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Biochimica et Biophysica Acta 1833 (2013) 2425–2429

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Biochimica et Biophysica Acta

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Review Forming disulfides in the endoplasmic reticulum☆

Ojore B.V. Oka, Neil J. Bulleid ⁎

Institute of Molecular, Cellular and Systems Biology, College of Medical Veterinary and Life Sciences, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, UK

article info abstract

Article history: disulfide bonds are an important co- and post-translational modification for entering the secretory Received 10 December 2012 pathway. They are covalent interactions between two cysteine residues which support structural stability and pro- Received in revised form 5 February 2013 mote the assembly of multi-protein complexes. In the mammalian endoplasmic reticulum (ER), disulfide bond for- Accepted 10 February 2013 mation is achieved by the combined action of two types of enzyme: one capable of forming disulfides de novo and Available online 20 February 2013 another able to introduce these disulfides into substrates. The initial process of introducing disulfides into substrate proteins is catalyzed by the protein disulfide isomerase (PDI) oxidoreductases which become reduced and, there- Keywords: fi Disulfide bond fore, have to be re-oxidized to allow for further rounds of disul de exchange. This review will discuss the various Thiol-disulfide exchange pathways operating in the ER that facilitate oxidation of the PDI oxidoreductases and ultimately catalyze disulfide Protein disulfide isomerase (PDI) bond formation in substrate proteins. This article is part of a Special Issue entitled: Functional and structural diversity Oxidoreductase of endoplasmic reticulum. Peroxidase © 2013 Elsevier B.V. All rights reserved. Sulfenylation

1. Introduction These functions illustrate the importance of disulfide bonds in numerous cellular processes and highlight the need for a proper understanding of Newly synthesized proteins fold into functional 3-dimensional con- the proteins involved and the mechanisms regulating their formation. formations stabilized by van der Waals interactions, salt bridges, hydro- In the last few decades, significant progress has been made toward gen bonds and the hydrophobic effect. In addition to these non-covalent unraveling the mechanistic details of protein disulfide bond formation interactions, a large number of proteins in the secretory pathway, or in prokaryotic and eukaryotic organisms. Thus we now know that those destined as cell surface receptors, form disulfides; covalent bonds enzyme-catalyzed disulfide bonds can be formed in a number of spe- formed between the side chains of two cysteine residues. Protein disul- cialized cellular locations, including the endoplasmic reticulum, the fide bonds are primarily formed as a result of a thiol-disulfide exchange bacterial periplasm [8] and the inter-membrane space of the mitochon- reaction, with PDI exchanging its active site disulfide with its substrate. dria [9]. While significant progress has been made in understanding the Potentially any two cysteine residues in close spatial proximity can enzyme-catalyzed oxidative pathways operating in the ER, no corre- form a disulfide bond. Therefore, the formation of a native disulfide sponding reduction pathways operating in this organelle have been bond in a polypeptide containing several cysteine residues can be- identified to date. come problematic. Non-native disulfides can prevent correct folding and have to be reduced or isomerized to form the correct, native 2. Environment of the ER and disulfide bond formation disulfides [1,2]. Hence, the formation of native disulfidesisoftenarate- limiting step during the folding and maturation of many disulfide- The environment of the ER is distinct from the cytosol in two impor- containing secretory proteins [3,4]. tant aspects that ensure the formation of disulfide bonds in proteins Disulfide bonds are crucial for the biosynthesis and function of many translocated across the ER membrane; namely the presence of an opti- proteins. They promote structural stability, facilitate the assembly of mized redox buffer and disulfide exchange proteins. Like the cytosol, multi-protein complexes and can modulate redox-dependent functions the ER contains a redox buffer, maintained by the cysteine-containing in response to changes in the cell [5].Disulfide bonds can also mediate tripeptide, glutathione (γ-L-glutamyl-L-cysteinylglycine). However, un- the formation of productive folding intermediates, and have been like the cytosol the glutathione buffer is much more oxidizing. In the cy- shown in the ER to promote thiol-mediated protein retention [6,7]. tosol, glutathione is predominantly in the reduced form (GSH) with only very low concentrations of oxidized glutathione (GSSG) [10].A high GSH:GSSG ratio is maintained by a robust reductive pathway consisting of glutathione reductase coupled to NADPH. Such a system, ☆ This article is part of a Special Issue entitled: Functional and structural diversity of in general, prevents disulfide formation. In addition, and endoplasmic reticulum. fi ⁎ Corresponding author. Tel.: +44 141 330 3870; fax: +44 141 330 5481. thioredoxin reductase coupled to NADPH act to reduce any disul des E-mail address: [email protected] (N.J. Bulleid). formed within cytosolic proteins [11]. In contrast, the ER contains no

