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Peter G W Gettins - One of the best experts on this subject based on the ideXlab platform.
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maspin binds to urokinase type and tissue type plasminogen activator through exosite exosite interactions
Journal of Biological Chemistry, 2007Co-Authors: Maher Alayyoubi, Bradford S Schwartz, Peter G W GettinsAbstract:Abstract Maspin is a member of the serpin family with a reactive center loop that is incompatible with Proteinase Inhibition by the serpin conformational change mechanism. Despite this there are reports that maspin might regulate uPA-dependent processes in vivo. Using exogenous and endogenous fluorescence, we demonstrate here that maspin can bind uPA and tPA in both single-chain and double-chain forms, with Kd values between 300 and 600 nm. Binding is at an exosite on maspin close to, but outside of, the reactive center loop and is therefore insensitive to mutation of Arg340 within the reactive center loop. The binding site on tPA does not involve the Proteinase active site, with the result that maspin can bind to S195A tPA that is already complexed to plasminogen activator inhibitor-1. The ability of maspin to bind these Proteinases without involvement of the reactive center loop leaves the latter free to engage in additional, as yet unidentified, maspin-protein interactions that may serve to regulate the properties of the exosite-bound Proteinase. This may help to reconcile apparently conflicting studies that demonstrate the importance of the reactive center loop in certain maspin functions, despite the inability of maspin to directly inhibit tPA or uPA catalytic activity in in vitro assays through engagement between its reactive center loop and the active site of the Proteinase.
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active site distortion is sufficient for Proteinase Inhibition by serpins structure of the covalent complex of α1 Proteinase inhibitor with porcine pancreatic elastase
Journal of Biological Chemistry, 2006Co-Authors: Alexey Dementiev, Jozsef Dobo, Peter G W GettinsAbstract:We report here the x-ray structure of a covalent serpin-Proteinase complex, α1-Proteinase inhibitor (α1PI) with porcine pancreatic elastase (PPE), which differs from the only other x-ray structure of such a complex, that of α1PI with trypsin, in showing nearly complete definition of the Proteinase. α1PI complexes with trypsin, PPE, and human neutrophil elastase (HNE) showed similar rates of deacylation and enhanced susceptibility to proteolysis by exogenous Proteinases in solution. The differences between the two x-ray structures therefore cannot arise from intrinsic differences in the Inhibition mechanism. However, self-proteolysis of purified complex resulted in rapid cleavage of the trypsin complex, slower cleavage of the PPE complex, and only minimal cleavage of the HNE complex. This suggests that the earlier α1 PI-trypsin complex may have been proteolyzed and that the present structure is more likely to be representative of serpin-Proteinase complexes. The present structure shows that active site distortion alone is sufficient for Inhibition and suggests that enhanced proteolysis is not necessarily exploited in vivo.
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heterogeneity of recombinant human antithrombin iii expressed in baby hamster kidney cells effect of glycosylation differences on heparin binding and structure
Journal of Biological Chemistry, 1993Co-Authors: Bingqi Fan, Brenda C Crews, Illarion V Turko, J Choay, G Zettlmeissl, Peter G W GettinsAbstract:To determine the effects of differences in glycosylation on the structure and functional properties of recombinant human antithrombin (rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three forms of rHAT, I-III, were isolated which differed in affinity for heparin. Form I had the lowest affinity and contained a high proportion of highly branched complex carbohydrate. Form II had higher affinity and contained both complex and high mannose-type chains. Form III had the highest affinity and was similar to form II in the type of carbohydrate present, but had a lower level of glycosylation, consistent with the absence of carbohydrate at one of the four glycosylation sites. 1H NMR spectra of plasma HAT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced almost identical NMR perturbation difference spectra. The only functional difference found was in the rates of inactivation of factor Xa. Forms II and III gave second order rate constants similar to that of plasma HAT, whereas form I gave a biphasic Inhibition, with the first phase having a rate about four times that of the other forms. We conclude that carbohydrate heterogeneity does not alter the structure of the HAT polypeptide or the heparin-induced conformational change, but does affect the heparin affinity and can alter the rate of Proteinase Inhibition.
John Paul Watts - One of the best experts on this subject based on the ideXlab platform.
