Endogenous S-nitrosylation Research Articles

Physiological role for S-nitrosylation of RyR1 in skeletal muscle function and development

Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca2+ release from the sarcoplasmic reticulum through the ryanodine receptor/Ca2+-release channel RyR1. The RyR1 contains ∼100 Cys thiols of which ∼30 comprise an allosteric network subject to posttranslational modification by S-nitrosylation, S-palmitoylation and S-oxidation. However, the role and function of these modifications is not understood. Although aberrant S-nitrosylation of multiple unidentified sites has been associated with dystrophic diseases, malignant hyperthermia and other myopathic syndromes, S-nitrosylation in physiological situations is reportedly specific to a single (1 of ∼100) Cys in RyR1, Cys3636 in a manner gated by pO2. Using mice expressing a form of RyR1 with a Cys3636→Ala point mutation to prevent S-nitrosylation at this site, we showed that Cys3636 was the principal target of endogenous S-nitrosylation during normal muscle function. The absence of Cys3636 S-nitrosylation suppressed stimulus-evoked Ca2+ release at physiological pO2 (at least in part by altering the regulation of RyR1 by Ca2+/calmodulin), eliminated pO2 coupling, and diminished skeletal myocyte contractility in vitro and measures of muscle strength in vivo. Furthermore, we found that abrogation of Cys3636 S-nitrosylation resulted in a developmental defect reflected in diminished myofiber diameter, altered fiber subtypes, and altered expression of genes implicated in muscle development and atrophy. Thus, our findings establish a physiological role for pO2-coupled S-nitrosylation of RyR1 in skeletal muscle contractility and development and provide foundation for future studies of RyR1 modifications in physiology and disease.

An improved sulfur-nitroso-proteome strategy for global profiling of sulfur-nitrosylated proteins and sulfur-nitrosylation sites in mice

Comprehensive sulfur-nitrosylation (SNO) proteome coverage in complex biological systems remains challenging as a result of the low level of endogenous S-nitrosylation and its chemical instability. Herein, we optimized the synthesis route of SNOTRAP (SNO trapping by triaryl phosphine) probe and the proteomics pipeline (including preventing over-alkylation, sample washing, trypsin digestion). Preventing overalkylation was found to be the key factor resulting in a higher number of identified SNO proteins by evaluating various experimental conditions. With the improved SNOTRAP-based proteomic pipeline, we achieved an improvement of ∼10-fold on identification efficiency, and identified 1181 SNO proteins (1714 SNO sites) in mouse brain, representing the largest repository of endogenous S-nitrosylation. Moreover, we identified the consensus motif of SNO sites, suggesting the correlation with local hydrophobicity, acid-base catalysis, and the surrounding secondary structures for modification of specific cysteines by NO. Collectively, we provide a universal pipeline for the high-coverage identification of low-abundance SNO proteins with high enrichment efficiency, high specificity (98%), good reproducibility, and easy implementation, contributing to the elucidation of the mechanism(s) of nitrosative stress in multiple diseases.

Proteome profiling of endogenous and potential S-nitrosylation in colorectal cancer.

As a common cancer with high incidence rate and mortality, colorectal cancer (CRC) is seriously threatening human health. S-nitrosylation (SNO) proteins mediated by nitric oxide (NO) has important implications in the genesis, progression, and apoptosis of CRC. It's worth noting that the SNO proteins also play an important role in the tumor endocrine and metabolic pathways of CRC. In this study, the protein extracts of human tissues and cell lines were treated by biotin switch technology and magnetic beads enrichment. The proteomic results of endogenous and potential SNO proteins were analyzed by mass spectrometry (MS). Through the comparison and analysis of MS results, Gene Ontology (GO) analysis, and literatures, some endogenous and potential SNO proteins were identified in CRC, which were closely related to the tumor endocrine and metabolic pathways, the apoptotic signaling pathways, protein maturation, and other biological processes of the proliferation and apoptosis of CRC cells. A total of 19 proteins containing potential or endogenous SNO sites were detected in both human cancer tissue and SW 480 cells. Through the cross validation of MS results, GO analysis, and literatures, several SNO proteins were identified frequently in CRC, such as the actin, cytoplasmic 1 (ACTB), peroxiredoxin-4 (PRDX4), protein S100A8 (S100A8), pyruvate kinase PKM (PKM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which were closely related to the tumor endocrine and metabolic pathways and the apoptotic signaling pathways of CRC. Different CRC cells and tissues contained potential and endogenous SNO modified proteins. In addition, some SNO proteins could participate in the proliferation, metastasis and apoptosis of CRC by regulating the tumor endocrine and metabolic pathways.

