STAGE SPECIFIC REMODELLING OF PROTOSOAN PARASITES

原生动物寄生虫进入细胞后不久， 形态学变化，包括整个细胞器的重组， 细胞骨架 这些大规模的修改必须包括选择性的 细胞质组分的蛋白水解。 在真核细胞中， 细胞质蛋白在蛋白酶体中降解，高度保守 在人类、古细菌、植物和酵母中发现的细胞器。 蛋白酶体具有多种功能，包括降解 调节蛋白，从而提供了一种快速转换的机制， 细胞程序 Lactacystin是一种从大肠杆菌中分离出的微生物代谢产物 链霉菌属的培养液，是一种特定的不可逆的 蛋白酶体抑制剂。 它抑制了 将克氏锥虫的锥鞭毛体转化为无鞭毛体样生物， 疟疾子孢子向肝期的转化， 内阿米巴包囊。 我们建议：1）研究 蛋白酶体和溶酶体在T. Cruzi; 2）确定 寄生虫短寿命和长寿命降解的主要部位 蛋白质; 3）开发一个无细胞系统来研究ATP-蛋白酶体- 依赖途径。Cruzi; 4）记录 寄生虫重构过程中的泛素（Ub）-蛋白酶体途径; 5） 在超微结构水平上确定蛋白酶体在 寄生虫，并验证他们是否与cytochelleton相关。 6)克隆T细胞的α和β亚基。Cruzi蛋白酶体;以及 7)探讨lactacystin在大肠埃希氏菌感染和发展中的作用， T.哺乳动物细胞中的Cruzi。 本提案的长期目标 是为了阐明蛋白酶体依赖性， 寄生虫在其生命周期中的阶段特异性转化。 这些 基础研究可以为工程突变体提供理论基础 不能启动寄生虫所需程序的蛋白酶体 传染性 此外，我们的研究可能会发现 在哺乳动物和寄生虫蛋白酶体途径之间，并导致 成功的治疗

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.