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Query: UNIPROT:P06889 (Mol)
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Phylogenetic analysis of 18S rRNA sequences from the families Trypanosomatidae and Bodonidae (Eugelenozoa: Kinetoplastida) was conducted using a variety of methods. Unlike previous analyses using unrooted trees and/or smaller numbers of sequences, the analysis did not support monophyly of the genus Trypanosoma, which includes the major human parasites T. cruzi (cause of Chagas' disease) and T. brucei (cause of African sleeping sickness). The section Salivaria of the genus Trypanosoma fell outside a cluster that includes the section Stercoraria of the genus Trypanosoma, along with members of the genera Leishmania, Endotrypanum, Leptomonas, Herpetomonas, Phytomonas, Crithidia, and Blastocrithidia. The phylogenetic analysis also indicated that the genera Bodo, Cryptobia, Leptomonas, Herpetomonas, Crithidia, and Blastocrithidia are polyphyletic. The results suggested that parasitism of vertebrates has probably arisen independently a number of times within the Trypanosomatidae.
Mol Biol Evol 2003 Apr
PMID:Phylogeny of Trypanosomatidae and Bodonidae (Kinetoplastida) based on 18S rRNA: evidence for paraphyly of Trypanosoma and six other genera. 1267 43

The process of Trypanosoma cruzi metacyclogenesis involves the transformation of noninfective epimastigotes into metacyclic trypomastigotes, which are the pathogenic form. The analysis of stage-specific genes during T. cruzi metacyclogenesis may provide insight into the mechanisms involved in the regulation of gene expression in trypanosomatids. It may also improve the understanding of the mechanisms responsible for the pathology of Chagas disease, and could lead to the identification of new targets for chemotherapy of this disease. We have demonstrated that during metacyclogenesis the expression of several genes is controlled at the translational level by an alternative regulatory mechanism. This mechanism may involve the mobilization of mRNA to the translation machinery. We have been using self-made T. cruzi microarrays to investigate the role of polysomal mobilization in modulating gene expression during metacyclogenesis.
Genet Mol Res 2003 Mar 31
PMID:Stage-specific gene expression during Trypanosoma cruzi metacyclogenesis. 1291 12

Trypanosoma cruzi, the flagellate protozoan which is the causative agent of the American trypanosomiasis, Chagas disease has carboxypeptidase activity. The enzyme has been purified to protein homogeneity, and shown to be a lysosomal monomeric glycoprotein with a molecular mass of about 54kDa. The enzyme has an optimum acidic pH (4.5 with furyl acryloyl-Phe-Phe as substrate), is highly specific for hydrophobic C-terminal amino acid residues, and is strongly inhibited by 3,4-dichloroisocoumarin (IC(50) value 0.3 microM). The enzyme is encoded by a number of genes arrayed in head-to-tail tandems; one of these genes has been cloned and sequenced. Sequence comparisons indicate that the enzyme belongs to the C group of serine carboxypeptidases, within the S10 serine peptidase family, and shows the higher similarity to plant and yeast enzymes. The residues involved in catalysis and most of those involved in substrate binding are conserved in the T. cruzi enzyme as well as 8 out of 10 Cys residues known to be involved in disulfide bridges in the yeast enzyme. This is the first report of an S10 family enzyme in trypanosomatids. The presence of serine carboxypeptidases is not restricted to T. cruzi, being possibly a general character of trypanosomatids.
Mol Biochem Parasitol 2003 Sep
PMID:Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi. 1296 8

Trypanosoma cruzi, the causative agent of Chagas disease, remains a significant public health concern throughout South and Central America. Although much is known about immune control of T. cruzi and in particular the importance of recognition of parasite-infected cells, relatively little is known about the target antigens of these protective immune responses. For instance, few of the genes expressed in the intracellular amastigote stage have been identified. To gain insight into the molecular events, at the level of mRNA abundance, involved in this critical point in the parasite life-cycle, we used DNA microarrays of 4400 sequences from T. cruzi ORF-selected and random, genomic sequencing libraries to determine relative mRNA abundances in trypomastigotes and developing amastigotes. Results from six hybridizations using independently generated parasite samples consistently identified 60 probes that detected genes upregulated within 2h after extracellular trypomastigotes were induced, in vitro, to differentiate into amastigotes. Sequence analysis from these 60 probes identified 14 known and 25 novel T. cruzi genes. The general direction of regulation was confirmed by quantitative RT-PCR for seven of the array-identified, amastigote upregulated, known genes. This work demonstrates the feasibility of computational and microarray approaches to gene discovery in T. cruzi, an organism for which a fully assembled and annotated genome sequence is not yet available and in which control of transcription initiation is believed to be absent. Moreover, this work is the first report of amastigote up regulation for 38 genes, thus expanding considerably the pool of genes known to be upregulated in this important yet poorly-studied stage of the T. cruzi life-cycle.
Mol Biochem Parasitol 2003 Sep
PMID:Microarray profiling of gene expression during trypomastigote to amastigote transition in Trypanosoma cruzi. 1296 12

