Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of basic residues of the 70-80-loop, Arg(74), Arg(75), and Lys(78) (chymotrypsin numbering) in the catalytic function of activated protein C (APC) was investigated by expressing mutants of protein C in which these residues were replaced with Ala in three separate constructs. Following purification to homogeneity and activation by thrombin, the catalytic properties of the mutants were characterized with respect to their ability to cleave the chromogenic substrate Spectrozyme PCa, react with protein C inhibitor (PCI), and inactivate factor Va. Relative to wild-type APC, the mutants cleaved Spectrozyme PCa with identical or improved catalytic efficiencies. Similarly, PCI inhibited mutants with identical or improved second-order rate constants (k(2)) in the absence of heparin. However, the heparin-catalyzed inhibition of mutants by PCI was impaired approximately 10-fold. Analysis of k(2) values by a ternary complex model revealed that the affinities of mutants for heparin were impaired to a similar extent. Moreover, analysis of the NaCl gradient elution profiles of APC derivatives from Heparin-Sepharose supported this conclusion. An oligosaccharide containing 14 residues efficiently catalyzed the PCI inhibition of APC by a template mechanism. Further studies revealed that the ability of Arg(74) and Arg(75) mutants to inactivate factor Va was markedly impaired. We conclude that basic residues of the 70-80-loop are critical for the catalytic function of APC.
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PMID:Contribution of basic residues of the 70-80-loop to heparin binding and anticoagulant function of activated protein C. 1199 10

DPC423, 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide, is a synthetic, orally bioavailable, competitive, and selective inhibitor of human coagulation factor Xa (K(i) [nM]: factor Xa, 0.15; trypsin, 60; thrombin, 6000; plasma kallikrein, 61; activated protein C, 1800; factor IXa, 2200; factor VIIa, >15,000; chymotrypsin, >17,000; urokinase, >19,000; plasmin, >35,000; tissue plasminogen activator, >45,000; complement factor I, 44,000 [IC(50)]). In vitro, DPC423 produced anticoagulant effects in human plasma in which it doubled prothrombin time, activated partial thromboplastin time, and Heptest clotting time at 3.1 +/- 0.4, 3.1 +/- 0.4, and 1.1 +/- 0.5 microM, respectively. In dogs, DPC423 had a good pharmacokinetic profile with an oral bioavailability of 57%, a plasma clearance of 0.24 L/kg/h, and a plasma half-life of 7.5 h. In rabbit and rat models of arteriovenous shunt thrombosis, DPC423 was an effective antithrombotic agent with an IC(50) of 150 and 470 nM, respectively. The antithrombotic effect of DPC423 is likely to be related to the inhibition of factor Xa but not to the inhibition of thrombin or due to direct inhibition of platelet aggregation. Therefore, based on potency, selectivity, efficacy, and oral bioavailability, DPC423 was selected for clinical development as an oral anticoagulant for the potential treatment of thrombotic disorders. Preliminary human data suggest that DPC423 is orally bioavailable in humans and has a long plasma half-life.
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PMID:Nonpeptide factor Xa inhibitors: DPC423, a highly potent and orally bioavailable pyrazole antithrombotic agent. 1217 91

Activated protein C (APC) is a vitamin K-dependent anticoagulant serine protease in plasma that downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. In addition to its anticoagulant function, APC also exhibits potent profibrinolytic and anti-inflammatory properties. The proteolytic activity of APC in plasma is slowly inhibited by three serpins: protein C inhibitor, plasminogen activator inhibitor-1, and alpha(1)-antitrypsin. Recent structural and mutagenesis data have indicated that basic residues of three exposed surface loops known as the 39-loop (Lys(37)-Lys(39)), 60-loop (Lys(62), Lys(63)), and 70-80-loop (Arg(74), Arg(75), and Lys(78)) (chymotrypsin numbering) constitute an anion-binding exosite in APC that interacts with these macromolecular substrates and inhibitors. Moreover, this exosite plays a critical role in the thrombomodulin-dependent activation of the zymogen protein C by thrombin. This article briefly reviews how the binding of physiological protein and polysaccharide cofactors on this exosite modulates the protein C anticoagulant pathway in plasma.
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PMID:Exosite-dependent regulation of the protein C anticoagulant pathway. 1255 95

The role of lysines 37-39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and alpha(1)-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by alpha(1)-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with alpha(1)-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI-heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI-APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by alpha(1)-antitrypsin, two important blood plasma serpins.
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PMID:Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by alpha(1)-antitrypsin. 1281 96

