Structure and Function of the Trypanosome Flagellar Membrane

描述(申请人提供)：锥虫鞭毛膜的结构和功能在原虫寄生虫克氏锥虫和布氏锥虫的鞭毛中发现了许多密切相关的钙结合蛋白：克氏锥虫的24 kDa鞭毛钙结合蛋白(FCaBP)和布氏锥虫的24 kDa(TB-24)、25 kDa(TB-17)和44 kDa(TB-44)的calfagin蛋白家族。这些蛋白质通过在其氨基末端添加肉豆蔻酸酯和棕榈酸酯来修饰，从而介导定位到鞭毛膜。在之前的资金支持期间，已经确定了钙鞭毛蛋白的各种性质：(I)它们与钙结合并经历钙诱导的构象变化，(Ii)它们在鞭毛膜上的定位依赖于膜甾醇的浓度，(Iii)它们似乎通过钙依赖的结合和伙伴蛋白的假定调节来发挥作用，(Iv)它们具有与钙调蛋白相似但又不同的独特的三维结构，以及(V)它们是鞭毛内运输(IFT)装置的组成部分和/或底物。此外，从这项工作中得到的相关发现包括：(I)鞭毛膜在化学上不同于膜，在脂筏中显示出高度的液体有序性和富集性，(Ii)鞭毛Dna J(Hsp40)蛋白参与了细胞孔的生物发生，(Iii)鞘磷脂的合成是布鲁氏锥虫正常胞质分裂和细胞器分离所必需的。我们对FCaBP和calFlagins的研究已经开始阐明这些蛋白在鞭毛功能和生物发生中的功能，并揭示了鞭毛膜作为一个新的平台来招募双酰化蛋白和参与IFT。来自同事的信息表明，这些基本特性可能是所有纤毛结构(鞭毛和感觉性纤毛)共同的，因此锥虫可能是一个强大的模式系统，一般情况下确定纤毛功能。我们建议在五年的时间里扩展我们对这些蛋白质和鞭毛膜的研究，具体目标如下：(1)确定鞭毛蛋白靶向的分子决定因素。(2)研究鞭毛内转运和鞭毛膜转运之间的相互作用。(3)测定鞭毛膜的脂类成分及其对蛋白质靶向的贡献。 公共卫生相关性：2亿多人面临感染克氏锥虫和布氏锥虫的风险，克氏锥虫是恰加斯病的寄生病原体，布氏锥虫是非洲昏睡病的病原体。现有的治疗锥虫感染的药物效果不是很好，存在疗效差、毒性高的问题。我们的研究旨在确定锥虫细胞生物学的新过程，可用于开发治疗恰加斯病和非洲昏睡病的新药。此外，我们提出的鞭毛-纤毛膜靶向研究与许多人类疾病有关，统称为“纤毛疾病”。纤毛疾病会导致肾脏疾病、糖尿病、癌症、发育障碍和失明，其中一些人类疾病很可能是由我们在这项研究项目中研究的过程中的缺陷引起的。

DESCRIPTION (provided by applicant): Structure and Function of the Trypanosome Flagellar Membrane A number of closely related calcium-binding proteins are found in the flagella of the protozoan parasites Trypanosoma cruzi and Trypanosoma brucei: a 24 kDa flagellar calcium binding protein (FCaBP) of T. cruzi and a family of 24 kDa (Tb-24), 25 kDa (Tb-17), and 44 kDa (Tb-44) calfagin proteins of Trypanosoma brucei. These proteins are modified by the addition of myristate and palmitate at their amino termini, which mediate localization to the flagellar membrane. Various properties of the calflagins have been determined during the previous funding period: (i) they bind calcium and undergo calcium-induced changes in conformation, (ii) their localization to the flagellar membrane is dependent on the concentration of membrane sterols, (iii) they appear to function through the calcium-dependent binding and presumed regulation of partner proteins, (iv) they have unique three-dimensional structures that are similar to but distinct from calmodulin and (v) they are components of and/or substrates for the intraflagellar transport (IFT) apparatus. Additional, related discoveries arising from from this work include the following: (i) the flagellar membrane is chemically distinct from the pellicular membrane and shows a high degree of liquid order and enrichment in lipid rafts, (ii) a flagellar DnaJ (hsp40) protein is involved in the biogenesis of the cytostome and (iii) sphingolipid synthesis is essential for normal cytokinesis and organelle segregation in T. brucei. Our work on FCaBP and the calflagins has begun to illuminate the functions of these proteins in flagellar function and biogenesis and has revealed the flagellar membrane as a novel platform for the recruitment of dually acylated proteins and participation in IFT. Information from colleagues indicate that these basic properties may be common to all ciliary structures (flagella and sensory cilia) and thus the trypanosome may be a powerful model system for the determination of ciliary function generally. We propose to extend our studies of these proteins specifically and the flagellar membrane generally in a five-year research effort having the following Specific Aims: (1) To define the molecular determinants of flagellar protein targeting. (2) To examine the interaction between intraflagellar transport and flagellar membrane trafficking. (3) To determine the lipid composition of the flagellar membrane and its contribution to protein targeting. PUBLIC HEALTH RELEVANCE: Over 200 million people are at risk of infection Trypanosoma cruzi, the parasitic agent of Chagas disease and Trypanosoma brucei, the agent of African sleeping sickness. The drugs available for treating trypanosome infections are not very effective and suffer from poor efficacy and high toxicity. Our studies are aimed at identifying novel process of trypanosome cell biology that can be exploited for the development of new drugs for Chagas disease and African sleeping sickness. Further, our proposed studies of flagellar- ciliary membrane targeting are relevant to many human diseases, collectively known as the "ciliopathies." The ciliopathies cause kidney disease, diabetes, cancer, developmental disorders, and blindness, and some of these human diseases are likely to be caused by defects in the processes we are studying in this research project.