Eighteen month old spontaneously hypertensive rats (SHR-rats) showed myocardial dysfunction and autoantibodies directed against the beta1-adrenoceptor similarly as known in human dilated cardiomyopathy or Chagas' disease. The agonist-like antibodies were able to activate the beta1-adrenoceptor mediated signal transduction cascade in cultured rat cardiomyocytes and induced a long-lasting stimulatory effect resulting in a harmful adrenergic overdrive. The antibodies recognized an epitope of the second extracellular loop of the beta1-adrenoceptor identical to that epitope identified in Chagas' disease. In conclusion, our assumption is supported that old SHR-rat are an useful animal model for investigating the role of anti-beta1-adrenoceptor antibodies in the induction of human cardiomyopathy.
Mol Cell Biochem 2003 Sep
PMID:Functional and structural characterization of anti-beta1-adrenoceptor autoantibodies of spontaneously hypertensive rats. 1457 6

The neuraminidase/trans-sialidase of Trypanosoma cruzi, the agent of Chagas' disease, promotes differentiation and survival of growth factor-deprived neuronal and glial cells. To gain further insights into the possible neuroprotection of this parasite-derived counterpart of neurotrophic factors (PDNF), we sought to determine whether it mimics growth factors in a cellular model of neurodegenerative diseases. Ascertaining cell viability by morphology, vital dye exclusion, mitochondrial reducing function, and absence of DNA fragmentation, we show here that PDNF rescues from death two dopaminergic neuronal cell lines and one differentiated immortalized mesencephalic neurons exposed to the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and its toxic metabolite, 1-methyl-4-phenylpyridinium (MPP+), both widely used in models of Parkinson's disease. We further show that PDNF promoted survival at concentrations comparable to bona fide growth factors in a MAPK/Erk activation-dependent manner. PDNF also strongly suppresses the overproduction of MPTP-induced reactive oxygen species (ROS), and the activation of both initiator caspase-9 and effector caspase-3. This down-regulation of ROS and caspases explains, at least in part, the PDNF-induced salvaging of the dopaminergic cells from the Parkinsonism-promoting toxin, confirming the novel and striking functional mimicry by the trypanosome neuraminidase of host growth factors in a cellular model of neurodegeneration.
Brain Res Mol Brain Res 2003 Nov 06
PMID:PDNF, a human parasite-derived mimic of neurotrophic factors, prevents caspase activation, free radical formation, and death of dopaminergic cells exposed to the Parkinsonism-inducing neurotoxin MPP+. 1459 29

Trypanosoma cruzi infection, transmitted by insect vectors or blood transfusions, is an important cause of morbidity and mortality in many Latin American countries. Treatments are toxic and frequently ineffective in curing patients with chronic manifestations of the infection (Chagas disease). Potentially exploitable chemotherapeutic targets of T. cruzi are enzymes of the sterol biosynthesis pathway. In particular, the P450 enzyme, lanosterol 14alpha-demethylase, has been implicated as the target of azole antifungal drugs that have potent anti-T. cruzi activity. In the work reported here, the T. cruzi lanosterol 14alpha-demethylase (Tc14DM) gene was cloned by degenerate PCR. The gene was found to be expressed in both insect and mammalian life-cycle stages of the parasite. Tc14DM was able to complement the function of the homologous gene in yeast (erg11) as demonstrated by restored ergosterol production in an erg11-deficient yeast strain. When the yeast strain was co-transfected with the P450 reductase gene from Trypanosoma brucei, the amount of ergosterol production was increased, indicating that the endogenous yeast P450 reductase was an inefficient partner with Tc14DM. Heterologous expression of Tc14DM in the baculovirus/Sf9 system resulted in a 52kDa product. The protein was observed to have the characteristic absorbance spectra of a P450 enzyme. A typical Type II binding spectrum was produced when the imidazole compound, ketoconazole, was mixed with the Tc14DM, demonstrating that ketoconazole binds the enzyme.
Mol Biochem Parasitol 2003 Dec
PMID:Cloning and analysis of Trypanosoma cruzi lanosterol 14alpha-demethylase. 1459 67

