Effect Of Protein Disulfide Isomerase Research Articles

Synergistic promotion of transient transgene expression in CHO cells by PDI/XBP-1s co-transfection and mild hypothermia.

Transient gene expression system is an important tool for rapid production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, their low productivity is the main hurdle to overcome. An effective approach through which to obtain high protein yield involves targeting transcriptional, post-transcriptional events (PTEs), and culture conditions. Here, we investigated the effects of protein disulfide isomerase (PDI) and spliced X-box binding protein 1 (XBP-1s) co-overexpression combined with mild hypothermia on the transient yields of recombinant proteins in CHO cells. The results showed that the gene of interest (GOI) and the PDI/XBP-1s helper vector at a co-transfection ratio of 10:1 could obviously increase transient expression level of recombinant protein in CHO cells. However, PDI/XBP-1s overexpression had no significance effect on the mRNA levels of the recombinant protein, suggesting that it targeted PTEs. Moreover, the increased production was due to the enhancing of cell specific productivity, not related to cell growth, viability, and cell cycle. In addition, combined PDI/XBP-1s co-overexpression and mild hypothermia could further improve Adalimumab expression, compared to the control/37°C and PDI/XBP-1s/37°C, the Adalimumab volume yield of PDI/XBP-1s/33°C increased by 203% and 142%, respectively. Mild hypothermia resulted in 3.52- and 2.33-fold increase in the relative mRNA levels of PDI and XBP-1s, respectively. In conclusion, the combination of PDI/XBP-1s overexpression and culture temperature optimization can achieve higher transient expression of recombinant protein, which provides a synergetic strategy to improve transient production of recombinant protein in CHO cells.

Cysteine Sulfenylation of Protein Disulfide Isomerase Links Oxidative Stress to Thrombosis

Oxidative stress increases the risk of clinically significant thrombosis in the setting of inflammation, malignancy, infection, and dyslipidemia. Oxidants generated during oxidative stress are prothrombotic; however, the mechanisms by which oxidants induce thrombus formation is poorly understood. Protein disulfide isomerase (PDI) is a thiol isomerase that serves an important role in thrombus formation. It has a domain structure of a-b-b'-a', in which the aand a' domains are catalytic and the b and b' are substrate binding. A flexible x linker is between the b' and a'domains. PDI is uniquely sensitive to oxidative cysteine modification within the CGHC catalytic motif of its a and a'domains. Yet whether PDI cysteine modifications mediates thrombosis in an oxidative environment has not previously been studied. We hypothesized that oxidized PDI links oxidative stress to a prothrombotic phenotype through post-translational modification of proteins. We have previously shown that dyslipidemia promotes hydrogen peroxide generation from platelets and oxidizes proteins. Using hydrogen peroxide as a model oxidant, we found that oxidation of recombinant PDI promoted the loss of free thiols and inhibited reductase activity in a dose-dependent manner (IC50= 3.3 ± 0.12 µM). To understand how free thiols were lost, we used a benzothiazine-based nucleophilic probe, termed BTD, to label sulfenic acid. The transient cysteine sulfenic acid (S-OH) was detected by BTD labeling in wildtype PDI following exposure to hydrogen peroxide, but not in a PDI mutant in which all active site cysteines had been mutated to alanine. PDI exposure to oxidized lipoprotein particles (OxLDL) or 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, also promoted its sulfenylation. Using single cysteine mutants of PDI to identify the sulfenylation site, we found that Cys56 was preferentially sulfenylated as mutation of this cysteine prevented further labeling of the sulfenic acid probe. To assess whether interdomain interactions influenced sulfenylation, we evaluated BTD labeling in isolated fragments of PDI. Sulfenylation was identified in the a domain, consistent with Cys56 labeling, and was significantly enhanced in the ab fragment, implicating an interaction between the a and b domains in sulfenylation. No labeling was observed in an isolated b'xa' fragment. Further analysis of structural elements revealed that a conserved Arg120 within the a domain regulates Cys56 reactivity. Indeed, mutation of Arg120 to Ala prevented sulfenylation by peroxides compared to the wildtype control. Maleimide labeling showed that sulfenylated PDI is an intermediary that resolves into disulfided PDI. To determine whether this disulfided PDI promotes oxidase activity, we used a denatured and reduced RNAse that refolds when disulfides are transferred from oxidized PDI to the RNAse. PDI oxidized by hydrogen peroxide promoted RNAse refolding, whereas reduced PDI did not. A selective inhibitor of sulfenylation, arsenite, prevented RNAse refolding, indicating that hydrogen peroxide-induced oxidation of PDI proceeds through a sulfenic acid intermediate. Since PDI is secreted from cells, we evaluated whether activated cells release sulfenylated PDI using both human vein endothelial cells (HUVECs) and platelets. Sulfenylated PDI was increased in the cultured media of HUVECs stimulated with thrombin and in the releasate of platelets stimulated via PAR1 or glycoprotein VI. OxLDL exposure sensitizes platelets to activation by low doses of agonists. Inhibition of PDI using a neutralizing antibody inhibited augmentation of platelet aggregation by oxLDL, suggesting that oxidation of PDI by oxLDL promotes platelet activation. The ability of oxLDL to enhance the prothrombotic effect of PDI was tested in vivo in a murine model of laser-induced thrombus formation. Mice were infused with either control IgG or IgG directed at PDI, followed by the infusion of oxLDL. OxLDL significantly enhanced thrombus formation in this model. The effect of oxLDL was inhibited by anti-PDI IgG but not control IgG antibody or buffer, implicating PDI as an effector of oxLDL-augmented thrombus formation. In conclusion, our study demonstrates sulfenylation of PDI and shows that PDI sulfenylation retains PDI oxidase activity. Oxidation of PDI by oxidants such as oxLDL occurs in platelets and in endothelium and promotes thrombosis in the setting of oxidative stress.

