S-nitrosylation Levels Research Articles

Oral nitrite circumvents antiseptic mouthwash-induced disruption of enterosalivary circuit of nitrate and promotes nitrosation and blood pressure lowering effect

The nitric oxide (NO•) metabolites nitrite and nitrate exert antihypertensive effects by mechanisms that involve gastric formation of S-nitrosothiols. However, while the use of antiseptic mouthwash (AM) is known to attenuate the responses to nitrate by disrupting its enterosalivary cycle, there is little information about whether AM attenuates the effects of orally administered nitrite. We hypothesized that the antihypertensive effects of orally administered nitrite would not be prevented by AM because, in contrast to oral nitrate, oral nitrite could promote S-nitrosothiols formation in the stomach without intereference by AM. Chronic effects of oral nitrite or nitrate were studied in two-kidney, one-clip (2K1C) hypertensive rats (and normotensive controls) treated with AM (or vehicle) once/day. We found that orally administered nitrite exerts antihypertensive effects that were not affected by AM. This finding contrasts with lack of antihypertensive responses to oral nitrate in 2K1C hypertensive rats treated with AM. Nitrite and nitrate treatments increased plasma nitrites, nitrates, and S-nitrosothiols concentrations. However, while treatment with AM attenuated the increases in plasma nitrite concentrations after both nitrite and nitrate treatments, AM attenuated the increases in S-nitrosothiols in nitrate-treated rats, but not in nitrite-treated rats. Moreover, AM attenuated vascular S-nitrosylation (detected by the SNO-RAC method) after nitrate, but not after nitrite treatment. Significant correlations were found between the hypotensive responses and S-nitrosothiols, and vascular S-nitrosylation levels. These results show for the first time that oral nitrite exerts antihypertensive effects notwithstanding the fact that antiseptic mouthwash disrupts the enterosalivary circulation of nitrate. Our results support a major role for S-nitrosothiols formation resulting in vascular S-nitrosylation as a key mechanism for the antihypertensive effects of both oral nitrite and nitrate.

S-nitrosylation of Inhibitor-κB kinase: Identifying Novel Targets of Curcumin

In this study, we investigated the preventive effects of curcumin using LPS (Lipopolysaccharides)-induced Raw264.7 cells and the potential role of curcumin in regulation of anti-inflammation through S-nitrosylation. Western blot presented the protein expression of iNOS can be reduced by treated curcumin with 5, 10 and 15 μM separately for 12 and 24h. Consistently, pro-inflammatory cytokines, such as IL-1β, IL-6, TNFα and IFN-γ was also repressed. Moreover, 5, 10 and 15 μM curcumin reduced the amount of nitrite and nitrate in LPS-induced Raw264.7 cells maintained total S-nitrosylation level on proteins at 12h, the similar results was also observed at 24h that indicating curcumin inhibited NO oxidation. Furthermore, the protection of S-nitrosylation on IKKβ in LPS-induced Raw264.7 cells at 12h by curcumin caused the repression of IκB phosphorylation and NF-κB activation. In conclusion, this study verified that curcumin-mediated S-nitrosylation may be as an important regulator for anti-inflammation in LPS-induced Raw264.7 cells.

Hydrogen Sulfide Modulates the S-Nitrosoproteome and the Mitochondrial Morphology in Endothelial Cells.

Hydrogen sulfide (H2S) is one of the endogenous gaseous molecules promoting the production of nitric oxide (NO) which has cardioprotective functions. However, the role of the H2S-mediated protein S-nitrosoproteome and its subsequent physiological response remains unclear. Endothelial cells EAhy 926 were treated with 50 μM of H2S for 2 hours. The NO bound S-nitrosoproteins were purified by a biotin-switch and then digested by trypsin. Resulting peptides from control and H2S treatment were separately labeled by isobaric tag for relative and absolute quantitation 114/115, quantified by liquid chromatography tandem-mass spectrometry and analyzed by ingenuity pathway analysis (IPA) software. The microP software was applied to analyze the morphological changes of mitochondria. With the treatment of H2S, 416 S-nitrosylated proteins were identified. IPA analysis showed that these proteins were involved in five signaling pathways. The NO-bound cysteine residues and the S-nitrosylation levels (115/114) were shown for ten S-nitrosoproteins. Western blot further verified the S-nitrosylation of thioredoxin-dependant peroxide reductase, cytochrome c oxidase and cytochrome b-c1 complex that are involved in the mitochondrial signaling pathway. H2O2-induced mitochondrial swelling can be reduced by the pretreatment of H2S. The H2S-mediated endothelial S-nitrosoproteome has been confirmed. In the present study, we have proposed the cardioprotective role of H2S via maintaining mitochondrial homeostasis.

