Protein Disulfide Isomerase A3 Research Articles

SUMOylation of Pdia3 exacerbates proinsulin misfolding and ER stress in pancreatic beta cells.

SUMOylation has long been recognized to regulate multiple biological processes in pancreatic beta cells, but its impact on proinsulin disulfide maturation and endoplasmic reticulum (ER) stress remains elusive. Herein, we conducted comparative proteomic analyses of SUMOylated proteins in primary mouse/human islets following proinflammatory cytokine stimulation. Cytokine challenge rendered beta cells to undergo a SUMOylation turnover manifested by the changes of SUMOylation substrates and SUMOylation levels for multiple substrates. Our data support that SUMOylation may play a crucial role to regulate proinsulin misfolding and ER stress at least by targeting Protein Disulfide Isomerase a3 (Pdia3). SUMOylation regulates Pdia3 enzymatic activity, subcellular localization, and protein binding ability. Furthermore, SUMOylation of Pdia3 exacerbated proinsulin misfolding and ER stress, and repressed Stat3 activation. In contrast, disruption of Pdia3 SUMOylation markedly rescued the outcomes. Collectively, our study expands the understanding how SUMOylation regulates ER stress in beta cells, which shed light on developing potential strategies against beta cell dysfunction.

PDIA3 Expression in Glioblastoma Modulates Macrophage/Microglia Pro-Tumor Activation.

The glioblastoma (GB) microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs) that are still poorly characterized. A potential role on the mechanisms regulating GAM activity might be played by the endoplasmic reticulum protein ERp57/PDIA3 (protein disulfide-isomerase A3), the modulation of which has been reported in a variety of cancers. Moreover, by using The Cancer Genome Atlas database, we found that overexpression of PDIA3 correlated with about 55% reduction of overall survival of glioma patients. Therefore, we analyzed the expression of ERp57/PDIA3 using specimens obtained after surgery from 18 GB patients. Immunohistochemical analysis of tumor samples revealed ERp57/PDIA3 expression in GB cells as well as in GAMs. The ERp57/PDIA3 levels were higher in GAMs than in the microglia present in the surrounding parenchyma. Therefore, we studied the role of PDIA3 modulation in microglia–glioma interaction, based on the ability of conditioned media collected from human GB cells to induce the activation of microglial cells. The results indicated that reduced PDIA3 expression/activity in GB cells significantly limited the microglia pro-tumor polarization towards the M2 phenotype and the production of pro-inflammatory factors. Our data support a role of PDIA3 expression in GB-mediated protumor activation of microglia.

A Gallium(III) Complex that Engages Protein Disulfide Isomerase A3 (PDIA3) as an Anticancer Target.

Gallium(III)-based drugs have gained momentum in cancer therapy due to their iron-dependent anticancer activity. Judicious choice of ligands is critical for improved oral bioavailability, antitumor efficacy, and distinct mechanisms from simple GaIII salts. We describe GaIII complexes with planar tetradentate salen ligands [salen=2,3-bis[(4-dialkylamino-2-hydroxybenzylidene)amino]but-2-enedinitrile)] and labile axial solvent ligands, which display tumor growth inhibition in vitro and in vivo comparable to cisplatin. Confocal fluorescence microscopy, western blotting, mRNA profiling, chemical proteomics, and surface plasmon resonance (SPR) studies provide compelling evidence that PDIA3, a member of the protein disulfide isomerase (PDI) family involved in endoplasmic reticulum (ER) stress, is a direct target of Ga-1. This work offers a new route to designing and synthesizing Ga-based drugs, and also reveals that PDIA3 is an important anticancer target.

A Gallium(III) Complex that Engages Protein Disulfide Isomerase A3 (PDIA3) as an Anticancer Target

AbstractGallium(III)‐based drugs have gained momentum in cancer therapy due to their iron‐dependent anticancer activity. Judicious choice of ligands is critical for improved oral bioavailability, antitumor efficacy, and distinct mechanisms from simple GaIII salts. We describe GaIII complexes with planar tetradentate salen ligands [salen=2,3‐bis[(4‐dialkylamino‐2‐hydroxybenzylidene)amino]but‐2‐enedinitrile)] and labile axial solvent ligands, which display tumor growth inhibition in vitro and in vivo comparable to cisplatin. Confocal fluorescence microscopy, western blotting, mRNA profiling, chemical proteomics, and surface plasmon resonance (SPR) studies provide compelling evidence that PDIA3, a member of the protein disulfide isomerase (PDI) family involved in endoplasmic reticulum (ER) stress, is a direct target of Ga‐1. This work offers a new route to designing and synthesizing Ga‐based drugs, and also reveals that PDIA3 is an important anticancer target.

Comparative Platelet Releasate Proteomic Profiling of Acute Coronary Syndrome versus Stable Coronary Artery Disease.

Upon activation, platelets release a host of soluble and vesicular signals, collectively termed the “platelet releasate” (PR). The contents of this PR play a significant role in haemostasis, inflammation, and pathologic sequelae. Despite this, proteomic studies investigating the PR in coronary artery disease have not been performed. Here, we undertook a comparative label-free quantitative (LFQ) proteomic profiling of the 1 U/ml thrombin-induced PR from 13 acute coronary syndrome vs. 14 stable angina pectoris patients using a tandem mass spectrometry approach. Data are available via ProteomeXchange with identifier PXD009356. 318 PR proteins were identified across both cohorts with 9 proteins found to be differentially released, including tetranectin (CLEC3B), protein disulfide-isomerase-A3 (PDIA3), coagulation factor V (F5), and fibronectin (FN1). Strikingly, these 9 differential proteins were all associated with the gene ontology cellular component term “extracellular vesicle” and reduced levels of EVs were detected in the corresponding plasma of ST-segment elevation myocardial infarction (STEMI) patients. Network analysis revealed 3 proteins either reduced (F5; FN1) or absent (CLEC3B) in the PR of STEMI patients that are strongly connected to both the clotting cascade and major druggable targets on platelets. This moderated proteomic signature may prove useful for non-invasive risk assessment of the progression of coronary artery disease. These data further contribute to the growing evidence-base of using the platelet releasate as a predictor of pathological state and disease severity.

Proteomics analysis of plasma protein changes in patent ductus arteriosus patients

ObjectivePatent ductus arteriosus (PDA) is a congenital heart defect with an unclear etiology that occurs commonly among newborns. Adequately understanding the molecular pathogenesis of PDA can contribute to improved treatment and prevention. Plasma proteins may provide evidence to explore the molecular mechanisms of abnormal cardiac development.MethodsIsobaric tags for relative and absolute quantitation (iTRAQ) proteomics technology was used to measure different plasma proteins in PDA patients (n = 4) and controls (n = 4). The candidate protein was validated by ELISA and Western blot (WB) assays in a larger sample. Validation of the location and expression of this protein was performed in mouse heart sections.ResultsThere were three downregulated proteins and eight upregulated proteins identified in the iTRAQ proteomics data. Among these, protein disulfide-isomerase A6 (PDIA6) was further analyzed for validation. The plasma PDIA6 concentrations (3.2 ± 0.7 ng/ml) in PDA patients were significantly lower than those in normal controls (5.8 ± 1.2 ng/ml). In addition, a WB assay also supported these results. PDIA6 was widely expressed in mouse heart outflow tract on embryonic day 14.5.ConclusionPlasma proteomics profiles suggested novel candidate molecular markers for PDA. The findings may allow development of a new strategy to investigate the mechanism and etiology of PDA.

A proteomic approach towards understanding crypoprotective action of Me2SO on the CHO cell proteome

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.

Essential cell-extrinsic requirement for PDIA6 in lymphoid and myeloid development.

In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell-independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.

