title: Low reduction potential of Ero1alpha regulatory disulphides ensures tight control of substrate oxidation.
abstract: Formation of disulphide bonds within the mammalian endoplasmic reticulum (ER) requires the combined activities of Ero1alpha and protein disulphide isomerase (PDI). As Ero1alpha produces hydrogen peroxide during oxidation, regulation of its activity is critical in preventing ER-generated oxidative stress. Here, we have expressed and purified recombinant human Ero1alpha and shown that it has activity towards thioredoxin and PDI. The activity towards PDI required the inclusion of glutathione to ensure sustained oxidation. By carrying out site-directed mutagenesis of cysteine residues, we show that Ero1alpha is regulated by non-catalytic disulphides. The midpoint reduction potential (E degrees') of the regulatory disulphides was calculated to be approximately -275 mV making them stable in the redox conditions prevalent in the ER. The stable regulatory disulphides were only partially reduced by PDI (E degrees' approximately -180 mV), suggesting either that this is a mechanism for preventing excessive Ero1alpha activity and oxidation of PDI or that additional factors are required for Ero1alpha activation within the mammalian ER.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK.
authors.forename: Karl M
authors.initials: KM
authors.lastname: Baker
authors.forename: Seema
authors.initials: S
authors.lastname: Chakravarthi
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Langton
authors.forename: Alyson M
authors.initials: AM
authors.lastname: Sheppard
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Bulleid
mesh_terms: D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D013879:Thioredoxins; C405327:ERO1A protein, human; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases