STAGE SPECIFIC REMODELLING OF PROTOSOAN PARASITES

原虫寄生虫的阶段特异性重塑

• 批准号: 2837426
• 负责人: Victor Nussenzweig
• 金额: $ 31.68万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2837426
• 关键词: DNA; SDS polyacrylamide gel electrophoresis; Trypanosoma cruzi; affinity chromatography; carbohydrate structure; cell migration; enzyme activity; enzyme inhibitors; enzyme linked immunosorbent assay; exo alpha sialidase; flow cytometry; gene expression; high performance liquid chromatography; host organism interaction; intracellular parasitism; ion exchange chromatography; laboratory mouse; laboratory rabbit; membrane proteins; molecular cloning; nucleic acid structure; oligosaccharides; protein structure function; sialate

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.

原生动物寄生虫进入细胞后不久，他们经历了深刻的 形态学变化，包括整个细胞器和 细胞骨架。 这些巨大的修改必须包括选择性 细胞质成分的蛋白水解。 在真核细胞中大多 细胞质蛋白在蛋白酶体中降解，高度保守 在人类，考古细菌，植物和酵母中发现的细胞器。 蛋白酶体具有各种功能，包括降解 调节蛋白，因此提供了快速切换的机制 蜂窝程序。 乳酸，一种从中分离的微生物代谢物 链霉菌的培养汤是一种特定且不可逆的 蛋白酶体的抑制剂。 它抑制了 锥虫锥虫的锥虫变成类似amastigote的生物， 疟疾子孢子转化为肝脏阶段和 Entamoeba的百科全书。 我们建议：1）研究 Cruzi重塑的蛋白酶体和溶酶体； 2）确定 寄生虫短而长的降解的主要地点 蛋白质； 3）开发一个无细胞的系统来研究ATP-蛋白酶体 T. Cruzi的依赖途径； 4）记录激活 泛素（UB） - 寄生虫重塑期间的蛋白酶体途径； 5）到 在超微结构一级确定蛋白酶体的定位 寄生虫，并验证它们是否与Cytoskelleton相关。 6）克隆克鲁齐蛋白酶体的一些α和β亚基；和 7）研究乳酸在感染和发育中的影响 哺乳动物细胞中的T. cruzi。 该提议的长期目标 是为了阐明蛋白酶体依赖性的机制， 寄生虫在生命周期中的特定阶段转化。 这些 基础研究可能为工程突变体提供基本原理 无法打开寄生虫所需程序的蛋白酶体 感染力。 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pathways之间，并导致 成功的疗法。