Crystal structure of reduced and of oxidized peroxiredoxin IV enzyme reveals a stable oxidized decamer and a non-disulfide-bonded intermediate in the catalytic cycle.

Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue.

Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulum.

Disulphide formation in the endoplasmic reticulum (ER) is catalysed by members of the protein disulphide isomerase (PDI) family. These enzymes can be oxidized by the flavoprotein ER oxidoreductin 1 (Ero1), which couples disulphide formation with reduction of oxygen to form hydrogen peroxide (H(2)O(2)). The H(2)O(2) produced can be metabolized by ER-localized peroxiredoxin IV (PrxIV). Continuous catalytic activity of PrxIV depends on reduction of a disulphide within the active site to form a free thiol, which can then react with H(2)O(2). Here, we demonstrate that several members of the PDI family are able to directly reduce this PrxIV disulphide and in the process become oxidized. Furthermore, we show that altering cellular expression of these proteins within the ER influences the efficiency with which PrxIV can be recycled. The oxidation of PDI family members by PrxIV is a highly efficient process and demonstrates how oxidation by H(2)O(2) can be coupled to disulphide formation. Oxidation of PDI by PrxIV may therefore increase efficiency of disulphide formation by Ero1 and also allows disulphide formation via alternative sources of H(2)O(2).

Peroxiredoxin IV protects cells from oxidative stress by removing H2O2 produced during disulphide formation.

Disulphide 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 reduction potential of the active site disulfides of human protein disulfide isomerase limits oxidation of the enzyme by Ero1α.

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.

Molecular mechanisms regulating oxidative activity of the Ero1 family in the endoplasmic reticulum.

Formation of disulfide bonds in the endoplasmic reticulum (ER) is catalyzed by the ER oxidoreductin (Ero1) family of sulfhydryl oxidases. Ero1 oxidizes protein disulfide isomerase (PDI), which, in turn, introduces disulfides into ER client proteins. To maintain an oxidized state, Ero1 couples disulfide transfer to PDI with reduction of molecular oxygen, forming hydrogen peroxide. Thus, Ero1 activity constitutes a potential source of ER-derived oxidative stress. Intricate feedback mechanisms have evolved to prevent Ero1 hyperactivity. Central to these mechanisms are noncatalytic cysteines, which form regulatory disulfides and influence catalytic activity of Ero1 in relation to local redox conditions. Here we focus on the distinct regulatory disulfides modulating Ero1 activities in the yeast and mammalian ER. In addition to considering effects on the Ero1 catalytic cycle, we consider the implications of these mechanisms with regard to function of Ero1 isoforms and the roles of Ero1 during responses to ER stress.

Substrate specificity of the oxidoreductase ERp57 is determined primarily by its interaction with calnexin and calreticulin.

The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins.

LuxS-independent formation of AI-2 from ribulose-5-phosphate.

BACKGROUND: In many bacteria, the signal molecule AI-2 is generated from its precursor S-ribosyl-L-homocysteine in a reaction catalysed by the enzyme LuxS. However, generation of AI-2-like activity has also been reported for organisms lacking the luxS gene and the existence of alternative pathways for AI-2 formation in Escherichia coli has recently been predicted by stochastic modelling. Here, we investigate the possibility that spontaneous conversion of ribulose-5-phosphate could be responsible for AI-2 generation in the absence of luxS. RESULTS: Buffered solutions of ribulose-5-phosphate, but not ribose-5-phosphate, were found to contain high levels of AI-2 activity following incubation at concentrations similar to those reported in vivo. To test whether this process contributes to AI-2 formation by bacterial cells in vivo, an improved Vibrio harveyi bioassay was used. In agreement with previous studies, culture supernatants of E. coli and Staphylococcus aureus luxS mutants were found not to contain detectable levels of AI-2 activity. However, low activities were detected in an E. coli pgi-eda-edd-luxS mutant, a strain which degrades glucose entirely via the oxidative pentose phosphate pathway, with ribulose-5-phosphate as an obligatory intermediate. CONCLUSION: Our results suggest that LuxS-independent formation of AI-2, via spontaneous conversion of ribulose-5-phosphate, may indeed occur in vivo. It does not contribute to AI-2 formation in wildtype E. coli and S. aureus under the conditions tested, but may be responsible for the AI-2-like activities reported for other organisms lacking the luxS gene.

Peroxiredoxin IV is an endoplasmic reticulum-localized enzyme forming oligomeric complexes in human cells.

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.