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Purification and characterization of a trypanothione-glutathione thioltransferase from Trypanosoma cruzi.

ABSTRACT: Although trypanothione [T(S)2] is the major thiol component in trypanosomatidae, significant amounts of glutathione are present in Trypanosoma cruzi. This could be explained by the existence of enzymes using glutathione or both glutathione and T(S)2 as cofactors. To assess these hypotheses, a cytosolic fraction of T. cruzi epimastigotes was subjected to affinity chromatography columns using as ligands either S-hexylglutathione or a non-reducible analogue of trypanothione disulphide. A similar protein of 52 kDa was eluted in both cases. Its partial amino acid sequence indicated that it was identical with the protein encoded by the TcAc2 cDNA previously described [Schoneck, Plumas-Marty, Taibi et al. (1994) Biol. Cell 80, 1-10]. This protein showed no significant glutathione transferase activity but surprisingly catalysed the thiol-disulphide exchange between dihydrotrypanothione and glutathione disulphide. The kinetic parameters were in the same range as those determined for trypanothione reductase toward its natural substrate. This trypanothione-glutathione thioltransferase provides a new target for a specific chemotherapy against Chagas' disease and may constitute a link between the glutathione-based metabolism of the host and the trypanothione-based metabolism of the parasite.

SUBMITTER: Moutiez M

PROVIDER: S-EPMC1135913 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Trypanothione-dependent glyoxalase I in Trypanosoma cruzi.

Project description:The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.

| S-EPMC1652828 | biostudies-literature

Trypanosoma cruzi adenylate cyclase activity. Purification and characterization.

Project description:Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.

| S-EPMC1146537 | biostudies-other

Comparative structural, kinetic and inhibitor studies of Trypanosoma brucei trypanothione reductase with T. cruzi.

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| S-EPMC2789240 | biostudies-literature

Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi.

Project description:BackgroundChagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol-redox metabolite in trypanosomatids.Methods and resultsWe utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36-71% higher resistance to H2O2 (50-1000 μM) and heavy metal (1-500 μM) toxicity. Treatment with TryS inhibitors (5-30 μM) abolished the proliferation and survival advantages against H2O2 pressure in a dose-dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment.ConclusionsTryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti-trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.

| S-EPMC6331241 | biostudies-literature

Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate.

Project description:Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD-orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%.

| S-EPMC1991314 | biostudies-literature

Relevance of Trypanothione Reductase Inhibitors on Trypanosoma cruzi Infection: A Systematic Review, Meta-Analysis, and In Silico Integrated Approach.

Project description:Due to the rudimentary antioxidant defenses in Trypanosoma cruzi, disruptors of redox balance are promising candidates for new antitrypanosomal drugs. We developed an integrated model based on systematic review, meta-analyses, and molecular modeling to evaluate the effect of trypanothione reductase (TR) inhibitors in T. cruzi infections. Our findings indicated that the TR inhibitors analyzed were effective in reducing parasitemia and mortality due to Trypanosoma cruzi infection in animal models. The most investigated drugs (clomipramine and thioridazine) showed no beneficial effects on the occurrence of infection-related electrocardiographic abnormalities or the affinity and density of cardiac ?-adrenergic receptors. The affinity between the tested ligands and the active site of TR was confirmed by molecular docking. However, the molecular affinity score was unable to explain TR inhibition and T. cruzi death in vitro or the antiparasitic potential of these drugs when tested in preclinical models of T. cruzi infection. The divergence of in silico, in vitro, and in vivo findings indicated that the anti-T. cruzi effects of the analyzed drugs were not restricted to TR inhibition. As in vivo studies on TR inhibitors are still scarce and exhibit methodological limitations, mechanistic and highly controlled studies are required to improve the quality of evidence.

| S-EPMC6220389 | biostudies-literature

Trypanosoma cruzi CLB vs Trypanosoma rangeli and Trypanosoma conorhini

Project description:Comparative genomic analysis of T. cruzi CLB vs Trypanosoma rangeli (strains SC, Choachí, C23, H14, R1625 and PIT10) and Trypanosoma conorhini

2023-04-11 | GSE229354 | GEO

Biochemical and genetic characterization of Trypanosoma cruzi N-myristoyltransferase.

Project description:Co- and post-translational N-myristoylation is known to play a role in the correct subcellular localization of specific proteins in eukaryotes. The enzyme that catalyses this reaction, NMT (N-myristoyltransferase), has been pharmacologically validated as a drug target in the African trypanosome, Trypanosoma brucei. In the present study, we evaluate NMT as a potential drug target in Trypanosoma cruzi, the causative agent of Chagas' disease, using chemical and genetic approaches. Replacement of both allelic copies of TcNMT (T. cruzi NMT) was only possible in the presence of a constitutively expressed ectopic copy of the gene, indicating that this gene is essential for survival of T. cruzi epimastigotes. The pyrazole sulphonamide NMT inhibitor DDD85646 is 13-23-fold less potent against recombinant TcNMT than TbNMT (T. brucei NMT), with Ki values of 12.7 and 22.8 nM respectively, by scintillation proximity or coupled assay methods. DDD85646 also inhibits growth of T. cruzi epimastigotes (EC50=6.9 μM), but is ~1000-fold less potent than that reported for T. brucei. On-target activity is demonstrated by shifts in cell potency in lines that over- and under-express NMT and by inhibition of intracellular N-myristoylation of several proteins in a dose-dependent manner. Collectively, our findings suggest that N-myristoylation is an essential and druggable target in T. cruzi.

| S-EPMC3969225 | biostudies-literature

Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins.

Project description:BackgroundTolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen.Methods/principle findingsGenome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis.Conclusion/significanceThis study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.

| S-EPMC6435186 | biostudies-literature

Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi.

Project description:Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.

| S-EPMC1134199 | biostudies-other

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