Abstract
Oxidative protein folding in the ER is driven mainly by oxidases of the endoplasmic reticulum oxidoreductin 1 (Ero1) family. Their action is regulated to avoid cell stress, including hyperoxidation. Previously published regulatory mechanisms are based on the rearrangement of active site and regulatory disulfides. In this study, we identify two novel regulatory mechanisms. First, both human Ero1 isoforms exist in a dynamic mixed disulfide complex with protein disulfide isomerase, which involves cysteines (Cys166 in Ero1α and Cys165 in Ero1β) that have previously been regarded as being nonfunctional. Second, our kinetic studies reveal that Ero1 not only has a high affinity for molecular oxygen as the terminal acceptor of electrons but also that there is a high cooperativity of binding (Hill coefficient >3). This allows Ero1 to maintain high activity under hypoxic conditions, without compromising cellular viability under hyper-hypoxic conditions. These data, together with novel mechanistic details of differences in activation between the two human Ero1 isoforms, provide important new insights into the catalytic cycle of human Ero1 and how they have been fine-tuned to operate at low oxygen concentrations.
Highlights
Control of the redox homeostasis of the ER is of fundamental importance for the effective formation of native disulfide bonds
To examine the kinetics of in vivo–folded wild-type human Ero1α and Ero1β, both human endoplasmic reticulum oxidoreductin 1 (Ero1) paralogs were expressed in Escherichia coli using a system for the production of folded disulfide-bonded proteins, which includes protein disulfide isomerase (PDI) as a catalyst of disulfide bond isomerization (Nguyen et al, 2011a; Gaciarz et al, 2016)
Reversephase HPLC analysis indicated that circa 97% of the Ero1α was in a single, oxidized, redox state, whereas for Ero1β, 78% was oxidized and 22% lacked at least one disulfide (Fig S2)
Summary
Control of the redox homeostasis of the ER is of fundamental importance for the effective formation of native disulfide bonds It is manipulated primarily by oxidative enzymes of the flavindependent endoplasmic reticulum oxidoreductin 1 (Ero1) family (Frand & Kaiser, 1998; Pollard et al, 1998; Cabibbo et al, 2000; Pagani et al, 2000) and by a glutathione buffer formed from a mixture of reduced glutathione (GSH) and oxidized glutathione (GSSG) in a molar ratio of 3:1–6:1 (Hwang et al, 1992; Bass et al, 2004; Dixon et al, 2008). PDI has been suggested to modulate this regulatory switch in a feedback mechanism manner by possessing the capability to activate (Appenzeller-Herzog et al, 2008) and possibly inactivate (Shepherd et al, 2014; Zhang et al, 2014) Ero. The exact mechanisms leading to inactivation of Ero are still unknown with auto-oxidation of Ero regulatory disulfides reported (Zhang et al, 2014)
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