Post-translational modifications mediated by reactive nitrogen species: Nitrosative stress responses or components of signal transduction pathways?

In animal cells, nitric oxide and NO-derived molecules have been shown to mediate post-translational modifications such as S-nitrosylation and protein tyrosine nitration which are associated with cell signalling and pathological processes, respectively. In plant cells, knowledge of the function of these post-translational modifications under physiological and stress conditions is still very rudimentary. In this addendum, we briefly examine how reactive nitrogen species (RNS) can exert important effects on proteins that could mediate signalling processes in plants.Key words: nitrosative stress, nitric oxide synthase, S-nitrosoglutathione, nitro-tyrosine, post-translational modifications, S-nitrosylation     

Perturbations in nitric oxide homeostasis promote Arabidopsis disease susceptibility towards Phytophthora parasitica

Phytophthora species can infect hundreds of different plants, including many important crops, causing a number of agriculturally relevant diseases. A key feature of attempted pathogen infection is the rapid production of the redox active molecule nitric oxide (NO). However, the potential role(s) of NO in plant resistance against Phytophthora is relatively unexplored. Here we show that the level of NO accumulation is crucial for basal resistance in Arabidopsis against Phytophthora parasitica. Counterintuitively, both relatively low or relatively high NO accumulation leads to reduced resistance against P. parasitica. S-nitrosylation, the addition of a NO group to a protein cysteine thiol to form an S-nitrosothiol, is an important route for NO bioactivity and this process is regulated predominantly by S-nitrosoglutathione reductase 1 (GSNOR1). Loss-of-function mutations in GSNOR1 disable both salicylic acid accumulation and associated signalling, and also the production of reactive oxygen species, leading to susceptibility towards P. parasitica. Significantly, we also demonstrate that secreted proteins from P. parasitica can inhibit Arabidopsis GSNOR1 activity.     

Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD⁺ as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD⁺, the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD⁺ and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.     

Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress

The nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) molecules play important roles in the redox homeostasis of plant cells. Using tomato (Solanum lycopersicum) plants grown with 120 mM NaCl, we studied the redox state of NADPH and GSH as well as ascorbate, nitric oxide (NO) and S-nitrosoglutathione (GSNO) content and the activity of the principal enzymes involved in the metabolism of these molecules in roots. Salinity caused a significant reduction in growth parameters and an increase in oxidative parameters such as lipid peroxidation and protein oxidation. Salinity also led to an overall decrease in the content of these redox molecules and in the enzymatic activities of the main NADPH-generating dehydrogenases, S-nitrosoglutathione reductase and catalase. However, NO content as well as gluthahione reductase and glutathione peroxidase activity increased under salinity stress. These findings indicate that salinity drastically affects redox and NO homeostasis in tomato roots. In our view, these molecules, which show the interaction between ROS and RNS metabolisms, could be excellent parameters for evaluating the physiological conditions of plants under adverse stress conditions.     

Involvements of <Emphasis Type="Italic">S</Emphasis>-nitrosylation and denitrosylation in the production of polyphenols by <Emphasis Type="Italic">Inonotus obliquus</Emphasis>

Nitric oxide (NO) has been evidenced to mediate biosynthesis of polyphenols in Inonotus obliquus. However, it remains unknown how NO regulates their biosynthesis. Here we show that higher cellular NO levels coincided with higher accumulation of S-nitrosothiols (SNO; the products of NO combined with a specific residue in glutathione or proteins) and polyphenols, and higher activity of denitrosylated S-nitrosoglutathione reductase (GSNOR) and thioredoxin reductase (TrxR). This homeostasis was breached by GSNOR or TrxR inhibitors. Inhibiting GSNOR boosted TrxR activity, but reduced SNO formation, coinciding with an enhanced production of polyphenols. Likewise, inhibiting TrxR increased GSNOR activity and SNO production, but downregulated accumulation of polyphenols. Inhibiting GSNOR or TrxR also modified the polyphenolic profiles of I. obliquus. Suppressing GSNOR-enhanced biosynthesis of phelligridins C and H, inoscavin C and methyl inoscavin B, but reduced that of phelligridin D, methyl inoscavin A, davallialactone and methyl davallialactone, the typical polyphenols in I. obliquus. Similarly, downregulating TrxR increased production of phelligridin D, methyl inoscavin A, davallialactone, and methyl davallialactone, but shrinking that of phelligridins C and H, methyl inoscavin B and inoscavin C. Thus, in I. obliquus, the state of S-nitrosylation and denitrosylation affects not only the accumulation of polyphenols, but also their metabolic profiles.     

