Protein Disulfide Isomerase P5 Research Articles

A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization

P5, also known as PDIA6, is a PDI family member involved in the ER quality control. Here, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. Unlike conventional leucine zipper motifs with leucine residues every two helical turns on ∼30-residue parallel α helices, this adhesive motif includes periodic repeats of leucine/valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α helices. The P5 dimerization interface is further stabilized by several reciprocal salt bridges and C-capping interactions between protomers. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as aberrant proteins that induce the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via intermolecular disulfide bond reduction and its Ca2+-dependent regulation of chaperone function invitro. Thus, the leucine-valine adhesive motif supports structure and function of P5.

Functional analysis of protein disulfide isomeraseP5 in glioblastoma cells as a novel anticancer target.

P5, which is a member of the protein disulfide isomerase family, possesses isomerase and chaperone activity invitro; however, the physiological functions of this enzyme in cells remain unclear. To understand the important roles of P5 in cancer cells, the present study examined its expression on the surface of normal and cancer cell lines by flow cytometry using an affinity‑purified anti‑P5 antibody labeled with 6‑(fluorescein‑5‑carboxamido) hexanoic acid succinimidyl ester. P5 expression was increased on the surface of various cancer cell lines, including leukemia cells, and glioblastoma, breast, colon, ovarian and uterine cervical cancer cells, compared with normal cells. However, P5 was constantly expressed within both normal and cancer cell lysates, and its total expression levels were not significantly different between the cells. P5 knockdown in glioblastoma cells by small interfering RNA affected Bip promoter activation during cancer cell growth, and significantly inhibited cancer cell growth and migration. Immunoprecipitation using an anti‑P5 antibody in cancer and normal cells demonstrated that vimentin was bound to P5, predominantly in U251 glioblastoma cells. P5 knockdown in glioblastoma cells did not affect the protein expression levels of vimentin; however, it did affect the expression of numerous epithelial‑mesenchymal transition markers, including Snail and Slug. These results suggested that P5 may serve an important role in cancer cell growth, and may be considered an attractive and potent target for the treatment of glioblastoma.

Decreased levels of PDI and P5 in oligodendrocytes in Alzheimer's disease.

Protein disulfide isomerase (PDI) is a chaperone protein located in the endoplasmic reticulum (ER). Nitric oxide-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to protein accumulation and activation of the unfolded protein response. Protein disulfide isomerase P5 (P5) is a member of the PDI family that mostly localizes to the ER lumen. Both S-nitrosylated PDI and S-nitrosylated P5 are found in Alzheimer's disease (AD) brain. Previously, we showed that expression of the ER stress marker, growth arrest, and DNA damage protein (GADD34) was significantly increased in neurons and oligodendrocytes in AD brain. In the present study, we showed that PDI and P5 levels were significantly decreased in oligodendrocytes in the brains of AD patients and an AD mouse model. Interestingly, these decreases were evident before the animals displayed typical AD pathology. Because we previously showed that small short interfering RNA knockdown of PDI or P5 could affect the viability of neuronal cells under ER stress, dysfunction of PDI and P5 under ER stress could cause apoptosis of neuronal cells. In summary, we showed that the levels of PDI and P5 were significantly decreased in the oligodendrocytes of AD patients. This phenomenon was also found in an AD mouse model before the animals displayed AD pathology. The overall findings suggest that oligodendrocytes may play important roles in AD pathogenesis.

Conserved C272/278 in b domain regulate the function of PDI-P5 to make lysozymes trypsin-resistant forms via significant intermolecular disulfide cross-linking

Protein disulfide isomerase-P5 (P5) is thought to have important functions as an oxidoreductase, however, molecular functions of P5 have not been fully elucidated. We have reported that P5 has insulin reductase activity and inhibits lysozyme refolding by formation of lysozyme multimers with hypermolecular mass inactivated by intermolecular disulfides (hyLYS) in oxidative refolding of reduced denatured lysozyme. To explore the role of each domain of P5, we investigated the effects of domain deletion and Cys-Ala mutants of P5 on insulin reduction and the oxidative refolding of the lysozyme. The mutants of catalytic cysteines, C36/39A, C171/174A, and C36/39/171/174A inhibited the lysozyme refolding almost similarly to P5, and even b domain without catalytic cysteines showed moderate inhibitory effect, suggesting that the b domain played a key role in the inhibition. Western blotting analysis of the refolding products indicated that the catalytic cysteines in both the a and a′ domains cross-linked lysozyme comparably to form hyLYS resistant to trypsin, in which b domain was suggested to capture lysozyme for the significant sulfhydryl oxidation. The mutant of the conserved cysteines in b domain, C272/278A, did not form hyLYS, however, showed predominant reductase activity, implying that P5 functioned as a potent sulfhydryl oxidase and a predominant reductase depending on the circumstance around C272/278. These results provide new insight into the molecular basis of P5 function.

