Domain Of Protein Disulfide Isomerase Research Articles

Mudanpioside C Discovered from Paeonia suffruticosa Andr. Acts as a Protein Disulfide Isomerase Inhibitor with Antithrombotic Activities.

Paeonia suffruticosa Andr. is a well-known landscape plant worldwide and also holds significant importance in China due to its medicinal and dietary properties. Previous studies have found that Cortex Moutan (CM), the dried root bark of P. suffruticosa, showed antiplatelet and cardioprotective effects, although the underlying mechanism and active compounds remain to be revealed. In this study, protein disulfide isomerase (PDI) inhibitors in CM were identified using a ligand-fishing method combined with the UHPLC-Q-TOF-MS assay. Further, their binding sites and inhibitory activities toward PDI were validated. The antiplatelet aggregation and antithrombotic activity were investigated. The results showed that two structurally similar compounds in CM were identified as the inhibitor for PDI with IC50 at 3.22 μM and 16.73 μM; among them Mudanpioside C (MC) is the most effective PDI inhibitor. Molecular docking, site-directed mutagenesis, and MST assay unequivocally demonstrated the specific binding of MC to the b'-x domain of PDI (Kd = 3.9 μM), acting as a potent PDI inhibitor by interacting with key amino acids K263, D292, and N298 within the b'-x domain. Meanwhile, MC could dose-dependently suppress collagen-induced platelet aggregation and interfere with platelet activation, adhesion, and spreading. Administration of MC can significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings present a promising perspective on the antithrombotic properties of CM and highlight the potential application of MC as lead compounds for targeting PDI in thrombosis therapy.

Galloylated Polyphenols Represent a New Class of Antithrombotic Antagonists of Thiol Isomerases

Protein disulfide isomerase (PDI) family thiol isomerases are multidomain oxidoreductases that function in thrombus formation. PDI antagonism or targeted genetic deletion blocks thrombus formation in several murine models of thrombosis. PDI has a domain structure of a-b-b'-a' in which the a and a' domains contain the Cys-Gly-His-Cys catalytic motifs responsible for disulfide shuffling and the b and b' domains are the substrate binding domains. We have previously identified flavonoid quercetins and bepristats as two structure independent classes of PDI inhibitors that act by binding the hydrophobic pocket in the b' domain and eliciting allosteric modifications that impair oxidoreductase activity. While screening a flavonoid library for inhibitors of the SARS-CoV-2 main protease (Mpro), we identified a Mpro inhibitor termed PGHG that blocked PDI reductase activity with an IC 50 of 1.5+0.6 µM. When tested against isolated domains of PDI, PGHG inhibited a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Supportive of an activity at the a and/or a' domains, PGHG inhibited other vascular thiol isomerases including ERp5, ERp46, ERp57, and ERp72 with IC 50s of 1.5, 3.9, 3.3, and 7.8 µM, respectively. When tested in a cremaster muscle murine model of thrombus formation, PGHG (25 mg/kg) inhibited platelet accumulation to 18+6% of control levels and fibrin formation to 21+3% of controls (Fig. 1). In contrast, when tested in the mice tail transection model of bleeding, PGHG did not prolong the time to cessation of bleeding nor the total amount of blood loss. These results show that PGHG can inhibit thrombus formation without promoting bleeding. To better understand the mechanism by which PGHG inhibits PDI, we evaluated other galloylated polyphenols for their ability to inhibit PDI (Fig. 2). These studies identified galloylated polyphenols with structurally unrelated backbones, including theaflavin 3-3'-digallate (TFDG) and tannic acid (TA), that inhibited PDI. In contrast, related galloylated analogs such as epigallocatechin gallate (EGCG), and epicatechin-3-gallate (EGC) did not inhibit PDI. Similarly, the isolated backbones of active compounds, including pinocembrin (backbone of PGHG), theaflavin (backbone of TFDG), and gallate (backbone of TA) failed to inhibit PDI. Like PGHG, TFDG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it, like PGHG, acts on the a and a' domains of PDI. These structure-activity relationships show that a subgroup of galloylated polyphenols can inhibit PDI via interactions with the a and/or a' domains. Thrombosis is a common complication of COVID19. Our evaluation of plasma PDI levels in patients with mild (n = 16), moderate (n = 21), and severe (n = 23) COVID19 showed that PDI levels increase according to the level of disease severity. Since previous studies have identified galloylated polyphenols that inhibit SARS-CoV-2 replication in vitro and in vivo (Furukawa, Sci Rep, 2021), we evaluated the PGHG for its ability to inhibit the proteolytic activity of SARS-CoV-2 Mpro and to block SARS-CoV-2 in a Vero E6-based viral replication assay. PGHG inhibited Mpro with an IC 50 of 6.5 µM and SARS-CoV-2 replication with an IC 50 of 4.9 µM. To better understand the interaction of PGHG with Mpro, we performed all-atom, explicit water molecular dynamic simulations . These simulations showed an overall binding energy (ΔG total) of -58.64+3.17 and demonstrated hydrogen bonding between the galloyl group of PGHG and Mpro Cys145, the active site cysteine of the protease. Similarly, published molecular dynamic simulations of TA with Mpro show hydrogen bonding of TA with Mpro Cys145 (Haddad et al., Int J Mol Sci, 2022). These observations suggest a mechanism wherein steric interactions of select galloylated polyphenols position a galloyl group for interactions with active site cysteines, as occur in both PDI and Mpro, inhibiting their activity. These studies thus identify a group of orally available galloylated polyphenols that modify vascular thiol isomerases, inhibit thrombus formation, and block SARS-CoV-2 replication. Galloyltransferases are found in many plants and galloylated polyphenols are abundant in commonly consumed beverages such as teas. Members of this class of compounds may be useful in the setting of COVID19 as inhibitors of thrombosis and viral replication.

