The CXXCXXC motif determines the folding, structure and stability of human Ero1-Lalpha

Benham, Adam M.; Cabibbo, Andrea; Fassio, Anna; Bulleid, Neil J.; Sitia, Roberto; Braakman, Ineke

doi:10.1093/emboj/19.17.4493

Cited by 118 publications

(143 citation statements)

References 44 publications

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“…The polyclonal antibody against CRT was purchased from Affinity BioReagents. The BiP antibody was purchased from Stressgen, and the PDI antibody has been described 37 . Immunoprecipitated HA was resolved on nonreducing and reducing 7.5% (w/v) SDS-PAGE.…”

Section: Immunoprecipitation and Sds-pagementioning

confidence: 99%

A critical step in the folding of influenza virus HA determined with a novel folding assay

Maggioni

Liscaljet

Braakman

2005

Nat Struct Mol Biol

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Most principles of protein folding emerged from refolding studies in vitro on small, soluble proteins, because large ones tend to misfold and aggregate. We developed a folding assay allowing the study of large proteins in detergent such that the extent of cellular assistance required for proper folding can be determined. We identified a critical step in the in vivo folding pathway of influenza virus hemagglutinin (HA). Only the formation of the first few disulfides in the top domain of HA required the intact endoplasmic reticulum. After that, HA proceeded to fold efficiently in a very dilute solution, despite its size and complexity. This study paves the way for detailed structural analyses during the folding of complex proteins.Protein folding is the process by which linear polypeptide chains acquire their biologically active three-dimensional conformation. Although all information needed to reach the native conformation is encoded by the amino acid sequence 1 , protein folding inside the cell generally is assisted by molecular chaperones and folding enzymes 2-4 . They mainly protect nascent chains and newly synthesized proteins from misfolding and aggregation. Chaperones and folding enzymes reside in almost every compartment of eukaryotic cells.The endoplasmic reticulum (ER) is highly specialized for the folding of membrane and secretory proteins. One of the bestcharacterized model proteins used to study protein folding in the ER is the influenza virus HA. This protein is not only crucial for influenza virus infection but also an important target for the immune system. HA is a multidomain glycoprotein of 84 kDa with six disulfide bonds and seven N-linked glycans. It folds in the ER, trimerizes and is transported to the plasma membrane, where it is incorporated into new virions. The folding of HA has been studied extensively both in complete cells [5][6][7][8][9][10][11] and in microsomes 12,13 , using radioactive labeling of newly synthesized HA or truncated ribosome-bound nascent chains 14 . Although these studies yielded fairly detailed information on the HA folding process, further molecular insight would require biophysical studies on purified protein, which would disregard the role of the cell.To start bridging the gap between folding studies in vitro and in intact cells, we developed an assay that allows the study of post-translational HA folding in a cell-free system without membranes. We replaced the chase period in the well-established pulse-chase folding assay 6 with a folding analysis in solution: the protein is translated and translocated in intact cells, after which post-translational folding is studied in a detergent cell lysate. Using this 'in vitro chase' assay, we found that HA can fold in a detergent cell lysate without ER assistance, provided that the first folding step occurs in an intact ER. RESULTS HA can fold in a detergent cell lysateWhen studied with the pulse-chase assay in intact cells, newly synthesized HA ran as a single band in a reducing SDS-PAGE gel (Fig. 1a, R) and as three ban...

