Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum

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The structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum Masaki Okumura, Hiroshi Kadokura, Kenji Inaba

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Received date: 10 November 2014 Revised date: 22 January 2015 Accepted date: 9 February 2015 Cite this article as: Masaki Okumura, Hiroshi Kadokura, Kenji Inaba, The structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum Masaki Okumura1,2, Hiroshi Kadokura1,2, and Kenji Inaba1,* 1

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University,

Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan. 2

These authors contributed equally to this work.

*

Corresponding author: Kenji Inaba, Institute of Multidisciplinary Research for

Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.

E-mail:

[email protected];

Tel.:

+81-22-217-5604;

Fax:

+81-22-217-5605. Abstract The endoplasmic reticulum (ER) is an essential cellular compartment in which an enormous number of secretory and cell surface membrane proteins are synthesized and subjected to co-translational or post-translational modifications, such as glycosylation and disulfide bond formation. Proper maintenance of ER protein homeostasis (sometimes termed proteostasis) is essential to avoid cellular stresses and diseases caused by abnormal proteins. Accumulating knowledge of cysteine-based redox reactions catalyzed by members of the protein disulfide isomerase (PDI) family has revealed that these enzymes play pivotal roles in productive protein folding accompanied by disulfide formation, as well as efficient ER-associated degradation accompanied by disulfide reduction. Each of PDIs forms a protein-protein interaction with a preferential partner to fulfill a distinct function. Multiple redox pathways that utilize PDIs appear to function synergistically to attain the highest quality and


 

productivity of the ER, even under various stress conditions. This review describes the structures, physiological functions, and cooperative actions of several essential PDIs, and provides important insights into the elaborate proteostatic mechanisms that have evolved in the extremely active and stress-sensitive ER.

Keywords: ER quality control, oxidative protein folding, ER-associated degradation, disulfide bond, PDI family

Protein folding coupled with disulfide bond formation takes place primarily in the endoplasmic reticulum (ER) and contributes to the maturation and stabilization of secretory and cell surface membrane proteins. More than 20 members of the protein disulfide isomerase family (referred to as “PDIs” in this review article) have been identified in the mammalian ER, and some of these enzymes are involved in protein quality control in the early secretory compartment [1, 2]. Although PDIs have diverse amino acid sequences and functionalities, they all contain at least one thioredoxin (Trx)-like domain [3-5]. Several factors can regulate the reactivities and substrate specificities of PDIs, including the number, locations, and redox potentials of redox-active Cys-Xaa-Xaa-Cys motifs and the pKa values of both the N-terminal and C-terminal cysteines at the motif. Further, the three-dimensional arrangement of the Trx-like domains and the hydrophobicity or electrostatic potential of the molecular surface can distinguish the functionality of PDIs. Importantly, PDIs most likely work in conjunction with partner proteins, thereby generating specific functions in response to various stresses and demands for the enormous production of secretory proteins, such as immunoglobulins and insulin.  

This review summarizes the ways in which PDIs fulfill their distinct functions and work cooperatively to maintain ER quality control. The first part of the review describes the latest insights into the structures and physiological functions of PDIs, focusing on productive protein folding involving disulfide bond formation, and aberrant protein degradation involving disulfide bond reduction. The second part of the review discusses the concerted actions and diverse redox cascades exerted by PDIs and their upstream oxidants/reductants, and raises important issues to reach a comprehensive understanding of the cellular mechanisms underlying the maintenance of protein and redox homeostasis in the mammalian ER.

1. Structures and functions of PDIs

Structures and functions of PDI and ERp57 Nearly fifty years ago, PDI was identified as an efficient catalyst of disulfide bond formation that is contained abundantly in the microsome fraction [6]. PDI is, though ubiquitous, highly expressed in secretory cells [7]. Canonical PDI comprises four Trx-like domains named a, b, b’, and a’ (in order from the N-terminus), which form a compact U-shape with a hydrophobic central cleft (Fig. 1) [8, 9]. Domains a and a’ contain a catalytic Cys-Gly-His-Cys sequence and are redox-active; these two active sites face each other across the central cleft. Domains b and b’ are redox-inactive due to the lack of a Cys-Xaa-Xaa-Cys sequence, although domain b’ is responsible for substrate binding [7, 10]. Several lines of evidence demonstrate that PDI recognizes partially structured intermediates bound to its central hydrophobic cleft in domain b’ and promotes their native folding by the cooperative action of the two mutually facing  

