Protein Disulfide Isomerase Reductase Activity Research Articles

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.

Isoquercetin for thromboinflammation in sickle cell disease: a randomized double-blind placebo-controlled trial

AbstractData from a small trial in patients with cancer suggest that isoquercetin (IQ) treatment lowered thrombosis biomarkers and prevented clinical thrombosis, but, to our knowledge, no studies of IQ have been conducted to target thromboinflammation in adults with sickle cell disease (SCD). We conducted a randomized, double-blind, placebo-controlled trial in adults with steady-state SCD (hemoglobin SS [HbSS], HbSβ0thal, HbSβ+thal, or HbSC). The primary outcome was the change in plasma soluble P-selectin (sP-selectin) after treatment compared with baseline, analyzed in the intention-to-treat population. Between November 2019 and July 2022, 46 patients (aged 40 ± 11 years, 56% female, 75% under hydroxyurea treatment) were randomized to receive IQ (n = 23) or placebo (n = 23). IQ was well tolerated and all the adverse events (AEs; n = 21) or serious AEs (n = 14) recorded were not attributable to the study drug. The mean posttreatment change for sP-selectin showed no significant difference between the treatment groups (IQ, 0.10 ± 6.53 vs placebo, 0.74 ± 4.54; P = .64). In patients treated with IQ, whole-blood coagulation (P = .03) and collagen-induced platelet aggregation (P = .03) were significantly reduced from the baseline. Inducible mononuclear cell tissue factor gene expression and plasma protein disulfide isomerase reductase activity were also significantly inhibited (P = .003 and P = .02, respectively). Short-term fixed-dose IQ in patients with SCD was safe with no off-target bleeding and was associated with changes from the baseline in the appropriate direction for several biomarkers of thromboinflammation. The trial was registered at www.clinicaltrials.gov as #NCT04514510.

Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation

BackgroundOxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines. ObjectivesWe hypothesized that PDI is sulfenylated during oxidative stress, contributing to the prothrombotic potential of PDI. MethodsBiochemical and enzymatic assays using purified proteins, platelet and endothelial cell assays, and in vivo murine thrombosis studies were used to evaluate the role of oxidative stress in PDI sulfenylation and prothrombotic activity. ResultsPDI exposure to oxidants resulted in the loss of PDI reductase activity and simultaneously promoted sulfenylated PDI generation. Following exposure to oxidants, sulfenylated PDI spontaneously converted to disulfided PDI. PDI oxidized in this manner was able to transfer disulfides to protein substrates. Inhibition of sulfenylation impaired disulfide formation by oxidants, indicating that sulfenylation is an intermediate during PDI oxidation. Agonist-induced activation of platelets and endothelium resulted in the release of sulfenylated PDI. PDI was also sulfenylated by oxidized low-density lipoprotein (oxLDL). In an in vivo model of thrombus formation, oxLDL markedly promoted platelet accumulation following an arteriolar injury. PDI oxidoreductase inhibition blocked oxLDL-mediated augmentation of thrombosis. ConclusionPDI sulfenylation is a critical posttranslational modification that is an intermediate during disulfide PDI formation in the setting of oxidative stress. Oxidants generated by vascular cells during activation promote PDI sulfenylation, and interference with PDI during oxidative stress impairs thrombus formation.

Effects of Isoquercetin on Plasma Protein Disulfide Isomerase Activity and Extracellular Vesicle Tissue Factor Antigen/Activity in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) results from a single mutation in the β-globin gene that causes hemoglobin polymerization, red cell sickling, vascular injury, and recurrent vaso-occlusive crises (VOC). Vascular protein disulfide isomerase (PDI) is shown to be critical for thrombus formation by facilitating neutrophil recruitment, platelet activation and vascular fibrin deposition, all processes implicated in sickle vasculopathy. Since plasma PDI is elevated in SCD and cell-surface associated PDI could promote tissue factor (TF) dependent thrombin generation, we investigated the effects of Isoquercetin (IQ) in patients with steady state SCD. Methods: RBC surface associated PDI was quantified by western blotting. Inhibitory effect of IQ on LPS-induced TF expression on sickle monocytes in vitro was determined using flow cytometry. We also measured in vivo the effect of exposure of IQ or placebo for at least 28 days on plasma PDI protein, PDI reductase activity, total plasma extracellular vesicles (EVs), total plasma TF+ EVs, EV-TF-dependent procoagulant activity and markers of coagulation (D-dimer and Thrombin-antithrombin Complexes) using previously published methods. We used plasma from steady state SCD patients participating in a randomized controlled trial (NCT04514510) obtained at baseline and 28 days after exposure to either Isoquercetin 1000mg once daily (n=22) or placebo (n=22). The patients were in steady state, at least 60 days remote from VOC or blood transfusion and on a stable dose of hydroxyurea. The Wilcoxon rank-sum test was used to perform significance testing for differences in the percent change in outcomes between the Isoquercetin and placebo groups. Results: Sickle RBCs had higher cell-surface associated PDI compared to ethnic matched healthy controls (fold change 5.09; p=0.028). In confirmatory experiments with RBC ghosts, extracellular PDI was cell-surface associated. Pretreatment with Quercetin (the active metabolite of Isoquercetin) reduced LPS-induced cell-surface TF expression on sickle monocytes in vitro in a dose dependent manner (Fig 1). Following Isoquercetin administration, there was no change in the percent reduction in PDI inhibitory activity in the IQ group (mean: 6.6, 95%CI [-21.6, 34.9]) compared to placebo group (mean; -31, 95%CI [-94, 32.6], p=0.63). The percent change from baseline in PDI antigen also did not differ between the IQ or placebo groups (IQ mean: 1.2, 95%CI [-11.3, 13.6]; placebo mean: 12, 95%CI [-6, 30.4], p=0.52) (Fig 1). There was no correlation between the percent change in PDI activity and antigen from baseline after treatment with IQ (Spearman's Rho, IQ: r2 =0.28; p=0.20 placebo: r2=0.10; p=0.67). Unexpectedly, total plasma EVs showed no difference between the IQ (mean: 38.4; 95%CI [-4.6, 81.5]) and the placebo group (mean: 9, 95%CI [-18, 37.3]; p=0.27) (Table 1) suggesting that IQ has no obvious anti-inflammatory effects. Moreover, plasma EVs were predominantly derived from RBCs and platelets as published previously by us and others, indicating no membrane stabilizing effect on sickle RBCs and platelets. Inhibiting PDI reductase activity could allosterically reduce TF procoagulant activity, but this hypothesis was untestable because the plasma EV-TF dependent procoagulant activity in this patient population was undetectable, possibly reflecting effective disease control by hydroxyurea. Conclusions: The anti-inflammatory effects of flavonoid quercetin in vitro do not appear to translate into measurable clinical benefits in vivo following short term treatment with IQ in steady state SCD. Trials of higher dose IQ and/or prolonged exposure duration could yield greater inhibition of plasma PDI activity that might reveal detectable clinical antithrombotic effects. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Paricalcitol protects against hydrogen peroxide-induced injury in endothelial cells through suppression of apoptosis.

The vascular endothelium is one of the main targets of oxidative stress which plays an important role in the pathophysiology of vascular damage. Recent studies show that vitamin D can positively regulate endothelial functions in various chronic diseases and in cases of increased oxidative stress. In our study, we investigated the restorative and protective potentials of paricalcitol which is frequently used in patients with chronic renal failure, a vitamin D analogue, in human umbilical vein endothelial cells (HUVEC) before and after H2O2-induced oxidative stress. Paricalcitol treatment after the oxidative stress induced by H2O2 increased cell viability in endothelial cells depending on the dose that was used. While paricalcitol (500 nM) decreased caspase-3 activity and mitochondrial membrane potential loss, it increased nitric oxide (NO) production and reduced glutathione (GSH) levels. Paricalcitol treatment before oxidative stress increased cell viability. It increased NO production and mitochondrial membrane potential while significantly reducing caspase-3 activity. While paricalcitol caused a significant inhibition of protein disulfide isomerase (PDI) reductase activity in healthy endothelial cells, it did not cause a significant change on the PDI reductase activity under oxidative stress conditions. Present study showed that paricalcitol has restorative and protective effects on endothelial cells against oxidative injury, but these effects occur at high concentrations of paricalcitol.

Shining a light on SSP4: A comprehensive analysis and biological applications for the detection of sulfane sulfurs

Fluorescent probes are useful tools for the detection of sulfane sulfurs in biological systems. In this work, we report the development of SSP4, a widely used probe generated in our laboratory. We describe its evolution, preparation, and physical/chemical properties. Fluorescence analyses of SSP4 determined its high selectivity and sensitivity to sulfane sulfurs, even with the interfering presence of other species, such as amino acids and metal ions. Protocols for using SSP4 in a relatively quick and simple manner for the detection of persulfidated proteins, including papain, BSA, and GAPDH were developed. The method was then applied to human protein disulfide isomerase (PDI), leading to the discovery that persulfidation can occur at PDI's non-active site cysteines, and that PDI reductase activity is affected by sulfane sulfur treatment. Protocols for using SSP4 for the bioimaging of exogenous and endogenous sulfane sulfurs in different -cell lines were also established. These results should guide further applications of SSP4.

Identification of Antithrombotic Natural Products Targeting the Major Substrate Binding Pocket of Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is a vital oxidoreductase. Extracellular PDI promotes thrombus formation but does not affect physiological blood hemostasis. Inhibition of extracellular PDI has been demonstrated as a promising strategy for antithrombotic treatment. Herein, we focused on the major substrate binding site, a unique pocket in the PDI b' domain, and identified four natural products binding to PDI by combining virtual screening with tryptophan fluorescence-based assays against a customized natural product library. These hits all directly bound to the PDI-b' domain and inhibited the reductase activity of PDI. Among them, galangin showed the most prominent potency (5.9 μM) against PDI and as a broad-spectrum inhibitor for vascular thiol isomerases. In vivo studies manifested that galangin delayed the time of blood vessel occlusion in an electricity-induced mouse thrombosis model. Molecular docking and dynamics simulation further revealed that the hydroxyl-substituted benzopyrone moiety of galangin deeply inserted into the interface between the PDI-b' substrate-binding pocket and the a' domain. Together, these findings provide a potential antithrombotic drug candidate and demonstrate that the PDI b' domain is a critical domain for inhibitor development. Besides, we also report an innovative high-throughput screening method for the rapid discovery of PDI b' targeted inhibitors.

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.

Ehrlichia chaffeensis EplA Interaction With Host Cell Protein Disulfide Isomerase Promotes Infection.