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Section: Immunoprecipitation and Sds-pagementioning

confidence: 99%

A critical step in the folding of influenza virus HA determined with a novel folding assay

Maggioni

Liscaljet

Braakman

2005

Nat Struct Mol Biol

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“…A prerequisite for the catalytic activity of PDI is the presence of proteins of the Ero1 family within the ER Mezghrani et al 2001;Sevier and Kaiser 2008). These oxidoreductases, represented by Ero1α and Ero1β in human cells, mediate the re-oxidation of PDI after its enzymatic reaction by transferring oxidative equivalents using their CXXCXXC active sites (Benham et al 2000;Pagani et al 2000). The oxidative power of Ero1 proteins originate from their flavin adenine dinucleotide (FAD) prosthetic group that transfers electrons from cysteines to molecular oxygen (Wang et al 2009).…”

Section: Introductionmentioning

confidence: 99%

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

Gilady

Bui

Lynes

et al. 2010

Cell Stress and Chaperones

159

115

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Protein secretion from the endoplasmic reticulum (ER) requires the enzymatic activity of chaperones and oxidoreductases that fold polypeptides and form disulfide bonds within newly synthesized proteins. The best-characterized ER redox relay depends on the transfer of oxidizing equivalents from molecular oxygen through ER oxidoreductin 1 (Ero1) and protein disulfide isomerase to nascent polypeptides. The formation of disulfide bonds is, however, not the sole function of ER oxidoreductases, which are also important regulators of ER calcium homeostasis. Given the role of human Ero1α in the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, we hypothesized that Ero1α may have a redox-sensitive localization to specific domains of the ER. Our results show that within the ER, Ero1α is almost exclusively found on the mitochondria-associated membrane (MAM). The localization of Ero1α on the MAM is dependent on oxidizing conditions within the ER. Chemical reduction of the ER environment, but not ER stress in general leads to release of Ero1α from the MAM. In addition, the correct localization of Ero1α to the MAM also requires normoxic conditions, but not ongoing oxidative phosphorylation.

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“…The most abundant and best-studied member of this family is PDI, which is a major catalyst of disulfide bridge formation in newly synthesized secretory and membrane proteins. After donating its disulfide bridge to a substrate, PDI is reoxidized by Ero1-α, an ER-luminal protein that uses FAD and molecular oxygen in the reaction (15)(16)(17). Another member of the ER family of Trx-like proteins, ERp57, has been implicated in the oxidation of glycosylated substrates maintained in the calnexin/ calreticulin folding cycle (18).…”

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confidence: 99%

Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners

Schulman

Wang

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

134

159

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Vitamin K epoxide reductase (VKOR) sustains blood coagulation by reducing vitamin K epoxide to the hydroquinone, an essential cofactor for the γ-glutamyl carboxylation of many clotting factors. The physiological redox partner of VKOR remains uncertain, but is likely a thioredoxin-like protein. Here, we demonstrate that human VKOR has the same membrane topology as the enzyme from Synechococcus sp., whose crystal structure was recently determined. Our results suggest that, during the redox reaction, Cys43 in a luminal loop of human VKOR forms a transient disulfide bond with a thioredoxin (Trx)-like protein located in the lumen of the endoplasmic reticulum (ER). We screened for redox partners of VKOR among the large number of mammalian Trx-like ER proteins by testing a panel of these candidates for their ability to form this specific disulfide bond with human VKOR. Our results show that VKOR interacts strongly with TMX, an ER membrane-anchored Trx-like protein with a unique CPAC active site. Weaker interactions were observed with TMX4, a close relative of TMX, and ERp18, the smallest Trx-like protein of the ER. We performed a similar screen with Ero1-α, an ER-luminal protein that oxidizes the Trx-like protein disulfide isomerase. We found that Ero1-α interacts with most of the tested Trx-like proteins, although only poorly with the membrane-anchored members of the family. Taken together, our results demonstrate that human VKOR employs the same electron transfer pathway as its bacterial homologs and that VKORs generally prefer membrane-bound Trx-like redox partners.disulfide bond formation | warfarin | quinone | blood coagulation | electron transfer

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The CXXCXXC motif determines the folding, structure and stability of human Ero1-Lalpha

Cited by 118 publications

References 44 publications

A critical step in the folding of influenza virus HA determined with a novel folding assay

A critical step in the folding of influenza virus HA determined with a novel folding assay

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

Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners

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