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:3.4.21.37 (neutrophil elastase)
4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.
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PMID:Structure of the human neutrophil elastase gene. 290 87

Bacterial adherence is an important pathogenetic mechanism for airway colonization, but the influence of airway proteins on this phenomenon is largely unknown. We measured tracheal cell bacterial binding in 13 subjects with chronic tracheostomy and related these results to measurements of sputum IgA, elastase activity, and total protein from the same subjects. Tracheal cell adherence was related directly to sputum elastase activity (r = 0.61, p = 0.02); and elastase activity, primarily a serine protease, was higher in subjects colonized by Pseudomonas aeruginosa than in those without this finding (p = 0.02). Sputum levels of IgA/mg protein were related inversely to tracheal cell adherence (r = 0.64, p = 0.02). Sputum IgA concentrations, in turn, were affected by host nutritional status and airway elastase activity. Evidence that elastase can degrade sputum IgA was provided by an inverse relationship observed between these 2 proteins (r = 0.56, p = 0.04) and by in vitro mixing experiments showing fragmentation of IgA by purified neutrophil elastase. In addition, sucrose density gradient separation indicated IgA fragmentation to have occurred in vivo. These data suggest that, once adherence leads to airway colonization, the resulting inflammatory response may foster microbial growth by an elastase-dependent IgA cleavage and hence enhanced tracheal cell adherence.
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PMID:Influence of sputum IgA and elastase on tracheal cell bacterial adherence. 308 Sep 31

Human neutrophils contain large amounts of a neutral serine protease, human neutrophil elastase (HNE), which has been implicated as a mediator of acute and chronic lung injury. We found that this enzyme is effectively inhibited, at physiological ionic strength, by several synthetic non-base-paired polyribonucleotides. Among the most active of these is polyguanylic acid (poly G). Inhibitory activity is greatest with high-molecular-weight poly G fractions, but poly G fractions even as low as 60K Mr (app) are effective. Both amidolysis of synthetic elastase substrates, such as succinyl-ala-ala-ala-p-nitroanilide, and proteolysis of elastin are blocked. Poly G inhibits elastin proteolysis even when subsequently added to mixtures of elastin and HNE that have first been preincubated together for 10 min. Under these conditions, polyribosylribitol phosphate, a polyanion derived from Haemophilus influenzae capsular polysaccharide, is not inhibitory. Complex formation between HNE and poly G is dependent on ionic rather than covalent interactions, since it is blocked by 0.6 M NaCl but not by inactivation of the enzyme's catalytic-site serine residue with diisopropylfluorophosphate. However, nonspecific ionic interactions alone cannot explain complex formation, since pancreatic elastase and cathepsin G, an even more basic serine protease from human neutrophils, do not form complexes with poly G, even at low ionic strength. Moreover, in the presence of the amphiphiles taurocholic acid and glycocholic acid, HNE is much less effectively blocked by poly G. Peptide chloromethyl ketone-inactivate HNE (which has its extended substrate-binding pocket occupied by the peptidyl inactivator) also fails to form complexes with poly G. These results indicate that HNE may utilize both hydrophobic and ionic binding sites to couple with poly G, and suggest that these sites may be close to or within the extended substrate-binding pocket of the enzyme.
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PMID:Inhibition of human neutrophil elastase by polyguanylic acid and other synthetic RNA homopolymers. 325 33

Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder associated with a major reduction in serum A1AT levels. Clinically, A1AT deficiency is associated with emphysema in adults and, less commonly, liver disease in neonates. A1AT is a 52-kDa, 394-amino acid, single-chain glycoprotein normally present in serum at 150 to 350 mg/dl. The A1AT gene, composed of seven exons dispersed over 12 kb of chromosomal segment 14q31-32.3, is expressed in hepatocytes and mononuclear phagocytes. The A1AT protein, a member of the class of protease inhibitor proteins known as serpins (serine protease inhibitors), is a globular molecule composed of nine alpha-helices and three beta-pleated sheets. The major function of A1AT is to inhibit neutrophil elastase; A1AT does so through an active site centered around Met358 contained within an external stressed loop on the surface of the molecule. A1AT is a highly pleomorphic protein with greater than 75 variants determined at the protein and/or gene level. These variants can be categorized into four groups according to their serum A1AT level and function: normal, deficient, dysfunctional, and absent. There are two important salt bridges within the A1AT molecule (Glu342-Lys290; Glu263-Lys387); a mutation in the A1AT gene causing disruption of either salt bridge causes distinct molecular pathology resulting in reduced serum A1AT levels. Clinically relevant variants can be distinguished by a combination of isoelectric focusing of serum, restriction fragment length analysis of genomic DNA, oligonucleotide probes, and direct sequencing of the variant A1AT genes.
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PMID:Molecular basis of alpha-1-antitrypsin deficiency. 328 85

Characterization of glucocorticoid receptors in leukemia cells is important to understand mechanisms of glucocorticoid resistance but has been impeded by receptor fragmentation in cytosol extracts. We recently found that formation of 52- and 30-kilodalton (kD) glucocorticoid receptor fragments in cytosol of leukemia cells is due to proteolysis and is blocked by diisopropylfluorophosphate (DFP). In the present study, we identify a 28-kD serine protease in cytosol of leukemia cells that binds [3H]DFP and correlates with the formation of 52- and 30-kD receptor fragments. This protease is immunoprecipitated by antiserum to neutrophil elastase. Limited digestion of [3H]dexamethasone-21-mesylate-labeled receptors by purified neutrophil elastase produces 52- and 30-kD receptor fragments. Receptor fragmentation in the cytosol of leukemia cells in inhibited by methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethylketone, a highly specific inhibitor of neutrophil elastase. The addition of as few as 5% neutrophils to a lymphoid cell suspension provides sufficient elastase to produce receptor fragmentation. Our findings indicate that neutrophil elastase is responsible for receptor fragmentation in the cytosol of leukemia cells. The neutrophil elastase may be endogenous to the leukemia cells or may come from neutrophils that contaminate leukemia cell suspensions.
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PMID:Neutrophil elastase produces 52-kD and 30-kD glucocorticoid receptor fragments in the cytosol of human leukemia cells. 330 64

Human neutrophil elastase (NE) functions as a powerful serine protease capable of attacking a broad range of proteins. To examine the cellular site(s) of NE gene expression, a 0.65-kilobase cDNA (pPB15) complementary to the coding region of the NE gene was cloned from the cell line U937 using an oligonucleotide based on the known NE protein sequence. The sequence of pPB15 demonstrated that it coded for the 173 C-terminal residues of the 218 amino acids that comprise the mature NE protein, plus an additional 3' 60 base pairs prior to the in-frame stop codon, suggesting the NE mRNA contains sequences for a 20-residue C-terminal "pro" peptide that is not found in the mature protein. Northern analysis using 32P-labeled pPB15 as a probe revealed that neutrophils do not contain detectable NE mRNA transcripts despite the fact that this cell carries large amounts of this protein. Furthermore, resting and activated blood monocytes also contained no detectable NE mRNA transcripts, although these cells also carry detectable NE. In contrast, bone marrow precursor cells contained NE transcripts, suggesting the NE gene is expressed in blood precursor cells. In this regard, evaluation of HL-60 cells, a human cell line with myelomonocytic lineage features, demonstrated NE transcripts in resting cells and increased NE mRNA levels when the cells were induced toward the myelocytic lineage with dimethyl sulfoxide. However, when the HL-60 cells were induced toward the monocytic lineage with phorbol 12-myristate 13-acetate, NE transcripts were lost even though transcripts for interleukin-1 beta were plentiful. Together, these observations are consistent with the concept that the NE gene is not expressed in the blood cells that carry the protein, but in bone marrow precursors that express NE transcripts about the time of commitment to the myelocytic series.
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PMID:Myelomonocytic cell lineage expression of the neutrophil elastase gene. 342 32

In order to determine the acute effect of smoking on elastase in bronchoalveolar lavage fluid (BAL), we obtained BAL from 30 smokers twice, the first before smoking (after 8 h of abstinence) and the second 2 min to 1 h after the subjects had smoked either 2 or 4 cigarettes. Bronchoalveolar lavage was concentrated 100-fold and was assayed for elastaselike activity against succinyl-trialanyl-p-nitroanilide (SLAPN) as substrate. Elastolytic activity against insoluble 3H-elastin and immunologic neutrophil elastase levels tested with a sensitive enzyme-linked immunosorbent assay were determined in some subjects. No activity against insoluble 3H-elastin was detected using a sensitive assay capable of detecting subnanogram quantities of elastase. Total elastaselike activity in BAL against SLAPN was significantly (p less than 0.02) increased in smokers prior to smoking (47.4 +/- SD 20.2 ng human neutrophil elastase (HNE) equivalents) when compared with BAL from 7 nonsmokers (26.3 +/- SD 13.4 ng HNE); however, there was no significant change in enzyme activity in smokers' BAL after smoking. Assays with EDTA and phenyl-methyl-sulfonyl-fluoride as inhibitors suggested that approximately two thirds of elastaselike activity was due to a metalloprotease and that there was negligible serine protease activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Acute effect of smoking on elastaselike esterase activity and immunologic neutrophil elastase levels in bronchoalveolar lavage fluid. 363 79

The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing.
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PMID:Neutrophil-induced injury of rat pulmonary alveolar epithelial cells. 377

Elastase activity generated during lung defense against aerobic bacteria was studied in an animal model. Bronchoalveolar lavage (BAL) fluid from hamsters inoculated with bacteria was assayed for elastase activity at 0, 2, 4, 6, and 8 h after inoculation using a synthetic substrate of elastase, succinyl-trialanine-nitroanilide (SLAPN). Streptococcus pneumoniae type 25 inoculation led to a peak elastase activity of 0.72 +/- 0.27 X 10(-3) units, not significantly different from baseline (0.41 +/- 0.08 X 10(-3) units) or saline control (0.33 +/- 0.18 X 10(-3) units). In contrast, inoculation with Pseudomonas aeruginosa strain PAO-1 (a species known to produce elastase as well as other virulence factors) produced peak elastase activity of 3.0 +/- 1.2 X 10(-3) units in BAL fluid, significantly higher than either pneumococcus type 25 or saline control (p less than 0.025). Inoculation with Pseudomonas aeruginosa strain E-64, an isogenic mutant of PAO-1 that produces a nonfunctional elastase, led to peak levels similar to the PAO-1 strain, suggesting that the presence of bacterial elastase was not the primary factor in BAL fluid elastase activity. Total numbers of granulocytes in BAL fluid from pneumococcus-inoculated animals (144 +/- 31 X 10(6] was significantly higher (p less than 0.05) than from either the PAO-1 (74 +/- 31 X 10(6] or E-64 (99 +/- 27 X 10(6] strains of Pseudomonas, Use of selective enzyme inhibitors of elastase, diisopropyl fluorophosphate and disodium ethylenediaminetetraacetate, implied that the majority of elastase activity in BAL fluid was due to a serine protease, of which granulocyte elastase is the primary source.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pulmonary elastase activity in response to Streptococcus pneumoniae and Pseudomonas aeruginosa. 384 39

The time-dependent inactivation of several serine proteases including human leukocyte elastase, cathepsin G, rat mast cell proteases I and II, and human skin chymase by a number of 3-alkoxy-4-chloroisocoumarins, 3-alkoxy-4-chloro-7-nitroisocoumarins, and 3-alkoxy-7-amino-4-chloroisocoumarins at pH 7.5 and the inactivation of several trypsin-like enzymes including human thrombin and factor XIIa by 7-amino-4-chloro-3-ethoxyisocoumarin and 4-chloro-3-ethoxyisocoumarin are reported. The 3-alkoxy substituent of the isocoumarin is likely interacting with the S1 subsite of the enzyme since the most reactive inhibitor for a particular enzyme had a 3-substituent complementary to the enzyme's primary substrate specificity site (S1). Inactivation of several enzymes including human leukocyte elastase by the 3-alkoxy-7-amino-4-chlorisocoumarins is irreversible, and less than 3% activity is regained upon extensive dialysis of the inactivated enzyme. Addition of hydroxylamine to enzymes inactivated by the 3-alkoxy-7-amino-4-chloroisocoumarins results in a slow (t1/2 greater than 6.7 h) and incomplete (32-57%) regain in enzymatic activity at pH 7.5. Inactivation by the 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-4-chloro-7-nitroisocoumarins on the other hand is transient, and full enzyme activity is regained rapidly either upon standing, after dialysis, or upon the addition of buffered hydroxylamine. The rate of inactivation by the substituted isocoumarins is decreased when substrates or reversible inhibitors are present in the incubation mixture, which indicates active site involvement. The inactivation rates are dependent upon the pH of the reaction mixture, the isocoumarin ring system is opened concurrently with inactivation, and the reaction of 3-alkoxy-7-amino-4-chloroisocoumarins with porcine pancreatic elastase is shown to be stoichiometric. The results are consistent with a scheme where 3-alkoxy-7-amino-4-chloroisocoumarins react with the active site serine of a serine protease to give an acyl enzyme in which a reactive quinone imine methide can be released. Irreversible inactivation could then occur upon alkylation of an active site nucleophile (probably histidine-57) by the acyl quinone imine methide. The finding that hydroxylamine slowly catalyzes partial reactivation indicates that several inactivated enzyme species may exist. The 3-alkoxy-substituted 4-chloroisocoumarins and 4-chloro-7-nitroisocoumarins are simple acylating agents and do not give stable inactivated enzyme structures.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: new reactive mechanism-based inhibitors. 391 97


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