Análise bioquímica inicial do proteassoma em populações do Trypanosoma cruzi com diferentes fenótipos de resistência ao benzonidazol

AUTOR(ES)
DATA DE PUBLICAÇÃO

2010

RESUMO

Differences in susceptibility to benznidazole (Bz) and nifurtimox (NFX) among populations of Trypanosoma cruzi may explain, at least in part, the differences in efficacy of treatment of Chagas disease. The proteasome has an important role in the degradation of normal proteins, damaged, mutated, or denatured, including the degradation of regulatory proteins of different cellular pathways such as cell cycle, apoptosis and damaged proteins in response to stress. This study intended to examine whether the expression profile and the rate of proteasome-dependent intracellular proteolysis are affected by the phenotype of drug resistance. For this, we used populations of T. cruzi with the same genotype and resistance induced in vitro (17WTS / 17LER) and in vivo (BZS / BZR) and populations with different genotypes, a susceptible (CL) and naturally resistant (Colombian) to benznidazole. Initially, the levels of 20S proteasome, proteasome-like HslV, PA700 and PA26 were analyzed by Western blot. The results showed no effect of the phenotype of the strains on the relative amount of both the 20S proteasome, HslV, and their regulators. Subsequently, the 20S proteasome peptidase activities were assessed using fluorogenic substrates. We observed significant variations in activity chymotrypsin-like, trypsin-like, and caspase-like of proteasome, and only the trypsin-like activity was shown to correlate with the phenotype of resistance to benznidazole. Finally we evaluated the rate of intracellular proteolysis in the same populations in the presence of ATP, ATP and ubiquitin and ATP ubiquitin and MG-132, a classic proteasome 20 and 26S inhibitor. The results suggest that the addition of ubiquitin did not induce a real increase in the rate of proteolysis, in addition, there was an real increase in proteolysis after 90 minutes of induction in the presence of ATP, suggesting the coexistence mechanisms of proteolysis proteasome-dependent and independent of ubiquitin. To corroborate this hypothesis, we assessed the levels of oxidized and ubiquitinated proteins. The results suggest a similar profile of ubiquitinated protein and differential for the oxidized. Together, the present results suggest that the steady state in epimastigote is influenced by drug resistance. Future experiments will be performed to determine which proteins are the natural targets of this pathway.

ASSUNTO(S)

tripanossomo bioquimica resistência drogas

ACESSO AO ARTIGO

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