Mini-Review |
Address correspondence to Jonathan S. Weissman, Department of Cellular and Molecular Pharmacology and Department of Biochemistry and Biophysics, University of California, San Francisco, HHMI 600 16th St., Genentech Hall S472C, San Francisco, CA 94143-2240. Tel.: (415) 502-7642. Fax: (415) 502-8644. email: jsw1{at}itsa.ucsf.edu
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Abstract |
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Abbreviations used in this paper: FAD, flavin adenine dinucleotide; PDI, protein disulfide isomerase; ROS, reactive oxygen species; UPR, unfolded protein response.
Proteins that traverse the secretory pathway typically depend on disulfide bonds for their maturation and function. These bonds are often crucial for the stability of a final protein structure, and the mispairing of cysteine residues can prevent proteins from attaining their native conformation and lead to misfolding. Classic experiments by Anfinsen et al. (1961) provided evidence that disulfide formation is a spontaneous process and that the polypeptide itself is sufficient for achieving the native state in vitro. However, compared with other aspects of protein folding, disulfide-linked folding is slow due to its dependence on a redox reaction, which requires an electron acceptor. These considerations hinted that disulfide-linked folding is an assisted process in vivo, which was demonstrated by the discovery of dsbA mutants in Escherichia coli that exhibited compromised disulfide formation (Bardwell et al., 1991).
In eukaryotes, oxidative protein folding occurs in the ER. Studies using the classic substrate ribonuclease A led to the identification of protein disulfide isomerase (PDI), a protein that can rearrange incorrect disulfides as well as catalyze disulfide formation and reduction in vitro (Goldberger et al., 1963). Despite the ability of PDI to enhance the rate of disulfide-linked folding, how the ER disposes of electrons as a result of the oxidative disulfide formation reaction remained unknown. Over the past 40 yr, a number of different factors have been proposed to contribute to maintaining the oxidized environment of the ER, including the preferential secretion of reduced thiols and uptake of oxidized thiols, as well as a variety of different redox enzymes and small molecule oxidants (Ziegler and Poulsen, 1977; Hwang et al., 1992; Carelli et al., 1997; Frand et al., 2000). However, the physiological relevance of these to oxidative folding has been unclear due to a lack of genetic evidence.
A combination of genetic and biochemical studies using the yeast Saccharomyces cerevisiae, and more recently mammalian and plant systems, have begun to reveal the proteins and mechanisms behind this fundamental protein folding process. The conserved, ER-resident protein Ero1p plays an analogous role to the bacterial periplasmic protein DsbB in oxidative folding. Both Ero1p and DsbB specifically oxidize a thioredoxin-like protein (PDI in eukaryotes, DsbA in bacteria) that serves as an intermediary in the transfer of oxidizing equivalents to folding proteins (Bardwell et al., 1993; Frand and Kaiser, 1999; Tu et al., 2000). Molecular oxygen can serve as the terminal electron acceptor for disulfide formation in both prokaryotes and eukaryotes (Bader et al., 1999; Tu and Weissman, 2002). Under anaerobic conditions, the DsbBDsbA system can support disulfide formation via alternate electron acceptors, such as fumarate (Bader et al., 1999). However, while in bacteria oxidative folding is conveniently coupled to molecular oxygen through the respiratory chain, Ero1p uses a flavin-dependent reaction to pass electrons directly to molecular oxygen (Tu and Weissman, 2002). As a result, Ero1p activity may generate reactive oxygen species (ROS) that could contribute an additional source of cellular oxidative stress, suggesting that its activity must be regulated according to the folding load. Furthermore, the apparent specificity of Ero1p for PDI may allow the numerous homologues of PDI to remain in the reduced state in order to carry out separate redox functions aside from protein oxidation. This review will focus on the mechanism of Ero1p-catalyzed oxidative folding and its broader cell biological ramifications.
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The components of the eukaryotic oxidative folding machinery |
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PDI has long been known to aid the formation of disulfide bonds. It has been shown to catalyze disulfide bond formation and isomerization, as well as reduction, for a wide range of substrates in vitro (Freedman, 1989), but its role in vivo has been less clear. PDI is an essential protein that constitutes 2% of the protein in the ER and contains two thioredoxin-like Cys-Gly-His-Cys (CGHC) active sites (Goldberger et al., 1963; Laboissiere et al., 1995). The finding that Cys-Gly-His-Ser (CGHS) active site mutants of PDI result in sensitivity to DTT provided evidence of a role for PDI in the formation of disulfide bonds in vivo (Holst et al., 1997). This mutant PDI cannot function as an oxidant of proteins but can still catalyze the isomerization of protein disulfides. While these observations suggest that the essential role of PDI is to unscramble nonnative disulfide bonds (Laboissiere et al., 1995), the DTT-sensitivity phenotype of this mutant PDI argues that PDI normally plays an important role in catalyzing disulfide formation. Furthermore, in ero1-1 mutants, PDI accumulates in a reduced form, suggesting that Ero1p acts upstream of PDI in a pathway for disulfide formation in the ER (Frand and Kaiser, 1999).