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bicyclic peptidomimetic tetrahydrofuro 3 2 b pyrrol 3 one and hexahydrofuro 3 2 b pyridine 3 one based scaffolds synthesis and cysteinyl Proteinase Inhibition
Bioorganic & Medicinal Chemistry, 2004Co-Authors: Martin Quibell, Alex Benn, Nick Flinn, Tracy Monk, Manoj Ramjee, Yikang Wang, John Paul WattsAbstract:Abstract A stereoselective synthesis of (3a S ,6a R )-tetrahydrofuro[3,2- b ]pyrrol-3-ones and (3a S ,7a R )-hexahydrofuro[3,2- b ]pyridine-3-ones has been developed through Fmoc protected scaffolds 12 and 13 . A key design element within these novel bicyclic scaffolds, in particular the 5,5-fused system, was the inherent stability of the cis -fused geometry in comparison to that of the corresponding trans -fused. Since the bridgehead stereocentre situated β to the ketone was of a fixed and stable configuration, the fact that cis ring fusion is both kinetically and thermodynamically stable with respect to trans ring fusion provides chiral stability to the bridgehead stereocentre that is situated α to the ketone. To exemplify this principle, building blocks 12 and 13 were designed, prepared and utilised in a solid phase combinatorial synthesis of peptidomimetic inhibitors 10 , 45a – e , 11 and 46 . Both series were chirally stable with 5,5-series 10 and 45a – e exhibiting potent in vitro activity against a range of CAC1 cysteinyl Proteinases. Compound 10 , a potent and selective inhibitor of cathepsin K, possessed good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.
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functionalised 2 3 dimethyl 3 aminotetrahydrofuran 4 one and n 3 oxo hexahydrocyclopenta b furan 3a yl acylamide based scaffolds synthesis and cysteinyl Proteinase Inhibition
Bioorganic & Medicinal Chemistry, 2004Co-Authors: John Paul Watts, Alex Benn, Nick Flinn, Tracy Monk, Manoj Ramjee, Yikang Wang, Peter Ray, Martin QuibellAbstract:A stereoselective synthesis of functionalised (2R,3R)-2,3-dimethyl-3-amidotetrahydrofuran-4-one, its (2S,3R)-epimer and (3aR,6aR)-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide cysteinyl Proteinase inhibitors has been developed using Fmoc-protected scaffolds 6-8 in a solid-phase combinatorial strategy. Within these scaffolds, the introduction of an alkyl substituent alpha to the ketone affords chiral stability to an otherwise configurationally labile molecule. Preparation of scaffolds 6-8 required stereoselective syntheses of suitably protected alpha-diazomethylketone intermediates 9-11, derived from appropriately protected alpha-methylthreonines (2R,3R)-12, (2R,3S)-13 and a protected analogue of (1R,2R)-1-amino-2-hydroxycyclopentanecarboxylic acid 14. Application of standard methods for the preparation of amino acid alpha-diazomethylketones, through treatment of the mixed anhydride or pre-formed acyl fluorides of intermediates 12-14 with diazomethane, proved troublesome giving complex mixtures. However, the desired alpha-diazomethylketones were isolated and following a lithium chloride/acetic acid promoted insertion reaction provided scaffolds 6-8. Elaboration of 6-8 on the solid phase gave alpha,beta-dimethyl monocyclic ketone based inhibitors 38a-f, 39a,b,d,e,f and bicyclic inhibitors 40a-e that exhibited low micromolar activity against a variety of cysteinyl Proteinases.
Martin Quibell - One of the best experts on this subject based on the ideXlab platform.
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bicyclic peptidomimetic tetrahydrofuro 3 2 b pyrrol 3 one and hexahydrofuro 3 2 b pyridine 3 one based scaffolds synthesis and cysteinyl Proteinase Inhibition
Bioorganic & Medicinal Chemistry, 2004Co-Authors: Martin Quibell, Alex Benn, Nick Flinn, Tracy Monk, Manoj Ramjee, Yikang Wang, John Paul WattsAbstract:Abstract A stereoselective synthesis of (3a S ,6a R )-tetrahydrofuro[3,2- b ]pyrrol-3-ones and (3a S ,7a R )-hexahydrofuro[3,2- b ]pyridine-3-ones has been developed through Fmoc protected scaffolds 12 and 13 . A key design element within these novel bicyclic scaffolds, in particular the 5,5-fused system, was the inherent stability of the cis -fused geometry in comparison to that of the corresponding trans -fused. Since the bridgehead stereocentre situated β to the ketone was of a fixed and stable configuration, the fact that cis ring fusion is both kinetically and thermodynamically stable with respect to trans ring fusion provides chiral stability to the bridgehead stereocentre that is situated α to the ketone. To exemplify this principle, building blocks 12 and 13 were designed, prepared and utilised in a solid phase combinatorial synthesis of peptidomimetic inhibitors 10 , 45a – e , 11 and 46 . Both series were chirally stable with 5,5-series 10 and 45a – e exhibiting potent in vitro activity against a range of CAC1 cysteinyl Proteinases. Compound 10 , a potent and selective inhibitor of cathepsin K, possessed good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.
