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Protease Inhibitors (protease + inhibitor)
Kinds of Protease Inhibitors Selected AbstractsEFFECTS OF MUSCLE PROTEASES, ENDOGENOUS PROTEASE INHIBITORS AND MYOFIBRIL FRAGMENTATION ON POSTMORTEM AGING OF GOAT MEATJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2006N.S. NAGARAJ ABSTRACT The present study was conducted to evaluate the extent of postmortem proteolysis in longissimus dorsi, biceps femoris, semimembranosus and semitendinosus goat muscles on postmortem aging at an ambient (27C) temperature. The activities of calpains and calpastatin were determined after separation on a (diethylamino)ethyl,Sephacel column (Sigma, St. Louis, MO) and cathepsin (B, B + L and H) by carboxymethyl,Sepharose column (Sigma). The results showed that the decrease in calpain I and calpastatin activities was significantly higher than that of calpain II. Cathepsin B, B + L, H and cystatin were found to fall by 30,80% after 12 h, whereas cathepsin D decreased significantly in all the muscles. The disappearance of titin 1 and nebulin, and the appearance of a 30-kDa component were confirmed by Western blot analysis. The appearance of the 30-kDa component reported here explains the time-induced structural changes of myofibrils. The Z-line degradation had occurred by 6 h postmortem. Cathepsins are not stable compared to calpains during postmortem aging, and both enzymes may play a significant role in the proteolysis of myofibrillar proteins at ambient temperature. [source] Porcine Plasma Proteins as a Surimi Protease Inhibitor: Effects on Actomyosin GelationJOURNAL OF FOOD SCIENCE, Issue 4 2000W. Visessanguan ABSTRACT Effect of porcine plasma proteins (PPP) on thermal gelation of actomyosin in the presence and absence of fish proteinase was studied using a dynamic rheological test. Substantial decreases in development rate and magnitude of gel modulus were observed by the addition of proteinase to actomyosin gels. PPP was effective in protecting a myosin-heavy chain from proteolytic degradation, however, PPP itself interfered with the formation of actomyosin gel. Lower gel modulus was observed with actomyosin gels developed with higher concentrations of PPP added. Overall, PPP reversed the loss of gel modulus by the proteinase, however, the recovered gel modulus was only as high as those containing PPP only. These results implicated that, although PPP may revert autolytic activity in surimi, it interfaces with actomyosin gelation. [source] Novel Heterocyclic Analogues of the HIV-1 Protease Inhibitor, Ritonavir.CHEMINFORM, Issue 50 2004Perry T. Kaye Abstract For Abstract see ChemInform Abstract in Full Text. [source] Application of the Lewis Acid,Lewis Base Bifunctional Asymmetric Catalysts to Pharmaceutical Syntheses: Stereoselective Chiral Building Block Syntheses of Human Immunodeficiency Virus (HIV) Protease Inhibitor and ,3 -Adrenergic Receptor Agonist.CHEMINFORM, Issue 44 2003Hiroyuki Nogami Abstract For Abstract see ChemInform Abstract in Full Text. [source] HIV Protease Inhibitors and PneumocystisTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2004GIOVANNA FANTONI [source] Effect of Protease Inhibitors on Yield of HSV-1-Based Viral VectorsBIOTECHNOLOGY PROGRESS, Issue 3 2000James B. Wechuck The ability to obtain high titer replication-defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV-1-based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV-1 or possibly several other vectors. [source] Rapid Diversity-Oriented Synthesis in Microtiter Plates for In Situ Screening of HIV Protease InhibitorsCHEMBIOCHEM, Issue 11 2003Ashraf Brik Dr. Click and go: By using click chemistry based on a new triazole forming reaction condition (see scheme), over 100 triazole compounds generated in microtiter plates from a core structure were screened for HIV protease inhibition in situ without product isolation. Potent inhibitors, active at nanomolar concentrations, against the wild type and drug resistant mutants were identified. [source] Design of Mutation-resistant HIV Protease Inhibitors with the Substrate Envelope HypothesisCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2007Sripriya Chellappan There is a clinical need for HIV protease inhibitors that can evade resistance mutations. One possible approach to designing such inhibitors relies upon the crystallographic observation that the substrates of HIV protease occupy a rather constant region within the binding site. In particular, it has been hypothesized that inhibitors which lie within this region will tend to resist clinically relevant mutations. The present study offers the first prospective evaluation of this hypothesis, via computational design of inhibitors predicted to conform to the substrate envelope, followed by synthesis and evaluation against wild-type and mutant proteases, as well as structural studies of complexes of the designed inhibitors with HIV protease. The results support the utility of the substrate envelope hypothesis as a guide to the design of robust protease inhibitors. [source] Synthesis and Antiviral Activity of P1, Arylsulfonamide Azacyclic Urea HIV Protease Inhibitors.CHEMINFORM, Issue 49 2004Peggy P. Huang Abstract For Abstract see ChemInform Abstract in Full Text. [source] Potential Protease Inhibitors Based on a Functional Cyclic Sulfamide Scaffold.CHEMINFORM, Issue 44 2004Jiaying Zhong Abstract For Abstract see ChemInform Abstract in Full Text. [source] Structure,Activity Relationships of First Bishydroxymethyl-Substituted Cage Dimeric 4-Aryl-1,4-dihydropyridines as HIV-1 Protease Inhibitors.CHEMINFORM, Issue 38 2003Andreas Hilgeroth Abstract For Abstract see ChemInform Abstract in Full Text. [source] Design and Synthesis of Highly Potent HIV Protease Inhibitors with Activity Against Resistant Virus.CHEMINFORM, Issue 37 2003Zhijian Lu Abstract For Abstract see ChemInform Abstract in Full Text. [source] Syntheses of Templates Derived from Pyrrolidine trans-Lactams as Potential Serine Protease Inhibitors.CHEMINFORM, Issue 42 2002Simon J. F. Macdonald Abstract For Abstract see ChemInform Abstract in Full Text. [source] ChemInform Abstract: Synthesis and Biological Evaluation of First N-Alkyl syn Dimeric 4-Aryl-1,4-dihydropyridines as Competitive HIV-1 Protease Inhibitors.CHEMINFORM, Issue 42 2001Andreas Hilgeroth Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] ChemInform Abstract: (S)-Thiirancarboxylic Acid as a Reactive Building Block for a New Class of Cysteine Protease Inhibitors.CHEMINFORM, Issue 14 2001Tanja Schirmeister Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] ChemInform Abstract: 6-Hydroxy-1,3-dioxin-4-ones as Non-peptidic HIV Protease Inhibitors.CHEMINFORM, Issue 14 2001Yong Sup Lee Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Bis-Tetrahydrofuran: a Privileged Ligand for Darunavir and a New Generation of HIV Protease Inhibitors That Combat Drug ResistanceCHEMMEDCHEM, Issue 9 2006Two inhibitors that incorporate bis-THF as an effective high-affinity P2 ligand for the HIV-1 protease substrate binding site maintain impressive potency against mutant strains resistant to currently approved protease inhibitors. Crystallographic structures of protein,ligand complexes help to explain the superior antiviral property of these inhibitors and their potency against a wide spectrum of HIV-1 strains. [source] Unexpected Novel Binding Mode of Pyrrolidine-Based Aspartyl Protease Inhibitors: Design, Synthesis and Crystal Structure in Complex with HIV ProteaseCHEMMEDCHEM, Issue 1 2006Edgar Specker Dr. Abstract At present nine FDA-approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide-like character or the stabilization of a new and unexpected binding-competent conformation of the protein through a novel ligand-binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non-peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp,25 and 25,. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin,D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single-digit micromolar inhibition of HIV-1 protease and cathepsin,D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross-links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus. [source] The prevalence of lipodystrophy in an ambulant HIV-infected population: it all depends on the definitionHIV MEDICINE, Issue 3 2001VM Carter Objectives This study's objective was to determine the prevalence of body shape changes and metabolic abnormalities in an ambulant population with HIV infection. Three different definitions of lipodystrophy were used to assess these changes. Patients' anthropometric measures and dual-energy X-ray absorptiometry (DEXA) scans were compared in order to estimate fat distribution in this population. We sought to evaluate potential predictors for lipodystrophy according to each of the three definitions. Methods We performed a cross-sectional study in the outpatient clinic of a tertiary referral hospital in Melbourne, Australia. We enrolled a total of 167 HIV-infected ambulatory patients over 3 months in mid-1998. Data on 159 males, 149 of whom were receiving triple combination antiretroviral therapy, were evaluated. Anthropometric measures, clinical examination, self-report of body shape changes, biochemical measures and DEXA scan were used to assess lipodystrophy and risk factors for cardiovascular disease. Patients described body shape changes in the face, trunk, arms and legs. Laboratory parameters measured included fasting triglyceride (TG), cholesterol, high-density lipoproteins (HDL), glucose, insulin, CD4 cell count and plasma HIV RNA. Current and past antiretroviral therapies were ascertained. Results According to one proposed Australian national definition of lipodystrophy (LDNC), the prevalence of lipodystrophy in this population was 65%. This definition included an objective assessment with major and minor criteria. Patient-defined lipodystrophy (LDP), which involved a subjective assessment of thinning arms and legs and central adiposity, occurred in 19%. Patient-defined lipoatrophy (LAP), which involved a subjective assessment of thinning arms and legs without central adiposity, occurred in 21.3%. No change in body habitus was noted by 37% of the cohort. Hypercholesterolaemia was recorded in 44%, hypertriglyceridaemia in 52% and elevated insulin levels in 23%. Anthropometry was predictive of the per cent total body fat recorded by DEXA scan, but produced consistently lower values. In multivariate analysis, LDP and LAP were significantly associated with stavudine (d4T) use, while LAP was also associated with zidovudine (ZDV) treatment. There were no treatment associations with LDNC. Protease inhibitor (PI) exposure was associated with metabolic changes but not patient perceived body shape changes, while d4T and ZDV exposure was associated with increased triglycerides and reduced peripheral fat stores. Conclusions The prevalence of body shape changes in a single population varied depending on the definition applied. The LDNC definition overestimated body shape abnormalities in comparison with patient perception. LAP was associated with significantly lower fat stores measured by anthropometry and DEXA scan than those identified under the LDNC definition. In contrast to LDNC, LAP was associated with d4T exposure, nucleoside reverse transcriptase inhibitor (NRTI) and ZDV duration of use, but not PI use. Until a consensus definition for lipodystrophy is developed, including agreement on objective measurement and thresholds for abnormality, careful description of the individual components of the syndrome is required to enable cohort comparisons so that predictors of the syndrome can be assessed more accurately and outcome studies made feasible. [source] Enzymatic modification as a tool to improve the functional properties of heat-processed soy flourJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2008Cheruppanpullil Radha Abstract BACKGROUND: There are a number of antinutritional factors present in soybeans that exert a negative impact on the nutritional quality of the protein. Among those factors that are destroyed by heat treatment are protease inhibitors and lectins. Protease inhibitors show antinutritional effect and moreover the digestibility of the protein is limited by the presence of these antinutrients. The aims of the present study are (1) to study the effect of autoclaving on the trypsin inhibitor inactivation, nitrogen solubility and protein digestibility of defatted soy flour and (2) to study the effect of enzymatic modification on the functional properties of autoclaved soy flour. RESULTS: The solubility of the soy flour decreased with increase in autoclaving time. Partial hydrolysis of the autoclaved soy flour increased its acid solubility (pH 4.5) from 17% to 56% over a control value of 24% without affecting its functional properties. Inactivation of trypsin inhibitors improved the protein digestibility of soy flour from 25% to 95%. Particle size analysis of the autoclaved flour indicated the formation of soy protein aggregates, which resulted in poor solubility. The enzymatic modification of autoclaved soy flour resulted in its property as a good emulsifying agent with an emulsion capacity of 118 ± 4 mL. CONCLUSION: Enzymatic modification of the heat-processed soy flour increased its solubility and other functional attributes. The increased acid solubility would be advantageous in the utilization of soy proteins in acidic foods. Thus the autoclaved and partially modified soy flour is a potential source for specific functional foods. Copyright © 2007 Society of Chemical Industry [source] Evaluation of different protein sources for aquafeeds by an optimised pH-stat systemJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 7 2002Francisco J Alarcón Abstract This paper presents the results of some experiments oriented to optimise and standardise the measurement of protein hydrolysis by fish proteases using the pH-stat technique. Various factors affecting the degree of hydrolysis (DH) were considered (autohydrolysis of the protein sources, type of enzymes utilised, substrate/enzyme ratio and effect of inhibitors). The crude extracts obtained from fish digestive tissues showed their suitability for the determination of protein hydrolysis, rendering better results than those obtained using mixtures of commercial enzymes. DH values for a given protein were greatly affected by the substrate/enzyme ratio, since small modifications in protein concentration resulted in significant variations in DH. Protease inhibitors present in various plant protein sources produced a reduction in DH values. © 2002 Society of Chemical Industry [source] Protease inhibitors in bacteria: an emerging concept for the regulation of bacterial protein complexes?MOLECULAR MICROBIOLOGY, Issue 6 2006Wolfgang H. Schwarz Summary Serine protease inhibitors (serpins), the antagonists of serine proteases, were unknown in the bacterial kingdom until recently. Kang et al. in this issue of Molecular Microbiology report the cloning and functional analysis of the three serpin genes from the thermophilic anaerobic bacterium Clostridium thermocellum. Two of the serpins contain a dockerin module for location in the extracellular hydrolytic multienzyme complex, the cellulosome. The susceptibility of cellulosome to proteolytic degradation and the presence of a serine protease in the same complex provoke speculation that protease inhibitor/protease pairs could play hitherto unrecognized roles in protein stability and regulation in bacteria. [source] Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteasesPROTEIN SCIENCE, Issue 3 2009Marie-Louise Zani Abstract The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin-2 are endogeneous low-molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol-based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin-2 variant, trappin-2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin-2 A62L are tight-binding inhibitors of all three NSPs with subnanomolar Kis, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane-bound forms of all three NSPs. The trappin-2 A62L and elafin-SLPI chimeras, like wild-type elafin and trappin-2, can be covalently cross-linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti-inflammatory agents. [source] Protease inhibitors and reproduction of reniform nematode in pineappleANNALS OF APPLIED BIOLOGY, Issue 1 2009C. Rabovich Abstract Endogenous protease inhibitors (PIs) in the roots of Smooth Cayenne pineapple clones may affect the growth of the plant-parasitic nematode Rotylenchulus reniformis. In pineapple, reniform population densities remain atypically near preplant levels for 6,9 months after pineapple planting. A potted plant experiment was conducted to determine if the PI present in pineapple roots affected nematode reproduction and possibly account for the observed nematode field population dynamics. Pineapple PI activity increased for the first 6 months after planting and was higher in nematode-inoculated plants. Nematode reproduction, however, was not correlated to PI activity. In a second experiment, PI activity was concentrated in the portion of the roots nearest the pineapple butt where nematode population densities were highest. The behaviour of the PI in pineapple roots suggested a defensive role, such as systemic acquired resistance, against R. reniformis. [source] Molecular background of EPM1,Unverricht,Lundborg diseaseEPILEPSIA, Issue 4 2008Tarja Joensuu Summary Unverricht,Lundborg disease (EPM1) is an autosomal recessively inherited neurodegenerative disorder and the most common single cause of progressive myoclonus epilepsy worldwide. Mutations in the gene encoding cystatin B (CSTB), a cysteine protease inhibitor, are responsible for the primary defect underlying EPM1. Here, progress toward understanding the molecular mechanisms in EPM1 is reviewed. We summarize the current knowledge about the CSTB gene and mutations as well as the cellular biology of the CSTB protein with emphasis on data emerging from analysis of EPM1 patients. We shed light on the disease mechanisms of EPM1 based on characterization of the CSTB -deficient mouse model. [source] Altered Tryptophan Metabolism in the Brain of Cystatin B -Deficient Mice: A Model System for Progressive Myoclonus EpilepsyEPILEPSIA, Issue 10 2006Annika Vaarmann Summary:,Purpose: Progressive myoclonus epilepsy of the Unverricht,Lundborg type (EPM1) is a rare neurologic disorder, associated with mutations in the Cystatin B (Cstb) gene. Mice lacking Cstb, a cysteine protease inhibitor of the cathepsine family of proteases, provide a mammalian model for EPM1 by displaying similarly progressive ataxia, myoclonic seizures, and neurodegeneration. However, the linkage of Cstb deficit on the molecular level to pathologic features like myoclonic jerks or tonic,clonic seizures has remained unclear. We examined the tryptophan (TRP) metabolism, along the serotonin (5HT) and kynurenine (KYN) pathway in the brain of Cstb -deficient mice, in relation to their possible involvement in the seizure phenotype. Methods: TRP and its metabolites, along the 5HT and KYN pathways, were assayed in brain tissue by high-pressure liquid chromatography (HPLC) with electrochemical detection. The inverted wire grid and mild handling tests were used for evaluation of ataxia and myoclonic activity. Results: The Cstb -deficient mice had constitutively increased TRP, 5HT, and 5-hydroxyindole acetic acid (5HIAA) levels in the cerebral cortex and cerebellum and increased levels of KYN in the cerebellum. These neurochemical changes were accompanied with ataxia and an apparent myoclonic phenotype among the Cstb -deficient mice. Conclusions: Our findings suggest that secondary processes (i.e., overstimulation of serotoninergic transmission) on the cellular level, initiated by Cstb deficiency in specific brain regions, may be responsible for the myoclonic/seizure phenotype in EPM1. [source] Signaling events leading to the curative effect of cystatin on experimental visceral leishmaniasis: Involvement of ERK1/2, NF-,B and JAK/STAT pathwaysEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009Susanta Kar Abstract Curative effect of cystatin, a natural cystein protease inhibitor, on experimental visceral leishmaniasis was associated with strong upregulation of iNOS. The transductional mechanisms underlying this cellular response was investigated in the murine macrophage cell line RAW 264.7 and in the BALB/c mouse model of visceral leishmaniasis. Cystatin synergizes with IFN-, in inducing ERK1/2 phosphorylation and NF-,B DNA-binding activity. Pretreatment of cells with specific inhibitors of NF-,B or ERK1/2 pathway blocked the cystatin plus IFN-,-inducible NF-,B activity and markedly reduced the expression of iNOS at both mRNA and protein levels. Silencing of mitogen- and stress-activated protein kinase 1 significantly reduced cystatin-mediated NF-,B-dependent iNOS gene transcription suggesting the involvement of mitogen- and stress-activated protein kinase 1 activation in ERK1/2 signaling. DNA binding as well as silencing experiments revealed the requirement of IFN-,-mediated JAK-STAT activation even though cystatin did not modulate this signaling cascade by itself. In the in vivo situation, key steps in the activation cascade of NF-,B, including nuclear translocation of NF-,B subunits, I,B phosphorylation and I,B kinase, are all remarkably enhanced in Leishmania -infected mice by cystatin. Understanding the molecular mechanisms through which cystatin modulates macrophage effector responses will contribute to better define its potential for macrophage-associated diseases, in general. [source] Activation of hepatocyte growth factor activator zymogen (pro-HGFA) by human kallikrein 1-related peptidasesFEBS JOURNAL, Issue 5 2008Shoichiro Mukai Hepatocyte growth factor activator (HGFA) is a serine protease and a potent activator of prohepatocyte growth factor/scatter factor (pro-HGF/SF), a multifunctional growth factor that is critically involved in tissue morphogenesis, regeneration, and tumor progression. HGFA circulates as a zymogen (pro-HGFA) and is activated in response to tissue injury. Although thrombin is considered to be an activator of pro-HGFA, alternative pro-HGFA activation pathways in tumor microenvironments remain to be identified. In this study, we examined the effects of kallikrein 1-related peptidases (KLKs), a family of extracellular serine proteases, on the activation of pro-HGFA. Among the KLKs examined (KLK2, KLK3, KLK4 and KLK5), we identified KLK4 and KLK5 as novel activators of pro-HGFA. Using N-terminal sequencing, the cleavage site was identified as the normal processing site, Arg407,Ile408. The activation of pro-HGFA by KLK5 required a negatively charged substance such as dextran sulfate, whereas KLK4 could process pro-HGFA without dextran sulfate. KLK5 showed more efficient pro-HGFA processing than KLK4, and was expressed in 50% (13/25) of the tumor cell lines examined. HGFA processed by these KLKs efficiently activated pro-HGF/SF, and led to cellular scattering and invasion in vitro. The activities of both KLK4 and KLK5 were strongly inhibited by HGFA inhibitor type 1, an integral membrane Kunitz-type serine protease inhibitor that inhibits HGFA and other pro-HGF/SF-activating proteases. These data suggest that KLK4 and KLK5 mediate HGFA-induced activation of pro-HGF/SF within tumor tissue, which may thereafter trigger a series of events leading to tumor progression via the MET receptor. [source] Mutational analysis of plasminogen activator inhibitor-1FEBS JOURNAL, Issue 8 2003-helix F, Interactions of, its neighbouring structural elements regulates the activity, the rate of latency transition The serpin plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the plasminogen activating serine proteases tissue-type and urokinase-type plasminogen activator and, as such, an important regulator in turnover of extracellular matrix and in fibrinolysis. PAI-1 spontaneously loses its antiproteolytic activity by inserting its reactive centre loop (RCL) as strand 4 in ,-sheet A, thereby converting to the so-called latent state. We have investigated the importance of the amino acid sequence of ,-helix F (hF) and the connecting loop to s3A (hF/s3A-loop) for the rate of latency transition. We grafted regions of the hF/s3A-loop from antithrombin III and ,1 -protease inhibitor onto PAI-1, creating eight variants, and found that one of these reversions towards the serpin consensus decreased the rate of latency transition. We prepared 28 PAI-1 variants with individual residues in hF and ,-sheet A replaced by an alanine. We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case. Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour, suggesting that insertion of the RCL is held back during latency transition as well as during complex formation with target proteases. The data presented identify new determinants of PAI-1 latency transition and provide general insight into the characteristic loop,sheet interactions in serpins. [source] The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73FEBS JOURNAL, Issue 22 2002Alona Pavlova The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by ,,300-fold, >10-fold and ,,4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by ,,10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one amino-acid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, ,,45% of the total binding energy, for inhibition of cathepsin B. [source] | |||||||||||||||||||||||||||||||||||||