Biochemical and Functional Analysis of Trypanin

锥虫蛋白酶的生化和功能分析

• 批准号: 6637754
• 负责人: KENT L HILL
• 金额: $ 30.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6637754
• 关键词: Trypanosoma brucei rhodesiense; cell motility; cytoskeletal proteins; flagellum; gel electrophoresis; host organism interaction; immunoelectron microscopy; intracellular parasitism; laboratory mouse; mass spectrometry; pathologic process; protein protein interaction; protein structure function; trypanosomiasis; yeast two hybrid system

DESCRIPTION (provided by the applicant): African trypanosomes (Trypanosoma brucei) are protozoan parasites that cause a fatal disease known as African sleeping sickness in humans and related diseases in animals. These diseases have devastating health and economic consequences. These parasites are transmitted to the bloodstream of a mammalian host by the bite of a tsetse fly vector. Cell motility is suspected to play a pivotal role in disease pathogenesis and parasite development, but it has not previously been possible to investigate this relationship directly. We have isolated a trypanosome mutant that is incapable of directional cell motility but, surprisingly, is not paralyzed. This mutant lacks a protein called "trypanin" and the broad, long-term objectives of this proposal are to exploit trypanin (-) mutants and recombinant trypanin proteins as tools to advance our understanding of the mechanism and biology of flagellum based cell motility in T. brucei. Trypanin (-) mutants will also be used to examine the role of cell motility in disease pathogenesis in the mammalian host and parasite development and within the tsetse fly. The first two specific aims are to determine the biochemical properties of trypanin and a trypanin-related protein (TRP 1) and to determine whether these proteins interact with one another. Trypanin is part of a unique flagellum attachment complex that is required for T. brucei cell motility and the next specific aim is to employ a combined biochemical and genetic approach to isolate and identify additional components of this complex. Finally, because trypanin (-) mutants have an actively beating flagellum and are not paralyzed, they provide a unique opportunity to specifically examine the role of directional cell motility in the mammalian and insect hosts. Thus, the fourth specific aim is to determine whether loss of trypanin alters the course of infection in the mammalian host and/or blocks parasite development within the tsetse fly vector. T. brucei flagellum attachment complexes offer unique opportunities to investigate fundamental aspects of cytoskeleton-membrane interactions. Thus, in addition to providing information relevant to parasitic disease, these studies are expected to have broad relevance to eukaryotic cell biology.

描述（由申请人提供）：非洲锥虫（Trypanosoma brucei）是原生动物寄生虫，会导致人类致命的非洲昏睡病和动物的相关疾病。这些疾病对健康和经济造成毁灭性的后果。这些寄生虫通过采采蝇媒介的叮咬传播到哺乳动物宿主的血液中。细胞运动被怀疑在疾病发病机制和寄生虫发育中发挥着关键作用，但以前不可能直接研究这种关系。我们分离出一种锥虫突变体，它不能进行定向细胞运动，但令人惊讶的是，它并没有瘫痪。该突变体缺乏一种名为“台盼酸”的蛋白质，该提案的广泛、长期目标是利用台盼酸 (-) 突变体和重组台盼酸蛋白作为工具，以增进我们对布氏锥虫鞭毛细胞运动机制和生物学的理解。锥虫蛋白 (-) 突变体还将用于检查细胞运动在哺乳动物宿主和寄生虫发育以及采采蝇疾病发病机制中的作用。前两个具体目标是确定胰蛋白酶和胰蛋白酶相关蛋白 (TRP 1) 的生化特性，并确定这些蛋白是否彼此相互作用。锥虫蛋白酶是布氏锥虫细胞运动所需的独特鞭毛附着复合物的一部分，下一个具体目标是采用生化和遗传学相结合的方法来分离和鉴定该复合物的其他成分。最后，由于台盼酸 (-) 突变体具有活跃跳动的鞭毛并且不会麻痹，因此它们提供了一个独特的机会来专门研究定向细胞运动在哺乳动物和昆虫宿主中的作用。因此，第四个具体目标是确定胰蛋白酶的缺失是否会改变哺乳动物宿主的感染过程和/或阻止采采蝇载体内寄生虫的发育。 T. brucei 鞭毛附着复合物为研究细胞骨架-膜相互作用的基本方面提供了独特的机会。因此，除了提供与寄生虫病相关的信息外，这些研究预计与真核细胞生物学具有广泛的相关性。