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D. James Morré - One of the best experts on this subject based on the ideXlab platform.
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The Sulfonylurea-Inhibited NADH Oxidase Activity of HeLa Cell Plasma Membranes has Properties of a Protein Disulfide–Thiol Oxidoreductase with Protein Disulfide–Thiol Interchange Activity
Journal of Bioenergetics and Biomembranes, 1998Co-Authors: Pin-ju Chueh, Juliana Lawler, D. James MorréAbstract:Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of Protein thiols by reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that Protein Disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, Protein Disulfides of the membranes were reduced with a concomitant stoichiometric increase in Protein thiols. The increase in Protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC_50 of ca. 30 nM. LY181984, with an EC_50 of 30 nM, also inhibited a Protein Disulfide–thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent Disulfide reduction (NADH oxidation), and Protein Disulfide–thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding Protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p -chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the Protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as Protein Disulfide–thiol interchange coupled to cell enlargement.
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A Protein Disulfide-thiol interchange Protein with NADH: Protein Disulfide reductase (NADH oxidase) activity as a molecular target for low levels of exposure to organic solvents in plant growth
Human & Experimental Toxicology, 1998Co-Authors: D. James MorréAbstract:A number of solvents including ethyl, amyl, butyl, octyl and benzyl alcohols, ethylene glycol, ethyl acetate, acetone, diethyl ether, propylene oxide, rho-dioxane, benzene, xylene, chloroform and carbon tetrachloride stimulate the growth of plants or plant parts at low concentrations and inhibit at high concentrations. These same solvents, at low dilutions, stimulate the activity of a growth-related Protein Disulfide-thiol interchange Protein (TIP) with NADH: Protein Disulfide reductase (NADH oxidase) (NOX) activity with plasma membrane vesicles isolated from elongating regions cut from dark grown seedlings of soybeans. Based on these and other findings, we suggest the TIP/NOX Protein to be the molecular target of the biological effects of low levels of exposure (hormesis) involved in the stimulation of plant growth.
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A Protein Disulfide-thiol interchange activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984)
Biochimica et biophysica acta, 1997Co-Authors: Mark Sweeting, Elizabeth Jacobs, Rafael De Cabo, D. James MorréAbstract:Plasma membrane vesicles isolated from HeLa cells grown in suspension culture contain a Protein Disulfide-thiol interchange (Protein Disulfide-like) activity. The activity was estimated from the restoration of activity to inactive (scrambled) pancreatic RNAase. RNAase activity was measured either by hydrolysis of cCMP or by a decrease in acid precipitable yeast RNA. The ability of plasma membrane vesicles to restore activity to inactive (scrambled) pancreatic ribonuclease was inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984). The activity correlated with that of a cyanide-resistant NADH oxidase also associated with the plasma membrane vesicles that exhibited a similar pattern of drug response. The activity was stimulated by reduced glutathione and inhibited by oxidized glutathione but did not depend on either for activity. The antitumor sulfonylurea-inhibited activity was greatest in the presence of reduced glutathione and least in the presence of oxidized glutathione. The antitumor sulfonylurea-inhibited activity was unaffected by a monoclonal antibody to Protein Disulfide isomerase. Also the antitumor sulfonylurea-inhibited activity was unaffected by peptide antisera to the consensus active site sequence of Protein Disulfide isomerase. Thus the antitumor sulfonylurea-inhibited activity appeared to reside with a novel cell surface Protein capable of oxidation of both NADH and Protein thiols and of carrying out a Protein Disulfide isomerase-like Protein Disulfide-thiol interchange activity in the absence of NADH or other external reductants.
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Response of a Protein Disulfide isomerase-like activity of transitional endoplasmic reticulum to all-trans retinol
Life sciences, 1996Co-Authors: Elizabeth Jacobs, D. James Morré, Mark Sweeting, Rafael De CaboAbstract:Isolated membrane fractions enriched in vesicles of transitional endoplasmic reticulum from rat liver exhibited Protein Disulfide isomerase-like activity of low specific activity. Activity was measured as the ability to restore activity to reduced, denatured and oxidized (scrambled) RNase. Submicromolar concentrations of retinol either stimulated or inhibited this activity depending on the composition of the redox buffer. In the presence of 1 microM reduced glutathione, micromolar concentrations of retinol stimulated the activity while higher or lower concentrations were less effective. With scrambled RNase, retinol was largely without effect in the absence of reduced glutathione or in the presence of oxidized glutathione. In the presence of NADH, retinol inhibited the Protein Disulfide-like activity over the same range of concentrations where retinol stimulated in the presence of reduced glutathione. These responses were observed with scrambled and inactive RNase and with reduced and inactive RNase as substrates. Also inhibited by retinol in these membrane preparations was their ability to oxidize NADH. Thus the retinol-modulated Protein Disulfide isomerase activity appears to correlate with the presence in transitional endoplasmic reticulum of an activity capable of oxidizing NADH in the presence of potassium cyanide that also was inhibited by submicromolar concentrations of retinol.
