Endoplasmic reticulum (ER) oxidoreductin 1α (Ero1α) is a disulfide producer in the ER of mammalian cells. Besides four catalytic cysteines (Cys94, Cys99, Cys394, Cys397), Ero1α harbors four regulatory cysteines (Cys104, Cys131, Cys208, Cys241). These cysteines mediate the formation of inhibitory intramolecular disulfide bonds, which adapt the activation state of the enzyme to the redox environment in the ER through feedback signaling. Accordingly, disulfide production by Ero1α is accelerated by reducing conditions, which minimize the formation of inhibitory disulfides, or by mutations of regulatory cysteines. Here we report that reductive stimulation enhances Ero1α activity more potently than the mutation of cysteines. Specifically, mutation of Cys208/Cys241 does not mechanistically mimic reductive stimulation, as it lowers the turnover rate of Ero1α in presence of a reducing agent. The Cys208/Cys241 pair therefore fulfills a function during catalysis that reaches beyond negative regulation. In agreement, we identify a reciprocal crosstalk between the stabilities of the Cys208–Cys241 disulfide and the inhibitory disulfide bonds involving Cys104 and Cys131, which also controls the recruitment of the H2O2 scavenger GPx8 to Ero1α. Two possible mechanisms by which thiol–disulfide exchange at the Cys208/Cys241 pair stimulates the catalytic turnover under reducing conditions are discussed.

In the mammalian endoplasmic reticulum, oxidoreductin-1 (Ero1) generates protein disulfide bonds and transfers them specifically to canonical protein-disulfide isomerase (PDI) to sustain oxidative protein folding. This oxidative process is coupled to the reduction of O-2 to H2O2 on the bound flavin adenine dinucleotide cofactor. Because excessive thiol oxidation and H2O2 generation cause cell death, Ero1 activity must be properly regulated. In addition to the four catalytic cysteines (Cys(94), Cys(99), Cys(104), and Cys(131)) that are located in the flexible active site region, the Cys(208)-Cys(241) pair located at the base of another flexible loop is necessary for Ero1 regulation, although the mechanistic basis is not fully understood. The present study revealed that the Cys(208)-Cys(241) disulfide was reduced by PDI and other PDI family members during PDI oxidation. Differential scanning calorimetry and small angle X-ray scattering showed that mutation of Cys(208) and Cys(241) did not grossly affect the thermal stability or overall shape of Ero1, suggesting that redox regulation of this cysteine pair serves a functional role. Moreover, the flexible loop flanked by Cys(208) and Cys(241) provides a platform for functional interaction with PDI, which in turn enhances the oxidative activity of Ero1 through reduction of the Cys(208)-Cys(241) disulfide. We propose a mechanism of dual Ero1 regulation by dynamic redox interactions between PDI and the two Ero1 flexible loops that harbor the regulatory cysteines.

Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O2) and releases hydrogen peroxide (H2O2), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys208–Cys241 disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H2O2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O2 is reduced to H2O2. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys208/Cys241-dependent mixed-disulfide complex with Ero1α. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys208/Cys241 loop region. Supported by O2 diffusion simulations, these data describe the first enzymatically controlled O2 access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H2O2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.

Unbalanced endoplasmic reticulum (ER) homeostasis (ER stress) leads to increased generation of reactive oxygen species (ROS). Disulfide-bond formation in the ER by Ero1 family oxidases produces hydrogen peroxide (H2O2) and thereby constitutes one potential source of ER-stress-induced ROS. However, we demonstrate that Ero1α-derived H2O2 is rapidly cleared by glutathione peroxidase (GPx) 8. In 293 cells, GPx8 and reduced/activated forms of Ero1α co-reside in the rough ER subdomain. Loss of GPx8 causes ER stress, leakage of Ero1α-derived H2O2 to the cytosol, and cell death. In contrast, peroxiredoxin (Prx) IV, another H2O2-detoxifying rough ER enzyme, does not protect from Ero1α-mediated toxicity, as is currently proposed. Only when Ero1α-catalyzed H2O2 production is artificially maximized can PrxIV participate in its reduction. We conclude that the peroxidase activity of the described Ero1α–GPx8 complex prevents diffusion of Ero1α-derived H2O2 within and out of the rough ER. Along with the induction of GPX8 in ER-stressed cells, these findings question a ubiquitous role of Ero1α as a producer of cytoplasmic ROS under ER stress.

