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| Author(s): |
Izaltina Silva Jardim
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2001-03-30 |
| Examining board members: |
Francisco Juarez Ramalho Pinto;
Aldina Maria Prado Barral;
Angela Kaysel Cruz
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| Advisor: | Francisco Juarez Ramalho Pinto |
| Abstract | |
Proteasomes are multicatalitic and multisubunit endopeptidase complexes widely distributed in eukaryotic cells. These enzymes are central proteases in the cytosol and nucleus and are involved in removal of abnormal, misfolded or incorrectly assembled proteins, in processing and degradation of transcriptional regulators in stress response and in the processing of protein antigens. This multicatalytic proteinase complex is composed of a catalytic core, 20S proteasome, which have multiple proteolytic activities (trypsin-like, chymotrypsin-like, peptidylglutamtyl-peptide hydrolyzing, BrAAP and SNAAP). The 20S proteasome associates with the multisubunit complex 19S to produce the 26S proteasome. The 26S proteasome has specificity for ubiquitinylated protein substrates and hydrolyses ATP during proteolysis of ubiquitinylated proteins. In the present work we have purified a 20S form of proteasome from Leishmania chagasi and partially characterized it. The purified 20S proteasome has activity towards fluorogenic substrates that are cleaved by trypsin or chymotrypsin, and is sensitive to lactacystin, a specific inhibitor of the proteasome. We show that the L.chagasi proteasome the trypsin-like activity is higher than the chymotrypsin-like. Therefore the chymotrypsin-like activity is inhibited by lactacystin and the trypsin-like it is only partially inhibited. We show here that lactacystin blocks in vitro L chagasi promastigote replication at a final concentration of 50 µM. To evaluate the effect of proteasome inhibition on the infectivity and intracellular development of L. chagasi, murine macropages were challenged with promastigotes from early stationary phase treated with lactacystin. Infectivity of macrophages was the same in lactacystin-treated parasites as in the untreated ones. Contrarywise, the intracellular development of the parasite is impaired by pretreating promastigotes with lactacystin. These promastigotes were able to infect BALB/c peritoneal macrophages but they did not survive inside macrophages. These data indicate the important role of the proteasomes of L. chagasi promastigotes on the intracellular development and replication in host cells in vitro. (AU) | |