The role of protein disulphide-isomerase A3 as autoantigen in the pathogenesis of autoimmune thyroiditis and related brain damage in adult mice

Autoimmune thyroiditis (AIT)-related brain damage is one of most severe extrathyroidal manifestations of AIT, but the mechanism remains unclear. In this study, we confirmed that protein disulfide-isomerase A3 (PDIA3) is expressed in both thyroid and brain tissues of mouse, and found the significantly increased serum levels of anti-PDIA3 antibody (PDIA3Ab) in classical mouse models of thyroiditis. In addition, we investigated the PDIA3-specific autoimmune reaction in thyroid and brain tissues in a mouse model with high–serum PDIA3Ab induced by immunization with recombinant PDIA3 protein. PDIA3-immunized mice had elevated serum thyrotropin and impaired learning and memory. PDIA3-expressing cells had IgG deposition, and IgG colocalized with C3 in the thyroid and brain tissues of PDIA3-immunized mice, resulting in membrane attack complex formation. Our results suggest that PDIA3 protein may be a common autoantigen shared by the thyroid and brain tissues and involve in the thyroidal and intracerebral damage through activating complement system.

Differential roles of exogenous protein disulfide isomerase A3 on proliferating cell and neuroblast numbers in the normal and ischemic gerbils.

IntroductionWe examined the effects of exogenous protein disulfide isomerase A3 (PDIA3) on hippocampal neurogenesis in gerbils under control and ischemic damage.MethodsTo facilitate the delivery of PDIA3 to the brain, we constructed Tat‐PDIA3 protein and administered vehicle (10% glycerol) or Tat‐PDIA3 protein once a day for 28 days. On day 24 of vehicle or Tat‐PDIA3 treatment, ischemia was transiently induced by occlusion of both common carotid arteries for 5 min.ResultsAdministration of Tat‐PDIA3 significantly reduced ischemia‐induced spontaneous motor activity, and the number of NeuN‐positive nuclei in the Tat‐PDIA3‐treated ischemic group was significantly increased in the CA1 region compared to that in the vehicle‐treated ischemic group. Ki67‐ and DCX‐immunoreactive cells were significantly higher in the Tat‐PDIA3‐treated group compared to the vehicle‐treated control group. In vehicle‐ and Tat‐PDIA3‐treated ischemic groups, the number of Ki67‐ and DCX‐immunoreactive cells was significantly higher as compared to those in the vehicle‐ and Tat‐PDIA3‐treated control groups, respectively. In the dentate gyrus, the numbers of Ki67‐immunoreactive cells were comparable between vehicle‐ and Tat‐PDIA3‐treated ischemic groups, while more DCX‐immunoreactive cells were observed in the Tat‐PDIA3‐treated group. Transient forebrain ischemia increased the expression of phosphorylated cAMP‐response element‐binding protein (pCREB) in the dentate gyrus, but the administration of Tat‐PDIA3 robustly increased pCREB‐positive nuclei in the normal gerbils, but not in the ischemic gerbils. Brain‐derived neurotrophic factor (BDNF) mRNA expression was significantly increased in the Tat‐PDIA3‐treated group compared to that in the vehicle‐treated group. Transient forebrain ischemic increased BDNF mRNA levels in both vehicle‐ and Tat‐PDIA3‐treated groups, and there were no significant differences between groups.ConclusionsThese results suggest that Tat‐PDIA3 enhances cell proliferation and neuroblast numbers in the dentate gyrus in normal, but not in ischemic gerbils, by increasing BDNF mRNA and phosphorylation of pCREB.

P046 Vitamin D decreases PDIA3 and prevents the enhanced migration of fibroblasts from stricturing Crohn’s disease

Abstract Background Fibrosis is a common complication in Crohn’s disease (CD) patients and fibroblasts play an important role in the fibrogenic process. Low vitamin D (VD) levels and a defective VD-signalling pathway have been reported in CD. VD signals through both vitamin D receptor (VDR) and protein disulfide-isomerase A3 (PDIA3) and we have previously demonstrated that VDR protein levels are reduced in fibroblasts isolated from CD patients and that VD increased VDR expression in these cells (A-2080; ECCO 2019). We aim to analyse here the effect of VD on both PDIA3 protein levels and migration in CD fibroblasts. Methods We used intestinal fibroblasts isolated from surgical resections of the damaged mucosa of CD patients with stricturing behaviour (B2). Control fibroblasts were obtained from the non-damaged intestine of patients with colorectal cancer. Fibroblasts were treated with VD (100 nM) or its vehicle for 24 h and PDIA3 protein levels were measured by Western Blot. In the wound healing analysis, a single scraping was done in the centre of the fibroblasts monolayer and FBS-free medium was added to the cells, which allows us to determine the ability of fibroblasts to migrate and close the wound. Photos were taken at 0, 24 and 48 h. Results of wound healing were expressed as the percentage of the wound at each time point for the maximal wounded area (time 0, 100%). Statistical significance was measured by ANOVA or t-test. Results No significant differences in PDIA3 protein levels were detected between control and CD fibroblasts but VD significantly decreased PDIA3 expression in CD fibroblasts (Figure 1A). In the wound healing assay, we detected that CD-B2 fibroblasts migrate faster than control cells, resulting in a reduced wounding area, 48 h later (Figure 1B). Treatment of these CD-B2 cells with VD decreased their migration rate, and 48 h later cells exhibited a higher and significant wounding area than vehicle-treated cells (Figure 1C). Conclusion Vitamin D decreased PDIA3 expression and prevented the accelerated migration detected in intestinal fibroblasts from B2-CD patients which suggest an anti-fibrotic effect of VD mediated by a direct effect of this hormone on intestinal fibroblasts.

The Hydroxyproline Proteome of HeLa Cells with Emphasis on the Active Sites of Protein Disulfide Isomerases.

Hydroxyproline-containing proteins (other than collagens) are rare and difficult to identify. Only 14 such proteins have been found in the human proteome by biochemical methods despite the fact that the list includes examples of biological importance such as hypoxia-inducible factor and the 40S ribosomal protein S23 (RPS23), both of which have significant biological function of the post-translational modification. Comparison of multinotch search software Global-PTM-Discovery to conventional proteomic database search software gave a nine-fold improvement in correctly identifying non-collagen peptides containing hydroxyproline. Manual interpretation of MS-MS spectra refined this list to discover 36 unique peptides representing 24 unique hydroxyproline sites in 21 proteins, of which only Pro62 of RPS23 had been reported previously in UniProt. Eight of the sites were found to be conserved as prolines in nine species examined, ranging from humans to yeast. These include sites 51 and 395 in protein disulfide-isomerase (PDIA1) and sites 204 and 553 in protein disulfide-isomerase A4 (PDIA4). The apparent occupancy of these sites ranged from 72-89%, suggesting a structural and possibly functional role of these PTMs. Fifteen of the sites most likely contain 4R-hydroxyproline (Pro30 of serpin H1, Pro520 of aspartyl/asparaginyl β-hydroxylase, Pro223 of neutral α-glucosidase AB, Pro977 of hypoxia upregulated protein 1, Pro378 of protein ERGIC-53, Pro252 of protein CASC4, Pro545 of bromodomain-containing protein 2, Pro488 of bromodomain-containing protein 3, Pro130 of nucleolar RNA helicase 2, Pro51 of PDIA1, Pro395 of PDIA1, Pro404 of PDIA3, Pro89 of PDIA4, Pro204 of PDIA4, and Pro553 of PDIA4). The remaining sites could be either 4R-hydroxyproline or 3S-hydroxyproline. Recommendations are made to improve automated interpretation of proteomic data to improve future proteomic research whose goal is to mine more of the remaining dark matter of the proteome.

Protein disulfide isomerase A3: A potential regulatory factor of colon epithelial cells

Aim: This study aimed to investigate the effects of protein disulfide isomerase A3 (PDIA3) on the proliferation and apoptosis of colon epithelial cells, so as to explore its possible role in colon compensation of ultra-short bowel syndrome. Methods: The expression of PDIA3 gene in NCM460 colonic epithelial cells was upregulated and silenced by liposome transient transfection technique. The expression of PDIA3 protein was determined by Western blotting, the proliferation rate of NCM460 cells was detected by CCK8, and the apoptosis rate of NCM460 cells was determined by flow cytometry. Results: DNA sequencing and Western blotting results successfully verified that PDIA3 protein expression in NCM460 cells was upregulated and silenced by liposomal transfection. In the PDIA3 overexpression experiment, the proliferation rate of the experimental group was lower than that of the empty carrier group at 24 h, 48 h, and 72 h, and the apoptosis rate of the experimental group was higher than that of the empty carrier group (P Conclusion: PDIA3 inhibits the proliferation of human colonic epithelial cells (NCM460) and promotes their apoptosis, which may not be a key regulatory protein in colon compensation of ultra-short bowel syndrome.

Heat shock protein 90 is downregulated in calcific aortic valve disease

BackgroundCalcific aortic valve disease (CAVD) is an atheroinflammatory process; finally it leads to progressive calcification of the valve. There is no effective pharmacological treatment for CAVD and many of the underlying molecular mechanisms remain unknown. We conducted a proteomic study to reveal novel factors associated with CAVD.MethodsWe compared aortic valves from patients undergoing valvular replacement surgery due to non-calcified aortic insufficiency (control group, n = 5) to a stenotic group (n = 7) using two-dimensional difference gel electrophoresis (2D-DIGE). Protein spots were identified with mass spectrometry. Western blot and immunohistochemistry were used to validate the results in a separate patient cohort and Ingenuity Pathway Analysis (IPA) was exploited to predict the regulatory network of CAVD.ResultsWe detected an upregulation of complement 9 (C9), serum amyloid P-component (APCS) and transgelin as well as downregulation of heat shock protein (HSP90), protein disulfide isomerase A3 (PDIA3), annexin A2 (ANXA2) and galectin-1 in patients with aortic valve stenosis. The decreased protein expression of HSP90 was confirmed with Western blot.ConclusionsWe describe here a novel data set of proteomic changes associated with CAVD, including downregulation of the pro-inflammatory cytosolic protein, HSP90.

Increased circulating protein disulfide isomerase A4 (PDIA4) levels in metabolically unhealthy obese (MUHO) men

Increased circulating protein disulfide isomerase A4 (PDIA4) levels in metabolically unhealthy obese (MUHO) men

MiRNA deregulation can contribute to breakdown of MHC I class antigen processing and presenting machinery in breast cancer cells

Background: To attack tumors, cytotoxic CD8+ T cells need to recognize tumor antigen peptides loaded onto major histocompatibility complex (MHC) class I molecules. Antigen processing and presentation is multistep process that includes proteasome-dependent and independent cleavage of cytosolic proteins, transport of derived peptides into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAPs), peptide trimming by ER aminopeptidases (ERAPs), peptide loading onto MHC molecules with the help of calnexin, calreticulin, protein disulfide-isomerase A3 and tapasin, movement of vesicles with MHC-peptide complex via the Golgi apparatus to the cell surface. Being one of the main immune escape pathways, defects in this machinery are frequently observed in various tumors and correlate with tumor grade and aggressiveness. This research aims to identify how the tumor-related abnormalities in microRNA (miRNA) expression pattern can disrupt antigen processing and presentation in breast cancer cells.

RNA-seq reveals temporal differences in the transcriptome response to acute heat stress in the Atlantic salmon (Salmo salar)

Acute heat stress is common in aquaculture and can affect diverse physiological processes in fish; however, different species of fish have various mechanisms for heat stress adaptation. In this study, we profiled the transcriptome responses of the Atlantic salmon (Salmo salar) to heat stress at 23 °C for 6 or 24 h, compared with that of fish at a normal temperature of 13 °C. The liver was selected as the target tissue for this analysis. A total of 243 and 88 genes were differentially expressed after 6 and 24 h of heat stress, respectively. Of these, only 22 were common to both time points, and most of these common genes were molecular chaperones such as heat shock cognate 71 kDa protein and heat shock protein 90-alpha. Genes such as activating transcription factor 6, calreticulin, protein disulfide isomerase A3, and protein kinase R-like endoplasmic reticulum kinase-eukaryotic initiation factor 2-alpha were only up-regulated after 6 h of heat stress; most of these genes are involved in the endoplasmic reticulum stress pathway. Indeed, endoplasmic reticulum stress was identified at 6 h but not at 24 h, suggesting that stress response plays an important role in the adaptation of Atlantic salmon to acute heat stress. Other up-regulated genes at 6 h were related to the insulin and nucleotide oligomerization domain-like receptor signaling pathways, which directly eliminate misfolded proteins and sustain sugar and lipid homeostasis. At 24 h, heat stress influenced the expression of steroid and terpenoid backbone biosynthesis, which may influence the sexual development and differentiation of Atlantic salmon. Overall, our results elucidate the transcriptome mechanisms that contribute to short-term heat tolerance in the liver of Atlantic salmon.