C-terminal truncation modulates α-Synuclein’s cytotoxicity and aggregation by promoting the interactions with membrane and chaperone

α-Synuclein (α-syn) is the main protein component of Lewy bodies, the major pathological hallmarks of Parkinson’s disease (PD). C-terminally truncated α-syn is found in the brain of PD patients, reduces cell viability and tends to form fibrils. Nevertheless, little is known about the mechanisms underlying the role of C-terminal truncation on the cytotoxicity and aggregation of α-syn. Here, we use nuclear magnetic resonance spectroscopy to show that the truncation alters α-syn conformation, resulting in an attractive interaction of the N-terminus with membranes and molecular chaperone, protein disulfide isomerase (PDI). The truncated protein is more toxic to mitochondria than full-length protein and diminishes the effect of PDI on α-syn fibrillation. Our findings reveal a modulatory role for the C-terminus in the cytotoxicity and aggregation of α-syn by interfering with the N-terminus binding to membranes and chaperone, and provide a molecular basis for the pathological role of C-terminal truncation in PD pathogenesis.

Flavonoids as Protein Disulfide Isomerase Inhibitors: Key Molecular and Structural Features for the Interaction.

Quercetin-3-rutinoside (rutin) is a bioflavonoid that is common in foods. The finding that quercetin-3-rutinoside inhibits protein disulfide isomerase (PDI) and potently blocks thrombosis in vivo has enabled the evaluation of PDI inhibition in multiple animal models of thrombus formation and has prompted clinical studies of PDI inhibition in thrombosis. Nonetheless, how quercetin-3-rutinoside blocks PDI activity remains an unanswered question. Combining NMR spectroscopy, site-directed mutagenesis, and biological assays, we identified H256 as the key residue for PDI interacting with quercetin-3-rutinoside. Quercetin-3-rutinoside inhibited the activity of PDI (WT) but not PDI (H256A). Molecular dynamic simulations indicated that the flavonoid skeleton, but not the rutinoside conjugate, is embedded in the major binding pocket on the b' domain. Among several quercetin-3-rutinoside analogues tested, only compounds with a phenoxyl group at position 7 showed direct binding to PDI, further supporting our molecular model. Studies using purified coagulation factors showed that quercetin-3-rutinoside inhibited the augmenting effects of PDI (WT), but not PDI (H256A), on tissue factor (TF) activity. Quercetin-3-rutinoside also inhibited chemotherapy-induced TF activity enhancement on endothelial cells. Together, our studies show that residue H256 in PDI and the phenoxyl group at position 7 in quercetin-3-rutinoside are essential for inhibition of PDI by quercetin-3-rutinoside. These results provide new insight into the molecular mechanism by which flavonoids block PDI activity.

Glucose-induced oxidative stress leads to in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells

BackgroundDementia places a significant burden on both patients and caregivers. Since diabetes is a risk factor for dementia, it is imperative to identify the relationship between diabetes and cognitive disorders. Protein disulfide isomerase (PDI) is an enzyme for oxidative protein folding. PDI S-nitrosylation is observed in the brain tissues of Alzheimer's disease patients. The aim of this study is to clarify the relationship between PDI S-nitrosylation and diabetes. MethodsWe used SH-SY5Y cells cultured in high-glucose media. ResultsS-nitrosylated PDI level increased at 7 days and remained high till 28 days in SH-SY5Y cells cultured in high-glucose media. Using PDI wild-type- or PDI C343S-expressing SH-SY5Y cells, PDI C343 was identified as the site of glucose-induced S-nitrosylation. IRE1α and PERK were phosphorylated at day 14 in the SH-SY5Y cells cultured in high-glucose media, and the phosphorylated status was maintained to day 28. To determine the effect of S-nitrosylated PDI on endoplasmic reticulum stress signaling, SH-SY5Y cells were treated with S-nitrosocystein (SNOC) for 30 min, following which the medium was replaced with SNOC-free media and the cells were cultured for 24 h. Only phosphorylated IRE1α treated with SNOC was associated with PDI S-nitrosylation. Neohesperidin, a flavonoid in citrus fruits, is a natural antioxidant. The treatment with neohesperidin in the final 7 days of glucose loading reversed PDI S-nitrosylation and improved cell proliferation. ConclusionGlucose loading leads to S-nitrosylation of PDI C343 and induces neurodegeneration via IRE1α phosphorylation. General significanceThe results may be useful for designing curative treatment strategies for dementia.

Protein disulfide isomerase inhibits endoplasmic reticulum stress response and apoptosis via its oxidoreductase activity in colorectal cancer

Protein disulfide isomerase (PDI), a principal endoplasmic reticulum resident oxidoreductase chaperone, is known to play a role in malignancies. This study aims to explore the molecular mechanism by which PDI regulates endoplasmic reticulum stress and the apoptosis signaling pathway in colorectal cancer (CRC). We determined the expression of PDI in CRC tissues and adjacent normal tissues. Gain- and loss- of function assays were conducted to evaluate the effects of PDI on oxidative stress, endoplasmic reticulum stress, and apoptosis in CRC cells, as reflected by hydrogen peroxide (H2O2) level and the expression of related proteins. PDI protein expression was upregulated in CRC tissues. Small molecule inhibitor of PDI or PDI knockdown reduced CRC cell viability and induced apoptosis. Overexpression of wild-type PDI augmented the viability of CRC cells and inhibited endoplasmic reticulum stress response and apoptosis. Small molecule inhibitor of PDI or PDI knockdown increased intracellular H2O2 level and activated apoptosis signaling pathway, which could be reversed by wild-type PDI restoration. Moreover, the catalytic active site of C-terminal of PDI was found to be indispensable for the regulatory effects of PDI on H2O2 levels, apoptosis and cell viability in CRC cells. Collectively, PDI inhibits endoplasmic reticulum stress and apoptosis of CRC cells through its oxidoreductase activity, thereby promoting the malignancy of CRC.

Effects of protein disulfide isomerase on hyperglycemia and hypoxia/reoxygenation injury in H9c2 cardiomyocytes

Objective: To explore the effect of protein disulfide isomerase (PDI) in diabetic ischemic heart disease. Methods: We established an in vitro model of high glucose and hypoxia/reoxygenation in H9c2 rat myocardial cells. Cultured cells were divided into four groups: Control, high glucose (HG), hypoxia/reoxygenation (H/R) and HG+H/R. Changes in PDI expression mediated by PDI adenovirus(Ad-PDI) infection and siRNA(PDI-siRNA) transfection in myocardial cells were observed by inverted fluorescence microscopy. We also measured lactate dehydrogenase(LDH) activity and malondialdehyde(MDA) and high molecular weight(HMW)-APN concentrations. PDI, APN, cleaved caspase-3, and glucose regulated protein 78 (Grp78) protein expression were detected. Results: PDI expression was significantly decreased in the HG, H/R and HG+H/R groups compared to the Control group; however, LDH activity[(179.7±10.4) U/L、(218.4±18.4) U/L、(328.2±5.3) U/L vs (91.0±11.0) U/L], MDA concentration[(7.0±0.4) μmol/L、(10.0±1.0) μmol/L、(11.7±1.0) μmol/L vs (4.2±1.8) μmol/L], cleaved caspase-3, and Grp78 expression were increased. Interestingly, APN and HMW-APN expression were decreased [(2.01±0.21) μg/L、(1.64±0.27) μg/L、(1.20±0.14) μg/L vs (2.62±0.12) μg/L, all P<0.05]. Over expression of PDI attenuated high glucose and hypoxia/reoxygenation induced apoptosis and oxidative stress in H9c2 cardiomyocytes(all P<0.05), and simultaneously increased APN and HMW-APN expression [(2.86±0.03) μg/L vs (3.03±0.10) μg/L、(2.06±0.05) μg/L vs (2.31±0.06) μg/L、(1.83±0.07) μg/L vs (1.96±0.11) μg/L、(1.20±0.06) μg/L vs (1.39±0.09) μg/L]. PDI-siRNA transfection increased LDH activity, MDA concentration, and cleaved caspase-3 and Grp78 expression, and decreased APN and HMW-APN expression [(0.75±0.09) μg/L vs (0.59±0.09) μg/L、(0.62±0.04) μg/L vs (0.53±0.05) μg/L、(0.55±0.14) μg/L vs (0.51±0.12) μg/L、(0.48±0.12) μg/L vs (0.35±0.08) μg/L] in response to different treatments in cultured H9c2 cardiomyocytes (all P<0.05). Conclusion: PDI may regulate the expression of APN and HMW-APN, and play an important role in the function of diabetic ischemia-reperfusion cardiomyocytes.