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functionalised 2 3 dimethyl 3 aminotetrahydrofuran 4 one and n 3 oxo hexahydrocyclopenta b furan 3a yl acylamide based scaffolds synthesis and cysteinyl Proteinase Inhibition
Bioorganic & Medicinal Chemistry, 2004Co-Authors: John Paul Watts, Alex Benn, Nick Flinn, Tracy Monk, Manoj Ramjee, Yikang Wang, Peter Ray, Martin QuibellAbstract:A stereoselective synthesis of functionalised (2R,3R)-2,3-dimethyl-3-amidotetrahydrofuran-4-one, its (2S,3R)-epimer and (3aR,6aR)-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide cysteinyl Proteinase inhibitors has been developed using Fmoc-protected scaffolds 6-8 in a solid-phase combinatorial strategy. Within these scaffolds, the introduction of an alkyl substituent alpha to the ketone affords chiral stability to an otherwise configurationally labile molecule. Preparation of scaffolds 6-8 required stereoselective syntheses of suitably protected alpha-diazomethylketone intermediates 9-11, derived from appropriately protected alpha-methylthreonines (2R,3R)-12, (2R,3S)-13 and a protected analogue of (1R,2R)-1-amino-2-hydroxycyclopentanecarboxylic acid 14. Application of standard methods for the preparation of amino acid alpha-diazomethylketones, through treatment of the mixed anhydride or pre-formed acyl fluorides of intermediates 12-14 with diazomethane, proved troublesome giving complex mixtures. However, the desired alpha-diazomethylketones were isolated and following a lithium chloride/acetic acid promoted insertion reaction provided scaffolds 6-8. Elaboration of 6-8 on the solid phase gave alpha,beta-dimethyl monocyclic ketone based inhibitors 38a-f, 39a,b,d,e,f and bicyclic inhibitors 40a-e that exhibited low micromolar activity against a variety of cysteinyl Proteinases.
Frank C Church - One of the best experts on this subject based on the ideXlab platform.
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ROLE OF THE H HELIX IN HEPARIN BINDING TO PROTEIN C INHIBITOR
Journal of Biological Chemistry, 1994Co-Authors: Rebecca A. Shirk, Marc G L M Elisen, Joost C. M. Meijers, Frank C ChurchAbstract:Abstract Protein C inhibitor (PCI) is a plasma serine Proteinase inhibitor (serpin) that is a major physiological regulator of activated protein C. Inhibition of its target Proteinase is accelerated by heparin in a reaction that involves the binding of both inhibitor and Proteinase to heparin to form a ternary complex. This study was undertaken to understand the role of the H helix region (residues 264-278) of PCI in heparin binding and used (i) a recombinant truncated PCI fusion protein of the first 294 residues, (ii) H helix synthetic peptides containing single Arg/Lys-->Glu substitutions, and (iii) site-directed Ala mutagenesis of 4 basic residues (Arg-269, Lys-270, Lys-276, and Lys-277) in the H helix region of full-length recombinant PCI (rPCI) expressed in Baculovirus. The PCI fusion protein interfered in heparin-accelerated PCI-Proteinase Inhibition reactions, and it bound to heparin-Sepharose. Compared to the wild-type PCI fusion protein, deletion of the H helix from the fusion protein resulted in a reduction of both heparin-Sepharose binding and the ability to compete for heparin during PCI-Proteinase Inhibition reactions. Competition assays with H helix synthetic peptides revealed that the R269E altered peptide was the least effective at blocking heparin-catalyzed PCI-Proteinase Inhibition reactions. Compared with full-length active wild-type rPCI, R269A: K270A and K276A:K277A rPCI both had reduced heparin-Sepharose binding, but only R269A:K270A rPCI showed a loss of heparin-accelerated Proteinase Inhibition for both activated protein C and thrombin. We conclude that a major heparin-binding site of PCI is the H helix, unlike its heparin-binding serpin homologues antithrombin and heparin cofactor II, which bind heparin primarily through the D helix.
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general features of the heparin binding serpins antithrombin heparin cofactor ii and protein c inhibitor
Blood Coagulation & Fibrinolysis, 1993Co-Authors: C W Pratt, Frank C ChurchAbstract:The blood plasma serine Proteinase inhibitors (serpins) are glycoproteins whose activities are involved in many important homeostatic reactions. The heparin-dependent plasma serpins, antithrombin, heparin cofactor II and protein C inhibitor, regulate the Proteinases of blood coagulation. Heparin and some other glycosaminoglycans increase the rate of Proteinase Inhibition by these three plasma serpins. Proteinases recognize a specific peptide, termed the reactive site, near the carboxyl-terminus of serpins (for antithrombin and protein C inhibitor this is Arg-Ser and for heparin cofactor II this is Leu-Ser). Additionally, these three serpins contain unique structural elements that confer glycosaminoglycan binding activities. The therapeutic anticoagulant action of the glycosaminoglycan heparin is believed to depend partially on heparin-accelerated Inhibition of Proteinases by antithrombin. The physiological importance of specific proteogly-cans has been attributed to their recognition of these serpins and their biological 'activation' of these Proteinase inhibitors.