redox-active sites in domains a and a’ [11-15]. Thus, PDI likely functions as an efficient terminator of native oxidative protein folding; however, the mechanisms by which PDI distinguishes folded from misfolded states and adapts to various substrates of different shapes and sizes remain to be uncovered. In this context, one intriguing aspect of PDI is the redox-dependent rearrangement of its four Trx-like domains [9, 16]. According to the crystal structures of PDI, the distance between the active sites of domains a and a’ is 27.6 Å in the reduced state and 40.3 Å in the oxidized state, suggesting that PDI undergoes a closed/open conformational conversion depending on its redox state [9]. The intrinsically dynamic nature of PDI is likely the key to its efficient catalysis of native disulfide bond formation in a wide variety of substrate proteins. ERp57 is another PDI family member with an overall domain arrangement similar to that of PDI, in which the redox-inactive domains b and b’ are flanked by the redox-active domains a and a’ located at the N- and C-terminus, respectively (Fig. 1) [17]. The overall sequence identity between ERp57 and PDI is 33% [18], but it is extremely low (17%) in the b’ domain [19]. Like PDI, ERp57 shows a ubiquitous distribution in a wide range of tissues but expressed at slightly higher levels in secretory tissues than in other tissues [20]. Domain b’ interacts with the arm-like P-domains of the lectin chaperones calnexin (CNX) and calreticulin (CRT), which restore the native structures of unfolded/misfolded glycoproteins via the ‘calnexin cycle’ [21]. Extensive analyses of its physiological substrates indicated that, unlike PDI, ERp57 plays a specific role in the isomerization of non-native disulfide bonds in disulfide-rich glycoproteins with few secondary structure domains [22]. ERp57 also participates in the assembly of the major histocompatibility class I peptide-loading complex in concert with the chaperone-like protein tapasin [23], and promotes the host cell entry of simian  

virus 40 in collaboration with other ER quality control components [24].

Structure and functions of ERp46 ERp46 (also termed Endo-PDI) contains three Trx-like domains (Trx1, Trx2, and Trx3), each with a CGHC active site (Fig. 1). ERp46 is a stress survival factor that protects cells against apoptosis and is expressed preferentially in endothelial cells and pancreatic β-cells [25-27]. Under hypoxic conditions, the loss of ERp46 results in reduced secretion of adrenomedullin and endothelin-1, as well as reduced levels of membrane-bound CD105, suggesting that ERp46 may be involved in the oxidative folding of some secretory proteins [25]. ERp46 also modulates adiponectin signaling [28] and insulin production [26], and a recent study suggested that ERp46 catalyzes the formation of regulatory disulfides (Cys94–Cys131) in ER oxidoreductin1α (Ero1α) to switch off its oxidative activity [29]. Crystallographic and small-angle X-ray scattering studies revealed that the three Trx-like domains of ERp46 are linked by unusually long loops, do not undergo significant interdomain interactions, and are arranged in an extended manner to form an open V-shape (Fig. 1) [12]. This Trx-like domain arrangement has not been identified in other PDIs with known structures, indicating a radically different molecular architecture of ERp46. Whereas two redox-active sites face each other across the central cleft of the U-shaped PDI and ERp57 proteins, the redox-active sites of ERp46 are located separately on the molecular surface (Fig. 1). In agreement with these structural features, we found that ERp46 mutants with a single redox-active Trx-like domain introduced disulfide bonds to reduced denatured bovine pancreatic trypsin inhibitor (BPTI) at a similar rate to wild-type ERp46 [12]. No cooperative actions of the three Trx-like  

domains were observed for the wild-type protein [12], indicating that these three domains function independently in intact ERp46. Using a detailed in vitro folding assay, we revealed that, whereas PDI tends to recognize kinetically trapped intermediates and form native disulfides with high fidelity, ERp46 introduces disulfide bonds to unfolded substrates much more rapidly and promiscuously than PDI. Thus, ERp46 and PDI likely contribute to different stages of oxidative protein folding as catalysts with different functional roles (see the section entitled ‘Cooperation between two distinct disulfide bond formation pathways during oxidative protein folding’ for more details).

Structural basis of the functional interplays between PDIs and their specific oxidases ER oxidoreductin 1 (Ero1) generates a disulfide bond de novo by consuming a molecule of oxygen as an electron acceptor and concomitantly yielding a molecule of hydrogen peroxide as a byproduct. Ero1-generated disulfide bonds are first transferred to PDI, and then to substrate proteins [30, 31]. A protruding β-hairpin loop of Ero1Į binds closely to a hydrophobic pocket in domain b’ of PDI [19, 32]; this geometry seems suitable for the ability of Ero1Į to oxidize the C-terminal Trx domain of PDI (a’), due to the close proximity of the Ero1Į electron shuttle loop and the redox-active site of domain a’. In agreement with this proposal, deletion of the protruding β-hairpin loop or mutation of the Trp272 residue, which is located at the top of the loop and is highly conserved in the Ero1 family, compromised the ability of Ero1Į to oxidize PDI [32]. Recently, peroxiredoxin 4 (Prx4), glutathione peroxidase 7/8 (GPx7/8), and vitamin K epoxide oxidoreductase (VKOR) were identified as alternate oxidases of PDIs in the mammalian ER [33-36]. Despite the fact that Prx4 has the potential to react with a broad range of PDIs, ERp46 and P5 were identified as the preferential partners of this  

enzyme both in vitro and in living cells [12, 33, 37]. In the reduced form of Prx4, the peroxidatic cysteine (Cys127 in one chain) and the resolving cysteine (Cys248 in the neighboring chain) are masked by the C-terminal α-helix, making the redox interplay with PDIs unlikely. However, the C-terminal α-helix of Prx4 is highly disordered upon oxidation. Consequently the active-site disulfide formed between those two cysteines are exposed to the molecular surface to facilitate thiol-disulfide exchange between oxidized Prx4 and reduced PDIs [37-39]. Analysis of the crystal structure of the Trx2 domain of ERp46 in complex with the C-terminal peptide of Prx4 revealed that the His244–Ala250 segment of Prx4 is accommodated in the peptide-binding groove of the Trx2 domain of ERp46 through van der Waals interaction and a hydrogen bond between the guanidinium group of Arg281 of ERp46 Trx2 and the main chain carbonyl group of Glu246 in Prx4 [12, 37]. A similar mode of interaction was also found in the crystal structure of the a0 domain of P5 in complex with the C-terminal peptide of Prx4 [37]. Furthermore, Arg281 in the Trx2 domain of ERp46 (Arg118 in the a0 domain of P5) is highly conserved at the same position in Trx-like domains of other PDIs, suggesting a common mode of interaction of Prx4 with various PDIs [12, 37].

Structure and functions of ERp27 ERp27 is a 27.7 kDa ER-resident protein that is expressed in a variety of tissues, including the kidney, lung, and pancreas [40]; however, its physiological function is not fully understood. ERp27 is composed of two non-catalytic Trx-like domains named b and b’ [41, 42] (Fig. 1), which share 33.5% sequence homology with the corresponding domains of PDI [43]. Somatostatin, a model peptide that binds to domain b’ of PDI, also binds to the corresponding domain of ERp27 [10]. Intriguingly, ERp27 is thought  

to be capable of discerning between the distinct folded states of substrates and interacts preferentially with unfolded client proteins [42]. Although ERp27 is unable to catalyze thiol-based redox reactions on its own, it transfers unfolded substrates to other PDIs with redox activity. Accordingly, domain b’ of ERp27 contains a negatively charged loop with high sequence similarity to the P-domain of CRT, and the Asp-Glu-Trp-Asp motif in this domain interacts with the positively charged region in domain b’ of ERp57 (Fig. 1) [42, 43]. Similar motifs (Gln-Asp-Trp-Asp) are also present in CNX and CRT; therefore, ERp27 and CNX/CRT can be regarded as functional partners of ERp57, although the exact physiological roles of the ERp27-ERp57 complex are not fully understood. It is possible that, whereas CNX/CRT targets monoglucosylated trimming intermediates specifically, ERp27 may play a role in the quality control of an even wider range of proteins, including non-glycosylated proteins, in concert with ERp57 [42].

Structure and functions of ERdj5 It is widely accepted that PDIs play a role not only in oxidative protein folding, but also in the reduction of non-native disulfide bonds to accelerate the degradation of misfolded proteins. Presumably, the cleavage of disulfide bonds facilitates the retrograde translocation of misfolded proteins from the ER lumen to the cytosol and their subsequent degradation by proteasomes in the cytosol, in a process called ER-associated degradation (ERAD). The 90 kDa ERdj5 protein is the largest PDI family member that is ubiquitously expressed but particularly abundant in secretory tissues such as pancreas and testis [44, 45]. ERdj5 functions as a disulfide reductase that accelerates ERAD of misfolded glycoproteins in concert with ER-degradation enhancing Į-mannosidase-like  