0167-4889/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2013.02.007 2426 O.B.V. Oka, N.J. Bulleid / Biochimica et Biophysica Acta 1833 (2013) 2425–2429 obvious candidates for a glutathione or a thioredoxin reductase and as a Conformational differences between the reduced and oxidized consequence the GSH:GSSG ratio is much lower and disulfides can form. human PDI indicate a large inter-domain rotation around the a′b′ region In addition, most of the thioredoxin domain-containing proteins pres- resulting in a transition from an open (oxidized) to a closed (reduced) ent in the ER are more likely to form than break disulfides due to the structure (Fig. 1). In the open conformation the binding cleft could ac- more positive reduction potential of their active sites than cytosolic commodate larger polypeptides which may have folded but not formed thioredoxin. For example, the active site disulfide of thioredoxin has a their disulfides. In contrast, the binding cleft in the reduced protein is reduction potential of approximately −270 mV [12] whereas the active much smaller restricting binding to smaller or less folded polypeptides. site disulfide of PDI has a reduction potential of approximately − Such conformational changes are intriguing and may indicate a possible 165 mV [13]. This means that the thioredoxin disulfide is more stable mechanism to ensure the release of polypeptides from PDI once the di- than the PDI disulfide so PDI is more likely to exchange its disulfide sulfide transfer has occurred. Indeed a recent paper using a model single with a substrate protein rather than reduce an existing disulfide. domain protein with one disulfide bond indicates that protein folding In addition to providing an environment for disulfide formation, the occurs prior to disulfide formation [2]. While this work used a single ER contains proteins that are dedicated to protein folding [14–16].Such domain of PDI as the catalyst, the basic premise that folding occurs folding factors can form multi-protein complexes that act in concert to prior to disulfide formation would necessitate a large binding cleft allow folding and disulfide formation in newly synthesized proteins. within full-length PDI to accommodate the folded polypeptide Many of these folding factors, such as BiP, calnexin and calreticulin, do substrate. not catalyze protein folding, but rather shield newly synthesized pro- When PDI and other oxidoreductases introduce disulfides into newly teins from other competing ‘off pathways’ such as aggregation, thereby synthesized proteins, their active sites have to be re-oxidized to allow for maintaining newly synthesized polypeptides in a ‘folding competent’ further rounds of disulfide formation. This function is fulfilled by specific conformation [16]. ER-resident oxidases, which do not directly introduce disulfides into newly synthesized proteins. These enzymes catalyze the first step in di- sulfide formation by transferring oxidizing equivalents to the PDI pro- 3. PDI thiol-disulfide oxidoreductase family teins, which then introduce these disulfides into nascent polypeptides [32–35]. Disulfide bond formation in the ER is catalyzed by the PDI family of dithiol-disulfide oxidoreductases [17]. About 20 proteins have been assigned to this family to date; with 15 members containing the typical 4. ERO1 oxidative pathway thioredoxin-like fold, with characteristic CXXC motifs in their active site. The number and position of the thioredoxin domains and their ac- The first of the ER oxidative pathways for de novo disulfide forma- tive site chemistries vary depending on the particular enzyme [17]. tion to be identified involves the ER oxidoreductin 1 protein (Ero1). These proteins catalyze thiol-disulfide exchange reactions by acting as This enzyme was initially characterized in yeast where it was shown electron acceptors during disulfide bond formation (oxidation reaction) to be essential for disulfide formation in PDIp [36,37]. Mammals and or as electron donors during breaking of disulfides (reduction reaction). other vertebrates have two Ero1 paralogs, namely: Ero1α which is pres- The PDI proteins also catalyze isomerization reactions by rearranging ent in all tissues and Ero1β which shows some tissue specific expres- non-native to native disulfides. The ability of a particular PDI enzyme sion [38,39]. The mammalian Ero1 proteins are able to complement to act as an oxidant or a reductant depends in part on the redox potential yeast Ero1p temperature-sensitive mutants [38]. The Ero1 proteins oftheCXXCactivesite[13,18]. As mentioned previously, the majority of are flavoenzymes that use FAD as a cofactor to transfer electrons from PDI proteins contain active sites disulfides that are relatively unstable in PDI to molecular oxygen, forming hydrogen peroxide in the process comparison to the CXPC motif of cytosolic thioredoxin [19].Anotableex- [40–42]. Ero1 contains a cluster of cysteine residues responsible for its ception is ERdj5, which contains four CXXC motifs, with three of these catalytic cycle. A pair of cysteine residues, present on an unstructured containing the CXPC motif [20]. Consistent with the similarity to cytosol- flexible loop, and termed the shuttle cysteines, constitutes the ‘outer’ ic thioredoxin, the active site disulfides in ERdj5 are quite stable and this active site. At the C-terminus of the protein are two additional cysteines, enzyme has been shown to act as a reductant, breaking disulfides to fa- present in a CXXC motif, and lying in close proximity to the bound co- cilitate the unfolding of proteins destined for degradation in the cytosol factor FAD. These cysteines comprise the ‘inner’ active site. The first [21]. While it was thought the functions of some of the PDI proteins step in disulfide exchange between Ero1and PDI proteins involves the would be redundant because of the similarities in their active sites, flexible loop shuttle cysteines which directly accept electrons from there is now accumulating evidence for substrate specificities and de- PDI. Through an internal disulfide exchange reaction, these electrons fined roles [22]. are shuttled to the ‘inner’ active site which pass them onto the FAD, PDI was the first ER oxidoreductase to be extensively characterized. It and ultimately onto the final acceptor oxygen. In addition to disulfide is an essential enzyme in yeast, and has been shown to function as a chap- exchange reactions, the flexible loop has been suggested to contain a eroneaswellasintroducedisulfide bonds into substrates [23–25].PDI binding site for PDI [43]. contains four thioredoxin-like domains namely a, b, b′,anda′. Only the While Ero1 activity is important for disulfide bond formation, aanda′ domains contain the canonical CXXC active site motif [18].The unregulated Ero1 could generate high concentration of hydrogen low pKa of the first cysteine of the CXXC motif allows this residue to par- peroxide, which could affect cell viability. Overexpression of a ticipate in disulfide bond formation [26]. The crystal structure of both a re- deregulated yeast Ero1 mutant results in inhibition of cell growth [44] duced and oxidized version of the human protein has been solved [27,28]. while the overexpression of human Ero1α results in an unfolded protein Overall the four PDI domains form a twisted ‘U’ conformation, with the (stress) response [45]. Thus, the activity of the Ero1 proteins are regulated catalytic domains a and a′ situated at the top, facing each other; while via a negative feedback mechanism that involves pairs of non-catalytic the domains b and b′, are sandwiched between the catalytic domains, cysteine residues that restrict the movement of the ‘outer’ shuttle disul- on the inside of the ‘U’ [28]. The interior of the U is hydrophobic, particu- fide, thereby preventing disulfide exchange with the ‘inner’ active site larly at the b and b′ domains and is the principal binding site for peptides [41,43,44,46]. These regulatory disulfides need to be reduced to activate and misfolded regions of substrates [29]. However, other domains of PDI theenzymeandallowelectrontransferbetweentheactivesites.Onecur- have been implicated in substrate binding [30]. The presence of such hy- rent view is that PDI itself can regulate Ero1 activity by reducing the reg- drophobic regions explains how in addition to facilitating disulfide forma- ulatory disulfides when the ER PDI pool becomes predominantly reduced tion in newly synthesized proteins, PDI may also function as a chaperone, and reform the regulatory disulfides once sufficient oxidized PDI is inhibiting aggregation of misfolded proteins [31]. formed [46,47]. O.B.V. Oka, N.J. Bulleid / Biochimica et Biophysica Acta 1833 (2013) 2425–2429 2427

