Protein Disulfide Isomerase Family Proteins Research Articles

Regulation of plant ER oxidoreductin 1 (ERO1) activity for efficient oxidative protein folding

In the endoplasmic reticulum (ER), ER oxidoreductin 1 (ERO1) catalyzes intramolecular disulfide-bond formation within its substrates in coordination with protein-disulfide isomerase (PDI) and related enzymes. However, the molecular mechanisms that regulate the ERO1-PDI system in plants are unknown. Reduction of the regulatory disulfide bonds of the ERO1 from soybean, GmERO1a, is catalyzed by enzymes in five classes of PDI family proteins. Here, using recombinant proteins, vacuum-ultraviolet circular dichroism spectroscopy, biochemical and protein refolding assays, and quantitative immunoblotting, we found that GmERO1a activity is regulated by reduction of intramolecular disulfide bonds involving Cys-121 and Cys-146, which are located in a disordered region, similarly to their locations in human ERO1. Moreover, a GmERO1a variant in which Cys-121 and Cys-146 were replaced with Ala residues exhibited hyperactive oxidation. Soybean PDI family proteins differed in their ability to regulate GmERO1a. Unlike yeast and human ERO1s, for which PDI is the preferred substrate, GmERO1a directly transferred disulfide bonds to the specific active center of members of five classes of PDI family proteins. Of these proteins, GmPDIS-1, GmPDIS-2, GmPDIM, and GmPDIL7 (which are group II PDI family proteins) failed to catalyze effective oxidative folding of substrate RNase A when there was an unregulated supply of disulfide bonds from the C121A/C146A hyperactive mutant GmERO1a, because of its low disulfide-bond isomerization activity. We conclude that regulation of plant ERO1 activity is particularly important for effective oxidative protein folding by group II PDI family proteins.

Expression of protein disulfide isomerase family members correlates with tumor progression and patient survival in ovarian cancer

ObjectiveProtein disulfide isomerase (PDI) is an oxidoreductase that is overexpressed in several cancers. PDI family members (PDIs) play a role in various diseases including cancer. Select PDIs were reported as useful markers in other cancers but their expression in ovarian cancer has not been thoroughly assessed. We sought to evaluate the expression of PDI, PDIA6, PDIR, ERp57, ERp72 and AGR3 in ovarian cancer patient samples and examine their prognostic significance.MethodsTMA samples from 415 tissues collected from three cancer centers (UM, USC, and KCCRI) were used to assess the expression levels of PDI family proteins using IHC.ResultsWe observed significant increases in PDI (p = 9.16E-36), PDIA6 (p = 5.51E-33), PDIR (p = 1.81E-12), ERp57 (p = 9.13E-07), ERp72 (p = 3.65E-22), and AGR3 (p = 4.56E-24) expression in ovarian cancers compared to normal tissues. Expression of PDI family members also increases during disease progression (p <0.001). All PDI family members are overexpressed in serous ovarian cancer (p<0.001). However, PDI, PDIA6, PDIR, ERp72 and AGR3 are more significantly overexpressed (p<0.001) than ERp57 (p<0.05) in clear cell ovarian carcinoma. Importantly, overexpression of PDI family members is associated with poor survival in ovarian cancer (p = 0.045 for PDI, p = 0.047 for PDIR, p = 0.037 for ERp57, p = 0.046 for ERp72, p = 0.040 for AGR3) with the exception of PDIA6 (p = 0.381).ConclusionsOur findings demonstrate that select PDI family members (PDI, PDIR, ERp72, ERp57 and AGR3) are potential prognostic markers for ovarian cancer.

Effect of Subcellular Translocation of Protein Disulfide Isomerase on Tetrachlorobenzoquinone-Induced Signaling Shift from Endoplasmic Reticulum Stress to Apoptosis.