protein 1 (EDEM1) and binding immunoglobulin protein (BiP) [46]. ERdj5 consists of a BiP-binding J-domain at the N-terminus, and four redox-active (Trx1–4) and two redox-inactive (Trxb1 and Trxb2) Trx-like domains within a single plane (Fig. 2) [47]. All of the redox-active sites are solvent-exposed and located on the J-domain-residing side of the plane; this structural feature seems suitable for reduction of the substrate by ERdj5 and its subsequent transfer to BiP. The molecular architecture of ERdj5 can be divided into two parts, namely an N-terminal cluster containing the J-domain and four Trx-like domains (Trx1, Trxb1, Trxb2, and Trx2), and a C-terminal cluster containing two Trx-like domains (Trx3 and Trx4). These two clusters are connected by a linker loop of five amino acids with limited interactions, although a salt bridge is formed between Asp552 and Arg603. The central cleft of ERdj5 formed by the N-terminal and C-terminal clusters is much narrower and less hydrophobic than that of PDI, suggesting that it is not essential for substrate binding. A series of biochemical analyses allowed us to propose the following sequential model of the ERdj5-mediated ERAD pathway: (i) terminally misfolded glycoproteins are recognized by EDEM1, which associates with the C-terminal cluster of ERdj5; (ii) aberrant disulfide bonds of misfolded glycoproteins are reduced by highly reducing active sites in the C-terminal cluster of ERdj5; (iii) the extended polypeptide chain is captured by BiP, which binds to the J-domain of ERdj5 in an ATP-dependent manner; and (iv) upon ATP hydrolysis, BiP dissociates from ERdj5 and transfers the substrate to the retrograde translocation channel to be degraded by the proteasome in the cytosol [48]. Unlike glycoproteins, non-glycoproteins in misfolded states are presumably recognized by BiP and then transferred to ERdj5 without going through the

 

CNX/EDEM1 pathway [48]. After the reduction of non-native disulfide bonds by ERdj5, the substrate is transferred to SEL1L, an essential component of the ERAD complex, with the aid of BiP, and then dislocated into the cytosol. Hence, it is conceivable that ERdj5 plays a critical role in promoting ERAD pathways regardless of the presence or absence of glycosylation sites on substrate proteins. Recently, Bulleid group showed that the role of ERdj5 is not limited to protein degradation. This group first identified a number of proteins that can form mixed-disulfide complexes with this enzyme [49] and then analyzed the role of ERdj5 in the biosynthesis of the low-density lipoprotein receptor (LDLR), one of the identified proteins. ERdj5 was thus found to act as a reductase that breaks non-native disulfide bonds to promote the productive folding of the LDLR [49]. The latter stage of the oxidative folding of the LDLR appears to require both ERdj5 [49] and the oxygen-dependent system [50], although the mechanism by which ERdj5 cooperates with this system remains unclear.

Structure and functions of ERp44 ERp44, another critical member of the PDI family, was initially identified as a key element in the thiol-mediated retention of Ero1Į in the ER [51, 52]. The wide distribution of ERp44 in secretory cells implies a critical role of ERp44 in the early secretory compartment [53]. Indeed, this protein was shown to retrieve unassembled subunits of several proteins in the early secretory pathway, including IgM and adiponectin [51, 52, 54, 55]. Thus, ERp44 plays a significant role in the intracellular localization of essential ER enzymes and quality control of multi-subunit secretory proteins [56-58].
 

To fulfill the unique function of ERp44, the active-site Cys29 residue plays an essential role in capturing client proteins through mixed-disulfide bond formation, although non-covalent interactions are also significant. However, analysis of the crystal structure of ERp44 revealed that Cys29 and its nearby hydrophobic patch are concealed by the C-terminal tail, thereby preventing the binding of client proteins to this region [59]. This closed conformation of ERp44 may also hinder recognition of its C-terminal ER retention signal (RDEL: Arg-Asp-Glu-Leu) by the KDEL receptor. Notably, several lines of biochemical evidence suggest that Cys29 and its nearby hydrophobic patch are exposed at the molecular surface through the release of the C-terminal tail at lower pH values, resulting in an increased affinity of ERp44 for client proteins. Presumably, pH-dependent opening of the C-terminal tail is triggered by protonation of the thiol group of Cys29, which in turn leads to disruption of the hydrogen bond network formed by Cys29 and its surrounding residues [60]. More recently, histidine residues concentrated around the border between domain b’ and the C-terminal tail of ERp44 have been shown to participate in the regulation of its ability to retrieve client proteins in the early secretory pathway [61]. Such pH-dependent and histidine-associated regulations of the conformation and function of ERp44 likely underlie its roles in the efficient surveillance of maturation and assembly of multi-subunit secretory proteins.

