Glutathione Peroxidase 7 Utilizes Hydrogen Peroxide Generated by Ero1α to Promote Oxidative Protein Folding

Abstract

Aims: Ero1 flavoproteins catalyze oxidative folding in the endoplasmic reticulum (ER), consuming oxygen and generating hydrogen peroxide (H 2 O 2 ). The ER-localized glutathione peroxidase 7 (GPx7) shows protein disulfide isomerase (PDI)-dependent peroxidase activity in vitro. Our work aims at identifying the physiological role of GPx7 in the Ero1a/PDI oxidative folding pathway and at dissecting the reaction mechanisms of GPx7. Results: Our data show that GPx7 can utilize Ero1a-produced H 2 O 2 to accelerate oxidative folding of substrates both in vitro and in vivo. H 2 O 2 oxidizes Cys57 of GPx7 to sulfenic acid, which can be resolved by Cys86 to form an intramolecular disulfide bond. Both the disulfide form and sulfenic acid form of GPx7 can oxidize PDI for catalyzing oxidative folding. GPx7 prefers to interact with the a domain of PDI, and intramolecular cooperation between the two redox-active sites of PDI increases the activity of the Ero1a/GPx7/PDI triad. Innovation: Our in vitro and in vivo evidence provides mechanistic insights into how cells consume potentially harmful H 2 O 2 while optimizing oxidative protein folding via the Ero1a/GPx7/PDI triad. Cys57 can promote PDI oxidation in two ways, and Cys86 emerges as a novel noncanonical resolving cysteine. Conclusion: GPx7 promotes oxidative protein folding, directly utilizing Ero1a-generated H 2 O 2 in the early secretory compartment. Thus, the Ero1a/ GPx7/PDI triad generates two disulfide bonds and two H 2 O molecules at the expense of a single O 2 molecule. Antioxid. Redox Signal. 20, 545-556.

Metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase 7

“…To further confirm that GPX‐6 in C. elegans and GPx7 in human share functional similarities, we purified both proteins and measured their peroxidase activities by in vitro NADPH consumption assays. A unique feature of human GPx7 is that it uses H 2 O 2 to oxidize the ER‐localized protein disulfide isomerase (PDI) rather than glutathione itself (Nguyen et al., 2011; Wang et al., 2014). Like human GPx7, C. elegans GPX‐6 did not display any glutathione peroxidase activity but had greatly increased peroxidase activity when PDI was used as a substrate (Figure 6b).…”

“…Mature human GPx7 and C. elegans GPX‐6 proteins including an N‐terminal 6× His tag were purified with a nickel‐chelating column (GE Healthcare), and peroxidase activity was conducted as previously described (Wang et al., 2014). In brief, the decrease in absorbance at 340 nm due to NADPH (150 μ m ; Roche) consumption by glutathione reductase (0.24 U; Sigma) was monitored, in the presence of 150 μ m H 2 O 2 , 0.5 m m GSH (Sigma), and 10 μ m human PDI, with or without 10 μ m human GPx7 or C. elegans GPX‐6, respectively.…”

“…Although studies have reported that metformin regulates redox metabolism in mitochondria (Novelle, Ali, Dieguez, Bernier, & de Cabo, 2016), little is known about the effects of metformin on the redox signaling associated with the ER. In mammalian cells, there are three peroxidases located in the ER, namely glutathione peroxidase 7 (GPx7) (Wang, Zhang, Niu, Sitia, & Wang, 2014), glutathione peroxidase 8 (GPx8) (Ramming, Hansen, Nagata, Ellgaard, & Appenzeller‐Herzog, 2014), and peroxiredoxin 4 (Prx4) (Zito et al., 2010). Of these peroxidases, GPx7 plays a particularly crucial role as GPX7 ‐deficient cells display increased ROS levels and an accumulation of misfolded proteins (Peng et al., 2012; Wei et al., 2012); furthermore, GPX7 ‐knockout mice exhibit increased systemic oxidative stress, increased carcinogenesis, and shortened lifespan (Wei et al., 2012).…”

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

“…These enzymes transfer electrons from glutathione [33], protein thiols [128] or PDI [33,127] to hydrogen peroxide. The source of thiol-oxidizing hydrogen peroxide can be the Ero1-catalyzed reaction [32,127]. However, it was also observed that Ero1-independent luminal hydrogen peroxide generation -via gulonolactone oxidase activity -could also stimulate disulfide bond formation [45].…”

“…This enzyme family is represented by GPx7 and 8 in the ER lumen, which have been proved to be effective PDI peroxidases (i.e. they can use reduced PDI instead of GSH as electron donor) [30][31][32]. However, at least in case of Gpx7, GSH can be an alternative substrate in the reaction, with a relatively low rate [33].…”