Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

Abstract

The dynamic post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased O-GlcNAcylation of numerous proteins, and this increase 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 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, 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. 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.

Keywords: BioID; O-GlcNAcylation; fatty acid synthase (FAS); glycoprotein; mgea5; oxidative stress; post-translational modification (PTM); protein-protein interaction; proteomics; signal transduction.

Figure 1.

Oxidative stress increases OGT and OGA expression, OGA activity, and O-GlcNAc levels. U2OS cells were treated with vehicle (V) or H₂O₂ (2.5 mm, 1–3 h). n = 10, unless otherwise indicated. A, expression of OGT, OGA, and actin, as well as O-GlcNAc levels, was assessed in NETN lysates (5 μg) by Western blotting (WB). Protein load was assessed by total protein stain (colloidal Coomassie G-250) and by Western blotting (actin). Molecular mass (MW) markers are indicated. B, quantitation of actin normalized to total protein (G-250). C, quantitation of O-GlcNAc levels normalized to actin. D, quantitation of OGT expression normalized to actin. E, NETN lysates (5 μg) were assayed for OGT activity using [³H]UDP-GlcNAc (0.5 μCi) and CKII acceptor peptide (1 mm). n = 4, three technical replicates per assay. F, quantitation of OGA expression normalized to actin. G, NETN lysates (5 μg) were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 6, two technical replicates per assay. B–G, data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Dunnett's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0001 (****).

Figure 2.

OGA-mBirA fusion proteins express, maintain catalytic activity, and biotinylate proteins in vivo. A, schematic of the OGA-mBirA fusion proteins. CD and AD represent the catalytic (β-N-acetylglucosaminidase) domain and acetyltransferase-like domain of OGA, respectively. B–D, U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with or without biotin (25 μm, 16 h) or TMG (100 nm, 20 h) as indicated. Proteins were extracted in TCL buffer. B, equal amounts of protein (10 μg) were separated by SDS-PAGE, and the following were detected by Western blotting: OGA, HA, Myc, and actin. n = 3. C, desalted lysates were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 3, representative data from one experiment is shown. Error bars indicate the intra-assay standard deviation from two technical replicates. D, equal amounts of protein (4.5 μg) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 2. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Figure 3.

OGA-mBirA fusion proteins localize to and biotinylate proteins in the nucleus, cytoplasm, and mitochondria of U2OS cells. U2OS cells were transfected with pcDNA3.1 (A), OGA-mBirA-HA (B–D), or Myc-mBirA-OGA (E–G), treated with (A, C, D, F, and G) or without (B and E) biotin (25 μm, 16 h), and treated with (D and G) or without (A–C, E, and F) H₂O₂ (2.5 mm, 2 h). Cells were fixed, permeabilized, and stained for BirA and biotin. Nuclei and mitochondria were stained with Hoechst 33342 and MitoTracker Orange CMTMRos, respectively. White triangles indicate co-localization (orange) of MitoTracker and OGA (B and E). Images were acquired at ×63 magnification on a Zeiss Axio Examiner 710NLO-Meta multiphoton microscope. n = 3. Scale bar, 15 μm.

Figure 4.

OGA-mBirA-HA and Myc-mBirA-OGA biotinylate proximal proteins differentially in response to oxidative stress. U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with biotin (25 μm, 16 h) in the presence or absence of H₂O₂ (2.5 mm, 2 h). A, equal amounts of protein (5 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 4. B, densitometric total lane profiles for each lane from the biotin signal in A. Asterisks are used to highlight a subset of the biotinylated signals that are altered by oxidative stress. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Figure 5.

SILAC-BioID-MS/MS strategy used to identify the basal and oxidative stress-dependent interactome of OGA. A, U2OS cells were labeled with light, medium, or heavy isotopes of arginine and lysine for six generations. In experiment 1, cells were transfected with pcDNA3.1 (heavy) or OGA-mBirA-HA (light, medium), treated with biotin (25 μm, 16 h), and treated with vehicle (medium, heavy) or H₂O₂ (light; 2.5 mm, 2 h, n = 1). In experiment 2, Myc-mBirA-OGA was transfected in replacement of OGA-mBirA-HA. For each experiment, proteins were extracted in denaturing TCL buffer and combined in equal amounts. The biotinylated proteins were isolated on NeutrAvidin-agarose in denaturing conditions, eluted in 2% (w/v) SDS (95 °C), and precipitated with acetone. Peptides were generated by trypsin and LysC digestion, separated by basic reversed phase (bRP) fractionation, and identified by mass spectrometry (LC-ESI-MS/MS). Subsequently, protein-protein interactions were validated by co- IP and Western blotting. B, for the SILAC experiments (n = 1, each), equal amounts of protein (10 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, OGA, HA, Myc, and actin. Protein load was assessed by total protein stain (Sypro Ruby) and by Western blotting (actin). Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Figure 6.