Hydrogen sulfide inhibits development of atherosclerosis through up-regulating protein S-nitrosylation.

Hydrogen sulfide (H2S) is an important gaseous signaling molecule that serves many important regulatory roles in physiological and pathophysiological conditions. H2S exerts an anti-atherosclerotic effect through mediating the biological functions of nitric oxide (NO). However, its mechanism of action is unclear. The purpose of this study is to explore the effect mechanism of H2S on the development of atherosclerosis with regard to protein S-nitrosylation. A total of 45 male apoE-/- mice were randomly divided into three groups. Atherosclerosis was induced by Western diet (21% fat and 0.15% cholesterol) with/without administration of a H2S donor (NaHS) or an endogenous cystathionine γ-lyase inhibitor (d, l-propargylglycine) for 12 weeks. After 12 weeks, plasma lipid and plasma NO levels were measured. Aortic gross lesion area and histopathological features of aortic lesion were determined. Additionally, the level of S-nitrosylated proteins in vascular smooth muscle cells (VSMCs) was detected using immunofluorescence in aorta. Rat VSMCs were performed in an in vitro experiment. Inducible nitric oxide synthase (iNOS) protein expression, NO generation, protein S-nitrosylation, and cell proliferation and migration were measured. We found that H2S significantly reduced the aortic atherosclerotic lesion area (P=0.006) and inhibited lipid and macrophage accumulation (P=0.004, P=0.002) and VSMC proliferation (P=0.019) in apoE-/- mice. H2S could up-regulate levels of plasma NO and protein S-nitrosylation in aorta VSMCs. However, d, l- propargylglycine had the opposite effect, increasing the lesion area and the content of lipids and macrophages in the lesions of apoE-/- mice and down-regulating plasma NO levels and protein S-nitrosylation in aorta VSMCs. In vitro experiments, H2S could significantly reverse the reduction of iNOS expression and NO generation induced by oxidized low-density lipoprotein in VSMCs. Moreover, H2S could increase the protein S-nitrosylation level of VSMCs in a dose-dependent manner, and the effect could be inhibited by iNOS inhibitors. In addition, proliferation and migration of VSMCs could be inhibited by H2S in a dose-dependent manner, which could be blocked by an iNOS inhibitor or protein S-nitrosylation removal agent. Our data suggest that H2S could inhibit the development of atherosclerosis by up-regulating plasma NO and protein S-nitrosylation, thereby inhibiting the proliferation and migration of VSMCs.

Curcumin represses the activity of inhibitor-κB kinase in dextran sulfate sodium-induced colitis by S-nitrosylation

In this study, we investigated the preventive effects of curcumin using dextran sulfate sodium (DSS)-induced colitis and the potential role of curcumin in regulation of anti-inflammation through S-nitrosylation. After curcumin treatment for 6days, the body weight and disease activity index of DSS-induced mice was alleviated and the colonic length was also rescued. Western blot presented that the protein expression of iNOS can be reduced by curcumin. Consistently, mRNA level of iNOS and pro-inflammatory cytokines, such as TNFα, IL-1β, and IL-6, was also repressed. Moreover, Curcumin reduced the amount of nitrite in DSS-induced colitis but not affected total S-nitrosylation level on proteins on day 6, indicating that curcumin inhibited NO oxidation. Furthermore, the protection of S-nitrosylation on IKKβ in DSS-induced colitis for 6days by curcumin caused the repression of IκB phosphorylation and NF-κB activation. In conclusion, this study verified that curcumin-mediated S-nitrosylation may be as an important regulator for anti-inflammation in DSS-induced colitis of mice.