Rudolf Ladenstein - One of the best experts on this subject based on the ideXlab platform.
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Protein Disulfides and Protein Disulfide oxidoreductases in hyperthermophiles.
The FEBS journal, 2006Co-Authors: Rudolf Ladenstein, Bin RenAbstract:Disulfide bonds are required for the stability and function of a large number of Proteins. Recently, the results from genome analysis have suggested an important role for Disulfide bonds concerning the structural stabilization of intracellular Proteins from hyperthermophilic Archaea and Bacteria, contrary to the conventional view that structural Disulfide bonds are rare in Proteins from Archaea. A specific Protein, known as Protein Disulfide oxidoreductase (PDO) is recognized as a potential key player in intracellular Disulfide-shuffling in hyperthermophiles. The structure of this Protein shows a combination of two thioredoxin-related units with low sequence identity which together, in tandem-like manner, form a closed Protein domain. Each of these units contains a distinct CXXC active site motif. Due to their estimated conformational energies, both sites are likely to have different redox properties. The observed structural and functional characteristics suggest a relation to eukaryotic Protein Disulfide isomerase. Functional studies have revealed that both the archaeal and bacterial forms of this Protein show oxidative and reductive activity and are able to isomerize Protein Disulfides. The physiological substrates and reduction systems, however, are to date unknown. The variety of active site Disulfides found in PDOs from hyperthermophiles is puzzling. Nevertheless, the catalytic function of any PDO is expected to be correlated with the redox properties of its active site Disulfides CXXC and with the distinct nature of its redox environment. The residues around the two active sites form two grooves on the Protein surface. In analogy to a similar groove in thioredoxin, both grooves are suggested to constitute the substrate binding sites of PDO. The direct neighbourhood of the grooves and the different redox properties of both sites may favour sequential reactions in Protein Disulfide shuffling, like reduction followed by oxidation. A model for peptide binding by PDO is proposed to be derived from the analysis of crystal packing contacts mimicking substrate binding interactions. It is assumed, that PDO enzymes in hyperthermophilic Archaea and Bacteria may be part of a complex system involved in the maintenance of Protein Disulfide bonds. The regulation of Disulfide bond formation may be dependent on a distinct interplay of thermodynamic and kinetic effects, including functional asymmetry and substrate-mediated protection of the active sites, in analogy to the situation in Protein Disulfide isomerase. Numerous questions related to the function of PDO enzymes in hyperthermophiles remain unanswered to date, but can probably successfully be studied by a number of approaches, such as first-line genetic and in vivo studies.
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Functional properties of the Protein Disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a member of a novel Protein family related to Protein Disulfide-isomerase.
European journal of biochemistry, 2004Co-Authors: Emilia Pedone, Bin Ren, Rudolf Ladenstein, Mosè Rossi, Simonetta BartolucciAbstract:Protein Disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol-Disulfide exchange reactions with a CXXC sequence motif at their active site. A Disulfide oxidoreductase, a highly thermostable Protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain Protein Disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal Protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the Protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 degrees C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel Protein Disulfide oxidoreductase family.
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[8] Protein Disulfide oxidoreductase from Pyrococcus furiosus: Structural properties
Methods in enzymology, 2001Co-Authors: Bin Ren, Rudolf LadensteinAbstract:Publisher Summary The determination of the first three-dimensional structure of a Protein Disulfide oxidoreductase from the archaeon Pyrococcus furiosus ( Pf PDO) has provided the first view of active site Disulfides in an archaeal Protein. It reveals a unique structural form compared to those of Protein Disulfide oxidoreductases in bacteria and eukarya. Protein Disulfide oxidoreductases have been well studied in bacteria and eukarya. They are ubiquitous redox enzymes consisting of the families of thioredoxin, glutaredoxin, Protein Disulfide-isomerase (PDI), DsbA, and their homologs. These enzymes catalyze the formation, breakage, and rearrangement of Protein Disulfide bonds. These well-characterized Protein Disulfide oxidoreductases share a similarity in their three-dimensional structures: all of them use a common structural motif as the scaffold. The three-dimensional structure of intact PDI is not yet determined, but its amino acid sequence and tertiary domain structures have revealed it to be a multidomain Protein.
