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.     

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO(2)-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO(2)-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO(2)-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.     

S‐nitrosoglutathione spraying improves stomatal conductance,Rubisco activity and antioxidant defense in both leaves and roots of sugarcane plants under water deficit

Water deficit is a major environmental constraint on crop productivity and performance and nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. This study aims to test the hypothesis that leaf spraying of S‐nitrosoglutathione (GSNO), an NO donor, improves the antioxidant defense in both roots and leaves of sugarcane plants under water deficit, with positive consequences for photosynthesis. In addition, the roles of key photosynthetic enzymes ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) in maintaining CO₂ assimilation of GSNO‐sprayed plants under water deficit were evaluated. Sugarcane plants were sprayed with water or GSNO 100 μM and subjected to water deficit, by adding polyethylene glycol (PEG‐8000) to the nutrient solution. Sugarcane plants supplied with GSNO presented increases in the activity of antioxidant enzymes such as superoxide dismutase in leaves and catalase in roots, indicating higher antioxidant capacity under water deficit. Such adjustments induced by GSNO were sufficient to prevent oxidative damage in both organs and were associated with better leaf water status. As a consequence, GSNO spraying alleviated the negative impact of water deficit on stomatal conductance and photosynthetic rates, with plants also showing increases in Rubisco activity under water deficit.     

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.     

S-Nitrosoglutathione is a component of wound- and salicylic acid-induced systemic responses in Arabidopsis thaliana

S-Nitrosoglutathione (GSNO) is a bioactive, stable, and mobile reservoir of nitric oxide (NO), and an important player in defence responses to herbivory and pathogen attack in plants. It has been demonstrated previously that GSNO reductase (GSNOR) is the main enzyme responsible for the in vivo control of intracellular levels of GSNO. In this study, the role of S-nitrosothiols, in particular of GSNO, in systemic defence responses in Arabidopsis thaliana was investigated further. It was shown that GSNO levels increased rapidly and uniformly in injured Arabidopsis leaves, whereas in systemic leaves GSNO was first detected in vascular tissues and later spread over the parenchyma, suggesting that GSNO is involved in the transmission of the wound mobile signal through the vascular tissue. Moreover, GSNO accumulation was required to activate the jasmonic acid (JA)-dependent wound responses, whereas the alternative JA-independent wound-signalling pathway did not involve GSNO. Furthermore, extending previous work on the role of GSNOR in pathogenesis, it was shown that GSNO acts synergistically with salicylic acid in systemic acquired resistance activation. In conclusion, GSNOR appears to be a key regulator of systemic defence responses, in both wounding and pathogenesis.     

Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a k(cat)/K(m) value approx. 10 times the k(cat)/K(m) value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD(+)/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1-5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.     

S-Nitrosogluthathione reductase activity of amphioxus ADH3: insights into the nitric oxide metabolism

Nitric oxide (NO) is a signalling molecule involved in many physiological functions. An important via of NO action is through the S-nitrosylation of proteins, a post-translational modification that regulates the activity of enzymes, protein-protein interactions and signal transduction pathways. Alcohol dehydrogenase class III (ADH3) recognises S-nitrosoglutathione (GSNO), the main reservoir of non-protein S-nitrosothiol, and functions as an effective GSNO reductase (GSNOR) and as a safeguard against nitrosative stress. To investigate the evolutionary conservation of this metabolic role, we have produced recombinant Branchiostoma floridae ADH3. Pure preparations of ADH3 showed 2-fold higher activity as GSNOR than as formaldehyde dehydrogenase, the previously assumed main role for ADH3. To correlate ADH3 expression in the gut with areas of NO production, we analysed the tissue distribution of the nitric oxide synthase (NOS) enzyme in amphioxus larvae. Immunostaining of the NOS enzyme revealed expression in the gut and in the dorsal region of the club-shaped gland. Co-localization in the gut supports the ADH3 and NOS joint contribution to the NO/SNO homeostasis.     

Novel metabolism of nitrogen in plants

Our previous study showed that approximately one-third of the nitrogen of 15N-labeled NO2 taken up into plants was converted to a previously unknown organic nitrogen (hereafter designated UN) that was not recoverable by the Kjeldahl method (Morikawa et al., 2004). In this communication, we discuss metabolic and physiological relevance of the UN based on our newest experimental results. All of the 12 plant species were found to form UN derived from NO2 (about 10-30% of the total nitrogen derived from NO2). The UN was formed also from nitrate nitrogen in various plant species. Thus, UN is a common metabolite in plants. The amount of UN derived from NO2 was greatly increased in the transgenic tobacco clone 271 (Vaucheret et al., 1992) where the activity of nitrite reductase is suppressed less than 5% of that of the wild-type plant. On the other hand, the amount of this UN was significantly decreased by the overexpression of S-nitrosoglutathione reductase (GSNOR). These findings strongly suggest that nitrite and other reactive nitrogen species are involved in the formation of the UN, and that the UN-bearing compounds are metabolizable. A metabolic scheme for the formation of UN-bearing compounds was proposed, in which nitric oxide and peroxynitrite derived from NO2 or endogenous nitrogen oxides are involved for nitrosation and/or nitration of organic compounds in the cells to form nitroso and nitro compounds, including N-nitroso and S-nitroso ones. Participation of non-symbiotic haemoglobin bearing peroxidase-like activity (Sakamoto et al., 2004) and GSNOR (Sakamoto et al., 2002) in the metabolism of the UN was discussed. The UN-bearing compounds identified to date in the extracts of the leaves of Arabidopsis thaliana fumigated with NO2 include a delta2-1,2,3-thiadiazoline derivative (Miyawaki et al., 2004) and 4-nitro-beta-carotene.     

Proteomic analysis of defense response of wildtype Arabidopsis thaliana and plants with impaired NO- homeostasis

In recent years, nitric oxide (NO) has been recognized as a signalling molecule of plants, being involved in diverse processes like germination, root growth, stomatal closing, and responses to various stresses. A mechanism of how NO can regulate physiological processes is the modulation of cysteine residues of proteins (S-nitrosylation) by S-nitrosoglutathione (GSNO), a physiological NO donor. The concentration of GSNO and the level of S-nitrosylated proteins are regulated by GSNO reductase, which seems to play a major role in NO signalling. To investigate the importance of NO in plant defense response, we performed a proteomic analysis of Arabidopsis wildtype and GSNO-reductase knock-out plants infected with both the avirulent and virulent pathogen strains of Pseudomonas syringae. Using 2-D DIGE technology in combination with MS, we identified proteins, which are differentially accumulated during the infection process. We observed that both lines were more resistant to avirulent infections than to virulent infections mainly due to the accumulation of stress-, redox-, and defense-related proteins. Interestingly, after virulent infections, we also observed accumulation of defense-related proteins, but no or low accumulation of stress- and redox-related proteins, respectively. In summary, we present here the first detailed proteomic analysis of plant defense response.     

The role of S-nitrosoglutathione reductase (GSNOR) in human disease and therapy

S-nitrosoglutathione reductase (GSNOR), or ADH5, is an enzyme in the alcohol dehydrogenase (ADH) family. It is unique when compared to other ADH enzymes in that primary short-chain alcohols are not its principle substrate. GSNOR metabolizes S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (the spontaneous adduct of formaldehyde and glutathione), and some alcohols. GSNOR modulates reactive nitric oxide (?NO) availability in the cell by catalyzing the breakdown of GSNO, and indirectly regulates S-nitrosothiols (RSNOs) through GSNO-mediated protein S-nitrosation. The dysregulation of GSNOR can significantly alter cellular homeostasis, leading to disease. GSNOR plays an important regulatory role in smooth muscle relaxation, immune function, inflammation, neuronal development and cancer progression, among many other processes. In recent years, the therapeutic inhibition of GSNOR has been investigated to treat asthma, cystic fibrosis and interstitial lung disease (ILD). The direct action of ?NO on cellular pathways, as well as the important regulatory role of protein S-nitrosation, is closely tied to GSNOR regulation and defines this enzyme as an important therapeutic target.     

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.     

Metabolism of reactive nitrogen species in pea plants under abiotic stress conditions

Nitric oxide (*NO) is a key signaling molecule in different physiological processes of animals and plants. However, little is known about the metabolism of endogenous *NO and other reactive nitrogen species (RNS) in plants under abiotic stress conditions. Using pea plants exposed to six different abiotic stress conditions (high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding), several key components of the metabolism of RNS including the content of *NO, S-nitrosothiols (RSNOs) and nitrite plus nitrate, the enzyme activities of l-arginine-dependent nitric oxide synthase (NOS) and S-nitrosogluthathione reductase (GSNOR), and the profile of protein tyrosine nitration (NO(2)-Tyr) were analyzed in leaves. Low temperature was the stress that produced the highest increase of NOS and GSNOR activities, and this was accompanied by an increase in the content of total *NO and S-nitrosothiols, and an intensification of the immunoreactivity with an antibody against NO(2)-Tyr. Mechanical wounding, high temperature and light also had a clear activating effect on the different indicators of RNS metabolism in pea plants. However, the total content of nitrite and nitrate in leaves was not affected by any of these stresses. Considering that protein tyrosine nitration is a potential marker of nitrosative stress, the results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.     

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.

     

Effect of S-nitrosoglutathione on renal mitochondrial function: a new mechanism for reversible regulation of manganese superoxide dismutase activity?

Mitochondria are at the heart of all cellular processes as they provide the majority of the energy needed for various metabolic processes. Nitric oxide has been shown to have numerous roles in the regulation of mitochondrial function. Mitochondria have enormous pools of glutathione (GSH≈5–10 mM). Nitric oxide can react with glutathione to generate a physiological molecule, S-nitrosoglutathione (GSNO). The impact GSNO has on mitochondrial function has been intensively studied in recent years, and several mitochondrial electron transport chain complex proteins have been shown to be targeted by GSNO. In this study we investigated the effect of GSNO on mitochondrial function using normal rat proximal tubular kidney cells (NRK cells). GSNO treatment of NRK cells led to mitochondrial membrane depolarization and significant reduction in activities of mitochondrial complex IV and manganese superoxide dismutase enzyme (MnSOD). MnSOD is a critical endogenous antioxidant enzyme that scavenges excess superoxide radicals in the mitochondria. The decrease in MnSOD activity was not associated with a reduction in its protein levels and treatment of NRK cell lysate with dithiothreitol (a strong sulfhydryl-group-reducing agent) restored MnSOD activity to control values. GSNO is known to cause both S-nitrosylation and S-glutathionylation, which involve the addition of NO and GS groups, respectively, to protein sulfhydryl (SH) groups of cysteine residues. Endogenous GSH is an essential mediator in S-glutathionylation of cellular proteins, and the current studies revealed that GSH is required for MnSOD inactivation after GSNO or diamide treatment in rat kidney cells as well as in isolated kidneys. Further studies showed that GSNO led to glutathionylation of MnSOD; however, glutathionylated recombinant MnSOD was not inactivated. This suggests that a more complex pathway, possibly involving the participation of multiple proteins, leads to MnSOD inactivation after GSNO treatment. The major highlight of these studies is the fact that dithiothreitol can restore MnSOD activity after GSNO treatment. To our knowledge, this is the first study showing that MnSOD activity can be reversibly regulated in vivo, through a mechanism involving thiol residues.     

Nitric oxide signaling in yeast

As a cellular signaling molecule, nitric oxide (NO) is widely conserved from microorganisms, such as bacteria, yeasts, and fungi, to higher eukaryotes including plants and mammals. NO is mainly produced by NO synthase (NOS) or nitrite reductase (NIR) activity. There are several NO detoxification systems, including NO dioxygenase (NOD) and S-nitrosoglutathione reductase (GSNOR). NO homeostasis based on the balance between NO synthesis and degradation is important for the regulation of its physiological functions because an excess level of NO causes nitrosative stress due to the high reactivity of NO and NO-derived compounds. In yeast, NO may be involved in stress responses, but NO and its signaling have been poorly understood due to the lack of mammalian NOS orthologs in the genome. Even though the activities of NOS and NIR have been observed in yeast cells, the gene encoding NOS and the NO production mechanism catalyzed by NIR remain unclear. On the other hand, yeast cells employ NOD and GSNOR to maintain an intracellular redox balance following endogenous NO production, exogenous NO treatment, or environmental stresses. This article reviews NO metabolism (synthesis, degradation) and its regulation in yeast. The physiological roles of NO in yeast, including the oxidative stress response, are also discussed here. Such investigations into NO signaling are essential for understanding the NO-dependent genetic and physiological modulations. In addition to being responsible for the pathology and pharmacology of various degenerative diseases, NO signaling may be a potential target for the construction and engineering of industrial yeast strains.     

ADH IB Expression,but Not ADH III,Is Decreased in Human Lung Cancer

Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer.     

A novel mechanism underlying the susceptibility of neuronal cells to nitric oxide: the occurrence and regulation of protein S-nitrosylation is the checkpoint

The susceptibility of neuronal cells to nitric oxide (NO) is a key issue in NO-mediated neurotoxicity. However, the underlying mechanism remains unclear. As a cyclic guanosine monophosphate (cGMP)-independent NO signaling pathway, S -nitrosylation (or S -nitrosation) has been suggested to occur as a post-translational modification in parallel with O-phosphorylation. The underlying mechanism of the involvement of protein S -nitrosylation in the susceptibility of neuronal cells to NO has been little investigated. In this study, we focused on the role of S -nitrosothiols (RSNO) in the susceptibility of a cerebellar cell line R2 to NO. Our results showed the following: (i) S -nitrosoglutathione (GSNO) induced a burst of RSNO in GSH-depleted R2 cells, the majority of which were primarily contributed by the S -nitrosylation of proteins (Pro-SNOs), and was followed by severe neuronal necrosis; (ii) the elevation in the level of Pro-SNOs resulted from a dysfunction of S -nitroglutathione reductase (GSNOR) as a result of its substrate, GSNO, being unavailable in GSH-depleted cells. In the meantime, the suppression of GSNOR increased NO-mediated neurotoxicity in R2 cells, as well as in cerebellar granule neurons; (iii) Our results also demonstrate that the burst of RSNO is the "checkpoint" of cell fate: if RSNO can be reduced to free thiol proteins, cells will survive; if they are further oxidized, cells will die; and (iv) GSH-ethyl ester and Vitamin C protected R2 cells against GSNO neurotoxicity through two distinct mechanisms: by inhibiting the elevation of Pro-SNOs and by reducing Pro-SNOs to free thiol proteins, respectively. A novel mechanism underlying the susceptibility of neuronal cells to NO is proposed and some potential strategies to prevent the NO-mediated neurotoxicity are discussed.