Low reduction potential of Ero1alpha regulatory disulphides ensures tight control of substrate oxidation.

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.

Intracellular catalysis of disulfide bond formation by the human sulfhydryl oxidase, QSOX1.

The discovery that the flavoprotein oxidase, Erv2p, provides oxidizing potential for disulfide bond formation in yeast, has led to investigations into the roles of the mammalian homologues of this protein. Mammalian homologues of Erv2p include QSOX (sulfhydryl oxidases) from human lung fibroblasts, guinea-pig endometrial cells and rat seminal vesicles. In the present study we show that, when expressed in mammalian cells, the longer version of human QSOX1 protein (hQSOX1a) is a transmembrane protein localized primarily to the Golgi apparatus.

ERp57 is essential for efficient folding of glycoproteins sharing common structural domains.

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate.

The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress.

Glutathione is a ubiquitous molecule found in all parts of the cell where it fulfils a range of functions from detoxification to protection from oxidative damage. It provides the main redox buffer for cells and as such has been implicated in the formation of native disulphide bonds. However, the discovery of the enzyme Ero1 has called into question the exact role of glutathione in this process.

Glutathione is required to regulate the formation of native disulfide bonds within proteins entering the secretory pathway.

The formation of native disulfide bonds is an essential event in the folding and maturation of proteins entering the secretory pathway. For native disulfides to form efficiently an oxidative pathway is required for disulfide bond formation and a reductive pathway is required to ensure isomerization of non-native disulfide bonds. The oxidative pathway involves the oxidation of substrate proteins by PDI, which in turn is oxidized by endoplasmic reticulum oxidase (Ero1).