# Structural Biochemistry/Chemical Bonding/ Disulfide bonds/Multiple Ways to make Disulfides
## Overview

Researchers initially thought that enzyme ERO1, endoplasmic reticulum oxidoreductin 1, couples oxygen reduction to de novo formation of disulfides, but it was recently discovered that mammals that are deficient in this enzyme still survive and form disulfides. This suggests that there exist alternative pathways to forming disulfides. Discoveries have found that peroxiredoxin 4 is involved in peroxide removal and disulfide formation. Many other different pathways for disulfide formation in the mammalian ER include quiescin sulfhydryl oxidase, ER-localized protein disulfide isomerase peroxidases and vitamin K epoxide reductase. These various pathways for forming disulfides are regulated by glutathione.

## Disulfide formation in the endoplasmic reticulum

Many important secretory and cell-surface proteins like antibodies, plasma membrane receptors and channels, extracellular matrix proteins, and blood clotting factors contain disulfide bonds because disulfides heighten protein stability and control redox-dependent functions. Disulfides in the endoplasmic reticulum undergo a procedure catalyzed by membranes of the PDI, protein disulfide isomerase, family and then undergo co-translational translocation to the ER. As the polypeptide starts to fold, cysteine residues that come into close contact form disulfides, even if they do not form in the end product. These disulfides that do not end up folding into the final product are sources of problems for misfolded proteins but may also serve as intermediates in normal folding processes. In order to obtain the final correctly folded disulfide, incorrectly folded disulfides must be disintegrated in a reaction catalyzed by the PDI family. The PDI family thus plays a vital role in the course for the correct formation and reduction of disulfides in order for proper folding of proteins that enter the endoplasmic reticulum
The correspondence of a disulfide between the enzyme and substrate is needed to cause a catalytic reaction to form a disulfide. Since PDI family members each accommodate at least one thioredoxin domain, a CXXC motif alternates between dithiol and disulfide states at their active site. When a disulfide is transferred to the substrate protein, a reduction of the active site occurs. It is necessary that the active site be deoxidized in order for the enzyme to carry out further oxidation. It was initially thought that GSSG, glutathione disulfide, was relevant in how the active site is reoxidized. In Vitro experiments were conducted and showed that a GSH/GSSG ratio similar to that found in the endoplasmic reticulum would efficiently oxidize active site cysteines in PDI, thus transferring disulfides onto substrate proteins. These experiments didn’t show how disulfides were introduced de novo. With the revelation of ERO1p, an enzyme in yeast called endoplasmic oxidoreduction, it was found that this enzyme was necessary for the formation of disulfide. ERO1p plays an important role in oxidizing PDI instead of secreting proteins or low-molecular-weight molecules like GSH and catalyzing oxidation by coupling de novo disulfide formation to the reduction of oxygen to hydrogen peroxide. While securely regulated in order to prevent overproduction of reactive oxygen species, ERO1 showed how disulfides could be formed de novo and also identified the ultimate electron acceptor for the pathway.

## Ero 1 is essential in yeast but not in higher eukaryotes

Knockout of the gene encoding ERO1p in various organisms showed different conclusions of its importance. When the gene encoding ERO1p was knocked out in yeast, it demonstrated its importance as an essential protein in yeast. Knockouts in higher eukaryotes showed different results. When knocked out in D. mlanogasterI, this led to a relatively mild phenotype with a certain problem in its folding of the cell-surface receptor Notch. Two ERO1 paralogs, pair of genes ERO1α and ERO1β, exist in mice and humans. When knocked out of ERO1 β, a defect in the folding of proinsulin occurred. ERO1α is thought to drive disulfide development in other tissues. It was found that a double knockout of ERO1α and ERO1β did not result in a more severe reaction than when ERO1 β is knocked out by itself. This showed that there existed an ERO1-independent pathway for the formation of disulfides in mammalian cells and double knockout cells helped re-establish normal endoplasmic redox conditions.

## Potential pathways to generate disulfides de novo in the ER

PRDX4
Since H2O2 is also generated when a reaction is catalyzed by ERO1, this indicates that additional proteins might be present in the endoplasmic reticulum to remove this reactive oxygen species. Enzymes called peroxiredoxins metabolize H2O2 ensuing disulfide formation. PRDx4 is active in both H2O2 removal and disulfide formation. Studies show a new responsibility for PRDx4 in de novo disulfide formation. Since PRDX4 and several PDI family members within the ER and equivalent, this shows that the enzyme is an abundant endoplasmic resident protein. Under circumstances where PRDX4 was efficiently reduced by some PDI family members during incubation of the two proteins at equal concentrations, some PDI protein was oxidized. This showed that even though GSH was a reductant on its own, when a PDI is included, the efficiency of PRDx4 reduction is enhanced. This proposes that disulfide exchanged between PRDx4 and GSH to form GSSP depends on the presence of PDI. If PRDX4 were to be reduced by PDI family members, a rapid disulfide formation in secretory proteins could occur. These results are confirmed by in vitro evidence. Since PRDX4 enhances a temperature-sensitive mutant of ERO1, viability and disulfide formation in yeast occurs at non-permissive temperatures. However, overexpression of PDI family members can also lead to decrease or increase in the ability of PRDX4 to be reduced in the endoplasmic reticulum. In conclusion, by combining the ERO1 and PRDX4 pathways, every oxygen molecule that is reduced causes two disulfides to be introduced, therefore making the whole process more effective than using ERO1 alone.
GPX7 and GPX8
GPX7 and GPX8, homologous enzymes, belong to the family of thioredoxin GPX-like peroxidases that can also reduce H2O2. They are PDI peroxidases that couple the reduction of H2O2 to oxidation of certain PDI family members. In the presence of these GPXs, certain PDI family members are easily oxidized. With the presence of both GPX and PDI, oxidative refolding of a reduced model protein mediated by H2O2 proceeds faster. Physical associations between ERO1α and both GPXs in cells are shown by bimolecular fluorescence complementation. With the addition of GPX7, the in vitro rate of oxygen consumed by ERO1α increased, indicating a more efficient process in its presence. These biochemical results show an important role for GPX7 and GPX8 in disulfide formation.
QSOX
Erx2p, a sulfhydrl oxidase, when overexpressed could suppress the ero1-1 mutation. This different pathway shows that alternative proteins could potentially fulfill the essential function of ERO1 in yeast and other organisms. QSOX, a flavoprotein known to introduce disulfides into proteins in vitro, is similar to Erv2p. QSOX plays a part in catalyzing de novo disulfide formation by coupling disulfide oxidation to the reduction of oxygen in order to form H2O2. Unlike ERO1, which specially oxidizes only PDI family members, QSOX has a much broader substrate specificity that can introduce disulfides into protein substrates. However, PDI has the ability to greatly enhance native disulfide formation since QSOX cannot isomerize non-native disulfides. QSOX has the ability to complement Δero1 yeast strain when overexpressed. This indicates that QSOX is involved in disulfide formation when in vivo. When ERO1 and QSOX are both knocked down, a more severe phenotype occurs than when ERO1 is knocked down alone indicating that QSOX might provide some function when ERO1 is absent. QSOX currently remains a candidate for de novo disulfide formation independent of ERO1 due to its promiscuous substrate specificity and location in the secretory pathway.
VKOR
The VKOR enzyme exhibits another potential ERO1-independent pathway for disulfide formation . VKOR is a four transmembrane helix protein in the endoplasmic reticulum. VKOR functions by catalyzing the two steps in the reduction of vitamin K epoxide so as to generate vitamin K hydroquinone. When vitamin K epoxide is reduced, a CXXC motif in VKOR is then oxidized to form a disulfide bond. Members in VKOR family exchange this disulfide with thiredoxin-like oxidoreductases in order to oxidize substrate proteins. Since human VKOR does not contain a thioredoxin domain, PDI family members instead serve as VKOR substrates. WHen overexpression occurs with active-site CXXA mutants, VKOR can be trapped in a mixed-disulfide complex with PDI family members, mainly the transmembrane-bound TMX and TMX4. Results of these experiments show that VKOR sustaining disulfide formation is still unclear but proves a potential pathway.

