Many secretory proteins such as antibodies, blood clotting factors and plasma
membrane receptors contain disulfide bonds. The formation of disulfide bonds in these secretory proteins is n essential step in attaining the functional native structure. In the endoplasmic reticulum (ER) of human cells, disulfide bonds are predominantly generated by the two isoforms of the ER oxidoreductin-1 (Ero1) family: Ero1α and Ero1β. Both enzymes oxidize the active-site cysteines in protein disulfide isomerases (PDIs), which in turn introduce disulfide bonds into newly synthesized proteins. Ero1 is re-oxidized by molecular oxygen and this step generates hydrogen peroxide: a
reactive oxygen species.
Intramolecular disulfide bonds tightly regulate the oxidase activity of Ero1α.
Whereas the regulatory mechanisms that regulate Ero1α activity are well understood, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. The work presented here shows that overexpression of a hyperactive mutant (C104A/C131A) of Ero1α leads to hyperoxidation of the ER oxidoreductase ERp57 and induces the unfolded protein response (UPR). These effects are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, analysis of the global transcriptome showed that the cell responds to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Collectively, these 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.
Compared to Ero1α, Ero1β activity is less tightly regulated. However, the
redox regulation of Ero1β is incompletely understood. The work presented here suggests that the regulatory disulfide bonds in Ero1α (Cys94-Cys131 and Cys99-Cys104) are conserved in Ero1β (Cys90-Cys130 and Cys95-Cys100). In contrast to a previous report, the present work shows that Cys262 in Ero1β is most likely not engaged in a regulatory disulfide bond. Collectively, these findings indicate that features other than a distinct pattern of disulfide bonds determine the loose redox regulation of Ero1β relative to Ero1α.
Recently, Ero1-complementary pathways for disulfide bond formation have
been identified. The ER-localized glutathione peroxidase 7 and 8 (GPx7 and GPx8) are able to oxidize PDI in vitro. This suggests that these enzymes are PDI peroxidases and may comprise an as yet unidentified pathway for disulfide bond formation in cells. However, the results presented here indicate that GPx7 and GPx8 do not comprise a major pathway for disulfide bond formation in the ER in cells deficient in Ero1 and peroxiredoxin-4. Interestingly, depletion of GPx8 in cells induced expression of an antioxidant response marker only in the presence of Ero1. These findings imply that GPx8 is an important scavenger of Ero1-generated hydrogen peroxide, and thus provides a critical function in negotiating oxidative stress originating from disulfide bond formation.
In conclusion, the results presented here suggest that Ero1-generated hydrogen peroxide is efficiently detoxified in the ER, and this work therefore provides novel insights into the intimate relationship between oxidative protein folding and oxidative stress.