The role of nitric oxide signalling in response to salt stress in Chlamydomonas reinhardtii

Nitric oxide signal and GSNOR activity play an essential role for Chlamydomonas reinhardtii response to salt stress. The unicellular alga Chlamydomonas reinhardtii is one of the most important model organisms phylogenetically situated between higher plants and animals. In the present study, we used comparative proteomics and physiological approaches to study the mechanisms underlying the response to salt stress in C. reinhardtii. We identified 74 proteins that accumulated differentially after salt stress, including oxidative enzymes and enzymes associated with nitric oxide (NO) metabolism, cell damage, and cell autophagy processes. A set of antioxidant enzymes, as well as S-nitrosoglutathione reductase (GSNOR) activity, were induced to balance the cellular redox status during short-term salt stress. Enzymes involved in DNA repair and cell autophagy also contribute to adaptation to short-term salt stress. However, under long-term salt stress, antioxidant enzymes and GSNOR were gradually inactivated through protein S-nitrosylation, leading to oxidative damage and a reduction in cell viability. Modulating the protein S-nitrosylation levels by suppressing GSNOR activity or adding thioredoxin affected the plant's adaptation to salt stress, through altering the redox status and DNA damage and autophagy levels. Based on these data, we propose that unicellular algae use multiple strategies to adapt to salt stress, and that, during this process, GSNOR activity and protein S-nitrosylation levels play important roles.

Nitric oxide and S-nitrosoglutathione function additively during plant immunity.

Nitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes. A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation. Here we show that endogenous NO accumulation in Arabidopsis, resulting from loss-of-function mutations in NO Overexpression 1 (NOX1), led to disabled Resistance (R) gene-mediated protection, basal resistance and defence against nonadapted pathogens. In nox1 plants both salicylic acid (SA) synthesis and signalling were suppressed, reducing SA-dependent defence gene expression. Significantly, expression of a GSNOR1 transgene complemented the SNO-dependent phenotypes of paraquat resistant 2-1 (par2-1) plants but not the NO-related characters of the nox1-1 line. Furthermore, atgsnor1-3 nox1-1 double mutants supported greater bacterial titres than either of the corresponding single mutants. Our findings imply that GSNO and NO, two pivotal redox signalling molecules, exhibit additive functions and, by extension, may have distinct or overlapping molecular targets during both immunity and development.

Modulation of Protein S-Nitrosylation by Isoprene Emission in Poplar.

Researchers have been examining the biological function(s) of isoprene in isoprene-emitting (IE) species for two decades. There is overwhelming evidence that leaf-internal isoprene increases the thermotolerance of plants and protects them against oxidative stress, thus mitigating a wide range of abiotic stresses. However, the mechanisms of abiotic stress mitigation by isoprene are still under debate. Here, we assessed the impact of isoprene on the emission of nitric oxide (NO) and the S-nitroso-proteome of IE and non-isoprene-emitting (NE) gray poplar (Populus × canescens) after acute ozone fumigation. The short-term oxidative stress induced a rapid and strong emission of NO in NE compared with IE genotypes. Whereas IE and NE plants exhibited under nonstressful conditions only slight differences in their S-nitrosylation pattern, the in vivo S-nitroso-proteome of the NE genotype was more susceptible to ozone-induced changes compared with the IE plants. The results suggest that the nitrosative pressure (NO burst) is higher in NE plants, underlining the proposed molecular dialogue between isoprene and the free radical NO Proteins belonging to the photosynthetic light and dark reactions, the tricarboxylic acid cycle, protein metabolism, and redox regulation exhibited increased S-nitrosylation in NE samples compared with IE plants upon oxidative stress. Because the posttranslational modification of proteins via S-nitrosylation often impacts enzymatic activities, our data suggest that isoprene indirectly regulates the production of reactive oxygen species (ROS) via the control of the S-nitrosylation level of ROS-metabolizing enzymes, thus modulating the extent and velocity at which the ROS and NO signaling molecules are generated within a plant cell.

Detection of S-Nitrosoglutathione Reductase Activity in Plants.

S-nitrosoglutathione reductase (GSNOR) is considered a key enzyme in the regulation of intracellular levels of S-nitrosoglutathione and protein S-nitrosylation. As a part of nitric oxide catabolism, GSNOR catalyzes the irreversible decomposition of GSNO to oxidized glutathione. GSNOR is involved in the regulation of plant growth and development, mediated by NO-dependent signaling mechanisms, and is known to play important roles in plant responses to various abiotic and biotic stress conditions. Here we present optimized protocols to determine GSNOR enzyme activities in plant samples by spectrophotometric measurements and by activity staining after the native polyacrylamide gel electrophoresis.

Abstract 19429: Trx1 Regulates Nitroso-redox Balance to Stimulate Autophagy and Cell Survival in Cardiomyocytes

Thioredoxin-1 (Trx1) is cardioprotective during oxidative stress. In addition to acting as an oxidoreductase, Trx1 also trans-nitrosylates other proteins. However, the role of Trx1-dependent S-nitrosylation in cardiomyocytes (CMs) is unknown. We investigated how Trx1 affects protein S-nitrosylation (SNO) and the nitroso-redox balance to regulate CM survival during glucose deprivation (GD). Mass spectrometry (MS/MS) analysis showed that Trx1 is S-nitrosylated at Cys73. Protein SNO levels were increased after 4 hrs of GD (1.64 fold, p<0.05; biotin switch assay). Overexpression of Trx1WT increased (3.94 fold), whereas knockdown of Trx1 (0.66 fold, p<0.01) or overexpression of Trx1C73S (0.77 fold, p<0.01) decreased, total protein SNO in response to GD, suggesting that Trx1 Cys73 regulates protein SNO in CMs during GD. Overexpression of Trx1 increased CM survival 24 hrs after GD (1.42 fold vs LacZ, p<0.05). Conversely, shTrx1 (2.13 fold vs control, p<0.01) or Trx1C73S (1.73 fold vs LacZ, p<0.01) increased cell death during GD. Either knockdown of Trx1 (LC3-II/Tubulin, 0.55 fold vs control) or overexpression of Trx1C73S (vs LacZ: LC3-II/Tubulin, 0.60 fold; autophagosomes, 0.83 fold, p<0.005; autolysosomes, 0.62 fold, p<0.005) significantly decreased autophagosome formation during GD. Increased interaction between Trx1 and a key autophagy protein, Atg7, was observed during GD and oxidative stress, as evaluated with the Trx1C35S trapping mutant. However, addition of DTT abolished this interaction, suggesting that Trx1 and Atg7 interact with one another via a disulfide linkage. By biotin switch assay and MS/MS analyses, we found that SNO-Trx1 can trans-nitrosylate Atg7 in vitro and that Atg7 is S-nitrosylated during GD in CMs. By labeling reduced and oxidized Cys residues in Atg7 with iodoacetamide and MMTS respectively, we determined that the level of Atg7 oxidation did not increase during oxidative stress in CMs, suggesting that Trx1 trans-nitrosylates Atg7 to prevent irreversible oxidative damage in Atg7. Taken all together, our results indicate that Trx1 promotes trans-nitrosylation of cellular proteins such as Atg7 to regulate the nitroso-redox balance and thus promote cell survival by stimulating autophagy during energy stress in CMs.

Abstract 12098: S-nitrosoglutathione Reductase Deficiency Prevents Neurological Injury After Cardiac Arrest and Cardiopulmonary Resuscitation

Introduction: Nitric oxide (NO) exerts many of its effects via protein S-nitrosylation (SNO). Protein SNO levels are critically controlled by the activity of denitrosylase S-nitrosoglutathione reductase (GSNOR). The objective of this study was to examine the impact of enhanced protein SNO induced by GSNOR deficiency on the outcomes of cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in mice. Methods and Results: Wild-type (WT) mice and mice deficient for GSNOR (GSNOR-/-) were subjected to CA for 8 min whereupon CPR was performed with chest compression and mechanical ventilation. There were no differences in characteristics before CA and during CPR except for lower mean blood pressure in GSNOR-/- mice at baseline. WT mice that were subjected to CA/CPR exhibited a poor 6-day survival rate (16.7%), depressed neurological function, and increased hydrogen peroxide production in the brain. In contrast, GSNOR-/- mice exhibited better neurological recovery, attenuated hydrogen peroxide production, and markedly improved 6-day survival rate (66.7%, P<0.05 vs WT). The protective effects of GSNOR deficiency were associated with markedly reduced mitochondria complex I activity in the brain at 5 min after CPR compared with sham-operated GSNOR-/- mice as evidenced by the ATP turnover rate in isolated cerebral mitochondria. Administration of a mitochondria-targeted S-nitrosothiol MitoSNO to WT mice 1min before CPR similarly depressed cerebral mitochondrial complex I activity and improved neurological outcomes, and survival rate. These results suggest that SNO-based inhibition of mitochondrial activity prevents ischemic neurological injury after CA and CPR. Conclusions: GSNOR-deficiency improves outcomes after CA and CPR in mice at least in part by modulating mitochondrial complex I activity and reducing reactive oxygen species generation during early reperfusion phase. Pharmacological augmentation of protein SNO may limit long term neurological impairment and death after CA/CPR.

Roles of sodium hydrosulfide and sodium nitroprusside as priming molecules during drought acclimation in citrus plants.

Emerging evidence suggests that the gaseous molecules hydrogen sulfide (H2S) and nitric oxide (NO) enhances plant acclimation to stress; however, the underlying mechanism remains unclear. In this work, we explored if pretreatment of citrus roots with NaHS (a H2S donor) or sodium nitroprusside (SNP, a NO donor) for 2days (d) could elicit long-lasting priming effects to subsequent exposure to PEG-associated drought stress for 21d following a 5d acclimation period. Detailed physiological study documented that both pretreatments primed plants against drought stress. Analysis of the level of nitrite, NOx, S-nitrosoglutahione reductase, Tyr-nitration and S-nitrosylation along with the expression of genes involved in NO-generation suggested that the nitrosative status of leaves and roots was altered by NaHS and SNP. Using a proteomic approach we characterized S-nitrosylated proteins in citrus leaves exposed to chemical treatments, including well known and novel S-nitrosylated targets. Mass spectrometry analysis also enabled the identification of 42 differentially expressed proteins in PEG alone-treated plants. Several PEG-responsive proteins were down-regulated, especially photosynthetic proteins. Finally, the identification of specific proteins that were regulated by NaHS and SNP under PEG conditions provides novel insight into long-term drought priming in plants and in a fruit crop such as citrus in particular.

Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress

BackgroundS-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. MethodsIn this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. ResultsWe found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. ConclusionsInhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. General significanceIdentification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

S-Nitrosylation of Calcium-Handling Proteins in Cardiac Adrenergic Signaling and Hypertrophy.

The regulation of calcium (Ca(2+)) homeostasis by β-adrenergic receptor (βAR) activation provides the essential underpinnings of sympathetic regulation of myocardial function, as well as a basis for understanding molecular events that result in hypertrophic signaling and heart failure. Sympathetic stimulation of the βAR not only induces protein phosphorylation but also activates nitric oxide-dependent signaling, which modulates cardiac contractility. Nonetheless, the role of nitric oxide in βAR-dependent regulation of Ca(2+) handling has not yet been explicated fully. To elucidate the role of protein S-nitrosylation, a major transducer of nitric oxide bioactivity, on βAR-dependent alterations in cardiomyocyte Ca(2+) handling and hypertrophy. Using transgenic mice to titrate the levels of protein S-nitrosylation, we uncovered major roles for protein S-nitrosylation, in general, and for phospholamban and cardiac troponin C S-nitrosylation, in particular, in βAR-dependent regulation of Ca(2+) homeostasis. Notably, S-nitrosylation of phospholamban consequent upon βAR stimulation is necessary for the inhibitory pentamerization of phospholamban, which activates sarcoplasmic reticulum Ca(2+)-ATPase and increases cytosolic Ca(2+) transients. Coincident S-nitrosylation of cardiac troponin C decreases myocardial sensitivity to Ca(2+). During chronic adrenergic stimulation, global reductions in cellular S-nitrosylation mitigate hypertrophic signaling resulting from Ca(2+) overload. S-Nitrosylation operates in concert with phosphorylation to regulate many cardiac Ca(2+)-handling proteins, including phospholamban and cardiac troponin C, thereby playing an essential and previously unrecognized role in cardiac Ca(2+) homeostasis. Manipulation of the S-nitrosylation level may prove therapeutic in heart failure.

