ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.     

DNA-binding activity of the ERp57 C-terminal domain is related to a redox-dependent conformational change

ERp57, a member of the protein-disulfide isomerase family, although mainly localized in the endoplasmic reticulum is here shown to have a nuclear distribution. We previously showed the DNA-binding properties of ERp57, its association with the internal nuclear matrix, and identified the C-terminal region, containing the a' domain, as being directly involved in the DNA-binding activity. In this work, we demonstrate that its DNA-binding properties are strongly dependent on the redox state of the a' domain active site. Site-directed mutagenesis experiments on the first cysteine residue of the -CGHC-thioredoxin-like active site lead to a mutant domain (C406S) lacking DNA-binding activity. Biochemical studies on the recombinant domain revealed a conformational change associated with the redox-dependent formation of a homodimer, having two disulfide bridges between the cysteine residues of two a' domain active sites. The formation of intermolecular disulfide bridges rather than intramolecular oxidation of active site cysteines is important to generate species with DNA-binding properties. Thus, in the absence of any dedicated motif within the protein sequence, this structural rearrangement might be responsible for the DNA-binding properties of the C-terminal domain. Moreover, NADH-dependent thioredoxin reductase is active on intermolecular disulfides of the a' domain, allowing the control of dimeric protein content as well as its DNA-binding activity. A similar behavior was also observed for whole ERp57.     

Tapasin and ERp57 form a stable disulfide-linked dimer within the MHC class I peptide-loading complex

We previously showed that the major histocompatibility complex (MHC) class I chaperone tapasin can be detected as a mixed disulfide with the thiol-oxidoreductase ERp57. Here we show that tapasin is a unique and preferred substrate, a substantial majority of which is disulfide-linked to ERp57 within the cell. Tapasin upregulation by interferon-gamma induces sequestration of the vast majority of ERp57 into the MHC class I peptide-loading complex. The rate of tapasin-ERp57 conjugate formation is unaffected by the absence of beta2-microglubulin (beta2m), and is independent of calnexin or calreticulin interactions with monoglucosylated N-linked glycans. The heterodimer forms spontaneously in vitro upon mixing recombinant ERp57 and tapasin. Noncovalent interactions between the native proteins inhibit the reductase activity of the thioredoxin CXXC motif within the N-terminal a domain of ERp57 to maintain its interaction with tapasin. Disruption of these interactions by denaturation allows reduction to proceed. Thus, tapasin association specifically inhibits the escape pathway required for disulfide-bond isomerization within conventional protein substrates, suggesting a specific structural role for ERp57 within the MHC class I peptide-loading complex.     

ERp16, an endoplasmic reticulum-resident thiol-disulfide oxidoreductase: biochemical properties and role in apoptosis induced by endoplasmic reticulum stress

We have characterized the properties and putative role of a mammalian thioredoxin-like protein, ERp16 (previously designated ERp18, ERp19, or hTLP19). The predicted amino acid sequence of the 172-residue human protein contains an NH(2)-terminal signal peptide, a thioredoxin-like domain with an active site motif (CGAC), and a COOH-terminal endoplasmic reticulum (ER) retention sequence (EDEL). Analyses indicated that the mature protein (comprising 146 residues) is generated by cleavage of the 26-residue signal peptide and is localized in the lumen of the ER. Biochemical experiments with the recombinant mature protein revealed it to be a thioldisulfide oxidoreductase. Its redox potential was about -165 mV; its active site cysteine residue Cys(66) was nucleophilic with a pK(a) value of approximately 6.6; it catalyzed the formation, reduction, and isomerization of disulfide bonds, with the unusual CGAC active site motif being responsible for these activities; and it existed as a dimer and underwent a redox-dependent conformational change. The observations that the redox potential of ERp16 (-165 mV) was within the range of that of the ER (-135 to -185 mV) and that ERp16 catalyzed disulfide isomerization of scrambled ribonuclease A suggest a role for ERp16 in protein disulfide isomerization in the ER. Expression of ERp16 in HeLa cells inhibited the induction of apoptosis by agents that elicit ER stress, including brefeldin A, tunicamycin, and dithiothreitol. In contrast, expression of a catalytically inactive mutant of ERp16 potentiated such apoptosis, as did depletion of ERp16 by RNA interference. Our results suggest that ERp16 mediates disulfide bond formation in the ER and plays an important role in cellular defense against prolonged ER stress.     

