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Philip J. Hogg - One of the best experts on this subject based on the ideXlab platform.
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Protein Disulfide Isomerase in Thrombosis.
Seminars in thrombosis and hemostasis, 2015Co-Authors: Joyce Chiu, Freda Passam, Diego Butera, Philip J. HoggAbstract:Protein Disulfide Isomerase (PDI) is a 57-kDa oxidoreductase that facilitates cysteine thiol reactions inside and outside the cell. It mediates reduction or oxidation of Protein Disulfide bonds, thiol/Disulfide exchange reactions, and transfer of NO from one Protein thiol to another. It also has chaperone properties. PDI is actively secreted by most, if not all, of the cell types involved in thrombosis, binds to integrins on the cell surface, and circulates as a soluble Protein in blood. It plays a critical role in thrombosis in mice and presumably the same role in human thrombosis. Eight Proteins involved in thrombosis have been identified as PDI substrates; however, the role of this oxidoreductase in this process is not fully understood. Novel small-molecule PDI inhibitors have been developed and are being evaluated as antithrombotics in clinical trials. This combination of ongoing laboratory and clinical studies will greatly accelerate the pace of discovery and targeting of PDI function in thrombosis.
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Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by Protein Disulfide Isomerase.
Biochimica et Biophysica Acta, 1998Co-Authors: Kylie A. Hotchkiss, Lisa J. Matthias, Philip J. HoggAbstract:Thrombospondin-1 is a matrix Protein that inhibits proliferation, motility and sprouting of endothelial cells in vitro and angiogenesis in vivo. One mechanism by which thrombospondin-1 may influence endothelial cell biology is through interaction with the endothelial cell αvβ3 integrin receptor. This interaction is mediated via a cryptic Arg-Gly-Asp sequence in the C-terminal Ca2+-binding region of thrombospondin-1. Exposure of the Arg-Gly-Asp sequence is controlled by Disulfide interchange events in the Ca2+-binding loops and C-globular domain. Limited reduction of thrombospondin-1 by dithiothreitol exposes the Arg-Gly-Asp sequence which can bind to the αvβ3 integrin receptor and support endothelial cell spreading (X. Sun, K. Skorstengaard, D.F. Mosher, J. Cell Biol. 118 (1992) 693–701). Our aim was to identify possible physiological reductants that can mediate Arg-Gly-Asp exposure. We now report that Protein Disulfide Isomerase, which is known to catalyze Disulfide interchange in thrombospondin-1 and change its enzyme inhibitory properties and its binding to monoclonal antibodies, was secreted by bovine aortic endothelial cells and deposited on the cell surface. There was an average of ∼2.2 fg of Protein Disulfide Isomerase on the surface of a bovine aortic endothelial cell. Treatment of thrombospondin-1 with purified Protein Disulfide Isomerase enhanced adhesion of endothelial cells to thrombospondin-1 in an Arg-Gly-Asp-dependent manner through the αvβ3 integrin receptor and supported cell spreading. Both Ca2+-depleted and Ca2+-replete thrombospondin-1 were substrates for Protein Disulfide Isomerase. These results suggest that endothelial cell derived Protein Disulfide Isomerase may regulate Arg-Gly-Asp-dependent binding of thrombospondin-1.
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Catalysis of Disulfide isomerization in thrombospondin 1 by Protein Disulfide Isomerase
Biochemistry, 1996Co-Authors: Kylie A. Hotchkiss, Colin N. Chesterman, Philip J. HoggAbstract:Thrombospondin 1 is a multidomain glycoProtein from platelets and most cells that participates in diverse biological processes. The structure and some functional properties of thrombospondin 1 are regulated by Disulfide interchange in the Ca(2+)-binding repeats and C-globular domain. The recent identification of the enzyme, Protein Disulfide Isomerase, on the platelet surface suggested that Protein Disulfide Isomerase may catalyze Disulfide isomerization in platelet thrombospondin 1. Protein Disulfide Isomerase was found to form Disulfide-linked complexes with thrombospondin 1, which is consistent with Protein Disulfide Isomerase-mediated rearrangement of Disulfide bonds in thrombospondin 1. To quantitate Disulfide interchange in thrombospondin 1, perturbation of the enzyme inhibitory properties of platelet thrombospondin 1 were measured, specifically changes in the apparent dissociation constant for inhibition of neutrophil cathepsin G by thrombospondin 1. The inhibition constant increased > or = 10-14-fold following incubation of either Ca(2+)-replete or Ca(2+)-depleted thrombospondin 1 with Protein Disulfide Isomerase and reduced glutathione. The rate of Protein Disulfide Isomerase-catalyzed Disulfide interchange in thrombospondin 1 increased linearly with Protein Disulfide Isomerase concentration and the K(m) for reduced glutathione was 0.4 +/- 0.2 mM. Disulfide isomerization in both platelet and fibroblast thrombospondin 1 was probed by measuring perturbation in epitopes for two anti-thrombospondin 1 monoclonal antibodies. Antibody D4.6 binds to the C-terminal Ca(2+)-binding domains which are involved in Disulfide interchange, whereas antibody HB8432 binds toward the N-terminus of the thrombospondin 1 subunit. In accordance with the location of these epitopes, incubation of platelet thrombospondin 1 or fibroblast thrombospondin 1 with Protein Disulfide Isomerase and reduced glutathione resulted in 2-fold enhancement of binding of D4.6, whereas binding of HB8432 did not significantly change. In summary, Protein Disulfide Isomerase catalyzes Disulfide interchange in thrombospondin 1 which alters binding of neutrophil cathepsin G and antibody D4.6 to thrombospondin 1.
Mehdi Chenik - One of the best experts on this subject based on the ideXlab platform.
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A high-throughput turbidometric assay for screening inhibitors of Leishmania major Protein Disulfide Isomerase.
Journal of Biomolecular Screening, 2011Co-Authors: Noureddine Ben Khalaf, Geraldine De Muylder, Joseline Ratnam, James H. Mckerrow, Kenny Kean-hooi Ang, Michelle Arkin, Mehdi ChenikAbstract:The use of a high-throughput technique to perform a pilot screen for Leishmania major Protein Disulfide Isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, Protein Disulfide Isomerase (PDI) plays a crucial role in Protein folding by catalyzing the rearrangement of Disulfide bonds in substrate Proteins following their synthesis. LmPDI displays similar domain structure organization and functional properties to other PDI family members and is involved in Leishmania virulence. The authors used a method based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol. The screen of a small library of 1920 compounds was performed in a 384-well format and led to the identification of 27 compounds with inhibitory activity against LmPDI. The authors further tested the cytotoxicity of these compounds using Jurkat cells as well as their effect on Leishmania donovani amastigotes using high-content analysis. Results show hexachlorophene and a mixture of theaflavin monogallates inhibit Leishmania multiplication in infected macrophages derived from THP-1 cells, although the inhibitory effect on LmPDI enzymatic activity does not necessarily correlate with the antileishmanial activity.
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A high-throughput turbidometric assay for screening inhibitors of Leishmania major Protein Disulfide Isomerase.
Journal of biomolecular screening, 2011Co-Authors: Noureddine Ben Khalaf, Geraldine De Muylder, Joseline Ratnam, Kenny K. H. Ang, Michelle R. Arkin, James H. Mckerrow, Mehdi ChenikAbstract:The use of a high-throughput technique to perform a pilot screen for Leishmania major Protein Disulfide Isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, Protein Disulfide Isomerase (PDI) plays a crucial role in Protein folding by catalyzing the rearrangement of Disulfide bonds in substrate Proteins following their synthesis. LmPDI displays similar domain structure organization and functional properties to other PDI family members and is involved in Leishmania virulence. The authors used a method based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol. The screen of a small library of 1920 compounds was performed in a 384-well format and led to the identification of 27 compounds with inhibitory activity against LmPDI. The authors further tested the cytotoxicity of these compounds using Jurkat cells as well as their effect on Leishmania donovani amastigotes using high-content analysis. Results show hexachlorophene and a mixture of theaflavin monogall...
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A high-throughput turbidometric assay for screening inhibitors of Leishmania major Protein Disulfide Isomerase.
Journal of biomolecular screening, 2011Co-Authors: Noureddine Ben Khalaf, Geraldine De Muylder, Joseline Ratnam, Kenny K. H. Ang, Michelle R. Arkin, James H. Mckerrow, Mehdi ChenikAbstract:The use of a high-throughput technique to perform a pilot screen for Leishmania major Protein Disulfide Isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, Protein disulfi...
Brent R Stockwell - One of the best experts on this subject based on the ideXlab platform.