Discovery of protein disulfide isomerase P5 inhibitors that reduce the secretion of MICA from cancer cells.

In order to regulate the activity of P5, which is a member of the protein disulfide isomerase family, we screened a chemical compound library for P5-specific inhibitors, and identified two candidate compounds (anacardic acid and NSC74859). Interestingly, anacardic acid inhibited the reductase activity of P5, but did not inhibit the activity of protein disulfide isomerase (PDI), thiol-disulfide oxidoreductase ERp57, or thioredoxin. NSC74859 inhibited all these enzymes. When we examined the effects of these compounds on the secretion of soluble major histocompatibility complex class-I-related gene A (MICA) from cancer cells, anacardic acid was found to decrease secretion. In addition, anacardic acid was found to reduce the concentration of glutathione up-regulated by the anticancer drug 17-demethoxygeldanamycin in cancer cells. These results suggest that anacardic acid can both inhibit P5 reductase activity and decrease the secretion of soluble MICA from cancer cells. It might be a novel and potent anticancer treatment by targeting P5 on the surface of cancer cells.

Protein Disulfide Isomerase P5-Immunopositive Inclusions in Patients with Alzheimer's Disease

Amyloid plaques and neurofibrillary tangles (NFTs) are the major pathological characteristics of Alzheimer's disease (AD). NFTs are composed of tubular filaments and paired helical filaments containing polymerized hyperphosphorylated tau protein. Another feature of AD is excessive generation of nitric oxide (NO). Protein disulfide isomerase (PDI) is a chaperon protein located in the endoplasmic reticulum (ER). It was recently reported that NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. In addition, we previously reported the presence of PDI-immunopositive NFTs in AD. Here, we found that protein disulfide isomerase P5 (P5), which is a member of the PDI protein family, was co-localized with tau in NFTs. To our knowledge, this is the first report of P5-immunopositive inclusion in AD. Furthermore, we showed that S-nitrosylated P5 was present and the expression level of P5 was decreased in AD brains compared with that of control brains. We also demonstrated that the knock-down of PDI or P5 by siRNA could affect the viability of SH-SY5Y cells under ER stress. Previously, the observation of S-nitrosylated PDI in AD was reported. NO may inhibit P5 by inducing S-nitrosylation in the same manner as PDI, which inhibits its enzymatic activity allowing protein misfolding to occur in AD. The accumulation of misfolded proteins induces ER stress and may cause apoptosis of neuronal cells through S-nitrosylation and down-regulation of PDI and P5 in AD.

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.

XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). We have investigated here the contribution of the UPR transcription factors XBP-1, ATF6alpha, and ATF6beta to UPR target gene expression. Gene profiling of cell lines lacking these factors yielded several XBP-1-dependent UPR target genes, all of which appear to act in the ER. These included the DnaJ/Hsp40-like genes, p58(IPK), ERdj4, and HEDJ, as well as EDEM, protein disulfide isomerase-P5, and ribosome-associated membrane protein 4 (RAMP4), whereas expression of BiP was only modestly dependent on XBP-1. Surprisingly, given previous reports that enforced expression of ATF6alpha induced a subset of UPR target genes, cells deficient in ATF6alpha, ATF6beta, or both had minimal defects in upregulating UPR target genes by gene profiling analysis, suggesting the presence of compensatory mechanism(s) for ATF6 in the UPR. Since cells lacking both XBP-1 and ATF6alpha had significantly impaired induction of select UPR target genes and ERSE reporter activation, XBP-1 and ATF6alpha may serve partially redundant functions. No UPR target genes that required ATF6beta were identified, nor, in contrast to XBP-1 and ATF6alpha, did the activity of the UPRE or ERSE promoters require ATF6beta, suggesting a minor role for it during the UPR. Collectively, these results suggest that the IRE1/XBP-1 pathway is required for efficient protein folding, maturation, and degradation in the ER and imply the existence of subsets of UPR target genes as defined by their dependence on XBP-1. Further, our observations suggest the existence of additional, as-yet-unknown, key regulators of the UPR.

A protein disulfide isomerase expressed in the embryonic midline is required for left/right asymmetries.

Although the vertebrate embryonic midline plays a critical role in determining the left/right asymmetric development of multiple organs, few genes expressed in the midline are known to function specifically in establishing laterality patterning. Here we show that a gene encoding protein disulfide isomerase P5 (PDI-P5) is expressed at high levels in the organizer and axial mesoderm and is required for establishing left/right asymmetries in the zebrafish embryo. pdi-p5 was discovered in a screen to detect genes down-regulated in the zebrafish midline mutant one-eyed pinhead and expressed predominantly in midline tissues of wild-type embryos. Depletion of the pdi-p5 product with morpholino antisense oligonucleotides results in loss of the asymmetric development of the heart, liver, pancreas, and gut. In addition, PDI-P5 depletion results in bilateral expression of all genes known to be expressed asymmetrically in the lateral plate mesoderm and the brain during embryogenesis. The laterality defects caused by pdi-p5 antisense treatment arise solely due to loss of the PDI-P5 protein, as they are reversed when treated embryos are supplied with an exogenous source of the PDI-P5 protein. Thus the spectrum of laterality defects resulting from depletion of the PDI-P5 protein fully recapitulates that resulting from loss of the midline. As loss of PDI-P5 does not appear to interfere with other aspects of midline development or function, we propose that PDI-P5 is specifically involved in the production of midline-derived signals required to establish left/right asymmetry.

Matrix assisted laser desorption/ionization-time of flight-mass spectrometry analysis of proteins detected by anti-phosphotyrosine antibody on two-dimensional-gels of fibrolast cell lysates after tumor necrosis factor-α stimulation

We describe efficient methods for using functional proteomics analysis to study signal transduction pathways in murine fibroblast L929 cells following stimulation with tumor necrosis factor (TNF)-α. After stimulation with TNF-α, cellular proteins of L929 cells were extracted with a lysis buffer containing 0.3% sodium dodecyl sulfate (SDS) for 10—30 min time intervals, and were separated by two-dimensional (2-D) electrophoresis followed by immunoblot analysis with anti-phosphotyrosine antibody and alkaline phosphatase-anti IgG antibody conjugate. To improve detection sensitivity by immunoblot analysis we used a chemifluorescent substrate for alkaline phosphatase. One hundred protein spots were detected in the TNF-α stimulated L929 cell extract by immunoblot analysis. The use of chemifluorescence allowed us to quantitate immunoblotted spots with fluoroscanner so that we were able to detect time-dependent changes of a number of immunoblotted spots. Protein spots on a silver-stained 2-D gel corresponding to those detected by immunoblot analysis were subjected to in-gel trypsin digestion- matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry analysis, respectively. Twenty-one proteins detected by immunoblot analysis were identified by MS-Fit database search analysis. Among them, the proteins that show time-dependent changes in staining intensity include vimentin, tubulin beta-chain, eukaryotic translation initiation factor 1A, chromatin assembly factor 1 (P48 subunit), probable protein disulfide isomerase P5, and several other proteins. Vimentin and tubulin beta-chain have been reported to be phosphorylated at tyrosine residues and involved in the signal transduction pathway induced by TNF-α. However, the other proteins have no previously known function in the signal transduction pathway. Thus, the methods used in this study seem to be suitable for the identification of time-dependent changes in many proteins that are involved in signal transduction. Usefulness of the method for comprehensive analysis of the proteins involved in signal transduction pathway and the limitations of the method are discussed.

Matrix assisted laser desorption/ionization-time of flight-mass spectrometry analysis of proteins detected by anti-phosphotyrosine antibody on two-dimensional-gels of fibrolast cell lysates after tumor necrosis factor-alpha stimulation.

We describe efficient methods for using functional proteomics analysis to study signal transduction pathways in murine fibroblast L929 cells following stimulation with tumor necrosis factor (TNF)-alpha. After stimulation with TNF-alpha, cellular proteins of L929 cells were extracted with a lysis buffer containing 0.3% sodium dodecyl sulfate (SDS) for 10-30 min time intervals, and were separated by two-dimensional (2-D) electrophoresis followed by immunoblot analysis with anti-phosphotyrosine antibody and alkaline phosphatase-anti IgG antibody conjugate. To improve detection sensitivity by immunoblot analysis we used a chemifluorescent substrate for alkaline phosphatase. One hundred protein spots were detected in the TNF-alpha stimulated L929 cell extract by immunoblot analysis. The use of chemifluorescence allowed us to quantitate immunoblotted spots with fluoroscanner so that we were able to detect time-dependent changes of a number of immunoblotted spots. Protein spots on a silver-stained 2-D gel corresponding to those detected by immunoblot analysis were subjected to in-gel trypsin digestion- matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry analysis, respectively. Twenty-one proteins detected by immunoblot analysis were identified by MS-Fit database search analysis. Among them, the proteins that show time-dependent changes in staining intensity include vimentin, tubulin beta-chain, eukaryotic translation initiation factor 1A, chromatin assembly factor 1 (P48 subunit), probable protein disulfide isomerase P5, and several other proteins. Vimentin and tubulin beta-chain have been reported to be phosphorylated at tyrosine residues and involved in the signal transduction pathway induced by TNF-alpha. However, the other proteins have no previously known function in the signal transduction pathway. Thus, the methods used in this study seem to be suitable for the identification of time-dependent changes in many proteins that are involved in signal transduction. Usefulness of the method for comprehensive analysis of the proteins involved in signal transduction pathway and the limitations of the method are discussed.