Human Synthesis and preparation of histone3 modification peptides
Ontology highlight
ABSTRACT:
INSTRUMENT(S): 4800 Proteomics Analyzer
ORGANISM(S): Homo Sapiens (human)
DISEASE(S): Breast Cancer
SUBMITTER: 
Josh Haipeng Lei
LAB HEAD: Hong Yan Sun
PROVIDER: PXD032850 | Pride | 2022-05-20
REPOSITORIES: Pride
Dataset's files
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Publications
FGFR2-BRD4 Axis Regulates Transcriptional Networks of Histone 3 Modification and Synergy Between Its Inhibitors and PD-1/PD-L1 in a TNBC Mouse Model.
Lei Josh Haipeng JH Zhang Lei L Wang Zhenyi Z Peltier Raoul R Xie Yusheng Y Chen Ganchao G Lin Shiqi S Miao Kai K Deng Chu-Xia CX Sun Hongyan H
Frontiers in immunology 20220425
Epigenetic reprogramming is an independent mode of gene expression that often involves changes in the transcription and chromatin structure due to tumor initiation and development. In this study, we developed a specifically modified peptide array and searched for a recognized epigenetic reader. Our results demonstrated that BRD4 is not only an acetylation reader but of propionylation as well. We also studied the quantitative binding affinities between modified peptides and epigenetic regulators ...[more]
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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.
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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.
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