Our previous studies illustrated tetrachlorobenzoquinone (TCBQ)-caused toxicities in neuron-like cells which imply its association with neurodegenerative disorders. Although it is known that TCBQ induces oxidative damage that in turn results in endoplasmic reticulum (ER) stress and apoptosis, it is unclear how TCBQ triggers the signaling switch from pro-survival (to restore cellular homeostasis) to pro-death (trigger apoptosis). Protein disulfide isomerase family proteins (PDIs) regulate the progress of various neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. We tested the hypothesis that subcellular translocation of PDIs implicates the survival/death signaling switch by inducing mitochondrial outer membrane permeabilization (MOMP). The rat pheochromocytoma PC12 cells were exposed to TCBQ, and the concentration-dependent ER stress was observed upon TCBQ treatment, as indicated by the increase in inositol-requiring kinase/endonuclease 1α (IRE1α) phosphorylation, C/EBP homologous protein (CHOP) expression, X-box-binding protein 1 (XBP1) splicing, and caspase 12 activation. Interestingly, pharmacological (or siRNA) abrogation of PDIA1/PDIA3 aggravated the loss of cell viability induced by the relatively low concentration (10 μM) of TCBQ. However, inhibition of PDIA1/PDIA3 rescued the high concentration (20 μM) of TCBQ-induced cell death. Further mechanistic study illustrated that PDIs initially acted to restore cellular homeostasis to pro-survival but that the constant ER stress promoted the signaling switch to pro-apoptotis by the release of PDIA1/PDIA3 from the ER lumen to induce Bak-dependent MOMP. Our findings suggested that subcellular translocation of PDIs determined the "life or death" fate of PC12 cells to TCBQ-induced oxidative insult.

HPW-RX40 prevents human platelet activation by attenuating cell surface protein disulfide isomerases

Protein disulfide isomerase (PDI) present at platelet surfaces has been considered to play an important role in the conformational change and activation of the integrin glycoprotein IIb/IIIa (GPIIb/IIIa) and thus enhances platelet aggregation. Growing evidences indicated that platelet surface PDI may serve as a potential target for developing of a new class of antithrombotic agents. In the present study, we investigated the effects of HPW-RX40, a chemical derivative of β-nitrostyrene, on platelet activation and PDI activity. HPW-RX40 inhibited platelet aggregation, GPIIb/IIIa activation, and P-selectin expression in human platelets. Moreover, HPW-RX40 reduced thrombus formation in human whole blood under flow conditions, and protects mice from FeCl3-induced carotid artery occlusion. HPW-RX40 inhibited the activity of recombinant PDI family proteins (PDI, ERp57, and ERp5) as well as suppressed cell surface PDI activity of platelets in a reversible manner. Exogenous addition of PDI attenuated the inhibitory effect of HPW-RX40 on GPIIb/IIIa activation. Structure-based molecular docking simulations indicated that HPW-RX40 binds to the active site of PDI by forming hydrogen bonds. In addition, HPW-RX40 neither affected the cell viability nor induced endoplasmic reticulum stress in human cancer A549 and MDA-MB-231 cells. Taken together, our results suggest that HPW-RX40 is a reversible and non-cytotoxic PDI inhibitor with antiplatelet effects, and it may have a potential for development of novel antithrombotic agents.

Identification and characterization of GmPDIL7, a soybean ER membrane-bound protein disulfide isomerase family protein.

The nucleotide sequence data for the GmPDIL7 cDNA are available in the DNA Data Bank of Japan (DDBJ) databases under the accession numbers LC158001. Protein disulfide isomerase: EC 5.3.4.1.

Extracellular Thiol Isomerases and Their Role in Thrombus Formation.

The mammalian endoplasmic reticulum (ER) houses a large family of twenty thioredoxin-like proteins of which protein disulfide isomerase (PDI) is the archetypal member. Although the PDI family is best known for its role in oxidative protein folding of secretory proteins in the ER, these thioredoxin-like proteins fulfill ever-expanding roles, both within the secretory pathway and beyond. Secreted PDI family proteins have now been shown to serve a critical role in platelet thrombus formation and fibrin generation. Utilizing intravital microscopy to visualize thrombus formation in mice, we have demonstrated the presence of extracellular PDI antigen during thrombus formation following injury of the vascular wall. Inhibition of PDI abrogates thrombus formation in vivo (16, 26, 46, 55). These observations have been extended to other PDI family members, including ERp57 (39, 116, 118, 123) and ERp5 (77). The vascular thiol isomerases are those PDI family members secreted from platelets and/or endothelium (40): PDI, ERp57, ERp5, ERp72, ERp44, ERp29, and TMX3. We focus here on PDI (16, 46, 55), ERp57 (39, 116, 118, 123), and ERp5 (77), which have been implicated in thrombus formation in vivo. It would appear that a system of thiol isomerase redox catalysts has been hijacked from the ER to regulate thrombus formation in the vasculature. How this redox system is trafficked to and regulated at the cell surface, the identity of extracellular substrates, why so many thiol isomerases are required, and which thiol isomerase functions are necessary are critical unanswered questions in understanding the role of thiol isomerases in thrombus formation.