2. Partnerships of PDIs in protein and redox homeostasis

Multiple disulfide bond formation pathways in the ER of mammalian cells It has long been assumed that the Ero1α/Ero1β system is the major mechanism of oxidizing PDI family members in the mammalian ER [62]. However, as discussed

 

above (in the section entitled ‘Structural basis of the functional interplays between PDIs and their specific oxidases’), alternate enzymes that oxidize PDI family members have recently been discovered (Fig. 3A), including Prx4 [33, 34] and VKOR [36, 63, 64]. The ER peroxidases GPx7 and GPx8 may also be capable of introducing disulfide bonds [35]. In fact, a recent study suggested that GPx7 uses Ero1α-derived hydrogen peroxide to promote the oxidative folding of proteins in vitro and in vivo [65]. Furthermore, another study suggested that GPx8 is dedicated to preventing the leakage of Ero1α-derived hydrogen peroxide from the ER [66]. However, the net contribution of GPx7 and GPx8 to oxidative protein folding in the ER of living cells remains elusive. The presence of multiple enzymes capable of oxidizing PDIs suggests a high complexity of the disulfide bond formation network in mammalian cells.

Roles of molecular oxygen in oxidative protein folding Recent studies demonstrated that a variety of small molecule oxidants can serve as electron acceptors to oxidize PDIs in enzyme-catalyzed in vitro reactions. These molecules include molecular oxygen (used by Ero1α and Ero1β), hydrogen peroxide (used by Prx4, GPx7, and GPx8), and vitamin K epoxide (used by VKOR) (Fig. 3A). Molecular oxygen is particularly important for the function of the ER because hypoxia causes ER stress [67]. More recently, the role of molecular oxygen in the ER of living cells was studied extensively by Wouters’ group [50]. This group analyzed the transcription of genes with functions related to the ER and found that the mRNAs encoding Ero1α and PDI were the only two transcripts that were significantly and substantially up-regulated by hypoxia, but not by ER stress caused by exposure to tunicamycin or thapsigargin. This
 

finding suggests that disulfide bond formation may be specifically limiting under conditions of ER stress caused by oxygen deprivation. Furthermore, Wouters’ group showed that oxygen depletion halted or retarded the maturation of disulfide-containing proteins but not that of a protein lacking disulfide bonds, indicating the importance of oxygen to the maturation of disulfide bond-containing proteins. Oxidative folding of the LDLR proceeds via two steps; initially, disulfide bonds are introduced into the protein co-translationally, giving rise to a collapsed form containing non-native disulfide bonds. This step is then followed by post-translational reactions that involve isomerization of the non-native disulfide bonds, resulting in the formation of the correctly folded protein [49, 68]. Notably, the initial co-translational oxidation proceeds rapidly under hypoxic conditions, but hypoxia inhibits the post-translational disulfide bond formation/isomerization step, indicating that the folding pathway of the LDLR contains oxygen-independent and -dependent steps. Finally, hypoxia severely impairs overall protein secretion, suggesting that oxygen is required for the secretion of a number of proteins. Overall, these findings highlight the necessity of molecular oxygen for the oxidative folding of a large majority of disulfide bond-containing proteins [50].

Cooperation between two distinct disulfide bond formation pathways during oxidative protein folding The need to introduce disulfide bonds into a large variety of proteins in the ER likely requires enzymes with different substrate specificities, which may explain why the ER of mammalian cells has more than 20 PDI family members (Fig. 3A). However, an alternative explanation of the requirement for so many PDIs came from our recent
 

studies [12, 37]. We showed that, in combination with its preferred partners ERp46 and P5, Prx4 mediates rapid and error-prone disulfide bond formation in model substrate proteins. This finding is in marked contrast to the Ero1α-PDI pathway, which mediates slow but precise disulfide bond formation. Remarkably, the presence of both systems in a tube synergistically accelerates the overall reaction [12, 37]. Based on these findings, we propose that the initial rapid and error-prone disulfide bond formation driven by Prx4 and ERp46/P5 is followed by the slow but precise disulfide bond formation mediated by Ero1 and PDI (Fig. 4). This model, which was established based on in vitro analyses, parallels the observations made in living cells by Koritzinsky et al. [50]. They showed that disulfide bonds were rapidly introduced into LDLR co-translationally in an oxygen-independent manner, which were followed by the oxygen-dependent post-translational disulfide bond formation/isomerization during the folding of the protein. Taken together, our model and the observations of Koritzinsky et al. imply that the initial rapid but promiscuous disulfide bond formation driven by Prx4 and ERp46/P5 takes place independently of molecular oxygen during protein translation, resulting in the formation of intermediates with non-native disulfide bonds. The non-native disulfide bonds are then repaired post-translationally by reactions that involve the PDI-Ero1α system. In this way, the two distinct disulfide bond formation systems may cooperate to enable the rapid and precise production of multi-disulfide proteins in the ER [12] (Fig. 4). Whether or not the actual folding of proteins proceeds in this manner in living cells requires further validation. It could be assumed that the accurate introduction of disulfide bonds into proteins in a single step would save time and enzymatic resources; however, as described above, cells appear to adopt a two-step mechanism to promote the oxidative folding of proteins.
 