Walter Kisiel - One of the best experts on this subject based on the ideXlab platform.
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human tissue factor pathway inhibitor 2 induces caspase mediated apoptosis in a human fibrosarcoma cell line
Apoptosis, 2008Co-Authors: Prakasha Kempaiah, Walter KisielAbstract:Human TFPI-2 is an extracellular matrix-associated Kunitz-type serine Proteinase inhibitor. We previously demonstrated that a human fibrosarcoma cell line, HT-1080, does not express TFPI-2, but genetic restoration of TFPI-2 expression in these cells markedly inhibited their growth and metastasis in vivo. In the present study, either full-length recombinant TFPI-2, or its mutated first Kunitz-type domain (R24K KD1), were offered to HT-1080 cells, and the degree of apoptosis assessed by nuclear fragmentation, ethidium bromide and acridine orange staining, fluorescence activated cell sorting, immunoblotting and gene expression profiling. R24K KD1 induced apoptosis in 69% of HT-1080 cells in a 48 h period compared to 39% for TFPI-2, while a KD1 preparation lacking a reactive site arginine/lysine residue (R24Q KD1) produced only an 18% apoptosis rate, suggesting that the observed apoptosis was related to Proteinase Inhibition. Immunoblotting experiments indicated increased caspase 3 and 9 activation, up-regulation of pro-apoptotic Bax and suppression of anti-apoptotic Bcl-2 protein. Finally, microarray analyses of R24K KD1-treated cells indicated elevated expression of several pro-apoptotic genes and under-expression of anti-apoptotic genes. Collectively, our results demonstrate that treatment of HT-1080 cells exogenously with either TFPI-2 or R24K KD1 activates caspase-mediated, pro-apoptotic signaling pathways resulting in apoptosis.
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human tissue factor pathway inhibitor 2 induces caspase mediated apoptosis in a human fibrosarcoma cell line ht 1080
Blood, 2007Co-Authors: Prakasha Kempaiah, Walter KisielAbstract:Human tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa extracellular matrix-associated Kunitz-type serine Proteinase inhibitor that regulates the plasmin and trypsin- mediated activation of zymogen matrix metalloProteinases and growth factors essential for tumor growth, invasion, and metastasis. We previously demonstrated that HT-1080 human fibrosarcoma cells do not express TFPI-2, but restoration of TFPI-2 expression in these cells by stable transfection with the human TFPI-2 cDNA markedly inhibited their growth and metastasis in-vivo. In this study, 1 μM concentrations of either recombinant human TFPI-2 or its mutated first Kunitz-type domain (R24K KD1), were offered to HT-1080 cells, incubated for 48 h, and the degree of apoptosis assessed by nuclear fragmentation, ethidium bromide/acridine orange (EB/AO) staining, fluorescence-activated cell sorting (FACS) and immunoblotting. Agarose gel electrophoresis of DNA from HT-1080 cells treated with either TFPI-2 or R24K KD1 for 48h indicated DNA fragmentation with a ladder pattern typically associated with apoptosis. EB/AO staining of TFPI-2- and R24K KD1- treated cells revealed that 40–70% of the cells were apoptotic in relation to vehicle-treated cells as judged by fluoresence microscopy. Co-administration of TFPI-2 with polymyxin B produced similar results, suggesting that apoptosis was not endotoxin-dependent. Consistent with our earlier studies showing its enhanced inhibitory activity relative to TFPI-2, R24K KD1 was able to induce apoptosis in 68% of HT-1080 cells after 48h of treatment compared to 39% for the parent TFPI-2 at an equivalent concentration. Moreover, HT-1080 cells treated with a KD1 preparation lacking the reactive site arginine residue (R24Q KD1) produced only an 18% apoptosis rate, thereby linking the observed apoptosis with serine Proteinase Inhibition. FACS analysis, using propidium iodide and annexin-V labeling, revealed similar apoptotic rates to that seen by EB/AO staining assays. In addition, immunoblotting experiments of vehicle and TFPI-2-treated cells indicated increased caspase-3 activation in TFPI-2-treated cells, thus providing evidence that apoptosis is caspase-mediated. We also observed up-regulation of the proapoptotic Bax protein by immunoblotting following treatment of HT-1080 cells with either TFPI-2 or R24K KD1. Taken together, our results demonstrate that treatment of HT-1080 cells with exogenous TFPI-2 or R24K KD1 activates caspase-mediated, proapoptotic signaling pathways and induces apoptosis. In addition, our data provides suggestive evidence that peritumor application of either TFPI-2 or R24K KD1 may facilitate tumor apoptosis in-vivo.
