Quantification of sulfenic acid modifications in an atherosclerotic model
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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: PXD003365 | Pride | 2016-10-10
REPOSITORIES: Pride
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