Shortly after protozoan parasites enter cells they undergo profound morphological changes, including restructuring of entire organelles and the cytoskeleton. These massive modifications must include selective proteolysis of cytoplasmic components. In eukaryotic cells most cytoplasmic proteins are degraded in proteasomes, highly conserved organelles found in humans, archaebacteria, plants and yeast. Proteasomes have various functions, including the degradation of regulatory proteins, thus providing a mechanism for rapidly switching cellular programs. Lactacystin, a microbial metabolite isolated from cultured broth of Streptomyces sp., is a specific and irreversible inhibitor of proteasomes. It inhibits the transformation of trypomastigotes of Trypanosoma cruzi into amastigote-like organisms, the transformation of malaria sporozoites into liver stages and encystation of Entamoeba. We propose: 1) to study the roles of proteasomes and lysosomes in remodeling of T. Cruzi; 2) to determine the main site(s) of degradation of the parasite's short and long -lived proteins; 3) to develop a cell-free system to study the ATP-proteasome- dependent pathway in T. Cruzi; 4) to document the activation of the ubiquitin (Ub)- proteasome pathway during parasite remodeling; 5) to determine at the ultrastructural level the localization of proteasomes in the parasites, and verify if they are associated with the cytoskelleton. 6) to clone some alpha and beta subunits of T. Cruzi proteasomes; and 7) to study the effect of lactacystin in the infection and development of T. Cruzi in mammalian cells. The long-term objective of this proposal is to clarify the mechanisms involved in the proteasome-dependent, stage-specific transformation of parasites during their life cycle. These basic studies may provide the rationale for engineering mutant proteasomes that cannot switch on the programs required for parasite infectivity. In addition, our studies are likely to uncover differences between the mammalian and parasite proteasome-pathways, and lead to successful therapies.
