Multiple ways to make disulfides

Review

Multiple ways to make disulfides Neil J. Bulleid1 and Lars Ellgaard2 1

Institute of Molecular, Cellular and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK 2 Department of Biology, University of Copenhagen, DK2200 Copenhagen, Denmark

Our concept of how disulfides form in proteins entering the secretory pathway has changed dramatically in recent years. The discovery of endoplasmic reticulum (ER) oxidoreductin 1 (ERO1) was followed by the demonstration that this enzyme couples oxygen reduction to de novo formation of disulfides. However, mammals deficient in ERO1 survive and form disulfides, which suggests the presence of alternative pathways. It has recently been shown that peroxiredoxin 4 is involved in peroxide removal and disulfide formation. Other less well-characterized pathways involving quiescin sulfhydryl oxidase, ER-localized protein disulfide isomerase peroxidases and vitamin K epoxide reductase might all contribute to disulfide formation. Here we discuss these various pathways for disulfide formation in the mammalian ER and highlight the central role played by glutathione in regulating this process. Disulfide formation in the endoplasmic reticulum Most secretory and cell-surface proteins contain disulfide bonds. This large subset of proteins includes biologically important molecules such as antibodies, plasma membrane receptors and channels, extracellular matrix proteins, blood clotting factors and a number of hormones. Disulfides enhance protein stability and regulate redoxdependent functions. In the endoplasmic reticulum (ER), disulfides are formed in a process catalyzed by members of the protein disulfide isomerase (PDI) family (see Glossary). Following co-translational translocation into the ER, a polypeptide will begin to fold. During folding, cysteine residues that come into close proximity can form disulfides, even if they are not linked in the final native structure. Such non-native disulfides are prevalent in misfolded proteins, but can also be intermediates in normal folding [1,2]. For native disulfides to form, non-native disulfides must be broken in a reaction that is also catalyzed by members of the PDI family. Hence, the PDI family plays a crucial role in both the formation and reduction of disulfides for correct folding of proteins entering the ER (Figure 1) [3–5]. The catalytic reaction that results in formation of a disulfide involves exchange of a disulfide between the enzyme and substrate. PDI family members each contain at least one thioredoxin domain [3,6]. At their active site, a CXXC motif shuttles between dithiol and disulfide states. Disulfide transfer to the substrate protein will result in reduction of the active site, which must be reoxidized for the enzyme to carry out further oxidation (Figure 1). How the active site is reoxidized has been the focus of much Corresponding authors: Bulleid, N.J. ([email protected]); Ellgaard, L. ([email protected]).