Ehrlichia chaffeensis is an obligate intracellular bacterium that invades monocytes to cause the emerging and potentially severe disease, monocytic ehrlichiosis. Ehrlichial invasion of host cells, a process that is essential for the bacterium's survival and pathogenesis, is incompletely understood. In this study, we identified ECH_0377, henceforth designated as EplA (E. chaffeensis PDI ligand A) as an E. chaffeensis adhesin that interacts with host cell protein disulfide isomerase (PDI) to mediate bacterial entry into host cells. EplA is an outer membrane protein that E. chaffeensis expresses during growth in THP-1 monocytic cells. Canine sera confirmed to be positive for exposure to Ehrlichia spp. recognized recombinant EplA, indicating that it is expressed during infection in vivo. EplA antiserum inhibited the bacterium's ability to infect monocytic cells. The EplA-PDI interaction was confirmed via co-immunoprecipitation. Treating host cell surfaces with antibodies that inhibit PDI and/or thioredoxin-1 thiol reductase activity impaired E. chaffeensis infection. Chemical reduction of host cell surfaces, but not bacterial surfaces with tris(2-carboxyethyl)phosphine (TCEP) restored ehrlichial infectivity in the presence of the PDI-neutralizing antibody. Antisera specific for EplA C-terminal residues 95-104 (EplA95−104) or outer membrane protein A amino acids 53-68 (OmpA53−68) reduced E. chaffeensis infection of THP-1 cells. Notably, TCEP rescued ehrlichial infectivity of bacteria that had been treated with anti-EplA95−104, but not anti-EcOmpA53−68. These results demonstrate that EplA contributes to E. chaffeensis infection of monocytic cells by engaging PDI and exploiting the enzyme's reduction of host cell surface disulfide bonds in an EplA C-terminus-dependent manner and identify EplA95−104 and EcOmpA53−68 as novel ehrlichial receptor binding domains.

Nitroxide 4-hydroxy-2,2′,6,6′-tetramethylpiperidine 1-oxyl (Tempol) inhibits the reductase activity of protein disulfide isomerase via covalent binding to the Cys400 residue on CXXC redox motif at the a′active site

Background and aimOxidative stress arising from inflammatory processes is a serious cause of cell and tissue damage. Tempol is an efficient antioxidant with superoxide dismutase-like activity. The purpose of this paper is to address the inhibition of protein disulfide isomerase (PDI), an essential redox chaperone whose active sites contain the Cys-Gly-His-Cys (CXXC) motif, by the nitroxide Tempol. ResultsIn the presence of Tempol (5–120 μM), the reductase activity of PDI was reversibly affected both in vitro and in activated mice neutrophils, with an IC50 of 22.9 ± 10.8 μM. Inhibitory activity was confirmed by using both the insulin method and fluorescent formation of eosin-glutathione (E-GSH). The capacity of Tempol to bind the enzyme was determined by EPR and mass spectrometry. EPR Tempol signal decreased in the presence of PDI while remained unaffected when PDI thiols were previously blocked with NEM. When total protein was analyzed, 1 and 4 molecules of Tempol were bound to the protein. However, only one was found to be covalently bound to PDI at the a′active site. More specifically, Cys400 was modified by Tempol. ConclusionWe have shown that the nitroxide Tempol acts as an inhibitor of PDI through covalent binding to the Cys400 of the protein structure. Since PDI is coupled with the assembly of the NADPH oxidase complex of phagocytes, these findings reveal a novel action of Tempol that presents potential clinical applications for therapeutic intervention to target PDI knockdown in pathological processes in which this protein is engaged.

Succination of Protein Disulfide Isomerase Links Mitochondrial Stress and Endoplasmic Reticulum Stress in the Adipocyte During Diabetes.

Protein succination by fumarate increases in the adipose tissue of diabetic mice and in adipocytes matured in high glucose as a result of glucotoxicity-driven mitochondrial stress. The endoplasmic reticulum (ER) oxidoreductase protein disulfide isomerase (PDI) is succinated in adipocytes that are matured in high glucose, and in this study we investigated whether succination would alter PDI oxidoreductase activity, directly linking mitochondrial stress and ER stress. Protein succination and the ER stress marker C/EBP homologous protein (CHOP) were diminished after pharmaceutical targeting of mitochondrial stress with the chemical uncoupler niclosamide in adipocytes matured in high-glucose concentrations. PDI was succinated by fumarate on both CXXC-containing active sites, contributing to reduced enzymatic activity. Succinated PDI decreased reductase activity in adipocytes matured in high glucose, and in db/db epididymal adipose tissue, in association with increased levels of CHOP. PDI succination was increased in fumarase knockdown adipocytes, leading to reduced PDI oxidoreductase activity, increased CHOP levels, and pro-inflammatory cytokine secretion, confirming the specific role of elevated fumarate levels in contributing to ER stress. In addition, PDI succination and ER stress were decreased, and PDI reductase activity was restored when exposure to chronic high glucose was limited, highlighting the importance of calorie restriction in the improvement of adipocyte metabolic function. These experiments identify PDI succination as a novel biochemical mechanism linking altered mitochondrial metabolism to ER stress in the adipocyte during diabetes. The current study demonstrates that early biochemical changes in mitochondrial metabolism have important implications for the development of adipocyte stress. Antioxid. Redox Signal. 27, 1281-1296.

Polyphenol Rich Extract of Syzygium cumini (L.) Skeels Leaves Decreases Platelet Aggregation and Inhibits Reductase Activity of Protein Disulfide Isomerase.

Platelet agreggation results from conformational changes in αIIbβIII integrin, which are dependent on protein disulfide isomerase (PDI) activity. Recently, PDI inhibition has been proposed as an antithrombotic mechanism, for that polyphenolic compounds have emerged as potential inhibitors. Our group has demonstrated that Syzygium cumini (L.) Skeels (Myrtacea) leaf contains multiple polyphenols, such as: gallic acid, miricetyn‐3‐a‐arabinoside, mirycetin deoxiexoside and quercetin‐3‐glycoside; which enable this species for anti‐platelet activity. Therefore, this study aimed to evaluate the effects of polyphenol rich extract (ERP) from S. cumini leaf on platelet aggregation and PDI reductase activity. Platelet‐rich plasma (PRP) from healthy volunteers (n=5) was incubated with ERP for 25 min at concentrations ranging from 10 to 1000μg/mL, previously to activation with 2.5 or 5μM ADP. ERP at the higher concentration inhibited platelet aggregation in 66 and 33%, respectively. Under thrombin stimulus (0.01 and 0.02 U/mL), ERP (1000 μg/mL) reduced platelet aggregation in ca. 35%. Moreover, ERP also inhibited PMA‐induced platelet aggregation by 29% in its higher concentration. To analyze the ERP effect on αIIbβIII integrin activation, flow citometry protocols were conducted in washed platelets similarly pre‐treated with ERP (10, 100 and 1000μg/mL) before activation with thrombin (0.02U/mL) and tagged with PAC–1 FITC antibody. ERP dose‐dependently reduced the integrin activation in presence of 10 μg/mL (21%), 100 μg/mL (27%) and 1000 μg/mL (33%). Finally, ERP effects on PDI reductase activity were also assessed at concentrations ranging from 0.1 to 100 μg/mL. A maximum inhibitory effect on PDI reductase activity of 77% was found for the higher concentration. Therefore, our data supports that ERP‐containing polyphenols from S. cumini leaf reduce platelet aggregation, probably through PDI inhibition, which demonstrates its prominent anti‐platelet property.Support or Funding InformationFinancial support: FAPEMA; UFMA

Novel antiplatelet role for a protein disulfide isomerase‐targeted peptide: evidence of covalent binding to the C‐terminal CGHC redox motif

Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.

Protein Succination‐A Biomarker of Mitochondrial Stress in Adipose Tissue Inhibits Protein Function and is Regulated by Nutrient Balance

