Non-native proteins inhibit the ER oxidoreductin 1 (Ero1)–protein disulfide-isomerase relay when protein folding capacity is exceeded

Antti Moilanen,Lloyd W Ruddock

doi:10.1074/jbc.ra119.011766

Abstract

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER. Oxidative protein folding needs to be regulated to avoid hyperoxidation. The folding capacity of the ER is regulated by the unfolded protein response (UPR) and ER-associated degradation (ERAD). The UPR is triggered by unfolded protein stress and leads to up-regulation of cellular components such as chaperones and folding catalysts. These components relieve stress by increasing folding capacity and up-regulating ERAD components that remove non-native proteins. Although oxidative protein folding and the UPR/ERAD pathways each are well-understood, very little is known about any direct cross-talk between them. In this study, we carried out comprehensive in vitro activity and binding assays, indicating that the oxidative protein folding relay formed by ER oxidoreductin 1 (Ero1), and protein disulfide-isomerase can be inactivated by a feedback inhibition mechanism involving unfolded proteins and folding intermediates when their levels exceed the folding capacity of the system. This mechanism allows client proteins to remain mainly in the reduced state and thereby minimizes potential futile oxidation–reduction cycles and may also enhance ERAD, which requires reduced protein substrates. Relief from excess levels of non-native proteins by increasing the levels of folding factors removed the feedback inhibition. These results reveal regulatory cross-talk between the oxidative protein folding and UPR and ERAD pathways.

Highlights

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER
Human ER oxidoreductin 1 (Ero1)␣ requires high concentrations of reduced substrate protein disulfide-isomerase (PDI) for redox activation, as well as for catalytic turnover [12]. Both events are mediated by the same molecular determinant, the protruding ␤-hairpin of Ero1␣ interacting with the PDI-mediated activation and catalysis of Ero1 are inhibited by peptides
Because our results suggested that non-native protein and peptide binding inhibited both Ero1␣ activation and the Ero1␣– PDI catalytic cycle, it is plausible that 17␤-E2 and bisphenol A (BPA) might have the same effect

Summary

Introduction

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER. We carried out comprehensive in vitro activity and binding assays, indicating that the oxidative protein folding relay formed by ER oxidoreductin 1 (Ero1), and protein disulfideisomerase can be inactivated by a feedback inhibition mechanism involving unfolded proteins and folding intermediates when their levels exceed the folding capacity of the system. This mechanism allows client proteins to remain mainly in the reduced state and thereby minimizes potential futile oxidation–reduction cycles and may enhance ERAD, which requires reduced protein substrates. The catalytic oxidation of PDI as well as the activation steps, have been suggested to be mediated by the same molecular determinant: a protruding ␤-hairpin with a tryptophan at the tip (Trp272) in Ero that binds to the b؅ substrate-binding site of PDI [11, 12]

Methods

Results

Conclusion