Protein Disulfide Isomerase A3 Precursor Research Articles

Therapeutic Options of Crystallin Mu and Protein Disulfide Isomerase A3 for Cuprizone-Induced Demyelination in Mouse Hippocampus

This study investigates the changes in hippocampal proteomic profiles during demyelination and remyelination using the cuprizone model. Employing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry for protein profiling, we observed significant alterations in the expression of ketimine reductase mu-crystallin (CRYM) and protein disulfide isomerase A3 precursor (PDIA3) following exposure to and subsequent withdrawal from cuprizone. Immunohistochemical staining validated these protein expression patterns in the hippocampus, revealing that both PDIA3 and CRYM were downregulated in the hippocampal CA1 region during demyelination and upregulated during remyelination. Additionally, we explored the potential protective effects of CRYM and PDIA3 against cuprizone-induced demyelination by synthesizing cell-permeable Tat peptide-fusion proteins (Tat-CRYM and Tat-PDIA3) to facilitate their crossing through the blood–brain barrier. Our results indicated that administering Tat-CRYM and Tat-PDIA3 mitigated the reduction in proliferating cell and differentiated neuroblast counts compared to the group receiving cuprizone alone. Notably, Tat-PDIA3 demonstrated significant effects in enhancing myelin basic protein expression alongside phosphorylation of CREB in the hippocampus, suggesting its potential therapeutic role in the prevention or treatment of demyelination, and by extension, in conditions such as multiple sclerosis.

Expression of protein disulfide isomerase A3 precursor in colorectal cancer.

IntroductionOver 20% of colorectal cancer (CRC) patients seek medical attention for the first time when they are in the advanced stages of CRC. Thus, early and reliable detection of CRC is critical to early diagnosis of CRC. Protein disulfide isomerase A3 precursor (PDIA3) has been implicated in various types of cancers. However, little is known about PDIA3 in CRC.MethodsIn this study, we screened PDIA3 expression in CRC tissues and cell lines. Small interfering RNA (siRNA) was introduced into SW480 cells to knockdown PDIA3 expression. The effect of PDIA3 in cell growth was evaluated.ResultsSignificant upregulation of PDIA3 expression was found in CRC tissues as compared with adjacent non-cancer tissues, and was found in established CRC cell lines (SW480, HCT116, CACO2, NCM460 and HT-29). In SW480 cells, knockdown of PDIA3 expression with siRNA resulted in subcellular morphological change, reduced cell proliferation and increased apoptosis.ConclusionPDIA3 inhibition could suppress CRC, likely through inducing apoptosis. PDIA3 could be a potential therapeutic target for CRC.

Changes in the expression of selected antioxidative proteins in roe deer (Capreolus capreolus) epididymis in different periods of the rutting season

Abstract One of the factors determining the homeostasis of biological systems is the balance between the formation of reactive oxygen species (ROS) and the activity of the antioxidative defence system. Regarding seasonal variations in secretory activity in different parts of roe buck’s (Capreolus capreolus) epididymis, the aim of present study was to compare the expression of selected antioxidative proteins in three periods of the rutting season. Using proteomics methods (2D PAGE , tandem mass spectrometry MS/MS) 5 antioxidative enzymes were identified for the first time in different segments of the epididymis of the roe buck. The highest expression of these enzymes was found in the rutting season. Findings indicated that the antioxidative function of the buck’s epididymis, regardless of the period of the rutting season, but particularly in the rutting season, is maintained mainly because of the presence of three conservative polypeptides: glutathione S-transferase (GST), protein disulfide isomerase A3 (PDIA3), and PDIA3 precursor. Moreover, a protective role against the harmful products of redox reactions is played during the discussed periods by peroxiredoxin-2 (PRDX2), identified in the cauda of the epididymis. In the tissue of the corpus and caput of the epididymis its expression was only found in the rutting season. The expression of biliverdin reductase A (BRVA ) in the epididymis was only observed in the rutting season.

PDIA3 Knockdown Exacerbates Free Fatty Acid-Induced Hepatocyte Steatosis and Apoptosis.

Nonalcoholic fatty liver disease (NAFLD) has emerged as one of the most common chronic liver disease over the past decades. Endoplasmic reticulum stress (ERS) plays a pivotal role during the development of NAFLD. This study aims to analyze the potential role of protein disulfide isomerase A3 precursor (PDIA3), one of the ER chaperones, in free fatty acid-induced cell model of NAFLD. Human liver L02 cell line was treated with sodium palmitate for 24 hours, which developed severe intracellular lipid accumulation. The increased protein level of PDIA3 was detected via immunoblotting analysis in the fat loaded cell models of NAFLD. siRNA-mediated knockdown of PDIA3 in L02 cells not only increased the cellular lipid accumulation, but also exacerbated hepatocytes apoptosis induced by sodium palmitate. Further investigation revealed that knockdown of PDIA3 up-regulated protein expression of fatty acid synthase (FAS), a key enzyme involved in fatty acid synthesis. PDIA3 knockdown also up-regulated key molecules of ERS pathway, including glucose-regulated protein 78 (GRP78), phospho-PKR-like ER kinase (p-PERK), and C/EBP homologous protein (CHOP). Our results suggested that ER chaperone PDIA3 plays a pivotal role in FFA-induced hepatocyte steatosis and apoptosis.