Peroxidases are enzymes that reduce hydroperoxide substrates. In many cases, hydroperoxide reduction is coupled to the formation of a disulfide bond, which is transferred onto specific acceptor molecules, the so-called reducing substrates. As such, peroxidases control the spatiotemporal distribution of diffusible second messengers such as hydrogen peroxide (H 2 O 2 ) and generate new disulfides. Members of two families of peroxidases, peroxiredoxins (Prxs) and glutathione peroxidases (GPxs), reside in different subcellular compartments or are secreted from cells. This review discusses the properties and physiological roles of PrxIV, GPx7, and GPx8 in the endoplasmic reticulum (ER) of higher eukaryotic cells where H 2 O 2 and—possibly—lipid hydroperoxides are regularly produced. Different peroxide sources and reducing substrates for ER peroxidases are critically evaluated. Peroxidase-catalyzed detoxification of hydroperoxides coupled to the productive use of disulfides, for instance, in the ER-associated process of oxidative protein folding, appears to emerge as a common theme. Nonetheless, in vitro and in vivo studies have demonstrated that individual peroxidases serve specific, nonoverlapping roles in ER physiology.

Upon chronic up-regulation of proinsulin synthesis, misfolded proinsulin can accumulate in the endoplasmic reticulum (ER) of pancreatic beta-cells, promoting ER stress and type 2 diabetes mellitus. In Mutant Ins-gene-induced Diabetes of Youth (MIDY), misfolded mutant proinsulin impairs ER exit of co-expressed wild-type proinsulin, limiting insulin production and leading to eventual beta-cell death. In this study we have investigated the hypothesis that increased expression of ER oxidoreductin-1 alpha (Ero1 alpha), despite its established role in the generation of H2O2, might nevertheless be beneficial in limiting proinsulin misfolding and its adverse downstream consequences. Increased Ero1 alpha expression is effective in promoting wild-type proinsulin export from cells co-expressing misfolded mutant proinsulin. In addition, we find that upon increased Ero1 alpha expression, some of the MIDY mutants themselves are directly rescued from ER retention. Secretory rescue of proinsulin-G(B23)V is correlated with improved oxidative folding of mutant proinsulin. Indeed, using three different variants of Ero1 alpha, we find that expression of either wild-type or an Ero1 alpha variant lacking regulatory disulfides can rescue mutant proinsulin-G(B23)V, in parallel with its ability to provide an oxidizing environment in the ER lumen, whereas beneficial effects were less apparent for a redox-inactive form of Ero1. Increased expression of protein disulfide isomerase antagonizes the rescue provided by oxidatively active Ero1. Importantly, ER stress induced by misfolded proinsulin was limited by increased expression of Ero1 alpha, suggesting that enhancing the oxidative folding of proinsulin may be a viable therapeutic strategy in the treatment of type 2 diabetes.

Results: Overexpression of Ero1 α partially rescues secretion of mutant proinsulin-G(B23)V. Conclusion:

SIGNIFICANCE The oxidative process of disulfide-bond formation is essential for the folding of most secretory and membrane proteins in the endoplasmic reticulum (ER). It is driven by electron relay pathways that transfer two electrons derived from the fusion of two adjacent cysteinyl side chains onto various types of chemical oxidants. The conserved, ER-resident endoplasmic oxidoreductin 1 (Ero1) sulfhydryl oxidases that reduce molecular oxygen to generate an active-site disulfide represent one of these pathways. In mammals, two family members exist, Ero1α and Ero1β. RECENT ADVANCES The two mammalian Ero1 enzymes differ in transcriptional and post-translational regulation, tissue distribution, and catalytic turnover. A specific protein-protein interaction between either isoform and protein disulfide isomerase (PDI) facilitates the propagation of disulfides from Ero1 via PDI to nascent polypeptides, and inbuilt oxidative shutdown mechanisms in Ero1α and Ero1β prevent excessive oxidation of PDI. CRITICAL ISSUES Besides disulfide-bond generation, Ero1α also regulates calcium release from the ER and the secretion of disulfide-linked oligomers through its reversible association with the chaperone ERp44. This review explores the functional repertoire and possible redundancy of mammalian Ero1 enzymes. FUTURE DIRECTIONS Systematic analyses of different knockout mouse models will be the most promising strategy to shed new light on unique and tissue-specific roles of Ero1α and Ero1β. Moreover, in-depth characterization of the known physical interactions of Ero1 with peroxidases and PDI family members will help broaden our functional and mechanistic understanding of Ero1 enzymes.

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.