SH SH a a' binding + b SH b' SH oxidized PDI a open conformation a' unfolded b SH polypeptide b' PDI /substrate mixed disulfide

folding HS a a' + b b' oxidized reduced PDI SH substrate closed conformation HS a a' b b'

HS substrate a disulfide release a' exchange b b'

Fig. 1. Possible sequence of events during disulfide formation. Schematic depicting one potential scenario for the oxidative folding of an unfolded polypeptide in the presence of PDI. PDI is known to bind to polypeptide chains within the bb′ domains at the base of its U-shaped open conformation (binding). Free thiols within the polypeptide can then form a mixed disulfide with either active site of PDI (here depicted as via the a′ domain). The formation of this mixed disulfide would potentially prevent the formation of non-native disulfides which are less likely to form once the polypeptide folds which brings the correct disulfides into close proximity (folding). A disulfide exchange reaction then takes place, forming the disulfide within the substrate and reducing PDI. The substrate is then released as PDI changes conformation to the closed form. Figure adapted from [2,27].

5. Peroxiredoxin IV pathway (SO3H) acid. While the sulfonic acid form is irreversible, in the cytosol the sulfinic acid can be reduced by the ATP-dependent sulfiredoxin [51]. PrxIV is an ER-localized member of the 2-cys peroxiredoxins, a family Whether a similar reaction can occur within the ER is debatable as to of enzymes which protect cells from peroxide-induced oxidative stress date no ER localized sulfiredoxin has been identified.

