Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0034067 (emphysema)
 11,506 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Isocoumarins are potent mechanism-based heterocyclic irreversible inhibitors for a variety of serine proteases. Most serine proteases are inhibited by the general serine protease inhibitor 3,4-dichloroisocoumarin, whereas isocoumarins containing hydrophobic 7-acylamino groups are potent inhibitors for human leukocyte elastase and those containing 7-alkylureidogroups are inhibitors for procine pancreatic elastase. Isocoumarins containing basic side chains that resemble arginine are potent inhibitors for trypsin-like enzymes. A number of 3-alkoxy-4-chloro-7-guanidinoisocoumarins are potent inhibitors of bovine thrombin, human factor Xa, human factor XIa, human factor XIIa, human plasma kallikrein, porcine pancreatic kallikrein, and bovine trypsin. Another cathionic derivative, 4-chloro-3-(2-isothiureidoethoxy) isocoumarin, is less reactive toward many of these enzymes but is an extremely potent inhibitor of human plasma kallikrein. Several guanidinoisocoumarins have been tested as anticoagulants in human plasma and are effective at prolonging the prothrombin time. The mechanism of inhibition by this class of heterocyclic inactivators involves formation of an acyl enzyme by reaction of the active site serine with the isocoumarin carbonyl group. Isocoumarins with 7-amino or 7-guanidino groups will then decompose further to quinone imine methide intermediates, which react further with an active site residue (probably His-57) to form stable inhibited enzyme derivatives. Isocoumarins should be useful in further investigations of the physiological function of serine proteases and may have future therapeutic utility for the treatment of emphysema and coagulation disorders.
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PMID:Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design. 265 46

The primary function of alpha 1-antitrypsin (alpha 1-AT), an antiprotease produced by the liver, is the inhibition of neutrophil elastase, a protease capable of hydrolysing most connective tissue components. The importance of alpha 1-AT is demonstrated by the high incidence of early-onset emphysema in individuals with hereditary alpha 1-AT deficiency (Type PiZZ), in whom serum levels of alpha 1-AT are 10-20% of normal. Oxidants in tobacco smoke can inactivate alpha 1-AT in vitro, and studies have shown that alpha 1-AT from the lungs of individuals who smoke cigarettes may also be partially inactivated, perhaps explaining the high incidence of emphysema associated with cigarette smoking. Oxidative inactivation is probably due to modification of the Met residue (Met358) at the P1 subsite position of the elastase binding site of the protein. To study the possibility of modulating the biological properties of alpha 1-AT, we have introduced selected sequence modifications at the reactive site by in vitro mutation of a cloned alpha 1-AT complementary DNA. We describe here the characterization of two alpha 1-AT analogues produced in Escherichia coli. The first, alpha 1-AT(Met385----Val), is not only fully active as an elastase inhibitor but is also resistant to oxidative inactivation. The other, alpha 1-AT(Met358----Arg), no longer inhibits elastase but is an efficient thrombin inhibitor. The active site of the latter is identical to that of the alpha 1-AT (Pittsburgh) variant, which was associated with a fatal bleeding disorder.
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PMID:Synthesis in E. coli of alpha 1-antitrypsin variants of therapeutic potential for emphysema and thrombosis. 388 Aug 73

alpha 1-Antitrypsin is the most abundant of several serum protease inhibitors. Its deficiency is associated with an increased incidence of emphysema in adults, jaundice in newborns, and childhood cirrhosis. We describe an automated functional assay for the Instrumentation Laboratory's Multistat III Microcentrifugal Analyzer with N-alpha-benzoyl-DL-arginine-p-nitroanilide as trypsin substrate. The assay is standardized in terms of moles of trypsin active sites inhibited per liter of serum, by use of a chromogenic titrant for trypsin active sites, p-nitrophenyl-p'-guanidinobenzoate. The method is rapid, precise, and independent of trypsin supplier, and results correlate well with those by a manual chromogenic and a nephelometric assay.
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PMID:Automated assay for alpha 1-antitrypsin with N-alpha-benzoyl-DL-arginine-p-nitroanilide as trypsin substrate and standardized with p-nitrophenyl-p'-guanidinobenzoate as titrant for trypsin active sites. 628 Aug 95

Nitrogen dioxide (NO2), an air pollutant produced by burning fossil fuels and a component of cigarette smoke, is thought to contribute to the pathogenesis of pulmonary diseases, such as emphysema. In order to gain information on the mechanism by which NO2 damages the lung and proteins vital to its function, as well as its reaction with proteins in general, in vitro exposures of alpha-1-proteinase inhibitor (alpha 1PI), elastin, poly-L-lysine, and poly-L-arginine were performed. The ability of alpha 1PI to inhibit its natural physiological target, human neutrophil elastase (HNE), declined with exposure to 54% of the control value at molar ratios of NO2:alpha 1PI of 400:1 and greater. Exposure of alpha 1PI to NO2 resulted in a 50% loss of immunoreactivity with either monoclonal or polyclonal antibodies in an enzyme-linked immunosorbent assay at molar ratios of NO2:alpha 1PI of 100:1 and greater. The results of parallel O-phthalaldehyde and bicinchoninic acid protein assays as well as amino acid analysis on control and NO2-exposed alpha 1PI suggested a reactivity of NO2 with lysine residues. Elastin and poly-L-lysine were labeled by reductive methylation of amino groups with [3H]HCHO prior to treatment with NO2 in aqueous solutions at physiological pH. NO2 exposure of elastin resulted in the solubilization of 84% of the associated radioactivity of which 79% was identified as [3H]methyllysine by amino acid analysis. After NO2 exposure of poly-L-[3H]lysine, gel filtration chromatography revealed that the 50,000 M(r) poly-L-[3H]lysine had been degraded to small peptides of 1-3000 M(r). Similarly, after NO2 exposure of unlabeled poly-L-arginine, gel filtration chromatography, and total peptide analysis revealed that the 47,500 M(r) peptide was also partially degraded to peptides. These results suggest that NO2 reacts with the epsilon-amino groups of Lys residues (primary amines) and with the amide nitrogen (secondary amines) of surface-exposed Lys and Arg residues in the peptide backbone to result in peptide bond cleavage. These findings are the first indication of NO2-mediated peptide degradation and provide additional data on the potential of NO2 to damage proteins vital to the function of the lung in an in vitro exposure system.
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PMID:Nitrogen dioxide reactivity with proteins: effects on activity and immunoreactivity with alpha-1-proteinase inhibitor and implications for NO2-mediated peptide degradation. 832 82

Elastinolytic enzymes derived from alveolar macrophages (AM) are considered to play an important role in the development of emphysema associated with cigarette smoking. In this study, the enzyme activity and mRNA expression of cathepsin L were quantitated in AM and bronchoalveolar lavage (BAL) fluid obtained from current smokers and compared with those from nonsmokers. Activity was measured with the synthetic substrate Z-Phe-Arg-MCA combined with a novel cathepsin B inhibitor, CA-074. We found that the specific activity of cathepsin L was significantly elevated in BAL cells from smokers (7.1 +/- 0.7 mumol/mg protein/h, mean +/- SEM) compared with cells from nonsmokers (2.9 +/- 0.3) (p < 0.01). The expression of cathepsin L mRNA in BAL cells as determined by dot-blot analysis was also higher in BAL cells from smokers, which was comparable to the increase in the enzyme activity. About 5 to 6% of the specific activity of cathepsin L in BAL cell lysates was detected in unconcentrated BAL fluid; specific activity was also significantly higher in samples from smokers (0.38 +/- 0.04 mumol/mg protein/h) than from nonsmokers (0.14 +/- 0.02). In addition, procathepsin L (42 kD) and the mature form of cathepsin L (33 kD) were demonstrated in BAL fluid by immunoblot analyses. These data suggest that cigarette smoking induces mRNA expression and the synthesis of cathepsin L in AM and the release of procathepsin from AM into extracellular milieu. Furthermore, increased activity levels of cathepsin L in extracellular compartments may contribute to the proteolysis of elastin in the process of lung destruction associated with cigarette smoking.
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PMID:Cathepsin L activity is increased in alveolar macrophages and bronchoalveolar lavage fluid of smokers. 850 70

Proteinase 3 is a human polymorphonuclear leukocyte serine proteinase that degrades elastin in vitro and causes emphysema when administered by intratracheal insufflation into hamsters. Proteinase 3, stored in the azurophilic granules, is expressed in progenitor cells of myeloid origin. In the present study, the biosynthesis, processing, and intracellular transport of the enzyme was investigated in the human myelomonocytic cell line U937. Proteinase 3 is initially identified as a 35-kDa precursor and converted into the 29-kDa mature form within 3 h. By using a combination of techniques including amino-terminal sequencing, we identified the 35-kDa form as a zymogen containing an activation dipeptide but lacking the amino-terminal 25 residues, presumably the result of cleavage by a signal peptidase. Tunicamycin treatment and alkalinization of acidic cell compartments with NH4Cl did not prevent the processing of the proteinase 3 zymogen into the mature form, suggesting that the enzyme is targeted to the cytoplasmic granules by a mechanism other than the mannose 6-phosphate receptor. Brefeldin A inhibited the zymogen processing, suggesting that the dipeptide cleavage occurred in a post-Golgi organelle. The enzyme responsible for the removal of the dipeptide is a cysteine proteinase since E-64d, a class-specific inhibitor, prevented processing. However, treatment of cells with a dipeptidyl peptidase I inhibitor, Gly-Phe-diazomethyl ketone and with the lysosomotropic agents, NH4Cl and chloroquine, did not prevent dipeptide cleavage, indicating that the processing enzyme for proteinase 3 is not dipeptidyl peptidase I. In contrast, Gly-Phe-diazomethyl ketone inhibited cleavage of the dipeptide from cathepsin G. This indicates that processing of proteinase 3 is distinct from that of cathepsin G. Proteinase 3 is also processed at the COOH-terminal extension. Cleavage takes place next to Arg-222, suggesting that a trypsin-like proteinase is involved in the COOH-terminal processing.
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PMID:Biosynthesis and processing of proteinase 3 in U937 cells. Processing pathways are distinct from those of cathepsin G. 862 89

Neutrophil accumulation in the lung is implicated in the pathogenesis of pulmonary emphysema and chronic bronchitis associated with cigarette smoking. To determine whether nicotine contributes to this accumulation through the prolongation of neutrophil survival, we examined the survival rates of isolated neutrophils cultured with or without nicotine. We found that nicotine prolonged neutrophil survival in a dose-dependent fashion, with a maximum effect at 10(-6) mol/L. The survival rate at 72 hours was 35.6% +/- 1.2% in medium with 10(-6) mol/L nicotine, compared with 15.5% +/- 0.5% in control medium (mean +/- SEM; p < 0.01), as determined by trypan blue dye exclusion. This prolongation was brought about by suppression of apoptosis, as evidenced by both transmission electron and fluorescence microscopy, and was associated with the preservation of neutrophil functions such as chemotaxis and O2- generation. The prolongation of survival caused by nicotine was abrogated by the addition of Pro-Lys-Arg-NH2, a competitive inhibitor of the specific binding of nicotine to noncholinergic receptors on neutrophils. However, the prolongation of survival caused by nicotine was not suppressed in the presence of K-252b, an inhibitor of protein kinase C. These findings suggest that nicotine prolongs neutrophil survival through noncholinergic nicotine receptors and new protein synthesis, without activation of protein kinase C.
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PMID:Nicotine prolongs neutrophil survival by suppressing apoptosis. 863 47

alpha 1-Antitrypsin is the archetypal member of the serine proteinase inhibitor or serpin superfamily. Members of the family show structural homology based on a dominant A beta-sheet and a mobile reactive centre loop. Our recent crystal structure of alpha 1-antitrypsin stabilized with a point mutation showed the loop to be in a canonical inhibitory conformation in the absence of significant insertion into the A beta-sheet. It could be argued that the stabilizing mutation may induce the reactive centre loop to adopt an artificial, and unrepresentative, conformation and the finding seems to be at variance with studies assessing rates of peptide insertion into the A beta-sheet and limited proteolysis of the reactive loop. Here we present a 2.9 A structure of recombinant wild-type alpha 1-antitrypsin with no stabilizing mutations. Again, the reactive loop is in a canonical conformation in the absence of significant insertion into the A beta-sheet. A stabilizing salt bridge between P5 glutamate and arginine residues 196, 223 and 281, already identified in the mutant, provides strong evidence that this conformation is not an artefact of crystallization but represents the conformation of the circulating inhibitor in vivo. Comparison with the structure of alpha 1-antitrypsin stabilized with the Phe51Leu mutation indicates that the increased thermal stability of the mutant results from enhanced packing of aromatic residues in the hydrophobic core of the molecule. The structure of wild-type alpha 1-antitrypsin reveals a hydrophobic pocket between s2A and helices D and E that is filled on reactive loop insertion and the formation of biologically relevant loop-sheet polymers. This pocket may provide a target for rational drug design to prevent the formation of polymers and the associated plasma deficiency, liver cirrhosis and emphysema.
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PMID:Wild-type alpha 1-antitrypsin is in the canonical inhibitory conformation. 946 20

Human neutrophil elastase (HNE, IEC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH2-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH2-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH2-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH2-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.
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PMID:Enzymatic and molecular biochemical characterizations of human neutrophil elastases and a cathepsin G-like enzyme. 1110 Nov 39

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.
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PMID:Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure. 1157 28


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