Identification of target antigens of anti-endothelial cell antibodies in patients with anti-neutrophil cytoplasmic antibody-associated vasculitides: A proteomic approach

Anti-endothelial cell antibodies (AECAs) have been reported to cause endothelial cell dysfunction, but their specific targets have never been identified in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs). Proteins from human umbilical vein endothelial cells (HUVECs) were separated by 2-dimensional electrophoresis (2-DE). 2-D immunoblots were used to compare serum IgG reactivities from 30 patients with AAV and 12 healthy controls (HCs). Proteins identified as target antigens by MALDI- TOF-TOF mass spectrometry included lamin A/C, vimentin, α-enolase, far upstream binding protein 2 (FUBP2) and protein disulfide-isomerase A3 precursor (PDIA3). Antibodies targeting lamin A, vimentin, α-enolase, FUBP2 and PDIA3 were identified in 57.1%, 64.3%, 35.7%, 50% and 0% of patients with microscopic polyangiitis and 8%, 3.3%, 7.2%, 0% and 6.7% of HCs respectively. IgG from patients with microscopic polyangiitis had stronger reactivity against HUVEC than other groups and HCs and induced stronger Erk phosphorylation in HUVECs than IgG from HCs.

Correlation between TGF‐β1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain‐like protease

Severe acute respiratory syndrome (SARS) coronavirus (SARS‐CoV) papain‐like protease (PLpro), a deubiquitinating enzyme, demonstrates inactivation of interferon (IFN) regulatory factor 3 and NF‐κB, reduction of IFN induction, and suppression of type I IFN signaling pathway. This study investigates cytokine expression and proteomic change induced by SARS‐CoV PLpro in human promonocyte cells. PLpro significantly increased TGF‐β1 mRNA expression (greater than fourfold) and protein production (greater than threefold). Proteomic analysis, Western blot, and quantitative real‐time PCR assays indicated PLpro upregulating TGF‐β1‐associated genes: HSP27, protein disulfide isomerase A3 precursor, glial fibrillary acidic protein, vimentin, retinal dehydrogenase 2, and glutathione transferase omega‐1. PLpro‐activated ubiquitin proteasome pathway via upregulation of ubiquitin‐conjugating enzyme E2–25k and proteasome subunit alpha type 5. Proteasome inhibitor MG‐132 significantly reduced expression of TGF‐β1 and vimentin. PLpro upregulated HSP27, linking with activation of p38 MAPK and ERK1/2 signaling. Treatment with SB203580 and U0126 reduced PLpro‐induced expression of TGF‐β1, vimentin, and type I collagen. Results point to SARS‐CoV PLpro triggering TGF‐β1 production via ubiquitin proteasome, p38 MAPK, and ERK1/2‐mediated signaling.

Rat hippocampal proteomic alterations following intrahippocampal injection of amyloid beta peptide (1–40)

Amyloid beta peptide 1–40 (Aβ 1–40) is closely associated with the progressive neuronal loss and cognitive decline observed in Alzheimer's disease (AD). This study aimed to establish a proteomic strategy for the profiling of AD tissues for disease-specific changes in protein abundance. Intrahippocampal injection of Aβ 1–40 induced spatial memory and learning decline in rats. Proteomic analysis revealed the changes in protein expression in the rat hippocampus treated with Aβ 1–40. Four proteins of interest which was in abundance was significantly altered in Aβ 1–40-treated rats were identified by peptide mass fingerprint (PMF). These proteins corresponded to synapsin Ib, protein disulfide-isomerase A3 precursor, tubulin β chain and ATP synthase β subunit. Our results provide new insights into the relationship between Aβ and the pathogenesis of AD, and suggest potential targets for the therapy of AD.