Unravelling the reactive oxygen and reactive nitrogen signalling networks in plants.

Unravelling the reactive oxygen and reactive nitrogen signalling networks in plants.

Hypercysteinemia promotes atherosclerosis by reducing protein S-nitrosylation

Protein S-nitrosylation plays important role in the regulation of cardiovascular functions in nitric oxide (NO) Pathway. Hypercysteinemia (HHcy) is an independently risk factor for atherosclerosis. We hypothesized that HHcy promotes atherosclerosis by reducing level of vascular protein S-nitrosylation. The aim of present study is to investigate effect of HHcy on vascular protein S-nitrosylation. A total of 45 male apoE−/− mice were randomly divided into three groups. The control group was fed a Western-type diet. The HHcy group was fed a diet containing 4.4% l-methionine, and the HHcy+NONOate group was fed a diet containing 4.4% l-methionine and administrated NONOate (ip). Human umbilical vein endothelial cells were performed for in vitro experiment. Plasma lipids were measured every 4 weeks. After 12 weeks, aortic atherosclerotic lesion areas were detected as well as cellular components. The levels of plasma homocysteine (Hcy) and NO were measured. S-nitrosylation was detected using immunofluorescence, and further confirmed by biotin switch method. We found that compared with the control group, Hcy levels, and atherosclerotic plaque, and content of vascular smooth muscle cells and macrophages in lesions significantly increased, and levels of NO significantly decreased in the HHcy group. However, NONOate reverses this effect. In addition, Hcy significantly reduced protein S-nitrosylation in human umbilical vein endothelial cells. This reduction of protein S-nitrosylation was accompanied by reduced levels of NO. Our results suggested that Hcy promoted atherosclerosis by inhibiting vascular protein S-nitrosylation.

NO Better Way to Protect the Heart during Ischemia-Reperfusion: To be in the Right Place at the Right Time.