Expression and characterization of protein disulfide isomerase family proteins in bread wheat.

BackgroundThe major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds. In eukaryotes, the formation of most intramolecular disulfide bonds in the ER is thought to be catalyzed by protein disulfide isomerase (PDI) family proteins. The cDNAs that encode eight groups of bread wheat (Triticum aestivum L.) PDI family proteins have been cloned, and their expression levels in developing wheat grains have been determined. The purpose of the present study was to characterize the enzymatic properties of the wheat PDI family proteins and clarify their expression patterns in wheat caryopses.ResultsPDI family cDNAs, which are categorized into group I (TaPDIL1Aα, TaPDIL1Aβ, TaPDIL1Aγ, TaPDIL1Aδ, and TaPDIL1B), group II (TaPDIL2), group III (TaPDIL3A), group IV (TaPDIL4D), and group V (TaPDIL5A), were cloned. The expression levels of recombinant TaPDIL1Aα, TaPDIL1B, TaPDIL2, TaPDIL3A, TaPDIL4D, and TaPDIL5A in Escherichia coli were established from the cloned cDNAs. All recombinant proteins were expressed in soluble forms and purified. Aside from TaPDIL3A, the recombinant proteins exhibited oxidative refolding activity on reduced and denatured ribonuclease A. Five groups of PDI family proteins were distributed throughout wheat caryopses, and expression levels of these proteins were higher during grain filling than in the late stage of maturing. Localization of these proteins in the ER was confirmed by fluorescent immunostaining of the immature caryopses. In mature grains, the five groups of PDI family proteins remained in the aleurone cells and the protein matrix of the starchy endosperm.ConclusionsHigh expression of PDI family proteins during grain filling in the starchy endosperm suggest that these proteins play an important role in forming intramolecular disulfide bonds in seed storage proteins. In addition, these PDI family proteins that remain in the aleurone layers of mature grains likely assist in folding newly synthesized hydrolytic enzymes during germination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0460-2) contains supplementary material, which is available to authorized users.

Endoplasmic Reticulum Protein (ERp) 29 Binds As Strongly As Protein Disulfide Isomerase (PDI) to Bisphenol A

Bisphenol A (BPA), which is used in polycarbonate and epoxy resins, affects the development or function of the central nervous system. Previously, we isolated a BPA-binding protein from rat brain, identified it as protein disulfide isomerase (PDI), and found that BPA binds to the b' domain of PDI and inhibits its activity. There are 20 kinds of PDI family proteins in mammalian endoplasmic reticulum. The member proteins each have a different length and domain arrangement. Here we investigated the binding of BPA and T3 to ERp29, ERp57, and ERp72, which each have the b or b' domain. BPA/T3 binding of ERp57 and that of ERp72 were lower than that of PDI, and BPA did not inhibit the oxidase or reductase activity of these proteins. On the other hand, BPA and T3 bound to ERp29 as strongly as to PDI. The CD spectrum of PDI was changed in the presence of BPA in a dose-dependent manner, while that of ERp29 was not, suggesting that BPA did not affect the conformation of ERp29. We found that PDI suppresses GH expression in rat GH3 cells stimulated by thyroid hormone (T3) overexpression of PDI and that ERp57 reduced the GH level, but overexpression of ERp29 did not change GH expression. These results suggested that affinity to T3 does not involve the reduction of the T3 response. In this study, ERp29 was first identified as a BPA-binding protein but is not involved in the T3 response of GH3 cells.

Ero1–PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum

The protein folding machinery of the endoplasmic reticulum (ER) ensures that proteins entering the eukaryotic secretory pathway acquire appropriate post-translational modifications and reach a stably folded state. An important component of this protein folding process is the supply of disulfide bonds. These are introduced into client proteins by ER resident oxidoreductases, including ER oxidoreductin 1 (Ero1). Ero1 is usually considered to function in a linear pathway, by ‘donating’ a disulfide bond to protein disulfide isomerase (PDI) and receiving electrons that are passed on to the terminal electron acceptor molecular oxygen. PDI engages with a range of clients as the direct catalyst of disulfide bond formation, isomerization or reduction. In this paper, we will consider the interactions of Ero1 with PDI family proteins and chaperones, highlighting the effect that redox flux has on Ero1 partnerships. In addition, we will discuss whether higher order protein complexes play a role in Ero1 function.

The Protein Disulfide Isomerase Family: Key Players in Health and Disease

Protein disulfide isomerase (PDI) and its homologs have essential roles in the oxidative folding and chaperone-mediated quality control of proteins in the secretory pathway. In this review, the importance of PDI in health and disease will be examined, using examples from the fields of lipid homeostasis, hemostasis, infectious disease, cancer, neurodegeneration, and infertility. Recent structural studies, coupled with cell biological, biochemical, and clinical approaches, have demonstrated that PDI family proteins are involved in a wide range of physiological and disease processes. Critical issues in the field include understanding how and why a PDI family member is involved in a given disease, and defining the physiological client specificity of the various PDI proteins when they are expressed in different tissues. Future directions are likely to include the development of new and more specific reagents to study and manipulate PDI family function. The development of conditional mouse models in concert with clinical data will help us to understand the in vivo function of the different PDIs at the organism level. Taken together with advances in structural biology and biochemical studies, this should help us to further understand and modify PDIs' functional interactions.

Progranulin, a Glycoprotein Deficient in Frontotemporal Dementia, Is a Novel Substrate of Several Protein Disulfide Isomerase Family Proteins

The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER) and Golgi. Using chemical crosslinking, immunoprecipitation, and mass spectrometry, we found that progranulin is bound to a network of ER Ca2+-binding chaperones including BiP, calreticulin, GRP94, and four members of the protein disulfide isomerase (PDI) family. Loss of ERp57 inhibits progranulin secretion. Thus, progranulin is a novel substrate of several PDI family proteins and modulation of the ER chaperone network may be a therapeutic target for controlling progranulin secretion.

Relationship between Endogenous Protein Disulfide Isomerase Family Proteins and Glutenin Macropolymer

The effects of endogenous protein disulfide isomerase (PDI) family proteins on the properties of gluten proteins in dough during breadmaking were determined using bacitracin, an inhibitor of PDI. Bread loaf volume in the presence of bacitracin was increased to 118% of that in the absence of bacitracin. The addition of bacitracin caused a decrease in the extension tolerance of the dough. The amount of sodium dodecyl sulfate (SDS)-insoluble glutenin macropolymer (GMP) in dough decreased to approximately 70% of that in flour during the 20 min of mixing for doughmaking. The addition of bacitracin to dough caused a dramatic GMP decrease, corresponding to ∼20-30% of that in flour during the 20 min of mixing. The decrease in GMP was compensated by an increase in SDS-soluble glutenin polymer. Taken together, these results suggest that the endogenous PDI family proteins in flour suppress the depolymerization of GMP during dough mixing.

Oxidative Activity of Yeast Ero1p on Protein Disulfide Isomerase and Related Oxidoreductases of the Endoplasmic Reticulum

The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.

