His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening

Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohé L (1997) Biol Chem 378: 827–836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 1 l fermentation broth of A ₆₀₀ 2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b(+)/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k _cat of 1.7 s⁻¹ and limiting K _m values of 51.8 μM and 1.7 μM for t-butyl hydroperoxide and CfTXN2H6, respectively. Received: 3 September 1999 / Accepted: 1 November 1999     

Oligomerization dynamics and functionality of Trypanosoma cruzi cytosolic tryparedoxin peroxidase as peroxidase and molecular chaperone

BackgroundTrypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin that plays an important role in coping with host cell oxidative response during the infection process, for which it has been described as a virulence factor.MethodsFour residues corresponding to c-TXNPx catalytic and solvent-exposed cysteines were individually mutated to serine by site-specific mutagenesis. Susceptibility to redox treatments and oligomeric dynamics were investigated by western-blot and gel filtration chromatography. Chaperone and peroxidase activities were determined.ResultsIn this study we demonstrated that c-TXNPx exists as different oligomeric forms, from decameric to high molecular mass aggregates. Moreover, c-TXNPx functions as a dual-function protein acting both as a peroxidase and as a molecular chaperone. Its chaperone function was shown to be independent of the presence of catalytic cysteines, even in the reduced and decameric forms, although it is enhanced when the protein is overoxidized leading to the formation of high molecular mass aggregates.Conclusionsc-TXNPx has chaperone activity which does not depend on the redox state. c-TXNPx does not undergo the dimer-decamer transition in the oxidized state described for other peroxiredoxins. Overoxidized c-TXNPx exists as different oligomeric forms from decamer to high molecular mass aggregates which are in a very slow dynamic equilibrium. The non-catalytic C57 residue may have a role in the maintenance of the decameric form, but seems not to have an alternative C_P and C_R role.General significanceThis study provides novel insights into some key aspects of the oligomerization dynamics and function of c-TXNPx.     

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain

Trypanosoma cruzi is the etiological agent of Chagas’ disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T. cruzi strain Y solved at 2.2 Å resolution. DHODH is a flavin mononucleotide containing enzyme, which catalyses the oxidation of l-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Genetic studies have shown that DHODH is essential for T. cruzi survival, validating the idea that this enzyme can be considered an attractive target for the development of antichagasic drugs. In our work, a detailed analysis of T. cruzi DHODH crystal structure has allowed us to suggest potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design.     

Crystal structure of chagasin, the endogenous cysteine-protease inhibitor from Trypanosoma cruzi

Trypanosoma cruzi chagasin belongs to a recently discovered family of cysteine protease inhibitors found in lower eukaryotes and prokaryotes but not in mammals. Chagasin binds tightly to cruzain, the major lysosomal T. cruzi cysteine protease, involved with infectivity and survival of the parasite in mammalian host cells. In the scope of a project to characterize proteins diferentially expressed during T. cruzi metacyclogenesis, we have determined the crystal structure of chagasin, which is now the first X-ray structure of a chagasin-like cysteine protease inhibitor to be reported. The structure was solved by the SIRAS method and refined at 1.7A resolution and a comparison with the two NMR structures available revealed some differences in the loops involved in binding to cysteine proteases. The highly flexible loop 4 could be entirely modeled and residues 29-33 from loop 2 form a 3(10)-helix structure that may be important to stabilize the loop conformation. Chagasin crystal structure was docked to the highest resolution structure available of cruzain and a model of chagasin-cruzain interaction was analyzed. The knowledge of the chagasin crystal structure may contribute to the elucidation of the molecular mechanism involved in the inhibition of cruzain and other T. cruzi cysteine proteases.     

Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.     