S‐(2‐succino)cysteine (2SC) is an irreversible post‐translational modification formed by the reaction of fumarate with protein cysteines, also known as protein succination. 2SC is a biomarker of glucotoxicity driven mitochondrial stress in the adipocyte. Here, we investigated if 2SC is a mechanistic mediator of adipocyte stress via modification of the critical chaperone protein disulfide isomerase (PDI) and examined if 2SC levels could be regulated by caloric restriction.Fumarase knockdown or control 3T3‐L1 adipocytes were matured in normal (5 mM) or high (30 mM) glucose for 8 days, or in 30 mM glucose for 4 days and then switched to 5 mM glucose for 4 days. Adipocyte lysates and adipose tissue protein from control and diabetic mice were analyzed by immunoblotting with an anti‐2SC antibody. The fluorescent PDI specific substrate DiEosinGSSG was utilized to assess PDI enzymatic activity in adipocytes. LC‐MS/MS mass spectrometry was utilized to identify the specific site of recombinant PDI succination.2SC significantly increases in adipocytes matured in high glucose and in the adipose tissue of diabetic mice. Succination significantly lowers PDI reductase activity in fumarase knockdown adipocytes and in the adipose tissue of diabetic mice versus control. MS/MS analysis identified an important PDI active site cysteine as a target of succination. 2SC is turned over in adipocytes when maturation medium is returned to normal glucose.2SC inhibits PDI reductase activity facilitating adipocyte stress as PDI is no longer able to re‐fold proteins within the adipocyte. The turnover of succinated proteins when nutrient balance is restored illustrates the importance of a balanced diet in preventing obesity and diabetes‐related dysfunction in adipose tissue. Our data demonstrates a novel biochemical mechanism mediating nutrient induced stress in the adipocyte, and that this stress is ameliorated when reasonable caloric restriction is employed, ensuring healthy functional adipose tissue.Support or Funding InformationNational Institute of Health 1F31DK108559‐01(AMM), 1R56DK105087‐01(NF) and 1‐11‐JF‐13 from the American Diabetes Association (NF).

Adverse Outcomes Associated with Cigarette Smoke Radicals Related to Damage to Protein-disulfide Isomerase

Identification of factors contributing to the development of chronic obstructive pulmonary disease (COPD) is crucial for developing new treatments. An increase in the levels of protein-disulfide isomerase (PDI), a multifaceted endoplasmic reticulum resident chaperone, has been demonstrated in human smokers, presumably as a protective adaptation to cigarette smoke (CS) exposure. We found a similar increase in the levels of PDI in the murine model of COPD. We also found abnormally high levels (4-6 times) of oxidized and sulfenilated forms of PDI in the lungs of murine smokers compared with non-smokers. PDI oxidation progressively increases with age. We begin to delineate the possible role of an increased ratio of oxidized PDI in the age-related onset of COPD by investigating the impact of exposure to CS radicals, such as acrolein (AC), hydroxyquinones (HQ), peroxynitrites (PN), and hydrogen peroxide, on their ability to induce unfolded protein response (UPR) and their effects on the structure and function of PDIs. Exposure to AC, HQ, PN, and CS resulted in cysteine and tyrosine nitrosylation leading to an altered three-dimensional structure of the PDI due to a decrease in helical content and formation of a more random coil structure, resulting in protein unfolding, inhibition of PDI reductase and isomerase activity in vitro and in vivo, and subsequent induction of endoplasmic reticulum stress response. Addition of glutathione prevented the induction of UPR, and AC and HQ induced structural changes in PDI. Exposure to PN and glutathione resulted in conjugation of PDI possibly at active site tyrosine residues. The findings presented here propose a new role of PDI in the pathogenesis of COPD and its age-dependent onset.

Development Of Second Generation Thiol Isomerase Inhibitors To Prevent Thrombus Formation

Protein disulfide isomerase (PDI) is a widely expressed oxidoreductase that catalyzes the rearrangement of intramolecular disulfide bonds during protein maturation in the endoplasmic reticulum. However, PDI can also be secreted from both platelets and endothelial cells during thrombus formation. Inhibition of extracellular PDI in the vasculature using neutralizing antibodies or bacitracin blocks thrombus formation in pre-clinical studies. Our previous experiments identified quercetin-3-rutinoside as an inhibitor of PDI and a potent antithrombotic. Recently, we performed a high-throughput screen of the MLSMR library through the Molecular Libraries Probe Production Centers Network (MLPCN) to identify more potent and selective inhibitors of PDI. We now compare the activity of the most potent lead, ML359, to previously described PDI inhibitors and assess its antithrombotic activity.ML359 inhibited the reductase activity of PDI in the insulin turbidometric assay with an IC50 of 0.3-0.6 microM. By comparison, quercetin-3-rutinoside and juniferdin inhibited PDI with IC50s of 6-10 microM, while bacitracin inhibited PDI reductase activity with an IC50 of 100 microM. ML359 demonstrated no confirmed activity at <10 microM in any of the other of 380 biological assays in which it has been tested within the MLPCN. Additionally, ML359 was selective for PDI, failing to inhibit ERp5, ERp57, ERP72, thioredoxin or thioredoxin reductase activity. Inhibition of PDI by the ML359 was entirely reversible and it did not demonstrate toxicity in a HeLa cell assay.In preparation for pre-clinical studies, further evaluation of the biophysical properties of ML359 was performed. ML359 demonstrated acceptable solubility in PBS, human plasma and GSH. However, it showed poor stability in mouse plasma as well as in presence of liver microsomes. To optimize and improve these biophysical properties of the ML359, over 85 analogs of the lead were synthesized. Modulation of the ethyl substituent of the ester group with bulky isopropyl or t-butyl substituents increased the mouse plasma stability from ∼5% in ML359 to ∼75% and ∼95% in the analogs, respectively. These substitutions only minimally altered the potency of the compound (0.6-0.9 microM). The ML359 analogs demonstrated increased inhibitory activity in platelet aggregation assays, with inhibition at 30 microM increasing from 25% for ML359 to 97 and 100% for the isopropyl and t-butyl substituted analogs, respectively. The ability of ML359 to prevent thrombus formation in a laser injury mouse model was examined. Thrombus formation was monitored following laser injury of cremaster arterioles and the effect of ML359 infusion on platelet accumulation evaluated. ML359 exposure resulted in a significantly smaller thrombus that that observed in the animals infused with vehicle alone.ML359 and its active analogs have enhanced properties compared to the first generation PDI antagonists. These compounds are substantially more potent than previously described PDI inhibitors such as bacitracin and show significant potential as second generation inhibitors to probe PDI function in biological systems. ML359 analogs could ultimately be developed as antithrombotics that target PDI. Disclosures:No relevant conflicts of interest to declare.

Inhibitory Autoantibodies to Protein Disulfide Isomerase From Systemic Lupus Erythematosus Patients Are Prothrombotic

