This study confirmed in mice observations made earlier in cultured cells on the redundancy between the ERO1 enzymes (ERO1α and ERO1β) and PRDX4 in initiating a disulfide relay in the ER of mammals. However, contrary to expectation, the kinetic defect in oxidative protein folding induced by mutation in these three upstream ER thiol oxidases was rather modest. Instead, the phenotype of the mutant mice and the biochemical characterization of their cells and tissues revealed an unanticipated consequence of altered ER thiol redox kinetics on intracellular ascorbate metabolism.
Free cysteine thiols that are not converted quickly enough to disulfides are exposed to an alternative fate: oxidation to sulfenic acid. Reduction of the excess cysteinyl-sulfenic acid side chains back to the free thiol converts ascorbate to an unstable oxidized derivative. The subtle kinetic defect in disulfide bond formation depletes the mutant ER of an essential factor (vitamin C), promoting an unconventional form of scurvy with profound defects in the extracellular matrix. This study therefore provides a remarkable example of the emerging links between altered ER protein metabolism and intermediary metabolism.
ERO1 and PRDX4 are biochemically distinct enzymes, but they have in common the ability to initiate a disulfide relay in the ER (Tavender and Bulleid, 2010
; Zito et al., 2010b
mutant mice and single gene defects in the Ero1
isoforms have no evident abnormality in the extracellular matrix. The defect is first apparent in the compound Ero1αm/m; Ero1βm/m
mice and is dramatic in triply compromised Ero1αm/m; Ero1βm/m; Prdx4m/y
mice, providing formal genetic evidence that the three ER thiol oxidases have partially redundant roles. The progressive compromise of the extracellular matrix with compounding of mutations is mirrored by the progressive retention of procollagen and in the progressive increase in levels of cysteinyl-sulfenic acid in mutant cultured fibroblasts. These genotype-phenotype correlations thus argue that a kinetic defect in disulfide bond formation, common to the three mutations, underlies the aforementioned abnormalities in ER function.
Disulfide formation and oxidation to sulfenic acid are alternative fates of cysteinyl thiols in the ER lumen. The combined defect in disulfide relay and in the conversion of H2O2 (to water and a disulfide) synergistically biases the competition in favor of cysteinyl thiols in the TM cells and readily explains the strong dimedone reactivity of protein samples from TM cells. However, the accumulation of cysteinyl thiols in the DM cells (which have no known defect in ER peroxidase activity) and the absence of such a defect in the PRDX4 mutant cells, which lack a peroxidase, suggest that merely a kinetic delay in disulfide formation is sufficient to expose free thiols to this alternative fate.
The transfer of two electrons from a free thiol to peroxide produces cysteinyl sulfenic acid. ERO1 generates H2
in course of disulfide bond formation (Gross et al., 2006
). However, both the previously noted redundancy of ERO1 and PRDX4 (Zito et al., 2010b
; Rutkevich and Williams, 2012
) and the abundance of cysteinyl sulfenic acid in the ER of DM
cells, noted here, exonerate ERO1 as a major contributor to ER peroxide content in mammals; alternative sources, such as mitochondrial respiration and ER-localized NADPH oxidase(s), may dominate the production of peroxide.
Formation of cysteinyl sulfenic acid on one thiol followed by its resolution by a second thiol creates a disulfide (and a water molecule). In the presence of reduced PDI, the disulfide can isomerise to a correctly placed protein disulfide, or it can be reduced back to the dithiol, initiating a PDI-mediated disulfide relay (Karala et al., 2009
). However, both processes are slow, and the cysteinyl sulfenic acid, an intermediate in the pathway to disulfide bond formation, is subject to alternative fates. Our observations suggest that by providing an efficient cysteinyl sulfenic acid-independent route to disulfide bond formation, the ERO1- and PRDX4-initiated disulfide relay pre-empts this alternative. Thus, the ER of the mutant cells is not paradoxically hyperoxidizing but rather coherently misoxidizing.
