Biochemical evidence that regulation of Ero1β activity in human cells does not involve the isoform-specific cysteine 262

Abstract

In the ER (endoplasmic reticulum) of human cells, disulfide bonds are predominantly generated by the two isoforms of Ero1 (ER oxidoreductin-1): Ero1α and Ero1β. The activity of Ero1α is tightly regulated through the formation of intramolecular disulfide bonds to help ensure balanced ER redox conditions. Ero1β is less tightly regulated, but the molecular details underlying control of activity are not as well characterized as for Ero1α. Ero1β contains an additional cysteine residue (Cys262), which has been suggested to engage in an isoform-specific regulatory disulfide bond with Cys100. However, we show that the two regulatory disulfide bonds in Ero1α are likely conserved in Ero1β (Cys90–Cys130 and Cys95–Cys100). Molecular modelling of the Ero1β structure predicted that the side chain of Cys262 is completely buried. Indeed, we found this cysteine to be reduced and partially protected from alkylation in the ER of living cells. Furthermore, mutation of Cys100–but not of Cys262–rendered Ero1β hyperactive in cells, as did mutation of Cys130. Ero1β hyperactivity induced the UPR (unfolded protein response) and resulted in oxidative perturbation of the ER redox state. We propose that features other than a distinct pattern of regulatory disulfide bonds determine the loose redox regulation of Ero1β relative to Ero1α.

Polyamine Metabolism and Oxidative Protein Folding in the ER as ROS-Producing Systems Neglected in Virology

“…Other intramolecular disulfide bonds also exist that are implicated in regulation of Ero1p activity [ 175 ]. Similar intra- and intermolecular disulfide-dithiol exchange has been demonstrated for human Ero1α and Ero1β isoforms [ 176 , 177 , 178 ]. Kinetic analysis revealed that formation of the correct regulatory S–S bond within Ero1α in the presence of O 2 or H 2 O 2 is slow, but is significantly enhanced in the presence of PDI family members [ 179 ].…”

Ero1‐Pdi1 module‐catalysed dimerization of a nucleotide sugar transporter, FonNst2, regulates virulence of Fusarium oxysporum on watermelon

“…The Ero1‐Pdi1 module is critical to the formation of disulfide bonds of substrate proteins in yeast and mammals (Anelli et al ., 2002; Vitu et al ., 2010; Hansen et al ., 2014). Considering that FonNst2 exists as dimers (Fig.…”

Molecular analysis of human Ero1 reveals novel regulatory mechanisms for oxidative protein folding

“…Addition of DTT to the Ero1α complex results in a mix of activated Ero1 states, whereas addition of DTT and GuHCl results in the formation of the fully reduced state. Ero1β is expected to have a free thiol ( Hansen et al, 2014 ); hence, the nonreduced protein was treated with 20 mM NEM to avoid disulfide rearrangement during the analysis.…”

“…Before rpHPLC analysis, 100 μg of purified Ero1α complex diluted in 500 µl of 20 mM sodium phosphate, pH 7.0, was treated with or without 2 mM DTT or 5.5 M guanidinium chloride plus 2 mM DTT for 15 min at RT. Similarly, 100 μg of Ero1β complex was treated with guanidinium chloride in the presence of either 2 mM DTT or 20 mM NEM to avoid potential disulfide rearrangement with the Ero1β-specific unpaired Cys262 ( Hansen et al, 2014 ). The samples were injected and bound to a μPRC C2/C18 ST 4.6/100 column (Amersham Biosciences) in a mobile phase of 0.1% TFA.…”

“… (A) Disulfide patterns of Ero1α OX2 ( Appenzeller-Herzog et al, 2008 ; Inaba et al, 2010 ; Hansen et al, 2012 ) and Ero1β oxidized (OX) redox states ( Hansen et al, 2014 ). Cysteines are shown as circles with reported functions indicated with colors.…”