Thiol Isomerases Orchestrate Thrombosis and Hemostasis.

Significance: Since protein disulfide isomerase (PDI) was first described in 1963, researchers have shown conclusively that PDI and sibling proteins are quintessential for thrombus formation. PDI, endoplasmic reticulum protein (ERp)5, ERp57, and ERp72 are released from platelets and vascular cells and interact with integrin αIIbβ3 on the outer surface of platelets. Recent Advances: At the cell surface they influence protein folding and function, propagating thrombosis and maintaining hemostasis. TMX1, which is a transmembrane thiol isomerase, is the first family member shown to negatively regulate platelets. Targets of thiol isomerases have been identified, including integrin α2β1, Von Willebrand Factor, GpIbα, nicotinamide adenine dinucleotide phosphate oxidase (Nox)-1, Nox-2, and tissue factor, all of which are pro-thrombotic, and several of which are on the cell surface. In spite of this, PDI can paradoxically catalyze the delivery of nitric oxide to platelets, which decrease thrombus formation. Critical Issues: Although the overall effect of PDI is to positively regulate platelet activation, it is still unclear how thiol isomerases function in pro-thrombotic states, such as obesity, diabetes, and cancer. In parallel, there has been a surge in the development of novel thiol isomerase inhibitors, which display selectivity, potency and modulate thrombosis and hemostasis. The availability of selective thiol isomerase inhibitors has culminated in clinical trials, with promising outcomes for the prevention of cancer-associated thrombosis. Future Directions: Altogether, thiol isomerases are perceived as an orchestrating force that regulates thrombus development. In the current review, we will explore the history of PDI in cardiovascular biology, detail known mechanisms of action, and summarize known thiol isomerase inhibitors.

Protein Disulfide Isomerase Disrupts and Detoxifies Aggregates of Amyloid Beta Peptide

Aggregated fibrils of amyloid beta (Aβ) peptide induce the neurotoxicity characteristic of Alzheimer's Disease. The inhibition of Aβ aggregation could therefore represent a therapeutic target for Alzheimer's Disease. Protein disulfide isomerase (PDI) is an oxidoreductase and chaperone that has been shown to effectively inhibit the formation of Aβ fibrils when present at the onset of aggregation. However, the potential disruption of ongoing Aβ fibrillization by PDI has not been demonstrated. We examined the disruptive effect of PDI on Aβ aggregation using a Thioflavin‐T fluorescence assay to monitor Aβ fibrillization. PDI prevented the aggregation of monomeric Aβ at a sub‐stoichiometric 1:10 molar ratio of PDI:Aβ. Fibrillization was partially inhibited by a 1:50 molar ratio of PDI:Aβ and was not affected by a 1:100 molar ratio of PDI:Aβ. As such, a 1:10 molar ratio of PDI:Aβ was used for all subsequent experiments. We next found that PDI could also disrupt Aβ fibrillization when added 2, 4, or 10 h after the initiation of aggregation. Cell culture studies using an MTS cell viability assay further demonstrated that PDI can protect neuronal cells from extracellular aggregates of Aβ when added 2 h after the initiation of aggregation. These findings demonstrate PDI is able to disrupt Aβ aggregation at sub‐stoichiometric molar ratios and support the potential use of PDI as a novel therapy for Alzheimer's Disease.Support or Funding InformationFlorida Department of Health, Ed and Ethel Moore Alzheimer's Disease Research Program Grant 8AZ12This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein Disulfide Isomerase Silence Inhibits Inflammatory Functions of Macrophages by Suppressing Reactive Oxygen Species and NF-κB Pathway.

Macrophages play an essential role in inflammation. Protein disulfide isomerase (PDI) is central to the redox system, which is closely linked with the inflammatory function of macrophages. However, the relationship between PDI and inflammation is still unknown. In this study, we tested the effects of PDI on inflammatory responses in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Using CRISPR/Cas9 system, we found that PDI knockout suppressed migration, M1 polarization, and secretion of tumor necrosis factor-α (TNF-α) and interluekin-6 (IL-6). The repression of these inflammatory processes was accompanied by decreased production of reactive oxygen species (ROS). PDI ablation also inactivated the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activated the phosphorylation of NF-κB inhibitor alpha (IκBα). These findings demonstrate that PDI knockout inhibits the inflammatory function of macrophages by decreasing ROS production and inactivating NF-κB pathway.