[48]. PrxIV utilizes H2O2 as a terminal electron acceptor and couples its The catalytic activities of the Ero1 proteins generally contribute to breakdown to disulfide bond formation in the PDI proteins [32,35]. the ER H2O2 pool under normal physiological conditions. While the Structurally, PrxIV is similar to other 2-cys peroxiredoxins, consisting of Ero1-generated peroxide appears to be important for the oxidation of

five dimers arranged in a donut-like ring structure, with each polypep- PrxIV [52], the enzyme also utilizes other sources of H2O2 as clearly tide in the dimer containing an active site peroxidatic and a resolving suggested from studies of double Ero1 knockout MEFs [35]. Although cysteine [49]. the origin of this H2O2 is currently obscure, potential sources include Indication of a role for PrxIV in disulfide formation came from a study mitochondria respiration, the peroxisomal oxidation of fatty acids and which showed that knockdown of PrxIV in MEFs lacking Ero1α/β the enzymatic activities of the ER-localized NADPH oxidase [53]. resulted in a lack of disulfide bond formation and subsequent cell death [35]. This result was supported by an independent study which showed 6. Glutathione peroxidase7/8 pathway that reduced PDI proteins can recycle the H2O2-induced disulfide bond in PrxIV; thereby coupling H2O2-mediated oxidation to disulfide forma- The glutathione peroxidases (GPxs) are found in all forms of life and tion in substrate proteins [32]. In addition, mice deficient of both Ero1 generally utilize GSH for the reduction of H2O2. So far eight GPXs have paralogs and PrxIV suffer from ascorbic acid deficiency probably caused been identified in humans, including the ER-localized GPx7 and GPx8 by reaction of H2O2 with this vitamin [50]. This result demonstrates [34]. Although classified as GPxs, both GPx7 and GPx8 exhibit poor glu- boththepresenceofH2O2 intheERintheabsenceofEro1andthecrucial tathione peroxidase activities, but have been shown to exhibit PDI per- role played by PrxIV in metabolizing this reactive oxygen species. oxidase activity. Both enzymes were shown to interact with Ero1 and to

The firststepinthebreakdownofH2O2 involves the sulfenylation of promote the introduction of disulfide bonds into reduced substrates in the peroxidatic cysteine (Cys 124) to sulfenic acid (SOH) which prompts the presence of PDI and H2O2 [34]. These reactions suggest an important a conformation change allowing the modified peroxidatic cysteine, to role for these proteins in disulfide bond formation. As typical peroxi- react with the nucleophilic resolving cysteine (Cys 245), resulting in an dases, it is possible that GPx7 and GPx8 reaction mechanism proceeds interchain disulfide bond. This disulfide can be reduced by PDI proteins, via a sulfenylated cysteine intermediate formed by the oxidation of including PDI, ERp46 and P5, via a typical thiol-disulfide exchange mech- theactivesitecysteinebyH2O2. The sulfenylated cysteine could then anism [32]. The PrxIV pathway ensures that for every oxygen molecule react with a reduced cysteine in the PDI active site, to form a transient consumed during Ero1-mediated oxidation of PDI proteins, two disulfide mixed-disulfide bond. The mixed disulfide is resolved by the second bonds can be generated in newly synthesized proteins. The sulfenylated cysteine in the PDI CXXC, to yield a disulfide bond in PDI and reduced cysteine can be further oxidized to the sulfinic (SO2H) or even sulfonic GPx. An alternative mechanism has recently been proposed whereby 2428 O.B.V. Oka, N.J. Bulleid / Biochimica et Biophysica Acta 1833 (2013) 2425–2429 a second cysteine within Gpx7/8 resolves the sulphenylated cysteine to their unique roles not only in disulfide formation but in general ER qual- form an intra-chain disulfide. The intrachain disulphide could then be ity control. reduced by PDI thereby recycling Gpx7/8 for further rounds of reaction with H2O2. Such a mechanism has recently been suggested for Gpx7 Acknowledgement [54] although in this work Gpx7 is reported to catalyze the formation fi of a disul de bond within the ER chaperone BiP thereby enhancing its This work was supported by a grant from The Wellcome Trust chaperone activity. (#088053).

