Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase

The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Zn2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.     

QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer “subunit” has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.     

Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase

The yeast ERV1 gene encodes a small polypeptide of 189 amino acids that is essential for mitochondrial function and for the viability of the cell. In this study we report the enzymatic activity of this protein as a flavin-linked sulfhydryl oxidase catalyzing the formation of disulfide bridges. Deletion of the amino-terminal part of Erv1p shows that the enzyme activity is located in the 15 kDa carboxy-terminal domain of the protein. This fragment of Erv1p still binds FAD and catalyzes the formation of disulfide bonds but is no longer able to form dimers like the complete protein. The carboxy-terminal fragment contains a conserved CXXC motif that is present in all homologous proteins from yeast to human. Thus Erv1p represents the first FAD-linked sulfhydryl oxidase from yeast and the first of these enzymes that is involved in mitochondrial biogenesis.     

A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p.

Erv2p is an FAD-dependent sulfhydryl oxidase that can promote disulfide bond formation during protein biosynthesis in the yeast endoplasmic reticulum. The structure of Erv2p, determined by X-ray crystallography to 1.5 A resolution, reveals a helix-rich dimer with no global resemblance to other known FAD-binding proteins or thiol oxidoreductases. Two pairs of cysteine residues are required for Erv2p activity. The first (Cys-Gly-Glu-Cys) is adjacent to the isoalloxazine ring of the FAD. The second (Cys-Gly-Cys) is part of a flexible C-terminal segment that can swing into the vicinity of the first cysteine pair in the opposite subunit of the dimer and may shuttle electrons between substrate protein dithiols and the FAD-proximal disulfide.     

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.     

Exploring the Smallest Active Fragment of HsQSOX1b and Finding a Highly Efficient Oxidative Engine

Human quiescin-sulfhydryl oxidase 1 isoform b (HsQSOX1b) is a highly efficient, multiple-domain enzyme that directly inserts disulfide bonds into client protein. However, previous studies have focused mainly on the catalytic activity of the whole protein rather than its domain structure. In this research, we dissected the structure and function of HsQSOX1b and explored its mechanism as a highly efficient sulfhydryl oxidase by analyzing the truncated variants. The results showed that the first HsQSOX1b thioredoxin domain was essential for thiol oxidase activity. The smallest active fragment (SAQ) was identified to consist of a helix-rich region (HRR) and an essential for respiration and viability/augmenter of liver regeneration (ERV/ALR) domain, which remained highly active to oxidize an artificial non-thiol substrate but not small molecular and protein thiols. Our study clearly demonstrated that SAQ is a highly efficient oxidative engine, which shows high efficiency in the de novo disulfide formation and oxygen reduction and that this more efficient oxidative engine is necessary for the highly efficient catalysis of QSOXs compared to Erv1 and Erv2. This study will help address the roles of different HsQSOX1b domains in de novo disulfide formation and encourage the engineering of more efficient QSOX variants for the in vitro folding of disulfide-containing proteins.     

Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.     

Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat/ K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.     

An Inhibitory Antibody Blocks the First Step in the Dithiol/Disulfide Relay Mechanism of the Enzyme QSOX1

Quiescin sulfhydryl oxidase 1 (QSOX1) is a catalyst of disulfide bond formation that undergoes regulated secretion from fibroblasts and is over-produced in adenocarcinomas and other cancers. We have recently shown that QSOX1 is required for incorporation of particular laminin isoforms into the extracellular matrix (ECM) of cultured fibroblasts and, as a consequence, for tumor cell adhesion to and penetration of the ECM. The known role of laminins in integrin-mediated cell survival and motility suggests that controlling QSOX1 activity may provide a novel means of combating metastatic disease. With this motivation, we developed a monoclonal antibody that inhibits the activity of human QSOX1. Here, we present the biochemical and structural characterization of this antibody and demonstrate that it is a tight-binding inhibitor that blocks one of the redox-active sites in the enzyme, but not the site at which de novo disulfides are generated catalytically. Sulfhydryl oxidase activity is thus prevented without direct binding of the sulfhydryl oxidase domain, confirming the model for the interdomain QSOX1 electron transfer mechanism originally surmised based on mutagenesis and protein dissection. In addition, we developed a single-chain variant of the antibody and show that it is a potent QSOX1 inhibitor. The QSOX1 inhibitory antibody will be a valuable tool in studying the role of ECM composition and architecture in cell migration, and the recombinant version may be further developed for potential therapeutic applications based on manipulation of the tumor microenvironment.     

Protein substrate discrimination in the quiescin sulfhydryl oxidase (QSOX) family

This work explores the substrate specificity of the quiescin sulfhydryl oxidase (QSOX) family of disulfide-generating flavoenzymes to provide enzymological context for investigation of the physiological roles of these facile catalysts of oxidative protein folding. QSOX enzymes are generally unable to form disulfide bonds within well-structured proteins. Use of a temperature-sensitive mutant of ubiquitin-conjugating enzyme 4 (Ubc4') as a model substrate shows that QSOX activity correlates with the unfolding of Ubc4' monitored by circular dichroism. Fusion of Ubc4' with the more stable glutathione-S-transferase domain demonstrates that QSOX can selectively introduce disulfides into the less stable domain of the fusion protein. In terms of intermolecular disulfide bond generation, QSOX is unable to cross-link well-folded globular proteins via their surface thiols. However, the construction of a septuple mutant of RNase A, retaining a single cysteine residue, demonstrates that flexible protein monomers can be directly coupled by the oxidase. Steady- and pre-steady-state kinetic experiments, combined with static fluorescence approaches, indicate that while QSOX is an efficient catalyst for disulfide bond formation between mobile elements of structure, it does not appear to have a significant binding site for unfolded proteins. These aspects of protein substrate discrimination by QSOX family members are rationalized in terms of the stringent steric requirements for disulfide exchange reactions.     

Vitamin K epoxide reductase contributes to protein disulfide formation and redox homeostasis within the endoplasmic reticulum

The transfer of oxidizing equivalents from the endoplasmic reticulum (ER) oxidoreductin (Ero1) oxidase to protein disulfide isomerase is an important pathway leading to disulfide formation in nascent proteins within the ER. However, Ero1-deficient mouse cells still support oxidative protein folding, which led to the discovery that peroxiredoxin IV (PRDX4) catalyzes a parallel oxidation pathway. To identify additional pathways, we used RNA interference in human hepatoma cells and evaluated the relative contributions to oxidative protein folding and ER redox homeostasis of Ero1, PRDX4, and the candidate oxidants quiescin-sulfhydryl oxidase 1 (QSOX1) and vitamin K epoxide reductase (VKOR). We show that Ero1 is primarily responsible for maintaining cell growth, protein secretion, and recovery from a reductive challenge. We further show by combined depletion with Ero1 that PRDX4 and, for the first time, VKOR contribute to ER oxidation and that depletion of all three activities results in cell death. Of importance, Ero1, PRDX4, or VKOR was individually capable of supporting cell viability, secretion, and recovery after reductive challenge in the near absence of the other two activities. In contrast, no involvement of QSOX1 in ER oxidative processes could be detected. These findings establish VKOR as a significant contributor to disulfide bond formation within the ER.     

Functional characterization of Mia40p, the central component of the disulfide relay system of the mitochondrial intermembrane space

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.     

Involvement of sulfhydryl oxidase QSOX1 in the protection of cells against oxidative stress-induced apoptosis

The QSOX1 protein, belonging to a new class of FAD-linked Quiescin/Sulfhydryl oxidase, catalyzes disulfide bond formation. To give new insight into the biological function of QSOX1, we studied its involvement in oxidative stress-induced apoptosis and cell recovery of PC12 cells. By real time RT-PCR and flow cytometric analysis, we show that the QSOX1 mRNA and protein levels increased late after the beginning of oxidative treatment and were sustained for 72 h. These levels were still high when the PC12 cells were not dying but had resumed proliferation. The kinetics of QSOX1 expression suggest a more protective effect of QSOX1 rather than an involvement of this protein in apoptosis. Human breast cancer MCF-7 cell lines overexpressing the guinea pig QSOX1 protein submitted to the same treatments appeared less sensitive to cell death than the MCF-7 control cells. The protective effect is partly due to a preservation of the mitochondrial polarization generally lost after an oxidative stress. These results strengthen our hypothesis of a protective role of QSOX1 against apoptosis.     

The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.     

QSOX1 Inhibits Autophagic Flux in Breast Cancer Cells

The QSOX1 protein (Quiescin Sulfhydryl oxidase 1) catalyzes the formation of disulfide bonds and is involved in the folding and stability of proteins. More recently, QSOX1 has been associated with tumorigenesis and protection against cellular stress. It has been demonstrated in our laboratory that QSOX1 reduces proliferation, migration and invasion of breast cancer cells in vitro and reduces tumor growth in vivo. In addition, QSOX1 expression has been shown to be induced by oxidative or ER stress and to prevent cell death linked to these stressors. Given the function of QSOX1 in these two processes, which have been previously linked to autophagy, we wondered whether QSOX1 might be regulated by autophagy inducers and play a role in this catabolic process. To answer this question, we used in vitro models of breast cancer cells in which QSOX1 was overexpressed (MCF-7) or extinguished (MDA-MB-231). We first showed that QSOX1 expression is induced following amino acid starvation and maintains cellular homeostasis. Our results also indicated that QSOX1 inhibits autophagy through the inhibition of autophagosome/lysosome fusion. Moreover, we demonstrated that inhibitors of autophagy mimic the effect of QSOX1 on cell invasion, suggesting that its role in this process is linked to the autophagy pathway. Previously published data demonstrated that extinction of QSOX1 promotes tumor growth in NOG mice. In this study, we further demonstrated that QSOX1 null tumors present lower levels of the p62 protein. Altogether, our results demonstrate for the first time a role of QSOX1 in autophagy in breast cancer cells and tumors.     

Erv1 and Cytochrome c Mediate Rapid Electron Transfer via A Collision-Type Interaction

Being essential for oxidative protein folding in the mitochondrial intermembrane space, the mitochondrial disulfide relay relies on the electron transfer (ET) from the sulfhydryl oxidase Erv1 to cytochrome c (Cc). Using solution NMR spectroscopy, we demonstrate that while the yeast Cc-Erv1 system is functionally active, no observable binding of the protein partners takes place. The transient interaction between Erv1 and Cc can be rationalized by molecular modeling, suggesting that a large surface area of Erv1 can sustain a fast ET to Cc via a collision-type mechanism, without the need for a canonical protein complex formation. We suggest that, by preventing the direct ET to molecular oxygen (O₂), the collision-type Cc-Erv1 interaction plays a role in protecting the organism against reactive oxygen species.     

Disulfide bond generation in mammalian blood serum: detection and purification of quiescin-sulfhydryl oxidase

A sensitive new plate-reader assay has been developed showing that adult mammalian blood serum contains circulating soluble sulfhydryl oxidase activity that can introduce disulfide bonds into reduced proteins with the reduction of oxygen to hydrogen peroxide. The activity was purified 5000-fold to >90% homogeneity from bovine serum and found by mass spectrometry to be consistent with the short isoform of quiescin-sulfhydryl oxidase 1 (QSOX1). This FAD-dependent enzyme is present at comparable activity levels in fetal and adult commercial bovine sera. Thus cell culture media that are routinely supplemented with either fetal or adult bovine sera will contain this facile catalyst of protein thiol oxidation. QSOX1 is present at approximately 25 nM in pooled normal adult human serum. Examination of the unusual kinetics of QSOX1 toward cysteine and glutathione at low micromolar concentrations suggests that circulating QSOX1 is unlikely to significantly contribute to the oxidation of these monothiols in plasma. However, the ability of QSOX1 to rapidly oxidize conformationally mobile protein thiols suggests a possible contribution to the redox status of exofacial and soluble proteins in blood plasma. Recent proteomic studies showing that plasma QSOX1 can be utilized in the diagnosis of pancreatic cancer and acute decompensated heart failure, together with the overexpression of this secreted enzyme in a number of solid tumors, suggest that the robust QSOX assay developed here may be useful in the quantitation of enzyme levels in a wide range of biological fluids.     

In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins

The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40–substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40–substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space of mitochondria.     

Disulfide Bond Formation: Sulfhydryl Oxidase ALR Controls Mitochondrial Biogenesis of Human MIA40

The conserved MIA pathway is responsible for the import and oxidative folding of proteins destined for the intermembrane space of mitochondria. In contrast to a wealth of information obtained from studies with yeast, the function of the MIA pathway in higher eukaryotes has remained enigmatic. Here, we took advantage of the molecular understanding of the MIA pathway in yeast and designed a model of the human MIA pathway. The yeast model for MIA consists of two critical components, the disulfide bond carrier Mia40 and sulfhydryl oxidase Erv1/ALR. Human MIA40 and ALR substituted for their yeast counterparts in the essential function for the oxidative biogenesis of mitochondrial intermembrane space proteins. In addition, the sulfhydryl oxidases ALR/Erv1 were found to be involved in the mitochondrial localization of human MIA40. Furthermore, the defective accumulation of human MIA40 in mitochondria underlies a recently identified disease that is caused by amino acid exchange in ALR. Thus, human ALR is an important factor that controls not only the ability of MIA40 to bind and oxidize protein clients but also the localization of human MIA40 in mitochondria.     

Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin

Endoplasmic reticulum (ER) oxidation 1 (ERO1) transfers disulfides to protein disulfide isomerase (PDI) and is essential for oxidative protein folding in simple eukaryotes such as yeast and worms. Surprisingly, ERO1-deficient mammalian cells exhibit only a modest delay in disulfide bond formation. To identify ERO1-independent pathways to disulfide bond formation, we purified PDI oxidants with a trapping mutant of PDI. Peroxiredoxin IV (PRDX4) stood out in this list, as the related cytosolic peroxiredoxins are known to form disulfides in the presence of hydroperoxides. Mouse embryo fibroblasts lacking ERO1 were intolerant of PRDX4 knockdown. Introduction of wild-type mammalian PRDX4 into the ER rescued the temperature-sensitive phenotype of an ero1 yeast mutation. In the presence of an H(2)O(2)-generating system, purified PRDX4 oxidized PDI and reconstituted oxidative folding of RNase A. These observations implicate ER-localized PRDX4 in a previously unanticipated, parallel, ERO1-independent pathway that couples hydroperoxide production to oxidative protein folding in mammalian cells.