It is unclear why cells use this seemingly wasteful reaction process; however, similar mechanisms are used for other cellular processes, including DNA replication. In these processes, rapid but error-prone synthesis is followed by a precise proofreading process [69]. Therefore, we suggest that the two-step mechanism proposed here could be a general strategy to achieve biological processes while ensuring high accuracy and speed.

Ero1α-PDI combination may constitute a core complex that delivers the oxidizing equivalent to other PDI family members Recently, Nagata’s group reported evidence of redox communication among PDIs, suggesting the presence of an even more complicated but efficient disulfide bond formation network in the mammalian ER [70]. According to Nagata’s model, canonical PDI regulates the activities of other PDI family members by delivering oxidizing equivalents from Ero1Į to these enzymes (Fig. 3B). This model was based on the following observations: Firstly, consistent with our findings [19, 37], PDI was the preferred substrate of Ero1α. Secondly, among the PDI family members examined, PDI was most effective in converting the inactive oxidized form (Ox2) of Ero1Į to its active oxidized form (Ox1), indicating that PDI is an efficient activator of Ero1α. Thirdly, PDI formed disulfide-linked complexes with other PDI family members within cells, including P5, ERp46, and ERp57. Fourthly, Ero1α-mediated oxidation of ERp46, ERp57, and P5 was accelerated significantly in the presence of PDI. Finally, this acceleration required the CXXC active site in domain a’ of PDI, which is the domain preferentially oxidized by Ero1α. These findings led Araki et al. to propose that Ero1α oxidizes PDI, which in turn oxidizes other PDI family members in an accelerated
 

manner. Thus, Ero1α-PDI likely constitutes a core complex that provides oxidizing equivalents to other PDI family members in a widespread and rapid manner [70] (Fig. 3B). Although the concept of this model is interesting, further experiments are required to characterize the role of the Ero1α-PDI axis as a hub of the ER redox network in more detail.

Quiescin sulfhydryl oxidase 1 introduces disulfide bonds into extracellular proteins using molecular oxygen Quiescin sulfhydryl oxidase 1 (QSOX1) is a disulfide catalyst equipped with Trx-like and Erv1 domains [71]. Disulfide bonds generated by the Erv1 domain using molecular oxygen as an electron acceptor are transferred to the Trx-like domain and then donated to a pair of cysteines on the substrates of QSOX1. This enzyme represents an atypical disulfide catalyst in that it is localized to the Golgi or secreted from cells. Of note, QSOX1 is not called a PDI family member because this protein does not reside in the ER. Although QSOX1 can introduce disulfide bonds into model substrate proteins with concomitant reduction of molecular oxygen to hydrogen peroxide in vitro [72], the role of this protein as a disulfide enzyme in vivo remained unclear. Recently, Ilani et al. [73] studied the physiological function of QSOX1 and demonstrated that it is required for the formation of the extracellular matrix. This finding raises some interesting questions, particularly regarding the modification of the molecular targets of QSOX1. These targets will be subjected to oxidation in the extracellular matrix; however, during their trip to the extracellular space, they must pass through the ER, which harbors a variety of disulfide-introducing enzymes. It will be interesting to determine the mechanism by which the substrates of QSOX1 avoid oxidation by ER enzymes but are susceptible to
 

oxidation by QSOX1 once they reach the extracellular space.

Reduction of non-native disulfide bonds in the ER of mammalian cells Previous in-depth studies of the bacterial periplasm demonstrated that the reductive pathway mediated by the disulfide bond isomerases DsbC and DsbD is essential for the efficient reduction of non-native disulfide bonds that are formed during the oxidative folding of proteins, and that the ultimate source of the reducing power for this system is derived from NADPH in the cytosol [74]. In yeast, inactivation of the glutathione synthetic pathway can suppress the disulfide bond formation defect caused by a temperature sensitive mutation in the ERO1 gene, indicating that glutathione acts as a net reductant in the ER to counteract the oxidizing activity of Ero1p pathway [75]. This finding led to the proposal that glutathione reduces the non-native disulfide bonds of substrates or reduces PDIp (PDI in yeast), allowing this enzyme to act as a reductase or isomerase. The mammalian ER contains up to 15 mM glutathione [76], which is delivered to the ER from the cytosol by unknown mechanisms. Importantly, in the cytosol, glutathione is kept in the reduced form by glutathione reductase. In contrast, in the ER, the ratio of glutathione to glutathione disulfide (the oxidized form of glutathione) is much lower compared with that in the cytosol, suggesting that incoming glutathione is continuously oxidized to glutathione disulfide in the ER [76]. Based on these facts and some other findings [76, 77], it is widely believed that its reduced form plays crucial roles in protein homeostasis in the mammalian ER by serving as a terminal electron donor to reduce PDI family members or substrates [78]. Ron’s group tested this long-held conviction by expressing a variant of a glutathione-degrading enzyme in the ER lumen to successfully eliminate glutathione
 

