Fatty acid synthase inhibits the O-GlcNAcase during oxidative stress

Abstract

The dynamic post-translational modification linked β--acetylglucosamine (-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased GlcNAcylation of numerous proteins, and this increase is thought to promote cell survival. The mechanisms by which theGlcNAc transferase (OGT) and the GlcNAcase (OGA), the enzymes that add and removeGlcNAc, respectively, are regulated during oxidative stress to alter GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevatedGlcNAc 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 GlcNAc. To identify potential protein partners, we utilized BioID proximity biotinylation in combination withtable sotopicabeling of mino acids inell 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 GlcNAcylation. 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 cellularGlcNAcylation.

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors

“…Prior proteomic analysis has focused on discovery of OGT substrates, leading to more than 4,000 O-GlcNAc-modified proteins identified to date (50). Many of these proteins have links to DNA damage and repair and their dynamic O-GlcNAcylation is implicated in the cellular response to diverse forms of stress (23,(51)(52)(53). Despite many leads, functional analysis has lagged considerably, leaving roles for O-GlcNAcylation in DDR poorly defined.…”

Transcriptional regulation of O-GlcNAc homeostasis is disrupted in pancreatic cancer

“…OGA activity assay was performed as described previously (58). Briefly, duplicate 50-l reactions containing whole cell lysate, 50 mM sodium cacodylate, pH 6.4, 0.3% BSA, 100 mM N-acetyl-D-galactosamine (GalNAc), and 1 mM 4-methylumbelliferyl (4MU)-GlcNAc or 4MU-GalNAc (Sigma) were set up in black flat-bottomed 96-well plates.…”

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O‐GlcNAc‐Cycling Enzymes

“…One study combined this with stable isotopicl abeling of amino acids in cell culture (SILAC) to analyze the proteins enriched by OGA quantitatively. [76] They identified 90 proteins that were potential interacting partners of OGA and validatedt hat one of them,f attya cid synthase, reduces OGA activity upon binding. Some considerations should be taken into account forO GA BioID methods.…”