Inhibition of Sars-Cov-2 Viral Replication and In Vivo Thrombus Formation By a Novel Plant Flavonoid

Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3“-galloyl-4”,6“-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh , inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

De-dimerization of PTB is catalyzed by PDI and is involved in the regulation of p53 translation.

Polypyrimidine tract-binding protein (PTB) is an RNA binding protein existing both as dimer and monomer and shuttling between nucleus and cytoplasm. However, the regulation of PTB dimerization and the relationship between their functions and subcellular localization are unknown. Here we find that PTB presents as dimer and monomer in nucleus and cytoplasm respectively, and a disulfide bond involving Cysteine 23 is critical for the dimerization of PTB. Additionally, protein disulfide isomerase (PDI) is identified to be the enzyme that catalyzes the de-dimerization of PTB, which is dependent on the CGHC active site of the a’ domain of PDI. Furthermore, upon DNA damage induced by topoisomerase inhibitors, PTB is demonstrated to be de-dimerized with cytoplasmic accumulation. Finally, cytoplasmic PTB is found to associate with the ribosome and enhances the translation of p53. Collectively, these findings uncover a previously unrecognized mechanism of PTB dimerization, and shed light on the de-dimerization of PTB functionally linking to cytoplasmic localization and translational regulation.

Rutin Increases Shear-Dependent TGF-β1 Activation By Interacting with Putative PDI Binding Sites: Role of Oxidation in Shear-Dependent TGF-β1 Activation