Nitric Oxide Enhances Salt Tolerance in Tomato Seedlings by Regulating Endogenous S-nitrosylation Levels

Salinity impairs plant growth and development, thereby leading to low yield and inferior quality of crops. Nitric oxide (NO) has emerged as an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. In this study, tomato seedlings of Lycopersicum esculentum L. “Micro-Tom” treated with 150 mM sodium chloride (NaCl) conducted decreased plant height, total root length, and leaf area by 25.43%, 24.87%, and 33.67%, respectively. While nitrosoglutathione (GSNO) pretreatment ameliorated salt toxicity in a dose-dependent manner and 10 µM GSNO exhibited the most significant mitigation effect. It increased the plant height, total root length, and leaf area of tomato seedlings, which was 31.44%, 20.56%, and 51.21% higher than NaCl treatment alone, respectively. However, NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium (cPTIO) treatment reversed the positive effect of NO under salt stress, implying that NO is essential for the enhancement of salt tolerance. Additionally, NaCl?+?GSNO treatment effectively decreased O^2? production and H₂O₂ content, increased the levels of soluble sugar, glycinebetaine, proline, and chlorophyll, and enhanced the activities of antioxidant enzymes and the content of antioxidants in tomato seedlings in comparison with NaCl treatment, whereas NaCl?+?cPTIO treatment significantly reversed the effect of NO under salt stress. Moreover, we found that GSNO treatment increased endogenous NO content, S-nitrosoglutathione reductase (GSNOR) activity, GSNOR expression and total S-nitrosylated level, and decreased S-nitrosothiol (SNO) content under salt stress, implicating that S-nitrosylation might be involved in NO-enhanced salt tolerance in tomatoes. Altogether, these results suggest that NO confers salt tolerance in tomato seedlings probably by the promotion of photosynthesis and osmotic balance, the enhancement of antioxidant capability and the increase of protein S-nitrosylation levels.

     

Immunolocalization of S-nitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in pea leaf organelles

S-Nitrosoglutathione (GSNO) is a nitrosothiol which plays a major role in the metabolism of NO in higher plants mediating signaling processes. Protein tyrosine nitration (NO₂–Tyr) is a post-translational modification which contributes to protein regulation. The subcellular localization of GSNO, S-nitrosoglutathione reductase (GSNOR), an enzyme which catalyzes its decomposition and protein tyrosine nitration was studied in pea (Pisum sativum L.) leaf plants with the aid of the electron microscopy immunogold-labeling technique. Our findings show that GSNO, GSNOR and nitrated proteins are present in the different subcellular compartments of leaf cells which include chloroplasts, cytosol, mitochondria, and peroxisomes. Given that pea peroxisomes are one of the cell compartments where nitric oxide (NO) has been thoroughly studied, our results provide additional insights into the metabolism of NO in this organelle where NO and GSNO could function as signal molecules in cross talk between the different cell compartments.     

Structure-activity relationships of pyrrole based S-nitrosoglutathione reductase inhibitors: pyrrole regioisomers and propionic acid replacement

S-Nitrosoglutathione reductase (GSNOR) is a member of the alcohol dehydrogenase family (ADH) that regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). GSNO and SNOs are implicated in the pathogenesis of many diseases including those in respiratory, cardiovascular, and gastrointestinal systems. The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious GSNOR inhibitor which is currently undergoing clinical development. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on scaffold modification and propionic acid replacement. We identified equally potent and novel GSNOR inhibitors having pyrrole regioisomers as scaffolds using a structure based approach.     

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys: IMPLICATIONS FOR ACTIVITY,ALLOSTERY, AND REGULATION*

Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS₄ site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions.     

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-Nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1–1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1^C40A, Panx1^C346A, and Panx1^C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1^C40A/C346A) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.     