SILAC-BioID-MS/MS reveals numerous basal and stress-induced OGA-interacting proteins. Venn diagrams illustrate the following: A, basal and stress-induced interactors identified in the OGA-mBirA-HA screen. B, basal and stress-induced interactors identified in the Myc-mBirA-OGA screen. C, basal interactors identified in the HA and Myc datasets. D, stress-induced interactors identified in the HA and Myc datasets. E, interactors identified above background in the basal and stressed samples (regardless of fold change upon stress) in the HA and Myc datasets. F, PANTHER gene list analysis indicating the protein classes represented in the OGA interactome.

Figure 7.

Oxidative stress induces the association of OGA with FAS, FLNA, HSC70, and OGT. U2OS cells were treated with Vehicle (V) or H₂O₂ (2.5 mm, 1–3 h). n = 3. A, anti-OGA antibody (IP: OGA; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous OGA from NETN cell lysates (500 μg), of which 1.5–2% (input) and 30–40% (immunoprecipitate) were analyzed by SDS-PAGE. OGA, FAS, FLNA, HSC70, OGT (positive control), and actin (loading/negative control) were detected by Western blotting. B, anti-FAS antibody (IP: FAS; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous FAS from NETN cell lysates (250 μg), of which 3% (input) and 60% (immunoprecipitate) were analyzed by SDS-PAGE. FAS, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. C, U2OS cells were transfected with pcDNA3.1 (control) or pCMV-SPORT6 V5-FAS (test). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (300 μg), of which 1.7% (input) and 33.3% (immunoprecipitate) were analyzed by SDS-PAGE. V5, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. A–C, FAS (CST) and FAS (NB) represent anti-FAS antibody from Cell Signaling Technology and Novus Biologicals, respectively. To ensure that images were in the linear range, Western blot exposures from the input and immunoprecipitated fractions are often different. The exposure lengths for the test and control isotype antibody immunoprecipitates are always identical. The migration of molecular mass (MW) markers is indicated.

Figure 8.

OGA exhibits reduced catalytic activity when bound to FAS. U2OS cells stably overexpressing pcDNA3.1 (control) or pcDNA3.1 V5-FAS (test) were treated with vehicle (V) or H₂O₂ (2.5 mm, 2 h). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (1.6 mg). n = 3. A and B, V5, OGA, and actin (loading/negative control) were detected by Western blotting. Western blots for each antibody were exposed for equal lengths of time. A, analysis of the inputs and unbound fractions. B, analysis of the bound fractions (31.25%). C, OGA activity was measured in the inputs and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min and then normalized to the pcDNA3.1 vehicle-treated sample. D, OGA activity was assessed in the input, bound (on-bead, 31.25%), and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min/μg using densitometric analysis of OGA in the input serial dilutions and the bound fractions, and then normalized to the pcDNA3.1 vehicle-treated sample. Only the data from FAS-overexpressing cells treated with H₂O₂ are shown, as OGA protein and activity was absent in the bound fraction of the other samples. Data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Tukey's MCT, and differences were considered statistically significant at p ≤ 0.0001 (****). The migration of molecular mass (MW) markers is indicated.

Figure 9.

FAS overexpression increases O-GlcNAcylation during oxidative stress. U2OS cells stably overexpressing pcDNA3.1 or pcDNA3.1 V5-FAS were treated with vehicle (V) or H₂O₂ (2.5 mm, 1–3 h). n = 5. A, NETN lysates (∼7.5 μg) were analyzed by SDS-PAGE. O-GlcNAc, OGA, OGT, V5, and actin were detected by Western blotting. Total protein stain (colloidal Coomassie G-250) was used to assess protein load. The migration of molecular mass (MW) markers is indicated. B, quantitation of O-GlcNAc levels normalized to G-250. C, quantitation of OGA expression normalized to G-250. D, quantitation of OGT expression normalized to G-250. B–D, data are presented as the mean ± S.E. Significance was determined by RM-2ANOVA followed by Sidak's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

Figure 10.

Proposed model for the regulation of OGA during oxidative stress resulting in elevated levels of O-GlcNAc. O-GlcNAc levels become elevated during oxidative stress, and this is associated with cytoprotection. Counterintuitively, in U2OS cells exposed to oxidative stress, OGT activity remains constant, whereas OGA activity and expression are elevated. Our data support a model in which OGA forms complexes with other proteins (?), such as FAS, that regulate its activity leading to a stress-induced elevation of O-GlcNAc.