Hyperactivity of the Ero1α oxidase elicits endoplasmic reticulum stress but no broad antioxidant response

Abstract

Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.

FIGURE 1.

Identification of the Cys⁹⁹–Cys¹⁰⁴ disulfide bond in Ero1α. A, shown is a schematic representation of the disulfide pattern in Ero1α OX2 based on (13, 15) and this study. The cysteine residues are shown as yellow, green (outer active site), or blue (inner active site) circles with amino acid numbering and disulfides as thick gray (likely structural), black (active site), or red (reported regulatory function based on Refs – and this study) lines. The thick orange line at Cys¹⁶⁶ indicates the connection to a potential (but unidentified) disulfide partner. The flexible loop regions are colored in light blue. The three identified peptides after trypsin cleavage are shown in gray boxes. B and C, shown are mass spectra of Ero1α tryptic peptides treated with the alkylating agent IAM without prior reduction (B) or reduced with DTE and alkylated with IAM (C). Peak numbers are shown above the peaks. See “Results” and Table 1 for details.

FIGURE 2.

The regulatory Cys⁹⁴–Cys¹³¹ disulfide in Ero1α is destabilized when the Cys⁹⁹–Cys¹⁰⁴ disulfide is absent. A, expression of His- and Myc-tagged Ero1 variants was induced with doxycycline for 24 h, and cells were NEM-treated to prevent post-lysis thiol-disulfide exchange reactions. Equal amounts of protein from lysates were analyzed by reducing SDS-PAGE and Western blotting (WB) using αMyc (Ero1α) and αActin (loading control) to compare expression levels of Ero1α variants. B, cell lysates were obtained as described for A, and the SDS-PAGE mobility of the Ero1α variants was analyzed under non-reducing (Non-red) or reducing (Red) conditions by αHis Western blotting. The mock cell line is stably transfected with an empty vector and functions as a background control. The vertical hairline denotes removal of one lane, and the asterisk indicates an uncharacterized redox form between OX1 and OX2. C, shown is a schematic representation of the proposed disulfide pattern (as in Fig. 1A) in the analyzed Ero1α Cys-to-Ala variants as inferred from gel mobility of monomeric species (see ”Results” for details). For the sake of clarity, the N-terminal region until Cys-85 was omitted. A dashed line denotes the presence of the disulfide in only a fraction of the monomeric species, and a question mark indicates that the presence of the disulfide is unknown.

FIGURE 3.

Deregulation of Ero1α perturbs ER redox conditions and induces the UPR. A, where indicated, Ero1α-WT or Ero1α-C104A/C131A cells were induced with dox for 24 h and co-treated with 5 mm NAC for the last 18 h. Before lysis, cells were treated with NEM to alkylate free thiols. After cell lysis, cysteines present in disulfides were reduced and decorated with AMS. Such AMS modification of active-site cysteines originally present in the oxidized state gives rise to slower SDS-PAGE mobility compared with the (NEM-decorated) pool of ERp57 containing reduced active-site cysteines. The cellular redox state of ERp57 was visualized by Western blotting (WB). DTT and Diamide (Dia)-treated cells were used to show the mobility of fully oxidized (OX) and reduced (RED) ERp57. B, expression levels of Myc-tagged Ero1α variants were analyzed by Western blotting using α-actin as the loading control. C and D, cells were treated as described for A, and the expression levels of BiP and HERP were analyzed by Western blotting using α-actin as the loading control. Cells treated with 5 μm thapsigargin (Tg) or 2.5 μg/ml tunicamycin (Tm) for 20 h were used to generate positive control lysates for UPR induction.

FIGURE 4.

The glutathione redox buffer counteracts Ero1α hyperactivity. A, where indicated, Ero1α-WT or Ero1α-C104A/C131A cells were induced with dox and treated with 1 mm BSO for 24 h. The cellular redox state of ERp57 was visualized by Western blotting (WB) as described in Fig. 3A. Dia, diamide. B, cell viability of Ero1α-WT and C104A/C131A cells, which where indicated were treated with dox and/or BSO for 48 h, was assessed by a water-soluble tetrazolium-1 assay (mean ± S.D., n = 3). Treatment with 5 μm thapsigargin (Tg) for 20 h was used as positive control for cytotoxicity (n = 2). Absorbance values were normalized to untreated cells. Statistical significance (p ≤ 0.05) was assessed by performing Student's unpaired t test (two tailed, heteroscedastic) on log 2-transformed -fold changes.

FIGURE 5.

The global transcriptional response to deregulated Ero1α activity. A, shown is a heatmap of the 159 genes found by a two-way analysis of variance to have significant changes in expression levels between non-induced (−dox) and induced (+dox) cells (see supplemental Table S2). The colors reflect expression intensities (transformed to mean of 0 and S.D. of 1) for each gene, with red and blue colors corresponding to high and low expression intensities, respectively. Genes are ranked by -fold change (+dox/−dox) in Ero1α-C104A/C131A cells. Numbers 1–3 denote independent biological replicates. B, shown is a density plot of log 2-transformed -fold changes (+dox/−dox) in absolute values for the 159 significant genes shown in A. C, shown is a scatter plot of log 2-transformed -fold changes (+dox/−dox) of the 159 significant genes in Ero1α-WT and Ero1α-C104A/C131A cells shown in A and B. Both the full view of the 159 genes (the inserted scatter plot) and an enlarged view (155 genes) are shown. The green line indicates no difference in -fold change between Ero1α-WT and Ero1α-C104A/C131A cells, whereas the dashed red and blue lines signify a difference of 0.3 and −0.3, respectively, between the log 2-transformed -fold changes (+dox/−dox) for Ero1α-C104A/C131A and Ero1α-WT. For the genes above the dashed red lane (see also supplemental Table S3), the associated gene names are either shown on top of the data point or in the vicinity of the data point indicated by an arrow. Numbers in italics denote probeset IDs of unannotated genes.

FIGURE 6.

CRELD1, CRELD2, and c18orf45 are transcriptionally up-regulated by the UPR. A, shown is relative abundance of mRNA analyzed by real-time quantitative PCR on RNA extracted from HEK293 cells. Cells were treated with solvent (0.025% DMSO) or 2.5 μg/ml tunicamycin in 0.025% DMSO for 8 h. mRNA levels were normalized to the housekeeping gene GAPDH (mean ± S.D., n = 3). ERp90 and ERLIN1 both serve as controls for genes that are largely unaffected by ER stress (47), whereas BiP and HERP serve as positive controls for UPR target genes. The expression level of the housekeeping gene HPRT1 serves as a control for the normalization to GAPDH expression. Statistical significance (p ≤ 0.05) was assessed by performing Student's unpaired t test (two tailed, heteroscedastic) on log 2-transformed -fold changes; *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; n.s., not significant. B, HEK293 cells were treated with solvent (0.04% ethanol) or 5 μm thapsigargin in 0.04% ethanol for 8 h and analyzed as described in A.