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

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

H2S-induced NO/SNO positively promotes betulin production in Betula platyphylla

S-nitrosoglutathione reductase (GSNOR) is the pivotal enzyme that mediates cellular levels of nitric oxide (NO) and S-nitrosylation (SNO) in plants. We have previously reported that GSNOR deficiency enhanced betulin production. However, the physiological function of NO/SNO in plants remains unclear, especially for SNO. This study aimed to reveal the role of hydrogen sulfide (H2S)-induced NO/SNO on betulin production in birch (Betula platyphylla). Results showed that the H2S donor sodium hydrosulfide(NaHS)enhanced the production rates of NO, SNO and betulin in birch suspension cells to maximum levels, which was 92.59%, 50.09% and 122.58% higher than that in the control, at 24 h after 1 mmol·L−1 NaHS treatment. NaHS also promoted NO burst, SNO production and the gene expression of farnesyl pyrophosphate synthase, lupeol synthase and squalene synthetase related to betulin synthesis in birch GSNOR transgenic plants by RNA interference silencing. Similarly, pretreatment of the birch suspension cells with the GSNOR inhibitor 3-(5-(4-(1H-imidazol-1-yl) phenyl)-1-(4-carbamoyl-2-methylphenyl)- 1H-pyrrol-2-yl) propionic acid (N6022) promoted NaHS-triggered NO/SNO and betulin production, and the betulin content under NaHS and N6022 treatment was significantly higher than that under NaHS treatment or NaHS and NO specific scavenger 2-(4-carboxyphenyl)-4,4,5,5 -tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). However, pretreatment of the birch suspension cells with cPTIO or NO synthase inhibitor (sodium azide or NG-nitro-L-Arg methyl ester) significantly decreased NaHS-triggered NO/SNO and betulin production. Overall, the above results first verified that endogenous SNO mediated hydrogen sulfide-induced betulin production at the pharmacological and genetic levels, and the homeostasis of NO/SNO mediated hydrogen sulfide-induced betulin production. This study provides new insights into the homeostasis of NO/SNO in plant secondary metabolite production.

Protein Cysteinyl-S-Nitrosylation: Analysis and Quantification.

The thiol moiety of cysteine residues can undergo a number of biologic modifications including oxidation, sulfenylation, nitrosylation, persulfidation, metalation, and other modifications. These modifications can control biological function, including gain as well as loss of function. Herein, we focus attention on the proteomic analysis of S-nitrosylation in health and disease. We describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. We present several studies where suitability of this approach for investigating endogenous S-nitrosylation is addressed.

An Interplay of S-Nitrosylation and Metal Ion Binding for Astrocytic S100B Protein.

Mammalian S100B protein plays multiple important roles in cellular brain processes. The protein is a clinically used marker for several pathologies including brain injury, neurodegeneration and cancer. High levels of S100B released by astrocytes in Down syndrome patients are responsible for reduced neurogenesis of neural progenitor cells and induction of cell death in neurons. Despite increasing understanding of S100B biology, there are still many questions concerning the detailed molecular mechanisms that determine specific activities of S100B. Elevated overexpression of S100B protein is often synchronized with increased nitric oxide-related activity. In this work we show S100B is a target of exogenous S-nitrosylation in rat brain protein lysate and identify endogenous S-nitrosylation of S100B in a cellular model of astrocytes. Biochemical studies are presented indicating S-nitrosylation tunes the conformation of S100B and modulates its Ca2+ and Zn2+ binding properties. Our in vitro results suggest that the possibility of endogenous S-nitrosylation should be taken into account in the further studies of in vivo S100B protein activity, especially under conditions of increased NO-related activity.

