Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:029742 (skin tryptase)
 38 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human lung tryptase (HLT), a trypsin-like serine proteinase stored as an active enzyme in association with heparin in mast cell granules, is released into the extracellular environment when mast cells are activated. Tryptases are unusual in that they form tetramers and bind heparin. As there are no known endogenous tryptase inhibitors, loss of heparin and dissociation of the active tetrameric enzyme to inactive monomers has been proposed as the mechanism of control. Activity and intrinsic fluorescence were used to measure the stabilization of HLT by NaCl, glycerol, and heparin. At physiological salt concentrations in the absence of heparin, activity decayed rapidly (t1/2 = 1-4 min at 37 degrees C) to an intermediate that could be immediately reactivated by heparin. But protein structural changes, as measured by intrinsic fluorescence, were much slower (t1/2 = 16 min), indicating that the intermediate continued to exist as a tetramer that slowly changed to a monomer. HLT tetramers, either active or inactive, were stabilized by 2 M NaCl, 20% glycerol, and heparin. Maximum stabilization was obtained with approximately 1 mol of heparin per HLT subunit. Heparan sulfate also stabilized HLT activity and active HLT was bound to and recovered from cartilage. Subunits of the inactive intermediate appeared to be loosely associated as demonstrated by the rapid disappearance of the tetramer in gel filtration studies in 1 M NaCl (t1/2 = 1.8 min), but the tetramer was stable in lower ionic strength buffers containing heparin. Fluorescence anisotropy measurements in the absence of heparin were also consistent with a slow (t1/2 = 22 min) transition from tetramer to monomer, and native polyacrylamide gel electrophoresis provided additional evidence for a tetrameric intermediate. HLT monomers isolated by gel filtration were minimally active in the presence of heparin. These data show that heparin-free HLT rapidly converts to an "inactive", loose tetrameric intermediate that can be reactivated with heparin or slowly dissociate to less active monomers and that tryptase released from mast cells is likely to remain active in association with heparin or other extracellular components. Thus, tryptase affinity for glycosaminoglycans and substrate specificity limitations are the primary factors controlling the proteolytic functions of these enzymes.
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PMID:Inactivation of human lung tryptase: evidence for a re-activatable tetrameric intermediate and active monomers. 888 30

The mast cell plays a pivotal role in the early asthmatic response via release of mediators, which directly influence airway smooth muscle tone. Canine mast cell tryptase has been reported to potentiate the contractile response of canine isolated airways to histamine. The aim of this study was to investigate whether human mast cell tryptase potentiated contractile responses in human isolated bronchi. The effect of tryptase differed according to the sensitization status of the bronchi. In lung tissue from sensitized patients (those whose bronchial tissue contracted in response to the application of any of four common antigens) 90 ng.mL-1 of human purified lung tryptase markedly potentiated the contractile response to histamine. The maximal response as a percentage of maximal contraction to acetylcholine was 80 +/- 8% in control tissues and 119 +/- 6% in tryptase treated tissues (n = 4; p < 0.05). Tryptase, at a dose of 200 ng.mL-1, also potentiated responses but to a lesser degree, 100 +/- 5% (n = 4; p < 0.05). In nonsensitized bronchi, neither 90 nor 200 ng.mL-1 tryptase had any significant effect on histamine responses. The increased response in the presence of tryptase in sensitized tissue was inhibited by the calcium voltage-dependent channel antagonist, verapamil (10(-6) M). We have shown, for the first time, that human mast cell tryptase potentiates contraction in sensitized bronchi via a calcium-related mechanism. These findings provide a link between a mast cell derived product and in vitro human airway hyperresponsiveness.
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PMID:Mast cell tryptase potentiates histamine-induced contraction in human sensitized bronchus. 903 89

The x-ray crystal structure of recombinant leech-derived tryptase inhibitor (rLDTI) has been solved to a resolution of 1.9 A in complex with porcine trypsin. The nonclassical Kazal-type inhibitor exhibits the same overall architecture as that observed in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase tryptase. The conformation of the binding region of rLDTI suggests that tryptase has a restricted active site cleft. The basic amino terminus of rLDTI, apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent in tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the tryptase molecule. A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a role in inhibition of human lung tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch is in accordance with the known low molecular weight inhibitors of tryptase.
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PMID:The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. Implications for the structure of human mast cell tryptase and its inhibition. 924 60