Acute myocardial infarction (MI) is one of the leading causes of mortality worldwide. MI is the heart muscle irreversible death secondary to prolonged ischemia. Over the last few decades, medical progress in how and when to restore blood flow to the ischemic area have markedly improved patient survival. Although early heart reperfusion is acknowledged to be the most effective way to limit infarct size, post-ischemic reperfusion is associated with detrimental effects, such as myocardial stunning, ventricular arrhythmias, microvascular dysfunction, and cell death. The molecular mechanisms of these reperfusion injuries remain to be elucidated and their management is very challenging. Among the various therapeutic molecular approaches proposed by experimental studies, nitric oxide (NO) role in protecting heart against MI and reperfusion injuries has been widely assessed and discussed (1–4). NO is a gasotransmitter that is abundantly produced in the cardiovascular system mainly by the NO synthase (NOS) enzymes system. Two isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS), are constitutively expressed in both myocardium and vessels, whereas inducible NOS (iNOS) is detected only in pathological conditions, such as inflammatory and/or oxidative stress. Both eNOS and nNOS are low-NO output Ca2+-dependent enzymes, while iNOS is a high-NO output Ca2+-independent enzyme. In physiological conditions, NOS form homodimers (“coupled” NOS) that catalyze NO production from l-arginine and O2 through electron transfer from NADPH on the reductase domain of one monomer to the oxidase domain of the second monomer. In pathological conditions, such as in the absence of the essential cofactor tetrahydrobiopterin (BH4), eNOS can be “uncoupled” to produce O2− instead of NO. In stress conditions, NO protects tissues through two distinct pathways. In the first one, NO activates the soluble guanylate cyclase (sGC) that initiates cyclic guanosine monophosphate (cGMP) production, leading to the activation of protein kinase G (PKG). As sGC is the major cell receptor for NO and the NO/sGC/cGMP/PKG pathway plays a critical role in both myocardium excitation–contraction coupling and cardiovascular function regulation (5–8), NO cardioprotective role was first attributed to PKG activation (9–11). However, a second pathway in which proteins are directly modified by NO addition to sulfhydryl residues, a process known as S-nitrosylation (SNO), has recently emerged in the scientific literature. Although PKG activation pathway has been largely involved in NO-mediated cardioprotection (11–13), SNO is now taking the front stage and is considered to be a key player in cardioprotection through (i) the transient modification of protein activity and/or (ii) their protection from irreversible oxidation (14–17). Indeed, Sun et al. (18) showed that reduced heart vulnerability to ischemia–reperfusion (IR) following acute ischemic preconditioning is mainly related to SNO signaling and not to PKG activation through the NO–SGC–cGMP pathway. Accordingly, we found that in exercise training-induced cardioprotection against IR injuries, protein SNO level, but not cGMP level, increased during early reperfusion (19). The same year, Methner et al. (20), using a Cre/loxP approach to selectively ablate type I PKG in cardiomyocytes, demonstrated that ischemic post-conditioning reduced infarct size in these mice like in wild type controls. Moreover, they showed that the cardioprotective effect against IR injury of mitochondria-targeted S-nitrosothiol (MitoSNO), which allows NO and S-nitrosothiol accumulation in mitochondria, was comparable in mice that specifically lack PKG in cardiomyocytes and in controls. This indicates that MitoSNO cardioprotective effect is independent of PKG. The current literature strongly supports NO implication in cardioprotection. However, the mechanism is still debated and whether increased NO availability during IR is cytoprotective remains to be demonstrated. Here, we discuss how NO might contribute to protect heart and particularly the importance of NO (i) localization, (ii) concentration, and (iii) time of availability during IR.

Abstract 20500: Trx1 Promotes Trans-Nitrosylation of Cellular Proteins to Stimulate Autophagy and Cell Survival

Thioredoxin-1 (Trx1) is cardioprotective during oxidative stress, mainly through its antioxidant activity. Trx1 is also S-nitrosylated and, in turn, trans-nitrosylates other proteins. However, the role of Trx1-dependent S-nitrosylation in cardiomyocytes (CMs) is unknown. Here, we investigated the role of Trx1-mediated protein S-nitrosylation in the regulation of CM survival during stress in vitro. Using biotin-switch assays, we found that wild-type Trx1 (Trx1WT) is S-nitrosylated at baseline, but the extent of S-nitrosylation was attenuated in Trx1C73S, suggesting that Trx1 is S-nitrosylated at Cys73. Trx1WT and Trx1C73S do not differ in their redox activity, as determined by Amplex Red assays. Cellular protein S-nitrosylation levels were increased after 4 hours of glucose deprivation (GD), an energy stress condition (1.64±0.27 fold, p<0.05), as determined by biotin switch assays. Overexpression of Trx1WT increased (3.94-fold), whereas knockdown of Trx1 (0.66±0.01 fold, p<0.01) or overexpression of Trx1C73S (0.77±0.02 fold, p<0.01) decreased, total protein S-nitrosylation in response to GD. These results suggest that Trx1C73 regulates protein S-nitrosylation in CMs during GD. Overexpression of Trx1 increased CM survival after 24 hours of GD (1.42±0.08 fold vs LacZ, p<0.05), as evaluated with propidium iodide assays. Conversely, shTrx1 (2.13±0.05 fold vs control, p<0.01) or Trx1C73S (1.73±0.034 fold vs LacZ, p<0.01) increased cell death during GD. Either knockdown of Trx1 (LC3-II/Tubulin: 0.55 fold vs control) or overexpression of Trx1C73S (vs LacZ: LC3-II/Tubulin, 0.60 fold; autophagosomes, 0.83±0.16-fold, p<0.005; autolysosomes, 0.62±0.13-fold, p<0.005) significantly decreased autophagy during GD. Mechanistically, Trx1 co-immunoprecipitates with Atg7, an E1-like protein which plays a critical role in mediating autophagy. Using mass spectroscopy analyses, we found that SNO-Trx1 can trans-nitrosylate Atg7 in vitro. These results suggest that Trx1 trans-nitrosylates Atg7 during GD. Taken all together, our results indicate that Trx1 promotes trans-nitrosylation of cellular proteins, including Atg7, and autophagy, thereby promoting cell survival during energy stress in CMs.