TGF-β1 is a very potent cytokine that, when activated, exerts both physiological and pathological functions, but the mechanism of activation remains unclear. Platelets contain 40-100-fold more TGF-β1 than other cell types and release TGF-β1 in an inactive latent complex comprised of mature TGF-β1 bound with latency-associated peptide and latent-TGF-β1 binding protein 1. We previously demonstrated that shear force activates latent TGF-β1 released from platelets in vitro, and that thiol-disulfide exchange contributes to this process. We also showed that protein disulfide isomerase (PDI) regulates shear-mediated TGF-β1 activation differentially, depending on whether PDI remains in its oxidized or reduced state. To test whether oxidation is required for latent TGF-β1 activation, we subjected platelet releasates to physiological shear/stirring for 2 hours in a closed chamber with room air or argon gas. We found ~10-fold increase in TGF-β1 activation compared to unsheared samples in room air, while the samples sheared under argon gas resulted in ~25% lower TGF-β1 activation (21 ± 1.0 % in room air vs. 16 ± 2.0 % of total TGF-β1 in argon gas, p=0.03). We also found that adding H2O2 to the platelet releasate resulted in increased shear-dependent TGF-β1 activation (active TGF-β1 was 16 ± 2.0 % in sheared samples and 22 ± 2.2 % of total TGF-β1 in sheared with H2O2 samples, p=0.03), indicating that oxidation during shear contributes to TGF-β1 activation. Since PDI can catalyze both oxidation and reduction of substrates, and because we previously showed that the reduced form of PDI inhibited shear-dependent TGF-β1 activation, we hypothesized that blocking the reduction function of PDI may increase shear-dependent TGF-β1 activation. To test this hypothesis, we assessed the effect of quercetin-3-rutinoside (rutin), a specific PDI inhibitor identified by high throughput screening, on shear-induced TGF-β1 activation. Rutin increased shear-induced TGF-β1 activation in a dose-dependent manner in that > 2.0 fold higher TGF-β1 activity was observed with 25 uM rutin, a concentration that blocked 55 to 80% of insulin reduction by PDI, confirming that rutin increases shear-dependent TGF-β1 activation by blocking the reduction function of PDI. We then assessed how rutin blocks PDI reduction function using PyMol modeling to identify the most likely interactions between rutin and PDI domains. After adjusting for the possible rotations of likely bond orientations, and considering minimum steric forces, the best model identified strong pi-pi interactions between Trp396 of the b' domain of PDI with the quercetin moiety of rutin (Figure-1). This interaction may stabilize the active site sulfhydryls of PDI so they maintain an inactive conformation, which buries the active site sulfhydryls, causing a loss in their solvent accessibility and thus inhibiting PDI's reductase function and increasing TGF-β1 activation. We conclude that thiol isomerases differentially regulate shear-dependent TGF-β1 activation. Thus, thiol-disulfide exchange regulator, such as PDI may be a potential target for suppressing or enhancing TGF-β1 activation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Protein Disulfide Isomerase Ameliorates Vaso-Occlusion and Thrombosis in a Mouse Model of Sickle Cell Disease

