Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.37 (neutrophil elastase)
 4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.
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PMID:Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry. 1097 67

The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extracts. The HiTI purified by affinity chromatography on trypsin-Sepharose has a molecular mass of 7029Da by MALDI-TOF mass spectrometry. HiTI inhibited bovine trypsin, human neutrophil elastase, and a trypsin-like enzyme purified from H. i. irritans abdomen with dissociation constants of 0.57, 1.30, and 0.20nM, respectively. The HiTI partial amino acid sequence allowed its classification into the BPTI-Kunitz-type family. An HiTI cDNA fragment was cloned in the pGEMT vector using RT-PCR. The translated amino acid sequence of HiTI cDNA confirmed a unique Kunitz-type-domain protein. Our results suggest that HiTI could control some endogenous enzyme, e.g., the H. i. irritans trypsin-like protein.
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PMID:Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae). 1517 17

There is great interest in the use of the sheep as a model for the investigation of inflammation in the lung. The serine antiproteases secretory leukoprotease inhibitor (SLPI) and elafin are important "alarm antiproteases" in the lung and have potentially important roles in the innate immune response. SLPI was first characterized in man and subsequently in murine, porcine, and rat tissues. Here we present the first data concerning the gene and cDNA sequence encoding for the ovine ortholog of SLPI, a protein of 132 amino acids with 66% sequence identity at the amino acid level with human SLPI. A 24-amino-acid signal sequence signifies that, like the other mammalian orthologs, ovine SLPI is a secreted protein. Tissue distribution of expression is demonstrated by reverse transcription polymerase chain reaction (RT-PCR) and shows features similar to SLPI expression in other mammals, specifically at mucosal surfaces such as the upper respiratory and intestinal tracts, and also the skin, liver, and kidney. This distribution lends credence to SLPI having important roles in innate immunity. We have also cloned the ovine SLPI cDNA into an expression vector and expressed the ovine SLPI protein in vitro. This has enabled us to demonstrate that ovine SLPI is correctly processed (Western blot analysis and SELDI-TOF mass spectrometry analysis) and has biological antihuman neutrophil elastase activity. In summary, the ovine ortholog of SLPI shows similarities to other members of the SLPI family and has all the features of a modulator of innate immunity.
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PMID:Characterization of the ovine ortholog of secretory leukoprotease inhibitor. 1618 Jan 44

Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 degrees C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI-I, CmPI-II and CmPI-III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI-I and CmPI-II was confirmed, while CmPI-III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI-I and CmPI-II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI-I (6 amino acids) and CmPI-II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI-II. Both inhibitors, CmPI-I and CmPI-II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (Ki) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI-I and CmPI-II are the first human neutrophil elastase inhibitors described in a mollusk.
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PMID:Purification and partial characterization of human neutrophil elastase inhibitors from the marine snail Cenchritis muricatus (Mollusca). 1654 27

BmSI-7 and BmSI-6, two Boophilus microplus subtilisin inhibitors (BmSI) were purified and characterized from eggs. The inhibitors isolated by classical purification methods presented molecular masses of 7408 and 7271Da, respectively, by MALDI-TOF-MS. Both BmSI-7 and BmSI-6 inhibited neutrophil elastase (K(i) 0.4 and 0.3nM) and subtilisin A (K(i) 1.4nM for both inhibitors). They also strongly inhibited Pr1 proteases from the fungus Metarhizium anisopliae; BmSI-7 (K(i) 50nM) and BmSI-6 (K(i) 2.2nM). The BmSI-7 full length cDNA was obtained using amino acid sequence information of BmSI-7 peptides generated by proteolytic digestion. BmSI-7 belongs to trypsin inhibitor like cysteine rich domain family (TIL), and it is transcribed in ovary, fat body, gut, salivary gland and haemocytes. BmSI-7 is the first TIL inhibitor described with inhibitory activity toward subtilisin A and Pr1 proteases of entomopathogenic fungi.
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PMID:BmSI-7, a novel subtilisin inhibitor from Boophilus microplus, with activity toward Pr1 proteases from the fungus Metarhizium anisopliae. 1788 50

