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PubMed1991

Effect of maturation on activities of various proteases and protease inhibitors in the muscle of Ayu (Plecoglossus altivelis).

1. Increases in activities of muscle muticatalytic proteinase, modori-inducing proteinase (latent trypsin-like proteinase), cathepsin B and L-like proteases and cathepsin D were observed more markedly for male fish than female fish, in the spawning stage. 2. Decreases in inhibitory activities of muscle serine and cysteine protease inhibitors were observed more markedly for male fish than female fish in the spawning stage.

Amino Acid Sequence↗
PubMed1991

Engineering of proteases and protease inhibition.

Proteases are unquestionably the single most studied class of enzymes and yet many questions still remain about their mechanisms and roles. Protein engineering offers the opportunity to provide some of the answers. In this review, recent advances towards the understanding of stability, mechanism, specificity and regulation of proteases and their inhibitors are outlined. In addition, the application of this increased understanding is also discussed.

Animals↗
PubMed2004

P1' oxadiazole protease inhibitors with excellent activity against native and protease inhibitor-resistant HIV-1.

HIV-1 protease inhibitors (PI's) bearing 1,3,4-oxadiazoles at the P1' position were prepared by a novel method involving the diastereoselective installation of a carboxylic acid and conversion to the P1' heterocycle. The compounds are picomolar inhibitors of native HIV-1 protease, with most of the compounds maintaining excellent antiviral activity against a panel of PI-resistant strains.

Cell Line, Tumor↗
PubMed2004

A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis suaveolens L.) inhibits proteases from the larger grain borer Prostephanus truncatus (Coleoptera: Bostrichidae).

A novel trypsin inhibitor purified from chan seeds (Hyptis suaveolens, Lamiaceae) was purified and characterized. Its apparent molecular mass was 8700 Da with an isoelectric point of 3.4. Its N-terminal sequence showed a high content of acidic amino acids (seven out of 18 residues). Its inhibitory activity was potent toward all trypsin-like proteases extracted from the gut of the insect Prostephanus truncatus (Coleoptera: Bostrichidae), a very important pest of maize. This activity was highly specific, because among proteases from seven different insects, only those from P. truncatus and Manduca sexta (Lepidoptera: Sphingidae) were inhibited. This inhibitor has potential to enhance the defense mechanism of maize against the attack of P. truncatus.

Animals↗
PubMed2005

Inhibitor-assisted refolding of protease: a protease inhibitor as an intramolecular chaperone.

Pleurotus ostrearus proteinase A inhibitor 1 (POIA1), which was discovered as a protease inhibitor, is unique in that it shows sequence homology to the propeptide of subtilisin, which functions as an intramolecular of a cognate protease. In this study, we demonstrate that POIA1 can function as an intramolecular chaperone of subtilisin by in vitro and in vivo experiments. The specific cleavage between POIA1 and the mature region of subtilisin BPN' occurred in a refolding process of a chimera protein, and Bacillus cells transformed with a chimera gene formed a halo on a skim milk plate. The mutational analyses of POIA1 in the chimera protein suggested that the tertiary structure of POIA1 is required for such a function, and that an increase in its ability to bind to subtilisin BPN' makes POIA1 a more effective intramolecular chaperone.

Alanine↗
PubMed2002

Inhibition of Candida albicans secreted aspartic protease by a novel series of peptidomimetics, also active on the HIV-1 protease.

Nineteen reduced amide, monohydroxy- or dihydroxyethylene-based transition-state peptidomimetics, known to be good inhibitors of the aspartic protease of HIV-1, were tested against a secreted aspartic protease (Sap2), purified from the culture medium of a virulent strain of Candida albicans. Ten of these compounds exhibited IC(50)s against Sap2 lower than 15 microM; the best inhibitor, Kyn-Val-Phe-Psi[OH-OH]-Phe-Val-Kyn, when added to the C. albicans culture, repressed the hydrolysis of bovine serum albumin (BSA), contained in the culture medium, and inhibited the growth of the fungus.

Aspartic Acid Endopeptidases↗
PubMed1998

Proteases and protease inhibitors in cancer.

The second conference on 'Proteases and protease inhibitors in cancer' was organized by the American Association for Cancer Research (AACR) and Acta Pathologica Microbiologica et Immunologica Scandinavica (APMIS). To understand the role of proteinases and to develop relevant synthetic inhibitors to stop or slow the progression of cancer, an integrated view on the role of proteinases and proteinase inhibitors in required. This is the main goal of this series of conferences. Approximately 300 people attended this conference, including many highly regarded scientists in the field. In total, 40 invited lectures were presented and 120 posters were displayed during two well-attended sessions.

Endopeptidases↗
PubMed2001

[Proteases, natural protease inhibitors and activation of the machinery of antigen presentation in dendritic cells].

Dendritic cells play a central role in the immune response due to their exceptionally strong capacity for presenting antigens to naive T lymphocytes. Recent evidences have demonstrated that dendritic cells (DCs) exhibit a remarkable pattern of differentiation (maturation) that is accompanied by striking changes in morphology, organization and function. The hallmark of DC maturation is the major reorganization of the MHC class II molecule intracellular transport which is in part regulated by endosomal proteases activation. The central role of the endosomal proteases in generating antigenic peptides and controlling MHC class II traffic clearly defines these enzymes as an important area of investigation.

Animals↗
PubMed2001

Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1.

Understanding infertility and sterility requires knowledge of the molecular mechanisms underlying sexual reproduction. We have found that male mice deficient for the gene encoding the protease inhibitor protease nexin-1 (PN-1) show a marked impairment in fertility from the onset of sexual maturity. Absence of PN-1 results in altered semen protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation. Progressive morphological changes of the seminal vesicles also are observed. Consistent with these findings, abnormal PN-1 expression was found in the semen of men displaying seminal dysfunction. The data demonstrate that the level of extracellular proteolytic activity is a critical element in controlling male fertility.

Amyloid beta-Protein Precursor↗
PubMed2004

The active site of a lon protease from Methanococcus jannaschii distinctly differs from the canonical catalytic Dyad of Lon proteases.

ATP-dependent Lon proteases catalyze the degradation of various regulatory proteins and abnormal proteins within cells. Methanococcus jannaschii Lon (Mj-Lon) is a homologue of Escherichia coli Lon (Ec-Lon) but has two transmembrane helices within its N-terminal ATPase domain. We solved the crystal structure of the proteolytic domain of Mj-Lon using multiwavelength anomalous dispersion, refining it to 1.9-angstroms resolution. The structure displays an overall fold conserved in the proteolytic domain of Ec-Lon; however, the active site shows uniquely configured catalytic Ser-Lys-Asp residues that are not seen in Ec-Lon, which contains a catalytic dyad. In Mj-Lon, the C-terminal half of the beta4-alpha2 segment is an alpha-helix, whereas it is a beta-strand in Ec-Lon. Consequently, the configurations of the active sites differ due to the formation of a salt bridge between Asp-547 and Lys-593 in Mj-Lon. Moreover, unlike Ec-Lon, Mj-Lon has a buried cavity in the region of the active site containing three water molecules, one of which is hydrogen-bonded to catalytic Ser-550. The geometry and environment of the active site residues in Mj-Lon suggest that the charged Lys-593 assists in lowering the pK(a) of the Ser-550 hydroxyl group via its electrostatic potential, and the water in the cavity acts as a proton acceptor during catalysis. Extensive sequence alignment and comparison of the structures of the proteolytic domains clearly indicate that Lon proteases can be classified into two groups depending on active site configuration and the presence of DGPSA or (D/E)GDSA consensus sequences, as represented by Ec-Lon and Mj-Lon.

Amino Acid Sequence↗
PubMed1997

In vitro effect of alpha1-acid glycoprotein on the anti-human immunodeficiency virus (HIV) activity of the protease inhibitor CGP 61755: a comparative study with other relevant HIV protease inhibitors.

Protein binding can impair the potency of human immunodeficiency virus (HIV) protease inhibitors. Therefore, the activity of a novel compound, CGP 61755, was studied in the absence or presence of alpha1-acid glycoprotein (alpha1AGP). In MT-2 cells, the activity loss was 4-fold (EC90 without alpha1AGP, 29 nM vs. 122 nM with alpha1AGP). In primary lymphocytes, the loss was 8-fold (EC90, 45 nM vs. 364 nM). In identical experiments, the activity loss in MT-2 cells and lymphocytes was 2- and 3-fold, respectively, for indinavir, 11- and 10-fold for saquinavir, and 11- and 48-fold for ritonavir. For SC-52151, a 17-fold loss was seen in MT-2 cells, whereas no EC90 with alpha1AGP was reached in lymphocytes. This study demonstrates that the impact of alpha1AGP on in vitro activity varies greatly among different HIV protease inhibitors. The magnitude of such differences is greater in human lymphocytes than in a standard cell line.

Anti-HIV Agents↗
PubMed2001

Synergistic antiadipogenic effects of HIV type 1 protease inhibitors with tumor necrosis factor alpha: suppression of extracellular insulin action mediated by extracellular matrix-degrading proteases.

Long-term use of HIV-1 protease inhibitors (PIs) is associated with a lipodystrophy syndrome. To delineate the associated mechanisms, adipogenesis was determined in 3T3-L1 cells in the presence or absence of either indinavir (2-50 microg/ml) or ritonavir (0.4-10 microg/ml). A concentration-dependent decrease in both lipid (4-59%) and triglyceride (11-49%) levels was seen after 10 days of exposure. Simultaneous treatment with TNF-alpha showed a synergistic suppression in lipid levels by 45-95% at 10 U/ml and almost complete suppression at 100 U/ml. The effect of PIs on insulin-induced lipogenesis was monitored by [(14)C)]glucose incorporation into lipids, which was suppressed by 21-86% in a concentration-dependent manner. Insulin-sensitizing agent, troglitazone (80 and 400 nM), effectively blocked the PI-mediated adipogenic suppression. Preadipocyte factor 1 gene (pref-1) expression, as monitored by RT-PCR, was downregulated (4- to 6-fold) within 48 hr after insulin stimulation; however, a smaller decrease (1.2- to 1.8-fold) was observed in PI-exposed cells. The decrease in proteolytic activity of matrix metalloproteases (MMP-2 and MMP-9) during adipogenesis was reversed on exposure to the PIs. Similarly, the plasminolytic activity was increased and plasminogen activator inhibitor (PAI) activity was decreased in supernatants from PI-treated cells. The insulin-mediated induction (3- to 4-fold) of PAI-1 and PAI-2 message was suppressed on exposure to PIs, which was reversed by troglitazone treatment. Thus, the HIV-1 PIs may suppress adipogenesis by disrupting the concerted actions of host proteases that regulate ECM integrity required for initiation of differentiation.

Adipocytes↗
PubMed2006

Successful treatment with atazanavir and lopinavir/ritonavir combination therapy in protease inhibitor-susceptible and protease inhibitor-resistant HIV-infected patients.

The combination of atazanavir (ATV) plus lopinavir/ritonavir (LPV/r) has been used in practice. However, clinical data supporting its use are limited. The objective of this study was to evaluate the efficacy and tolerability of regimens with ATV + LPV/r in protease inhibitor (PI)-susceptible and PI-resistant patients. A retrospective review of 2703 charts was performed to identify all patients who received ATV + LPV/r. From June 2003 to January 2005, 33 patients received ATV + LPV/r with nucleoside reverse transcriptase inhibitors (NRTIs) for 3 months or more. Virologic success (HIV-RNA < 400 copies per milliliter) was achieved in 30 patients (91%) in a median of 10 weeks (range, 2-68). Nineteen of the 23 patients (83%) who had ultrasensitive viral load (VL) assays were nondetectable. Among patients with 6 or more protease resistance (PR) mutations (PI-resistant), 11 of 14 (79%) achieved virologic success. Eleven of those received phenotypic testing (10 Virtual Phenotype, VircoLab, Baltimore, MD). Despite predicted phenotypic resistance to ATV (6 patients) and LPV/r (7 patients), virologic success was achieved in 4 of 6 (67%) and 4 of 7 (57%), respectively. The 3 PI-resistant patients who were virologic failures had extensive prior LPV/r use, 8-11 PR mutations, and predicted phenotypic resistance to LPV/r, but 2 of 3 had CD4 increases with ATV + LPV/r. Overall, 28 patients (85%) continue to tolerate ATV + LPV/r for a median of 32 weeks follow-up (range, 12-76). Combination ATV + LPV/r with NRTIs appears safe, tolerable, and efficacious in PI-resistant patients (>/=6 PR mutations) and predicted phenotypic resistance to ATV and LPV/r. Further studies of ATV + LPV/r in HIV-treatment are warranted.

Adult↗
PubMed2005

TMC114, a novel human immunodeficiency virus type 1 protease inhibitor active against protease inhibitor-resistant viruses, including a broad range of clinical isolates.

The purpose of this study was to characterize the antiviral activity, cytotoxicity, and mechanism of action of TMC114, a novel human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI). TMC114 exhibited potent anti-HIV activity with a 50% effective concentration (EC50) of 1 to 5 nM and a 90% effective concentration of 2.7 to 13 nM. TMC114 exhibited no cytotoxicity at concentrations up to 100 muM (selectivity index, >20,000). All viruses in a panel of 19 recombinant clinical isolates carrying multiple protease mutations and demonstrating resistance to an average of five other PIs, were susceptible to TMC114, defined as a fold change in EC50 of <4. TMC114 was also effective against the majority of 1,501 PI-resistant recombinant viruses derived from recent clinical samples, with EC50s of <10 nM for 75% of the samples. In sequential passage experiments using HIV-1 LAI, two mutations (R41T and K70E) were selected. One selected virus showed a 10-fold reduction in susceptibility to TMC114, but <10-fold reductions in susceptibility to the current PIs (atazanavir was not assessed), except saquinavir. However, when the selected mutations were introduced into a laboratory strain by site-directed mutagenesis, they had no effect on susceptibility to TMC114 or other PIs. There was no evidence of antagonism between TMC114 and any currently available PIs or reverse transcriptase inhibitors. Combinations with ritonavir, nelfinavir, and amprenavir showed some evidence of synergy. These results suggest that TMC114 is a potential candidate for the treatment of both naive and PI-experienced patients with HIV.

Cell Line↗
PubMed1997

Characterization of proteases and protease inhibitors in the rat stomach.

Previously our laboratory reported increased activity of the thiol proteinase cathepsin B in gastric juice after ethanol-induced mucosal injury. In this study we measured proteinase activity (PA) and proteinase inhibitory activity (PIA) with the general substrates hemoglobin, azocasein, and bovine serum albumin (BSA) at optimal pH (2.0, 5.6, and 7.4) of aspartic, cysteine, and serine proteinases. Homogenates of glandular stomach mucosa and gastric juice from fasted rats were incubated in the presence or absence of specific inhibitors and sulfhydryl (SH) alkylators N-ethylmaleimide and iodoacetate. PIA was measured after acid and heat inactivation of endogenous proteinases and addition of 20 micrograms/ml pepsin, 20 or 100 micrograms/ml thiol proteinase papain, or 20 micrograms/ml trypsin for 5 min before digestion at 37 degrees C. The highest proteolytic activity was found at pH 2.0 (pepsin) in juice and mucosal homogenate, but proteases were also found at pH 5.6 and 7.4, where pepsin was inactive. Pepstatin inhibited most proteolytic activity at pH 2.0. The SH protease inhibitor leupeptin diminished PA mainly at pH 5.6. N-ethylmaleimide or iodoacetate substantially reduced the PA in acidic milieu, with maximum effect at pH 5.6. Endogenous PIA, expressed as inhibition of the effect of 1 microgram of pepsin, papain, and trypsin on BSA, was 13.1, 1.4, and 9.2% in gastric mucosa and 15.3, 22.5, and 6.2% in gastric juice at pH 2.0, 5.6, and 7.4, respectively. We have concluded that 1) endogenous proteinases and inhibitors in rat stomach can be measured using BSA and hemoglobin as substrates, 2) of the proteinases found in the stomach, 98% was pepsin at pH 2.0 and up to 27% or 17% was SH sensitive at pH 5.6 or 7.4, respectively, and 3) proteinases and their specific endogenous inhibitors may play a role in gastric mucosal injury and protection.

Alkylating Agents↗
PubMed2005

Comprehensive transcriptome of proteases and protease inhibitors in vascular cells.

BACKGROUND AND PURPOSE: Smooth muscle cells, endothelial cells, and macrophages are essential components of the vasculature, of which the homeostatic gene expression participate importantly in the maintenance of vascular wall integrity. The pathogenesis of vascular diseases, such as cerebral ischemia, atherosclerosis, and abdominal aortic aneurysms, often associates with inflammation and altered gene expression, including proteolytic enzymes that play multiple and important roles in extracellular matrix degradation, cell proliferation and migration, and latent enzyme or growth factor activation. METHODS: Human saphenous vein smooth muscle cells, endothelial cells, and monocyte-derived macrophages from 3 independent donors were stimulated with interleukin 1beta, interferon gamma, tumor necrosis factor alpha, basic fibroblast growth factor, and vascular endothelial growth factor, 5 common proinflammatory mediators often found in diseased human microvessels and macrovessels. Quantitative real-time PCR was used to examine the mRNA levels of 49 proteolytic enzymes and their inhibitors, selected from 4 protease families, in these vascular cells. CONCLUSIONS: Although primary cultured cells from different donors may behave differently in response to these proinflammatory cytokines, data from this study revealed a broad view of vascular cell protease expression profiles under inflammatory conditions, critical to studies of inflammation-associated vascular tissue remodeling.

Aortic Aneurysm, Abdominal↗
PubMed1994

Studies of human immunodeficiency virus type 1 (HIV-1) protease inhibitors. III. Structure-activity relationship of HIV-1 protease inhibitors containing cyclohexylalanylalanine hydroxyethylene dipeptide isostere.

Systematic replacement of the P4-P2 subsites of substrate-based human immunodeficiency virus type 1 protease (HIV-1 PR) inhibitors containing cyclohexylalanylalanine hydroxyethylene dipeptide isostere (Cha-psi [H.E.]-Ala) at positions corresponding to the scissile sites of substrates was carried out. The structure-activity relationship revealed that compounds with the combination of hydrophilic P3 and beta-branched hydrophobic P2 amino acids generally showed strong inhibitory activity against HIV-1 PR. In particular, compounds 4 (Boc-Orn-Val-Cha-psi [H.E.]-Ala-NHBun; Bu(n) = n-butyl, Ki = 11 nM) and 6 (Z-Orn-Val-Cha-psi [H.E.]-Ala-NHBun, Ki = 8 nM) exhibited good enzyme selectivity, possessing no significant inhibitory activities toward closely related aspartic proteases, pepsin, cathepsin D, and renin. As a possible model system for (anti-Mo-MSV/MLV complex (Mo-MSV = Moloney murine sarcoma virus; MLV = murine leukemia virus)) activity was investigated. Both compounds were found to inhibit moderately the focus formation of Mo-MSV/MLV complex in NIH3T3 cells (compound 4, IC50 = 1.8 microM; compound 6, IC50 = 1.0 microM).

Amino Acid Sequence↗
PubMed1987

Pancreatic pseudocysts: a biochemical evaluation of proteases and protease inhibitors in plasma.

A biochemical evaluation was performed on plasma from eight patients developing a pancreatic pseudocyst during acute pancreatitis attacks and from six patients with a known pseudocyst. Patients developing an acute pancreatic pseudocyst had high levels of activated trypsin in complex with alpha 1-protease inhibitor, together with a probable activation of the kinin, complement, coagulation and fibrinolytic systems. Profound changes were also seen in several protease inhibitors, indicating consumption of the inhibitors. The changes did, however, not differ from those seen in severe acute pancreatitis attacks in which no pseudocyst developed. Patients with chronic pancreatic pseudocysts had biochemical changes similar to those seen in moderate pancreatitis attacks, without any overt cascade system activation. At convalescence, however, these patients had biochemical signs of leakage from the pancreas and an ongoing proteolytic activity.

Acute Disease↗