research over the last 15 years. A role for glutathione disulfide (GSSG) in this process was initially thought to be paramount. In vitro experiments showed that a GSH/ GSSG ratio similar to that found in the ER could efficiently oxidize active-site cysteines in PDI, which could then transfer disulfides onto substrate proteins [3]. However, this concept did not address the question of how disulfides are generated de novo. A solution was provided by the discovery of an enzyme in yeast, called ER oxidoreductin or Ero1p, which was shown to be essential for disulfide formation [7,8]. Importantly, Ero1p oxidizes PDI rather than secreted proteins or low-molecular-weight molecules such as GSH [9,10]. Further experiments revealed that Ero1p and the mammalian homologues ERO1a and ERO1b are able to catalyze oxidation by coupling de novo disulfide formation to the reduction of oxygen to hydrogen peroxide (H2O2) [11–13]. All forms of ERO1 are tightly regulated, possibly to prevent overproduction of reactive oxygen species (ROS) (Box 1). The discovery of Ero1 neatly provided an answer to how disulfides could be formed de novo and identified the ultimate electron acceptor for the pathway. Ero1 is essential in yeast but not in higher eukaryotes Knockout of the gene encoding Ero1p demonstrated that it is an essential protein in yeast [7,8]. However, when Glossary Glutathione: tripeptide with the sequence g-Glu-Cys-Gly that exists in a reduced (GSH) and an oxidized (GSSG; the disulfide-linked dimer) form. GSH is synthesized in the cytosol and is present throughout the cell in millimolar concentrations, which make it an important cellular thiol-disulfide redox buffer. Glutathionylation: covalent modification of a free cysteine with GSH through a disulfide bond. Glutathionylation protects thiols from undergoing oxidation to sulfenic, sulfinic and sulfonic acids (see below). Microsomes: isolated membrane-bound vesicles derived from the ER. Microsomes are capable of translocating and modifying proteins essentially as intact ER membranes because they contain all the key components associated with these functions. Protein disulfide isomerases (PDIs): family of thiol-disulfide oxidoreductases of the ER. Their unifying feature is the presence of one or more thioredoxin-like domains. Approximately 20 members of the family are known in mammalian cells. Reactive oxygen species (ROS): diverse group of small molecules such as nitric oxide (NO) and derivatives, hypochlorous acid (HOCl), superoxide (O2–) and hydrogen peroxide (H2O2). ROS function in signal transduction and antimicrobial defense, but have the capacity to induce oxidative damage to a broad range of biomolecules such as lipids, DNA and proteins [43]. Redox balance: equilibrium between reduction and oxidation reactions. In the ER, redox balance is likely to be determined by the GSH/GSSG ratio. Sulfenylation: oxidation of a thiol to sulfenic acid (–SOH). Although this modification is reversible, further oxidation to sulfinic acid (–SO2H) and sulfonic acid (–SO3H) is irreversible and potentially damaging to protein function. Sulfhydryl oxidases: enzymes that catalyze disulfide bond formation using molecular oxygen in a reaction that generates hydrogen peroxide. The flavoproteins Ero1 and QSOX both belong to this class of enzymes.

0968-0004/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2011.05.004 Trends in Biochemical Sciences, September 2011, Vol. 36, No. 9

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Non-native disulfide

SH

S S

SH SH

SH SH

Disulphide exchange

SH

Isomerization

S S

SH SH

PDI

PDI

Reduced substrate Active-site reoxidation

PDI SH SH PDI SH

Native disulfide

S S

Ti BS

Figure 1. PDI family members exchange disulfides with substrate proteins. Cysteine pairs in newly synthesized proteins entering the ER form disulfides following a disulfide exchange reaction with a member of the PDI family of proteins. After disulfide exchange, the PDI active site needs to be reoxidized in a process involving de novo disulfide formation. Note that both non-native and native disulfides can be formed as a result of disulfide exchange with the PDI family member. Non-native disulfides will be subjected to isomerization, either through rounds of reduction and oxidation or by direct isomerization to form the native disulfide. It is important to point out that reactions involving free cysteines – depicted here and in subsequent figures in their protonated state (–SH) – take place via the thiolate anion (–S–) state. Reduced PDI family member, green; oxidized PDI family member, orange; substrate, grey.

knockout experiments were carried out in higher eukaryotes, a more complicated picture emerged. Ero1 knockout in D. melanogaster led to a relatively mild phenotype with a specific defect in folding of the cell-surface receptor Notch [14]. In mice (and humans) there are two ERO1 paralogs, ERO1a and ERO1b [15,16]. Knockout of ERO1b resulted in a defect in the folding of proinsulin [17]. This result was perhaps not unexpected, because ERO1b expression within the pancreas is localized to insulin-producing cells and it was assumed that ERO1a might drive disulfide formation in other tissues. It was, therefore, very surprising to find that double knockout of ERO1a and b did not yield a more severe phenotype than ERO1b knockout alone [17]. This result argued strongly for an ERO1-independent pathway for disulfide formation in mammalian cells. In support, double knockout cells re-established normal ER redox conditions after a strong reductive challenge, albeit at a slower rate than in wild-type cells [18]. At least four additional pathways have the potential to generate disulfides de novo in the ER (Figure 2). These pathways are defined by their key enzyme(s): peroxiredoxin (PRDX) 4 [19,20], glutathione peroxidase (GPX) 7 and GPX8 [21], quiescin sulfhydryl oxidase (QSOX) [22] and vitamin K epoxide reductase (VKOR) [23]. Role of PRDX4 in disulfide formation The fact that the reaction catalyzed by ERO1 produces H2O2 suggested that additional proteins might be present in the ER to remove this ROS. Throughout the cell, a group of enzymes called peroxiredoxins metabolize H2O2, which results in disulfide formation. The mechanism of disulfide 486

