Tissue-specific Expression and Dimerization of the Endoplasmic Reticulum Oxidoreductase Ero1β

Abstract

Endoplasmic reticulum oxidoreductases (Eros) are essential for the formation of disulfide bonds. Understanding disulfide bond catalysis in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. Mammals express two related Ero proteins, Ero1␣ and Ero1␤. Ero1␤ is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1␤ can form homodimers and mixed heterodimers with Ero1␣, in addition to Ero-PDI dimers. Ero-Ero dimers require the Ero active site, occur in vivo, and can be modeled onto the Ero1p crystal structure. Our data indicate that the Ero1␤ protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1␤ occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1␤ expression is high, but is inversely correlated with PDI and PDIp levels, demonstrating that cell-specific differences exist in the regulation of oxidative protein folding in vivo.Protein folding in the ER 5 attracts considerable interest because the failure of a protein to fold can lead to a host of genetic and acquired diseases (1), ranging from cystic fibrosis to ␣1 anti-trypsin deficiency (2). Professional secretory cells in particular must regulate the synthesis of their ER membranes and chaperones to cope with the demands of increased protein production. This is achieved through ER to nucleus signaling pathways, mediated by the trans-membrane associated proteins Ire1␣, PERK, and ATF6 (3). ATF6 and Ire1␣ induce the transcription of XBP1 and the splicing of its mRNA, culminating in the expression of UPR target genes (4). XBP1 is required for B cell maturation into antibody-producing plasma cells (5), and recently, XBP1 and chronic unfolded protein responses have been implicated in obesity and the onset of type 2 diabetes (6), suggesting that targeting physiological unfolded protein responses may have therapeutic value in this disease.Disulfide bond formation is an essential component of the protein folding process, and disulfide bonds are required for structural stability, enzymatic function, and regulation of protein activity (7). The catalytic events involving the oxidation, reduction, and isomerization of disulfide bonds take place in the ER. During protein oxidation, PDI introduces native disulfide bonds into substrate proteins, and is reoxidized by the Ero proteins (Ero1p in yeast, Ero1␣ and Ero1␤ in humans) (8 -11). In yeast, Pdi1p is capable of both oxidizing and isomerizing disulfide bonds, although the relative importance of each function has been debated (12). In humans, PDI also contributes to collagen biosynthesis as a component of the prolyl-4-hydroxlase complex (13) and can act as a component of the ER degradation machinery, particularly with respect to the unfolding and retro-translocation of toxins (14). Numerous PDI homologues exist in yeast (Mpd1p, Mpd2p, Eps1p, and Eug...

Protein Folding and Quality Control in the ER

“…Concomitantly, hydrogen peroxide (H 2 O 2 ) is thought to be produced from oxygen as an electron acceptor (Enyedi et al 2010). Ero1a is expressed broadly in multiple human tissuetypes, whereas Ero1b is well-expressed only in certain tissue-types, such as the pancreas (Dias-Gunasekara et al 2005). Interestingly, Ero1a activity is regulated by the isomerization/reduction of intramolecular disulfide bonds that are exerted by PDI monitoring the redox state of the ER, whereas Ero1b seems to be less tightly regulated and shows higher oxidase activity in vitro (Inaba et al 2010;Tavender and Bulleid 2010a;Wang et al 2011).…”

Mutations in the FAD Binding Domain Cause Stress-induced Misoxidation of the Endoplasmic Reticulum Oxidoreductase Ero1β

“…Limited Proteolysis of Ero1␤G252S and Ero1␤H254Y-We have previously characterized the human Ero1␤ protein and shown that Ero1␤ homodimerises and interacts with PDI by both non-covalent and covalent (disulfide-dependent) interactions (22). Ero1␤ is functionally equivalent to Ero1p (21), and both Ero1␤ and Ero1p bind to PDI (31) and homodimerise (32).…”

“…The polyclonal rabbit anti-sera against PDI (28) and Ero1␤ (22) have been described. The mouse monoclonal antibody HA-7 (Sigma), the anti-Myc monoclonal antibody 9B11, and the anti-Myc polyclonal antibody (both Cell Signaling) were commercially available.…”

“…The Ero1␤C390A-Myc mutant has been described previously (22). Site-directed mutagenesis (QuikChange, Stratagene) was used to create Myc-and HAtagged G252S and H254Y mutants with the following forward primers: G252S, GAGTCTTCTATAAGCTTATATCGTCA-CTTCAT GCTAGCATCAATTTAC; H254Y, CTATAAGCT-TATATCGGGACTTTATGCTAGCATCAATTTACATCT-ATG.…”

Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

“…However, the shuttling of PDI/Ero1α complexes into the vicinity of mitochondria would result in the higher availability of mitochondrial ATP and FAD, both of which are required for efficient oxidative protein folding (Papp et al 2005). Alternatively, PDI could be re-oxidized by the other Ero1 family member, Ero1β, which is present in non-detectable levels in HEK 293 and HeLa cells (Dias-Gunasekara et al 2005. Since PDI is found abundantly in all domains of the ER, such a mechanism definitely remains a possibility.…”