Defining the Interactome and Functional Regulation of the O-GlcNAcase Enzyme During Oxidative Stress

The essential post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. O-GlcNAc is dynamically added and removed from proteins by the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), respectively. Dysregulation of O-GlcNAc-cycling is implicated in the etiology of numerous diseases, including cancer, neurodegeneration, and metabolic dysfunction. Underpinning these observations, O-GlcNAc regulates nearly every major cellular process including transcription, translation, protein degradation, protein localization, and the cell cycle. Furthermore, many forms of cellular stress and injury, including oxidative stress, elicit an increase in O-GlcNAcylation on numerous proteins, which has been demonstrated to promote cell survival. However, the mechanisms by which OGT and OGA are regulated during oxidative stress to alter O-GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevated O-GlcNAc levels in U2OS cells but has little impact on the activity of OGT. In contrast, the expression and activity of OGA are enhanced. We hypothesized that this seeming paradox could be explained by proteins that bind to and control the local activity or substrate targeting of OGA, thereby resulting in the observed stress-induced elevations of O-GlcNAc. To identify potential protein partners and regulators, we utilized BioID proximity biotinylation in combination with Stable Isotopic Labeling of Amino Acids in Cell Culture (SILAC). This analysis revealed 90 OGA-interacting partners, many of which exhibited increased binding to OGA upon stress. The associations of OGA with fatty acid synthase (FAS), filamin-A, heat shock cognate 70 kDa protein, and OGT were confirmed by co-immunoprecipitation. The pool of OGA bound to FAS demonstrated a substantial (∼85%) reduction in specific activity, suggesting that FAS inhibits OGA. Consistent with this observation, FAS overexpression augmented stress-induced O-GlcNAcylation on a subset of proteins. Although the mechanism by which FAS sequesters OGA remains unknown, these data suggest that FAS fine-tunes the cell's response to stress and injury by remodeling cellular O-GlcNAcylation. Finally, our studies characterized the use of two commercially available and three non-commercially available antibodies for detecting and enriching full-length OGA from lysates of mouse and human origin in order to facilitate future studies probing the regulation of OGA in disease