formation is quite distinct from ERO1 catalysis, and involves initial oxidation of the active-site cysteine to sulfenic acid, followed by reaction of the sulfenylated cysteine with a second cysteine, present in an adjacent polypeptide, to form an interchain disulfide bond (Figure 3). This disulfide can then be exchanged with a thioredoxindomain-containing protein. It has been shown that a ubiquitously expressed [24], ER-localized peroxiredoxin, PRDX4, is active in both H2O2 removal and disulfide formation. Evidence of a role for PRDX4 in de novo disulfide formation comes from in vitro and in vivo studies. It was shown that relative concentrations of PRDX4 and several PDI family members within the ER are equivalent, which indicates that the enzyme is indeed an abundant ER resident protein [19]. Disulfidebonded PRDX4 was efficiently reduced by some, but not all, PDI family members when the two proteins were incubated together at equal concentrations, during which the PDI protein was oxidized [19]. Strikingly, GSH was a modest reductant on its own, but when a PDI family member was included the efficiency of PRDX4 reduction was enhanced [19]. Under these conditions, the PDI family member itself was efficiently reduced, which suggests that disulfide exchange between PRDX4 and GSH to form GSSG depends on the presence of PDI. Finally, PRDX4 catalyzed H2O2and PDI-dependent oxidative refolding of reduced RNase [20]. Such evidence indicates that reduction of PRDX4 by PDI family members could lead to rapid disulfide formation in secretory proteins, either directly or via GSSG. The in vivo evidence confirms these conclusions. It was shown that Prdx4 complements a temperature-sensitive mutant of Ero1 (ero1-1) allowing both viability and disulfide formation in yeast at non-permissive temperatures [20]. Knockdown of PRDX4 in cells derived from ERO1a and ERO1b double knockout mice resulted in both a decrease in cell viability and a reducing redox balance [20]. In addition, knockdown or overexpression of PDI family members in mammalian cells led to a decrease or increase, respectively, in the ability of PRDX4 to be reduced in an intact ER [19]. In summary, PRDX4 can efficiently oxidize PDI family members in vivo and its depletion can exacerbate the phenotype of mouse cells derived from double knockout of ERO1a and ERO1b. Combining the ERO1 and PRDX4 pathways means that for every oxygen molecule reduced, two disulfides are introduced, thereby making the whole process more efficient than if ERO1 acted alone. The PDI peroxidases GPX7 and GPX8 PRDX4 is not the only ER enzyme that can reduce H2O2. The ER also harbors two homologous enzymes, GPX7 and GPX8 [25], that belong to the family of thioredoxin GPXlike peroxidases [26]. More specifically, they are PDI peroxidases that can couple the reduction of H2O2 to oxidation of some PDI family members (Figure 2) [21]. Whereas GSH on its own is a poor substrate for peroxide-mediated oxidation by GPX7 and GPX8, certain PDI family members are readily oxidized in the presence of GPXs. In addition, oxidative refolding of a reduced model protein mediated by H2O2 proceeds significantly faster when performed with both PDI and a GPX present compared to either of these enzymes or H2O2 alone. Moreover, bimolecular

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Box 1. Intramolecular disulfide bonds regulate Ero1 activity The consequence of coupling of PDI oxidation to oxygen reduction by ERO1 is that hydrogen peroxide is produced. In a cell that is active in the formation of disulfide-bonded proteins, this production of hydrogen peroxide has the potential to cause damage to proteins, lipids and DNA due to oxidation [70]. In addition, PDI catalyzes isomerization of non-native disulfide bonds and therefore needs to be present in the cell in a reduced state. Thus, ERO1 activity needs to be tightly regulated to prevent ROS build-up and to limit PDI oxidation. Regulation of ERO1 occurs via a feedback mechanism involving the formation of non-catalytic disulfides that need to be reduced to activate the enzyme [12,55,61]. It is thought that in Ero1p the regulatory disulfides restrict movement of the so-called shuttle

[(Box_1)TD$FIG]

Ero1p OX (yeast)

40 52

90 100105

3 0 6 14 15 16

disulfide, which shuttles electrons between the substrate and FADproximal cysteines (Figure I). This movement restriction prevents interaction with the substrate or with cysteine residues close to the FAD group [61,71]. In ERO1a, regulatory disulfides are formed between non-catalytic and catalytic cysteines, thereby preventing activity [12,55,72]. Although we do not know what reduces or reoxidizes these disulfides, redox balance within the ER is probably what determines the ERO1 activation status. Hence, if the ER becomes more oxidizing, ERO1 is inactivated by the formation of regulatory disulfides and vice versa. It has been shown that the sensor molecule that reports the ER redox status to ERO1a is PDI [18]. Thus, oxidized PDI turns off ERO1a, whereas reduced PDI activates it.

8

20

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? ERO1α OX2 (human)

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Key:

85 94 99 104

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1

13

6

16

8 1 20 24

Inner active site (FAD adjacent)

1 4 7 39 39 39

Structural/unknown

Regulatory

Ti BS

Figure I. Disulfide connectivity in Ero1p and ERO1a. Circles represent cysteine residues (numbered as indicated). The function of each residue is shown by the color. Regulatory disulfides are shown as black lines connecting yellow cysteines. Orange, flexible loop region; black, four a-helices, which together form the catalytic core structure that binds the FAD moiety. The exact nature (structural or regulatory) of the C143–C166 disulfide in Ero1p is unclear (indicated by the broken line). ERO1a C166 was modified with an unknown molecule (indicated by the question mark). The figure is based on the crystal structure of both proteins, as well as biochemical data [12,55,71–73].

fluorescence complementation suggested a physical association between ERO1a and both GPX7 and GPX8 in cells [21]. In accordance, the in vitro rate of oxygen consumption by ERO1a (as a measure of PDI oxidation) increased on addition of GPX7, which indicates a more efficient process in its presence. These biochemical data suggest a role for GPX7 and GPX8 in disulfide formation, although cell biological support for this idea is currently lacking. QSOX: an oxidase in search of a function A search for proteins that could suppress the ero1-1 mutation identified a sulfhydryl oxidase called Erv2p, which, when overexpressed, could complement a Dero1 strain [27]. Clearly this alternative pathway for disulfide formation does not operate under normal physiological conditions, but its existence indicated that other proteins could fulfill the essential function of Ero1 in yeast and, by conjecture, in other organisms. Erv2p is related to mammalian QSOX, which is known to introduce disulfides into proteins in vitro, although its physiological role remains unclear. Like ERO1 and Erv2, QSOX is a flavoprotein that catalyses de novo disulfide formation by coupling disulfide oxidation to the reduction of oxygen to form H2O2 [22]. However, unlike ERO1, which only oxidizes PDI family members, QSOX has broad substrate specificity and therefore does not require PDI to introduce disulfides into protein substrates (Figure 2). The efficiency of native disulfide formation is,

