Caracteriza??o das subunidades catal?ticas do proteassoma 20S em cepas de Trypanosoma cruzi suscept?veis e resistentes ao benzonidazol.

A doen?a de Chagas ? causada pelo protozo?rio Trypanosoma cruzi. Segundo a WHO existem 16-18 milh?es de pessoas infectadas com este protozo?rio na Am?rica Latina. Devido ao envolvimento do proteassoma na biologia celular do T. cruzi e a baixa efici?ncia das drogas utilizadas no tratamento de indiv?duos com doen?a de Chagas, entendemos que a caracteriza??o das subunidades catal?ticas do proteassoma 20S, poder? ajudar no desenvolvimento de futuros quimioter?picos contra essa doen?a. Neste sentido, este trabalho mostra a caracteriza??o molecular e bioqu?mica das subunidades catal?ticas do proteassoma 20S em um padr?o de cepas do T. cruzi com sensibilidade e resist?ncia induzida ?in vitro? ao benzonidazol (17WTS e 17LER), sensibilidade e resist?ncia selecionada ?in vivo? ao benzonidazol (BZS e BZR) e cepas naturalmente sens?veis e resistentes a essa droga (CL e Colombiana, respectivamente). As an?lises de localiza??o cromoss?mica utilizando a t?cnica de eletroforese em gel de campo alternado, mostraram v?rios perfis de hibridiza??o, sugerindo que estes genes n?o est?o associados com o fen?tipo de resist?ncia do T. cruzi a drogas. Al?m disso, utilizando a t?cnica de Southern blot, mostramos que estes genes est?o presentes em no m?nimo duas c?pias por genoma hapl?ide do parasito. A express?o desses genes foi avaliada atrav?s da t?cnica de Northern blot e os n?veis do proteassoma 20S identificados utilizando Western blot e anticorpos contra subunidades alfa. Podemos constatar que enquanto os n?veis de mRNA n?o variaram entre as cepas, uma maior quantidade de proteassoma 20S foi detectado nas cepas sens?veis (17WTS, BZS e CL) quando comparado as cepas resistentes (17LER, BZR e Colombiana). Al?m disso, tamb?m n?o detectamos ac?mulo de conjugados poliubiquitinados, sugerindo que o processo de poliubiquitina??o e degrada??o de prote?nas intracelulares dependente do proteassoma 26S s?o fortemente regulados no padr?o de cepas de T. cruzi estudadas. A medida da atividade peptid?sica utilizando substratos espec?ficos para as subunidade Beta-1, -2 e -5, mostraram uma atividade peptid?sica principal semelhante ? tripsina em todas as cepas analisadas, entretanto, as cepas 17WTS e 17LER tamb?m apresentaram uma alta atividade peptid?sica semelhante ? quimotripsina. Os resultados obtidos at? o momento, sugerem que modifica??es p?straducionais s?o mais eficientes na fun??o do proteassoma 20S do que a regula??o ao n?vel transcricional ou p?s-transcricional, na forma epimastigota do T. cruzi.Chagas disease is caused by Trypanosoma cruzi. According to WHO, in Latin America, 16-18 million people is infected with this protozoan. Due the involvement of the proteasome in the cellular biology of the T. cruzi and the low efficiency of the drugs used in the individuals treatment with Chagas disease, we believed that the characterization of the catalytic subunits of the 20S proteasome, would help in the development of future drugs that could be used in the therapeutics of the Chagas disease. This work shows the molecular and biochemical characterization of the catalytic subunits of the 20S proteasome in a T. cruzi population with in vitro induced (17WTS and 17LER), in vivo (BZS and BZR) and strains naturally sensitive and resistant to benznidazole (CL and Colombian). Chromosomal localization by using the pulse field gel electrophoresis and hybridization profiles with specific probes showed several pattern in the different strains, suggesting that these genes are not associated with the drugs phenotype resistance in T. cruzi. Southern blot analyses showed that these genes are present in at least two copies for genome haploid of the parasite. The expression of those genes was evaluated through of Northern blot analyses and the levels of the 20S proteasome identified using Western blot and antibodies against alpha subunits. The mRNA levels did not vary among the strains, but a larger level of 20S proteasome were detected in the sensitive strains (17WTS, BZS and CL) when compared the resistant strains (17LER, BZR and Colombian). The absent of accumulation of poly-ubiquitin conjugated, suggest that the poly-ubiquitination pathway and degradation by 26S proteasome, are strongly regulated in the strains of T. cruzi studied. The measure of the peptidase activity using specific substrate for the subunit Beta-1, -2 and -5, showed that the main proteolytic activity is tripsin-like in all strains. Moreover, the strains 17WTS and 17LER also presented a high quimotripsin-like activity. These results obtained until the moment suggest that, in the epimastigote development stage of the T. cruzi the post-translation modifications are more efficient by the regulation of 20S proteasome function than the transcriptional or posttranscriptional modification