Molecular characterization and interactome analysis of Trypanosoma cruzi tryparedoxin 1

Trypanosoma cruzi tryparedoxin 1 (TcTXN1) is an oxidoreductase belonging to the thioredoxin superfamily, which mediates electron transfer between trypanothione and peroxiredoxins. In trypanosomes TXNs, and not thioredoxins, constitute the oxido-reductases of peroxiredoxins. Since, to date, there is no information concerning TcTXN1 substrates in T. cruzi, the aim of this work was to characterize TcTXN1 in two aspects: expression throughout T. cruzi life cycle and subcellular localization; and the study of TcTXN1 interacting-proteins. We demonstrate that TcTXN1 is a cytosolic and constitutively expressed protein in T. cruzi. In order to start to unravel the redox interactome of T. cruzi we designed an active site mutant protein lacking the resolving cysteine, and validated the complex formation in vitro between the mutated TcTXN1 and a known partner, the cytosolic peroxiredoxin. Through the expression of this mutant protein in parasites with an additional 6xHis-tag, heterodisulfide complexes were isolated by affinity chromatography and identified by 2-DE/MS. This allowed us to identify fifteen TcTXN1 proteins which are involved in two main processes: oxidative metabolism and protein synthesis and degradation. Our approach led us to the discovery of several putatively TcTXN1-interacting proteins thereby contributing to our understanding of the redox interactome of T. cruzi.     

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp₂₃₃^•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.     

The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.     

Crystal structure of the Trypanosoma cruzi trypanothione reductase·mepacrine complex

The three-dimensional structure of the complex between Trypanosoma cruzi trypanothione reductase (TR) (EC 1.6.4.8) and the antiparasitic drug mepacrine (quinacrine) has been solved at 2.9 Å resolution. Mepacrine is a competitive inhibitor of TR but does not affect human glutathione reductase (GR), a closely related host enzyme. Of particular importance for inhibitor binding are four amino acid residues in the disulfide substrate-binding site of TR that are not conserved in human GR, namely, Glu-18 (Ala-34 in GR), Trp-21 (Arg-37), Ser-109 (Ile-113), and Met-113 (Asn-117). The acridine ring of mepacrine is fixed at the active site close to the hydrophobic wall formed by Trp-21 and Met-113. Specific pairwise interactions between functional groups of the drug and amino acid side chains include the ring nitrogen and Met-113, the chlorine atom and Trp-21, and the oxymethyl group and Ser-109. The alkylamino chain of mepacrine points into the inner region of the active site and is held in position by a solvent-mediated hydrogen bond to Glu-18. The structure of the complex shows for the first time the atomic interactions between TR and an inhibitory ligand. This is a crucial step towards the rational design of inhibitors that might be suited as drugs against Chagas' disease. © 1996 Wiley-Liss, Inc.     

Biochemical characterization of a low-affinity arginine permease from the parasite Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, uses arginine for several metabolic processes, including energy reserves management. In the present work, a novel low-affinity arginine transport system has been studied. Maximum velocity (97 pmol min(-1) per 10(7) cells), and an estimate for the apparent Km value (350 microM) of this arginine transporter, were 6-fold and 80-fold higher respectively, when compared with the previously described high-affinity arginine transport system. This transport activity seems to be H+ -mediated, presents a broad specificity by other amino acids such as methionine, and is regulated along the parasite growth curve and life cycle.     

Trypanosoma brucei and Trypanosoma cruzi tryparedoxin peroxidases catalytically detoxify peroxynitrite via oxidation of fast reacting thiols

