Quantification of sulfenic acid on model protein
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Mammalia Gallus Gallus (chicken) Lagomorpha
TISSUE(S): Whole Body
SUBMITTER: 
Ru Li
LAB HEAD: Juergen Kast
PROVIDER: PXD003348 | Pride | 2016-10-10
REPOSITORIES: Pride
Publications
Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin β<sub>2</sub> on Monocytes via NADPH Oxidase.
Li Ru R Klockenbusch Cordula C Lin Liwen L Jiang Honghui H Lin Shujun S Kast Juergen J
Journal of proteome research 20161007 12
Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions but also release the contents of their granules, commonly called "platelet releasate". It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previ ...[more]
Similar Datasets
Project description:Redox imbalance in cells, due to the accumulation of reactive oxygen species (ROS), has been implicated in the pathogenesis of several diseases, including atherosclerosis. However, the molecular details of redox regulation in atherosclerosis remain to be established.1 During the progression of atherosclerotic plaque, NADPH oxidase (NOX) that are expressed in vascular cells, particularly in phagocytic cells like monocytes, mainly contribute to the production of ROS.2 ROS with exogenous or endogenous sources is able to modify DNA, lipids to proteins. Cysteine residue in protein, despite being one of the least abundant amino acid residues, is a major target of ROS-mediated cell-signaling modulation3 The ROS-mediated regulation is accomplished through a variety of reversible and irreversible oxidative modifications on cysteines such as sulfenic acid, S-nitrosylation, S-glutathionylation, sulfonic acid. Among the reversible cysteine oxidative modifications, sulfenic acid has emerged as an important post-translational modification in proteins.4 Sulfenic acid, as the principle product of the reaction between a thiol and hydrogen peroxide, is a pivotal reactive intermediate under physiological and oxidative stress conditions. Several studies have identified the catalytic or regulatory sulfenic acid formation in proteins such as NADH peroxidase, peroxiredoxins, and protein tyrosine phosphatase.4 Pinpointing the precise location of sulfenic acid in proteins enables us to track down the reactive site where oxidation is initiated. However, the detection of sulfenic acid is challenging as sulfenic acids is highly reactive and in most situations they are rapidly converted back to the reduced state or further oxidized to a more stable state. Nonetheless, certain protein microenvironments, such as the presence of polar uncharged residues, particularly threonine, and the absence of the charged residues close to the sulfenic acid sites, promote sulfenic acid formation and stabilization.5 To conduct proteomic study of this significant oxidative modification, various analytical methods have been developed to enrich proteins or peptides bearing sulfenic acid.6 Launched by Jaffrey in 2001, the biotin switch assay has been extensively adapted to identify various cysteine modifications in complex biological samples.7 We previously combined the biotin switch assay with stable isotope labeling by amino acid in cell culture (SILAC) to quantify total reversible cysteine oxidation and applied it on an atherosclerotic model with human platelets and monocytic THP-1 cells. In the development of atherosclerosis, activated platele facilitates the recruitment of monocytes to the sites of inflamed vascular endothelium not only via P-selectin-PSGL-1 mediated direct binding, but also via releasing the contents of their granules, which are defined as platelet releasate (PR). We demonstrated that thrombin- or LPA- induced PR leads to ROS production in THP-1 cells which in turn alters fundamental biological processes like glycolysis.8 To further digging into the redox regulation mechanism in the atherosclerotic model, we extend the modified biotin switch assay to quantify sulfenic acid. The Eaton group has modified the biotin switch assay using m-arsenite to selectively reduce sulfenic acid.9 Here we combined SILAC and m-arsenite in the biotin switch assay and quantified more than 100 sulfenic acid sites in the model system. Bioinformatics analysis of these proteins with sulfenic acid modification underlined the involvement of integrin β2 in leukocytes transendothelium migration. LFA-1 (αLβ2) and Mac-1 (αMβ2) are two integrin β2 complexes that are most relevant to the migration of monocyte on the endothelial cells.10 We validated the activation of LFA-1 and Mac-1 in primary monocytes upon platelet releasate treatment. Moreover, the activation of LFA-1 and Mac-1 were shown to be independent of P-selectin-PSGL1 interaction on monocytes. In a word, analysis of sulfenic acid reveals a new angle of the crosstalk between platelet releasate and monocytes in the early stage of atherosclerosis.