however, greatly enhanced in the presence of PDI because QSOX cannot isomerize non-native disulfides [28]. Mammalian QSOX consists of an Erv2-like domain harboring the oxidase activity, along with two thioredoxin domains [29]. It is thought that the ability to bypass the disulfide exchange reaction catalyzed by PDI is due to the ability of the protein to shuttle disulfides between the Erv2-like domain and the first thioredoxin domain, which can then exchange its disulfide with substrate proteins. Evidence to suggest that QSOX is involved in disulfide formation in vivo comes from its ability to complement a Dero1 yeast strain when overexpressed [30]. In addition, knockdown of the QSOX ortholog in D. melanogaster resulted in defective secretion of EGF-domain-containing proteins [14]. However, there was no observable phenotype at the organismal level following QSOX knockdown, which suggests that this enzyme plays a redundant or nonessential role in the fruit fly. Combined ERO1 and QSOX knockdown led to a more severe phenotype than ERO1 knockdown alone, which indicates that QSOX might well provide some function when ERO1 is absent. In mammalian cells, substrates of QSOX are still unknown, but its steady-state subcellular location in the Golgi apparatus seems at odds with a function in the ER [31]. Given its promiscuous substrate specificity and location in the secretory pathway, QSOX remains a candidate for de novo disulfide formation independent of ERO1. 487

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O2

ERO1FADH2

H2 O2

ERO1 FAD SH SH

H2 O2

PRDX4 H2 O2

2H 2O

QSOX FAD

PRDX4 S

O2

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SH H2 O2

H 2O

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GPX7/8 SOH

ROS/Ascox

Substrate

GPX7/8

Ascorbate

SH SH

SH SH S

S

PDI Substrate

DHA SH SH Vitamin K epoxide

VKOR VKOR

Vitamin K H 2 S

S Ti BS

Figure 2. Disulfides can be introduced into PDI family members and substrate proteins via multiple pathways. De novo disulfide formation can occur via several mechanisms. For ERO1 and PRDX4, biochemical and cell biological data support their role in the processes as depicted here, whereas all other pathways are biochemically supported but lack solid cell biological evidence in mammalian cells (indicated by the broken-lined boxes). Note that QSOX does not oxidize PDI, but rather oxidizes the substrate directly. ERO1 and QSOX are sulfhydryl oxidases that couple reduction of oxygen to disulfide formation via oxidation of enzyme-bound FADH2. Peroxiredoxin and glutathione peroxidases utilize oxidation of a cysteine residue to a highly reactive sulfenic acid, which reacts with a free thiol to form a disulfide. VKOR can accept electrons from PDI to regenerate vitamin K during g-glutamyl carboxylation [23]. Dehydroascorbate (DHA) can accept electrons from PDI (or substrate; not shown) to regenerate ascorbate [47]. Glutathione also plays an important role in maintaining balanced ER redox conditions (not shown; see the text for details). The reduced form of the molecules is shown in red and the oxidized form in blue, except for the PDI family member (green, reduced; orange, oxidized). Black arrows indicate the flow of oxidizing equivalents, and grey arrows the flow of reducing equivalents (i.e. electrons). ROS, reactive oxygen species; Ascox, ascorbate oxidase.

VKOR: a specific oxidase for transmembrane PDI family members? A fourth potential ERO1-independent pathway for disulfide formation involves the VKOR enzyme. Human VKOR is a four transmembrane helix protein of the ER [23] that can catalyze the two steps in the reduction of vitamin K epoxide to generate vitamin K hydroquinone [32]. The latter is an essential co-factor for the g-glutamyl carboxylase enzyme that catalyzes g-carboxylation of glutamate residues, a post-translational modification crucial for the function of blood-clotting factors [33]. On reduction of vitamin K epoxide, a CXXC motif in VKOR is oxidized to form a disulfide bond [34]. In bacteria, members of the VKOR family exchange this disulfide with thioredoxin-like oxidoreductases that in turn oxidize substrate proteins [35]. The thioredoxin domain can be located on an additional transmembrane helix of the VKOR protein itself [36], a feature that positions it favorably for oxidation by 488