IntroductionSickle cell disease (SCD) results from a single mutation in the β-globin gene. Altered erythrocyte physiology in SCD results in severe vascular complications, including vaso-occlusion (VOC) and painful crises along with various other manifestations, such as thrombosis and end organ damage. Extracellular protein disulfide isomerase (PDI) plays a critical role in the regulation of neutrophil recruitment, platelet activation and fibrin formation, processes implicated in the thrombo-inflammatory vasculopathy in SCD. We investigated the effect of a PDI inhibitor, isoquercetin, in combination with hydroxyurea, in TNFα-induced VOC and thrombosis in a mouse model of SCD. In addition, the effect of chronic PDI inhibition by isoquercetin on thrombo-inflammatory vasculopathy was also examined.MethodsHumanized TOWNES knock-in mice, containing human normal (βA/βA, control) or sickle (βS/βS, SCD) hemoglobin β chains, were used as a mouse model of SCD. PDI inhibition in the plasma was achieved by oral gavage or daily diet feeding of isoquercetin. Intravital microscopy was performed to determine vaso-occlusive events in the venules and thrombus formation in the arterioles in TNFα-stimulated mice. Plasma levels of PDI and parameters for coagulation and vascular inflammation were determined by ELISA. Neutrophil accumulation in the organs was determined as the myeloperoxidase (MPO) levels in tissue homogenates.ResultsCompared to control mice, SCD mice have elevated plasma PDI levels. Gavage of isoquercetin resulted in reduced PDI activity in the plasma of treated mice (38±10% inhibition, p <0.05). Isoquercetin ingestion increased leukocyte rolling flux fraction in TNFα-treated SCD mice as monitored using intravital microscopy, whereas the influence on leukocyte adhesion and shear rate was minimal. When a reversal agent for PDI inhibitor, b'x (the isolated substrate binding domain of PDI) was co-infused, the effect of isoquercetin on leukocyte rolling was reversed, suggesting this effect of isoquercetin is mediated by PDI inhibition. We also determined the effect of isoquercetin in combination with hydroxyurea, a treatment with established efficacy in SCD, on the vaso-occlusive events in our model. We confirmed that hydroxyurea treatment elevated the plasma nitric oxide level as determined by plasma nitrite/nitrate content (11.5±1.9 μM vs 5.0±1.9 μM, p <0.05). Consistent with previous studies, hydroxyurea decreased leukocyte adhesion while the rolling flux fraction was not changed. However, combined treatment with isoquercetin and hydroxyurea in SCD mice increased the rolling flux fraction (15±3% vs 6±1%, p <0.05), decreased leukocyte adhesion (158±13 mm-2 vs 289±24 mm-2, p <0.05), and increased vessel shear rate (302±18 s-1 vs 122±11 s-1, p <0.05). We investigated in vivo thrombus formation in the arterioles of TNFα-treated SCD mice using intravital microscopy following laser injury. Compared to the control mice, SCD mice have significantly enhanced fibrin generation but normal platelet accumulation. Both isoquercetin and hydroxyurea inhibited TNFα-induced platelet accumulation (62±36% AUC-Area Under the Curve with isoquercetin and 48±17% AUC with hydroxyurea of untreated SCD) and fibrin generation (30±6% AUC with isoquercetin and 44±9% AUC with hydroxyurea of untreated SCD). To study the effect of chronic isoquercetin treatment in SCD, control and SCD mice were fed isoquercetin for 4 weeks. Daily consumption of isoquercetin resulted in reduction of plasma PDI activity (18±5% inhibition, p <0.05). Consistent with the intravital microscopic data, chronic PDI inhibition reduced neutrophil migration and accumulation into the lung, as determined by the MPO level (481±85 ng/mg vs 829±140 ng/mg, p <0.05) in the tissue homogenate. Chronic treatment with isoquercetin also reduced plasma fibrinogen degradation products (FDP) (102±15 μg/ml vs 183±25 μg/ml, p <0.05) in SCD mice. In addition, a correlation (Pearson r=0.651) was observed between reducing plasma FDP and lung MPO levels.ConclusionsAcute and chronic PDI inhibition by isoquercetin may provide beneficial effect on vascular complications in a mouse model of SCD. PDI inhibition has the potential to ameliorate thrombo-inflammatory syndromes associated with SCD. These studies justify a longitudinal trial of PDI inhibitors in SCD. DisclosuresNo relevant conflicts of interest to declare.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase

Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail. In this study, we identified that in vitro and in vivo S-nitrosylation of C343 in the b’ domain of PDI occurs. Reduced recombinant human PDI (hPDI) reacted quickly with S-nitrosocompounds, with an observed increase in the expected S-nitrosylated species and the appearance of the disulfide state of the active sites. Both Mononitrosylated and dinitrosylated were observed from the S-nitrosylation of hPDI. Dinitrosylated species were S-nitrosylated both cysteines at active site. But, at least in part, mononitrosylated species were S-nitrosylated on cysteine 343 in the substrate binding b’ domain. Although active site S-nitrosylation is reversible by reduced glutathione, S-nitrosylation of C343 is comparative stable. S-nitrosylation of PDI in SH-SY5Y cells was observed after the S-nitrosocysteine (SNOC) treatment and S-nitrosylated PDI was still detected 24 h after removing SNOC. While wild-type PDI was S-nitrosylated, the level of S-nitrosylation of the C343S mutant in over-expressed cells was substantially lower and only wild-type PDI of S-nitrosylation remained 24 h after removing SNOC in over-expressed cells. Both of in vitro and in vivo results suggested that S-nitrosylation of C343 in PDI may be the causative effect on physiological changes in neurodegerenative disease patients, and may be useful for the drug development for neurodegenerative diseases.

