title: Fatty acid synthase inhibits theO-GlcNAcase during oxidative stress.
abstract: The dynamic post-translational modificationO-linked β-N-acetylglucosamine (O-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increasedO-GlcNAcylation of numerous proteins, and this increase is thought to promote cell survival. The mechanisms by which theO-GlcNAc transferase (OGT) and theO-GlcNAcase (OGA), the enzymes that add and removeO-GlcNAc, respectively, are regulated during oxidative stress to alterO-GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevatedO-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 ofO-GlcNAc. To identify potential protein partners, we utilized BioID proximity biotinylation in combination withstableisotopiclabeling ofamino acids incell 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-inducedO-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 cellularO-GlcNAcylation.
authors.affiliation: From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Groves
authors.affiliation: From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.|the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, and.
authors.forename: Austin O
authors.initials: AO
authors.lastname: Maduka
authors.affiliation: From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.|the Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Robert N
authors.initials: RN
authors.lastname: O'Meally
authors.affiliation: From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.|the Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Cole
authors.affiliation: From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, nzachara@jhmi.edu.
authors.forename: Natasha E
authors.initials: NE
authors.lastname: Zachara
mesh_terms: D000818:Animals; D019921:Biotinylation; D002384:Catalysis; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D064429:Fatty Acid Synthases / Q000378:metabolism*; D064448:Filamins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D018384:Oxidative Stress; D040901:Proteomics; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
chemical_list: C578663:FLNA protein, human; D064448:Filamins; C529499:FlnA protein, mouse; D006023:Glycoproteins; D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C494372:Hspa8 protein, mouse; D007527:Isoenzymes; D064429:Fatty Acid Synthases; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase
keywords: BioID; O-GlcNAcylation; fatty acid synthase (FAS); glycoprotein; mgea5; oxidative stress; post-translational modification (PTM); protein-protein interaction; proteomics; signal transduction