the internal CXXC motif [37]. Human VKOR is devoid of a thioredoxin domain, but instead PDI family members can serve as VKOR substrates (Figure 2) [23,38]. For instance, when overexpressed together with active-site CXXA mutants, VKOR can be trapped in a mixed-disulfide complex with select PDI family members, predominantly the transmembrane-bound TMX and TMX4 [23]. A similar preference for interaction with TMX and TMX4 was not observed for overexpressed ERO1a. These experiments implicated VKOR in oxidation of a specific subset of PDIs, although a cellular role in sustaining disulfide formation is still unclear. Relative contribution and interplay between oxidative pathways We now know a number of potential pathways for disulfide formation, but our understanding of their relative contribution is still limited. The ERO1 pathway is likely to be the

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SH SH S

HS HS

PRDX4 decamer

S

PDI SH SH

SH per resHS

SH res

HS HS

per HS

H 2 O2

S

SOH SH

H2 O H 2O2

HS HS

PDI

S S

H2O

S

PDI SH SH

H2 O

HS HOS

S

SOH SH

S

PDI

S

S 2H2 O Ti BS

Figure 3. PRDX4 reaction mechanism. PRDX4 exists as a decamer comprising five dimers. The peroxidatic cysteine (per) in PRDX4 reacts with hydrogen peroxide to become sulfenylated. This sulfenylated cysteine (SOH) can then react with a resolving cysteine (res) present on an adjacent polypeptide to form a disulfide. There is one peroxidatic and one resolving cysteine per subunit, so two disulfides can form per dimer. A reduced PDI family member (green) is then oxidized (orange) following disulfide exchange with PRDX4.

major pathway for disulfide formation under normal physiological conditions. It is clear that the enzyme is active in the ER and can produce hydrogen peroxide, as evidenced by TRDX4 hyperoxidation in cells expressing a deregulated form of ERO1 [39]. In addition, although the pathway is not essential in mammals, its absence does compromise disulfide formation [20]. Moreover, ERO1 determines the redox status of PDI in cells and forms mixed disulfides with PDI, evidence that clearly demonstrates the ability of ERO1 to oxidize PDI [18,40]. The ability of animals devoid of ERO1 to survive and form disulfide-bonded proteins clearly indicates that other pathways exist, but their relative contributions are not known. If the PRDX4-dependent pathway provided a significant contribution to disulfide formation, we might predict that the Prdx4 knockout mouse should have a severe phenotype. The Prdx4 knockout mouse is viable, but is sterile owing to oxidative stress in the testis [24]. This result shows that the PRDX4 pathway is not essential for survival, but is crucial for correct function of specific tissues. The presence of PRDX4 in higher eukaryotes would explain the mild phenotypes of ERO1 knockouts if an alternative source of hydrogen peroxide is available to drive the PRDX4 pathway. This source might involve leakage of H2O2 produced by the mitochondrial electron transport chain to the ER lumen [41] or de novo production of H2O2 by the NADPH-dependent oxidase NOX4 [42]. Clearly, more work is necessary to identify whether either of these sources provides the H2O2 necessary to drive disulfide formation. Hence, although PRDX4 might well provide an alternative pathway to ERO1 for disulfide formation, the relative importance of this pathway remains to be established. The same is true for GPx7 and GPx8. At endogenous level these enzymes can apparently not substitute for

