Stress-induced interactome of the O-GlcNAcase by SILAC-BioID-MS/MS

ABSTRACT: O-linked-β-N-acetylglucosamine (O-GlcNAc) dynamically modifies and regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased O-GlcNAcylation on numerous proteins and this is thought to promote cell survival. The mechanisms by which the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), the enzymes that add and remove O-GlcNAc respectively, are regulated leading to oxidative stress-induced changes in 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 protein interactors of OGA may control the local activity or substrate targeting of this enzyme, resulting in stress-induced elevations of O-GlcNAc. We utilized the BioID proximity biotinylation technique in combination with Stable Isotope Labeling of Amino Acids in Cell culture (SILAC) to define the basal and oxidative stress-dependent interactomes of OGA. Our study revealed 90 OGA-interacting partners, many of which exhibit increased binding upon oxidative 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 demonstrates a substantial (~85%) reduction in catalytic activity, suggesting that FAS is an inhibitor of OGA. Consistent with this observation, FAS overexpression augments stress-induced O-GlcNAcylation. Together, these data suggest that O-GlcNAcylation may be one downstream effector of FAS that fine-tunes the cell’s response to stress and injury.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Jennifer Groves  

LAB HEAD: Natasha Elizabeth Zachara

PROVIDER: PXD005039 | Pride | 2017-03-14

REPOSITORIES: Pride