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

NO regulates a variety of physiological processes, including cell proliferation, differentiation, and inflammation. S-nitrosylation, a NO-mediated reversible protein modification, leads to changes in the activity and function of proteins. In particular, the role of S-nitrosylation during adipogenesis is largely unknown. We hypothesized that the normal physiological levels of NO, but not the excess levels generated under severe conditions, such as inflammation, may be critically involved in the proper regulation of adipogenesis. We found that endogenous S-nitrosylation of proteins was required for adipocyte differentiation. By performing a biotin-switch assay, we identified FAS, a key lipogenic enzyme in adipocytes, as a target of S-nitrosylation during adipogenesis. Interestingly, we also observed that the dimerization of FAS increased in parallel with the amount of S-nitrosylated FAS during adipogenesis. In addition, we found that exogenous NO enhanced the dimerization and the enzymatic activity of FAS. Moreover, site-directed mutagenesis of three predicted S-nitrosylation sites indicated that S-nitrosylation of FAS at Cys(1471)and Cys(2091), but not at Cys(1127), increased its enzymatic activity. Taken together, these results suggest that the S-nitrosylation of FAS at normal physiological levels of NO increases its activity through dimerization and may contribute to the proper regulation of adipogenesis.

P76 - Measuring site occupancy: a new perspective on cysteine oxidation

Site occupancy is an extremely important aspect of quantification of protein modifications. Knowing the degree of modification of each oxidised cysteine residue is critical to understanding the biological role of these modifications. Yet modification site occupancy is very often overlooked, in part because there are very few analytical tools that allow such measurements. Here we present a new strategy, which provides quantitative analysis of cysteine S-nitrosylation (SNO) and S-sulfenylation (SOH) simultaneously at the resolution of single cysteine and allows for determination of relative oxidation occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance.We make use of high resolution mass spectrometry. The method relies on differential reduction of “total” cysteines, SNO cysteines and SOH cysteines with TCEP, sodium ascorbate and sodium arsenite respectively followed by iodoTMTTM alkylation. Enrichment of iodoTMTTM-containing peptides is performed using anti-TMT antibody. In vivo model of mild oxidative stress in Escherichia coli is used. To induce endogenous SNO bacteria were grown anaerobically in minimal media supplemented with fumarate or nitrate. Short-term treatment with submilimolar levels of hydrogen peroxide were used to induce SOH.We have quantified 114 SNO/SOH modified peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site occupancy in cellular redox response.

Global Analysis of S-nitrosylation Sites in the Wild Type (APP) Transgenic Mouse Brain-Clues for Synaptic Pathology

Alzheimer's disease (AD) is characterized by an early synaptic loss, which strongly correlates with the severity of dementia. The pathogenesis and causes of characteristic AD symptoms are not fully understood. Defects in various cellular cascades were suggested, including the imbalance in production of reactive oxygen and nitrogen species. Alterations in S-nitrosylation of several proteins were previously demonstrated in various AD animal models and patients. In this work, using combined biotin-switch affinity/nano-LC-MS/MS and bioinformatic approaches we profiled endogenous S-nitrosylation of brain synaptosomal proteins from wild type and transgenic mice overexpressing mutated human Amyloid Precursor Protein (hAPP). Our data suggest involvement of S-nitrosylation in the regulation of 138 synaptic proteins, including MAGUK, CamkII, or synaptotagmins. Thirty-eight proteins were differentially S-nitrosylated in hAPP mice only. Ninety-five S-nitrosylated peptides were identified for the first time (40% of total, including 33 peptides exclusively in hAPP synaptosomes). We verified differential S-nitrosylation of 10 (26% of all identified) synaptosomal proteins from hAPP mice, by Western blotting with specific antibodies. Functional enrichment analysis linked S-nitrosylated proteins to various cellular pathways, including: glycolysis, gluconeogenesis, calcium homeostasis, ion, and vesicle transport, suggesting a basic role of this post-translational modification in the regulation of synapses. The linkage of SNO-proteins to axonal guidance and other processes related to APP metabolism exclusively in the hAPP brain, implicates S-nitrosylation in the pathogenesis of Alzheimer's disease.

Decoding the s-nitrosoproteomic atlas in individualized human colorectal cancer tissues using a label-free quantitation strategy.

The abnormal S-nitrosylation induced by the overexpression and activation of inducible nitric oxide synthase (iNOS) modulates many human diseases, such as inflammation and cancer. To delineate the pathophysiological S-nitrosoproteome in cancer patients, we report an individualized S-nitrosoproteomic strategy with a label-free method for the site-specific quantification of S-nitrosylation in paired tumor and adjacent normal tissues from 11 patients with colorectal cancer (CRC). This study provides not only the first endogenous human S-nitrosoproteomic atlas but also the first individualized human tissue analysis, identifying 174 S-nitrosylation sites in 94 proteins. Fourteen novel S-nitrosylation sites with a high frequency of elevated levels in 11 individual patients were identified. An individualized S-nitrosylation quantitation analysis revealed that the detected changes in S-nitrosylation were regulated by both the expression level and the more dramatic post-translational S-nitrosylation of the targeted proteins, such as thioredoxin, annexin A4, and peroxiredoxin-4. These endogenous S-nitrosylated proteins illustrate the network of inflammation/cancer-related and redox reactions mediated by various S-nitrosylation sources, including iNOS, transnitrosylase, or iron-sulfur centers. Given the demonstrated sensitivity of individualized tissue analysis, this label-free approach may facilitate the study of the vastly under-represented S-nitrosoproteome and enable a better understanding of the effect of endogenous S-nitrosylation in cancer.

Attenuated response of L-type calcium current to nitric oxide in atrial fibrillation

Nitric oxide (NO) synthesized by cardiomyocytes plays an important role in the regulation of cardiac function. Here, we studied the impact of NO signalling on calcium influx in human right atrial myocytes and its relation to atrial fibrillation (AF). Right atrial appendages (RAAs) were obtained from patients in sinus rhythm (SR) and AF. The biotin-switch technique was used to evaluate endogenous S-nitrosylation of the α1C subunit of L-type calcium channels. Comparing SR to AF, S-nitrosylation of Ca(2+) channels was similar. Direct effects of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) on L-type calcium current (ICa,L) were studied in cardiomyocytes with standard voltage-clamp techniques. In SR, ICa,L increased with SNAP (100 µM) by 48%, n/N = 117/56, P < 0.001. The SNAP effect on ICa,L involved activation of soluble guanylate cyclase and protein kinase A. Specific inhibition of phosphodiesterase (PDE)3 with cilostamide (1 µM) enhanced ICa,L to a similar extent as SNAP. However, when cAMP was elevated by PDE3 inhibition or β-adrenoceptor stimulation, SNAP reduced ICa,L, pointing to cGMP-cAMP cross-regulation. In AF, the stimulatory effect of SNAP on ICa,L was attenuated, while its inhibitory effect on isoprenaline- or cilostamide-stimulated current was preserved. cGMP elevation with SNAP was comparable between the SR and AF group. Moreover, the expression of PDE3 and soluble guanylate cyclase was not reduced in AF. NO exerts dual effects on ICa,L in SR with an increase of basal and inhibition of cAMP-stimulated current, and in AF NO inhibits only stimulated ICa,L. We conclude that in AF, cGMP regulation of PDE2 is preserved, but regulation of PDE3 is lost.

S-nitrosylation regulates mitochondrial quality control via activation of parkin

Parkin, a ubiquitin E3 ligase of the ring between ring fingers family, has been implicated in mitochondrial quality control. A series of recent reports have suggested that the recruitment of parkin is regulated by phosphorylation. However, the molecular mechanism that activates parkin to induce mitochondrial degradation is not well understood. Here, and in contrast to previous reports that S-nitrosylation of parkin is exclusively inhibitory, we identify a previously unrecognized site of S-nitrosylation in parkin (Cys323) that induces mitochondrial degradation. We demonstrate that endogenous S-nitrosylation of parkin is in fact responsible for activation of its E3 ligase activity to induce aggregation and degradation. We further demonstrate that mitochondrial uncoupling agents result in denitrosylation of parkin, and that prevention of denitrosylation restores mitochondrial degradation. Our data indicates that NO both positive effects on mitochondrial quality control, and suggest that targeted S-nitrosylation could provide a novel therapeutic strategy against Parkinson's disease.

Abstract 239: Nitric Oxide Suppresses the Angiogeneic Potential of Mesenchymal Stem Cells