ERp57 is a multifunctional thiol-disulfide oxidoreductase

The thiol-disulfide oxidoreductase ERp57 is a soluble protein of the endoplasmic reticulum and the closest known homologue of protein disulfide isomerase. The protein interacts with the two lectin chaperones calnexin and calreticulin and thereby promotes the oxidative folding of newly synthesized glycoproteins. Here we have characterized several fundamental structural and functional properties of ERp57 in vitro, such as the domain organization, shape, redox potential, and the ability to catalyze different thiol-disulfide exchange reactions. Like protein disulfide isomerase, we find ERp57 to be comprised of four structural domains. The protein has an elongated shape of 3.4 +/- 0.1 nm in diameter and 16.8 +/- 0.5 nm in length. The two redox-active a and a' domains were determined to have redox potentials of -0.167 and -0.156 V, respectively. Furthermore, ERp57 was shown to efficiently catalyze disulfide reduction, disulfide isomerization, and dithiol oxidation in substrate proteins. The implications of these findings for the function of the protein in vivo are discussed.     

Crystal structure of the bb' domains of the protein disulfide isomerase ERp57

The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.     

Formation of a major histocompatibility complex class I tapasin disulfide indicates a change in spatial organization of the peptide-loading complex during assembly

The assembly and peptide loading of major histocompatibility complex Class I molecules within the endoplasmic reticulum are essential for antigen presentation at the cell surface and are facilitated by the peptide-loading complex. The formation of a mixed disulfide between the heavy chain of Class I and components of the loading complex (ERp57, protein disulfide isomerase, and tapasin) suggests that these molecules are involved in the redox regulation of components during assembly and peptide loading. We demonstrate here that a disulfide formed between heavy chain and tapasin can occur between cysteine residues located in the cytosolic regions of these proteins following translation of heavy chain in an in vitro translation system. The formation of this disulfide occurs after assembly into the loading complex and is coincident with the stabilization of the alpha2 disulfide bond within the peptide binding grove. A ternary complex between heavy chain, ERp57, and tapasin was observed and shown to be stabilized by a disulfide between both tapasinheavy chain and tapasin-ERp57. No disulfides were observed between ERp57 and heavy chain within the loading complex. The results provide a detailed evaluation of the various transient disulfides formed within the peptide-loading complex during biosynthesis. In addition, the absence of the disulfide between tapasin and heavy chain in TAP-deficient cells indicates that a change in the spatial organization of tapasin and heavy chain occurs following assembly into the loading complex.     

Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.     

Functions of ERp57 in the folding and assembly of major histocompatibility complex class I molecules

ERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain alpha(3) domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-beta(2)-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and beta(2) -microglobulin does not depend on an oxidized alpha(3) domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process.     

PDI family protein ERp29 forms 1:1 complex with lectin chaperone calreticulin

Lectin chaperone calreticulin is well known to interact with ERp57 which is one of PDI family proteins. The interaction of ERp57 with calreticulin is believed to assist disulfide bond formation of nascent glycoprotein in the ER. Various kinds of PDI family proteins are present in the ER, however, their precise roles have been unclear. In this study, interaction assay between PDI family proteins and calreticulin by SPR analysis was performed. Our analysis revealed for the first time formation of a 1:1 complex between ERp29 and calreticulin. The dissociation constant of interaction between ERp29 and calreticulin was shown to be almost identical to ERp57–calreticulin interaction. We speculate that the recognition site of ERp29 within calreticulin is different from that of ERp57.     

Functional characterization of ERp18, a new endoplasmic reticulum-located thioredoxin superfamily member

Native disulfide bond formation in the endoplasmic reticulum is a critical process in the maturation of many secreted and outer membrane proteins. Although a large number of proteins have been implicated in this process, it is clear that our current understanding is far from complete. Here we describe the functional characterization of a new 18-kDa protein (ERp18) related to protein-disulfide isomerase. We show that ERp18 is located in the endoplasmic reticulum and that it contains a single catalytic domain with an unusual CGAC active site motif and a probable insertion between beta3 and alpha3 of the thioredoxin fold. From circular dichroism and NMR measurements, ERp18 is well structured and undergoes only a minor conformational change upon dithioldisulfide exchange in the active site. Guanidinium chloride denaturation curves indicate that the reduced form of the protein is more stable than the oxidized form, suggesting that it is involved in disulfide bond formation. Furthermore, in vitro ERp18 possesses significant peptide thiol-disulfide oxidase activity, which is dependent on the presence of both active site cysteine residues. This activity differs from that of the human PDI family in that under standard assay conditions it is limited by substrate oxidation and not by enzyme reoxidation. A putative physiological role for Erp18 in native disulfide bond formation is discussed.     