The epithelial Na(+) channel (ENaC) that mediates regulated Na(+) reabsorption by epithelial cells in the kidney and lungs can be activated by endogenous proteases such as channel activating protease 1 and exogenous proteases such as trypsin and neutrophil elastase (NE). The mechanism by which exogenous proteases activate the channel is unknown. To test the hypothesis that residues on ENaC mediate protease-dependent channel activation wild-type and mutant ENaC were stably expressed in the FRT epithelial cell line using a tripromoter human ENaC construct, and protease-induced short-circuit current activation was measured in aprotinin-treated cells. The amiloride-sensitive short circuit current (I(Na)) was stimulated by aldosterone (1.5-fold) and dexamethasone (8-fold). Dexamethasone-treated cells were used for all subsequent studies. The serum protease inhibitor aprotinin decreased baseline I(Na) by approximately 50% and I(Na) could be restored to baseline control values by the exogenous addition of trypsin, NE, and porcine pancreatic elastase (PE) but not by thrombin. All protease experiments were thus performed after exposure to aprotinin. Because NE recognition of substrates occurs with a preference for binding valines at the active site, several valines in the extracellular loops of alpha and gamma ENaC were sequentially substituted with glycines. This scan yielded two valine residues in gamma ENaC at positions 182 and 193 that resulted in inhibited responses to NE when simultaneously changed to other amino acids. The mutations resulted in decreased rates of activation and decreased activated steady-state current levels. There was an approximately 20-fold difference in activation efficiency of NE against wild-type ENaC compared to a mutant with glycine substitutions at positions 182 and 193. However, the mutants remain susceptible to activation by trypsin and the related elastase, PE. Alanine is the preferred P(1) position residue for PE and substitution of alanine 190 in the gamma subunit eliminated I(Na) activation by PE. Further, substitution with a novel thrombin consensus sequence (LVPRG) beginning at residue 186 in the gamma subunit (gamma(Th)) allowed for I(Na) activation by thrombin, whereas wild-type ENaC was unresponsive. MALDI-TOF mass spectrometric evaluation of proteolytic digests of a 23-mer peptide encompassing the identified residues (T(176)-S(198)) showed that hydrolysis occurred between residues V193 and M194 for NE and between A190 and S191 for PE. In vitro translation studies demonstrated thrombin cleaved the gamma(Th) but not the wild-type gamma subunit. These results demonstrate that gamma subunit valines 182 and 193 are critical for channel activation by NE, alanine 190 is critical for channel activation by PE, and that channel activation can be achieved by inserting a novel thrombin consensus sequence. These results support the conclusion that protease binding and perhaps cleavage of the gamma subunit results in ENaC activation.
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PMID:A segment of gamma ENaC mediates elastase activation of Na+ transport. 1799 93

The somatic cell count (SCC) in milk is associated with increasing proteolytic degradation of caseins and it has been suggested that enzymes derived from somatic cells contribute to a lower yield and poorer quality of cheese. It is essential to increase the knowledge on naturally occurring milk proteinase activities to better understand how to improve the technological quality of milk. The aim of this work was to identify peptides actually present in milk as a result of proteolysis at different levels of SCC and to assign these peptides to potential responsible proteases where possible. Peptide fractions were prepared from acid whey by ultrafiltration at a molecular cut-off value of 10 000 Da. The peptides were separated using capillary reversed phase high performance liquid chromatography (RP-HPLC) and identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (MALDI-TOF MS/MS). Peptides identified ranged in mass from 1023 to 2000 Da, and originated from alphaS1-, alphaS2- or beta-casein. Possible responsible proteases that could be suggested when examining the peptide cleavage sites included plasmin, cathepsin B, D and leukocyte elastase. The results indicated that plasmin was primarily responsible for the observed proteolysis in milk at low cell count, whereas the cathepsins and elastase became implicated at elevated cell count. Specificity and activity of cathepsins and elastase has earlier mainly been studied in model systems, whereas less is known about their activities in milk itself. This is also the first indication of involvement of elastase in milk proteolysis through the unequivocal determination of cleavage sites.
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PMID:Identification of peptides in milk as a result of proteolysis at different levels of somatic cell counts using LC MALDI MS/MS detection. 1822 1

The first step in the activation of plasma factor XIII (FXIII) is the cleavage of R37-G38 bond in FXIII-A subunit (FXIII-A) by thrombin, which makes the subsequent formation of an active transglutaminase possible. No active truncated form of FXIII-A, other than G38-FXIII-A, has been identified. In contrast to thrombin, which has a preference toward arginine residues, human neutrophil elastase (HNE) cleaves peptide bonds at small side-chain aliphatic amino acids, preferably at valine. As there are several valine residues close to the thrombin cleavage-site, we tested if an active truncated FXIII-A was formed during fragmentation of FXIII by HNE. It was demonstrated by Western blotting and transglutaminase assay that HNE induced a limited cleavage of FXIII-A resulting in the activation of both plasma and cellular FXIII; the maximal transglutaminase activities were 52.5% and 67.4% of thrombin-activated FXIII, respectively. After the relatively rapid activation a much slower inactivation occurred. HNE-activated FXIII cross-linked fibrin gamma- and alpha-chains in the clot formed by batroxobin moojeni. MALDI-TOF analysis of the cleaved fragments and N-terminal Edman degradation of the truncated protein identified V39-N40 as the primary cleavage-site and N40-FXIII-A as the active form. No primary cleavage occurred at V34, V35, V47, V50 residues. FXIII-A V34L polymorphism, which increases the rate of FXIII-A cleavage by thrombin, was without effect on FXIII activation by HNE. Molecular modeling located the primary HNE cleavage-site in the middle of the flexible and accessible Q32-L45 loop and showed that other neighboring valine residues were in less favorable position.
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PMID:Cleavage of factor XIII by human neutrophil elastase results in a novel active truncated form of factor XIII A subunit. 1839 24

Eight new cyanopeptolins (insulapeptolides A-H) were obtained from the cyanobacterium Nostoc insulare. Their isolation was guided by their bioactivity toward the target enzyme human leukocyte elastase, molecular biological investigations, and MALDI-TOF analysis. These peptides are selective inhibitors of human leukocyte elastase with activities in the nanomolar range. Insulapeptolide D was the most potent compound with an IC(50) value of 85 nM (K(i) value of 36 nM).
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PMID:New peptolides from the cyanobacterium Nostoc insulare as selective and potent inhibitors of human leukocyte elastase. 1892 17

Frankincense preparations, used in folk medicine to cure inflammatory diseases, showed anti-inflammatory effectiveness in animal models and clinical trials. Boswellic acids (BAs) constitute major pharmacological principles of frankincense, but their targets and the underlying molecular modes of action are still unclear. Using a BA-affinity Sepharose matrix, a 26-kDa protein was selectively precipitated from human neutrophils and identified as the lysosomal protease cathepsin G (catG) by mass spectrometry (MALDI-TOF) and by immunological analysis. In rigid automated molecular docking experiments BAs tightly bound to the active center of catG, occupying the same part of the binding site as the synthetic catG inhibitor JNJ-10311795 (2-[3-[methyl[1-(2-naphthoyl)piperidin-4-yl]amino]carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid). BAs potently suppressed the proteolytic activity of catG (IC(50) of approximately 600 nM) in a competitive and reversible manner. Related serine proteases were significantly less sensitive against BAs (leukocyte elastase, chymotrypsin, proteinase-3) or not affected (tryptase, chymase). BAs inhibited chemoinvasion but not chemotaxis of challenged neutrophils, and they suppressed Ca(2+) mobilization in human platelets induced by isolated catG or by catG released from activated neutrophils. Finally, oral administration of defined frankincense extracts significantly reduced catG activities in human blood ex vivo vs placebo. In conclusion, we show that catG is a functional and pharmacologically relevant target of BAs, and interference with catG could explain some of the anti-inflammatory properties of frankincense.
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PMID:Identification of human cathepsin G as a functional target of boswellic acids from the anti-inflammatory remedy frankincense. 1964 70


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