Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protease-activated receptor 2 (PAR2) is the second member of a new subfamily of G-protein coupled receptors: the protease-activated receptors (PARs). At present, four different PARs have been cloned and all of them share the same basic mechanism of activation. A serine protease cleaves the extended, extracellular N-terminus of the receptor at a specific site within the protein chain to expose an N-terminal tethered ligand domain, which binds to and activates the cleaved receptor. In this manner, trypsin and mast cell beta-tryptase activate PAR2. PARs are single use receptors because proteolytic activation is irreversible and the cleaved receptors are degraded in lysosomes. Thus, PARs play important roles in emergency situations, such as trauma and inflammation. Emerging evidence indicates that PAR2 is involved in the cardiovascular, pulmonary and gastrointestinal systems, where it controls inflammation and nociception. Work with selective agonists and knockout animals suggests a contribution of PAR2 to certain inflammatory diseases. Therefore, selective antagonists or agonists of these receptors may be useful therapeutic agents for the treatment of human diseases.
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PMID:Proteinase-activated receptor-2: physiological and pathophysiological roles. 1531 91

Bowman-Birk inhibitor proteins (BBIs), which are potent inhibitors of chymotrypsin-like proteases, do not inhibit human beta-tryptase despite this protein having a chymotrypsin-like fold. We have reported previously that, in contrast, BBI-derived peptides (whose sequences incorporate the solvent exposed reactive site loop motif) are able to inhibit human beta-tryptase. This is due to their small size, which allows them to access the restricted active site(s) of tryptase, which has an unusual tetrameric arrangement with four active sites flanking a central pore. In this paper, we have examined the possibility of creating additional interactions within this pore by adding extensions to the BBI-peptide motif. We have taken the core disulfide-bridged sequence SCTKSIPPQCY and examined a series of extensions, at both the C- and N-termini, that bear a second positively charged Lys residue at their end. The aim was to construct inhibitors that could make additional interactions in tryptase by spanning the gap between adjacent active sites in the enzyme, producing a double-headed inhibitor; a positively charged group was used as the dominant specificity of this enzyme is for a positively charged P1 residue. Both N- and C-terminal extensions are found to produce inhibitors of much increased potency, with a strong dependence of potency on chain length. Moreover, it was found that the C- and N-terminal extensions were able to synergise, with their combination on the same peptide producing an even better inhibitor with a potency 10(4)-fold greater than the original sequence. We suggest that the C- and N-terminal extensions are picking up interactions with separate additional sites on the tryptase, making the doubly extended BBI peptide a tri-functional tryptase inhibitor.
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PMID:Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides: creation of a new tri-functional inhibitor. 1551 50

Tryptase, a tetrameric serine protease, is a main constituent of the secretory granules in human mast cells, where it is stored in complex with heparin or chondroitin sulfate proteoglycan. Human tryptase has been implicated in a variety of clinical conditions including asthma, but the mechanisms that lead to its tetramerization/activation have not been extensively investigated. Here we addressed the activation mechanisms for human betaI and betaII-tryptase, which differ in that betaI-tryptase is N-glycosylated at Asn102 whereas betaII-tryptase has a Lys residue at position 102, and consequently lacks the corresponding N-glycosylation. We found that both tryptases were dependent on heparin for activation/tetramerization, but whereas betaI-tryptase activation preferentially occurred at acidic pH, betaII-tryptase activation was less pH-dependent. Both betaI and betaII-tryptase bound strongly to heparin-Sepharose at acidic pH but with lower affinity at neutral pH. Further, while addition of heparin to betaI-tryptase predominantly resulted in formation of active tetrameric enzyme, betaII-tryptase showed a tendency to form inactive aggregates. betaI and betaII-tryptase were similar in that the minimal heparin size to induce activation was an octasaccharide and in that the interaction with heparin and structurally related polysaccharides was dependent on high anionic charge density rather than on specific structural motifs. Addition of decasaccharides to both betaI and betaII-tryptase resulted in the formation of active monomeric enzyme, whereas intact heparin promoted assembly of tetrameric enzyme. This, together with a bell-shaped dose response curve for heparin-induced activation, suggests that the mechanism for tetramerization involves bridging of individual tryptase monomers by heparin. Taken together, this study indicates a key role for heparin in the activation of human beta-tryptase.
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PMID:Structural requirements and mechanism for heparin-dependent activation and tetramerization of human betaI- and betaII-tryptase. 1556 16

beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits. We have recently identified a potent bivalent inhibitor (K(i)=18 nM), based on the bifunctional scaffold cyclo-(-D-Asp-L-Asp-) and the arginine mimetic dl-3-aminomethyl-phenylalanine methyl ester as a ligand for S1 pockets that takes advantage of the this unique tetrameric geometry. To generate an affinity matrix, the bivalent ligand was modified and immobilized on a Sepharose matrix by use of the PEG derivative Jeffamine ED 900 as spacer. This matrix selectively recognizes and binds beta-tryptase from crude protein mixtures and thus is useful as a geometry-driven means of isolating and purifying human mast cell tryptases.
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PMID:Affinity chromatography of tryptases: design, synthesis and characterization of a novel matrix-bound bivalent inhibitor. 1559 13

Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests tryptase beta is the most important form released during mast cell activation in allergic diseases. It is shown to have numerous pro-inflammatory cellular activities in vitro, and in animal models tryptase provokes broncho-constriction and induces a cellular inflammatory infiltrate characteristic of human asthma. Screening of in-house inhibitors of factor Xa (a closely related serine protease) identified beta-amidoester benzamidines as potent inhibitors of recombinant human betaII tryptase. X-ray structure driven template modification and exchange of the benzamidine to optimize potency and pharmacokinetic properties gave selective, potent and orally bioavailable 4-(3-aminomethyl phenyl)piperidinyl-1-amides.
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PMID:Structure based design of 4-(3-aminomethylphenyl)piperidinyl-1-amides: novel, potent, selective, and orally bioavailable inhibitors of betaII tryptase. 1578 96

Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.
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PMID:X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition. 1641 69

The novel tetrameric structure of human beta-tryptase faces each active site into the central pore, thereby restricting access of most biologic protease inhibitors. The mechanism by which the anti-tryptase mAb B12 inhibits human beta-tryptase peptidase and proteolytic activities at neutral pH, but augments proteolytic activity at acidic pH, was examined. At neutral pH, B12-beta-tryptase complexes are inactive. At acidic pH, B12 (intact and Fab) minimally affects peptidase activity when added to beta-tryptase tetramers, but does induce susceptibility to inhibition by soybean trypsin inhibitor and antithrombin III. Surprisingly, B12 Fab-beta-tryptase complexes formed at both neutral and acidic pH exhibit the apparent molecular mass of a complex with 1 beta-tryptase monomer and 1 Fab by gel filtration. B12 does not compete with heparin for binding to tryptase at either neutral or acidic pH. Thus, B12 directly disrupts beta-tryptase tetramers to monomers that are inactive at neutral pH, whereas at acidic pH, are active and more accessible to protein inhibitors and substrates.
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PMID:The B12 anti-tryptase monoclonal antibody disrupts the tetrameric structure of heparin-stabilized beta-tryptase to form monomers that are inactive at neutral pH and active at acidic pH. 1649 76

Recent studies have shown that a lack of eosinophils in asthmatic airway smooth muscle (ASM) bundles in contrast to the large number of mast cells is a key feature of asthma. We hypothesized that this is caused by beta-tryptase, the predominant mast cell-specific protease, abrogating the eosinophil chemotactic activities of ASM cell-derived eosinophil chemoattractants such as eotaxin and RANTES. We studied the effect of beta-tryptase on the immunoreactivities of human ASM cell-derived and recombinant eotaxin and other recombinant chemokines that are known to be produced by human ASM cells. We report in this study that purified beta-tryptase markedly reduced the immunoreactivity of human ASM cell-derived and recombinant eotaxin, but had no effect on eotaxin mRNA expression. The effect was mimicked by recombinant human beta-tryptase in the presence of heparin and was reversed by heat inactivation and the protease inhibitor leupeptin, suggesting that the proteolytic activity of tryptase is required. beta-Tryptase also exerted similar effects on recombinant RANTES, but not on the other chemokines and cytokines that were screened. Furthermore, a chemotaxis assay revealed that recombinant eotaxin and RANTES induced eosinophil migration concentration-dependently, which was abrogated by pretreatment of these chemokines with beta-tryptase. Another mast cell protease chymase also markedly reduced the immunoreactivity of eotaxin, but had no effect on RANTES and other chemokines and did not affect the influence of beta-tryptase on RANTES. These findings suggest that mast cell beta-tryptase selectively cleaves ASM-derived eotaxin and RANTES and abrogates their chemotactic activities, thus providing an explanation for the eosinophil paucity in asthmatic ASM bundles.
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PMID:Mast cell beta-tryptase selectively cleaves eotaxin and RANTES and abrogates their eosinophil chemotactic activities. 1651 49

Since beta-tryptase is considered a critical mediator of asthma, potent tryptase inhibitors may be useful as new agents for the treatment of asthma. We investigated 4-substituted benzylamine derivatives and obtained M58539 (15h) as a potent inhibitor of beta-tryptase (IC50 = 5.0 nM) with high selectivity against other serine proteases, low molecular weight, clog P value less than 5, lack of amidino and guanidino groups, and independence of Zn2+ ion.
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PMID:Synthesis and evaluation of 4-substituted benzylamine derivatives as beta-tryptase inhibitors. 1654 Mar 15

The key event of allergic inflammation, allergen-induced crosslinking of mast cell-bound IgE antibodies, is accompanied by release of inflammatory mediators, cytokines, and proteases, in particular beta-tryptase. We provide evidence that protease-mediated cleavage of allergens represents a mechanism that regulates allergen-induced mast cell activation. When used in molar ratios as they occur in vivo, purified beta-tryptase cleaved major grass and birch pollen allergens, resulting in defined peptide fragments as mapped by mass spectrometry. Tryptase-cleaved allergens showed reduced IgE reactivity and allergenic activity. The biological relevance is demonstrated by the fact that lysates from activated human mast cells containing tryptase levels as they occur in vivo cleaved allergens. Additionally, protamine, an inhibitor of heparin-dependent effector cell proteases, augmented allergen-induced release of mediators from effector cells. Protease-mediated allergen cleavage may represent an important mechanism for terminating allergen-induced effector cell activation.
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PMID:Allergen cleavage by effector cell-derived proteases regulates allergic inflammation. 1658 63


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