7. Vitamin K epoxide reductase (VKOR) pathway References

The VKOR enzyme provides an additional pathway for disulfide bond [1] A. Jansens, E. van Duijn, I. Braakman, Coordinated nonvectorial folding in a newly formation. Homologues of the human enzyme have been identified in nu- synthesized multidomain protein, Science 298 (2002) 2401–2403. merous organisms, where they are implicated in disulfide bond formation [2] P. Kosuri, J. Alegre-Cebollada, J. Feng, A. Kaplan, A. Ingles-Prieto, C.L. Badilla, B.R. Stockwell, J.M. Sanchez-Ruiz, A. Holmgren, J.M. Fernandez, Protein folding drives [55]. VKOR is an important component of the ER membrane-resident vi- disulfide formation, Cell 151 (2012) 794–806. tamin K-dependent γ-carboxylation system which plays a key role in [3] A. Matagne, C.M. Dobson, The folding process of hen lysozyme: a perspective blood coagulation. It is a polytopic transmembrane protein, containing from the ‘new view’, Cell. Mol. Life Sci. 54 (1998) 363–371. [4]B.vandenBerg,E.W.Chung,C.V.Robinson,P.L.Mateo,C.M.Dobson,Theoxidative two conserved cysteine pairs; one that is membrane embedded, and refolding of hen lysozyme and its catalysis by protein disulfide isomerase, EMBO J. 18 the other present on a loop region. These cysteines catalyze redox reac- (1999) 4794–4803. tions as part of the catalytic cycle. VKOR reduces vitamin K or its epoxide [5] P.J. Hogg, Disulfide bonds as switches for protein function, Trends Biochem. Sci. – fi 28 (2003) 210 214. derivative resulting in the formation of a disul de in the membrane- [6] A.S. Robinson, J. King, Disulphide-bonded intermediate on the folding and assembly embedded CXXC motif. Recycling of the enzyme involves a disulfide pathway of a non-disulphide bonded protein, Nat. Struct. Biol. 4 (1997) 450–455. exchange reaction between the CXXC disulfide and a cysteine pair [7] T. Anelli, M. Alessio, A. Bachi, L. Bergamelli, G. Bertoli, S. Camerini, A. Mezghrani, E. Ruffato, T. Simmen, R. Sitia, Thiol-mediated protein retention in the endoplasmic in the loop region [55], which ultimately can accept electrons from reticulum: the role of ERp44, EMBO J. 22 (2003) 5015–5022. thioredoxin-like proteins [55,56].Conflicting membrane topologies, [8] H. Kadokura, J. Beckwith, Mechanisms of oxidative protein folding in the bacterial with implications for the location of the conserved loop cysteines, cell envelope, Antioxid. Redox Signal. 13 (2010) 1231–1246. fi have been proposed for human VKOR. In the four transmembrane [9] J. Riemer, N. Bulleid, J.M. Herrmann, Disul de formation in the ER and mitochon- dria: two solutions to a common process, Science 324 (2009) 1284–1287. helix model the loop cysteines are exposed in the ER lumen and can, [10] C. Hwang, A.J. Sinskey, H.F. Lodish, Oxidized redox state of glutathione in the en- therefore, accept electrons from PDI family proteins [55].Insupportof doplasmic reticulum, Science 257 (1992) 1496–1502. fi [11] E.S. Arner, Focus on mammalian thioredoxin reductases—important selenoproteins this model, VKOR was shown to form mixed disul des with PDI-like – fi with versatile functions, Biochim. Biophys. Acta 1790 (2009) 495 526. proteins. Speci cally, it was reported to interact with the membrane an- [12] G. Krause, A. Holmgren, Substitution of the conserved tryptophan 31 in Escherichia coli chored TMX and less so with the closely-related TMX4 and the soluble thioredoxin by site-directed mutagenesis and structure–functionanalysis,J.Biol.Chem. ERp18 [55]. In contrast, the three transmembrane helix model places 266 (1991) 4056–4066. [13] J.E. Chambers, T.J. Tavender, O.B. Oka, S. Warwood, D. Knight, N.J. Bulleid, The reduction the loop cysteines in the cytosol, making cytosolic thioredoxin the likely potential of the active site disulfides of human protein disulfide isomerase limits oxida- electron donor [57,58]. On reduction of vitamin K or its epoxide, the tion of the enzyme by Ero1alpha, J. Biol. Chem. 285 (2010) 29200–29207. membrane-embedded Cys132 and Cys135 of the CXXC motif become [14] T. Anelli, R. Sitia, Protein quality control in the early secretory pathway, EMBO J. 27 – fi (2008) 315 327. oxidized [59,60]. The CXXC disul de accepts electrons from the loop [15] D.N. Hebert, M. Molinari, In and out of the ER: protein folding, quality control, Cys43 and Cys51 pair, which become oxidized. Ultimately, the Cys43– degradation, and related human diseases, Physiol. Rev. 87 (2007) 1377–1408. Cys51 disulfide is transferred to thioredoxin-like proteins. [16] I. Braakman, N.J. Bulleid, Protein folding and modification in the mammalian endo- fi plasmic reticulum, Annu. Rev. Biochem. 