Drug design strategies based on Comparative Molecular Field Analysis (CoMFA) have been used to predict the activity of new compounds. The major advantage of this approach is that it permits the analysis of a large number of quantitative descriptors and uses chemometric methods such as partial least squares (PLS) to correlate changes in bioactivity with changes in chemical structure. Because it is often difficult to rationalize all variables affecting the binding affinity of compounds using CoMFA solely, the program GRID was used to describe ligands in terms of their molecular interaction fields, MIFs. The program VolSurf that is able to compress the relevant information present in 3D maps into a few descriptors can treat these GRID fields. The binding affinities of a new set of compounds consisting of 13 coumarins, for one of which the three-dimensional ligand-enzyme bound structure is known, were studied. A final model based on the mentioned programs was independently validated by synthesizing and testing new coumarin derivatives. By relying on our knowledge of the real physical data (i.e., combining crystallographic and binding affinity results), it is also shown that ligand-based design agrees with structure-based design. The compound with the highest binding affinity was the coumarin chalepin, isolated from Rutaceae species, with an IC50 value of 55.5 microM towards the enzyme glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) from glycosomes of the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. The proposed models from GRID MIFs have revealed the importance of lipophilic interactions in modulating the inhibition, but without excluding the dependence on stereo-electronic properties as found from CoMFA fields.
J Comput Aided Mol Des
PMID:3D QSAR studies on binding affinities of coumarin natural products for glycosomal GAPDH of Trypanosoma cruzi. 1463 21

As an intracellular parasite, Trypanosoma cruzi is exposed to reactive oxygen species. The study of the proteins involved in the hydroperoxide detoxification cascade, tryparedoxin peroxidase included, may lead to the development of a more specific chemotherapy for Chagas'disease. In this work, the involvement of TcCPX in T. cruzi resistance to oxidant-mediated injury was investigated. At low concentrations of hydrogen peroxide cell proliferation was stimulated and parasites increased their resistance to sub-lethal doses of H2O2 (100 microM) if previously treated with a non-toxic concentration of H2O2 (20 microM). Incubation of cells with different H2O2 concentrations induced a dose-dependent increase in TcCPX levels, as detected by Western blotting analysis. The increase in TcCPX levels in the presence of high H2O2 concentrations possibly reflects an initial cell attempt to promote detoxification. To further demonstrate TcCPX involvement in T. cruzi response to oxidative stress, TcCPX overexpressing cells were produced. Compared to pTEX transformed cells, pTEX-TcCPX mutant cells showed a higher mRNA level (129%), without a corresponding increase in protein production (11%), suggesting that regulation of gene expression occurs at post-transcriptional levels. Furthermore, parasite treatment with 200 microM H2O2 for 30 min, led to an increase in mRNA (192%), but not in protein levels (24%). Higher mRNA levels correlated to protein levels were observed only after longer H2O2 incubation periods (1-2 h), suggesting that protein translation occurs accordingly to parasite needs. An increase in glucose-6-phosphate dehydrogenase activity was observed in pTEX-TcCPX epimastigotes that could provide cells with extra reducing power and a higher growth index.
Mol Biochem Parasitol 2004 Jan
PMID:Trypanosoma cruzi response to the oxidative stress generated by hydrogen peroxide. 1466 10

Enzymes that salvage 6-oxopurines, including hypoxanthine phosphoribosyltransferases (HPRTs), are potential targets for drugs in the treatment of diseases caused by protozoan parasites. For this reason, a number of high-resolution X-ray crystal structures of the HPRTs from protozoa have been reported. Although these structures did not reveal why HPRTs need to form dimers for catalysis, they revealed the existence of potentially relevant interactions involving residues in a loop of amino acid residues adjacent to the dimer interface, but the contributions of these interactions to catalysis remained poorly understood. The loop, referred to as active-site loop I, contains an unusual non-proline cis-peptide and is composed of residues that are structurally analogous with Leu67, Lys68, and Gly69 in the human HPRT. Functional analyses of site-directed mutations (K68D, K68E, K68N, K68P, and K68R) in the HPRT from Trypanosoma cruzi, etiologic agent of Chagas' disease, show that the side-chain at position 68 can differentially influence the K(m) values for all four substrates as well as the k(cat) values for both IMP formation and pyrophosphorolysis. Also, the results for the K68P mutant are inconsistent with a cis-trans peptide isomerization-assisted catalytic mechanism. These data, together with the results of structural studies of the K68R mutant, reveal that the side-chain of residue 68 does not participate directly in reaction chemistry, but it strongly influences the relative efficiencies for IMP formation and pyrophosphorolysis, and the prevalence of lysine at position 68 in the HPRT of the majority of eukaryotes is consistent with there being a biological role for nucleotide pyrophosphorolysis.
J Mol Biol 2004 Jan 23
PMID:Interactions at the dimer interface influence the relative efficiencies for purine nucleotide synthesis and pyrophosphorolysis in a phosphoribosyltransferase. 1469 88


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