Dataset Information

Quantification of sulfenic acid modifications in an atherosclerotic model-2

ABSTRACT: 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.

INSTRUMENT(S): maXis

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Monocyte

SUBMITTER: Ru Li

LAB HEAD: Juergen Kast

PROVIDER: PXD003363 | Pride | 2016-10-10

REPOSITORIES: Pride

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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]

PMID: 27690452

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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.

Project description:Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by PCR and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change .2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change .0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analysing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-sepcific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential. Microarray hybridizations (Agilent: Whole Human Genome Oligo Microarray; 41k unique probe) were carried out with 5 µg Cy3 labeled cRNA and 5 µg Cy5 labeled cRNA, both prepared from each sample.

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