Positive contribution of ERdj5/JPDI to endoplasmic reticulum protein quality control in the salivary gland

In eukaryotic cells, most membrane and secretory proteins are modified post-translationally in the ER (endoplasmic reticulum) for correct folding and assembly. Disulfide-bond formation is one of the important modifications affecting folding and is catalysed by the PDI (protein disulfide isomerase) family proteins. ERdj5 [also known as JPDI (J-domain-containing PDI-like protein)] is a member of the PDI family proteins and has been reported to act as a reductase in ERAD (ER-associated degradation). However, the role of ERdj5 at the whole-body level remains unclear. Therefore in the present study we generated ERdj5-knockout mice {the mouse gene of ERdj5 is known as Dnajc10 [DnaJ (Hsp40) homologue, subfamily C, member 10]} and analysed them. Although ERdj5-knockout mice were viable and healthy, the ER stress response was activated in the salivary gland of the knockout mice more than that of control mice. Furthermore, in ERdj5-knockout cells, the expression of exogenous ERdj5 mitigated the ER stress caused by overproduction of alpha-amylase, which is one of the most abundant proteins in saliva and has five intramolecular disulfide bonds. This effect was dependent on the thioredoxin-like motifs of ERdj5. Thus we suggest that ERdj5 contributes to ER protein quality control in the salivary gland.

Molecular cloning and characterization of soybean protein disulfide isomerase family proteins with nonclassic active center motifs

Protein disulfide isomerase (PDI) and other PDI family proteins are members of the thioredoxin superfamily and are thought to play important roles in disulfide bond formation and isomerization in the endoplasmic reticulum (ER). The exact functions of PDI family proteins in plants remain unknown. In this study, we cloned two novel PDI family genes from soybean leaf (Glycine max L. Merrill cv. Jack). The cDNAs encode proteins of 520 and 523 amino acids, and have been denoted GmPDIL-3a and GmPDIL-3b, respectively. GmPDIL-3a and GmPDIL-3b are the first plant ER PDI family proteins reported to contain the nonclassic redox center motif CXXS/C, and both proteins are ubiquitously expressed in the plant body. However, recombinant GmPDIL-3a and GmPDIL-3b did not function as oxidoreductases or as molecular chaperones in vitro, although a proportion of each protein formed complexes in both thiol-dependent and thiol-independent ways in the ER. Expression of GmPDIL-3a and GmPDIL-3b in the cotyledon increased during seed maturation when synthesis of storage proteins was initiated. These results suggest that GmPDIL-3a and GmPDIL-3b may play important roles in the maturation of the cotyledon by mechanisms distinct from those of other PDI family proteins.

Protein disulfide isomerase–like proteins play opposing roles during retrotranslocation

Misfolded proteins in the endoplasmic reticulum (ER) are retained in the organelle or retrotranslocated to the cytosol for proteasomal degradation. ER chaperones that guide these opposing processes are largely unknown. We developed a semipermeabilized cell system to study the retrotranslocation of cholera toxin (CT), a toxic agent that crosses the ER membrane to reach the cytosol during intoxication. We found that protein disulfide isomerase (PDI) facilitates CT retrotranslocation, whereas ERp72, a PDI-like protein, mediates its ER retention. In vitro analysis revealed that PDI and ERp72 alter CT's conformation in a manner consistent with their roles in retrotranslocation and ER retention. Moreover, we found that PDI's and ERp72's opposing functions operate on endogenous ER misfolded proteins. Thus, our data identify PDI family proteins that play opposing roles in ER quality control and establish an assay to further delineate the mechanism of CT retrotranslocation.

ERp57 binds competitively to protein disulfide isomerase and calreticulin

In this study, we screened for protein disulfide isomerase (PDI)-binding proteins in bovine liver microsomes under strict salt concentrations, using affinity column chromatography. One main band observed using SDS–PAGE was identified as ERp57 (one of the PDI family proteins) by LC-MS/MS analysis. The K D value of PDI binding to ERp57 was calculated as 5.46 × 10 −6 M with the BIACORE system. The interactions between PDI and ERp57 occurred specifically at their a and b domains, respectively. Interestingly, low concentrations of ERp57 enhanced the chaperone activity of PDI, while high concentrations interfered with chaperone activity. On the other hand, ERp57 did not affect the isomerase activity of PDI. Additionally, following pre-incubation of ERp57 with calreticulin (CRT), decreased interactions were observed between ERp57 and PDI, and vice versa. Based on the data, we propose that once ERp57 binds to PDI or CRT, the resultant complex inhibits further interactions. Therefore, ERp57 selectively forms a protein-folding complex with PDI or CRT in ER.

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.