2016-10-10 | PXD003354 | Pride
Project description:Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions, but also release the contents of their granules, commonly called “platelet releasate”. It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin β2. Flow cytometry validated the activation of LFA-1 (αLβ2) and Mac-1 (αMβ2), two subfamilies of integrin β2 complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.
2016-10-10 | PXD004862 | Pride
Project description:Physiological stimuli such as thrombin or pathological stimuli such as lysophosphatidic acid (LPA) activate platelets. The activated platelets bind to monocyte through P-selectin-PSGL-1 interactions, but also release the contents of their granules, which were defined as platelet releasate. It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS has been viewed as a potential regulator of protein function. In previous study, we employed a model of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that been influenced by reversible cysteine oxidation. To gain better understanding of the redox regulation of monocyte in atherosclerosis, we now extend the modified biotin switch to quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite in the modified biotin-switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites further highlighted biological processes of monocyte transendothelial migration including integrin β2. Flow cytometry validated the activation of LFA-1 (αLβ2) and Mac-1 (αMβ2), two subfamilies of integrin β2 complexes on human primary monocyte upon platelet releasate treatments. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Moreover, the production of ROS and the activation of LFA-1 in human primary monocyte induced by platelet releasate were independent of P-selectin-PSGL-1 interaction. The modified biotin switch assay proved to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.
2016-10-10 | PXD003363 | Pride
Project description:Physiological stimuli such as thrombin or pathological stimuli such as lysophosphatidic acid (LPA) activate platelets. The activated platelets bind to monocyte through P-selectin-PSGL-1 interactions, but also release the contents of their granules, which were defined as platelet releasate. It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS has been viewed as a potential regulator of protein function. In previous study, we employed a model of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that been influenced by reversible cysteine oxidation. To gain better understanding of the redox regulation of monocyte in atherosclerosis, we now extend the modified biotin switch to quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite in the modified biotin-switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites further highlighted biological processes of monocyte transendothelial migration including integrin β2. Flow cytometry validated the activation of LFA-1 (αLβ2) and Mac-1 (αMβ2), two subfamilies of integrin β2 complexes on human primary monocyte upon platelet releasate treatments. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Moreover, the production of ROS and the activation of LFA-1 in human primary monocyte induced by platelet releasate were independent of P-selectin-PSGL-1 interaction. The modified biotin switch assay proved to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.
2016-10-10 | PXD003365 | Pride
Project description:Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively-fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1alpha, ERO1beta and PRDX4, compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly-modest delay in disulfide bond formation in secreted proteins but a profound, five-fold lower procollagen 4 hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content, in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2 generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation and a non-canonical form of scurvy. double and triple mutants and wild type
2012-09-15 | E-GEOD-39492 | biostudies-arrayexpress
Project description:Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively-fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1alpha, ERO1beta and PRDX4, compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly-modest delay in disulfide bond formation in secreted proteins but a profound, five-fold lower procollagen 4 hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content, in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2 generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation and a non-canonical form of scurvy.
2012-09-15 | GSE39492 | GEO
Project description:The goal of this study was to identify yeast mutants with arsenite hypersensitivity. Keywords: dose response Matched pools of yeast knockout (YKO) mutants were grown in paired growth media lacking or containing arsenite at various concentrations. The relative abundances of the YKO mutants were profiled using microarrays. Three types of pools were used in this study: (1) heterozygous diploid mutants; (2) haploid mutants obtained as progeny from the heterozygous diploid pool; and (3) haploid mutants also containing a null mutation of the prefoldin gene PFD1.
2010-07-26 | E-GEOD-5973 | biostudies-arrayexpress
Project description:Background: Arsenite is one of the most toxic chemical substances known and is assumed to exert detrimental effects on viability even at lowest concentrations. By contrast and unlike higher concentrations, we here find that exposure to low-dose arsenite promotes growth of cultured mammalian cells. In the nematode C. elegans, low-dose arsenite promotes resistance against thermal and chemical stressors, and extends lifespan of this metazoan, whereas higher concentrations reduce longevity. While arsenite causes a transient increase in reactive oxygen species (ROS) levels in C. elegans, co-exposure to ROS scavengers prevents the lifespan-extending capabilities of arsenite, indicating that transiently increased ROS levels act as transducers of arsenite effects on lifespan, a process known as mitohormesis. The RNA-seq data comprises 2 biological replicates for worms exposed to 100nM Arsenite 48h after L4 and 2 biological replicates of the same age as controls 4 samples: 2 mRNA profiles of C.elegans 48h after L4 exposed to Arsenite; 2 mRNA profiles of C.elegans 48h after L4 as controls (H20). The N2 wild type (var. Bristol) strain was used.
2013-04-23 | E-GEOD-46257 | biostudies-arrayexpress
Project description:Exposure to high levels of arsenic in drinking water is associated with several types of cancers including lung, bladder and skin, as well as vascular disease and diabetes. Drinking water standards are based primarily on epidemiology and extrapolation from higher dose experiments, rather than measurements of phenotypic changes associated with chronic exposure to levels of arsenic similar to the current standard of 10ppb, and little is known about the difference between arsenic in food as opposed to arsenic in water. Measurement of phenotypic changes at low doses may be confounded by the effect of laboratory diet, in part because of trace amounts of arsenic in standard laboratory chows, but also because of broad metabolic changes in response to the chow itself. Finally, this series contrasts 8hr, 1mg/kg injected arsenic with the various chronic exposures, and also contrasts the acute effects of arsenic, dexamethasone or their combination. Male C57BL/6 mice were fed on two commercially available laboratory diets (LRD-5001 and AIN-76A) were chronically exposed, through drinking water or food, to environmentally relevant concentrations of sodium arsenite, or acutely exposed to dexamethasone. Experiment Overall Design: Male C57BL/6 mice, fed on two commercially available laboratory diets (LRD-5001 and AIN-76A), were chronically exposed through drinking water or food, to environmentally relevant concentrations of sodium arsenite. Another group animals, fed on the AIN 76A diet, was IP injected with dexamethasone (1 mg/kg), sodium arsenite (1mg/kg), both dexamethosone and arsenite, or saline alone.
2008-10-21 | E-GEOD-11056 | biostudies-arrayexpress
Project description:Exposure to high levels of arsenic in drinking water is associated with several types of cancers including lung, bladder and skin, as well as vascular disease and diabetes. Drinking water standards are based primarily on epidemiology and extrapolation from higher dose experiments, rather than measurements of phenotypic changes associated with; chronic exposure to levels of arsenic similar to the current standard of 10ppb, and little is known about the difference between arsenic in food as opposed to arsenic in water. Measurement of phenotypic changes at low doses may be confounded by the effect of laboratory diet, in part because of trace amounts of arsenic in standard laboratory chows,; but also because of broad metabolic changes in response to the chow itself. Finally, this series contrasts 8hr, 1mg/kg injected arsenic with the various chronic exposures, and also contrasts the acute effects of arsenic, dexamethasone or their combination. Male C57BL/6 mice were fed on two commercially available laboratory diets (LRD-5001 and AIN-76A) were chronically exposed, through drinking water or food, to environmentally relevant concentrations of sodium arsenite, or acutely exposed to dexamethasone. Experiment Overall Design: Male C57BL/6 mice, fed on two commercially available laboratory diets (LRD-5001 and AIN-76A), were chronically exposed through drinking water or food, to environmentally relevant concentrations of sodium arsenite. Experiment Overall Design: Another group animals, fed on the AIN 76A diet, was IP injected with dexamethasone (1 mg/kg), sodium arsenite (1mg/kg), both dexamethosone and arsenite, or saline alone.
2008-06-16 | E-GEOD-9630 | biostudies-arrayexpress