Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin β₂ on Monocytes via NADPH Oxidase

Abstract

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 β. Flow cytometry validated the activation of LFA-1 (αβ) and Mac-1 (αβ), two subfamilies of integrin β 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.

DeepCSO: a deep-learning network approach to predicting Cysteine S-sulphenylation sites

“…Sapiens and A. thaliana [9][10][11][12][13]. The data of the species were pre-processed and the related procedure was exemplified using the A. thaliana data, as listed below ( Figure 1A).…”

“…This modification has been reported to influence protein functions, regulate signal transduction and affect cell cycle across various species [1,[3][4][5][6][7][8]. So far, thousands of CSO sites have been identified in different species including the mammal Homo sapiens and the plant organism Arabidopsis thaliana using the chemoproteomics approach [9][10][11][12][13] (Summarized in Table S1). Nevertheless, highly efficient detection of the CSO sites remains a major methodological issue, due to the low abundance and dynamic level of CSO-containing proteins in vivo.…”

DeepCSO: A Deep-Learning Network Approach to Predicting Cysteine S-Sulphenylation Sites

“…The experimentally identified CSO sites were derived from two different organisms including H. Sapiens and A. thaliana (Yang et al, 2014;Li et al, 2016;Gupta et al, 2017;Akter et al, 2018;Huang et al, 2019). The data of the species were pre-processed and the related procedure was exemplified using the A. thaliana data, as listed below ( Figure 1A).…”

“…This modification has been reported to influence protein functions, regulate signal transduction and affect cell cycle (Van Breusegem and Dat, 2006;Men and Wang, 2007;Paulsen and Carroll, 2013;Hourihan et al, 2016;Choudhury et al, 2017;Mhamdi and Van Breusegem, 2018). So far, thousands of CSO sites have been identified from different species including the mammal Homo sapiens and the plant organism Arabidopsis thaliana using the chemoproteomics approach (Yang et al, 2014;Li et al, 2016;Gupta et al, 2017;Akter et al, 2018;Huang et al, 2019;summarized in Supplementary Table 1). Nevertheless, the CSO site detection remains a major methodological issue due to low abundance and dynamic level of CSO-containing proteins in vivo.…”

Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry

“…Indirect analysis of SOH by modified BST and specific reduction of SOH by arsenite was used for the study of a SILAC-labeled THP-1 human monocytic cell line treated with H 2 O 2 or activated human platelet releasate. More than 200 SOH peptides were quantified [209].…”