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

Marissa Martinez,Santosh Renuse,Simion Kreimer,Robert O’Meally,Peter Natov,Anil K Madugundu,Raja Sekhar Nirujogi,Raiha Tahir,Robert Cole,Akhilesh Pandey,Natasha E Zachara

doi:10.1016/j.mcpro.2021.100069

Abstract

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.

Highlights

G quantitative proteomics, the basal and injury-induced interactome of O-GlcNAc transferase (OGT) has been defined and validated
We observed a small decrease in protein O-GlcNAcylation followed by a modest, time-dependent increase in protein OGlcNAcylation (Fig. 1A)
We measured OGT and OGA enzymatic activity using 3H-UDP-GlcNAc transfer to either casein kinase II (CKII) or α-crystallin peptides and the conversion of 4-MUGlcNAc to 4-MU, respectively (Fig. 1B). Both OGT (CKII and α-crystallin) and OGA activities were moderately increased across all time points, but statistical significance was not reached (Fig. 1B)

Summary

Graphical Abstract

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. To provide insight into the regulation of OGT during injury, we have mapped the basal and oxidative stress–induced interactomes of OGT via endogenous enrichment coupled with orthogonal quantitative proteomics strategies for identification (stable isotopic labeling of amino acids in cell culture [SILAC]) and validation (parallel reaction monitoring [PRM]). Both approaches utilized mouse embryonic fibroblasts (MEFs), in which OGT can be inducibly deleted, and the stress response has been comprehensively studied [3, 20, 21]. These data suggest that varied and specific protein partners regulate the function of OGT in response to stress

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

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