Thioredoxin-interacting Protein (Txnip) Is a Feedback Regulator of S-Nitrosylation

Nitric oxide exerts a plethora of biological effects via protein S-nitrosylation, a redox-based reaction that converts a protein Cys thiol to a S-nitrosothiol. However, although the regulation of protein S-nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus-coupled protein denitrosylation. We now demonstrate that protein denitrosylation by thioredoxin is regulated dynamically by thioredoxin-interacting protein (Txnip), a thioredoxin inhibitor. Endogenously synthesized nitric oxide represses Txnip, thereby facilitating thioredoxin-mediated denitrosylation. Autoregulation of denitrosylation thus allows cells to survive nitrosative stress. Our findings reveal that denitrosylation of proteins is dynamically regulated, establish a physiological role for thioredoxin in protection from nitrosative stress, and suggest new approaches to manipulate cellular S-nitrosylation.     

Involvement of Reactive Nitrogen and Oxygen Species (RNS and ROS) in Sunflower-Mildew Interaction

Nitric oxide (·NO) has been shown to participate in plantresponse against pathogen infection; however, less is knownof the participation of other NO-derived molecules designatedas reactive nitrogen species (RNS). Using two sunflower (Helianthusannuus L.) cultivars with different sensitivity to infectionby the pathogen Plasmopara halstedii, we studied key componentsinvolved in RNS and ROS metabolism. We analyzed the superoxideradical production, hydrogen peroxide content, L-arginine-dependentnitric oxide synthase (NOS) and S-nitrosoglutathione reductase(GSNOR) activities. Furthermore, we examined the location andcontents of ·NO, S-nitrosothiols (RSNOs), S-nitrosoglutathione(GSNO) and protein 3-nitrotyrosine (NO₂-Tyr) by confocal laserscanning microscopy (CLSM) and biochemical analyses. In thesusceptible cultivar, the pathogen induces an increase in proteinsthat undergo tyrosine nitration accompanied by an augmentationin RSNOs. This rise of RSNOs seems to be independent of theenzymatic generation of ·NO because the L-arginine-dependentNOS activity is reduced after infection. These results suggestthat pathogens induce nitrosative stress in susceptible cultivars.In contrast, in the resistant cultivar, no increase of RSNOsor tyrosine nitration of proteins was observed, implying anabsence of nitrosative stress. Therefore, it is proposed thatthe increase of tyrosine nitration of proteins can be considereda general biological marker of nitrosative stress in plantsunder biotic conditions.     

Site-Specific Nitrosoproteomic Identification of Endogenously S-Nitrosylated Proteins in Arabidopsis

Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants.Nitric oxide (NO), a gaseous signaling molecule, plays important regulatory roles in higher plants, including seed dormancy and germination, root development and hypocotyl elongation, floral transition, senescence and cell death, phytohormone signaling, and responses to abiotic and biotic stresses (He et al., 2004; Besson-Bard et al., 2008; Hong et al., 2008; Neill et al., 2008; Leitner et al., 2009; Feng et al., 2013). S-Nitrosoglutathione (GSNO) is a major biologically active form of reactive nitrogen species (RNS) and functions as a primary NO donor. The endogenous GSNO homeostasis is highly dynamic, and the GSNO level is negatively regulated by GSNO reductase (GSNOR), an evolutionally conserved enzyme catalyzing irreversibly degrading GSNO (Liu et al., 2001). Mutations in the GSNOR gene cause the elevated GSNO level and consequently severe abnormalities under physiological and pathological conditions in various species (Liu et al., 2004; Feechan et al., 2005; Que et al., 2005; Lee et al., 2008; Chen et al., 2009; Moore et al., 2009; Kwon et al., 2012).In Arabidopsis (Arabidopsis thaliana), GSNOR1 is a single-copy gene, and the enzymatic activity of the encoded protein has been biochemically characterized (Sakamoto et al., 2002). Genetic studies revealed that the gsnor1-1 and gsnor1-2 mutants are gain-of-function mutations with increased GSNOR activity and a decreased cellular S-nitrosothiol level. Conversely, gsnor1-3 is a loss-of-function mutant with a significantly increased S-nitrosothiol level (Feechan et al., 2005). The defense responses mediated by distinct resistance (R) genes are significantly impaired in the gsnor1-3 mutant, and GSNOR1 functions as a positive regulator of the salicylic acid-regulated signaling network in the defense response (Feechan et al., 2005). In a genetic screen for thermotolerance-defective mutants, the sensitive to hot temperatures5 (hot5) mutant was characterized as having decreased heat acclimation and was shown to be allelic to gsnor1, indicating the importance of GSNOR1-regulated NO homeostasis in the regulation of the abiotic stress response (Lee et al., 2008). In an independent genetic screen for the oxidative stress-related mutants, the paraquat resistant2 (par2) mutant was also identified to be allelic to gsnor1, which showed an anti-cell death phenotype and multiple developmental defects, revealing the critical role of GSNOR1/HOT5/PAR2 in the regulation of oxidative stress-induced cell death (Chen et al., 2009). Similar to gsnor1-3, the hot5 and par2 allelic mutants also accumulate the significantly increased level of NO. As a result of this defect, these gsnor1/hot5/par2 mutants show a pleiotropic phenotype, with severe developmental abnormalities in both reproductive and vegetative stages (Lee et al., 2008; Chen et al., 2009; Kwon et al., 2012). These studies highlight the critical role of GSNOR1/HOT5/PAR2-modulated NO homeostasis in diverse physiological processes, including plant growth and development as well as in responses to both biotic and abiotic stresses. However, little is known about the underpinning molecular mechanisms of the NO-modulated signaling in various physiological processes.A major physiological effect of NO is executed by protein S-nitrosylation, a reversible posttranslational modification by covalent addition of an NO molecule onto a Cys thiol to form S-nitrosothiol (Jaffrey et al., 2001; Stamler et al., 2001). S-Nitrosothiols are dynamically labile in response to the intracellular redox status, allowing protein S-nitrosylation as a highly sensitive mechanism in the regulation of cellular signaling (Stamler et al., 2001; Hess et al., 2005). Emerging evidence indicates that S-nitrosylation regulates the function of the modified proteins by various mechanisms, including enzymatic activity, stability, subcellular localization, three-dimensional conformation changes, protein-protein interaction, and ligand binding (Hess et al., 2005; Wang et al., 2006; Astier et al., 2011; Gupta, 2011; Hess and Stamler, 2012). In Arabidopsis, S-nitrosylation has been shown as an important mechanism in regulating the stress responses. The activity of Met adenosyltransferase1 (MAT1), which catalyzes S-adenosyl-Met synthesis, was shown to be inhibited by S-nitrosylation (Lindermayr et al., 2006). S-nitrosylation negatively regulates the activity of a peroxynitrite detoxification enzyme, peroxiredoxin II E (PrxII E), and an NADPH oxidase, thereby modulating the oxidative stress in the defense response (Romero-Puertas et al., 2007; Yun et al., 2011). Moreover, S-nitrosylation has also been shown to regulate the conformational changes of NONEXPRESSOR OF PATHOGEN-RELATED1 (NPR1), a master regulator of the defense response, and the activity of SALICYLIC ACID-BINDING PROTEIN3 (SABP3), a key enzyme for salicylic acid biosynthesis (Tada et al., 2008; Wang et al., 2009). In addition, S-nitrosylation of TRANSPORT INHIBITOR RESPONSE1 (TIR1) and Arabidopsis Histidine Phosphotransfer Protein1 (AHP1), two key signaling components of the auxin and cytokinin pathways, respectively, plays an important role in regulating respective phytohormone signaling (Terrile et al., 2012; Feng et al., 2013). These studies illustrate the importance of S-nitrosylation in the regulation of diverse physiological processes in plants.S-Nitrosylation has been considered as one of the most important posttranslational modification mechanisms (Lane et al., 2001; Stamler et al., 2001; Hess et al., 2005). A growing number of S-nitrosylated proteins have been identified using the proteomic approach. To date, the S-nitrosoproteomic studies have identified more than 2,200 S-nitrosylated proteins, covering more than 4,100 S-nitrosylated Cys residues. Of those S-nitrosylated proteins, more than 95% were identified from mammals (Lee et al., 2012). Several proteomic studies in Arabidopsis identified a number of S-nitrosylated proteins (Lindermayr et al., 2005; Romero-Puertas et al., 2008; Palmieri et al., 2010; Fares et al., 2011; Puyaubert et al., 2014). In GSNO-treated cell suspension cultures and NO-treated leaves derived from Arabidopsis, 63 and 52 S-nitrosylated proteins were identified, which are involved in stress response, redox homeostasis, cytoskeleton organization, metabolic processes, and cellular signaling (Lindermayr et al., 2005). In an independent study, 16 S-nitrosylated proteins were identified from Arabidopsis seedlings undergoing the hypersensitive response (Romero-Puertas et al., 2008). In another independent analysis, 46 S-nitrosylated proteins were identified from cultured Arabidopsis suspension cells (Fares et al., 2011). In a more specific analysis, 11 mitochondria proteins were identified to be S-nitrosylated and/or glutathionylated (Palmieri et al., 2010). More recently, 62 endogenously S-nitrosylated proteins were identified from Arabidopsis seedlings (Puyaubert et al., 2014). Notably, a large number of the S-nitrosylated proteins are repeatedly identified in these analyses, thus confirming the validation of each study. Because of the labile nature of S-nitrosylation, most of the S-nitrosoproteomic studies used the protein samples treated with NO donors or the protein extracts prepared from NO donor-treated cells or tissues. The Arabidopsis gsnor1-3 mutants accumulate an excessive amount of NO (Feechan et al., 2005; Lee et al., 2008; Chen et al., 2009), and the identification of S-nitrosylated proteins in gsnor1-3 should depict a more comprehensive map of S-nitrosoproteome in Arabidopsis, and provide important clues on the molecular basis of the pleiotropic phenotype of the mutant.Because of the labile and dynamic nature of protein S-nitrosylation, large-scale identification of endogenously S-nitrosylated proteins remains technically challenging. At present, two major methods for identification of S-nitrosoproteome are shotgun and site-specific nitrosoproteomic analysis, both of which are based on the biotin-switch method and mass spectrometry (Jaffrey et al., 2001; Hao et al., 2006; Torta et al., 2008). In the shotgun analysis, S-nitrosylated proteins were first biotinylated, enriched by affinity-chromatography, and then identified by mass spectrometry. Although the method is relatively simple, the number of S-nitrosylated proteins identified by shotgun proteomics is often few due to various technical limitations (Torta et al., 2008). The identification capacity of nitrosoproteomics was greatly improved by the site-specific strategy, in which biotinylated proteins were first digested by trypsin and the enriched peptides were then characterized by mass spectrometry (Hao et al., 2006; Chen et al., 2010). Moreover, S-nitrosylated Cys residues can also be identified from site-specific nitrosoproteomic analysis.In this study, we performed a large-scale, site-specific proteomic analysis of endogenously S-nitrosylated proteins in Arabidopsis wild-type and gsnor1-3 seedlings, and identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the model plant species, representing the largest data set thus far reported in any organisms and providing important resources for future studies on S-nitrosylation-regulated signaling in plants.     

Protein S-nitrosylation: Role for nitric oxide signaling in neuronal death

Background

One of the signaling mechanisms mediated by nitric oxide (NO) is through S-nitrosylation, the reversible redox-based modification of cysteine residues, on target proteins that regulate a myriad of physiological and pathophysiological processes. In particular, an increasing number of studies have identified important roles for S-nitrosylation in regulating cell death.

Scope of review

The present review focuses on different targets and functional consequences associated with nitric oxide and protein S-nitrosylation during neuronal cell death.

Major conclusions

S-Nitrosylation exhibits double-edged effects dependent on the levels, spatiotemporal distribution, and origins of NO in the brain: in general Snitrosylation resulting from the basal low level of NO in cells exerts anti-cell death effects, whereas S-nitrosylation elicited by induced NO upon stressed conditions is implicated in pro-cell death effects.

General Significance

Dysregulated protein S-nitrosylation is implicated in the pathogenesis of several diseases including degenerative diseases of the central nervous system (CNS). Elucidating specific targets of S-nitrosylation as well as their regulatory mechanisms may aid in the development of therapeutic intervention in a wide range of brain diseases.