Neuroprotection by Endoplasmic Reticulum Stress-Induced HRD1 and Chaperones: Possible Therapeutic Targets for Alzheimer's and Parkinson's Disease.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are neurodegenerative disorders with a severe medical and social impact. Further insights from clinical and scientific studies are essential to develop effective therapies. Various stresses on the endoplasmic reticulum (ER) cause unfolded/misfolded proteins to aggregate, initiating unfolded protein responses (UPR), one of which is the induction of neuronal cell death. Some of the pathogenic factors for AD and PD are associated with UPR. ER molecules such as ubiquitin ligases (E3s) and chaperones are also produced during UPR to degrade and refold aberrant proteins that accumulate in the ER. In this review, we examine the role of HMG-CoA reductase degradation protein 1 (HRD1) and the chaperone protein-disulfide isomerase (PDI), which are both produced in the ER in response to stress. We discuss the importance of HRD1 in degrading amyloid precursor protein (APP) and Parkin-associated endothelin receptor-like receptor (Pael-R) to protect against neuronal death. PDI and the chemical chaperone 4-phenyl-butyrate also exert neuroprotective effects. We discuss the pathophysiological roles of ER stress, UPR, and the induction and neuroprotective effects of HRD1 and PDI, which may represent significant targets for novel AD and PD therapies.

Protein Disulfide Isomerase in Sickle Cell Disease

Protein disulfide isomerase (PDI), a multifunctional oxidoreductase that regulates thrombus formation, leukocyte adherence to the endothelium and nitric oxide delivery, is present at high levels and regulates KCNN4 (Gardos Channel) activity in erythrocytes from humans with Sickle Cell Disease (SCD) and sickle mouse models. We reported evidence of elevated erythrocyte-surface associated and circulating PDI activity in humans and mouse models of SCD when compared to either wild-type mice, transgenic mice expressing normal human globins or otherwise healthy individuals; results that suggest a novel role for PDI in the pathophysiology of SCD through unclear mechanisms. We studied the effects of deoxygenation (5% O2) on PDI activity in endothelial cells and sickle erythrocytes from humans with SCD and two sickle transgenic knockout mouse models expressing human sickle hemoglobin, BERK and βSAntilles. Our data shows that deoxygenation of human and mouse sickle erythrocytes increased surface-associated reductive capacity that was sensitive to monoclonal antibodies against PDI (mAb PDI [RL-90]). We then studied sickle human erythrocytes and showed that PDI inhibitors (quercentin-3-rutinoside [Q3R] and mAb PDI) significantly reduced deoxy-stimulated sickle cell dehydration and Gardos channel activity (n=5; P <0.03). We characterized erythrocyte density with a phthalate density-distribution assay, generated density distribution curves and calculated the D50. Both mAb PDI and Q3R significantly reduced D50 when compared to vehicle (1.113±0.002 to 1.102±0.001 [P <0.0001]; and 1.101±0.002 [P <0.0001]; respectively). In contrast, incubation with exogenous PDI (3 nM) increased cellular dehydration (from D50= 1.110 ± 0.001 to D50= 1.115 ±0.001, P <0.01). We also measured the effect of hypoxia and endothelin-1 (ET-1; 10nM) in the human vascular endothelial cell line, EA.hy926. We observed that hypoxia induced PDI secretion that was further enhanced by co-incubation with ET-1 (10nM; n=3; P <0.05). The selective inhibitor of ET-1 subtype B receptor antagonist, BQ788 blocked ET-1 stimulated PDI increases in these cells as was previously reported in red blood cells. Consistent with these results hypoxia was associated with increased mRNA expression of MCP-1, VEGF-a but not ICAM by qRT-PCR and Taqman probes (n=3; P <0.05). Of importance we observed that early cultures of mouse aortic endothelial cells from BERK mice showed similar results. We then evaluated the effects of PDI on erythrocyte hemolysis by exposing cells from patients with SCD to 20 mM 2-2'-azo-bis- (2-amidinopropane) dihydrochloride (AAPH) with or without Q3R or mAb PDI. AAPH-induced hemolysis was dose-dependently blocked by Q3R (IC50: 4.1nM; n=6, P <0.0001 compared to vehicle) or mAb PDI (IC50: 11nM; n=3). Irrelevant IgG did not affect hemolysis under these conditions. Experiments performed in blood from BERK or βSAntilles mice showed similar results. We then studied sickle mice that express varying levels of HbF; BERK (<1% HbF), BERKγM (25% HbF), and BERKγH (45% HbF). BERKγH had the lowest circulating and cell associated PDI activity among the three mouse types that was associated with lower circulating ET-1 levels (n=2; P <0.05). Consistent with these results we observed an inverse correlation between levels of HbF and PDI activity in cells from humans with SCD (n=4). Thus we posit that in SCD elevated erythrocyte PDI activity is important for cell survival and stability and that its inhibition may represent a novel therapeutic approach for improving both the hematological and vascular complications of SCD as it may not only increase sickle erythrocyte survival but may likewise interfere with cellular adhesion leading to reduced vaso-occlusive episodes. [Supported by NIH: HL090632 (AR) and HL096518 (JRR)] DisclosuresNo relevant conflicts of interest to declare.

