Localization and function in endoplasmic reticulum stress tolerance of ERdj3, a new member of Hsp40 family protein

Heat shock protein 40 (Hsp40) family proteins are known to bind to Hsp70 through their J-domain and regulate the function of Hsp70 by stimulating its adenosine triphosphatase activity. In the endoplasmic reticulum (ER), there are 5 Hsp40 family proteins known so far, 3 of which were recently identified. In this report, one of the novel Hsp40 cochaperones, ERdj3, was characterized in terms of its subcellular localization, stress response, and stress tolerance of cells. By using ERdj3-specific polyclonal antibody, endogenous ERdj3 protein was shown to reside in the ER as gene transfer–mediated exogenous ERdj3. Analysis of the expression level of endogenous ERdj3 protein revealed its moderate induction in response to various ER stressors, indicating its possible action as a stress protein in the ER. Subsequently, we analyzed whether this molecule was involved in ER stress tolerance of cells, as was the case with the ER-resident Hsp70 family protein BiP. Although overexpression of ERdj3 by gene transfection could not strengthen ER stress tolerance of neuroblastoma cells, reduction of ERdj3 expression by small interfering ribonucleic acid decreased the tolerance of cells, indicating that ERdj3 might have just a marginal role in the ER stress resistance of neuroblastoma cells. In contrast, overexpression of ERdj3 notably suppressed vero toxin–induced cell death. These data suggest that ERdj3 might have diverse roles in the ER, including that of the molecular cochaperone of BiP and an as yet unknown protective action against vero toxin.     

ERdj4 and ERdj5 are required for endoplasmic reticulum-associated protein degradation of misfolded surfactant protein C

Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. In this study, genes specifically induced in response to transient expression of two disease-associated mutations were identified by microarray analyses. Immunoglobulin heavy chain binding protein (BiP) and two heat shock protein 40 family members, endoplasmic reticulum-localized DnaJ homologues ERdj4 and ERdj5, were significantly elevated and exhibited prolonged and specific association with the misfolded proprotein; in contrast, ERdj3 interacted with BiP, but it did not associate with either wild-type or mutant SP-C. Misfolded SP-C, ERdj4, and ERdj5 coprecipitated with p97/VCP indicating that the cochaperones remain associated with the misfolded proprotein until it is dislocated to the cytosol. Knockdown of ERdj4 and ERdj5 expression increased ER retention and inhibited degradation of misfolded SP-C, but it had little effect on the wild-type protein. Transient expression of ERdj4 and ERdj5 in X-box binding protein 1(-/-) mouse embryonic fibroblasts substantially restored rapid degradation of mutant SP-C proprotein, whereas transfection of HPD mutants failed to rescue SP-C endoplasmic reticulum-associated protein degradation. ERdj4 and ERdj5 promote turnover of misfolded SP-C and this activity is dependent on their ability to stimulate BiP ATPase activity.     

Structural basis of an ERAD pathway mediated by the ER-resident protein disulfide reductase ERdj5

ER-associated degradation (ERAD) is an ER quality-control process that eliminates terminally misfolded proteins. ERdj5 was recently discovered to be a key ER-resident PDI family member protein that accelerates ERAD by reducing incorrect disulfide bonds in misfolded glycoproteins recognized by EDEM1. We here solved the crystal structure of full-length ERdj5, thereby revealing that ERdj5 contains the N-terminal J domain and six tandem thioredoxin domains that can be divided into the N- and C-terminal clusters. Our systematic biochemical analyses indicated that two thioredoxin domains that constitute the C-terminal cluster form the highly reducing platform that interacts with EDEM1 and reduces EDEM1-recruited substrates, leading to their facilitated degradation. The pulse-chase experiment further provided direct evidence for the sequential movement of an ERAD substrate from calnexin to the downstream EDEM1-ERdj5 complex, and then to the retrotranslocation channel, probably through BiP. We present a detailed molecular view of how ERdj5 mediates ERAD in concert with EDEM1.     

PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum

Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.     

Different Interaction Modes for Protein-disulfide Isomerase (PDI) as an Efficient Regulator and a Specific Substrate of Endoplasmic Reticulum Oxidoreductin-1α (Ero1α)

Protein-disulfide isomerase (PDI) and sulfhydryl oxidase endoplasmic reticulum oxidoreductin-1α (Ero1α) constitute the pivotal pathway for oxidative protein folding in the mammalian endoplasmic reticulum (ER). Ero1α oxidizes PDI to introduce disulfides into substrates, and PDI can feedback-regulate Ero1α activity. Here, we show the regulatory disulfide of Ero1α responds to the redox fluctuation in ER very sensitively, relying on the availability of redox active PDI. The regulation of Ero1α is rapidly facilitated by either a or a′ catalytic domain of PDI, independent of the substrate binding domain. On the other hand, activated Ero1α specifically binds to PDI via hydrophobic interactions and preferentially catalyzes the oxidation of domain a′. This asymmetry ensures PDI to function simultaneously as an oxidoreductase and an isomerase. In addition, several PDI family members are also characterized to be potent regulators of Ero1α. The novel modes for PDI as a competent regulator and a specific substrate of Ero1α govern efficient and faithful oxidative protein folding and maintain the ER redox homeostasis.     

Identification and characterization of a novel endoplasmic reticulum (ER) DnaJ homologue, which stimulates ATPase activity of BiP in vitro and is induced by ER stress

The activity of Hsp70 proteins is regulated by accessory proteins, which include members of the DnaJ-like protein family. Characterized by the presence of a highly conserved 70-amino acid J domain, DnaJ homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates. DnaJ homologues have been identified in most organelles where they are involved in nearly all aspects of protein synthesis and folding. Within the endoplasmic reticulum (ER), DnaJ homologues have also been shown to assist in the translocation, secretion, retro-translocation, and ER-associated degradation (ERAD) of secretory pathway proteins. By using bioinformatic methods, we identified a novel mammalian DnaJ homologue, ERdj4. It is the first ER-localized type II DnaJ homologue to be reported. The signal sequence of ERdj4 remains uncleaved and serves as a membrane anchor, orienting its J domain into the ER lumen. ERdj4 co-localized with GRP94 in the ER and associated with BiP in vivo when they were co-expressed in COS-1 cells. In vitro experiments demonstrated that the J domain of ERdj4 stimulated the ATPase activity of BiP in a concentration-dependent manner. However, mutation of the hallmark tripeptide HPD (His --> Gln) in the J domain totally abolished this activation. ERdj4 mRNA expression was detected in all human tissues examined but showed the highest level of the expression in the liver, kidney, and placenta. We found that ERdj4 was highly induced at both the mRNA and protein level in response to ER stress, indicating that this protein might be involved in either protein folding or ER-associated degradation.     

A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins

We demonstrate the existence of a large endoplasmic reticulum (ER)-localized multiprotein complex that is comprised of the molecular chaperones BiP; GRP94; CaBP1; protein disulfide isomerase (PDI); ERdj3, a recently identified ER Hsp40 cochaperone; cyclophilin B; ERp72; GRP170; UDP-glucosyltransferase; and SDF2-L1. This complex is associated with unassembled, incompletely folded immunoglobulin heavy chains. Except for ERdj3, and to a lesser extent PDI, this complex also forms in the absence of nascent protein synthesis and is found in a variety of cell types. Cross-linking studies reveal that the majority of these chaperones are included in the complex. Our data suggest that this subset of ER chaperones forms an ER network that can bind to unfolded protein substrates instead of existing as free pools that assembled onto substrate proteins. It is noticeable that most of the components of the calnexin/calreticulin system, which include some of the most abundant chaperones inside the ER, are either not detected in this complex or only very poorly represented. This study demonstrates an organization of ER chaperones and folding enzymes that has not been previously appreciated and suggests a spatial separation of the two chaperone systems that may account for the temporal interactions observed in other studies.     

Ero1-α and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases

Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α–associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a′ domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells.     

Protein disulfide–isomerase,a folding catalyst and a redox-regulated chaperone

Protein disulfide–isomerase (PDI) was the first protein-folding catalyst to be characterized, half a century ago. It plays critical roles in a variety of physiological events by displaying oxidoreductase and redox-regulated chaperone activities. This review provides a brief history of the identification of PDI as both an enzyme and a molecular chaperone and of the recent advances in studies on the structure and dynamics of PDI, the substrate binding and release, and the cooperation with its partners to catalyze oxidative protein folding and maintain ER redox homeostasis. In this review, we highlight the structural features of PDI, including the high interdomain flexibility, the multiple binding sites, the two synergic active sites, and the redox-dependent conformational changes.     