Proteomic Profiling of Acute Cardiac Allograft Rejection

Proteome analysis has emerged as a valuable tool for the study of large-scale protein expression profiles. Here, we applied this novel technology to identify specific biomarkers for acute cardiac allograft rejection. Hearts of C57BL/10 mice were placed in fully major histocompatibility complex-mismatched C3H/He recipients. Syngeneic transplants served as controls. Intragraft protein expression analysis was performed using fluorescence two-dimensional difference gel electrophoresis on day 6 posttransplant. Spots of interest were subsequently subjected to nanospray ionization tandem mass spectrometry (MS/MS) for protein identification. In addition, expression of selected proteins was confirmed by Western blot analysis and by immunohistochemistry. Two-dimensional difference gel electrophoresis enabled detection of 1541 protein spots. For 95 protein spots, the expression level during acute rejection differed by more than 1.5-fold from that observed in syngeneic grafts. Spots with significant differential regulation identified by tandem mass spectrometry were derived from peroxiredoxin 6, pyruvate kinase isozyme M2, coronin 1A, protein disulfide isomerase A3 precursor, and aconitate hydratase. These identified proteins may constitute novel biomarkers of acute cardiac allograft rejection and might hold great potential as surrogate markers for monitoring in vivo alloimmune response.

Proteomic Analyses of Contrast Media-Treated Mesangial Cell

Contrast Media is used in angiography, urography and tomography. It has been reported that contrast media nephrotoxicity incidence is 50% higher in patients with diabetic nephropathy undergoing coronary angiography and ap- proximately 15% of these patients developed renal failure. Proteomic analyses are a promising tool to study renal patho- physiology. In the future identification of biomarkers in renal diseases will develop therapeutic targets that will decrease the damage of acute renal failure. To identify proteins that were up or down-regulated in immortalized mesangial cells in a culture in the presence of contrast media we evaluated three groups: control, manitol used as control of osmolarity and Hexabrix TM . The cell homogenate from each group was submitted to the 2D-PAGE analysis, and proteins were identified by MALDI-TOF mass spectrometry. Some proteins were expressed only in the Hexabrix group (Proliferating Cell Nu- clear Antigen and Masp 1); others were up-regulated in the Manitol group (Translationally Controlled Tumor Protein), a protein was expressed only in the Control and Manitol (Annexin A3) groups, and we identified proteins that were ex- pressed in all three groups (Heat shock proteins 27 and 84, protein disulfide isomerase A3 precursor and Beta-actin 1). We believe that other substances present in Hexabrix, possibly the iodine, could be the regulatory factor of these proteins and the Hexabrix could be responsible for the nephrotoxicity observed in these cells.

Modification of the Hepatic Mitochondrial Proteome in Response to Ischemic Preconditioning following Ischemia-Reperfusion Injury of the Rat Liver

Background/Aim: Ischemic preconditioning (IPC) may reduce hepatic ischemia-reperfusion (IR) injury, but efficacy of IPC on mitochondrial proteome is not demonstrated. We investigated how IPC modifies the mitochondrial proteome after IR injury. Methods: Rats were subjected to 25 min of portal triad crossclamping (IR group, n = 8). In the IPC group (n = 8), 10 min of temporal portal triad clamping was performed before 25 min of portal clamping. Samples were obtained after 24 h. The mitochondrial inner-membrane potential was measured by the uptake of a lipophilic cationic carbocyanine probe and mitochondrial proteome was also investigated using 2-dimensional differential in-gel electrophoresis and liquid chromatography-tandem mass spectrometry. Results: Mitochondrial inner-membrane potential and glutathione were lower and serum transaminase was higher in the IPC group than in the IR group. The mitochondrial precursor of aldehyde dehydrogenase 2 and α-methylacyl-CoA-racemase were upregulated in the IPC group in comparison to the IR group. In contrast, protein disulfide-isomerase A3 precursor, 60S acid ribosomal protein P0, carbonic anhydrase 3 and superoxide dismutase were significantly more downregulated in the IPC group than in the IR group. Conclusions: A hepatoprotective effect by IPC was not shown; however, IPC caused significant up- or downregulation of several mitochondrial proteins.

Gel-Based Proteomics of Unilateral Irradiated Striatum after Gamma Knife Surgery

Gamma knife surgery (GKS) is used for the treatment of various brain disorders. The biological effects of focal gamma ray irradiation on targeted or surrounding areas in the brain are not well-known. In the present study, we evaluated protein expression changes in the unilateral irradiated (60 Gy) striatum in rat. Striata of irradiated and control brains were dissected 16 h post-irradiation for analysis by large-format two-dimensional gel electrophoresis (2-DGE). In parallel, we also examined the un-targeted contralateral striatum over the control for potential changes in proteins patterns that may have occurred due to the effects of irradiation to the unilateral striatum. A total of 17 reproducible and differentially expressed silver nitrate-stained protein spots in the irradiated striatum was detected on 2-D gel. Their subsequent analysis by tandem mass spectrometry (nESI-LC-MS/MS) resulted in the identification of 13 nonredundant proteins. Interestingly, out of these 13 changed proteins, 2 proteins were also detected in the contralateral striatum. Some of the significantly changed proteins identified were creatine kinase, protein disulfide isomerase A3 precursor (PDA3), and peroxiredoxin 2 (Prx2). Western analysis with anti-PDA3 and anti-Prx2 antibodies revealed 4 and 2 cross-reacting protein spots on 2-D gel blots. Interestingly, after GKS, in the irradiated and un-irradiated striata, these spots showed a shift toward the acidic side, suggesting post-translational modifications. Taken together, these results indicate that unilateral irradiation during GKS triggers molecular changes in the bilateral striata.

Proteomic investigation of 1,6-dimethoxyhexane testicular toxicity

The aliphatic ether 1,6-dimethoxyhexane (DMH) was previously identified as a testicular toxicant. Testis protein extracts from control and DMH-treated rats were subjected to two-dimensional gel electrophoresis for comparison of protein expression profiles. MALDI-ToF peptide mass fingerprinting of differentially expressed proteins resulted in the conclusive identification of heat shock-related 70 kDa protein 2 (HSP70.2), 60 kDa heat shock protein, mitochondrial precursor (HSP60) and protein disulfide isomerase A3 precursor (ERp60). The potential involvement of these proteins in chemically induced perturbation of spermatogenesis and their utility as biomarkers of testicular toxicity are discussed in light of the knowledge currently available from the literature.

Expression of glycolytic enzymes is increased in pancreatic cancerous tissues as evidenced by proteomic profiling by two-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry

The prognosis of patients with pancreatic cancer is very poor because of late diagnosis and the lack of response to various therapies. We tried to identify proteins that might be available for early diagnosis and effective therapies by proteomic profiling of pancreatic cancer tissues. Pancreatic cancerous and paired non-cancerous tissues obtained from surgical resections or autopsies of 10 patients were analyzed by two-dimensional gel electrophoresis. The differential display showed 11 spots whose expression was increased in cancerous tissues compared with the paired non-cancerous tissues. The liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) system identified the spots as alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase, transgelin, calmodulin, superoxide dismutase(Mn) mitochondrial precursor, glutathione S-transferase P, cyclophilin A, protein disulfide isomerase A3 precursor, and apolipoprotein A-I precursor. Two of the 11 spots were detected as GAPDH. We noticed that 4 of 11 spots were enzymes involved in glycolytic pathway. Increased glycolysis in cancer cells has been regarded as the effect of intratumoral hypoxia and is possibly associated with tumor invasion, metastasis or resistance to therapies. These glycolytic proteins and transgelin, were confirmed by Western blotting and immunohistochemistry.

Proteomic analysis of proteins regulated by TRPS1 transcription factor in DU145 prostate cancer cells

The aim of the present study was to identify proteins differentially regulated by TRPS1 in human prostate cancer cells in order to better understand the role of TRPS1 in prostate cancer development. The proteomes of androgen-independent DU145 prostate cancer cells, that do not express TRPS1 and of genetically engineered DU145 cells that stable and inducible express recombinant TRPS1 protein, were compared. Using two-dimensional electrophoresis followed by mass spectrometric analysis, 13 proteins that were differentially expressed between these two cell lines were identified. These proteins represent a dominant reduction of expression of antioxidant proteins, including superoxide dismutase, protein disulfide isomerase A3 precursor, endoplasmin precursor and annexin A2. Furthermore, regulation was observed for mitochondrion-associated proteins, glycolytic enzymes, a cytoskeleton-associated protein, a nuclear protein and proteins involved in apoptosis. Our data indicate that overexpression of TRPS1 protein is correlated with reduced protein expression of certain antioxidants. This suggests a possible involvement of TRPS1 in oxidative stress, and possibly in apoptosis in androgen-independent DU145 prostate cancer cells.

Life-long effects of perinatal asphyxia on stress-induced proteins and dynamin 1 in rat brain.

In previous work, we have shown that perinatal asphyxia (PA) in the rat leads to life-long neurotransmitter deficits and impairment of cognitive functions and behavior. This observation made us examine protein expression in hippocampus of rats with PA at the end of the life span. We applied a well-documented and characterized animal model of PA. Pups, normoxic and asphyxiated for 20 min, were brought up until the age of 24 months and then were sacrificed. Hippocampal tissue was dissected from the brains, and proteins were run on two-dimensional gel electrophoresis with in-gel digestion and subsequent identification of proteins by MALDI-TOF followed by quantification of protein spots by specific software. In hippocampus of rats with PA, the stress proteins protein disulfide isomerase A3 precursor and stress-induced phosphoprotein-1 were significantly increased, whereas the microtubule-associated protein dynamin-1 was significantly reduced. Increased stress protein levels may represent long-term effects of PA or, alternatively, could reflect conditioning of the stress protein machinery known to occur as a neuroprotective principle following hypoxic-ischemic conditions. Decreased dynamin-1 levels may be considered as a long-term effect on the exocytotic system possibly reflecting or leading to impaired neuronal transport and vesicle-trafficking in PA of the rat of advanced age.