Protein disulfide isomerase and Nox: new partners in redox signaling.

Reactive oxygen species (ROS) contribute to the pathogenesis of cardiovascular disease, including hypertension, atherosclerosis, cardiac hypertrophy, heart failure and restenosis. Thiol proteins and thiol oxidoreductases are key players in cell signaling, and their altered expression and/or activity has been associated with a disrupture in cardiac and vascular homeostasis. Protein disulfide isomerase (PDI) is a thiol oxidoreductase member of the thioredoxin family that has multiple roles in cellular function. Originally discovered in the endoplasmic reticulum (ER), PDI is essential for protein folding. However, it can also be found in the cytosol and closely associated with the surface of platelets, smooth muscle cells, neutrophils and endothelial cells. On the cell surface, PDI is imperative for platelet aggregation and transnitrosation, which are related to thrombosis and control of vascular tone by nitric oxide, respectively. Furthermore, PDI signaling contributes to redox-dependent events such as smooth muscle cell migration induced by PDGF and TNFα-dependent angiogenesis. Studies from our group have shown that intracellular PDI regulates the expression and activity of the NADPH oxidase family of proteins (Nox), which are enzymes dedicated to ROS generation. PDI acts as a new organizer of leukocyte Nox2 by redox dependently associating with p47phox and controlling its recruitment to the plasma membrane, an essential step for assembly of the active enzyme. Such multiple effects of PDI suggest that specific targeting of this oxidoreductase could represent a new approach in the treatment of vascular disease. In this review, we present a novel role for PDI as an adaptor protein involved in redox processes and Nox signaling and propose PDI as a potential therapeutic target in the treatment of atherosclerosis, thrombosis and hypertension.

Regulatory Effect of Protein Disulfide Isomerase on Platelet GPIbα Ectodomain Shedding

This study was aimed to investigate the regulatory effect of protein disulfide isomerase (PDI) on platelet GPIbα ectodomain shedding. The washed platelets were obtained from healthy volunteers. Platelets were incubated with PDI inhibitor bacitracin before stimulation with PMA (Phorbol-12-myristate-13-acetate), dibucaine and collagen. The N-terminal domain of GPIbα in supernatant was detected by Western blot, the GPIbα expression and the intraplatelet ROS levels were measured by flow cytometry. neither GC content nor GPIbα expression was changed after the washed platelets from the healthy donors were incubated only with PDI inhibitor. The washed platelets were incubated with PDI inhibitor before stimulation with different stimulin, PMA, dibucaine or collagen, and then GPIbα was cleaved and ROS levels were elevated more than that in the controls. PDI participates in the induced GPIbα ectodomein shedding, and the effect of PDI in this process maybe depend on the change of ROS level inside platelets. These results might provide a new point of view for the platelet drug development.

Protein Disulfide Isomerase Interacts with Tau Protein and Inhibits Its Fibrillization

BackgroundTau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI) is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization.Methodology/Principal FindingsAs evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau244–372) monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau244–372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau244–372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau244–372 fibrillization more strongly than full-length human PDI.Conclusions/SignificanceWe demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau fibrillization, and have applications for developing novel strategies for treatment and early diagnosis of Alzheimer disease.

