期刊
MOLECULAR & CELLULAR PROTEOMICS
卷 20, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.mcpro.2021.100069
关键词
-
资金
- NHLBI, National Institutes of Health [P01 HL107153, R01 HL139640]
- Dean's Office Microgrant Program
- NIGMS, National Institutes of Health [T32 GM007445]
- NCI, National Institutes of Health [P30 CA006973]
- NCI's Clinical Proteomic Tumor Analysis Consortium Initiative [U24CA210985]
- [S10OD021844]
The enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase are able to add and remove O-GlcNAc from thousands of substrates, controlling glycosylation in response to stress by affecting their activity, localization, and substrate specificity. Using quantitative proteomics, the interaction partners of OGT were identified, showing unique and specific interactors that change their association in response to stress.
The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-beta-D-glucosamine (O-GlcNAc) mitigates injury and promotes cytoprotection in a variety of stress models. The O-GlcNAc transferase (OGT) and the O-GlcNAcase are the sole enzymes that add and remove O-GlcNAc, respectively, from thousands of substrates. It remains unclear how just two enzymes can be specifically controlled to affect glycosylation of target proteins and signaling pathways both basally and in response to stress. Several lines of evidence suggest that protein interactors regulate these responses by affecting OGT and O-GlcNAcase activity, localization, and substrate specificity. To provide insight into the mechanisms by which OGT function is controlled, we have used quantitative proteomics to define OGT's basal and stress-induced interactomes. OGT and its interaction partners were immunoprecipitated from OGT WT, null, and hydrogen peroxide-treated cell lysates that had been isotopically labeled with light, medium, and heavy lysine and arginine (stable isotopic labeling of amino acids in cell culture). In total, more than 130 proteins were found to interact with OGT, many of which change their association upon hydrogen peroxide stress. These proteins include the major OGT cleavage and glycosylation substrate, host cell factor 1, which demonstrated a time-dependent dissociation after stress. To validate less well-characterized interactors, such as glyceraldehyde 3-phosphate dehydrogenase and histone deacetylase 1, we turned to parallel reaction monitoring, which recapitulated our discovery-based stable isotopic labeling of amino acids in cell culture approach. Although the majority of proteins identified are novel OGT interactors, 64% of them are previously characterized glycosylation targets that contain varied domain architecture and function. Together these data demonstrate that OGT interacts with unique and specific interactors in a stress-responsive manner.
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