosylation in multiple cell types and tissues.

There is substantial evidence that protein S-nitrosylation provides a significant route through which nitric oxide (NO)-derived bioactivity is conveyed. However, most examples of S-nitrosylation have been characterized on the basis of analysis in vitro, and relatively little progress has been made in assessing the participant forms of nitric-oxide synthase (NOS) or the dynamics of protein S-nitrosylation in situ. Here we utilize antibodies specific for the nitrosothiol (SNO) moiety to provide an immunohistochemical demonstration that protein S-nitrosylation is coupled to the activity of each of the major forms of NOS. In cultured endothelial cells, SNO-protein immunoreactivity increases in response to Ca(2+)-stimulated endothelial NOS (eNOS) activity, and in aortic rings, endothelium-derived and eNOS-mediated relaxation (EDRF) is coupled to increased protein S-nitrosylation in both endothelial and associated smooth muscle cells. In cultured macrophages, SNO-protein levels increase upon cytokine induction of induced NOS (iNOS), and in PC12 cells, increased protein S-nitrosylation is linked to nerve growth factor induction of neuronal NOS (nNOS). In addition, we describe developmental and pathophysiological increases in SNO-protein immunoreactivity within human lung. These results, which demonstrate Ca(2+), neurohumoral, growth factor, cytokine, and developmental regulation of protein S-nitrosylation that is coupled to NOS expression and activity, provide unique evidence for the proposition that this ubiquitous NO-derived post-translational protein modification serves as a major effector of NO-related bioactivity.     

S-nitrosylated S100A8: novel anti-inflammatory properties

S100A8 and S100A9, highly expressed by neutrophils, activated macrophages, and microvascular endothelial cells, are secreted during inflammatory processes. Our earlier studies showed S100A8 to be an avid scavenger of oxidants, and, together with its dependence on IL-10 for expression in macrophages, we postulated that this protein has a protective role. S-nitrosylation is an important posttranslational modification that regulates NO transport, cell signaling, and homeostasis. Relatively few proteins are targets of S-nitrosylation. To date, no inflammation-associated proteins with NO-shuttling capacity have been identified. We used HPLC and mass spectrometry to show that S100A8 and S100A9 were readily S-nitrosylated by NO donors. S-nitrosylated S100A8 (S100A8-SNO) was the preferred nitrosylated product. No S-nitrosylation occurred when the single Cys residue in S100A8 was mutated to Ala. S100A8-SNO in human neutrophils treated with NO donors was confirmed by the biotin switch assay. The stable adduct transnitrosylated hemoglobin, indicating a role in NO transport. S100A8-SNO suppressed mast cell activation by compound 48/80; intravital microscopy was used to demonstrate suppression of leukocyte adhesion and extravasation triggered by compound 48/80 in the rat mesenteric microcirculation. Although S100A8 is induced in macrophages by LPS or IFN-gamma, the combination, which activates inducible NO synthase, did not induce S100A8. Thus, the antimicrobial functions of NO generated under these circumstances would not be compromised by S100A8. Our results suggest that S100A8-SNO may regulate leukocyte-endothelial cell interactions in the microcirculation, and suppression of mast cell-mediated inflammation represents an additional anti-inflammatory property for S100A8.     

Say NO to neurodegeneration: role of S-nitrosylation in neurodegenerative disorders

Nitric oxide (NO) is an important signaling molecule that controls a wide range of biological processes. One of the signaling mechanisms of NO is through the S-nitrosylation of cysteine residues on proteins. S-nitrosylation is now regarded as an important redox signaling mechanism in the regulation of different cellular and physiological functions. However, deregulation of S-nitrosylation has also been linked to various human diseases such as neurodegenerative disorders. Nitrosative stress has long been considered as a major mediator in the development of neurodegeneration, but the molecular mechanism of how NO can contribute to neurodegeneration is not completely clear. Early studies suggested that nitration of proteins, which can induce protein aggregation might contribute to the neurodegenerative process. However, several recent studies suggest that S-nitrosylation of proteins that are important for neuronal survival contributes substantially in the development of various neurodegenerative disorders. Thus, in-depth understanding of the mechanism of neurodegeneration in relation to S-nitrosylation will be critical for the development of therapeutic treatment against these neurodegenerative diseases.     

Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry

S-nitrosylation, the formation of S-nitrosothiol (SNO), is an important reversible thiol oxidation event that has been increasingly recognized for its role in cell signaling. Although many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challenge because of the low abundance and labile nature of this modification. Herein we present further improvement and optimization of the recently reported resin-assisted cysteinyl peptide enrichment protocol for SNO identification and its application to mouse skeletal muscle to identify specific cysteine sites sensitive to S-nitrosylation by a quantitative reactivity profiling strategy. Our results indicate that the protein- and peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications. S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione, an NO donor, at two different concentrations (i.e., 10 and 100 μM). The reactivity profiling experiments led to the identification of 488 SNO-modified sites from 197 proteins with specificity of ∼95% at the unique peptide level, i.e., ∼95% of enriched peptides contain cysteine residues as the originally SNO-modified sites. Among these sites, 281 from 145 proteins were considered more sensitive to S-nitrosylation based on the ratios of observed SNO levels between the two treatments. These SNO-sensitive sites are more likely to be physiologically relevant. Many of the SNO-sensitive proteins are localized in mitochondria, contractile fiber, and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation. Moreover, these observed SNO-sensitive proteins are primarily involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis/gluconeogenesis, glutathione metabolism, and fatty acid metabolism, suggesting the importance of redox regulation in muscle metabolism and insulin action.     

S-nitrosylation of fatty acid synthase regulates its activity through dimerization

NO regulates a variety of physiological processes, including cell proliferation, differentiation, and inflammation. S-nitrosylation, a NO-mediated reversible protein modification, leads to changes in the activity and function of proteins. In particular, the role of S-nitrosylation during adipogenesis is largely unknown. We hypothesized that the normal physiological levels of NO, but not the excess levels generated under severe conditions, such as inflammation, may be critically involved in the proper regulation of adipogenesis. We found that endogenous S-nitrosylation of proteins was required for adipocyte differentiation. By performing a biotin-switch assay, we identified FAS, a key lipogenic enzyme in adipocytes, as a target of S-nitrosylation during adipogenesis. Interestingly, we also observed that the dimerization of FAS increased in parallel with the amount of S-nitrosylated FAS during adipogenesis. In addition, we found that exogenous NO enhanced the dimerization and the enzymatic activity of FAS. Moreover, site-directed mutagenesis of three predicted S-nitrosylation sites indicated that S-nitrosylation of FAS at Cys¹⁴⁷¹ and Cys²⁰⁹¹, but not at Cys¹¹²⁷, increased its enzymatic activity. Taken together, these results suggest that the S-nitrosylation of FAS at normal physiological levels of NO increases its activity through dimerization and may contribute to the proper regulation of adipogenesis.     

蛋白质巯基亚硝基化参与调控一氧化氮介导的心肌缺血预适应

一氧化氮（nitricoxide,NO）作为一种重要的信使分子参与缺血预适应（ischemic preconditioning,IPC）心肌保护。目前普遍认为NO通过经典的NO／cGMP依赖的信号转导途径调节线粒体ATP敏感性钾（ATP-sensitive potassium,KATP通道来发挥其保护作用,然而越来越多的数据表明NO还可能通过蛋白质巯基亚硝基化（S-nitrosylation）来发挥生理功能。蛋白质巯基亚硝基化,即蛋白质半胱氨酸巯基与NO基团形成共价键,是一种氧化还原依赖的蛋白质翻译后可逆修饰。蛋白质巯基亚硝基化不仅可以改变蛋白质的结构和功能,而且还可以阻抑目标半胱氨酸的进一步氧化修饰。IPC增加S-亚硝基硫醇（S-nitrosothi01）含量,引起蛋白质巯基亚硝基化。S-亚硝基硫醇还能发挥药理性预适应作用,抵抗心肌缺血,再灌注损伤。因此,蛋白质巯基亚硝基化是IPC心肌保护的一种重要途径,参与抵抗细胞内氧化应激和亚硝化应激（nitrosative stress）。     

Protein S-nitrosylation: a physiological signal for neuronal nitric oxide

Nitric oxide (NO) has been linked to numerous physiological and pathophysiological events that are not readily explained by the well established effects of NO on soluble guanylyl cyclase. Exogenous NO S-nitrosylates cysteine residues in proteins, but whether this is an important function of endogenous NO is unclear. Here, using a new proteomic approach, we identify a population of proteins that are endogenously S-nitrosylated, and demonstrate the loss of this modification in mice harbouring a genomic deletion of neuronal NO synthase (nNOS). Targets of NO include metabolic, structural and signalling proteins that may be effectors for neuronally generated NO. These findings establish protein S-nitrosylation as a physiological signalling mechanism for nNOS.