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.     

A Small Molecule Inhibitor of Endoplasmic Reticulum Oxidation 1 (ERO1) with Selectively Reversible Thiol Reactivity

Endoplasmic reticulum oxidation 1 (ERO1) is a conserved eukaryotic flavin adenine nucleotide-containing enzyme that promotes disulfide bond formation by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen. Although disulfide bond formation is an essential process, recent experiments suggest a surprisingly broad tolerance to genetic manipulations that attenuate the rate of disulfide bond formation and that a hyperoxidizing ER may place stressed cells at a disadvantage. In this study, we report on the development of a high throughput in vitro assay for mammalian ERO1α activity and its application to identify small molecule inhibitors. The inhibitor EN460 (IC₅₀, 1.9 μm) interacts selectively with the reduced, active form of ERO1α and prevents its reoxidation. Despite rapid and promiscuous reactivity with thiolates, EN460 exhibits selectivity for ERO1. This selectivity is explained by the rapid reversibility of the reaction of EN460 with unstructured thiols, in contrast to the formation of a stable bond with ERO1α followed by displacement of bound flavin adenine dinucleotide from the active site of the enzyme. Modest concentrations of EN460 and a functionally related inhibitor, QM295, promote signaling in the unfolded protein response and precondition cells against severe ER stress. Together, these observations point to the feasibility of targeting the enzymatic activity of ERO1α with small molecule inhibitors.     

ERO1-L, a human protein that favors disulfide bond formation in the endoplasmic reticulum

Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins. Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding. When expressed in mammalian cells, the product of the human ERO1-L gene co-localizes with ER markers and displays Endo-H-sensitive glycans. In isolated microsomes, ERO1-L behaves as a type II integral membrane protein. ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y. ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated. These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells.     

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.     

