STAGE SPECIFIC REMODELLING OF PROTOSOAN PARASITES

原生动物寄生虫进入细胞后不久，它们经历了深刻的 形态变化，包括整个细胞器和 细胞骨架。这些大规模的修改必须包括选择性的 细胞质成分的蛋白质分解。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切换的机制 手机节目。乳酸菌素，一种从大豆中分离的微生物代谢物 链霉菌发酵液是一种特异的不可逆链霉菌 蛋白酶体的抑制物。它抑制了细胞的转化 锥虫的锥虫鞭毛虫变成无鞭毛虫样生物体， 疟疾子孢子向肝期的转化和 内阿米巴的囊化。我们建议：1)研究 蛋白酶体和溶酶体在克氏锥虫重塑中的作用2)确定 寄生虫短寿命和长寿命退化的主要场所(S) 3)建立研究ATP-蛋白酶体的无细胞体系。 T.ruzi中的依赖途径；4)记录激活的 寄生虫重塑过程中的泛素(Ub)-蛋白酶体途径；5) 蛋白酶体在超微结构水平上的定位 寄生虫，并验证它们是否与细胞器有关。 6)克隆克氏毛滴虫蛋白酶体的一些α和β亚基； 7)研究lactacystin在鸡传染性支气管炎感染和发展中的作用 哺乳动物细胞中的克氏毛滴虫。这项提议的长期目标是 是为了阐明蛋白酶体依赖的机制， 寄生虫在其生命周期中特定阶段的转化。这些 基础研究可能为工程突变提供理论基础 不能开启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差异 在哺乳动物和寄生虫蛋白酶体通路之间，并导致 成功的治疗方法。

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.