AbstractAbstract 392Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by loss of self-tolerance, aberrant autoantibody production and multiple organ damage. Treating the disease symptoms (flares) has remarkably improved the survival of SLE patients, but thrombosis remains a major negative prognostic factor. Currently available tests do not predict thrombotic events; hence there is a need for identification of novel biomarkers of thrombosis associated with SLE and characterization of the relevant pathophysiologic mechanisms. We have shown that inhibition of cell-surface protein disulfide isomerase (PDI) can regulate the thrombotic properties of the endothelium in vitro, through exposure of phosphatidylserine (PS). We therefore hypothesized that inhibitory autoantibodies to PDI may promote or enhance the prothrombotic phenotype in SLE.We developed a quantitative ELISA to measure the titers of anti-PDI autoantibodies present in the sera and performed a cross-sectional analysis of sera from 217 SLE patients and 99 healthy controls enrolled in the Oklahoma Lupus Cohort. Low levels of PDI autoreactivity were detected in 54 out of 99 healthy controls tested, with a median of 4.185 AU and an interquartile range (IQR) of 2.25–4.18. Anti-PDI autoantibodies were detected in 107 out of 217 SLE sera tested, with significantly higher PDI autoreactivity (median 5.982, IQR 2.43–11.23, p = 0.042 Mann-Whitney test). We defined elevated anti-PDI titers as those to be higher than the 95th percentile of serum anti-PDI levels in the control group. Thirty-five out of the 217 SLE patients (16%) exhibited anti-PDI titers above the 95th percentile cut-off, an incidence significantly higher (p=0.0057, Fisher’s exact test) than the control group (5/99, 5% by cut-off definition). Interestingly, the incidence of patients with elevated anti-PDI autoreactivity was doubled in SLE patients with secondary antiphospholipid syndrome (APS), 20 out of 89 (22%), as compared to SLE patients without APS (15 out of 128, 11%, p = 0.04 Fisher’s exact test). Accordingly, we found a significant correlation between the titers of anti-PDI autoantibodies and the anti-cardiolipin IgG levels (Spearman r= 0.212, P = 0.003), indicating a link with the presence of antiphospholipid antibodies. Since the APS diagnosis involves both the detection of antiphospholipid antibodies and the presence of clinical thrombotic events, this might be an indication that anti-PDI autoantibodies are associated with thrombosis in SLE.Clinical history revealed that SLE patients with elevated PDI autoreactivity had higher SLE Disease Activity Index (SLEDAI) scores than those with normal anti-PDI levels (P = 0.0256, Mann-Whitney test), indicating a correlation between anti-PDI levels and disease activity. We observed significant inverse correlations between anti-PDI titers and complement levels as measured by the total hemolytic complement assay (Spearman r = −0.134, P = 0.049), C3 levels (Spearman r = −0.169, P = 0.013) and C4 levels (Spearman r = −0.215, P < 0.001). While the biological significance of this correlation is under investigation, activation of the classical complement pathway on the surface of PS-exposing cells has been documented in the literature.We have shown that PDI inhibition on endothelial surface promotes PS exposure and a thrombotic phenotype. We tested the inhibitory properties of anti-PDI autoantibodies and found that 50% of the SLE sera with elevated anti-PDI titers inhibited PDI reductase activity in vitro, thus indicating that these autoantibodies can modulate PDI function. Furthermore, affinity-purified anti-PDI autoantibodies from SLE sera were able to induce a prothrombotic phenotype on endothelial cells in vitro as evidenced by tissue factor decryption and enhanced prothrombinase activity, a direct indicator that anti-PDI autoantibodies induce anionic phospholipid exposure in vascular cells.In conclusion, we found an increased incidence of elevated anti-PDI titers in SLE, especially in patients with antiphospholipid syndrome, that correlate with disease activity. These autoantibodies inhibit PDI enzymatic activity and are prothrombotic in vitro. To our knowledge this is the first report of autoantibodies directly inducing the exposure of anionic phospholipids on vascular cells with mechanistic implications for the pathophysiology of SLE and APS. Disclosures:No relevant conflicts of interest to declare.

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents

Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.

Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase

Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI inhibitors.

Discovery of a Small Molecule PDI Inhibitor That Inhibits Reduction of HIV-1 Envelope Glycoprotein gp120

Protein disulfide isomerase (PDI) is a promiscuous protein with multifunctional properties. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction. The entry of HIV-1 into cells is facilitated by the PDI-catalyzed reductive cleavage of disulfide bonds in gp120. PDI is regarded as a potential drug target because of its reduction activity. We screened a chemical library of natural products for PDI-specific inhibitors in a high-throughput fashion and identified the natural compound juniferdin as the most potent inhibitor of PDI. Derivatives of juniferdin were synthesized, with compound 13 showing inhibitory activities comparable to those of juniferdin but reduced cytotoxicity. Both juniferdin and compound 13 inhibited PDI reductase activity in a dose-dependent manner, with IC(50) values of 156 and 167 nM, respectively. Our results also indicated that juniferdin and compound 13 exert their inhibitory activities specifically on PDI but do not significantly inhibit homologues of this protein family. Moreover, we found that both compounds can inhibit PDI-mediated reduction of HIV-1 envelope glycoprotein gp120.