Quercetin 3, 3′, 4′, 5, 7-O- pentasulfate (QPS): A novel activator of protein disulfide isomerase

AimsDesign and synthesis of a novel 3, 3′, 4′, 5, 7-O- pentasulfated Quercetin (QPS) as an activator of protein disulfide isomerase (PDI). Main MethodsBased on an in silico analysis we show that QPS binds to a and b domain of PDI (−7.4 kcal/mol) unlike PDI inhibitor quercetin 3-rutinoside (Q3R) that binds at substrate-binding domain b (−8.0 kcal/mol). QPS was synthesized and its structure validated using a combination of FTIR, NMR and mass spectral studies. Surprisingly, unlike Q3R QPS is shown to enhance the activity of purified recombinant PDI. Fluorometric and circular dichroism analysis showed that QPS binds to a unique site on PDI with marginal variations in its secondary structure. Key FindingsHypersulfation of quercetin reverses its inhibitory effect on PDI as addition of exogenous PDI and QPS in human plasma showed increase in rate of coagulation, unlike Q3R which shows a decrease. SignificanceThe results indicate QPS to be a novel enhancer of PDI activity that can influence the coagulation rates in plasma. QPS has therapeutic potential to control bleeding.

Design, Synthesis, and Biological Evaluation of Novel Allosteric Protein Disulfide Isomerase Inhibitors.

Protein disulfide isomerase (PDI) is responsible for nascent protein folding in the endoplasmic reticulum (ER) and is critical for glioblastoma survival. To improve the potency of lead PDI inhibitor BAP2 (( E)-3-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-1-yl)benzonitrile), we designed and synthesized 67 analogues. We determined that PDI inhibition relied on the A ring hydroxyl group of the chalcone scaffold and cLogP increase in the sulfonamide chain improved potency. Docking studies revealed that BAP2 and analogues bind to His256 in the b' domain of PDI, and mutation of His256 to Ala abolishes BAP2 analogue activity. BAP2 and optimized analogue 59 have modest thiol reactivity; however, we propose that PDI inhibition by BAP2 analogues depends on the b' domain. Importantly, analogues inhibit glioblastoma cell growth, induce ER stress, increase expression of G2M checkpoint proteins, and reduce expression of DNA repair proteins. Cumulatively, our results support inhibition of PDI as a novel strategy to treat glioblastoma.

The b′ domain of protein disulfide isomerase cooperates with the a and a′ domains to functionally interact with platelets