A unique pentasaccharide fragment of heparin can enhance the reactivity of antithrombin with coagulation proteases factors IXa and Xa by 300- to 600-fold through a conformational activation of the serpin, without having a significant effect on the reactivity of antithrombin with thrombin. In this study, it was hypothesized that differences in the structure of the autolysis loop of coagulation proteases (residues 143-154 in chymotrypsin numbering) may be responsible for their differential reactivity with the native and heparin-activated antithrombin. To test this hypothesis, the autolysis loops of both thrombin and the anticoagulant serine protease-activated protein C were replaced with the corresponding loop of factor Xa. Inhibition studies revealed that in contrast to the approximately 1.5-fold difference in the reactivity of thrombin with antithrombin in the absence and presence of pentasaccharide, the difference in reactivity was increased to approximately 37-fold for the mutant thrombin. In the case of the activated protein C mutant, similar to factor Xa, pentasaccharide accelerated the reaction 375-fold. These results suggest that structural differences in the autolysis loop of coagulation proteases play a key role in their differential reactivity with the native and heparin-activated conformations of antithrombin.
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PMID:Heparin-activated antithrombin interacts with the autolysis loop of target coagulation proteases. 1517 83

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.
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PMID:The conformation of the activation peptide of protein C is influenced by Ca2+ and Na+ binding. 1525 39

Wheat gluten causes gut inflammation in genetically predisposed individuals. We tested the hypothesis that wheat gluten is not only a target of adaptive immunity, but also modulates the function of APC. Dendritic cells (DC) derived from the bone marrow of BALB/c mice were exposed to chymotrypsin-treated wheat gluten. This induced DC maturation as estimated by all surface markers tested (MHC class II, CD40, CD54, and CD86). The effect was dose dependent, and, at 100 microg/ml gluten matched that caused by 10 ng/ml LPS. A role of endotoxin contamination was ruled out by demonstrating the resistance of wheat gluten effects to LPS antagonist polymyxin B. DC from LPS nonresponder strain C3H/HeJ were affected by wheat gluten, but not by LPS. Proteinase K-digested wheat gluten was unable to stimulate DC maturation. Wheat gluten induced a unique secretion pattern of selected cytokines and chemokines in DC. Classic pro- or anti-inflammatory mediators were not produced, in contrast to LPS. Rather, chemokines MIP-2 and keratinocyte-derived cytokine were secreted in large amounts. We conclude that wheat gluten lowers the threshold for immune responses by causing maturation of APC, by attracting leukocytes and increasing their reactivity state. In the presence of an appropriate genetic predisposition, this is expected to increase the risk of adverse immune reactions to wheat gluten or to other Ags presented.
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PMID:Wheat gluten causes dendritic cell maturation and chemokine secretion. 1526 26

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA(3). B and C hordein polypeptides and the salt-soluble proteins beta-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the alpha-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, beta-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of alpha-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for alpha-amylase, these hormones have the opposite effect on ASI mRNA levels.
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PMID:Differential synthesis in vitro of barley aleurone and starchy endosperm proteins. 1666 68

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an approximately 500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved approximately 10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.
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PMID:The role of autolysis loop in determining the specificity of coagulation proteases. 1766 41

We previously demonstrated that the substitution of the autolysis loop (residues 143-154 in chymotrypsin numbering) of APC with the corresponding loop of trypsin (APC-Tryp 143-154) has no influence on the proteolytic activity of the protease toward fVa, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma. To further investigate the role of the autolysis loop in APC and determine whether this loop is a target for modulation by protein S, we evaluated the activity of APC-Tryp 143-154 toward fVa and several plasma inhibitors both in the absence and presence of protein S. Furthermore, we evaluated the active-site topography of APC-Tryp 143-154 by determining the average distance of the closest approach (L) between a fluorescein dye tethered to a tripeptide inhibitor, attached to the active-site of APC-Tryp 143-154, and octadecylrhodamine dyes incorporated into PCPS vesicles both in the absence and presence of protein S. The activity of APC-Tryp 143-154 toward fVa was identical to that of wild-type APC both in the presence and absence of protein S. However, the reactivity of APC-Tryp 143-154 with plasma inhibitors was preferentially improved independent of protein S. The FRET analysis revealed a dramatic change in the active-site topography of APC both in the absence and presence of protein S. Anisotropy measurements revealed that the fluorescein dye has a remarkable degree of rotational freedom in the active-site of APC-Tryp 143-154. These results suggest that the autolysis loop of APC may not be a target for modulation by protein S. This loop, however, plays a critical role in restricting both the specificity and spatial environment of the active-site groove of APC.
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PMID:Autolysis loop restricts the specificity of activated protein C: analysis by FRET and functional assays. 1832 82


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