Chagasin, the endogenous cysteine-protease inhibitor of Trypanosoma cruzi, modulates parasite differentiation and invasion of mammalian cells (original) (raw)
Related papers
Infection and Immunity, 2004
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...
Current Medicinal Chemistry, 2009
This review aims to present different aspects related to cruzipain, one of the most important proteins of the etiological agent of Chagas disease that has been extensively studied in the last two decades, including all the particularities of the molecule as well as to highlight its participation in multiple relevant functions of the parasite to favour the cell invasion phenomena, to facilitate host tissues proteolytic degradation and to trigger the evasion mechanism from host immune response. Cruzipain has been related with parasite metabolism and identified as both an important candidate for vaccine development and for trypanocidal drug design. We have reported for the first time that this enzyme is a sulfated glycoprotein. Indeed, the sulfated oligosaccharides are main targets for immune responses and are involved in tissue damage in mice immunized in absence of infection contributing to get deeper into the knowledge of the molecule composition and helping to elucidate its role in the infection and/or pathogenesis of the disease. A whole view including all the aspects related to the major cysteine proteinase of Trypanosoma cruzi studied so far including recent advances as proteinase, antigen and glycoprotein will be discussed.
Molecular and Biochemical Parasitology, 2001
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 mM), R-Dm28 epimastigotes exhibited 13-fold (IC 50 20 mM) 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 www.elsevier.com/locate/parasitology Abbre6iations: Abz: S 0 1 6 6 -6 8 5 1 ( 0 0 ) 0 0 2 3 7 -1 48 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.
European Journal of Biochemistry, 1997
Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected 7: cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandenily repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life-cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non-transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteiiie proteinase inhibitor Cbz-Phe-Phe-CHN, (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.
Molecular and Biochemical Parasitology, 2000
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 mM), R-Dm28 epimastigotes exhibited 13-fold (IC 50 20 mM) 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 www.elsevier.com/locate/parasitology Abbre6iations: Abz: S 0 1 6 6 -6 8 5 1 ( 0 0 ) 0 0 2 3 7 -1 48 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.
Kininogenase Activity by the Major Cysteinyl Proteinase (Cruzipain) from Trypanosoma cruzi
Journal of Biological Chemistry, 1997
The major isoform of Trypanosoma cruzi cysteinyl proteinase (cruzipain) has generated Lys-bradykinin (Lys-BK or kallidin), a proinflammatory peptide, by proteolysis of kininogen. The releasing of this peptide was demonstrated by mass spectrometry, radioimmunoassay, and ileum contractile responses. The kinin-releasing activity was immunoabsorbed selectively by monoclonal antibodies to the characteristic COOH-terminal domain of cruzipain. To determine the hydrolysis steps that account for the kininogenase activity of cruzipain, we synthesized a fluorogenic peptide (o-aminobenzoyl-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 389-Ser 390-Ser-Arg-Ile-NH 2) based on the sequence Leu 373 to Ile 393 of the human high molecular weight kininogen. The hydrolysis products from this peptide were isolated by high performance liquid chromatography, and Lys-BK was characterized as the major released kinin by mass spectrometry. Intramolecularly quenched fluorogenic peptides spanning the Met 379-Lys 380 and Arg 389-Ser 390 bradykinin-flanking sequences were then used to assess the substrate specificity requirements of the parasite-derived protease compared with two COOH-terminal truncated recombinant isoforms (cruzain and cruzipain 2). In contrast to the high catalytic efficiency of parasite-derived cruzipain, the recombinant proteinases cleaved the bradykinin-flanking sites at markedly different rates. In addition, we also demonstrated that cruzipain activates plasmatic prekallikrein, which would be a second and indirect way of the parasite protease to release bradykinin.
Experimental Parasitology, 2002
Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as a 2 -macroglobulin (a2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of a2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both a-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native a2-M and PZP, respectively; (ii) limited proteolysis of bait region in a2-M and PZP, and (iii) covalent binding of cruzipain to PZP and a 2 -M. Conformational and structural changes experimented by a-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-a-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, a2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, a-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human a-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.
The Biochemical journal, 1996
A panel of intramolecularly quenched fluorogenic substrates containing the conserved QVVA and LVG inhibitory sequences of cystatin inhibitors was used to describe the specificity of the major cysteine proteinase of Trypanosoma cruzi (cruzipain or cruzain). This approach was based on the observations that: (1) cruzipain is strongly inhibited by chicken cystatin and rat T-kininogen, two representative members of cystatin families 2 and 3; (2) the QVVA- and LVG-containing substrates are specifically hydrolysed by papain-like proteinases; and (3) the cystatin-like motifs are similar to the proteolytically sensitive sequences in cruzipain that separate the pro-region and/or the C-terminal extension from the catalytic domain. Specificity constants (kcat/Km) were determined and compared with those of mammalian cathepsins B and L from rat liver lysosomes. Cruzipain and the mammalian proteinases cleaved cystatin-derived substrates at the same site, but their specificities differed significan...
The role of conserved residues of chagasin in the inhibition of cysteine peptidases
Febs Letters, 2008
We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10-100-fold increases in the K i for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.