Ascorbate is the active ingredient in vitamin C, whose absence from the diet results in human scurvy. Mice are able to synthesize ascorbate in their liver and thus do not require a dietary source of the vitamin. Ascorbate is taken up by cells, most likely as dehydroascorbate, which is rapidly converted back to ascorbate in the reducing environment of the cytosol (Linster and Van Schaftingen, 2007
). It is unclear how the active vitamin (ascorbate) makes its way into the ER lumen to exert its antiscorbutic effect. But our findings argue that, once in the ER, ascorbate can reduce cysteinyl-sulfenic acid side chains of ER proteins. The two electrons transferred in this process oxidize ascorbate to dehydroascorbate (Monteiro et al., 2007
). The dehydroascorbate could, in theory, reoxidize thiols to disulfides, contributing to the ER disulfide relay, but this process is too slow for effective recycling back to ascorbate (Saaranen et al., 2010
). In plants, reduced glutathione can be utilized to enzymatically generate ascorbate from dehydroascorbate, in the first step of the Halliwell-Asada cycle (reviewed in Noctor and Foyer, 1998
). However, it is unclear if such enzymatic activity is conserved in animal cells and, if so, whether it is present in the ER. Thus the pool of reduced GSH may well be kinetically isolated from dehydroascorbate produced in the ER and the competing nonenzymatic process, whereby dehydroascorbate hydrolyzed to 2,3-diketo-l-gulonate (2,3-DKG) likely predominates (Bode et al., 1990
). Unlike dehydroascorbate, which has some antiscorbutic activity (because it can be reduced to ascorbate), 2,3-DKG is a dead-end product, and its formation is a net loss of vitamin C.
The cysteinyl-sulfenic acid-driven consumption of ER ascorbate proposed above parallels a similar accelerated depletion of ascorbate reported in cells exposed to Ni+2
(Salnikow et al., 2004
). The parallels extend further, as heavy-metal-mediated ascorbate depletion occurs against a background of high concentration of reduced glutathione and also culminates in the inactivation of the cytoplasmic proline 4 hydroxylases, the PHD enzymes charged with oxygen sensing (Kaczmarek et al., 2007
). While our experiments outline the feasibility of direct depletion of ascorbate by excessive cysteinyl-sulfenic acid side chains of ER client proteins, we cannot exclude the contribution of other radicals generated in the mutant cells. Furthermore, abnormally high oxidized to reduced glutathione ratios have been recently noted in cells compromised in ERO1 (Appenzeller-Herzog et al., 2010
; Rutkevich and Williams, 2012
) and were confirmed here and suggest that depletion of reduced glutathione might favor a situation whereby nascent proteins, oxidized to sulfenic species, selectively exploit ascorbate as their reductant in the mutant cells. This issue also affects the interpretation of the in vitro demonstration of cysteinyl-sulfenic acid-driven consumption of ascorbate (E), which, because of the uncertainty surrounding the concentration of reduced glutathione in the ER, does not take into account the potential for a competing reduction pathway.
The defect in collagen biosynthesis and the abnormal extracellular collagen fibrils are associated with abnormalities of the skin of the ER oxidase-compromised mice, a conspicuous feature of which is an elevated TGF-β gene expression signature and elevated levels of the inflammatory cytokine IL-6. These observations are consistent with a role for the extracellular matrix in regulating TGF-β signaling (Ramirez and Rifkin, 2009
). They are also consistent with the developing notion that altered TGF-β signaling may contribute to the pleiotrophy of primary genetic defects in extracellular matrix proteins (Lindsay and Dietz, 2011
The chain of events linking perturbed ER redox to tissue level abnormalities in extracellular matrix was exposed here through targeted mutagenesis of ER thiol oxidases. However, ER stress, associated with delayed egress of client proteins from the ER and excessive oxidative stress (Malhotra et al., 2008
), accompanies other pathophysiological states and is especially prominent in the context of the metabolic syndrome of obesity and cardiovascular disease (Hotamisligil, 2010
). It is thus tempting to speculate on the possibility that intracellular ascorbate depletion by its reaction with cysteinyl sulfenic acid may starve some cells of vitamin C and thus contribute to altered secretion of extracellular proteins and tissue-level abnormalities in a range of disease states.