Aldosterone Stimulates a Degranulation Response in Human Neutrophils: Role of Protein Disulfide Isomerase

AbstractAbstract 1034There is growing evidence for an important role of aldosterone (ALDO) in inflammatory responses in addition to its well-described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). We studied the effects of ALDO on activation of ex vivo human polymorphonuclear leukocytes (PMNC). We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses and when incubated with ALDO (10−9 −10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 2 min using FURA-2AM fluorescence. We then studied the effect of ALDO on PMNC degranulation following incubations with ALDO (10−9 −10−7 M) for 30 min and observed a significant increase in β–glucuronidase release (P<0.001, n=3) by established fluorescent detection methods, an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). PMA and N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) were used as positive controls for PMNC activation. We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcripts by quantitative RT-PCR using TaqMan detection probes in these cells and as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence. To assess the degranulation response of these cells we quantified the in vitro release of myeloperoxidase (MPO) and observed that 10−8M ALDO was likewise associated with increased degranulation when compared to vehicle treated cells (AUC: 590±14 to 185±11, P<0.01, n=6). To characterize the mechanisms by which ALDO regulates the degranulation responses of these cells we studied the effects of Protein Disulfide Isomerase (PDI) on ALDO-stimulated cells. PDI catalyzes the oxidation or reduction of thiol/disulfide groups and modulates leukocyte function. Our results show that blockade of PDI, by bacitracin, led to a blunted ALDO-stimulated degranulation response in both cell types. Consistent with these observations, we show that in differentiated HL-60 cells, siRNA against PDI likewise led to reduced MPO responses (AUC: 590±14 to 290±13, P<0.01, n=6) that were associated with significantly reduced PDI mRNA levels but not with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls (0.55 ± 0.02, ΔΔCT of PDI siRNA relative to scrambled transfected cells, P<0.01, n=6). These results suggest that ALDO stimulates MPO release. MPO has been shown to be one of the predominant granule proteins associated with Neutrophil Extracelullar Traps (NETs), extracellular structures that contain chromatin (DNA and histones) that can also trap microorganisms. We studied the effects of ALDO following digestion of the NETs by DNAse, and observed that 30–35% of the total cellular MPO was NET-associated. We also observed that incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3), a responses that was blocked by pre-incubation of cells with 1 uM CA (P<0.03, n=3). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). Thus our results suggest that activation of MR by ALDO leads to degranulation and NET production in neutrophils that may contribute to the inflammatory responses associated with MR activation in vivo. Furthermore, the association between degranulation and NET release implicates PDI as a novel regulator of MPO generated NET production. Disclosures:No relevant conflicts of interest to declare.

Differential Effects of Protein Disulfide Isomerase (PDI) Inhibitors On the Expression of Tissue Factor Procoagulant Activity (TF PCA) by AML Blasts

Abstract 1112Acute myelogenous leukemia (AML) may be complicated by DIC. TF plays a critical role in AML-associated coagulopathy, and induction of apoptosis significantly increases TF PCA on leukemic blasts, mainly via phosphatidylserine (PS) membrane exposure. However, PDI, a thiol isomerase with oxidoreductase and chaperone activity, has also been implicated in cellular TF regulation. Particularly, PDI inhibitors have been shown to exert antithrombotic activity in animal models. Besides its predominant localization in the endoplasmic reticulum, PDI is present on cell surfaces, where it may represent a promising therapeutic target. We investigated the effect of PDI inhibitors on the expression of TF PCA by leukemic HL60 and THP1 cells to explore their potential as anticoagulant drugs for the prevention and/or treatment of AML-associated DIC. Using a fluorescence-based insulin reduction assay, we confirmed inhibition of recombinant human PDI by bacitracin and quercetin-3-rutinoside (also known as rutin and recently shown to be a specific PDI inhibitor) with IC50 values of 0.6 mM and 14 μM, respectively, showing >95% inhibition at 1 mM (bacitracin) and 50 μM (rutin). Significant insulin reductase activity was observed on HL60 cells, and this activity was inhibited by 75% and 49% using 1 mM bacitracin and 100 μM rutin, respectively, suggesting the presence of additional, PDI-independent thiol isomerase activity. Short-term treatment with 100 μM rutin for 15 min also inhibited TF PCA on HL60 cells by 37%. Importantly, the inhibitory effect of rutin on cell-associated PDI and TF activity was completely abolished by cell washing, confirming previous evidence that rutin is a reversible PDI inhibitor. When HL60 cells were exposed to rutin (100 μM) for 24 hrs, cell-associated TF PCA was increased 2.3-fold (P<0.01), an effect that was accompanied by enhanced PS exposure, as assessed by annexin V-FITC binding (positive cells, 32±11 vs. 10±4%; P<0.01), and increased PCA of cellular microparticles (MPs) isolated from culture supernatants, as evidenced by the thrombin generation parameters lag phase (LP, 14±1 vs. 19±4 min), peak thrombin (PT, 55±17 vs. 22±14 nM), and area under the curve (AUC, 1193±329 vs. 476±347 nM*min; P<0.01). Interestingly, treatment with 100 μM rutin also resulted in a 1.7-fold increase in total cellular TF antigen (P=0.07). The effects of long-term incubation with bacitracin (1 mM) were even more pronounced, involving an 8.3-fold and 4.6-fold increase in cell-associated TF PCA and total cellular TF antigen, respectively. PS exposure (45±9%) and shedding of procoagulant MPs (LP, 7±1 min; PT, 175±49 nM; AUC, 2756±402 nM*min) were also significantly increased. While neither short-term nor long-term exposure to rutin affected TF PCA on THP1 cells, co-incubation with rutin dose-dependently (10–100 μM) inhibited daunorubicin-induced TF PCA in this cell model, an effect that could not be explained by decreased PS exposure. Importantly, both the reaction pattern of HL60 and that of THP1 cells were reproduced ex vivo using myeloblasts from AML patients. In summary, our findings suggest a highly complex and context-dependent role of PDI in leukemic-cell TF PCA expression. While short-term exposure to rutin can reversibly inhibit both PDI and TF activity, long-term exposure may result in significantly increased cellular TF PCA and MP shedding, pointing to a possible role of PDI in PS homeostasis, cytoskeleton rearrangement, and/or TF recycling. In addition, induction of leukemic-cell apoptosis and necrosis by cytotoxic drugs, which is associated with an early loss in membrane integrity and enhanced accessibility of cytoplasmic enzymes, may involve an additional role of (intracellular) PDI in the efficient presentation of TF PCA by AML blasts. Disclosures:No relevant conflicts of interest to declare.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

Altered Oxido-Reductive State in the Diabetic Heart: Loss of Cardioprotection due to Protein Disulfide Isomerase

Diabetes is associated with an increased risk of heart failure, in part explained by endoplasmic reticulum stress and apoptosis. Protein disulfide isomerase (PDI) prevents stressed cardiomyocytes apoptosis. We hypothesized that diabetes impairs PDI function by an alteration in its oxido-reductive state. Myocardial biopsies harvested from the anterolateral left ventricular wall from diabetic (n = 7) and nondiabetic (n = 8) patients were used to assess PDI expression and cardiomyocyte death. A mouse model of diabetes (streptozotocin injection, 130 mg/mL) was used to study PDI expression and its redox state after ischemia/reperfusion injury induced by 30-min occlusion of the left anterior coronary artery followed by reperfusion. Transthoracic echocardiography was performed to assess cardiac remodeling after 1 wk. Western blot analysis was used to analyze PDI expression, and methoxy-polyethyleneglycol-maleimide was used to assess its redox state. Dehydroascorbate (DHA) administration was used to restore the PDI redox state. Diabetic patients had a greater number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells than nondiabetic patients despite a greater myocardial PDI expression suggesting altered PDI function. Diabetic mice had a worse postinfarction remodeling associated with an altered PDI redox state. DHA treatment restored functional PDI redox state and ameliorated post-myocardial infarction remodeling. An increase in PDI levels with a paradoxical decrease of its active form occurs in the diabetic heart after ischemia and may explain the lack of protective effects of PDI in diabetes. Restoration of PDI redox state prevents adverse remodeling. The potential significance of these findings deserves to be validated in a clinical setting.

Biological Functions of Protein Disulfide Isomerase as a Target of Phenolic Endocrine-disrupting Chemicals

Endocrine disrupting chemicals (EDCs) are widespread in the environment and suspected of interfering with endocrine homeostasis. Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl)propane, an EDC, has been widely used throughout the world as an industrial chemical. BPA is a developmental, neural, and reproductive toxicant that mimics estrogen and can interfere with growth and body function. In a recent study, a BPA-binding protein was isolated from P2 fractions of rat brain, and identified as protein disulfide isomerase (PDI). PDI has been studied extensively as a key enzyme involved in the formation of the correct pattern of disulfide bonds in proteins. PDI is also known to be a membrane-associated 3,3',5-triiodo-L-thyronine (T3)-binding protein. BPA inhibits the binding of T3 to PDI and isomerase activity of PDI. PDI has four consecutive domains, a, b, b', and a', each with a thioredoxin fold. The domains a and a' have a catalytically active Cys-Gly-His-Cys motif, while b and b' have substrate-binding sites. To address the chemical structural requirements of BPA for the inhibitory effect of PDI, several BPA analogs were tested, suggesting that the phenolic structure is important for BPA to affect PDI functions. To investigate the biological effects of BPA on PDI functions, GH3 cells were used. GH3 is a rat pituitary tumor cell line and releases growth hormone (GH) via the T3 receptor. Over-expression of PDI suppresses the T3-induced release of GH and when the cells were treated with BPA together with T3, the amount of GH released was much increased. Thus, PDI plays an important role in the hypothalamic-pituitary-thyroid axis. In this review, the possible involvement and biological significance especially in the central nervous system, of PDI as a target of phenolic environmental chemicals, are discussed.