from this compartment [79]. Surprisingly, glutathione depletion had no effect on the reduction-dependent folding of the LDLR or reduction-dependent ERAD of a misfolded form of an alpha-1 antitrypsin variant. Furthermore, glutathione depletion had no measurable effect on the induction of ER stress, which is activated by the accumulation of unfolded proteins in this compartment. These findings question the long-held belief that glutathione acts as a terminal electron donor in the mammalian ER and suggest the presence of alternative electron donors that maintain protein homeostasis in this cellular compartment [79]. Nevertheless, we surmise that the high concentration of glutathione in the ER must play some role in living cells under normal or stress conditions.

ERdj5 may interact with other PDI family members to regulate their activities Recently, we described a simple method to purify and identify the disulfide-linked partners of a PDI family member from animal tissues [80]. By combining acid-quenching and thiol-alkylation, we were able to stabilize presumed intermediates that formed between ERdj5, a PDI family member, and its potential substrates in mouse tissues. We purified these complexes from the mouse epididymis, at which ERdj5 is expressed at a high level, using an anti-ERdj5 antibody. A number of PDI family members were identified as presumed redox partners of ERdj5, including ERp57, P5, PDI, ERp72, and ERp44 [80]. Similarly, Oka et al. [49] identified these proteins as potential partners of ERdj5 using a different approach. Thus, these PDI family members appear to be the common partners of ERdj5. It is possible that, by forming mixed-disulfide complexes, ERdj5 donates electrons to other PDI family members, thereby regulating their activities. Because ERdj5 is unique among PDI family members in that it acts preferentially as a reductase of
 

disulfide bonds [46, 47, 49], it may be well suited to regulate the activities of other enzymes by donating electrons. It should be noted that, in addition to the specific PDI family members and LDLR (see the preceding section on LDLR), a large variety of proteins were identified as potential substrates of ERdj5 [49, 80]. Thus far, no specific structural similarities were found among these proteins. Further experiments are required to clarify the physiological meanings of the interactions between ERdj5 and those identified proteins.

Roles of PDI family members in the regulation of unfolded protein response pathways Upon ER stress, cells activate signaling pathways called unfolded protein response (UPR) to upregulate genes that cope with the ER stress. This cellular system is vital for the maintenance of ER homeostasis. In mammalian cells, UPR consists of three major branches that are mediated by ER stress sensors, ATF6, IRE1α and PERK, respectively. Recently, Higa et al. showed that a PDI family member is involved in the activation of the ATF6 pathway [81]. ATF6 is a transcriptional activator anchored to the ER membrane. Under normal conditions, ATF6 exists in the ER as disulfide bonded oligomers that are bound by BiP. Upon ER stress, BiP dissociates from the luminal domain of ATF6, which result in exposure of both the Golgi localization signal and the intermolecular disulfide bonds of ATF6 [82]. ATF6 is then reduced by an unknown mechanism and transported from the ER to the Golgi apparatus to be cleaved by the Golgi apparatus-localized proteases. Eventually, a soluble and active transcriptional activator is released from ATF6 [83]. To identify a factor involved in this process, Higa et al. set up an ATF6 activation screen using siRNAs [81]. Remarkably, PDIR (PDIA5), a PDI family member, was identified
 

as an activator of ATF6. Knocking down PDIR indeed caused defects in the disulfide rearrangement in ATF6, the packaging of ATF6 into coat protein complex II vesicles, the export of ATF6 from the ER, the cleavage of ATF6 in the Golgi apparatus, and the activation of the ATF6 target genes under ER stress. Thus, PDIR-dependent disulfide rearrangement in ATF6 appears to be crucial for the activation of ATF6 upon ER stress [81]. Interestingly, ATF6 is not the only ER stress sensor regulated by a PDI family member. Recent work by Eletto et al. showed that P5 (PDIA6) directly interacts with the activated form of IRE1α via specific cysteines and attenuates its activity by converting the disulfide-linked oligomers (the active form) of IRE1α to its monomers (the inactive form) [84]. Interestingly, the P5-dependent attenuation of IRE1α activity is indeed important because its prolonged activation that occurred in the absence of P5 led to increased apoptosis [84]. Of note, PERK, but not ATF6, was also inactivated by P5, indicating the specificity of P5 for the UPR pathways. More recently, Groenendyk et al. reported that P5 was required for the IRE1α activation caused by disrupted calcium homeostasis [85]. Thus, specific PDI family members play profound roles in the maintenance of ER homeostasis by regulating the activation and/or inactivation of ER stress sensors.

Concluding Remarks As described, PDIs participate in many important cellular events including the oxidative protein folding, the ER associated degradation, the retrieval of unassembled proteins in the early secretory pathway, and the regulation of UPR pathways. It is obvious that each PDIs has distinct physiological functions despite with some overlap or complementarity.  

Structural studies on PDIs have made remarkable progress, which revealed their different thioredoxin domain arrangement and different molecular surface features. However, information on structures of PDIs in complex with their substrates or functional partners are the essential milestones to fully understand how PDIs fulfill their distinguished functions to maintain the cellular homeostasis. Perhaps, PDIs undergo significant dynamics in conformation and cellular localization in response to various conditions and situations. Highly organized systems constituted by PDIs and their functional partners yield some higher order functions, and hence their disruptions are related to a number of diseases including neurodegenerative diseases, diabetes and cancer [86]. Thus, comprehensive understanding of the PDIs-involving systems at molecular, cellular and whole-body levels is of both fundamental and pathological significance and will greatly contribute to further development of present-day molecular cell biology and medical science.

Financial support: This work was supported by funding from the Next Generation World-Leading Researchers program (MEXT) and the Takeda Science Foundation (to K. Inaba), a Grant-in-Aid for Scientific Research (C) (to H. Kadokura), a Grant-in-Aid for Scientific Research on Innovative Areas (to K. Inaba and H. Kadokura), and a Grant-in-Aid for JSPS Fellows (to M. Okumura). The authors declare that there are no conflicts of interest related to this work.

Abbreviations: BiP, binding immunoglobulin protein; CNX, calnexin; CRT, calreticulin; EDEM1, ER-degradation enhancing α-mannosidase-like protein 1; ER, endoplasmic reticulum; ERAD, ER-associated degradation; Ero1, endoplasmic 
 

reticulum oxidoreductin1; GPx, glutathione peroxidase; LDLR, low-density lipoprotein receptor; Prx, peroxiredoxin; QSOX1, quiescin sulfhydryl oxidase 1; Trx, thioredoxin; UPR, unfolded protein response; VKOR, vitamin K epoxide oxidoreductase. References [1]

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Figure Legends

Fig. 1. The domain organizations and overall structures of PDI family members. In the upper panels, the redox-active and redox-inactive Trx-like domains are represented by red and blue boxes, respectively. The sequences of the active sites (Cys-Xaa-Xaa-Cys or Cys-Xaa-Xaa-Ser) are indicated in the red boxes. In the lower panels, the crystal structures of human PDI, ERp57, ERp27, and ERp44, and the solution structure of human ERp46, are represented by ribbon diagrams. The redox-active sites are indicated by yellow spheres. The positively charged region in domain b’ of ERp57 and the Asp-Glu-Trp-Asp sequence in domain b’ of ERp27 are shown as blue and magenta spheres, respectively. The putative structure of ERp44 with the open C-terminal tail at lower pH values is also shown, and the ER retention signal (Arg-Asp-Glu-Leu: RDEL) is indicated.

Fig. 2. The overall structure of ERdj5. A ribbon diagram representing the crystal structure of full-length ERdj5. The redox-active sites are represented by yellow spheres. The J-domain essential for BiP binding is shown in blue. Note that ERdj5 can be divided into an N-terminal and a C-terminal cluster. EDEM1 recruits misfolded glycoproteins and transfers them to ERdj5 through interaction with the C-terminal cluster.

Fig. 3. Pathways of protein disulfide bond formation. (A) An overview of the multiple pathways involved in oxidative protein folding in the ER of mammalian cells. The black arrows indicate the direction of flow of oxidizing   

equivalents and the red arrows denote the direction of electron flow. The use of dashed arrows indicates that the path is minor or not well established. (B) The Ero1Į-PDI complex transfers oxidizing equivalents to other PDI family members as a disulfide-generating and -delivering hub.

Fig. 4. Cooperation of distinct disulfide bond formation/isomerization pathways during oxidative folding of nascent chains in the mammalian ER. According to the proposed model, the Prx4-P5/ERp46 axis engages primarily in rapid but promiscuous disulfide bond introduction during the early stage of oxidative protein folding. By contrast, the Ero1-PDI axis or ERdj5 likely play a critical role in the later folding stage as donors of native disulfide bonds or efficient disulfide proofreaders. 

Highlights 1. PDI family members work distinctly but cooperatively to ensure the proteostasis. 2. Structures of the PDI family provide mechanistic insights into the ER homeostasis. 3. Structure analyses of the PDI family with their partners will be the milestones. 4. New physiological roles of the PDI family will be a frontier.