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A Protein Disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units
Nature structural biology, 1998Co-Authors: Bin Ren, Simonetta Bartolucci, Donatella De Pascale, Mosè Rossi, Gudrun Tibbelin, Rudolf LadensteinAbstract:Protein Disulfide bond formation is a rate limiting step in Protein folding and is catalyzed by enzymes belonging to the Protein Disulfide oxidoreductase superfamily, including Protein Disulfide isomerase (PDI) in eucarya and DsbA in bacteria. The first high resolution X-ray crystal structure of a Protein Disulfide oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus reveals structural details that suggest a relation to eukaryotic PDI. The Protein consists of two homologous structural units with low sequence identity. Each unit contains a thioredoxin fold with a distinct CXXC active site motif. The accessibilities of both active sites are rather different as are, very likely, their redox properties. The Protein shows the ability to catalyze the oxidation of dithiols as well as the reduction of Disulfide bridges.
Bin Ren - One of the best experts on this subject based on the ideXlab platform.
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Protein Disulfides and Protein Disulfide oxidoreductases in hyperthermophiles.
The FEBS journal, 2006Co-Authors: Rudolf Ladenstein, Bin RenAbstract:Disulfide bonds are required for the stability and function of a large number of Proteins. Recently, the results from genome analysis have suggested an important role for Disulfide bonds concerning the structural stabilization of intracellular Proteins from hyperthermophilic Archaea and Bacteria, contrary to the conventional view that structural Disulfide bonds are rare in Proteins from Archaea. A specific Protein, known as Protein Disulfide oxidoreductase (PDO) is recognized as a potential key player in intracellular Disulfide-shuffling in hyperthermophiles. The structure of this Protein shows a combination of two thioredoxin-related units with low sequence identity which together, in tandem-like manner, form a closed Protein domain. Each of these units contains a distinct CXXC active site motif. Due to their estimated conformational energies, both sites are likely to have different redox properties. The observed structural and functional characteristics suggest a relation to eukaryotic Protein Disulfide isomerase. Functional studies have revealed that both the archaeal and bacterial forms of this Protein show oxidative and reductive activity and are able to isomerize Protein Disulfides. The physiological substrates and reduction systems, however, are to date unknown. The variety of active site Disulfides found in PDOs from hyperthermophiles is puzzling. Nevertheless, the catalytic function of any PDO is expected to be correlated with the redox properties of its active site Disulfides CXXC and with the distinct nature of its redox environment. The residues around the two active sites form two grooves on the Protein surface. In analogy to a similar groove in thioredoxin, both grooves are suggested to constitute the substrate binding sites of PDO. The direct neighbourhood of the grooves and the different redox properties of both sites may favour sequential reactions in Protein Disulfide shuffling, like reduction followed by oxidation. A model for peptide binding by PDO is proposed to be derived from the analysis of crystal packing contacts mimicking substrate binding interactions. It is assumed, that PDO enzymes in hyperthermophilic Archaea and Bacteria may be part of a complex system involved in the maintenance of Protein Disulfide bonds. The regulation of Disulfide bond formation may be dependent on a distinct interplay of thermodynamic and kinetic effects, including functional asymmetry and substrate-mediated protection of the active sites, in analogy to the situation in Protein Disulfide isomerase. Numerous questions related to the function of PDO enzymes in hyperthermophiles remain unanswered to date, but can probably successfully be studied by a number of approaches, such as first-line genetic and in vivo studies.
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Functional properties of the Protein Disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a member of a novel Protein family related to Protein Disulfide-isomerase.
European journal of biochemistry, 2004Co-Authors: Emilia Pedone, Bin Ren, Rudolf Ladenstein, Mosè Rossi, Simonetta BartolucciAbstract:Protein Disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol-Disulfide exchange reactions with a CXXC sequence motif at their active site. A Disulfide oxidoreductase, a highly thermostable Protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain Protein Disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal Protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the Protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 degrees C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel Protein Disulfide oxidoreductase family.
