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At least 235 records · Page 13Linked to original sources
PubMed1985

Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor.

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.

Animals↗
PubMed2006

Impact of naturally occurring variants of HCV protease on the binding of different classes of protease inhibitors.

HCV drug discovery efforts have largely focused on genotype 1 virus due to its prevalence and relatively poor response to current therapy. However, patients infected with genotype 2 and 3 viruses account for a significant number of cases and would also benefit from new therapies. In vitro studies using two chemically distinct protease inhibitors with clinical potential showed that one, VX-950, was equally active on proteases from all three genotypes, whereas the other, BILN 2061, was significantly less active on genotype 2 and 3 proteases. Naturally occurring variation near the inhibitor binding site was identified based on sequence alignment of the protease region from genotype 1-3 sequences. Substitution of amino acids in genotype 1 based on genotype 2 and 3 has revealed residues which impact binding of BILN 2061. Substitution of residues 78-80, together with 122 and 132, accounted for most of the reduced sensitivity of genotype 2. The most critical position affecting inhibitor binding to genotype 3 protease was 168. Substitution of residues at positions 168, 123, and 132 fully accounted for the reduced sensitivity of genotype 3. Comparative studies of BILN 2061 and a closely related nonmacrocycle inhibitor suggested that the rigidity of BILN 2061, while conferring greater potency against genotype 1, rendered it more sensitive to variations near the binding site. Free energy perturbation analysis confirmed the experimental observations. The identification of naturally occurring variations which can affect inhibitor binding is an important step in the design of broad-spectrum, second generation protease inhibitors.

Binding Sites↗
PubMed2001

HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: a potential mechanism for protease inhibitor-induced hyperlipidemia.

Highly active anti-retroviral therapies, which incorporate HIV protease inhibitors, resolve many AIDS-defining illnesses. However, patients receiving protease inhibitors develop a marked lipodystrophy and hyperlipidemia. Using cultured human and rat hepatoma cells and primary hepatocytes from transgenic mice, we demonstrate that protease inhibitor treatment inhibits proteasomal degradation of nascent apolipoprotein B, the principal protein component of triglyceride and cholesterol-rich plasma lipoproteins. Unexpectedly, protease inhibitors also inhibited the secretion of apolipoprotein B. This was associated with inhibition of cholesteryl-ester synthesis and microsomal triglyceride transfer-protein activity. However, in the presence of oleic acid, which stimulates neutral-lipid biosynthesis, protease-inhibitor treatment increased secretion of apolipoprotein B-lipoproteins above controls. These findings suggest a molecular basis for protease-inhibitor-associated hyperlipidemia, a serious adverse effect of an otherwise efficacious treatment for HIV infection.

Animals↗
PubMed2001

Human immunodeficiency virus type 1 protease genotype predicts immune and viral responses to combination therapy with protease inhibitors (PIs) in PI-naive patients.

Protease genotype, as a variable in outcome to combination therapy for human immunodeficiency virus (HIV) type 1 infection, was evaluated among protease inhibitor-naive children and adolescents who had received extensive treatment with reverse-transcriptase inhibitors. After 24 weeks of combination therapy, 35% had viral and immune success (VSIS patients), 19% had viral and immune failure (VFIF patients), and 46% had viral failure but marked improvement in CD4 T cells (VFIS patients). Disease stage was the only pretherapy clinical variable associated with outcome (P=.02). Although reverse-transcriptase genotype was unrelated to outcome, pretherapy protease genotype was related significantly to therapy response (P=.005). Odds for immune or viral failure were 17.7 to 1 and 2.5 to 1, respectively, for protease genotype as a single variable. Protease genotype combined with disease stage and CD4 cell percentage predicted correct therapy response for 81% of patients (100% of VFIF, 78% of VSIS, and 75% of VFIS patiens). Naturally occurring amino acid polymorphisms in protease provide sensitive biomarkers for treatment response among inhibitor-naive patients with advanced HIV disease.

Adolescent↗
PubMed1994

Proteases and the emerging role of protease inhibitors in prohormone processing.

Peptide hormones and neurotransmitters constitute a large class of neurohumoral agents that mediate cell-cell communication in neuroendocrine systems. Their biosynthesis requires proteolytic processing of inactive protein precursors into active neuropeptides. Elucidation of the proteolytic components required for prohormone processing is important for identifying key proteases that may control the production of neuropeptides. This article compares the subtilisin-like PC1/3 and PC2 processing enzymes identified through molecular biological approaches, and several candidate processing enzymes identified biochemically, including the 'proopiomelanocortin converting enzyme' (PCE) and the 'prohormone thiol protease' (PTP), as well as others of different classes (aspartyl, cysteine, metallo, and serine proteases). A role for PTP in cellular proenkephalin processing is suggested by blockade of forskolin-stimulated (Met)enkephalin production by Ep453 that is converted intracellularly to E-64c, a selective cysteine protease inhibitor that potently inhibits PTP. A possible role for endogenous protease inhibitors in prohormone processing represents a new aspect of cellular mechanisms that may regulate neuropeptide biosynthesis. Future studies of the enzymology and molecular biology of processing enzymes and endogenous protease inhibitors will be necessary to elucidate mechanisms of prohormone processing.

Amino Acid Sequence↗
PubMed1989

The substrate of epidermal proteases--effects of in situ activation of epidermal thiol proteases on stratum granulosum cells.

In newborn rat skin, disulfide bonds exist in the cell membrane of the stratum corneum and in the cytoplasm of the stratum granulosum. However, in the latter region, they can be detected after ethanol fixed epidermis remains for 2 weeks at 4 degrees C. Thus, the idea arose that the visualization of the disulfide bonds in the stratum granulosum may be caused by proteases. Therefore, the in situ effects by both activation and inhibition of epidermal proteases were examined in tissue sections. Cryostat sections which were fixed in cold ethanol (-20 degrees C, 98%) were incubated either in PBS, pH 7.4, containing 10mM 2-ME as a control or in an epidermal homogenate (15,000 x g supernatant fraction containing 2-ME) at 37 degrees C for 30 minutes and 4 hours, respectively, in order to activate the epidermal proteases and then reacted with NEM, 2-ME, and DACM in steps. Many minute fluorescent particles were seen in the stratum granulosum under both of the above conditions. However, with incubation in PBS without 2-ME, no fluorescent particles were seen. In addition, when the tissue was treated with NEM and zinc chloride before incubation, no fluorescent particles were seen. O-phenanthrolin remarkably inhibited the appearance of these fluorescent particles. In contrast, leupeptin and antipain only slightly inhibited the appearance of these fluorescent particles, and pepstatin and phenylmethylsulfonyl fluoride didn't inhibit at all. In general, as NEM and zinc chloride strongly inhibit thiol proteases, these findings suggest that presence of the minute fluorescent particles in the stratum granulosum could be due to the effects of proteases, especially thiol proteases.

Animals↗
PubMed1992

Protease inhibitors in bronchoalveolar lavage fluid from neonates with special reference to secretory leukocyte protease inhibitor.

An imbalance of proteolytic enzymes and protease inhibitors may contribute to the development of bronchopulmonary dysplasia. We studied secretory leukocyte protease inhibitor (not previously addressed), and alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-macroglobulin and elastase. Albumin was used as an internal reference. Infants with pneumonia had higher concentrations of secretory leukocyte protease inhibitor (p = 0.02) and elastase (p = 0.04) in bronchoalveolar lavage fluid than those with respiratory distress syndrome; those who also developed bronchopulmonary dysplasia had intermediate values. A decreased concentration of alpha 1-antitrypsin was found in the second and third postnatal weeks (p = 0.002). Further detailed studies of the balance between proteases and protease inhibitors and of the importance of pulmonary infections in the pathogenesis of bronchopulmonary dysplasia are suggested. Secretory leukocyte protease inhibitor is important both as an elastase inhibitor of bronchial mucus and as a marker of infection in the bronchi.

Bronchoalveolar Lavage Fluid↗
PubMed1982

Inactivation of factor XIa by plasma protease inhibitors: predominant role of alpha 1-protease inhibitor and protective effect of high molecular weight kininogen.

Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, alpha(1)-protease inhibitor, antithrombin III, C1-inhibitor, and alpha(2)-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with alpha(1)-protease inhibitor, antithrombin III, and CI-inhibitor were 4.08, 10, and 14.6 M(-1) min(-1) x 10(3), respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 x 10(-1), 4.68 x 10(-2), and 2.4 x 10(-2) min(-1), respectively. These kinetic data predict that alpha(1)-protease inhibitor should account for 68%, antithrombin III for 16%, and C1-inhibitor and the equipotent alpha(2)-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions. Factor XI, the zymogen form of Factor XIa, circulates in plasma associated with the contact system cofactor, high molecular weight kininogen (HMW kininogen). Kinetic analysis indicated the existence of a reversible bimolecular Factor XIa-HMW kininogen complex with a dissociation constant (K(d)) = 0.17 muM. The light chain derived from HMW kininogen decreased the inactivation rate of Factor XIa by C1-inhibitor with a K(d) of 0.08 muM for a complex of Factor XIa and the light chain derived from HMW kininogen. The protective effect of HMW kininogen was confirmed by the finding that the inactivation rate of Factor XIa in kininogen-deficient plasma was increased over normal plasma. The present study confirms that alpha(1)-protease inhibitor is the major inhibitor of Factor XIa in plasma, and that the formation of a reversible complex between Factor XIa and HMW kininogen decreases the rate of inactivation of the enzyme by its inhibitors.

Antithrombin III↗
PubMed1996

Selective induction of apoptosis in Hep 3B cells by topoisomerase I inhibitors: evidence for a protease-dependent pathway that does not activate cysteine protease P32.

Progress in the treatment of hepatocellular carcinoma (HCC), a common tumor worldwide, has been disappointing. Inhibitors of topoisomerases are being widely studied as potential inducers of tumor cell apoptosis. Our aims were to determine whether topoisomerase-directed drugs would induce apoptosis in a human HCC cell line (Hep 3B) and, if so, to investigate the mechanism. The topoisomerase I poison camptothecin (CPT) induced apoptosis of Hep 3B cells in a time- and concentration-dependent manner. In contrast, the topoisomerase II poison etoposide failed to induce apoptosis despite the apparent stabilization of topoisomerase II-DNA complexes. Unexpectedly, CPT-induced apoptosis in this cell type occurred without any detectable cleavage of poly(ADP-ribose) polymerase or lamin B, polypeptides that are commonly cleaved in other cell types undergoing apoptosis. Likewise, Hep 3B cell apoptosis occurred without a detectable increase in interleukin-1beta-converting enzyme (ICE)-like or cysteine protease P32 (CPP32)-like protease activity. In contrast, trypsin-like protease activity (cleavage of Boc-Val-Leu-Lys-chloromethylaminocoumarin in situ) increased threefold in cells treated with CPT but not etoposide. Tosyl-lysyl chloromethyl ketone inhibited the trypsin-like protease activity and diminished CPT-induced apoptosis. These data demonstrate that (a) apoptosis is induced in Hep 3B cells after stabilization of topoisomerase I-DNA complexes but not after stabilization of topoisomerase II-DNA complexes as measured by alkaline filter elution; (b) Hep 3B cell apoptosis occurs without activation of ICE-like and CPP32-like protease activity; and (c) a trypsin-like protease activity appears to contribute to apoptosis in this cell type.

Apoptosis↗
PubMed1989

Proteases and protease inhibitors in immune complex otitis media in chinchillas.

Using an immune complex-induced (IC) otitis media model in chinchillas sensitized with human serum albumin (HSA), we measured the levels of protease activity, protease inhibitors, and HSA in the middle ear fluid (MEF). The effect of a corticosteroid agent (triamcinolone) on the degree of IC otitis media also was studied. The levels of protease inhibitors alpha 1-antitrypsin and alpha 2-macroglobulin in the MEF were significantly higher than in nonsensitized animals. Fibrinolytic (plasminogen activator [PA]) activity was detected in the MEF; however, only a small level of activity of nonspecific proteases was detected because of the significant level of antitryptic activity in the MEF. The levels of protease inhibitors and PA activity were significantly reduced by the steroid treatment. It is concluded that protease inhibitors play an important role in the protection of the middle ear mucosa and that corticosteroid treatment can reduce the severity of IC otitis media.

Animals↗
PubMed1982

Protease inhibitors and their relation to protease activity in human milk.

Protease inhibitors and protease (caseinolytic, elastinolytic and esterolytic) activity were analysed in 190 milk samples from 94 mothers from day 1 to day 160 after delivery. The main protease inhibitors in human milk are alpha 1-antichymotrypsin and alpha 1-antitrypsin. As measured by electroimmunoassay, the level of alpha 1-antichymotrypsin in day 1 colostrum was higher than that in normal serum. Trace amounts of inter-alpha-trypsin inhibitor, alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, or antileukoprotease could be demonstrated. According to their protease inhibiting activity, the 53 milk samples from day 1-3 could be divided into two groups. (1) Presence of protease inhibiting activity (n = 35). Both alpha 1-antitrypsin and alpha 1-antichymotrypsin appeared intact and were able to form complexes with added trypsin or chymotrypsin although the major part of alpha 1-antichymotrypsin showed a retarded electrophoretic mobility. The proteolytic inhibiting activity, in spite of the presence of immunoreactive inhibitors (n = 18). alpha 1-antichymotrypsin had a precipitate pattern similar to group 1, whereas alpha 1-antitrypsin had a major fraction with slightly retarded mobility and two minor peaks in the alpha 1-and beta-regions. These precipitate patterns were unchanged on addition of human trypsin or chymotrypsin compatible with the presence of nonreactive inhibitor only. These samples had a caseinolytic and esterolytic activity with an electrophoretic mobility in the beta-region. All samples from day 4 and later had a demonstrable protease inhibiting activity.

Chymotrypsin↗
PubMed1988

Influence of plasma protease inhibitors and the secretory leucocyte protease inhibitor on leucocyte elastase-induced consumption of selected plasma proteins in vitro in man.

Cleavage of C3, fibronectin, antithrombin III and alpha 2-antiplasmin in human plasma following the addition of increasing amounts of human leucocyte elastase was studied using an in vitro model. The cleavage was correlated with the degree of saturation of the plasma protease inhibitors alpha 2-macroglobulin and alpha 1-protease inhibitor and also with varying amounts of secretory leucocyte protease inhibitor. When alpha 1-protease inhibitor approached saturation, there was a prompt cleavage of all the four plasma proteins studied. The secretory leucocyte protease inhibitor was needed in a concentration of 6 mumol/l in the present model to block this consumption completely. This concentration also gave some protection of alpha 1-protease inhibitor and alpha 2-macroglobulin.

Antithrombin III↗
PubMed1988

Detection of alpha 2-macroglobulin, alpha 1-protease inhibitor, and neutral protease-antiprotease complexes within liver granulomas of Schistosoma mansoni-infected mice.

In schistosomiasis mansoni the parasite egg-induced granulomatous tissue inflammations resolve by fibrosis. Intralesional collagen synthesis and deposition are influenced by collagenase, elastase activity that is diminished at the chronic stage of the disease. To determine the cause of diminished neutral protease activity, the authors determined levels of the antiprotease/alpha 2-macroglobulin (alpha 2M) and alpha 1-protease inhibitor (alpha 1Pi) in extracts or secretions of liver granulomas of infected mice. By ELISA, both antiproteases were detected in granuloma-derived substances, as well as supernatants of cultured, adherent granuloma macrophages. In all samples, alpha 2M was the predominant inhibitor. Antiprotease levels were similar in granuloma-derived samples obtained from acutely and chronically infected mice. However, supernatants of cultured adherent macrophages isolated from granulomas of mice with acute infection contained levels of protease inhibitors several times higher than those of similar preparations obtained from chronically infected animals. Gel filtration of samples on Sephacryl S-200 columns did not separate collagenase and elastase from protease inhibitors. By chromatofocusing, a few inhibitor-free collagenase as well as enzyme-free alpha 2M and alpha 1Pi-active peaks were eluted. The bulk of the material that eluted at the acidic region contained protease-antiprotease activity indicating the presence of enzyme-inhibitor complexes. The intragranulomatous presence of antiproteases complexed with protease enzymes emphasizes their importance in the possible enhancement of fibrosis.

Animals↗
PubMed1994

Regulation of protease nexin-1 target protease specificity by collagen type IV.

Recent studies have shown that serine protease inhibitors can be regulated in their activity, specificity, and location by glycoprotein or extracellular matrix (ECM) co-factors. Protease nexin-1 (PN-1) is a member of the serpin superfamily of serine protease inhibitors which can rapidly inhibit thrombin, urokinase, and plasmin. PN-1 binds tightly to and is regulated by the ECM. This interaction accelerates the inhibition of thrombin by PN-1 and blocks urokinase and plasmin inhibition by PN-1. Previous work showed that heparan sulfate proteoglycan is largely responsible for the acceleration of thrombin inhibition by PN-1. Our current studies were directed at identifying ECM component(s) that decreased the ability of PN-1 to inhibit urokinase and plasmin. These studies showed that collagen type IV decreased the formation of SDS-stable complexes between urokinase or plasmin and PN-1 without affecting formation of complexes between thrombin and PN-1. The second order rate constant for inhibition of urokinase by PN-1 was markedly decreased with increasing collagen type IV, whereas the second order rate constant for inhibition of thrombin by PN-1 was unaffected by addition of collagen type IV. Other ECM components (collagen type I, vitronectin, fibronectin, and heat-denatured collagen type IV) did not affect complex formation or the rate of inhibition of proteases by PN-1, indicating that these effects were specific to collagen type IV. Binding of PN-1 to immobilized collagen type IV was demonstrated using an enzyme-linked immunosorbent assay; the concentration of PN-1 necessary to obtain 50% saturation of the immobilized collagen type IV binding sites was approximately 15 nM. Collagen type IV was also copurified with PN-1 from fibroblast-conditioned medium. These results demonstrate a novel regulation of serpin specificity in which an ECM co-factor decreased the inhibition of certain proteases by the serpin without affecting the inhibition of its target protease.

Amyloid beta-Protein Precursor↗
PubMed1998

Characterization of mouse mast cell protease-8, the first member of a novel subfamily of mouse mast cell serine proteases, distinct from both the classical chymases and tryptases.

Using a recently developed PCR-based strategy, a cDNA encoding a novel mouse mast cell (MC) serine protease (MMCP-8) was isolated and characterized. The MMCP-8 mRNA contains an open reading frame of 247 amino acids (aa), divided into a signal sequence of 18 aa followed by a 2-aa activation peptide (Gly-Glu) and a mature protease of 227 aa. The mature protease has an M(r) of 25072, excluding post-translational modifications, a net positive charge of +12 and six potential N-glycosylation sites. MMCP-8 showed a high degree of homology with mouse granzyme B in the critical regions for determining substrate cleavage specificity, indicating that MMCP-8, similar to granzyme B, preferentially cleaves after Asp residues. A comparative analysis of the aa sequence of MMCP-8 with other hematopoietic serine proteases shows that it is more closely related to cathepsin G and T cell granzymes than to the MC chymases. We therefore conclude that MMCP-8 belongs to a novel subfamily of mouse MC proteases distinct from both the classical chymases and tryptases. Southern blot analysis of BALB/c genomic DNA indicated that only one MMCP-8 gene (or MMCP-8 like gene) is present in the mouse genome. Northern blot analysis of rodent hematopoietic cell lines revealed high levels of MMCP-8 mRNA in a mouse connective tissue MC-like tumor line. However, MMCP-8 mRNA could not be detected in mouse liver, intestine, lung or ears, indicating very low expression in normal tissues. Analysis of the expression of different MMCP in the tissues of Schistosoma mansoni-infected BALB/c mice showed a strong increase in MMCP-8 levels in the lungs but not in the intestines of infected animals, suggesting the presence of a novel subpopulation of MC in the lungs that expressed MMCP-8, either alone or in combination with MMCP-5 and carboxypeptidase A. The dramatic increase in MMCP-1 and MMCP-2 levels but not of MMCP-8 in the intestines of parasitized animals also shows that MMCP-8 is not expressed in mucosal MC in the mouse. This latter is in clear contrast to what has been observed in the rat where the MMCP-8 homologues, RMCP-8, -9 and -10, can be considered as true mucosal MC proteases.

Amino Acid Sequence↗
PubMed2004

Is histoaspartic protease a serine protease with a pepsin-like fold?

The primary structure of the so-called histoaspartic protease from Plasmodium falciparum has a very high percentage of identity and homology with the pepsin-like enzyme plasmepsin II. A homology modeling approach was used to calculate the three-dimensional structure of the enzyme. Molecular dynamics (MD) simulations were applied to find those structural properties of the histoaspartic protease that had a tendency to remain stable during all runs. The results have shown that hydrogen-bonded residues Ser37-His34-Asp214 are arranged without any strain, in a manner that resembles the active site of a serine protease, while Ser38 and Asn39 take up positions appropriate to formation of an oxyanion hole. Although there are several important differences between the enzyme and plasmepsin II, all of the structural features associated with a typical pepsin-like aspartic protease are present in the final model of the histoaspartic protease. A possibility that this enzyme may function as a serine protease is discussed.

Animals↗
PubMed2006

Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins.

One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host-specific proteases. As an initial solution to this problem, we constructed a protease-deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine- and/or cysteine-type proteases in the culture medium. These findings also demonstrated that construction of a protease-deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required.

Biotechnology↗