MgrA nitrosylation assay in S. aureus XN108 WTand nos
Ontology highlight
ABSTRACT:
ORGANISM(S): Staphylococcus Aureus
SUBMITTER: 
Baolin Sun
PROVIDER: PXD057340 | iProX | Wed Oct 30 00:00:00 GMT 2024
REPOSITORIES: iProX
Similar Datasets
Project description:Nitrosylation assay of MgrA in wild-type and C12S knockout strains
2024-10-30 | PXD057338 |
Project description:Yeast protein microarrays were utilized to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). Large numbers of S-nitrosylated yeast proteins were identified after treatment with SNOs, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols
2009-12-01 | GSE19218 | GEO
Project description:Yeast protein microarrays were utilized to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). Large numbers of S-nitrosylated yeast proteins were identified after treatment with SNOs, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols Invitrogen yeast Protoarrays for kinase substrate identification (KSI) were treated with S-nitrosothiols and assayed for protein S-nitrosylation by using a modified biotin switch protocol. Slides were scanned and with a Genepix 4000b scanner (Molecular Devices) using Genepix Pro and analyzed by using Prospector Analyzer (Invitrogen). Results were validated using yeast cell lysates and recombinant, purified yeast proteins.
2010-05-19 | E-GEOD-19218 | biostudies-arrayexpress
Project description:Calcific aortic valve disease (CAVD) is an increasingly prevalent condition and endothelial dysfunction is implicated in its etiology. We previously identified nitric oxide (NO) as a calcification inhibitor by its activation of NOTCH1, which is genetically linked to human CAVD. Here, we show that NO rescues calcification by a S-nitrosylation-mediated mechanism in porcine aortic valve interstitial cells (pAVICs) and single cell RNA-seq demonstrated regulation of NOTCH pathway by NO. A unbiased proteomic approach to identify S-nitrosylated proteins in valve cells found enrichment of the ubiquitin proteasome pathway and implicated S-nitrosylation of USP9X in NOTCH regulation during calcification. Furthermore, S-nitrosylated USP9X was shown to deubiquitinate and stabilize MIB1 for NOTCH1 activation. Consistent with this, genetic deletion of Usp9x in mice demonstrated aortic valve disease and human calcified aortic valves displayed reduced S-nitrosylation of USP9X. These results demonstrate a novel mechanism by which S-nitrosylation dependent regulation of ubiquitin-associated pathway prevents CAVD.
2020-12-22 | GSE161123 | GEO
Project description:Drug-induced liver injury (DILI), especially acetaminophen overdose, is the leading cause of acute liver failure. Pregnane X receptor (PXR) is a nuclear receptor and the master regulator of drug metabolism. Aberrant activation of PXR plays a pathogenic role in the acetaminophen hepatotoxicity. Here, we aimed to examine the PXR S-nitrosylation (SNO) in response to acetaminophen. We found that PXR was S-nitrosylated in hepatocytes and the mouse livers after exposure to acetaminophen or S-nitrosoglutathione (GSNO). Mass-spectrometry and site-directed mutagenesis identified the cysteine 307 as the primary residue for SNO-modification. In hepatocytes, SNO suppressed both agonist (rifampicin and SR12813)-induced and constitutively active PXR (VP-PXR) activations. Furthermore, in acetaminophen overdosed mouse livers, PXR protein was decreased at the centrilobular regions overlapping with increased SNO. In PXR-deficient (PXR-/-) mice, replenishing the livers with the SNO-deficient PXR significantly aggravated hepatic necrosis and apoptosis, increased HMGB1 release, and exacerbated liver injury and inflammation. Particularly, we demonstrated that S-nitrosoglutathione reductase (GSNOR) inhibitor N6022 promoted hepatoprotection by increasing the levels of PXR S-nitrosylation. In conclusion, PXR is post-translationally modified by S-nitrosylation in hepatocytes in response to acetaminophen. This modification mitigated the acetaminophen-induced PXR hyperactivity. It may serve as a new target for therapeutical intervention.
