Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress

• Published in: Molecular & cellular proteomics Vol. 20; p. 100069
• Main Authors: Martinez, Marissa, Renuse, Santosh, Kreimer, Simion, O’Meally, Robert, Natov, Peter, Madugundu, Anil K., Nirujogi, Raja Sekhar, Tahir, Raiha, Cole, Robert, Pandey, Akhilesh, Zachara, Natasha E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2021; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cells, Cultured; Cytoprotection; Fibroblasts - metabolism; Glycosylation; Mice; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; Network; Oxidative Stress; Protein Interaction Maps; Proteomics; Quantitative proteomics; Oxidative stress; Null; HDAC1; FL; PRM; UAP1; H2O2; Bag6; Network; CKII; HSF1; TPR; HCF-1; TNKS1BP1; 4MU; Cytoprotection; eIF3b; FOXO; MEFs; Quantitative proteomics; SETD1A; OGA; TBST; Glycosylation; SILAC; O-GlcNAc; TEABC; GFAT; CARM1; TCL; OGT
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1016/j.mcpro.2021.100069

The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-β-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. [Display omitted] •O-GlcNAc levels change dynamically in response to injury.•Injury does not induce changes in activity of the enzymes that cycle O-GlcNAc.•Quantitative proteomics identified and validated interactors of OGT.•The interactome of OGT changes significantly in response to oxidative stress. The goal of these studies was to provide insight into the regulation of the O-GlcNAc transferase (OGT) basally and in response to oxidative stress, as well as the role that O-GlcNAc plays in promoting cytoprotection. Using quantitative proteomics, the basal and injury-induced interactome of OGT has been defined and validated. Protein interactors are anticipated to regulate either the activity or substrate targeting of OGT, or to be substrates of OGT, thus affecting cytoprotection.