RESUMO
Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH+ and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16α-brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In Trypanosoma cruzi, immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16α of the cyclopentane moiety yielded more potent T. cruzi G6PDH inhibitors than the corresponding ß-substituted analogues. For the 16α brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit µM EC50 against infective T. brucei and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH+-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.
Assuntos
Glucosefosfato Desidrogenase/metabolismo , Estágios do Ciclo de Vida , Esteroides/farmacologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimento , Androsterona/química , Androsterona/farmacologia , Sítios de Ligação , Citosol/enzimologia , Desidroepiandrosterona/química , Desidroepiandrosterona/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/química , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Modelos Moleculares , Oxirredução , Reprodutibilidade dos Testes , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
Several ortho-naphthoquinones (o-NQs) have trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Previously, we demonstrated that the aldo-keto reductase from this parasite (TcAKR) reduces o-NQs, such as ß-lapachone (ß-Lap) and 9,10-phenanthrenequinone (9,10-PQ), with concomitant reactive oxygen species (ROS) production. Recent characterization of TcAKR activity and expression in two T. cruzi strains, CL Brener and Nicaragua, showed that TcAKR expression is 2.2-fold higher in CL Brener than in Nicaragua. Here, we studied the trypanocidal effect and induction of several death phenotypes by ß-Lap and 9,10-PQ in epimastigotes of these two strains. The CL Brener strain was more resistant to both o-NQs than Nicaragua, indicating that greater TcAKR activity is unlikely to be a major influence on o-NQ toxicity. Evaluation of changes in ROS production, mitochondrial membrane potential, phosphatidylserine exposure and monodansylcadaverine labelling evidenced that ß-Lap and 9,10-PQ induce different death phenotypes depending on the combination of drug and T. cruzi strain analysed. To study whether TcAKR participates in o-NQ activation in intact parasites, ß-Lap and 9,10-PQ trypanocidal effect was next evaluated in TcAKR-overexpressing parasites. Only ß-Lap was more effective and induced greater ROS production in TcAKR-overexpressing epimastigotes than in controls, suggesting that TcAKR may participate in ß-Lap activation.
Assuntos
Aldo-Ceto Redutases/metabolismo , Naftoquinonas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Aldo-Ceto Redutases/genética , Animais , Chlorocebus aethiops , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fenótipo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Células VeroRESUMO
Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC50) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH- and NADH-dependent reductases.
Assuntos
Nitroimidazóis/metabolismo , Trypanosoma cruzi/enzimologia , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases , Doença de Chagas/tratamento farmacológico , Doença de Chagas/metabolismo , DNA de Protozoário/genética , Nitroimidazóis/farmacologia , Nitrorredutases/genética , Nitrorredutases/metabolismo , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologiaRESUMO
Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.
Assuntos
Doença de Chagas/parasitologia , Ciclinas/metabolismo , Trypanosoma cruzi/genética , Animais , Ciclo Celular , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Expressão Gênica , Hemaglutininas/genética , Hemaglutininas/metabolismo , Orthomyxoviridae/genética , Fosforilação , Proteólise , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão , Trypanosoma cruzi/citologia , Trypanosoma cruzi/metabolismoRESUMO
In eukaryotes, an oscillating network of protein kinase activities drives the order and timing of the cell cycle progression. Complexes formed by cyclins associated to cyclin-dependent kinases (CDKs) are the central components of this network. Cyclins act as the activating subunits and their abundance is regulated by different mechanisms in order to promote or prevent kinase activity. Protein synthesis, proteasomal degradation and/or differential subcellular compartmentalization modulate cyclin expression levels along the cell cycle. We describe in this work the characterization of Trypanosoma cruzi Cyclin 2 (TcCYC2), which contributes to a better understanding of the cell cycle regulation in this protozoan parasite. We found TcCYC2 exhibited cyclin function in a yeast complementation assay and over-expression of hemagglutinin tagged TcCYC2-HA rendered shorter duplication times and smaller cell sizes in the epimastigote form of the parasite. Analysis of synchronized cultures showed that over-expression of TcCYC2-HA altered the timing epimastigotes pass through G2/M boundary or cytokinesis. Taken together, our results showed that TcCYC2 is a functional cyclin whose over-expression modifies the dynamics of the cell cycle as well as the morphology of epimastigote forms of T. cruzi, suggesting it plays an important role in the cell cycle regulation machinery.
Assuntos
Ciclo Celular/fisiologia , Ciclinas/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma cruzi/fisiologia , Sequência de Aminoácidos , Ciclinas/química , Ciclinas/genética , Citoplasma/química , Citometria de Fluxo , Expressão Gênica , Teste de Complementação Genética , Filogenia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência , Transfecção , Trypanosoma cruzi/citologia , Trypanosoma cruzi/genéticaRESUMO
SUMOylation is a relevant protein post-translational modification in eukaryotes. The C terminus of proteolytically activated small ubiquitin-like modifier (SUMO) is covalently linked to a lysine residue of the target protein by an isopeptide bond, through a mechanism that includes an E1-activating enzyme, an E2-conjugating enzyme, and transfer to the target, sometimes with the assistance of a ligase. The modification is reversed by a protease, also responsible for SUMO maturation. A number of proteins have been identified as SUMO targets, participating in the regulation of cell cycle progression, transcription, translation, ubiquitination, and DNA repair. In this study, we report that orthologous genes corresponding to the SUMOylation pathway are present in the etiological agent of Chagas disease, Trypanosoma cruzi. Furthermore, the SUMOylation system is functionally active in this protozoan parasite, having the requirements for SUMO maturation and conjugation. Immunofluorescence analysis showed that T. cruzi SUMO (TcSUMO) is predominantly found in the nucleus. To identify SUMOylation targets and get an insight into their physiological roles we generated transfectant T. cruzi epimastigote lines expressing a double-tagged T. cruzi SUMO, and SUMOylated proteins were enriched by tandem affinity chromatography. By two-dimensional liquid chromatography-mass spectrometry a total of 236 proteins with diverse biological functions were identified as potential T. cruzi SUMO targets. Of these, metacaspase-3 was biochemically validated as a bona fide SUMOylation substrate. Proteomic studies in other organisms have reported that orthologs of putative T. cruzi SUMOylated proteins are similarly modified, indicating conserved functions for protein SUMOylation in this early divergent eukaryote.