Essentials Protein disulfide isomerase (PDI) interacts with the αIIbβ3 integrin on platelets We generated PDI domain fragments and full-length PDI containing point mutations PDI interacts with αIIbβ3 through the b' domain, with the a and a' domains contributing This is the first report demonstrating PDI binding to a native protein on intact cells SUMMARY: Background Protein disulfide isomerase (PDI) is an oxidoreductase consisting of four domains arranged in the order a-b-b'-a' with an x-linker between the b' and a' domains. PDI binds to αIIb β3 integrin on activated platelets, and potentiates activation of this integrin through the C-terminal CGHC active-site motif. How PDI binds to platelet αIIb β3 is unknown. Objective and methods We used PDI domain fragments and full-length PDI containing point mutations to study inhibition of Alexa488-labeled PDI binding to thrombin-activated platelets. The effect of the PDI variants on platelet aggregation was tested. Results Only PDI fragments containing the b' domain bound to activated platelets. A double mutant of the b' domain had decreased binding, confirming the essential role of the b' domain. Addition of mutations in the a and a' domains further decreased binding, suggesting that these domains contribute to the interaction of PDI with platelets. The ability of the b' domain to interact directly with αIIb β3 was demonstrated with surface plasmon resonance, with contributions from the a and a' domains. The abb'x PDI fragment that binds to platelets but lacks the critical C-terminal active site inhibited platelet aggregation and invivo thrombosis. Moreover, site mutations in the a, b' and a' domains that resulted in partial binding to platelets partially recovered aggregation of PDI-null platelets. PDI mutants that did not bind showed no recovery. Conclusion PDI functionally interacts with αIIb β3 on platelets through the substrate-binding b' domain, with the a and a' domains contributing to efficient binding.

Inhibition of protein disulfide isomerase in glioblastoma causes marked downregulation of DNA repair and DNA damage response genes.

Aberrant overexpression of endoplasmic reticulum (ER)-resident oxidoreductase protein disulfide isomerase (PDI) plays an important role in cancer progression. In this study, we demonstrate that PDI promotes glioblastoma (GBM) cell growth and describe a class of allosteric PDI inhibitors that are selective for PDI over other PDI family members.Methods: We performed a phenotypic screening triage campaign of over 20,000 diverse compounds to identify PDI inhibitors cytotoxic to cancer cells. From this screen, BAP2 emerged as a lead compound, and we assessed BAP2-PDI interactions with gel filtration, thiol-competition assays, and site-directed mutagenesis studies. To assess selectivity, we compared BAP2 activity across several PDI family members in the PDI reductase assay. Finally, we performed in vivo studies with a mouse xenograft model of GBM combining BAP2 and the standard of care (temozolomide and radiation), and identified affected gene pathways with nascent RNA sequencing (Bru-seq).Results: BAP2 and related analogs are novel PDI inhibitors that selectively inhibit PDIA1 and PDIp. Though BAP2 contains a weak Michael acceptor, interaction with PDI relies on Histidine 256 in the b' domain of PDI, suggesting allosteric binding. Furthermore, both in vitro and in vivo, BAP2 reduces cell and tumor growth. BAP2 alters the transcription of genes involved in the unfolded protein response, ER stress, apoptosis and DNA repair response.Conclusion: These results indicate that BAP2 has anti-tumor activity and the suppressive effect on DNA repair gene expression warrants combination with DNA damaging agents to treat GBM.

Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells

A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.

Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/μg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC₅₀ and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

Advances in vascular thiol isomerase function.

The present review will provide an overview of several recent advances in the field of vascular thiol isomerase function. The initial observation that protein disulfide isomerase (PDI) functions in thrombus formation occurred approximately a decade ago. At the time, there was little understanding regarding how PDI or other vascular thiol isomerases contribute to thrombosis. Although this problem is far from solved, the past few years have seen substantial progress in several areas that will be reviewed in this article. The relationship between PDI structure and its function has been investigated and applied to identify domains of PDI that are critical for thrombus formation. The mechanisms that direct thiol isomerase storage and release from platelets and endothelium have been studied. New techniques including kinetic-based trapping have identified substrates that vascular thiol isomerases modify during thrombus formation. Novel inhibitors of thiol isomerases have been developed that are useful both as tools to interrogate PDI function and as potential therapeutics. Human studies have been conducted to measure circulating PDI in disease states and evaluate the effect of oral administration of a PDI inhibitor on ex-vivo thrombin generation. Current findings indicate that thiol isomerase-mediated disulfide bond modification in receptors and plasma proteins is an important layer of control of thrombosis and vascular function more generally.