Disulfide Bond Formation in the Mammalian Endoplasmic Reticulum

The formation of disulfide bonds between cysteine residues occurs during the folding of many proteins that enter the secretory pathway. As the polypeptide chain collapses, cysteines brought into proximity can form covalent linkages during a process catalyzed by members of the protein disulfide isomerase family. There are multiple pathways in mammalian cells to ensure disulfides are introduced into proteins. Common requirements for this process include a disulfide exchange protein and a protein oxidase capable of forming disulfides de novo. In addition, any incorrect disulfides formed during the normal folding pathway are removed in a process involving disulfide exchange. The pathway for the reduction of disulfides remains poorly characterized. This work will cover the current knowledge in the field and discuss areas for future investigation.One of the characteristics of proteins that enter the secretory pathway is that they frequently contain covalent linkages called disulfide bonds within and between constituent polypeptide chains. The presence of these linkages is thought to confer stability when secreted proteins are exposed to the extracellular milieu or when membrane proteins are recycled through acidic endocytic compartments. In addition to structural disulfides it is now clear that a number of proteins use the formation and breaking of disulfides as a mechanism for regulation of activity (Schwertassek et al. 2007). Hence, it is important that we have a clear understanding of how correct disulfides are formed within proteins both during the protein folding process and to regulate protein function. The focus of this article will be on how correct disulfides are introduced into proteins within the secretory pathway, specifically within the endoplasmic reticulum (ER) during folding and assembly.The formation of disulfides within polypeptides begins as the protein is being cotranslationally translocated into the ER (Chen et al. 1995). The initial collapse of the polypeptide and formation of secondary structure brings cysteine residues into close enough proximity for them to form disulfides. Correct disulfide formation requires enzymes to both introduce disulfides between proximal cysteines and to reduce disulfides that form during folding but that are not present in the final native structure (Jansens et al. 2002). In addition, proteins that do not fold correctly are targeted for degradation and may require their disulfides to be broken before dislocation across the ER membrane into the cytosol (Ushioda et al. 2008). Hence, there must be a reduction and oxidation pathway present in the ER to ensure that native disulfides form and nonnative disulfides are broken during protein folding.Central to both reduction and oxidation pathways is the protein disulfide isomerase (PDI) family of enzymes (Ellgaard and Ruddock 2005) that are capable of exchanging disulfides with their substrate proteins (Fig. 1). Whether disulfide exchange results in the formation or breaking of a disulfide depends on the relative stability of the disulfides in the enzyme and substrate. To drive the formation of disulfides, the PDI family member must itself be oxidized. It is now clear that there are a number of ways for the disulfide exchange proteins to be oxidized by specific oxidases. Importantly, these oxidases do not introduce disulfides into nascent polypeptide chains; rather, they specifically oxidize members of the PDI family. The exception to this rule is the enzyme quiescin sulfydryl oxidase (QSOX; see below). The pathway for disulfide reduction is not as well characterized. It is known that the PDI family members can be reduced by the low molecular mass thiol glutathione (GSH) (Chakravarthi and Bulleid 2004; Jessop and Bulleid 2004; Molteni et al. 2004) but no enzymatic process for reduction has been identified. The glutathione redox balance within the ER is significantly more oxidized than in the cytosol (Hwang et al. 1992; Dixon et al. 2008), indicating that GSH is actively oxidized to glutathione disulfide either during the reduction of PDI family members or by reducing disulfides in nascent polypeptides directly. However, there is currently no clear indication as to how glutathione disulfide is itself reduced.Open in a separate windowFigure 1.PDI family of enzymes catalyzes disulfide exchange reactions in the endoplasmic reticulum. Nascent polypeptide chains are cotranslationally translocated across the ER membrane whereupon cysteines in close proximity can form disulfides. The reaction is catalyzed by members of the PDI family (depicted as PDI) by a disulfide exchange reaction resulting in the reduction of the PDI active site. If nonnative disulfides are formed these can be reduced by the reverse disulfide exchange reaction, resulting in the oxidation of the PDI active site.Both the formation and breaking of disulfides can be thought of as electron transport pathways that require suitable electron acceptors or donors to drive the flow of electrons. For the purposes of this article the two pathways will be discussed separately, but it should be appreciated that each pathway occurs within the same organelle so the possibility of crossover between them is real. Whether futile redox reactions occur between the pathways is unclear but any kinetic segregation of the pathways will be highlighted where it is known to occur.     

Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum.

Native protein disulfide bond formation in the endoplasmic reticulum (ER) requires protein disulfide isomerase (PDI) and Ero1p. Here we show that oxidizing equivalents flow from Ero1p to substrate proteins via PDI. PDI is predominantly oxidized in wild-type cells but is reduced in an ero1-1 mutant. Direct dithiol-disulfide exchange between PDI and Ero1p is indicated by the capture of PDI-Ero1p mixed disulfides. Mixed disulfides can also be detected between PDI and the ER precursor of carboxypeptidase Y (CPY). Further, PDI1 is required for the net formation of disulfide bonds in newly synthesized CPY, indicating that PDI functions as an oxidase in vivo. Together, these results define a pathway for protein disulfide bond formation in the ER. The PDI homolog Mpd2p is also oxidized by Ero1p.     

Molecular bases of cyclic and specific disulfide interchange between human ERO1alpha protein and protein-disulfide isomerase (PDI)

In the endoplasmic reticulum (ER) of human cells, ERO1α and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1α is essential to avoid ER hyperoxidation. To investigate how ERO1α oxidizes PDI selectively among more than 20 ER-resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding β-hairpin of ERO1α specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b'-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a'-domain. ERO1α associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1α to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1α with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1α-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1α activity may suggest its physiological role in ER redox and protein homeostasis.     

ERO1-β, a pancreas-specific disulfide oxidase,promotes insulin biogenesis and glucose homeostasis

Mammals have two genes encoding homologues of the endoplasmic reticulum (ER) disulfide oxidase ERO1 (ER oxidoreductin 1). ERO1-β is greatly enriched in the endocrine pancreas. We report in this study that homozygosity for a disrupting allele of Ero1lb selectively compromises oxidative folding of proinsulin and promotes glucose intolerance in mutant mice. Surprisingly, concomitant disruption of Ero1l, encoding the other ERO1 isoform, ERO1-α, does not exacerbate the ERO1-β deficiency phenotype. Although immunoglobulin-producing cells normally express both isoforms of ERO1, disulfide bond formation and immunoglobulin secretion proceed at nearly normal pace in the double mutant. Moreover, although the more reducing environment of their ER protects cultured ERO1-β knockdown Min6 cells from the toxicity of a misfolding-prone mutant Ins2^Akita, the diabetic phenotype and islet destruction promoted by Ins2^Akita are enhanced in ERO1-β compound mutant mice. These findings point to an unexpectedly selective function for ERO1-β in oxidative protein folding in insulin-producing cells that is required for glucose homeostasis in vivo.     

Oxidative Activity of Yeast Ero1p on Protein Disulfide Isomerase and Related Oxidoreductases of the Endoplasmic Reticulum

The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.     

Modulation of cellular disulfide-bond formation and the ER redox environment by feedback regulation of Ero1

Introduction of disulfide bonds into proteins entering the secretory pathway is catalyzed by Ero1p, which generates disulfide bonds de novo, and Pdi1p, which transfers disulfides to substrate proteins. A sufficiently oxidizing environment must be maintained in the endoplasmic reticulum (ER) to allow for disulfide formation, but a pool of reduced thiols is needed for isomerization of incorrectly paired disulfides. We have found that hyperoxidation of the ER is prevented by attenuation of Ero1p activity through noncatalytic cysteine pairs. Deregulated Ero1p mutants lacking certain cysteines show increased enzyme activity, a decreased lag phase in kinetic assays, and growth defects in vivo. We hypothesize that noncatalytic cysteine pairs in Ero1p sense the level of potential substrates in the ER and correspondingly modulate Ero1p activity as part of a homeostatic regulatory system governing the thiol-disulfide balance in the ER.     

Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1

The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys⁹⁰-Cys⁹⁷ disulfide in Pdi1p. The principal client-binding site in the Pdi1p b′ domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway.     

Deregulation of Pancreas-Specific Oxidoreductin ERO1β in the Pathogenesis of Diabetes Mellitus

A growing body of evidence has underlined the significance of endoplasmic reticulum (ER) stress in the pathogenesis of diabetes mellitus. ER oxidoreductin 1β (ERO1β) is a pancreas-specific disulfide oxidase that is known to be upregulated in response to ER stress and to promote protein folding in pancreatic β cells. It has recently been demonstrated that ERO1β promotes insulin biogenesis in β cells and thus contributes to physiological glucose homeostasis, though it is unknown if ERO1β is involved in the pathogenesis of diabetes mellitus. Here we show that in diabetic model mice, ERO1β expression is paradoxically decreased in β cells despite the indications of increased ER stress. However, overexpression of ERO1β in β cells led to the upregulation of unfolded protein response genes and markedly enlarged ER lumens, indicating that ERO1β overexpression caused ER stress in the β cells. Insulin contents were decreased in the β cells that overexpressed ERO1β, leading to impaired insulin secretion in response to glucose stimulation. These data indicate the importance of the fine-tuning of the ER redox state, the disturbance of which would compromise the function of β cells in insulin synthesis and thus contribute to the pathogenesis of diabetes mellitus.     

