Efeito do PSI na inibição do proteassoma 20S de Trypanosoma cruzi

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

In recent decades the proteases of protozoa parasites have emerged as potential therapeutic targets. One of them, the 20S proteasome, is a self-compartimentalizing protease that plays a crucial role in cell biology regulating several cellular processes including proteins degradation, cell-cycle control, and antigens presentation. In this sense, the main objective of this work was to evaluate the pattern of gene expression as well as peptidase activities of this protease and the impact of proteasome inhibition by PSI, a potent and reversible inhibitor of chymotrypsin-like activity on the proliferation in vitro and in the development of experimental infection of Swiss mice inoculated with the Y, Berenice-62 e Berenice-78 strains of Trypanosma cruzi. The analysis by RT-PCR, using oligonucleotides specific to the catalitic subunits of proteasome 20S (similar to caspase, tripsin and chymotripinsin), total RNA of the epimastigote forms from the Y, Be-62 and Be-78 strains, and from the gene hypoxantine-guanine phosphoribosytransferase as a normalizer, showed equivalent levels of mRNA to these subunits. On the other hand, the levels of proteasome analyzed by Western immunoblotting using an antibody to alfa subunit (1, 2, 3, 5, 6 and 7) were higher for strain Be-78 when compared to the strains Y and Be-62. These differences in the proteasome amount in these strains were confirmed for measurement of proteolytic activities associated to these subunits using specific fluorogenic substrates and an enriched fraction of the proteasome obtained from the same strains. Our results showed that the main proteasome activity is trypsin-like activity in epimastigote forms of T. cruzi, suggesting that post- transcriptional regulation may modulates the proteolytic activity in T. cruzi. For an evaluation of the proteasome inhibition in parasite replication ability we used epimastigote, in the presence of 0033μM to 50μM of the PSI. It was observed a reduction of the proliferation of 90% for the Y and Be-78 strains when compared to control groups. However, the Be-62 strain presented 57% of proliferation reduction in the concentration of 0,033μM and 73% in the concentration of 0,083μM. These results suggest that a functional proteasome is essential for the replication of epimastigote forms. Subsequently, these parasites were analyzed by the scanning electron microscopy. We observed that the flagellum was detached from the parasite cell body; roughness appeared on the parasites cell membranes, besides several others body cell deformations. These results suggest that the proteasome inhibition led to unbalanced of turnover of the proteins involved in the maintenance of the cell. Finally the effect of proteasome inhibition in the development of experimental infection of Chagas Disease was examined. Initially, young (15/18g), female, outbred Swiss mice were intraperitoneally inoculated with 5x103 trypomastigotes of the Y, Be-62 and Be-78 strains in the presence of 100uM of PSI. A reduction of 68, 42 and 73% on the peaks of parasitemia of these strains were corroborating the initial biochemical differences observed between the proteasome of these strains. The analysis of the tissue parasitism inflammation in the spleen, liver, heart and skeletal muscle showed that with Y strain, the level degree of parasitism of mice infected with tripomastigote pre-incubated with PSI,. However, higher tissue parasitism and intense inflammatory processes were observed in all tissue infected with Be-62 strain. This pattern was not observed for Be- 78 strain that maintained the same tissue parasitism and inflammatory process when compared to the control group. Taken together, our results suggest that the proteasome is essential for the maintenance of the T. cruzi life cycle, since that the protease inhibition during epimastigote division, in addition to cell cycle blocking induces morphological changes, while the inhibition of infective forms leads to changes in the development of the experimental infection.

ASSUNTO(S)

proteassoma 20s trypanosoma cruzi biologia molecular protozoários inibição

ACESSO AO ARTIGO

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