ERdj4 protein is a soluble endoplasmic reticulum (ER) DnaJ family protein that interacts with ER-associated degradation machinery

Protein localization within cells regulates accessibility for interactions with co-factors and substrates. The endoplasmic reticulum (ER) BiP co-factor ERdj4 is up-regulated by ER stress and has been implicated in ER-associated degradation (ERAD) of multiple unfolded secretory proteins. Several other ERdj family members tend to interact selectively with nascent proteins, presumably because those ERdj proteins associate with the Sec61 translocon that facilitates entry of nascent proteins into the ER. How ERdj4 selects and targets terminally misfolded proteins for destruction remains poorly understood. In this study, we determined properties of ERdj4 that might aid in this function. ERdj4 was reported to retain its signal sequence and to be resistant to mild detergent extraction, suggesting that it was an integral membrane protein. However, live cell photobleaching analyses of GFP-tagged ERdj4 revealed that the protein exhibits diffusion coefficients uncommonly high for an ER integral membrane protein and more similar to the mobility of a soluble luminal protein. Biochemical characterization established that the ERdj4 signal sequence is cleaved to yield a soluble protein. Importantly, we found that both endogenous and overexpressed ERdj4 associate with the integral membrane protein, Derlin-1. Our findings now directly link ERdj4 to the ERAD machinery and suggest a model in which ERjd4 could help recruit clients from throughout the ER to ERAD sites.     

Protein disulfide isomerase in redox cell signaling and homeostasis

Thiol proteins may potentially act as redox signaling adaptor proteins, adjusting reactive oxygen species intermediates to specific signals and redox signals to cell homeostasis. In this review, we discuss redox effects of protein disulfide isomerase (PDI), a thioredoxin superfamily oxidoreductase from the endoplasmic reticulum (ER). Abundantly expressed PDI displays ubiquity, interactions with redox and nonredox proteins, versatile effects, and several posttranslational modifications. The PDI family contains >20 members with at least some apparent complementary actions. PDI has oxidoreductase, isomerase, and chaperone effects, the last not directly dependent on its thiols. PDI is a converging hub for pathways of disulfide bond introduction into ER-processed proteins, via hydrogen peroxide-generating mechanisms involving the oxidase Ero1α, as well as hydrogen peroxide-consuming reactions involving peroxiredoxin IV and the novel peroxidases Gpx7/8. PDI is a candidate pathway for coupling ER stress to oxidant generation. Emerging information suggests a convergence between PDI and Nox family NADPH oxidases. PDI silencing prevents Nox responses to angiotensin II and inhibits Akt phosphorylation in vascular cells and parasite phagocytosis in macrophages. PDI overexpression spontaneously enhances Nox activation and expression. In neutrophils, PDI redox-dependently associates with p47phox and supports the respiratory burst. At the cell surface, PDI exerts transnitrosation, thiol reductase, and apparent isomerase activities toward targets including adhesion and matrix proteins and proteases. Such effects mediate redox-dependent adhesion, coagulation/thrombosis, immune functions, and virus internalization. The route of PDI externalization remains elusive. Such multiple redox effects of PDI may contribute to its conspicuous expression and functional role in disease, rendering PDI family members putative redox cell signaling adaptors.     

The ERdj5-Sel1L complex facilitates cholera toxin retrotranslocation

Cholera toxin (CT) traffics from the host cell surface to the endoplasmic reticulum (ER), where the toxin''s catalytic CTA1 subunit retrotranslocates to the cytosol to induce toxicity. In the ER, CT is captured by the E3 ubiquitin ligase Hrd1 via an undefined mechanism to prepare for retrotranslocation. Using loss-of-function and gain-of-function approaches, we demonstrate that the ER-resident factor ERdj5 promotes CTA1 retrotranslocation, in part, via its J domain. This Hsp70 cochaperone regulates binding between CTA and the ER Hsp70 BiP, a chaperone previously implicated in toxin retrotranslocation. Importantly, ERdj5 interacts with the Hrd1 adaptor Sel1L directly through Sel1L''s N-terminal lumenal domain, thereby linking ERdj5 to the Hrd1 complex. Sel1L itself also binds CTA and facilitates toxin retrotranslocation. By contrast, EDEM1 and OS-9, two established Sel1L binding partners, do not play significant roles in CTA1 retrotranslocation. Our results thus identify two ER factors that promote ER-to-cytosol transport of CTA1. They also indicate that ERdj5, by binding to Sel1L, triggers BiP–toxin interaction proximal to the Hrd1 complex. We postulate this scenario enables the Hrd1-associated retrotranslocation machinery to capture the toxin efficiently once the toxin is released from BiP.     

A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2—an ER chaperone and member of the Trx superfamily—and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.     

Elevated expression of PDI family proteins during differentiation of mouse F9 teratocarcinoma cells

We investigated the expression of protein disulfide isomerase family proteins (PDI, ERp61, and ERp72) in mouse F9 teratocarcinoma cells during differentiation induced by treatment with retinoic acid and dibutyryl cAMP. Each member of this family was expressed at a constitutive level in undifferentiated F9 cells. During differentiation of F9 cells to parietal or visceral endodermal cells the protein level of all these enzymes increased, although the extent of this increase in both protein and mRNA levels varied among the enzymes. Certain proteins were found to be co-immunoprecipitated with PDI, ERp61, and ERp72 in the presence of a chemical crosslinker. Type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Thus, the induction of PDI family proteins during the differentiation of F9 cells and their association with different proteins may implicate specific functions of each member of this family despite the common redox activity capable of catalyzing the disulfide bond formation. J. Cell. Biochem. 68:436–445, 1998. © 1998 Wiley-Liss, Inc.     