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Inhibitors of Protein Disulfide Isomerase suppress apoptosis induced by misfolded Proteins
Nature Chemical Biology, 2010Co-Authors: Benjamin G Hoffstrom, Anna Kaplan, Reka Letso, Ralf S Schmid, Gregory J Turmel, Donald C Lo, Brent R StockwellAbstract:Expression of a Huntington's-disease variant of huntingtin Protein causes accumulation of the chaperone Protein Disulfide Isomerase. This Protein is the target of compounds obtained from screening for those that can alleviate cell death promoted by the mutant huntingtin, and represents a new connection between Protein misfolding and cell death. A hallmark of many neurodegenerative diseases is accumulation of misfolded Proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link Protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving Protein Disulfide Isomerase (PDI), a Protein chaperone that catalyzes isomerization, reduction and oxidation of Disulfides. Through a small molecule screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin Protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated ER membranes and trigger apoptotic cell death via mitochondrial outer-membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon 1 and Aβ peptides processed from the amyloid precursor Protein. This pro-apoptotic function of PDI represents a new mechanism linking Protein misfolding and apoptotic cell death.
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inhibitors of Protein Disulfide Isomerase suppress apoptosis induced by misfolded Proteins
Nature Chemical Biology, 2010Co-Authors: Benjamin G Hoffstrom, Anna Kaplan, Reka Letso, Ralf S Schmid, Gregory J Turmel, Brent R Stockwell, Donald C LoAbstract:Expression of a Huntington's-disease variant of huntingtin Protein causes accumulation of the chaperone Protein Disulfide Isomerase. This Protein is the target of compounds obtained from screening for those that can alleviate cell death promoted by the mutant huntingtin, and represents a new connection between Protein misfolding and cell death.
Osamu Asami - One of the best experts on this subject based on the ideXlab platform.
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Enhancement of the activity of renatured lysozyme by Protein Disulfide Isomerase.
Journal of bioscience and bioengineering, 2007Co-Authors: Daisuke Nohara, Hiroshi Hizikata, Osamu AsamiAbstract:The coexistence of Protein Disulfide Isomerase (PDI) in the oxidative refolding of a fully reduced hen egg white lysozyme brought about a final recovered activity significantly exceeding 100% in addition to the expected acceleration effect. This increase could not be explained by the simple increase produced by suppressing aggregation. After examination of the starting material and assay system, it was concluded that PDI enhances the activity of renatured lysozyme.
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Disulfide bond formation in refolding of thermophilic fungal Protein Disulfide Isomerase.
Journal of bioscience and bioengineering, 2001Co-Authors: Takushi Harada, Eiji Kurimoto, Kenrou Tokuhiro, Osamu Asami, Tomoya Sakai, Daisuke Nohara, Koichi KatoAbstract:Disulfide bond formation in the refolding of thermophilic fungal Protein Disulfide Isomerase (PDI) was investigated. It was revealed that (i) a Disulfide bond buried inside the molecule is preferentially formed and contributes to the thermal stability and the isomerizing power of PDI, and (ii) formation of Disulfide bonds in active sites located on the molecular surface causes deformation of the optimum conformation resulting in a decrease in the thermal stability.
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Thermophilic fungal Protein Disulfide Isomerase.
Methods in enzymology, 1998Co-Authors: Tsutomu Kajino, Osamu Asami, Chie Miyazaki, Masana Hirai, Yukio Yamada, Shigezo UdakaAbstract:Publisher Summary For the study the authors have isolated and characterized a thermostable Protein Disulfide Isomerase (PDI) from a thermophilic fungus, Humicola insolens . The cDNA encoding the fungal PDI has been cloned and expressed in Bacillus brevis. PDIs from vertebrates and yeast are relatively heat labile. Even in the case of an algal enzyme that is most stable of the known PDIs, the stability against heat is not enough for industrial use of the enzyme. Hence, there is continuing interest in finding new, stable PDIs. Refolding activity using scrambled ribonuclease (RNase) as a substrate is discussed. The fungal PDI cDNA is expressed in a heterologous Protein production system using B. brevis as a host. In the purification process, anion-exchange chromatography, lectin affinity chromatography, and high-performance liquid chromatography are discussed. The chapter also presents the data for purification of PDI from the fungus, Humicola insolens . To amplify the DNA fragment around the N-terminal consensus region of fungal PDI, two oligonucleotides corresponding to the amino acid sequence are synthesized and used as primers for a reverse transcriptase-mediated polymerase chain reaction (RT-PCR).
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Purification of Protein Disulfide Isomerase from a thermophilic fungus.
Bioscience biotechnology and biochemistry, 1993Co-Authors: Hidehiko Sugiyama, Osamu Asami, Tsutomu Kajino, Yukio Yamada, Chie Idekoba, Fumihiko Hoshino, Shigezo UdakaAbstract:A Protein Disulfide Isomerase (PDI) was purified to homogeneity from the thermophilic fungus Humicola insolens by a rapid three-step procedure, anion-exchange chromatography, concanavalin A-affinity chromatography, and reverse phase high performance liquid chromatography. Forty-oneμg of PDI was obtained from 100 g of wet mycelium. Concanavalin A-Sepharose chromatography is available for purification of the fungal PDI, indicating that the enzyme is also glycosylated like the yeast PDI. The fungal PDI exists as a dimer (2x60kDa), has a pI of 3.5, and is fairly heat-stable. The amino acid composition of the PDI is similar to those of yeast and bovine liver PDI, and the high content of acidic amino acid residues agrees with the lower acidic pI.
Robert Flaumenhaft - One of the best experts on this subject based on the ideXlab platform.
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inhibition of Protein Disulfide Isomerase in thrombosis
Basic & Clinical Pharmacology & Toxicology, 2016Co-Authors: Roelof H Bekendam, Robert FlaumenhaftAbstract:: This MiniReview addresses our current understanding of the mechanisms by which Protein Disulfide Isomerase (PDI) mediates thrombus formation and discusses the potential of blocking thrombosis by targeting PDI. Thiol Isomerases are ubiquitous oxidoreductases primarily localized to the endoplasmic reticulum (ER) where they serve a critical role in Protein folding. PDI is the founding member of the thiol Isomerase family. Although PDI is an essential intracellular enzyme, it can participate in pathological processes once released from cells. In particular, PDI serves a critical role in thrombus formation, the underlying cause of myocardial infarction and stroke. Both platelets and endothelial cells secrete PDI upon vascular injury. Secreted PDI appears to activate multiple extracellular substrates in the vasculature, enabling the initiation of thrombus formation. As an essential component of thrombus formation, extracellular PDI represents a new target for pharmacological inhibition of clinical thrombosis. Quercetin-3-rutinoside, a flavonol highly abundant in common foods, inhibits PDI and blocks thrombus formation both in vitro and in vivo. Such observations have prompted clinical trials targeting PDI in thrombotic diseases.
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Therapeutic Implications of Protein Disulfide Isomerase Inhibition in Thrombotic Disease
Arteriosclerosis thrombosis and vascular biology, 2014Co-Authors: Robert Flaumenhaft, Bruce Furie, Jeffrey I. ZwickerAbstract:The study of thrombus formation has increasingly applied in vivo tools such as genetically modified mice and intravital microscopy to the evaluation of molecular and cellular mechanisms of thrombosis. Among several unexpected findings of this approach was the discovery that Protein Disulfide Isomerase serves an essential role in thrombus formation at sites of vascular injury. The observation that the commonly ingested quercetin flavonoid, quercetin-3-rutinoside, inhibits Protein Disulfide Isomerase and blocks thrombus formation in preclinical studies has set the stage for clinical trials using Protein Disulfide Isomerase antagonists as antithrombotics. Although the mechanisms by which Protein Disulfide Isomerase facilitates platelet activation and fibrin formation have yet to be elucidated, Protein Disulfide Isomerase antagonists are currently being developed as antithrombotics. This review will consider what is known about the role of Protein Disulfide Isomerase in platelet accumulation and fibrin generation with a focus on pharmacological strategies for blocking Protein Disulfide Isomerase activity in the context of thrombus formation. Potential indications and clinical trial design for testing the efficacy of Protein Disulfide Isomerase inhibition to reduce the incidence of thrombosis will be considered.
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Protein Disulfide Isomerase as an antithrombotic target
Trends in cardiovascular medicine, 2013Co-Authors: Robert FlaumenhaftAbstract:Protein Disulfide Isomerase (PDI) is a ubiquitously expressed oxidoreductase required for proper Protein folding. It is highly concentrated in the endoplasmic reticulum, but can also be released into the extracellular environment. Several in vivo thrombosis models have demonstrated that vascular PDI secreted by platelets and endothelial cells is essential for normal thrombus formation. Inhibition of extracellular PDI thus represents a potential strategy for antithrombotic therapy. Yet this approach requires the discovery of well-tolerated PDI inhibitors. A recent high-throughput screening identified the commonly ingested flavonoid, quercetin-3-rutinoside, as an inhibitor of PDI. Quercetin-3-rutinoside blocked thrombus formation at concentrations that are commonly ingested as nutritional supplements. The observation that a compound with Generally Recognized As Safe status inhibits PDI and blocks thrombosis in animal models forms a rationale for clinical trials evaluating PDI inhibitors as a new class of antithrombotics.