80 (2011) 71–99. Support for a role of the VKOR pathway in disul de formation comes [17] L. Ellgaard, L.W. Ruddock, The human protein disulphide isomerase family: substrate from a study of the consequence of knockdown of the enzyme in combi- interactions and functional properties, EMBO Rep. 6 (2005) 28–32. nation with an Ero1 knockdown [33]. While depletion of VKOR on its own [18] F. Hatahet, L.W. Ruddock, Protein disulfide isomerase: a critical evaluation of its fi – had minimal effects, a combination of Ero1 α and β depletion with VKOR function in disul de bond formation, Antioxid. Redox Signal. 11 (2009) 2807 2850. [19] G. Krause, J. Lundstrom, J.L. Barea, C. Pueyo de la Cuesta, A. Holmgren, Mimicking caused a more severe delay in disulfideformationthanEro1α and β the active site of protein disulfide-isomerase by substitution of proline 34 in alone. In addition, VKOR was able to support the recovery of cells from Escherichia coli thioredoxin, J. Biol. Chem. 266 (1991) 9494–9500. a reductive challenge in the near absence of the Ero1 paralogues and [20] P.M. Cunnea, A. Miranda-Vizuete, G. Bertoli, T. Simmen, A.E. Damdimopoulos, S. Hermann,S.Leinonen,M.P.Huikko,J.A.Gustafsson,R.Sitia,G.Spyrou,ERdj5,anendo- PrxIV. plasmic reticulum (ER)-resident protein containing DnaJ and thioredoxin domains, is expressed in secretory cells or following ER stress, J. Biol. Chem. 278 (2003) 8. Conclusion 1059–1066. [21] R. Ushioda, J. Hoseki, K. Araki, G. Jansen, D.Y. Thomas, K. Nagata, ERdj5 is required as adisulfide reductase for degradation of misfolded proteins in the ER, Science 321 The past two decades have seen an increase in our understanding of (2008) 569–572. the various components and enzymatic pathways that operate in the [22] C.E. Jessop, T.J. Tavender, R.H. Watkins, J.E. Chambers, N.J. Bulleid, Substrate spec- ificity of the oxidoreductase ERp57 is determined primarily by its interaction ER. In this time, it has become apparent that multiple pathways exist with calnexin and calreticulin, J. Biol. Chem. 284 (2009) 2194–2202. to accomplish disulfide bond formation in the ER. Importantly, the real- [23] M.M. Lyles, H.F. Gilbert, Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the ization that H2O2 can be utilized to generate disulfides in newly synthe- oxidizing equivalents of the isomerase, Biochemistry 30 (1991) 619–625. sized proteins has to some extent, clarified how cells alleviate oxidative [24] R.B. Freedman, A.D. Dunn, L.W. Ruddock, Protein folding: a missing redox link in stress. While our understanding of the basic mechanisms involved in di- the endoplasmic reticulum, Curr. Biol. 8 (1998) R468–R470. sulfide bond formation has no doubt increased, a number of important [25] J. Winter, P. Klappa, R.B. Freedman, H. Lilie, R. Rudolph, Catalytic activity and fi questions remain unanswered. Significantly, the specific roles of the in- chaperone function of human protein-disul de isomerase are required for the ef- ficient refolding of proinsulin, J. Biol. Chem. 277 (2002) 310–317. dividual enzymes and components remain obscure. Furthermore, is [26] A.R. Karala, A.K. Lappi, L.W. Ruddock, Modulation of an active-site cysteine pKa allows there a functional relationship between the various oxidative path- PDI to act as a catalyst of both disulfide bond formation and isomerization, J. Mol. Biol. – ways? Whereas the PDI proteins have been identified as key players 396 (2010) 883 892. fi fi [27] C. Wang, W. Li, J. Ren, J. Fang, H. Ke, W. Gong, W. Feng, C.C. Wang, Structural in- in disul de formation, their speci c functions remain unknown. Do sights into the redox-regulated dynamic conformations of human protein disul- these proteins function singly or together in large multi-protein com- fide isomerase, Antioxid. Redox Signal. in press. (Electronic publication ahead of plexes? Although some studies have identified specific functions for Print). [28] G. Tian, S. Xiang, R. Noiva, W.J. Lennarz, H. Schindelin, The crystal structure of yeast some PDIs and preference for some members by certain oxidative path- protein disulfide isomerase suggests cooperativity between its active sites, Cell 124 ways [32,55], a detailed characterization is necessary to understand (2006) 61–73. O.B.V. Oka, N.J. Bulleid / Biochimica et Biophysica Acta 1833 (2013) 2425–2429 2429

