Disulfide bond formation in the mammalian endoplasmic reticulum

Abstract

The formation of disulfide bonds between cysteine residues occurs during the folding of many proteins that enter the secretory pathway. As the polypeptide chain collapses, cysteines brought into proximity can form covalent linkages during a process catalyzed by members of the protein disulfide isomerase family. There are multiple pathways in mammalian cells to ensure disulfides are introduced into proteins. Common requirements for this process include a disulfide exchange protein and a protein oxidase capable of forming disulfides de novo. In addition, any incorrect disulfides formed during the normal folding pathway are removed in a process involving disulfide exchange. The pathway for the reduction of disulfides remains poorly characterized. This work will cover the current knowledge in the field and discuss areas for future investigation.

Figure 1.

PDI family of enzymes catalyzes disulfide exchange reactions in the endoplasmic reticulum. Nascent polypeptide chains are cotranslationally translocated across the ER membrane whereupon cysteines in close proximity can form disulfides. The reaction is catalyzed by members of the PDI family (depicted as PDI) by a disulfide exchange reaction resulting in the reduction of the PDI active site. If nonnative disulfides are formed these can be reduced by the reverse disulfide exchange reaction, resulting in the oxidation of the PDI active site.

Figure 2.

Electron flow following the oxidation of PDI by Ero1. Ero1 accepts electrons from PDI (1) resulting in the oxidation of PDI and the reduction of the shuttle disulfide within Ero1. An internal disulfide exchange reaction then occurs within Ero1 (2) to transfer electrons to cysteines close to the bound FAD. FAD accepts electrons from the disulfide that is formed (3) and in the process is reduced to form FADH2. Oxygen is the ultimate electron acceptor (4) becoming reduced to liberate hydrogen peroxide.

Figure 3.

Alternative pathways for PDI oxidation involving PrxIV or Gpx7/8. For both pathways the initial reaction is the oxidation of the active site cysteine to form sulfenylated cysteine. This residue is resolved by a second cysteine within an adjacent subunit in PrxIV or within the PDI active site for Gpx7/8. Reduced PDI recycles PrxIV to regenerate a free thiol at the active site of PrxIV and in the process becomes oxidized. Any mixed disulfide formed between PDI and Gpx7/8 will be rapidly resolved by the second cysteine in the PDI active site to form oxidized PDI. The cysteine residues marked “res” and “per” in PrxIV refer to the resolving and peroxidatic cysteines, respectively.

Figure 4.

VKOR can also oxidize members of the PDI family. VKOR is a membrane protein that contains two cysteines within a transmembrane helix. These can form a disulfide following the donation of electrons to either vitamin K epoxide (KO) or vitamin K (K), generating vitamin K hydroquinone (KH2). The resulting disulfide can then exchange with two cysteines present within the luminal domain of VKOR. The resulting disulfide can be reduced by the PDI family members TMX1, TMX4, or ERp18, resulting in the oxidation of these proteins.