## Relative contribution and interplay between oxidative pathways

With all these potential pathways for disulfide formation, now their relative contribution has to be figured out. ERO1 pathway is an important pathway for disulfide formation under normal physiological conditions. TRDX4 hyperoxidation in cells expresses a deregulated form of ERO1 and shows that the enzyme is active in the ER and has the ability to produce hydrogen peroxide. Even though mammals do no necessarily need this pathway, if it didn’t exist, it would compromise disulfide formation. ERO1 is important in determining the redox status of PDI in cells and also forms mixed disulfides with PDI. This shows that ERO has the ability to oxidize PDI. If animals did not have ERO1 to survive and still formed disulfide-bonded proteins, this shows that other pathways exist. Their relative contributions still have to be explored.
PRDX4-dependent pathway doesn’t provide a significant contribution to disulfide formation. If it did, it would be predicted that the Prdx4 knockout mouse should have a severe phenotype and even though the Prdx4 knockout mouse is viable, it is sterile due to oxidative stress in the testis. PRDX4 pathway is then determined to be crucial for correct function of specific tissues though not essential for survival. If there were a higher presence of PRDX4 in higher eukaryotes, it would show mild phenotypes of ERO1 knockouts as an alternative source of hydrogen peroxide that is available in order to make the PRDX4 pathway work. More research is needed to determine which sources provide the H2O2 necessary to drive disulfide formation. The importance of the PRDX4 pathway needs to be determined, even if PRDX4 provides an alternative pathway to ERO1 for disulfide formation.
An endogenous level, enzymes GPx7 and GPx8 cannot substitute for PrdxIV in ERO1 knockout cells. These enzymes could have a similar function to PRDX4. Research shows that proteins of the peroxiredoxin family have a higher rate of reactivity towards H2O2than GPXs. Evidence shows that interaction between Ero1α with the GPXs in vivo might compensate for their relatively slower rates of reactivity. Further understanding of their characterization is needed of the enzymes in order to explain their roles in de novo disulfide formation. Current research shows that the cellular function of VKOR in disulfide formation remains essentially uncharacterized. In VKOR, the mixed disulfide trapped with PDI family members does suggest a role in the process though. The pathway of oxidation of PDIs by VKOR being coupled strictly to γ-carboxyglutamate formation is not important due to its low flux. This would not be the case if the hydroquinone could be reoxidized through an alternative electron acceptor. This could bring about a more important role in the pathway.
As shown in vitro experiments, direct oxidation by H2O2 of cysteines in PDI and substrate proteins to form disulfides could potentially occur. Although, these pathways are shown to not be important due to faster kinetics of substrate refolding when the reaction mixture contains H2O2 and PDI family members together with either of the PDI peroxidases or PRDX4. Dehydroascorbate is an addition source of oxidizing equivalents in the ER. DHa can be moved into the ER from the cytosol, generated in the ER, and like H2O2, DHA directly oxidizes PDI and unfolds reduced proteins in vitro . Due to its slow rate, the former pathway is shown to not be a main route for the reduction of DHA. A faster pathway occurs with a faster PDI-independent oxidation of protein substrates.

## References

Bulleid NJ, Ellgaard L. Trends Biochem Sci. 2011 Sep;36(9):485-92. Epub 2011 Jul 19. Review. PMID: 21778060 [PubMed - indexed for MEDLINE]a http://www.ncbi.nlm.nih.gov/pubmed/21778060