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[8] Protein Disulfide oxidoreductase from Pyrococcus furiosus: Structural properties
Methods in enzymology, 2001Co-Authors: Bin Ren, Rudolf LadensteinAbstract:Publisher Summary The determination of the first three-dimensional structure of a Protein Disulfide oxidoreductase from the archaeon Pyrococcus furiosus ( Pf PDO) has provided the first view of active site Disulfides in an archaeal Protein. It reveals a unique structural form compared to those of Protein Disulfide oxidoreductases in bacteria and eukarya. Protein Disulfide oxidoreductases have been well studied in bacteria and eukarya. They are ubiquitous redox enzymes consisting of the families of thioredoxin, glutaredoxin, Protein Disulfide-isomerase (PDI), DsbA, and their homologs. These enzymes catalyze the formation, breakage, and rearrangement of Protein Disulfide bonds. These well-characterized Protein Disulfide oxidoreductases share a similarity in their three-dimensional structures: all of them use a common structural motif as the scaffold. The three-dimensional structure of intact PDI is not yet determined, but its amino acid sequence and tertiary domain structures have revealed it to be a multidomain Protein.
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A Protein Disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units
Nature structural biology, 1998Co-Authors: Bin Ren, Simonetta Bartolucci, Donatella De Pascale, Mosè Rossi, Gudrun Tibbelin, Rudolf LadensteinAbstract:Protein Disulfide bond formation is a rate limiting step in Protein folding and is catalyzed by enzymes belonging to the Protein Disulfide oxidoreductase superfamily, including Protein Disulfide isomerase (PDI) in eucarya and DsbA in bacteria. The first high resolution X-ray crystal structure of a Protein Disulfide oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus reveals structural details that suggest a relation to eukaryotic PDI. The Protein consists of two homologous structural units with low sequence identity. Each unit contains a thioredoxin fold with a distinct CXXC active site motif. The accessibilities of both active sites are rather different as are, very likely, their redox properties. The Protein shows the ability to catalyze the oxidation of dithiols as well as the reduction of Disulfide bridges.
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.
Simonetta Bartolucci - One of the best experts on this subject based on the ideXlab platform.
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Functional properties of the Protein Disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a member of a novel Protein family related to Protein Disulfide-isomerase.
European journal of biochemistry, 2004Co-Authors: Emilia Pedone, Bin Ren, Rudolf Ladenstein, Mosè Rossi, Simonetta BartolucciAbstract:Protein Disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol-Disulfide exchange reactions with a CXXC sequence motif at their active site. A Disulfide oxidoreductase, a highly thermostable Protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain Protein Disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal Protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the Protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 degrees C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel Protein Disulfide oxidoreductase family.
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[7] Protein Disulfide oxidoreductase from Pyrococcus furiosus: Biochemical properties
Methods in enzymology, 2001Co-Authors: Simonetta Bartolucci, Donatella De Pascale, Mosè RossiAbstract:Publisher Summary Protein Disulfide oxidoreductases (PDI) are redox enzymes that catalyze dithiol-Disulfide exchange reactions in eukaryotic and bacterial cells. Several Proteins belonging to this superfamily have been identified and characterized; thioredoxin, glutaredoxin, Protein Disulfideisomerase (PDI), Disulfide bond forming (DsbA), and their homologs in prokaryotes are the most extensively studied members of this group. A highly thermostable Protein Disulfide oxidoreductase was first isolated from Sulfolobus solfataricus. From its ability to catalyze the reduction of insulin Disulfides in the presence of dithiothreitol (DTT), the Protein was considered a thioredoxin. The Protein showed an unusually high molecular mass of 25 kDa and from amino acid composition analysis contained four cysteine residues. A homologous Protein was subsequently purified from Pyrococcusfuriosus. More recently, the Protein was crystallized, and its three-dimensional (3-D) structure at high resolution revealed structural details suggesting it may be related to the multidomain eukaryotic PDI. For this reason, the Protein was more correctly named “Protein Disulfide oxidoreductase” from P furiosus ( Pf PDO). This chapter describes the purification of the Pf PDO, the cloning, and its gene expression in E. coli .
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A Protein Disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units
Nature structural biology, 1998Co-Authors: Bin Ren, Simonetta Bartolucci, Donatella De Pascale, Mosè Rossi, Gudrun Tibbelin, Rudolf LadensteinAbstract:Protein Disulfide bond formation is a rate limiting step in Protein folding and is catalyzed by enzymes belonging to the Protein Disulfide oxidoreductase superfamily, including Protein Disulfide isomerase (PDI) in eucarya and DsbA in bacteria. The first high resolution X-ray crystal structure of a Protein Disulfide oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus reveals structural details that suggest a relation to eukaryotic PDI. The Protein consists of two homologous structural units with low sequence identity. Each unit contains a thioredoxin fold with a distinct CXXC active site motif. The accessibilities of both active sites are rather different as are, very likely, their redox properties. The Protein shows the ability to catalyze the oxidation of dithiols as well as the reduction of Disulfide bridges.