[29] A.Y. Denisov, P. Maattanen, C. Dabrowski, G. Kozlov, D.Y. Thomas, K. Gehring, Solution [46] C. Appenzeller-Herzog, J. Riemer, B. Christensen, E.S. Sorensen, L. Ellgaard, A novel structure of the bb′ domains of human protein disulfide isomerase, FEBS J. 276 (2009) disulphide switch mechanism in Ero1alpha balances ER oxidation in human cells, 1440–1449. EMBO J. 27 (2008) 2977–2987. [30] P. Klappa, H.C. Hawkins, R.B. Freedman, Interactions between protein disulphide [47] S. Kim, D.P. Sideris, C.S. Sevier, C.A. Kaiser, Balanced Ero1 activation and inactivation isomerase and peptides, Eur. J. Biochem. 248 (1997) 37–42. establishes ER redox homeostasis, J. Cell Biol. 196 (2012) 713–725. [31] H. Cai, C.C. Wang, C.L. Tsou, Chaperone-like activity of protein disulfide isomerase in the [48] T.J. Tavender, A.M. Sheppard, N.J. Bulleid, Peroxiredoxin IV is an endoplasmic refolding of a protein with no disulfide bonds, J. Biol. Chem. 269 (1994) 24550–24552. reticulum-localized enzyme forming oligomeric complexes in human cells, [32] T.J. Tavender, J.J. Springate, N.J. Bulleid, Recycling of peroxiredoxin IV provides a Biochem. J. 411 (2008) 191–199. novel pathway for disulphide formation in the endoplasmic reticulum, EMBO J. 29 [49] Z. Cao, T.J. Tavender, A.W. Roszak, R.J. Cogdell, N.J. Bulleid, Crystal structure of re- (2010) 4185–4197. duced and of oxidized peroxiredoxin IV enzyme reveals a stable oxidized decamer [33] L.A. Rutkevich, D.B. Williams, Vitamin K epoxide reductase contributes to protein and a non-disulfide-bonded intermediate in the catalytic cycle, J. Biol. Chem. 286 disulfide formation and redox homeostasis within the endoplasmic reticulum, (2011) 42257–42266. Mol. Biol. Cell 23 (2012) 2017–2027. [50] E. Zito, H.G. Hansen, G.S. Yeo, J. Fujii, D. Ron, Endoplasmic reticulum thiol oxidase de- [34] V.D. Nguyen, M.J. Saaranen, A.R. Karala, A.K. Lappi, L. Wang, I.B. Raykhel, H.I. Alanen, ficiency leads to ascorbic acid depletion and noncanonical scurvy in mice, Mol. Cell K.E. Salo, C.C. Wang, L.W. Ruddock, Two endoplasmic reticulum PDI peroxidases in- 48 (2012) 39–51. crease the efficiency of the use of peroxide during disulfide bond formation, J. Mol. [51] W.T. Lowther, A.C. Haynes, Reduction of cysteine sulfinic acid in eukaryotic, typical Biol. 406 (2011) 503–515. 2-Cys peroxiredoxins by sulfiredoxin, Antioxid. Redox Signal. 15 (2011) 99–109. [35] E. Zito, E.P. Melo, Y. Yang, A. Wahlander, T.A. Neubert, D. Ron, Oxidative protein folding [52] T.J. Tavender, N.J. Bulleid, Peroxiredoxin IV protects cells from oxidative stress by