2024-03-06 | GSE243679 | GEO
Project description:HUVECs stimulated with HG+oxLDL or Mannitol+nLDL were subjected to the biotin-switch assay to enrich the S-nitrosylated proteins, the S-nitrosylated proteins were resolved by SDS-PAGE and gels were silver stained. Mass spectrometry was conducted to determine proteins with S-nitrosylation.
2021-06-03 | PXD025295 | Pride
Project description:ABSTRACT Rationale Premature senescence is conducive to aging and cardiovascular diseases. Nrf2 transcription factor, the master orchestrator of adoptive response to cellular stress, has been implicated in regulation of premature senescence in fibroblasts, neural and mesenchymal stem cells by transactivation of antioxidant gene expression. However, as we show here, human primary endothelial cells (ECs) devoid of Nrf2 and murine Nrf2 transcriptional knockout (tKO) aortas are senescent but do not encounter oxidative stress and damage, what contradicts this mechanism. Moreover, a molecular switch between normal, senescent and apoptotic fate remains unknown. Objective To elucidate the mechanism of Nrf2-related premature senescence of vascular system, to understand why Nrf2 deregulation does not cause oxidative stress exclusionary in ECs and to indicate a molecular switch determining ECs fate. Methods and Results Herein we evidence that ECs deficient in Nrf2 protein, or with limited Nrf2 activity in shear stress conditions, exhibit excessive S-nitrosylation of proteins. It is also a characteristic of Nrf2 tKO murine aortas, as determined by biotin switch assay in situ. Mass spectrometry analysis reveals that NOX4 is S-nitrosylated exclusively in ECs devoid of Nrf2. A functional role of S-nitrosylation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage. As a result Nrf2-deficient ECs preserve oxidative balance but are redirected to premature senescence. The same phenotype is seen in Nrf2 tKO aortas. These effects are mediated by Keap1, a direct binding partner of Nrf2 and repressor of its transcriptional activity, remaining in cytoplasm unrestrained by Nrf2. S-nitrosylation, followed by senescence, can also be triggered in smooth muscle cells (SMCs) by EC-derived paracrine induction of iNOS. Conclusions Collectively, Keap1-dependent S-nitrosylation of NOX4 hampers oxidative detriment in ECs with disturbed Nrf2 signaling and may provide defence in the adjacent aortic cells. Overabundance of unrestrained Keap1 in the cytoplasm determines fate of ECs.
2020-08-12 | PXD013640 | Pride
Project description:Protein S-nitrosylation, a post-translational modification consisting in the covalent binding of nitric oxide (NO) to a cysteine thiol moiety, plays a major role in cell signaling and is recognized to be involved in numerous physiological processes and diseases in mammals. The importance of nitrosylation in photosynthetic eukaryotes has been less studied. The aim of this study was to expand our knowledge on protein nitrosylation by performing a large scale proteomic analysis of proteins undergoing nitrosylation in vivo in Chlamydomonas reinhardtii cells under nitrosative stress. Using two complementary proteomic approaches, 492 nitrosylated proteins were identified. They participate in a wide range of biological processes and pathways including photosynthesis, carbohydrate metabolism, amino acid metabolism, translation, protein folding or degradation, cell motility and stress. Several proteins were confirmed in vitro by western blot, site-directed mutagenesis and activity measurements. Moreover, 392 sites of nitrosylation were also identified. These results strongly suggest that S-nitrosylation could constitute a major mechanism of regulation in C. reinhardtii under nitrosative stress conditions. This study constitutes the largest proteomic analysis of protein nitrosylation reported to date. The identification of 381 previously unrecognized targets of nitrosylation further extends our knowledge on the importance of this post-translational modification in photosynthetic eukaryotes.
2013-12-19 | PXD000569 | Pride
Project description:Nitric oxide (NO) is a pleiotropic signaling molecule that affects numerous biological functions. One unique posttranslational modification of proteins by NO is termed S-nitrosylation (SNO), but the role in modulating key proteins’ function in melanoma has not been fully characterized. In the present study, by conducting the biotin switch assay and mass spectrometry, we confirmed that p53 was S-nitrosylated under nitrosative stress. Further proteomic characterization showed that Cys242, Cys275, and Cys277 were S-nitrosylated in A375 cells treated with 100 µM GSNO.
2025-07-21 | PXD031807 | Pride