Conserved structural and functional properties of D-domain containing redox-active and -inactive protein disulfide isomerase-related protein chaperones

The structure and mode of binding of the endoplasmic reticulum protein disulfide isomerase-related proteins to their substrates is currently a focus of intensive research. We have recently determined the crystal structure of the Drosophila melanogaster protein disulfide isomerase-related protein Wind and have described two essential substrate binding sites within the protein, one within the thioredoxin b-domain and another within the C-terminal D-domain. Although a mammalian ortholog of Wind (ERp29/28) is known, conflicting interpretations of its structure and putative function have been postulated. Here, we have provided evidence indicating that ERp29 is indeed similar in both structure and function to its Drosophila ortholog. Using a site-directed mutagenesis approach, we have demonstrated that homodimerization of the b-domains is significantly reduced in vitro upon replacement of key residues at the predicted dimerization interface. Investigation of Wind-ERp29 fusion constructs showed that mutants of the D-domain of ERp29 prevent transport of a substrate protein (Pipe) in a manner consistent with the presence of a discrete, conserved peptide binding site in the D-domain. Finally, we have highlighted the general applicability of these findings by showing that the D-domain of a redox-active disulfide isomerase, from the slime mold Dictyostelium discoideum, can also functionally replace the Wind D-domain in vivo.     

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration

ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson’s disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.     

Major histocompatibility complex class I-ERp57-tapasin interactions within the peptide-loading complex

The endoplasmic reticulum-located multimolecular peptide-loading complex functions to load optimal peptides onto major histocompatibility complex (MHC) class I molecules for presentation to CD8(+) T lymphocytes. Two oxidoreductases, ERp57 and protein-disulfide isomerase, are known to be components of the peptide-loading complex. Within the peptide-loading complex ERp57 is normally found disulfide-linked to tapasin, through one of its two thioredoxin-like redox motifs. We describe here a novel trimeric complex that disulfide links together MHC class I heavy chain, ERp57 and tapasin, and that is found in association with the transporter associated with antigen processing peptide transporter. The trimeric complex normally represents a small subset of the total ERp57-tapasin pool but can be significantly increased by altering intracellular oxidizing conditions. Direct mutation of a conserved structural cysteine residue implicates an interaction between ERp57 and the MHC class I peptide-binding groove. Taken together, our studies demonstrate for the first time that ERp57 directly interacts with MHC class I molecules within the peptide-loading complex and suggest that ERp57 and protein-disulfide isomerase act in concert to regulate the redox status of MHC class I during antigen presentation.     

Association of ERp57 with mouse MHC class I molecules is tapasin dependent and mimics that of calreticulin and not calnexin

Before peptide binding in the endoplasmic reticulum, the class I heavy (H) chain-beta(2)-microglobulin complexes are detected in association with TAP and two chaperones, TPN and CRT. Recent studies have shown that the thiol-dependent reductase, ERp57, is also present in this peptide-loading complex. However, it remains controversial whether the association of ERp57 with MHC class I molecules precedes their combined association with the peptide-loading complex or whether ERp57 only associates with class I molecules in the presence of TPN. Resolution of this controversy could help determine the role of ERp57 in class I folding and/or assembly. To define the mouse class I H chain structures involved in interaction with ERp57, we tested chaperone association of L(d) mutations at residues 134 and 227/229 (previously implicated in TAP association), residues 86/88 (which ablate an N-linked glycan), and residue 101 (which disrupts a disulfide bond). The association of ERp57 with each of these mutant H chains showed a complete concordance with CRT, TAP, and TPN but not with calnexin. Furthermore, ERp57 failed to associate with H chain in TPN-deficient.220 cells. These combined data demonstrate that, during the assembly of the peptide-loading complex, the association of ERp57 with mouse class I is TPN dependent and parallels that of CRT and not calnexin.     

ERp57 and PDI: multifunctional protein disulfide isomerases with similar domain architectures but differing substrate-partner associations.

Secretory proteins become folded and acquire stabilizing disulfide bonds in the endoplasmic reticulum (ER). Correct disulfide bond formation is a key step in ER quality control (ERQC). Proteins with incorrect disulfide bonds are recognized by the quality control machinery and are retrotranslocated into the cytosol where they are degraded by the proteasome. The mammalian ER contains 17 disulfide isomerases and at least one of them, ERp57, works in conjunction with the ER lectin-like chaperones calnexin and calreticulin. The targeting of ERp57 to calnexin-calreticulin is mediated by its noncatalytic b' domain, and analogous domains in other disulfide isomerases likely determine their substrate and partner preferences. This review discusses some explanations for the multiplicity of disulfide isomerases and highlights structural differences in the b' domains of PDI and ERp57 as an example of how noncatalytic domains define specialized roles in oxidative folding.     

Endoplasmic reticulum protein 29 (ERp29): An emerging role in cancer

The endoplasmic reticulum protein 29 (ERp29) is a molecule that facilitates processing and transport of proteins in the early secretory pathway. Structural and functional analyses have suggested a biological role as a putative chaperone in the endoplasmic reticulum. The N-terminal domain of ERp29 resembles the thioredoxin domain of protein disulfide isomerase, but lacks its redox-active function due to the absence of an active motif consisting of double cysteines. In the context of carcinogenesis, the role of ERp29 in cancer progression has not been fully elucidated. However, recent studies indicate that high expression of ERp29 inversely correlates to tumor progression. In addition, over-expression of ERp29 significantly inhibits proliferation and suppresses tumorigenesis by modulating ER stress signaling and the mesenchymal-epithelial transition in breast cancer cells. In this review, we summarize the biological properties of ERp29 and its novel function as a tumor suppressor.     

The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death

The exposure of calreticulin (CRT) on the plasma membrane can precede anthracycline-induced apoptosis and is required for cell death to be perceived as immunogenic. Mass spectroscopy, immunofluorescence and immunoprecipitation experiments revealed that CRT co-translocates to the surface with another endoplasmic reticulum-sessile protein, the disulfide isomerase ERp57. The knockout and knockdown of CRT or ERp57 inhibited the anthracycline-induced translocation of ERp57 or CRT, respectively. CRT point mutants that fail to interact with ERp57 were unable to restore ERp57 translocation upon transfection into crt(-/-) cells, underscoring that a direct interaction between CRT and ERp57 is strictly required for their co-translocation to the surface. ERp57(low) tumor cells generated by retroviral introduction of an ERp57-specific shRNA exhibited a normal apoptotic response to anthracyclines in vitro, yet were resistant to anthracycline treatment in vivo. Moreover, ERp57(low) cancer cells (which failed to expose CRT) treated with anthracyclines were unable to elicit an anti-tumor response in conditions in which control cells were highly immunogenic. The failure of ERp57(low) cells to elicit immune responses and to respond to chemotherapy could be overcome by exogenous supply of recombinant CRT protein. These results indicate that tumors that possess an intrinsic defect in the CRT-translocating machinery become resistant to anthracycline chemotherapy due to their incapacity to elicit an anti-cancer immune response.     

Characterization of the redox activity and disulfide bond formation in apurinic/apyrimidinic endonuclease

Apurinic/apyrimidinic endonuclease (APE1) is an unusual nuclear redox factor in which the redox-active cysteines identified to date, C65 and C93, are surface inaccessible residues whose activities may be influenced by partial unfolding of APE1. To assess the role of the five remaining cysteines in APE1's redox activity, double-cysteine mutants were analyzed, excluding C65A, which is redox-inactive as a single mutant. C93A/C99A APE1 was found to be redox-inactive, whereas other double-cysteine mutants retained the same redox activity as that observed for C93A APE1. To determine whether these three cysteines, C65, C93, and C99, were sufficient for redox activity, all other cysteines were substituted with alanine, and this protein was shown to be fully redox-active. Mutants with impaired redox activity failed to stimulate cell proliferation, establishing an important role for APE1's redox activity in cell growth. Disulfide bond formation upon oxidation of APE1 was analyzed by proteolysis of the protein followed by mass spectrometry analysis. Within 5 min of exposure to hydrogen peroxide, a single disulfide bond formed between C65 and C138 followed by the formation of three additional disulfide bonds within 15 min; 10 total disulfide bonds formed within 1 h. A single mixed-disulfide bond involving C99 of APE1 was observed for the reaction of oxidized APE1 with thioredoxin (TRX). Disulfide-bonded APE1 or APE1-TRX species were further characterized by size exclusion chromatography and found to form large complexes. Taken together, our data suggest that APE1 is a unique redox factor with properties distinct from those of other redox factors.