by an endoplasmic reticulum-localized peroxiredoxin, Mol. Cell 40 (2010) 787–797. removing H2O2 produced during disulphide formation, J. Cell Sci. 123 (2010) [36] A.R. Frand, C.A. Kaiser, The ERO1 of yeast is required for oxidation of protein 2672–2679. dithiols in the endoplasmic reticulum, Mol. Cell 1 (1998) 161–170. [53] E. Zito, PRDX4, an endoplasmic reticulum-localized peroxiredoxin at the crossroads [37] M.G. Pollard, K.J. Travers, J.S. Weissman, Ero1p: a novel and ubiquitous protein with an between enzymatic oxidative protein folding and nonenzymatic protein oxidation, essential role in oxidative protein folding in the endoplasmic reticulum, Mol. Cell 1 Antioxid. Redox Signal. in press. (Electronic publication ahead of Print). (1998) 171–182. [54] P.C. Wei, Y.H. Hsieh, M.I. Su, X. Jiang, P.H. Hsu, W.T. Lo, J.Y. Weng, Y.M. Jeng, J.M. Wang, [38] A. Cabibbo, M. Pagani, M. Fabbri, M. Rocchi, M.R. Farmery, N.J. Bulleid, R. Sitia, P.L.Chen,Y.C.Chang,K.F.Lee,M.D.Tsai,J.Y.Shew,W.H.Lee,Lossoftheoxidativestress ERO1-L, a human protein that favors disulfide bond formation in the endoplasmic sensor NPGPx compromises GRP78 chaperone activity and induces systemic disease, reticulum, J. Biol. Chem. 275 (2000) 4827–4833. Mol. Cell 48 (2012) 747–759. [39] M. Pagani, M. Fabbri, C. Benedetti, A. Fassio, S. Pilati, N.J. Bulleid, A. Cabibbo, R. Sitia, En- [55] S. Schulman, B. Wang, W. Li, T.A. Rapoport, Vitamin K epoxide reductase prefers doplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in ER membrane-anchored thioredoxin-like redox partners, Proc. Natl. Acad. Sci. the course of the unfolded protein response, J. Biol. Chem. 275 (2000) 23685–23692. U. S. A. 107 (2010) 15027–15032. [40] B.P. Tu, J.S. Weissman, The FAD- and O(2)-dependent reaction cycle of Ero1-mediated [56] N. Wajih, S.M. Hutson, R. Wallin, Disulfide-dependent protein folding is linked to oxidative protein folding in the endoplasmic reticulum, Mol. Cell 10 (2002) 983–994. operation of the vitamin K cycle in the endoplasmic reticulum. A protein disulfide [41] K.M. Baker, S. Chakravarthi, K.P. Langton, A.M. Sheppard, H. Lu, N.J. Bulleid, Low isomerase-VKORC1 redox enzyme complex appears to be responsible for vitamin reduction potential of Ero1alpha regulatory disulphides ensures tight control of K1 2,3-epoxide reduction, J. Biol. Chem. 282 (2007) 2626–2635. substrate oxidation, EMBO J. 27 (2008) 2988–2997. [57] J.K. Tie, C. Nicchitta, G. von Heijne, D.W. Stafford, Membrane topology mapping of [42] E. Gross, C.S. Sevier, N. Heldman, E. Vitu, M. Bentzur, C.A. Kaiser, C. Thorpe, D. Fass, Gen- vitamin K epoxide reductase by in vitro translation/cotranslocation, J. Biol. Chem. erating disulfidesenzymatically:reactionproductsandelectronacceptorsoftheendo- 280 (2005) 16410–16416. plasmic reticulum thiol oxidase Ero1p, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 299–304. [58] J.K. Tie, D.Y. Jin, D.W. Stafford, Human vitamin K epoxide reductase and its bacte- [43] K. Inaba, S. Masui, H. Iida, S. Vavassori, R. Sitia, M. Suzuki, Crystal structures of rial homologue have different membrane topologies and reaction mechanisms, human Ero1alpha reveal the mechanisms of regulated and targeted oxidation of J. Biol. Chem. 287 (2012) 33945–33955. PDI, EMBO J. 29 (2010) 3330–3343. [59] N. Wajih, D.C. Sane, S.M. Hutson, R. Wallin, Engineering of a recombinant vitamin [44] C.S. Sevier, H. Qu, N. Heldman, E. Gross, D. Fass, C.A. Kaiser, Modulation of cellular K-dependent gamma-carboxylation system with enhanced gamma-carboxyglutamic disulfide-bond formation and the ER redox environment by feedback regulation acid forming capacity: evidence for a functional CXXC redox center in the system, of Ero1, Cell 129 (2007) 333–344. J. Biol. Chem. 280 (2005) 10540–10547. [45] H.G. Hansen, J.D. Schmidt, C.L. Soltoft, T. Ramming, H.M. Geertz-Hansen, B. [60] M.A. Rishavy, A. Usubalieva, K.W. Hallgren, K.L. Berkner, Novel insight into the mech- Christensen, E.S. Sorensen, A.S. Juncker, C. Appenzeller-Herzog, L. Ellgaard, Hy- anism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and peractivity of the Ero1alpha oxidase elicits endoplasmic reticulum stress but no Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin broad antioxidant response, J. Biol. Chem. 287 (2012) 39513–39523. K-dependent protein carboxylation, J. Biol. Chem. 286 (2011) 7267–7278.