PrdxIV in Ero1 knockout cells. Still, they might perform a function similar to that of PRDX4. Whereas proteins of the peroxiredoxin family have a higher rate of reactivity towards H2O2 than GPXs [43], the fact that Ero1a interacts with the GPXs in vivo might compensate for their relatively slower rates of reactivity. Clearly a more comprehensive characterization of these enzymes is required to evaluate their relative roles in de novo disulfide formation. As is the case for the GPXs, the cellular function of VKOR in disulfide formation remains essentially uncharacterized, although the mixed disulfide trapped with PDI family members suggests a role in the process. If oxidation of PDIs by VKOR is strictly coupled to g-carboxyglutamate formation, this pathway is predictably not of great significance owing to its low flux. If the hydroquinone could be reoxidized via an alternative electron acceptor (i.e. uncoupled from g-glutamyl carboxylase activity), a more prominent role might be envisaged. Direct oxidation by H2O2 of cysteines in PDI and substrate proteins to form disulfides might also occur as shown in vitro [44]. However, these pathways are likely to be of minor importance, as indicated by the faster kinetics of substrate refolding when the reaction mixture contains H2O2 and PDI family members together with either of the PDI peroxidases or PRDX4 (see above). Finally, an additional source of oxidizing equivalents in the ER is dehydroascorbate (DHA; an oxidized form of the antioxidant ascorbate). DHA is transported into the ER from the cytosol [45] and can also be generated in the ER [46]. Like H2O2, DHA directly oxidizes PDI [47–49] and unfolded reduced proteins in vitro (Figure 2) [47]. Because of the slow rate, the former reaction was judged not to constitute a main route for the reduction of DHA, whereas the faster PDI-independent oxidation of protein substrates might 489

Review have physiological relevance [47]. Importantly, however, as illustrated by these examples, an in-depth understanding of how the different pathways are interconnected necessitates detailed knowledge about the relative concentrations of all components and their reaction rates, for which data are limited at present. Role of glutathione in regulating ER redox status and disulfide formation The high concentration and fast reaction rates between GSH and PDI suggest a central function in oxidative folding and redox regulation in the ER. However, experimental challenges in measuring the concentration of GSH or GSSG in the ER or changes to the GSH/GSSG ratio hamper progress towards understanding its functions [50,51]. Although the GSH/GSSG ratio in the secretory pathway is much lower than in the cytosol [52], GSH still imposes an overall reducing load on the ER in yeast [53]. Accordingly, a decrease in cellular GSH levels can rescue growth of the ero1-1 strain [53]. GSH also counterbalances the activity of ERO1 in mammalian cells; in other words, GSSG is generated as an indirect consequence of ERO1 activity [53–55]. Moreover, GSH plays an important role in keeping activesite cysteines of PDI-family members partially reduced [54,56,57]. This facilitates substrate reduction and thereby helps to prevent the formation of non-native disulfides [54,56,57]. Recent data show that GSH levels in the ER are tightly balanced [18]. Thus, the normal cellular GSH/GSSG ratio is quickly reestablished after treatment with the reducing agent dithiothreitol (DTT). Likewise, a sixfold GSSG excess observed in cells overexpressing ERO1 at the onset of DTT washout was quickly restored to the normal level [18]. These results highlight two central features of ER redox control. First, the high total GSH concentration in the ER (probably in the millimolar range at an approximate GSH/GSSG ratio of 5:1 [52,58]) provides buffering capacity against both oxidative and reductive stress. In addition, protein thiols constitute a major cellular pool for buffering oxidative stress, whereupon they become glutathionylated [59]. However, even under normal cellular conditions the extent of protein glutathionylation in the ER is not clear [58,60] and at present it is difficult to assess its functional role. Second, an important factor in ER redox control is feedback regulation of ERO1 activity. Thus, Ero1p [61], ERO1a [12,55] and ERO1b [13] all contain regulatory disulfides that shut down enzyme activity when formed in response to hyperoxidizing conditions (Box 1). For ERO1a it has been demonstrated that regulatory disulfides respond to the redox state of PDI [55]. Hence, ERO1a activity is switched off when oxidized PDI is abundant. Conversely, when levels of reduced PDI are high, ERO1a remains active. This mechanism, whereby PDI relays to ERO1 the prevailing ER redox state, ensures that ERO1 does not generate disulfides (and H2O2) unless needed. Underlying the ability of PDI to serve as a sensor of ER redox conditions is most probably the fast reaction rate of its active sites with both GSSG and GSH [44,62,63], a feature that is shared with TMX3 and is thus potentially 490

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general for PDI family members [64]. Importantly, the reaction between GSSG and PDI proceeds much faster than the initial oxidation of a reduced model protein by GSSG [44]. Moreover, ERO1, PRDX4 and the GPX enzymes all clearly prefer PDI family members over GSH as substrates. QSOX also does not react with GSH at appreciable rates compared to an unfolded reduced protein [22]. The overall picture emerging is that oxidizing equivalents generated by most pathways described (Figure 2) are passed on to PDI family members, which in turn oxidize substrate proteins or – in a competing reaction – GSH. ER-derived microsomes can import GSH but are quite impermeable to GSSG [65]. This suggests that export of GSSG to the cytosol does not occur at substantial levels. Moreover, GSH reductase activity in microsomes could not be detected [66]. Which mechanism then prevents ER hyperoxidation? Although some GSSG must escape the ER by vesicular transport, this route did not contribute to resetting of ER redox conditions after oxidant treatment of cells [18]. An alternative mechanism could be the import of reducing equivalents from the cytosol (i.e. GSH and/or free cysteines) in newly synthesized proteins. However, neither decreasing cellular GSH levels nor inhibiting protein translation significantly changed the steady-state redox distribution of two PDI family members [18]. It is therefore most likely that GSSG simply reacts with substrate proteins or PDI family members. This implies that GSSG is not a waste product that must be discarded from the ER. Rather, it is productively used in oxidative folding, predominantly via PDI owing to the fast reaction rate. Overall, GSH is central for maintaining ER redox homeostasis. It quickly equilibrates with PDI to regulate ERO1 via feedback, which helps to prevent the futile generation of oxidizing equivalents that could occur if ERO1 activity were unconstrained [67]. An important purpose of this redox control is to prevent hyperoxidizing conditions from restricting disulfide isomerization and thus causing protein misfolding and accumulation in the ER. Protein misfolding can induce the unfolded protein response and eventually apoptosis [68]. The latter process can be mediated by ROS and although it is tempting to speculate that H2O2 generated by ERO1 could be the culprit, mitochondrial ROS produced as a result of signaling from the ER might rather be the source [51]. Concluding remarks After more than a decade with a strong focus on ERO1 structure and function – work that has provided crucial insight into disulfide bond formation and redox regulation in the ER – the existence of alternative pathways remains speculative. As exemplified by the characterization of PRDX4, the role of ERO1a (although clearly important) should not be overemphasized, and it is now evident that peroxide is used productively in the ER to promote oxidative folding. Taken together, recent work has reminded us that undiscovered pathways might still exist and has significantly expanded our understanding of the cellular mechanisms for disulfide formation in mammalian cells. A key challenge for the future will be to dissect the interplay between pathways for disulfide formation. It will

Review be interesting to learn whether the different pathways prefer specific PDI family members to pass on disulfides, as indicated by the preference of ERO1, PRDX4, GPX7, GPX8 and VKOR for subsets of PDI family members [18,19,21,23,69]. Thus, fine tuning among pathways could tailor a specific response, depending on the primary pathway used by the cell under a given set of conditions. Here, we have emphasized questions relating to the different pathways for disulfide formation and their interconnectivity. Resolution of these and other issues, such as the mechanisms of GSH and FAD transport into the ER, the potential presence of ER subcompartments with specialized redox environments, the importance of H2O2 generated independently of ERO1 under normal cellular conditions and the contribution of ER-generated ROS to oxidative stress, will be crucial in achieving a better understanding of the fascinating and diverse processes that generate native disulfides in the ER. Acknowledgements The authors wish to thank all laboratory members and J.R. Winther for critical reading of the manuscript, and H.G. Hansen for help with figures. This work was supported by funding from the Novo Nordisk Foundation, Carlsberg Foundation, Lundbeck Foundation and the Danish Research Council for Natural Sciences to L.E., and the Wellcome Trust (grant #088053) and the Scottish Universities Life Science Alliance to N.J.B.

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