Introduction: Mesenchymal stem cells (MSCs) participate in blood vessel formation which is crucial for tissue regeneration. However, the mechanism involved in regulating MSC angiogenesis remains elusive. We examined the role of Nitric Oxide (NO) in this process by comparing angiogenesis by MSCs derived from wild type mice and mice lacking S-nitrosoglutathione reductase (GSNOR-/-), an enzyme that increases intracellular GSNO and enhances endogenous S-nitrosylation (a ubiquitous redox-related modification of cysteine thiol by NO) Hypothesis: Nitric oxide signaling pathways are mediated via small molecular thiols that regulate MSC angiogenesis. Method: Bone marrow-derived MSCs were isolated from wild type and GSNOR-/- mice and grown for 7 days in endothelial growth media followed by 24h in Matrigel (in vitro angiogenesis assay) in the presence of either vehicle, 15µM L-NAME (a nitric oxide synthase (NOS) inhibitor) or 500µM SNAP (a nitric oxide donor). Human (h)MSCs were similarly treated with vehicle, 15µM L-NAME and 100µM SNAP. MSC NO production and NOS expression were assessed. Results: Both types of MSCs exhibited similar levels of NO production and NOS expression, however, in the Matrigel Assay, MSCs from GSNOR-/- mice formed fewer (29.9±12.0 vs.. 50.0±15.4, p&lt;0.001) and shorter (62.8 ± 34.6 µm vs. 105.9±57.0 µm, p&lt;0.001) tubes than MSCs derived from wild type mice. L-NAME treatment normalized GSNOR-/- tube number (49.9 ± 14.8, p&lt;0.001) and length (82.8±42.5 µm, p&lt;0.001). Treatment of wild type MSCs with SNAP impaired tube formation (15.6±9.0, p&lt;0.001) and length (57.1±24.9 µm, p&lt;0.001). Similarly, hMSCs treated with SNAP exhibited impaired tube formation, both in number (25.0±4.9 vs. 49.2±8.1, p&lt;0.001) and length (81±29.1 vs. 128.0±52.5 µm, p&lt;0.001) compared to vehicle treated MSCs. L-NAME had no effect on hMSCs . Conclusion: These findings are consistent with a paradoxical negative influence of NO/GSNO on vascular formation by MSCs and reveal a novel mechanism whereby NO/GSNO inhibits MSC dependent angiogenesis. These findings have implications for disease states characterized by excessive or dysregulated angiogenesis; moreover NO deficient environments may trigger MSC mediated angiogenesis.

Proteomics investigation of endogenous S-nitrosylation in Arabidopsis

S-Nitrosylation emerges as an important protein modification in many processes. However, most data were obtained at the protein level after addition of a NO donor, particularly in plants where information about the cysteines nitrosylated in these proteins is scarce. An adapted work-flow, combining the classical biotin switch method and labeling with isotope-coded affinity tags (ICAT), is proposed. Without addition of NO donor, a total of 53 endogenous nitrosocysteines was identified in Arabidopsis cells, in proteins belonging to all cell territories, including membranes, and covering a large panel of functions. This first repertoire of nitrosothiols in plants enabled also preliminary structural description. Three apolar motifs, not located in close vicinity of cysteines and accounting for half the dataset, were detected and are proposed to complement nitrosylation prediction algorithms, poorly trained with plant data to date. Analysis of changes induced by a brief salt stress showed that NaCl modified the nitrosylation level of a small proportion of endogenously nitrosylated proteins and did not concern all nitrosothiols in these proteins. The possible role of some NO targets in the response to salt stress was discussed.

Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies.

Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.

Identification of potential S‐nitrosylation sites in the myocardium

S-nitrosylation (SNO) is a reversible protein modification that results from the covalent attachment of nitric oxide to a thiol group of a cysteine residue. Numerous methodologies have been developed to detect SNO proteins, but very few SNO sites have been identified in complex samples. Therefore, the objective of this study is to identify potential SNO sites in the myocardium. We utilized SNO-resin assisted capture (SNO-RAC) with mass spectrometry in order to determine SNO sites. Briefly, crude mouse heart homogenate was incubated with or without the S-nitrosylating agent GSNO (1 mM) and a modified biotin switch was performed. SNO-RAC analysis identified 952 unique SNO proteins in GSNO-treated homogenates, many of which contained multiple SNO sites. These proteins all show the potential for endogenous SNO formation. Of the 952 SNO proteins identified with GSNO treatment, 116 were identified to be endogenously SNO in control homogenates in the absence of GSNO. Gene ontology analysis revealed that many different pathways are targeted by SNO, including myocardial contraction, metabolism, cellular signaling and cell death. This study provides novel information regarding the cardiac SNO proteome and provides additional information on the sites of SNO for key proteins involved in many different myocardial processes. Supported by NIH grants 1F32HL096142 and 5R01HL039752, and the NIH Intramural Program.

Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.

Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation (NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.