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].
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PMID:Oxyanion-mediated inhibition of serine proteases. 983 2

Human lung tryptase, a homotetrameric serine protease unique to mast cell secretory granules, has been implicated in the pathogenesis of asthma. A hypothesis that tethered symmetrical inhibitors might bridge two adjacent active sites was explored via a rationally designed series of bisbenzamidines. These compounds demonstrated a remarkable distanced-defined structure-activity relationship against human tryptase with one series possessing subnanomolar potencies. Additional evidence supporting the concept of active-site bridging is also presented.
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PMID:Potent selective nonpeptidic inhibitors of human lung tryptase. 1041 78

Mast cell proteases are believed to participate in the basement membrane destruction in blistering diseases. Thus, normal human skin specimens were incubated with purified human skin tryptase or compound 48/80 (a mast cell degranulator) for up to 24 h. Thereafter, the specimens were studied immunohistochemically. Tryptase caused, in the presence and absence of 1,10-phenanthroline, focal dermal-epidermal separation above laminin and almost complete disappearance of the staining of the extra domain A region of cellular fibronectin in and beneath the basement membrane. The immunopositivity of the cell-binding region of fibronectin, laminin, and collagens IV and VII, however, was unaltered. Compound 48/80 induced almost complete dermal-epidermal separation above intact laminin and only focal reduction in the extra domain A region of cellular fibronectin staining. These alterations by compound 48/80 were prevented partially by Nalpha-p-tosyl-L-lysine chloromethyl ketone or 1,10-phenanthroline alone but completely when both inhibitors were present suggesting the involvement of tryptic serine proteinases, probably also tryptase, and metalloproteinases. Preventive effect of N-tosyl-L-phenylalanine chloromethyl ketone was weak suggesting minor function of chymotryptic serine proteinases. When tryptase was incubated with heparin and pure plasma fibronectin, an abrupt decrease in the adherence of cultured keratinocytes on to plastic surface coated with these substances and a gradual plasma fibronectin cleavage to 173, 161, and 28 kDa fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis were found. In conclusion, tryptase can cause focal dermal-epidermal separation above laminin in skin specimens but it is not known to what extent the decreased keratinocyte adherence in vitro and fibronectin cleavage are related to this dermal-epidermal separation.
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PMID:Focal dermal-epidermal separation and fibronectin cleavage in basement membrane by human mast cell tryptase. 1050 42

Tryptase may be a key mediator in mast cell-mediated inflammatory reactions. When mast cells are activated, they release large amounts of these tetrameric trypsin-like serine proteases. Tryptase is present in a macromolecular complex with heparin proteoglycan where the interaction with heparin is known to be essential for maintaining enzymatic activity. Recent investigations have shown that tryptase has potent proinflammatory activity, and inhibitors of tryptase have been shown to modulate allergic reactions in vivo. Many of the tryptase inhibitors investigated previously are directed against the active site. In the present study we have investigated an alternative approach for tryptase regulation. We show that the heparin antagonists Polybrene and protamine are potent inhibitors of both human lung tryptase and of recombinant mouse tryptase (mouse mast cell protease 6). Protamine inhibited tryptase in a competitive manner whereas Polybrene showed noncompetitive inhibition kinetics. Treatment of tetrameric, active tryptase with Polybrene caused dissociation into monomers, accompanied by complete loss of enzymatic activity. The present report thus suggests that heparin antagonists potentially may be used in treatment of mast cell-mediated diseases such as asthma.
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PMID:Heparin antagonists are potent inhibitors of mast cell tryptase. 1140 83