Soluble expression and purification of bioactive interleukin 33 in E. coli

Interleukin-33 (IL-33) is one of the important alarmins of the immune system and possesses dual functions as an anti- or pro-inflammatory molecule. The production of this cytokine in E. coli is hampered by the insoluble expression in the cytoplasm, resulting in inclusion body formation. In this study, the expression of IL-33 was optimized by fusing the N-terminus of IL-33 with several solubilizing tags that act as chaperones for proper protein folding: maltose binding protein (MBP), b´a´ domain of protein disulfide isomerase (PDIb´a´) and glutathione Stransferase (GST). The expression of the fusion proteins was stimulated by 0.5 mM IPTG at different temperatures, 37, 30, 25, and 18°C. As a result, IL-33 was expressed highly and in soluble form in the cytoplasm of E. coli when fused with MBP or PDIb´a´ tags in the presence of 0.5 mM IPTG at 25 or 30°C. We describe a simple purification procedure of IL-33 from the PDIb´a´-IL-33 construct using immobilized metal affinity chromatography (IMACs) with supplementary of tobacco etch virus (TEV) protease for tag removal. The high bioactivity of purified IL-33 on the proliferation and activation of macrophages was confirmed by MTT and nitrite releasing assays using RAW 264.7 These data show an improved method for producing high grade and yield IL-33.

A Non-Classical Member of the Protein Disulfide Isomerase Family, PDI7 of Arabidopsis thaliana, Localizes to the cis-Golgi and Endoplasmic Reticulum Membranes.

Members of the protein disulfide isomerase (PDI)-C subfamily are chimeric proteins containing the thioredoxin (Trx) domain of PDIs, and the conserved N- and C-terminal Pfam domains of Erv41p/Erv46p-type cargo receptors. They are unique to plants and chromalveolates. The Arabidopsis genome encodes three PDI-C isoforms: PDI7, PDI12 and PDI13. Here we demonstrate that PDI7 is a 65 kDa integral membrane glycoprotein expressed throughout many Arabidopsis tissues. Using a PDI7-specific antibody, we show through immunoelectron microscopy that PDI7 localizes to the endoplasmic reticulum (ER) and Golgi membranes in wild-type root tip cells, and was also detected in vesicles. Tomographic modeling of the Golgi revealed that PDI7 was confined to the cis-Golgi, and accumulated primarily at the cis-most cisterna. Shoot apical meristem cells from transgenic plants overexpressing PDI7 exhibited a dramatic increase in anti-PDI7 labeling at the cis-Golgi. When N- or C-terminal fusions between PDI7 and the green fluorescent protein variant, GFP(S65T), were expressed in mesophyll protoplasts, the fusions co-localized with the ER marker, ER-mCherry. However, when GFP(S65T) was positioned internally within PDI7 (PDI7-GFPint), the fusion strongly co-localized with the cis-Golgi marker, mCherry-SYP31, and faintly labeled the ER. In contrast to the Golgi-resident fusion protein (Man49-mCherry), PDI7-GFPint did not redistribute to the ER after brefeldin A treatment. Protease protection experiments indicated that the Trx domain of PDI7 is located within the ER/Golgi lumen. We propose a model where PDI-C isoforms function as cargo receptors for proteins containing exposed cysteine residues, cycling them from the Golgi back to the ER.

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.

Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b′x Domain

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.

Redox-coupled structural changes of the catalytic a′ domain of protein disulfide isomerase

Protein disulfide isomerase functions as a folding catalyst in the endoplasmic reticulum. Its b′ and a′ domains provide substrate-binding sites and undergo a redox-dependent domain rearrangement coupled to an open–closed structural change. Here we determined the first solution structure of the a′ domain in its oxidized form and thereby demonstrate that oxidation of the a′ domain induces significant conformational changes not only in the vicinity of the active site but also in the distal b′-interfacial segment. Based on these findings, we propose that this conformational transition triggers the domain segregation coupled with the exposure of the hydrophobic surface.