Glyceraldehyde-3-phosphate dehydrogenase acts as a mitochondrial trans-S-nitrosylase in the heart.

Mitochondrial proteins have been shown to be common targets of S-nitrosylation (SNO), but the existence of a mitochondrial source of nitric oxide remains controversial. SNO is a nitric oxide-dependent thiol modification that can regulate protein function. Interestingly, trans-S-nitrosylation represents a potential pathway for the import of SNO into the mitochondria. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown to act as a nuclear trans-S-nitrosylase, has also been shown to enter mitochondria. However, the function of GAPDH in the mitochondria remains unknown. Therefore, we propose the hypothesis that S-nitrosylated GAPDH (SNO-GAPDH) interacts with mitochondrial proteins as a trans-S-nitrosylase. In accordance with this hypothesis, SNO-GAPDH should be detected in mitochondrial fractions, interact with mitochondrial proteins, and increase mitochondrial SNO levels. Our results demonstrate a four-fold increase in GAPDH levels in the mitochondrial fraction of mouse hearts subjected to ischemic preconditioning, which increases SNO-GAPDH levels. Co-immunoprecipitation studies performed in mouse hearts perfused with the S-nitrosylating agent S-nitrosoglutathione (GSNO), suggest that SNO promotes the interaction of GAPDH with mitochondrial protein targets. The addition of purified SNO-GAPDH to isolated mouse heart mitochondria demonstrated the ability of SNO-GAPDH to enter the mitochondrial matrix, and to increase SNO for many mitochondrial proteins. Further, the overexpression of GAPDH in HepG2 cells increased SNO for a number of different mitochondrial proteins, including heat shock protein 60, voltage-dependent anion channel 1, and acetyl-CoA acetyltransferase, thus supporting the role of GAPDH as a potential mitochondrial trans-S-nitrosylase. In further support of this hypothesis, many of the mitochondrial SNO proteins identified with GAPDH overexpression were no longer detected with GAPDH knock-down or mutation. Therefore, our results suggest that SNO-GAPDH can act as a mitochondrial trans-S-nitrosylase, thereby conferring the transfer of SNO from the cytosol to the mitochondria.

Effect of exogenous nitric oxide on antioxidative system and S-nitrosylation in leaves of Boehmeria nivea (L.) Gaud under cadmium stress.

Cadmium (Cd)-induced growth inhibition is one of the primary factors limiting phytoremediation effect of Boehmeria nivea (L.) Gaud in contaminated soil. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been evidenced to alleviate Cd toxicity in many plants. However, as an important mechanism of NO in orchestrating cellular functions, S-nitrosylation is still poorly understood in its relation with Cd tolerance of plants. In this study, higher exogenous NO levels were found to coincide with higher S-nitrosylation level expressed as content of S-nitrosothiols (SNO). The addition of low concentration (100 μM) SNP increased the SNO content, and it simultaneously induced an alleviating effect against Cd toxicity by enhancing the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) and reduced the accumulation of H2O2 as compared with Cd alone. Application of S-nitrosoglutathione reductase (GSNOR) inhibitors dodecanoic acid (DA) in 100 μM SNP group brought in an extra elevation in S-nitrosylation level and further reinforced the effect of SNP. While the additions of 400 μM SNP and 400 μM SNP + 50 μM DA further elevated the S-nitrosylation level, it markedly weakened the alleviating effect against Cd toxicity as compared with the addition of 100 μM SNP. This phenomenon could be owing to excess consumption of glutathione (GSH) to form SNO under high S-nitrosylation level. Therefore, the present study indicates that S-nitrosylation is involved in the ameliorating effect of SNP against Cd toxicity. This involvement exhibited a concentration-dependent property.