Tryptase, the major mast cell product, is considered to play an important role in airway inflammation and hyperresponsiveness. Tryptase produces different, sometimes opposite, effects on airway responsiveness (bronchoprotection and/or airway contraction). This study was designed to examine the effect of human lung tryptase and activation of protease-activated receptor (PAR)-2 by synthetic activated peptide (AP) SLIGKV-NH(2) on Ca(2+) signaling in human airway smooth muscle (HASM) cells. Immunocytochemistry revealed that PAR-2 was expressed by HASM cells. Tryptase (7.5--30 mU/ml) induced a concentration-dependent transient relative rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) that reached 207 +/- 32 nM (n = 10) measured by indo 1 spectrofluorometry. The protease inhibitors leupeptin or benzamidine (100 microM) abolished tryptase-induced [Ca(2+)](i) increase. Activation of PAR-2 by AP (1-100 microM) also induced a concentration-dependent transient rise in [Ca(2+)](i), whereas the reverse peptide produced no effect. There was a homologous desensitization of the [Ca(2+)](i) response on repeated stimulation with tryptase or AP. U-73122, a specific phospholipase C (PLC) antagonist, xestospongin, an inositol trisphosphate (IP(3))-receptor antagonist, or thapsigargin, a sarcoplamic Ca(2+)-ATPase inhibitor, abolished tryptase-induced [Ca(2+)](i) response, whereas Ca(2+) removal, in the additional presence of EGTA, had no effect. Calphostin C, a protein kinase C inhibitor, increased PAR-2 [Ca(2+)](i) response. Our results indicate that tryptase activates a [Ca(2+)](i) response, which appears as PAR-2 mediated in HASM cells. Signal transduction implicates the intracellular Ca(2+) store via PLC activation and thus via the IP(3) pathway. This study provides evidence that tryptase, which is increasingly recognized as an important mediator in airway inflammation and hyperresponsiveness, is also a potent direct agonist at the site of airway smooth muscle.
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PMID:Selected contribution: tryptase-induced PAR-2-mediated Ca(2+) signaling in human airway smooth muscle cells. 1145 17

Airway remodeling with smooth muscle cell (SMC) hyperplasia is a feature of chronic asthma. We investigated the potential for tryptase, the major secretory product of human mast cells, to act as a growth factor for human airway SMCs. Because this serine protease can activate proteinase-activated receptor-2 (PAR-2), we also examined the actions of SLIGKV, a peptide agonist of PAR-2. Incubation with lung tryptase provoked a twofold increase in [(3)H]thymidine incorporation; a similar increase in cell numbers was found when we used the MTS assay. The effect was catalytic site dependent, being abolished by the protease inhibitors leupeptin and benzamidine and by heat inactivation of the enzyme. Tryptase-induced DNA synthesis was inhibited by preincubation of the cells with pertussis toxin, calphostin C, or genistein. Transduction mechanisms are thus likely to involve a pertussis toxin-sensitive G protein, protein kinase C, and tyrosine kinase. SLIGKV elicited a response on SMCs similar to that of tryptase. Tryptase could provide an important stimulus for SMC proliferation in asthmatic airways, by acting on PAR-2.
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PMID:Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. 1150 38

We investigated the potential of human mast cell tryptase to induce relaxation of rat aorta. Trypsin and the selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2 stimulated robust relaxation of phenylephrine-precontracted rat aortic rings. However, human lung tryptase (1-100 nM) either in the presence or absence of heparin failed to induce any significant relaxation. Notwithstanding, incubation of the aorta with tryptase (100 nM), following the addition of a peptide corresponding to the cleavage/activation sequence of rat PAR2 (rPAR2), resulted in relaxation of precontracted tissue due to the proteolytic release of the PAR2-AP SLIGRL/ from the parent peptide. Thus, tryptase was enzymatically active in the bioassay system. Preincubation of aorta with neuraminidase to remove cell-surface sialic acid unmasked the ability of tryptase to induce relaxation of the aorta, but had no effect on relaxation induced by trypsin, SLIGRL-NH2, or acetylcholine (Ach). Like trypsin and SLIGRL-NH2, the tryptase-induced relaxation was inhibited by either removal of the endothelium or pretreatment of the tissue with NG-nitro-L-arginine methyl ester (L-NAME), suggesting an endothelium-derived nitric oxide mechanism. Interestingly, tryptase in the presence of heparin failed to induce relaxation of precontracted neuraminidase-treated rat aorta. We conclude that tryptase-induced relaxation of rat aorta, most likely via PAR2, is tightly regulated by heparin and cell-surface sialic acid.
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PMID:Restricted ability of human mast cell tryptase to activate proteinase-activated receptor-2 in rat aorta. 1245 65


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