Macrophage activation is one of the hallmarks observed in trypanosomiasis, and the parasites must cope with the resulting oxidative burden, which includes the production of peroxynitrite, an unusual peroxo-acid that acts as a strong oxidant and trypanocidal molecule. Cytosolic tryparedoxin peroxidase (cTXNPx) has been recently identified as essential for oxidative defense in trypanosomatids. This peroxiredoxin decomposes peroxides using tryparedoxin (TXN) as electron donor, which in turn is reduced by dihydrotrypanothione. In this work, we studied the kinetics of the reaction of peroxynitrite with the different thiol-containing components of the cytosolic tryparedoxin peroxidase system in T. brucei (Tb) and T. cruzi (Tc), namely trypanothione, TXN, and cTXNPx. We found that whereas peroxynitrite reacted with dihydrotrypanothione and TbTXN at moderate rates (7200 and 3500 m(-1) s(-1), respectively, at pH 7.4 and 37 degrees C) and within the range of typical thiols, the second order rate constants for the reaction of peroxynitrite with reduced TbcTXNPx and TccTXNPx were 9 x 10(5) and 7.2 x 10(5) m(-1) s(-1) at pH 7.4 and 37 degrees C, respectively. This reactivity was dependent on a highly reactive cTXNPx thiol group identified as cysteine 52. Competition experiments showed that TbcTXNPx inhibited other fast peroxynitrite-mediated processes, such as the oxidation of Mn(3+)-porphyrins. Moreover, steady-state kinetic studies indicate that peroxynitrite-dependent TbcTXNPx and TccTXNPx oxidation is readily reverted by TXN, supporting that these peroxiredoxins would be not only a preferential target for peroxynitrite reactivity but also be able to act catalytically in peroxynitrite decomposition in vivo.     

Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin.     

Calcineurin B of the human protozoan parasite Trypanosoma cruzi is involved in cell invasion

During Trypanosoma cruzi cell invasion, signal transduction pathways are triggered in parasite and host cells, leading to a rise in intracellular Ca(2+) concentration. We posed the question whether calcineurin (CaN), in particular the functional regulatory subunit CaNB, a Ca(2+)-binding EF-hand protein, was expressed in T. cruzi and whether it played a role in cell invasion. Here we report the cloning and characterization of CL strain CaNB gene, as well as the participation of CaNB in cell invasion. Treatment of metacyclic trypomastigotes (MT) or tissue-culture trypomastigotes (TCT) with the CaN inhibitors cyclosporin or cypermethrin strongly inhibited (62-64%) their entry into HeLa cells. In assays using anti-phospho-serine/threonine antibodies, a few proteins of MT were found to be dephosphorylated in a manner inhibitable by cyclosporin upon exposure to HeLa cell extract. The phosphatase activity of CaN was detected by a biochemical approach in both MT and TCT. Treatment of parasites with antisense phosphorothioate oligonucleotides directed to TcCaNB-CL, which reduced the expression of TcCaNB and affected TcCaN activity, resulted in approximately 50% inhibition of HeLa cell entry by MT or TCT. Given that TcCaNB-CL may play a key role in cell invasion and differs considerably in its primary structure from the human CaNB, it might be considered as a potential chemotherapeutic target.     

Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.     

Crystal structure of S-adenosyl-L-homocysteine hydrolase from the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of malarial parasite resistant to conventional drug therapy has stimulated searches for antimalarials with novel modes of action. S-Adenosyl-L-homocysteine hydrolase (SAHH) is a regulator of biological methylations. Inhibitors of SAHH affect the methylation status of nucleic acids, proteins, and small molecules. P.falciparum SAHH (PfSAHH) inhibitors are expected to provide a new type of chemotherapeutic agent against malaria. Despite the pressing need to develop selective PfSAHH inhibitors as therapeutic drugs, only the mammalian SAHH structures are currently available. Here, we report the crystal structure of PfSAHH complexed with the reaction product adenosine (Ado). Knowledge of the structure of the Ado complex in combination with a structural comparison with Homo sapiens SAHH (HsSAHH) revealed that a single substitution between the PfSAHH (Cys59) and HsSAHH (Thr60) accounts for the differential interactions with nucleoside inhibitors. To examine roles of the Cys59 in the interactions with nucleoside inhibitors, a mutant PfSAHH was prepared. A replacement of Cys59 by Thr results in mutant PfSAHH, which shows HsSAHH-like nucleoside inhibitor sensitivity. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.     

The short non-coding transcriptome of the protozoan parasite Trypanosoma cruzi

The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16-61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95-98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3' end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes.     

In vitro activity of Etanidazole against the protozoan parasite Trypanosoma cruzi

We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exerting its anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.     

Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi

Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism.