A NOVEL MICROTUBULE-ASSOCIATED PROTEIN IN TRYPANOSOMES

描述(改编自申请摘要)：非洲锥虫是 导致非洲锥虫病的原虫寄生虫，这是一种致命的疾病 造成毁灭性的健康和经济后果。中国的长期目标是 拟议的研究旨在增进我们对以下两个重要组成部分的理解 这些病原体的细胞结构：微管细胞骨架， 鞭毛袋是一种特殊的细胞器，起着至关重要的作用 在宿主和寄生虫的交流中。这两个结构都是 寄生虫在它们的哺乳动物宿主和昆虫媒介中存活，并 被认为是治疗干预的主要目标 锥虫病。我们已经证明了一种名为TLTF的蛋白质定位于 锥虫前鞭毛囊膜的胞浆面 蛋白质提取物和一种相关的人类蛋白质与微管相关 哺乳动物细胞。拟议研究的前两个目标是利用 结合体内和体外微管结合试验确定 这两种蛋白是真正的微管结合蛋白，并且 确定微管是否有助于靶向TLTF 锥虫。这项研究的另一个目的是利用生化和分子 用生物学方法确定两种反病毒之间的关系 TLTF与昆虫形式的锥虫体内存在的交叉反应蛋白。 其中一种蛋白的表达表现出戏剧性的发展 调节，表明该蛋白具有发育阶段特异性 功能。对TLTF靶向结构域的点突变分析表明 鞭毛袋中特定的蛋白质-蛋白质相互作用是 对于TLTF的正确靶向很重要。因此，这项研究的另一个目的是 使用免疫共沉淀和亲和捕获树脂来确定 TLTF与其他锥体蛋白相互作用，即某些TLTF “结合伙伴”可能是鞭毛袋的组成部分。除了……之外 有助于我们理解一种具有重要医学意义的细胞生物学 病原体，与TLTF密切相关的特性和以前的 未知的人类蛋白质使得这些研究的发现很可能 将在其他系统中具有广泛的生物学相关性。

DESCRIPTION (adapted from application abstract): African trypanosomes are protozoan parasites that cause African trypanosomiasis, a deadly disease with devastating health and economic consequences. The long-term goal of the proposed research is to advance our understanding of two vital components of the cellular architecture of these pathogens: the microtubule cytoskeleton, and the flagellar pocket, a specialized organelle that plays a critical role in host-parasite communication. Both of these structures are required for parasite survival in their mammalian host and insect vector, and are considered to be prime targets for therapeutic intervention in trypanosomiasis. We have shown that a protein called TLTF is localized to the cytoplasmic face of the anterior flagellar pocket membrane in trypanosome protein extracts and a related human protein associates with microtubules in mammalian cells. The first two aims of the proposed research are to utilize combined in vivo and in vitro microtubule binding assays to determine whether these two proteins are bona fide microtubule-binding proteins, and to determine whether or not microtubules contribute to targeting of TLTF in trypanosomes. Another aim of this research is to use biochemical and molecular biological approaches to determine whether the relationship between two anti- TLTF cross-reactive proteins that are present in insect-form trypanosomes. Expression of one of these proteins exhibits dramatic developmental regulation, suggesting that this protein has developmental stage-specific functions. Analysis of point mutations in the TLTF targeting domain suggests that specific protein-protein interactions at the flagellar pocket are important for proper targeting of TLTF. Hence, another aim of this research is to use co-immunoprecipitation and affinity capture resins to determine whether TLTF interacts with other trypanosomal proteins, i.e, whether some TLTF `binding partners` may be components of the flagellar pocket. In addition to contribution to our understanding of the cell biology of a medically important pathogen, the closely related properties of TLTF and a previously uncharacterized human protein make it likely that findings from these studies will have broad biological relevance in other systems.