Recently, the sulfhydryl oxidase Erv2p has been implicated as playing a role in oxidative folding in the ER parallel to that of Ero1p (Gerber et al., 2001; Sevier et al., 2001; Gross et al., 2002). Erv2p is a member of the ERV/ALR family of sulfhydryl oxidases (Thorpe et al., 2002), which have been found in a number of subcellular compartments. Although Erv2p can compensate for defects in Ero1p when overexpressed (Sevier et al., 2001) and can oxidize proteins directly in vitro (Gerber et al., 2001), under the conditions examined, deletion of ERV2 at most has a modest effect on growth (Sevier et al., 2001; Tu and Weissman, 2002). These observations established that Ero1p is the major player in protein oxidation and that the physiological role of Erv2p as a sulfhydryl oxidase is limited to a subset of nonessential proteins or growth conditions not yet examined. In yeast, there is an essential homologue of Erv2p (Erv1p) that is localized to the mitochondrial intermembrane space (Lange et al., 2001). The sulfhydryl oxidase activity of Erv1p may be important for proper mitochondrial homeostasis and the assembly of iron-sulfur cluster proteins (Lisowsky, 1994; Lee et al., 2000; Lange et al., 2001) perhaps by catalyzing disulfide bond formation, although the oxidative environment of the mitochondrial intermembrane space is not well characterized. Interestingly, a vaccinia virus protein of this ERV family has been implicated in the formation of disulfides in viral proteins during assembly in the host cytosol (Senkevich et al., 2000).
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The Ero1-dependent pathway for the transfer of oxidizing equivalents |
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The role of PDI and its homologues in oxidative folding |
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The inability of Ero1p to interact with several PDI variants suggests that it can discriminate between PDI and its homologues. Such discrimination could allow different PDI-related proteins to function as dedicated disulfide isomerases or reductases, which require cysteine residues in their thioredoxin-like active sites to be in the reduced form (Fig. 1). PDI itself probably also contributes to disulfide isomerization and reduction in vivo. In the bacterial system, it has been shown that the role of these PDI-related proteins is dictated by whether they can interact with upstream oxidases or reductases, rather than the redox potential of their active sites. For example, dimerization of the protein DsbC prevents oxidation by DsbB and allows it to function as a dedicated isomerase (Bader et al., 2001). Mutants of DsbC that disrupt dimerization become oxidized by DsbB and can serve the role of the protein oxidant DsbA (Bader et al., 2001). In addition, DsbC is normally maintained in the reduced form by the periplasmic membrane protein DsbD, which transfers the reducing power of cytosolic thioredoxin to DsbC (Rietsch et al., 1997). Some of the eukaryotic PDI homologues may similarly be kept in a reduced form by an upstream reductase. Resolving how and whether these PDI-related proteins interact with Ero1p should lend insight into their cellular roles and the determinants of function.
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The role of glutathione in oxidative folding |
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Genetic evidence in yeast has demonstrated that glutathione is dispensable for disulfide formation and instead functions as a net reductant in the ER (Cuozzo and Kaiser, 1999). In a screen for suppressors of the temperature sensitivity of the ero1-1 mutant, a deletion of GSH1, which is involved in the biosynthesis of glutathione, was found to strongly suppress the ero1-1 phenotype (Cuozzo and Kaiser, 1999). The interpretation of this observation is that absence of glutathione as a reductant results in less reduction of oxidized PDI and proteins, which allows a compromised ero1-1 oxidation system to support growth. In gsh1 strains, the oxidative folding of CPY proceeds with normal kinetics but is highly sensitive to oxidative stresses, consistent with the role of glutathione as a net reductant (Cuozzo and Kaiser, 1999).
What is the basis for the high GSSG content in the ER? Ero1p cannot directly oxidize GSH to GSSG (Tu et al., 2000). However, Ero1p and PDI can drive the oxidation of folding substrates even in the presence of reduced glutathione (GSH). Over time, a gradual production of GSSG resulting from GSH-mediated reduction of disulfides in PDI and folding proteins is observed in vitro (Tu et al., 2000) (Fig. 1). Thus, the abundance of GSSG in the ER is likely a consequence of Ero1p activity, and the GSH:GSSG redox buffer in the ER represents an equilibrium between the consequences of Ero1p-mediated oxidative and glutathione-mediated reductive processes. The kinetic shuttling of oxidizing equivalents by Ero1p and PDI that occurs independently of the bulk redox environment could explain how the ER supports rapid disulfide formation while maintaining the ability to reduce or rearrange incorrect disulfides, perhaps through glutathione and certain PDI homologues.
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The source of oxidizing potential for the ER |
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Further evidence that oxidative folding in eukaryotes is dependent on FAD came from the discovery that purified Ero1p itself is a novel FAD-binding protein (Tu et al., 2000) (Fig. 1). When FAD is added to purified Ero1p and PDI, these components together can support robust oxidative folding in vitro (Tu et al., 2000). Although FAD is crucial for sustained activity of Ero1p, it is not functioning as the terminal electron acceptor. Each FAD-bound Ero1p molecule can support multiple rounds of PDI oxidation, and an excess of free FAD cannot drive Ero1p-catalyzed disulfide formation under anaerobic conditions (Tu and Weissman, 2002). These observations suggested that molecular oxygen rather than FAD is functioning as the terminal electron acceptor (Tu and Weissman, 2002) (Fig. 1). In vitro experiments confirmed that Ero1p-catalyzed disulfide formation is compromised under anaerobic conditions, and that Ero1p directly consumes molecular oxygen during its reaction cycle (Tu and Weissman, 2002). Furthermore, the ero1-1 mutant is completely inviable under anaerobic conditions at normally permissive temperatures (Tu and Weissman, 2002). The efficient use of molecular oxygen as the terminal electron acceptor by FAD-bound Ero1p could explain how the oxidation of millimolar concentrations of PDI (Gilbert, 1990) is achieved despite FAD concentrations in the low micromolar range (Gliszczynska and Koziolowa, 1998). Ero1p likely uses alternate terminal electron acceptors under anaerobic conditions, though the identity of these acceptors remains unknown.
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The role of FAD in oxidative folding |
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Oxidative folding as a source of cellular oxidative stress |
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The fate of the molecular oxygen consumed by Ero1p remains unclear. A standard two-electron reduction of O2 produces hydrogen peroxide (H2O2). It does not appear that Ero1p is releasing stoichiometric amounts of hydrogen peroxide per disulfide formed, although substoichiometric amounts of hydrogen peroxide can be detected during its catalysis of disulfide formation (Tu and Weissman, 2002). However, on the cellular level, recent studies suggest that uncontrolled Ero1p oxidase activity could be a significant source of oxidative stress. Harding et al. (2003) have recently found that ER stress can lead to the acute production of reactive oxygen species. Stressing the ER in worms lacking the transmembrane kinase PERK, which phosphorylates eIF2 to decrease global translation upon ER stress (Harding et al., 1999), leads to a significant accumulation of peroxides in the cell, and lowering Ero1p function by RNAi largely eliminates this effect (Harding et al., 2003).
These observations indicate that Ero1p could be responsible for a significant proportion of ROS in the cell. A simple calculation indicates the plausibility of this hypothesis. Assuming 1/3 of all proteins are secretory proteins, one disulfide must be formed for every
500 amino acids translated. If the equivalent of approximately three ATP are consumed per amino acid translated (Stryer, 1995), then one disulfide is formed for every
1,500 ATP. ROS from ATP production occurs at 12% frequency (Stryer, 1995), and if approximately four to five ATP are produced per molecule of oxygen reduced, then one to two molecules of ROS are expected per 500 ATP produced through respiration. Assuming one molecule of ROS is produced per disulfide formed, Ero1p-mediated oxidation could account for up to
25% of cellular ROS produced during protein synthesis, which has been suggested by a recent study to be the major source of cellular energy consumption (Princiotta et al., 2003). ROS production by Ero1p appeared significant but substoichiometric, which may be a limitation of the detection method (Tu and Weissman, 2002). The mechanism and extent of ROS production by Ero1p needs to be explored further. Nonetheless, it is clear that disulfide formation could contribute a significant source of ROS, especially in specialized secretory cells. Many secretory proteins also contain large numbers of disulfides, and their proper formation may require multiple cycles of misoxidation, reduction, and reoxidation. In addition, Ero1p activity is the main source of oxidized glutathione (GSSG) in the cell (Cuozzo and Kaiser, 1999; Tu et al., 2000), which contributes an additional source of oxidative stress.
Thus, Ero1p activity may be a substantial source of oxidative stress, necessitating proper regulation of oxidative folding and the function of reductant systems in the ER. It is likely to be important for a cell to tie protein oxidation to its folding load, since without control of oxidative folding, the ER could become over-oxidized, leading to protein misfolding, the production of reactive oxygen species and oxidized glutathione, and the futile consumption of energy in the form of reducing equivalents. This production of oxidizing equivalents may also need to be controlled in order to facilitate the maintenance of PDI homologues and perhaps a portion of PDI itself in a reduced form, and to minimize the intrinsic toxicity caused by oxidative stress associated with disulfide formation. UPR induction is one mechanism to regulate oxidative folding, but there may be more direct means of regulation. As the Ero1p oxidation system is highly responsive to levels of free FAD in the cell, controlling the levels of free FAD available to Ero1p may be a posttranslational mechanism to regulate oxidative folding according to the cell's needs. Although speculative, it is interesting to note that RIB1, which controls the first step of riboflavin biosynthesis, is a target of the UPR (Travers et al., 2000). Moreover, preliminary experiments indicate that free FAD levels in yeast can vary according to its growth phase and conditions (unpublished data). Alternatively, free FAD levels in the ER could be controlled by a FAD-specific transporter.
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Conclusion |
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