The chloroplast protein disulfide isomerase RB60 reacts with a regulatory disulfide of the RNA-binding protein RB47

Biochemical studies have identified two proteins, RB47 and RB60, that are involved in the light-regulated translation of the psbA mRNA in the chloroplast of the unicellular alga Chlamydomonas reinhardtii. RB47, a member of the eukaryotic poly(A)-binding protein family, binds directly to the 5' untranslated region of the mRNA, whereas RB60, a protein disulfide isomerase (PDI), is thought to bind to RB47 and to modulate its activity via redox and phosphorylation events. Our present studies show that RB47 forms a single disulfide bridge that most probably involves Cys143 and Cys259. We found that RB60 reacts with high selectivity with the disulfide of RB47, suggesting that the redox states of these two redox partners are coupled. Kinetics analysis indicated that RB47 contains two fast reacting cysteines, of which at least one is sensitive to changes in pH conditions. The results support the notion that light controls the redox regulation of RB47 function via the coupling of RB47 and RB60 redox states, and suggest that light-induced changes in stromal pH might contribute to the regulation.     

Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds

Protein disulfide isomerase (PDI) oxidizes, reduces, and isomerizes disulfide bonds, modulates redox responses, and chaperones proteins. The Arabidopsis thaliana genome contains 12 PDI genes, but little is known about their subcellular locations and functions. We demonstrate that PDI5 is expressed in endothelial cells about to undergo programmed cell death (PCD) in developing seeds. PDI5 interacts with three different Cys proteases in yeast two-hybrid screens. One of these traffics together with PDI5 from the endoplasmic reticulum through the Golgi to vacuoles, and its recombinant form is functionally inhibited by recombinant PDI5 in vitro. Peak PDI5 expression in endothelial cells precedes PCD, whereas decreasing PDI5 levels coincide with the onset of PCD-related cellular changes, such as enlargement and subsequent collapse of protein storage vacuoles, lytic vacuole shrinkage and degradation, and nuclear condensation and fragmentation. Loss of PDI5 function leads to premature initiation of PCD during embryogenesis and to fewer, often nonviable, seeds. We propose that PDI5 is required for proper seed development and regulates the timing of PCD by chaperoning and inhibiting Cys proteases during their trafficking to vacuoles before PCD of the endothelial cells. During this transitional phase of endothelial cell development, the protein storage vacuoles become the de facto lytic vacuoles that mediate PCD.     

Evidence for mitochondrial localization of P5, a member of the protein disulphide isomerase family

This report demonstrates for the first time that P5, a member of the protein disulphide isomerase (PDI) family, is present in the mitochondria. Various organelles were screened for proteins bearing the CGHC motif using an affinity column conjugated with the phage antibody 5E, which cross-reacts with PDI family proteins. P5 was found in bovine liver mitochondrial extract and identified by Western blot analysis using anti-P5 antibody and by mass spectrometric analysis. Results of cell fractionation, proteinase sensitivity experiments and immuno-electron microscopy supported the mitochondrial localization of P5 and also indicated the presence of ERp57, another PDI family protein, in mitochondria. Our findings will be useful for the elucidation of the translocation mechanism of PDI family proteins and their roles in mitochondria.     

Overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the Newcastle disease virus fusion protein

Newcastle disease virus (NDV) fusion (F) protein directs membrane fusion, which is required for virus entry and cell-cell fusion. We have previously shown that free thiols are present in cell surface-expressed NDV F protein and that blocking the production of free thiols by thiol-disulfide exchange inhibitors inhibited the membrane fusion mediated by F protein (J Virol. 81:2328-2339, 2007). Extending these observations, we evaluated the role of the overexpression of two disulfide bond isomerases, protein disulfide isomerase (PDI) and ERdj5, in cell-cell fusion mediated by NDV glycoproteins. The overexpression of these isomerases resulted in significantly increased membrane fusion, as measured by syncytium formation and content mixing. The overexpression of these isomerases enhanced the production of free thiols in F protein when expressed without hemagglutination-neuraminidase (HN) protein but decreased free thiols in F protein expressed with HN protein. By evaluating the binding of conformation-sensitive antibodies, we found that the overexpression of these isomerases favored a postfusion conformation of surface-expressed F protein in the presence of HN protein. These results suggest that isomerases belonging to the PDI family catalyze the production of free thiols in F protein, and free thiols in F protein facilitate membrane fusion mediated by F protein.