Expression of protein disulfide isomeraseA3 and its clinicopathological association in gastric cancer.

Protein disulfide isomerase A3 (PDIA3) is a chaperone protein that supports the folding and processing of synthesized proteins. Its expression is associated with the prognosis of laryngeal cancer, hepatocellular carcinoma, diffuse glioma and uterine cervical cancer. In the present study, the expression levels of PDIA3 and its clinicopathological association were examined in 52 cases of gastric cancer (GC). The expression of PDIA3 was examined by immunohistochemistry and scored using a semi-quantitative method. According to the score, GC samples were classified into PDIA3‑High and PDIA3‑Low GC. PDIA3‑High GC samples were predominantly of the intestinal type. Multivariate survival analysis indicated that PDIA3 expression and cancer stage were independent factors. The overall survival of PDIA3‑High GC cases was significantly favorable compared with that of PDIA3‑Low GC cases, and this was more evident in cases at an advanced stage. In GC cell cultures, the PDIA3 and major histocompatibility complex (MHC) class I proteins were expressed in three out of the four assessed cell lines according to western blot analysis. Notably, the expression of MHC class I was increased by the stimulation of interferon γ. Co‑immunoprecipitation assays suggested the formation of a PDIA3 and MHC class I complex. The findings suggested that PDIA3 may be involved in the immune response of carcinoma cells. The improved prognosis in PDIA3‑High GC may be accounted for, in part, by sufficient antigen processing and expression of MHC class I, which can be mediated by PDIA3. It was suggested that PDIA3 serves an important role in the pathobiology of GC, and that PDIA3 is a useful marker for the prediction of prognosis.

Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma.

Protein disulfide‑isomeraseA3 (PDIA3) is a chaperone protein that modulates folding of newly synthesized glycoproteins and responds to endoplasmic reticulum (ER) stress. Previous studies reported that increased expression of PDIA3 in hepatocellular carcinoma (HCC) is a marker for poor prognosis. However, the mechanism remains poorly understood. The aim of the present study, therefore, was to understand the role of PDIA3 in HCC development. First, immunohistochemical staining of tissues from 53HCC cases revealed that HCC tissues with high PDIA3 expression exhibited a higher proliferation index and contained fewer apoptotic cells than those with low expression. In addition, the knockdown of PDIA3 significantly inhibited cell proliferation and induced apoptosis in HCC cell lines. These results suggest that PDIA3 regulates cell proliferation and apoptosis in HCC. An examination of whether PDIA3 knockdown induced apoptosis through ER stress revealed that PDIA3 knockdown did not increase ER stress marker, 78kDa glucose‑regulated protein, in HCC cell lines. Furthermore, the association between PDIA3 and the signal transducer and activator of transcription3 (STAT3) signaling pathway were investigated invitro and invivo. Immunofluorescence staining and co‑immunoprecipitation experiments revealed colocalization and binding, respectively, of PDIA3 and STAT3 in HCC cell lines. The knockdown of PDIA3 decreased the levels of phosphorylated STAT3 (P‑STAT3; Tyr705) and downstream proteins of the STAT3 signaling pathway: The anti‑apoptotic proteins (Bcl‑2‑like protein 1, induced myeloid leukemia cell differentiation protein Mcl‑1, survivin and X‑linked inhibitor of apoptosis protein). In addition, PDIA3 knockdown provided little inhibitory effect on cell proliferation in HCC cell lines treated with AG490, a tyrosine‑protein kinase JAK/STAT3 signaling inhibitor. Finally, an association was demonstrated between PDIA3 and P‑STAT3 expression following immunostaining of 35 HCC samples. Together, the present data suggest that PDIA3 promotes HCC progression through the STAT3 signaling pathway.

Lung epithelial protein disulfide isomerase A3 (PDIA3) plays an important role in influenza infection, inflammation, and airway mechanics

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons.