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Juan José Cazzulo - One of the best experts on this subject based on the ideXlab platform.

  • novel scaffolds for inhibition of Cruzipain identified from high throughput screening of anti kinetoplastid chemical boxes
    Scientific Reports, 2017
    Co-Authors: Emir Salassarduy, Juan José Cazzulo, Lionel Uran Landaburu, Joel X Karpiak, Kevin P Madauss, Fernan Aguero, Vanina E Alvarez
    Abstract:

    American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.

  • Substrate inhibition of Cruzipain is not affected by the C-terminal domain
    FEBS letters, 2016
    Co-Authors: Vito Turk, Veronika Stoka, Juan José Cazzulo, James H Mckerrow
    Abstract:

    Endogenous and recombinant Cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi, exhibit differences in the protein and circular dichroism spectra probably attributed to the absence of the C- terminal domain in the recombinant enzyme. Substrate hydrolysis of both molecules at 25°C and neutral pH obeyed Michaelis-Menten kinetics whereas significant substrate inhibition was observed above neutral pH. The results suggest that substrate inhibition of Cruzipain is pH-dependent, and that the C-terminal domain does not play an essential role in this process.

  • The major cysteine proteinase of trypanosoma cruzi : a valid target for chemotherapy of Chagas disease
    Current pharmaceutical design, 2016
    Co-Authors: Vito Turk, Veronika Stoka, Juan José Cazzulo
    Abstract:

    Trypanosoma cruzi, the causative agent of the American Trypanosomiasis, Chagasdisease, contains a major cysteine proteinase (CP), Cruzipain (also known as cruzain, or GP57/51). The enzyme is a member of the papain C1 family of CPs, with a specificity intermediate between those of cathepsin Land cathepsin B. The enzyme, which is expressed at different levels by different parasite stages, is encoded by a high number of genes (up to 130 in the Tul2 strain), which code for a pre-pro-enzyme. Mature Cruzipain consists of a catalytic moiety with high homology to cathepsins S and L, and a C-terminal domain, characteristic of Type I CPs of Trypanosomatids, and absent in all other C1 family CPs described so far. Irreversible inhibitors ofCruzipain (peptidyl diazomethylketones, peptidyl fluoromethylketones, peptidyl vinyl sulphones) are able to block the differentiation steps in the parasite's life cycle, and effectively kill the organism. Recently, a vinyl sulphone derivative (N-piperazine- Phe-hPhevinyl sulphone phenyl) which is an efficient inhibitor of Cruzipain and kills T. cruzi by inducing an accumulation of unprocessed Cruzipain in the Golgi cisternae, interfering with the secretory pathway, has been tested in vivo in a mice model (J.H. McKerrow et al.). The curative effects observed, as well as the good bioavailability of the inhibitor and its apparent lack of undesirable side effects, make it a promising lead compound for the development of new drugs for the chemotherapy of Chagas disease.

  • the peptidases of trypanosoma cruzi digestive enzymes virulence factors and mediators of autophagy and programmed cell death
    Biochimica et Biophysica Acta, 2012
    Co-Authors: Vanina E Alvarez, Gabriela T Niemirowicz, Juan José Cazzulo
    Abstract:

    Abstract Trypanosoma cruzi , the agent of the American Trypanosomiasis, Chagas disease, contains cysteine, serine, threonine, aspartyl and metallo peptidases. The most abundant among these enzymes is Cruzipain, a cysteine proteinase expressed as a mixture of isoforms, some of them membrane-bound. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship. Inhibitors of Cruzipain kill the parasite and cure infected mice, thus validating the enzyme as a very promising target for the development of new drugs against the disease. In addition, a 30 kDa cathepsin B-like enzyme, two metacaspases and two autophagins have been described. Serine peptidases described in the parasite include oligopeptidase B, a member of the prolyl oligopeptidase family involved in Ca 2+ -signaling during mammalian cell invasion; a prolyl endopeptidase (Tc80), against which inhibitors are being developed, and a lysosomal serine carboxypeptidase. Metallopeptidases homologous to the gp63 of Leishmania spp. are present, as well as two metallocarboxypeptidases belonging to the M32 family, previously found only in prokaryotes. The proteasome has properties similar to those of other eukaryotes, and its inhibition by lactacystin blocks some differentiation steps in the life cycle of the parasite. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

  • specific cleavage sites on human igg subclasses by Cruzipain the major cysteine proteinase from trypanosoma cruzi
    Molecular and Biochemical Parasitology, 2003
    Co-Authors: Patricia Berasain, Juan José Cazzulo, Carlos Carmona, Blas Frangione, Fernando Goni
    Abstract:

    Cruzipain, the major cysteine proteinase of Trypanosoma cruzi, might have other biological roles than its metabolic functions. In this report, we have explored the interaction of Cruzipain with molecules of the immune system. The enzyme was used to digest all human IgG subclasses at different pH values and lengths of time. At pH 7.3, all subclasses were readily split at the hinge region. Immunoblot and amino acid sequence analysis showed fragments of IgG1 and IgG3 to be compatible with Fab and Fc, whereas IgG2 and IgG4 rendered Fab2 and Fc. In all cases the fragments produced might impair the binding capacities and the effector functions of specific IgG. At these cleavage sites Cruzipain displays cathepsin L and/or cathepsin B activities and shows a clear preference for Pro at the P'2 position and polar residues at P1. Despite the activity of Cruzipain within the hinge, the enzyme also cleaved all heavy chains between the CH2 and CH3 domains; producing Fc'-like-fragments of 14 kDa. These fragments are potential candidates to block or saturate Fc receptors on immunocompetent cells. At mild acidic pH Cruzipain produced further degradation of the Fc of all subclasses, the Fd of IgG4 and partially the Fd of IgG1, with the consistent loss of any antibody activity. The L chains apparently were not affected. Thus, Cruzipain should be able to modulate, depending on the subclass selected and the pH of the environment, the production and the length of different biologically active/inactive IgG fragments.

Julio Scharfstein - One of the best experts on this subject based on the ideXlab platform.

  • angiotensin converting enzyme limits inflammation elicited by trypanosoma cruzi cysteine proteases a peripheral mechanism regulating adaptive immunity via the innate kinin pathway
    Biological Chemistry, 2008
    Co-Authors: Julio Scharfstein, Veronica Schmitz, Ana Carolina Monteiro, E Svensjo
    Abstract:

    Tissue injury by pathogens induces a stereotyped inflammatory response that alerts the innate immune system of the potential threat to host integrity. Here, we review knowledge emerging from investigations of the role of the kinin system in the mechanisms that link innate to the adaptive phase of immunity. Progress in this field started with results demonstrating that bradykinin is an endogenous danger signal that induces dendritic cell (DC) maturation via G protein-coupled bradykinin B2 receptors (B2R). The immunostimulatory role of kinins was recently confirmed in two different mouse models of Trypanosoma cruzi infection, a parasitic protozoan equipped with kinin-releasing cysteine proteases (Cruzipain). Infection by the intraperitoneal route showed that DCs from B2R-/- mice (susceptible phenotype) failed to sense kinin 'danger' signals proteolytically released by parasites, explaining why these mutant mice display lower frequencies of interferon-gamma-producing effector T-cells. Studies of the dynamics of inflammation in the subcutaneous model of infection revealed that the balance between Cruzipain and angiotensin-converting enzyme, respectively acting as kinin-generating and degrading enzymes, governs extent of DC maturation and TH1 development via the B2R-dependent innate pathway. Studies of the kinin role in immunity may shed light on the relationship between proteolytic networks and the cytokine circuits that guide T-cell development.

  • the propeptide of Cruzipain a potent selective inhibitor of the trypanosomal enzymes Cruzipain and brucipain and of the human enzyme cathepsin f
    FEBS Journal, 2007
    Co-Authors: Flavia C G Reis, Julio Scharfstein, Robert Menard, Tatiana F R Costa, Traian Sulea, Alessandra Mezzetti, Dieter Bromme, Ana Paula C. A. Lima
    Abstract:

    Papain-like cysteine proteases of pathogenic protozoa play important roles in parasite growth, differentiation and host cell invasion. The main cysteine proteases of Trypanosoma cruzi (Cruzipain) and of Trypanosoma brucei (brucipain) are validated targets for the development of new chemotherapies. These proteases are synthesized as precursors and activated upon removal of the N-terminal prodomain. Here we report potent and selective inhibition of Cruzipain and brucipain by the recombinant full-length prodomain of Cruzipain. The propeptide did not inhibit human cathepsins S, K or B or papain at the tested concentrations, and moderately inhibited human cathepsin V. Human cathepsin F was very efficiently inhibited (K(i) of 32 pm), an interesting finding indicating that Cruzipain propeptide is able to discriminate cathepsin F from other cathepsin L-like enzymes. Comparative structural modeling and analysis identified the interaction between the beta1p-alpha3p loop of the propeptide and the propeptide-binding loop of mature enzymes as a plausible cause of the observed inhibitory selectivity.

  • the substrate specificity of Cruzipain 2 a cysteine protease isoform from trypanosoma cruzi
    Fems Microbiology Letters, 2006
    Co-Authors: Flavia C G Reis, Julio Scharfstein, Luiz Juliano, Maria A Juliano, Wagner As Judice, Ana Paula Cd Lima
    Abstract:

    Papain-like cysteine proteases are important for the survival of the flagellated protozoa Trypanosoma cruzi, the causative agent of Chagas' Disease. The lysosomal cysteine protease designated as Cruzipain or cruzain, is the archetype of a multigene family of related isoforms. We investigated the substrate specificity of the Cruzipain 2 isoform using internally quenched fluorogenic substrates. We found that Cruzipain 2 and cruzain differ substantially regarding the specificity in the S2, S′1 and S′2 pockets. Our study indicates that Cruzipain 2 has a more restricted specificity than cruzain, suggesting that these isoforms might act on distinct natural substrates.

  • chagasin the endogenous cysteine protease inhibitor of trypanosoma cruzi modulates parasite differentiation and invasion of mammalian cells
    Journal of Cell Science, 2005
    Co-Authors: Camila C Santos, Julio Scharfstein, Celso Santanna, Amanda Terres, Narcisa L Cunhaesilva, Ana Paula C. A. Lima
    Abstract:

    Chagasin is a Trypanosoma cruzi protein that was recently characterized as a tight-binding inhibitor of papain-like cysteine proteases (CPs). Considering that parasite virulence and morphogenesis depend on the endogenous activity of lysosomal CPs of the Cruzipain family, we sought to determine whether chagasin and Cruzipain interact in the living cell. Ultrastructural studies showed that chagasin and Cruzipain both localize to the Golgi complex and reservosomes (lysosome-like organelles), whereas free chagasin was found in small intracellular vesicles, suggesting that chagasin trafficking pathways might intersect with those of Cruzipain. Taking advantage of the fact that sodium dodecyl sulphate and β-mercaptoethanol prevent binding between the isolated proteins but do not dismantle preformed Cruzipain-chagasin complexes, we obtained direct evidence that chagasin-Cruzipain complexes are indeed formed in epimastigotes. Chagasin transfectants (fourfold increase in CP inhibitory activity) displayed low rates of differentiation (metacyclogenesis) and exhibited increased resistance to a synthetic CP inhibitor. These phenotypic changes were accompanied by a drastic reduction of soluble Cruzipain activity and by upregulated secretion of Cruzipain-chagasin molecular complexes. Analysis of six T. cruzi strains revealed that expression levels of Cruzipain and chagasin are variable, but the molar ratios are fairly stable (∼50:1) in most strains, with the exception of the G strain (5:1), which is poorly infective. On the same vein, we found that trypomastigotes overexpressing chagasin are less infective than wild-type parasites in vitro. The deficiency of chagasin overexpressers is caused by lower activity of membrane-associated CPs, because membranes recovered from wild-type trypomastigotes restored infectivity and this effect was nullified by the CP inhibitor E-64. In summary, our studies suggest that chagasin regulates the endogenous activity of CP, thus indirectly modulating proteolytic functions that are essential for parasite differentiation and invasion of mammalian cells.

  • a new Cruzipain mediated pathway of human cell invasion by trypanosoma cruzi requires trypomastigote membranes
    Infection and Immunity, 2004
    Co-Authors: Isabela M Aparicio, Julio Scharfstein, Ana Paula C. A. Lima
    Abstract:

    The intracellular protozoan Trypanosoma cruzi causes Chagas' disease, a chronic illness associated with cardiomyopathy and digestive disorders. This pathogen invades mammalian cells by signaling them through multiple transduction pathways. We previously showed that Cruzipain, the main cysteine protease of T. cruzi, promotes host cell invasion by activating kinin receptors. Here, we report a Cruzipain-mediated invasion route that is not blocked by kinin receptor antagonists. By testing different strains of T. cruzi, we observed a correlation between the level of Cruzipain secreted by trypomastigotes and the capacity of the pathogen to invade host cells. Consistent with a role for Cruzipain, the cysteine protease inhibitor N-methylpiperazine-urea-Phe-homophenylalanine-vinylsulfone-benzene impaired the invasion of human smooth muscle cells by strains Dm28c and X10/6 but not by the G isolate. Cruzipain-rich supernatants of Dm28c trypomastigotes enhanced the infectivity of isolate G parasites twofold, an effect which was abolished by the cysteine protease inhibitor l-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane and by thapsigargin, a drug that induces depletion of the intracellular Ca2+ stores. The enhancement due to Dm28 supernatants was abolished upon Cruzipain immunodepletion, and the activity was restored by purified Cruzipain. In contrast, supernatants from isolate G trypomastigotes (with low levels of Cruzipain) or supernatants from Dm28c epimastigotes or purified Cruzipain alone did not enhance parasite invasion, indicating that the protease is required but not sufficient to engage this invasion pathway. We provide evidence that activation of this pathway requires Cruzipain-mediated processing of a trypomastigote molecule associated with parasite-shed membranes. Our results couple Cruzipain to host cell invasion through a kinin-independent route and further suggest that high-level Cruzipain expression may contribute to parasite infectivity.

Ana Paula C. A. Lima - One of the best experts on this subject based on the ideXlab platform.

  • Cruzipain promotes trypanosoma cruzi adhesion to rhodnius prolixus midgut
    PLOS Neglected Tropical Diseases, 2012
    Co-Authors: Livia A Uehara, Ana Paula C. A. Lima, André L.s. Santos, Marta H. Branquinha, Otacilio C Moreira, Ana Carolina Oliveira, Patricia Azambuja, Constanca Britto, Claudia M Davilalevy
    Abstract:

    Background: Trypanosoma cruzi is the etiological agent of Chagas’ disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction. Methodology/Principal Findings: Here, we have investigated whether Cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-Cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active Cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous Cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active Cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous Cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface Cruzipain. Conclusions/Significance: These data highlight, for the first time, that Cruzipain contributes to the interaction of T. cruzi with the insect host.

  • the propeptide of Cruzipain a potent selective inhibitor of the trypanosomal enzymes Cruzipain and brucipain and of the human enzyme cathepsin f
    FEBS Journal, 2007
    Co-Authors: Flavia C G Reis, Julio Scharfstein, Robert Menard, Tatiana F R Costa, Traian Sulea, Alessandra Mezzetti, Dieter Bromme, Ana Paula C. A. Lima
    Abstract:

    Papain-like cysteine proteases of pathogenic protozoa play important roles in parasite growth, differentiation and host cell invasion. The main cysteine proteases of Trypanosoma cruzi (Cruzipain) and of Trypanosoma brucei (brucipain) are validated targets for the development of new chemotherapies. These proteases are synthesized as precursors and activated upon removal of the N-terminal prodomain. Here we report potent and selective inhibition of Cruzipain and brucipain by the recombinant full-length prodomain of Cruzipain. The propeptide did not inhibit human cathepsins S, K or B or papain at the tested concentrations, and moderately inhibited human cathepsin V. Human cathepsin F was very efficiently inhibited (K(i) of 32 pm), an interesting finding indicating that Cruzipain propeptide is able to discriminate cathepsin F from other cathepsin L-like enzymes. Comparative structural modeling and analysis identified the interaction between the beta1p-alpha3p loop of the propeptide and the propeptide-binding loop of mature enzymes as a plausible cause of the observed inhibitory selectivity.

  • chagasin the endogenous cysteine protease inhibitor of trypanosoma cruzi modulates parasite differentiation and invasion of mammalian cells
    Journal of Cell Science, 2005
    Co-Authors: Camila C Santos, Julio Scharfstein, Celso Santanna, Amanda Terres, Narcisa L Cunhaesilva, Ana Paula C. A. Lima
    Abstract:

    Chagasin is a Trypanosoma cruzi protein that was recently characterized as a tight-binding inhibitor of papain-like cysteine proteases (CPs). Considering that parasite virulence and morphogenesis depend on the endogenous activity of lysosomal CPs of the Cruzipain family, we sought to determine whether chagasin and Cruzipain interact in the living cell. Ultrastructural studies showed that chagasin and Cruzipain both localize to the Golgi complex and reservosomes (lysosome-like organelles), whereas free chagasin was found in small intracellular vesicles, suggesting that chagasin trafficking pathways might intersect with those of Cruzipain. Taking advantage of the fact that sodium dodecyl sulphate and β-mercaptoethanol prevent binding between the isolated proteins but do not dismantle preformed Cruzipain-chagasin complexes, we obtained direct evidence that chagasin-Cruzipain complexes are indeed formed in epimastigotes. Chagasin transfectants (fourfold increase in CP inhibitory activity) displayed low rates of differentiation (metacyclogenesis) and exhibited increased resistance to a synthetic CP inhibitor. These phenotypic changes were accompanied by a drastic reduction of soluble Cruzipain activity and by upregulated secretion of Cruzipain-chagasin molecular complexes. Analysis of six T. cruzi strains revealed that expression levels of Cruzipain and chagasin are variable, but the molar ratios are fairly stable (∼50:1) in most strains, with the exception of the G strain (5:1), which is poorly infective. On the same vein, we found that trypomastigotes overexpressing chagasin are less infective than wild-type parasites in vitro. The deficiency of chagasin overexpressers is caused by lower activity of membrane-associated CPs, because membranes recovered from wild-type trypomastigotes restored infectivity and this effect was nullified by the CP inhibitor E-64. In summary, our studies suggest that chagasin regulates the endogenous activity of CP, thus indirectly modulating proteolytic functions that are essential for parasite differentiation and invasion of mammalian cells.

  • a new Cruzipain mediated pathway of human cell invasion by trypanosoma cruzi requires trypomastigote membranes
    Infection and Immunity, 2004
    Co-Authors: Isabela M Aparicio, Julio Scharfstein, Ana Paula C. A. Lima
    Abstract:

    The intracellular protozoan Trypanosoma cruzi causes Chagas' disease, a chronic illness associated with cardiomyopathy and digestive disorders. This pathogen invades mammalian cells by signaling them through multiple transduction pathways. We previously showed that Cruzipain, the main cysteine protease of T. cruzi, promotes host cell invasion by activating kinin receptors. Here, we report a Cruzipain-mediated invasion route that is not blocked by kinin receptor antagonists. By testing different strains of T. cruzi, we observed a correlation between the level of Cruzipain secreted by trypomastigotes and the capacity of the pathogen to invade host cells. Consistent with a role for Cruzipain, the cysteine protease inhibitor N-methylpiperazine-urea-Phe-homophenylalanine-vinylsulfone-benzene impaired the invasion of human smooth muscle cells by strains Dm28c and X10/6 but not by the G isolate. Cruzipain-rich supernatants of Dm28c trypomastigotes enhanced the infectivity of isolate G parasites twofold, an effect which was abolished by the cysteine protease inhibitor l-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane and by thapsigargin, a drug that induces depletion of the intracellular Ca2+ stores. The enhancement due to Dm28 supernatants was abolished upon Cruzipain immunodepletion, and the activity was restored by purified Cruzipain. In contrast, supernatants from isolate G trypomastigotes (with low levels of Cruzipain) or supernatants from Dm28c epimastigotes or purified Cruzipain alone did not enhance parasite invasion, indicating that the protease is required but not sufficient to engage this invasion pathway. We provide evidence that activation of this pathway requires Cruzipain-mediated processing of a trypomastigote molecule associated with parasite-shed membranes. Our results couple Cruzipain to host cell invasion through a kinin-independent route and further suggest that high-level Cruzipain expression may contribute to parasite infectivity.

  • heparan sulfate modulates kinin release by trypanosoma cruzi through the activity of Cruzipain
    Journal of Biological Chemistry, 2002
    Co-Authors: Ana Paula C. A. Lima, Veronica Schmitz, Isaura Y Hirata, Werner Mulleresterl, Jair Ribeiro Chagas, Paulo C Almeida, Luiz Juliano, Alvin H. Schmaier, Ivarne L.s. Tersariol, Julio Scharfstein
    Abstract:

    Abstract Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, Cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of Cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K i app) of HK about 10-fold. Conversely, the catalytic efficiency of Cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by Cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35-fold increase in Cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between Cruzipain, HK, and heparan sulfate proteoglycans.

Luiz Juliano - One of the best experts on this subject based on the ideXlab platform.

  • the substrate specificity of Cruzipain 2 a cysteine protease isoform from trypanosoma cruzi
    Fems Microbiology Letters, 2006
    Co-Authors: Flavia C G Reis, Julio Scharfstein, Luiz Juliano, Maria A Juliano, Wagner As Judice, Ana Paula Cd Lima
    Abstract:

    Papain-like cysteine proteases are important for the survival of the flagellated protozoa Trypanosoma cruzi, the causative agent of Chagas' Disease. The lysosomal cysteine protease designated as Cruzipain or cruzain, is the archetype of a multigene family of related isoforms. We investigated the substrate specificity of the Cruzipain 2 isoform using internally quenched fluorogenic substrates. We found that Cruzipain 2 and cruzain differ substantially regarding the specificity in the S2, S′1 and S′2 pockets. Our study indicates that Cruzipain 2 has a more restricted specificity than cruzain, suggesting that these isoforms might act on distinct natural substrates.

  • heparan sulfate modulates kinin release by trypanosoma cruzi through the activity of Cruzipain
    Journal of Biological Chemistry, 2002
    Co-Authors: Ana Paula C. A. Lima, Veronica Schmitz, Isaura Y Hirata, Werner Mulleresterl, Jair Ribeiro Chagas, Paulo C Almeida, Luiz Juliano, Alvin H. Schmaier, Ivarne L.s. Tersariol, Julio Scharfstein
    Abstract:

    Abstract Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, Cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of Cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K i app) of HK about 10-fold. Conversely, the catalytic efficiency of Cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by Cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35-fold increase in Cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between Cruzipain, HK, and heparan sulfate proteoglycans.

  • comparison of the specificity stability and individual rate constants with respective activation parameters for the peptidase activity of Cruzipain and its recombinant form cruzain from trypanosoma cruzi
    FEBS Journal, 2001
    Co-Authors: Wagner Alves De Souza Judice, Julio Scharfstein, Ivarne L.s. Tersariol, Maria A Juliano, Maria Helena S Cezari, Apca Lima, Jair R Chagas, Luiz Juliano
    Abstract:

    The Trypanosoma cruzi cysteine protease Cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural Cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2′ subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH–activity profile between the two enzymes was found, but in 1 m NaCl Cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for Cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k−1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis–Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci.9, 1589–1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E−1, which are significantly higher for Cruzipain. The corresponding ΔS1 and ΔS−1 were positive and significantly higher for Cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for Cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of Cruzipain.

  • altered expression of Cruzipain and a cathepsin b like target in a trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine proteinases
    Molecular and Biochemical Parasitology, 2000
    Co-Authors: Vladimir Yong, Veronica Schmitz, Ana Paula C. A. Lima, Luiz Juliano, Marcos A Vanniersantos, Gilles Lalmanach, Francis Gauthier, Julio Scharfstein
    Abstract:

    Abstract The therapeutic potential of synthetic inhibitors to the major cysteine-proteinase from Trypanosoma cruzi (cruzain or Cruzipain) was recently demonstrated in animal models of Chagas’ disease. A possible limitation of this strategy would be the emergence of parasite populations developing resistance to cysteine-proteinase inhibitors. Here, we describe the properties of a phenotypically stable T. cruzi cell line (R-Dm28) that displays increased resistance to Z -(SBz)Cys-Phe-CHN 2 , an irreversible cysteine-proteinase inhibitor which preferentially inactivates cathepsin L-like enzymes. Isolated from axenic cultures of the parental cells (IC 50 1.5 μM), R-Dm28 epimastigotes exhibited 13-fold (IC 50 20 μM) higher resistance to this inhibitor and did not display cross-resistance to unrelated trypanocidal drugs, such as benznidazol and nifurtimox. Western blotting (with mAb), affinity labeling (with biotin-LVG-CHN 2 ) and FACS analysis of R-Dm28 log-phase epimastigotes revealed that the Cruzipain target was expressed at lower levels, as compared with Dm28c. Interestingly, this deficit was paralleled by increased expression of an unrelated Mr 30 000 cysteine-proteinase whose activity was somewhat refractory to inhibition by Z -(SBz)Cys-Phe-CHN 2 . N-terminal sequencing of the affinity-purified biotin-LVG-proteinase complex allowed its identification as a cathepsin B-like enzyme. Increased antigenic deposits of this proteinase were found in the grossly enlarged and electron dense reservosomes from R-Dm28 epimastigotes. Our data suggest that R-Dm28 resistance to toxic effects induced by the synthetic inhibitor may result from decreased availability of the most sensitive cysteine-proteinase target, Cruzipain. The deficit in metabolic functions otherwise mediated by this cathepsin L-like proteinase is likely compensated by increased expression/accumulation of a cathepsin B-like target.

  • inhibition of Cruzipain visualized in a fluorescence quenched solid phase inhibitor library assay d amino acid inhibitors for Cruzipain cathepsin b and cathepsin l
    Journal of Peptide Science, 1998
    Co-Authors: Morten Meldal, Luiz Juliano, Maria A Juliano, Elaine Del Nery, Ib Svendsen, Julio Scharfstein
    Abstract:

    A PEGA-resin was derivatized with a 3:1 mixture of hydroxymethyl benzoic acid and Fmoc-Lys(Boc)-OH and the fluorogenic substrate Ac-Y(NO2)KLRFSKQK(Abz)–PEGA was assembled on the lysine using the active ester approach. Following esterification of the hydroxymethyl benzoic acid with Fmoc-Val-OH a library XXX-k/r-XXXV containing approximately 200,000 beads was assembled by split synthesis. The resulting ‘one bead, two peptides’ library was subjected to extensive hydrolysis with Cruzipain. One hundred darker beads were isolated and the 14 most persistently dark beads were collected and sequenced. The putative inhibitor peptides and several analogues were synthesized and found to be competitive μM to nM inhibitors of Cruzipain in solution. The inhibitory activity was found to be unspecific to Cruzipain when compared with cathepsins B and L and specific when compared with kallikrein. One of the inhibitors was docked into the active site of the cathepsin B and was found most probably to bind to the enzyme cavity in an unusual manner, owing to the inserted D-amino acid residue. © 1998 European Peptide Society and John Wiley & Sons, Ltd.

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  • Substrate inhibition of Cruzipain is not affected by the C-terminal domain
    FEBS letters, 2016
    Co-Authors: Vito Turk, Veronika Stoka, Juan José Cazzulo, James H Mckerrow
    Abstract:

    Endogenous and recombinant Cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi, exhibit differences in the protein and circular dichroism spectra probably attributed to the absence of the C- terminal domain in the recombinant enzyme. Substrate hydrolysis of both molecules at 25°C and neutral pH obeyed Michaelis-Menten kinetics whereas significant substrate inhibition was observed above neutral pH. The results suggest that substrate inhibition of Cruzipain is pH-dependent, and that the C-terminal domain does not play an essential role in this process.

  • The major cysteine proteinase of trypanosoma cruzi : a valid target for chemotherapy of Chagas disease
    Current pharmaceutical design, 2016
    Co-Authors: Vito Turk, Veronika Stoka, Juan José Cazzulo
    Abstract:

    Trypanosoma cruzi, the causative agent of the American Trypanosomiasis, Chagasdisease, contains a major cysteine proteinase (CP), Cruzipain (also known as cruzain, or GP57/51). The enzyme is a member of the papain C1 family of CPs, with a specificity intermediate between those of cathepsin Land cathepsin B. The enzyme, which is expressed at different levels by different parasite stages, is encoded by a high number of genes (up to 130 in the Tul2 strain), which code for a pre-pro-enzyme. Mature Cruzipain consists of a catalytic moiety with high homology to cathepsins S and L, and a C-terminal domain, characteristic of Type I CPs of Trypanosomatids, and absent in all other C1 family CPs described so far. Irreversible inhibitors ofCruzipain (peptidyl diazomethylketones, peptidyl fluoromethylketones, peptidyl vinyl sulphones) are able to block the differentiation steps in the parasite's life cycle, and effectively kill the organism. Recently, a vinyl sulphone derivative (N-piperazine- Phe-hPhevinyl sulphone phenyl) which is an efficient inhibitor of Cruzipain and kills T. cruzi by inducing an accumulation of unprocessed Cruzipain in the Golgi cisternae, interfering with the secretory pathway, has been tested in vivo in a mice model (J.H. McKerrow et al.). The curative effects observed, as well as the good bioavailability of the inhibitor and its apparent lack of undesirable side effects, make it a promising lead compound for the development of new drugs for the chemotherapy of Chagas disease.

  • the high stability of Cruzipain against ph induced inactivation is not dependent on its c terminal domain
    FEBS Letters, 2000
    Co-Authors: Veronika Stoka, Ingemar Björk, James H Mckerrow, Juan José Cazzulo, Boris Turk, Vito Turk
    Abstract:

    Unlike mammalian lysosomal cysteine proteases, the trypanosomal cysteine protease Cruzipain contains a 130-amino acid residue C-terminal domain, in addition to the catalytic domain, and it is stable at neutral pH. The endogenous (with C-terminal domain) and recombinant (without C-terminal domain) Cruzipains exhibit similar stabilities at both acid (kinac=3.1×10−3 s−1 and 4.4×10−3 s−1 at pH 2.75 for endogenous and recombinant Cruzipain, respectively) and alkaline pH (kinac=3.0×10−3 s−1 and 3.7×10−3 s−1 at pH 9.15 for endogenous and recombinant Cruzipain, respectively). The pH-induced inactivation, which is a highly pH dependent first order process, is irreversible and accompanied by significant changes of secondary and tertiary structure as revealed by circular dichroism measurements. The different stability of Cruzipain as compared to related proteases, is therefore due mainly to the different number, nature and distribution of charged residues within the catalytic domain and not due to addition of the C-terminal domain.

  • A fragment of the major histocompatibility complex class II - associated p41 invariant chain inhibits Cruzipain, the major cysteine proteinase from Trypanosoma cruzi
    FEBS letters, 1997
    Co-Authors: Vito Turk, Veronika Stoka, Galina Pungerčič, Tadeja Bevec, Juan José Cazzulo
    Abstract:

    A peptide fragment derived from the p41 form of the invariant chain (Ii) associated with the major histocompatibility complex (MHC) class II molecule has been shown to inhibit the mammalian lysosomal cysteine proteinase, cathepsin L, and to be a novel cysteine proteinase inhibitor, distinct from cystatins. Here we report that this same fragment also binds to and inhibits Cruzipain, the cathepsin L-like enzyme from the protozoan parasite Trypanosoma cruzi. The binding of the Ii fragment to Cruzipain is fast (k(ass) = 2.4 x 107 M-1 s-1) and tight (K(i) = 5.8 x 10-11 M). The inhibition is competitive. These results suggest the possibility of using the invariant chain as a model for the specific inhibition of Cruzipain in who, i.e. as a potential drug to combat Chagas' disease.

  • Cruzipain the major cysteine proteinase from the protozoan parasite trypanosoma cruzi
    Biological Chemistry, 1997
    Co-Authors: Juan José Cazzulo, Veronika Stoka, Vito Turk
    Abstract:

    Trypanosoma cruzi, the parasitic protozoan which causes the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), Cruzipain. The enzyme belongs to the papain family, but contains, as other CPs from Trypanosomatids, an unusual C-terminal extension. This C-terminal domain contains a number of post-translational modifications and is responsible for the immunodominant antigenic character of Cruzipain in natural human infections. In addition, this domain is probably the cause of most of the microheterogeneities found in natural Cruzipain. Irreversible inhibitors of CPs are able to block the parasite's life cycle at the differentiation steps, suggesting an essential role for CPs for parasite survival, and opening up possibilities of developing new chemotherapeutic agents against Chagas disease based on specific Cruzipain inhibitors.