Lipase maturation factor 1 affects redox homeostasis in the endoplasmic reticulum

Lipoprotein lipase (LPL) is a secreted lipase that clears triglycerides from the blood. Proper LPL folding and exit from the endoplasmic reticulum (ER) require lipase maturation factor 1 (LMF1), an ER‐resident transmembrane protein, but the mechanism involved is unknown. We used proteomics to identify LMF1‐binding partners necessary for LPL secretion in HEK293 cells and found these to include oxidoreductases and lectin chaperones, suggesting that LMF1 facilitates the formation of LPL's five disulfide bonds. In accordance with this role, we found that LPL aggregates in LMF1‐deficient cells due to the formation of incorrect intermolecular disulfide bonds. Cells lacking LMF1 were hypersensitive to depletion of glutathione, but not DTT treatment, suggesting that LMF1 helps reduce the ER. Accordingly, we found that loss of LMF1 results in a more oxidized ER. Our data show that LMF1 has a broader role than simply folding lipases, and we identified fibronectin and the low‐density lipoprotein receptor (LDLR) as novel LMF1 clients that contain multiple, non‐sequential disulfide bonds. We conclude that LMF1 is needed for secretion of some ER client proteins that require reduction of non‐native disulfides during their folding.     

Two pairs of conserved cysteines are required for the oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic reticulum

In the major pathway for protein disulfide-bond formation in the endoplasmic reticulum (ER), oxidizing equivalents flow from the conserved ER-membrane protein Ero1p to secretory proteins via protein disulfide isomerase (PDI). Herein, a mutational analysis of the yeast ERO1 gene identifies two pairs of conserved cysteines likely to form redox-active disulfide bonds in Ero1p. Cys100, Cys105, Cys352, and Cys355 of Ero1p are important for oxidative protein folding and for cell viability, whereas Cys90, Cys208, and Cys349 are dispensable for these functions. Substitution of Cys100 with alanine impedes the capture of Ero1p-Pdi1p mixed-disulfide complexes from yeast, and also blocks oxidation of Pdi1p in vivo. Cys352 and Cys355 are required to maintain the fully oxidized redox state of Ero1p, and also play an auxiliary role in thiol-disulfide exchange with Pdi1p. These results suggest a model for the function of Ero1p wherein Cys100 and Cys105 form a redox-active disulfide bond that engages directly in thiol-disulfide exchange with ER oxidoreductases. The Cys352-Cys355 disulfide could then serve to reoxidize the Cys100-Cys105 cysteine pair, possibly through an intramolecular thiol-disulfide exchange reaction.     

Oxidative protein folding: From thiol–disulfide exchange reactions to the redox poise of the endoplasmic reticulum

This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI_red:PDI_ox. The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC–MS–MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment.     

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.     

Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeast

Oxidative protein folding can exceed the cellular secretion machinery, inducing the unfolded protein response (UPR). Sustained endoplasmic reticulum (ER) stress leads to cell stress and disease, as described for Alzheimer, Parkinson, and diabetes mellitus, among others. It is currently assumed that the redox state of the ER is optimally balanced for formation of disulfide bonds using glutathione as the main redox buffer and that UPR causes a reduction of this organelle. The direct effect of oxidative protein folding in the ER, however, has not yet been dissected from UPR regulation. To measure in vivo redox conditions in the ER and cytosol of the yeast model organism Pichia pastoris we targeted redox-sensitive roGFP variants to the respective organelles. Thereby, we clearly demonstrate that induction of the UPR causes reduction of the cytosol in addition to ER reduction. Similarly, a more reduced redox state of the cytosol, but not of the ER, is observed during oxidative protein folding in the ER without UPR induction, as demonstrated by overexpressing genes of disulfide bond-rich secretory proteins such as porcine trypsinogen or protein disulfide isomerase (PDI1) and ER oxidase (ERO1). Cytosolic reduction seems not to be caused by the action of glutathione reductase (GLR1) and could not be compensated for by overexpression of cytosolic glutathione peroxidase (GPX1). Overexpression of GPX1 and PDI1 oxidizes the ER and increases the secretion of correctly folded proteins, demonstrating that oxidative protein folding per se is enhanced by a more oxidized ER and is counterbalanced by a more reduced cytosol. As the total glutathione concentration of these strains does not change significantly, but the ratio of GSH to GSSG is altered, either transport or redox signaling between the glutathione pools of ER and cytosol is assumed. These data clearly demonstrate that protein folding and ER stress have a severe impact on the cytosolic redox balance, which may be a major factor during development of folding-related diseases.