Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulumEro1 is thought to be the only oxidase that mediates the re-oxidation of protein disulphide isomerases (PDIs) during oxidative protein folding in the ER. This study reveals that peroxiredoxin IV can also directly oxidize PDI-family members and thus act as a second source of oxidizing equivalents for disulphide-bond formation in the ER.

SummaryDisulphide formation within the endoplasmic reticulum (ER) requires the activity of the ER oxidase Ero1, and as a consequence, generates hydrogen peroxide. The production of hydrogen peroxide is thought to lead to oxidative stress that ultimately results in apoptosis. Here, we show that mammalian peroxiredoxin IV (PrxIV) metabolises hydrogen peroxide produced by Ero1. We demonstrate that the presence of PrxIV within the ER provides a cytoprotective effect against stresses known to enhance Ero1 activity and perturb ER redox balance. Increased Ero1 activity and production of hydrogen peroxide led to preferential hyperoxidation of PrxIV relative to peroxiredoxins in other cellular compartments. The hyperoxidation was increased by the upregulation of Ero1 and by the expression of a hyperactive Ero1. These findings provide the first evidence for an enzymatic mechanism that facilitates peroxide removal from the ER, and show that the oxidation status of PrxIV acts as a marker for ER oxidative stress.

The peroxiredoxins are a ubiquitous family of proteins involved in protection against oxidative stress through the detoxification of cellular peroxides. In addition, the typical 2-Cys peroxiredoxins function in signalling of peroxide stress and as molecular chaperones, functions that are influenced by their oligomeric state. Of the human peroxiredoxins, Prx IV (peroxiredoxin IV) is unique in possessing an N-terminal signal peptide believed to allow secretion from the cell. Here, we present a characterization of Prx IV in human cells demonstrating that it is actually retained within the ER (endoplasmic reticulum). Stable knockdown of Prx IV expression led to detrimental effects on the viability of human HT1080 cells following treatment with exogenous H2O2. However, these effects were not consistent with a dose-dependent correlation between Prx IV expression and peroxide tolerance. Moreover, modulation of Prx IV expression showed no obvious effect on ER-associated stress, redox conditions or H2O2 turnover. Subsequent investigation demonstrated that Prx IV forms complex structures within the ER, consistent with the formation of homodecamers. Furthermore, Prx IV oligomeric interactions are stabilized by additional non-catalytic disulfide bonds, indicative of a primary role other than peroxide elimination.

Disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum is catalyzed by protein disulfide isomerase (PDI), which is itself thought to be directly oxidized by Ero1α. The activity of Ero1α is tightly regulated by the formation of noncatalytic disulfides, which need to be broken to activate the enzyme. Here, we have developed a novel PDI oxidation assay, which is able to simultaneously determine the redox status of the individual active sites of PDI. We have used this assay to confirm that when PDI is incubated with Ero1α, only one of the active sites of PDI becomes directly oxidized with a slow turnover rate. In contrast, a deregulated mutant of Ero1α was able to oxidize both PDI active sites at an equivalent rate to the wild type enzyme. When the active sites of PDI were mutated to decrease their reduction potential, both were now oxidized by wild type Ero1α with a 12-fold increase in activity. These results demonstrate that the specificity of Ero1α toward the active sites of PDI requires the presence of the regulatory disulfides. In addition, the rate of PDI oxidation is limited by the reduction potential of the PDI active site disulfide. The inability of Ero1α to oxidize PDI efficiently likely reflects the requirement for PDI to act as both an oxidase and an isomerase during the formation of native disulfides in proteins entering the secretory pathway.

Background: Peroxiredoxin IV metabolizes endoplasmic reticulum-derived hydrogen peroxide.Results: Peroxiredoxin IV structures reveal an unusually stable decamer and a sulfenylated intermediate in the enzymatic cycle.Conclusion: The enzymatic cycle of peroxiredoxin IV involves destabilization of the active site followed by formation of a stable disulfide-bonded decamer.Significance: Elucidating peroxiredoxin structures is required to understand how they function during oxidative stress.