Phosphoinositides in the T. brucei Endomembrane System

性状（由申请方提供）：布氏锥虫亚种是一种寄生原生动物，可引起人类非洲锥虫病（HAT，昏睡病）和牛和其他牲畜的长角线虫病。这种寄生虫在撒哈拉以南非洲的36个国家流行，采采蝇病媒控制计划是最近这种疾病下降的主要因素。尽管如此，该疾病在未经治疗时总是致命的，并且可用于治疗HAT的少数药物具有严重的局限性，例如延长的治疗方案、费用、难以储存的要求和包括死亡在内的严重副作用。了解T.布鲁氏菌对开发更好的药物至关重要，这将有助于实现世卫组织彻底消除HAT这一公共卫生问题的目标。T.可用于开发药物的布鲁氏菌生物学是内吞货物的贩运。血流形式的锥虫具有异常流线型的内膜系统，允许非常快速地摄取和回收或降解材料。该系统的一些方面已经被探索，例如小Rab GTP酶在调节不同隔室中的必要性。在其他真核生物中，内吞途径中的多个步骤由称为磷酸肌醇的信号脂质调节。磷脂酰肌醇可以在肌醇环的3、4和5位磷酸化形成7种不同的种类。通过激酶和磷酸酶的局部作用，单个磷酸肌醇被限制在特定的膜上。因此，特定磷酸肌醇的富集可以标记功能区域或膜结合区室的身份。下游功能由效应蛋白的随后募集产生，所述效应蛋白通过保守结构域（包括PH、PX和FYVE结构域）结合磷酸肌醇。PI（3）P和PI（3，5）P2特别涉及内吞细胞器之间的运输。尽管磷酸肌醇在其他系统中很重要，但对T.布鲁塞。本文旨在具体探讨PI（3）P和PI（3，5）P2在T中的作用。布鲁氏菌内吞运输。我们打算使用生物传感器映射这些磷酸肌醇的亚细胞定位，通过光学和电子显微镜。接下来，我们将使用敲低方法来独立地破坏PI（3）P和PI（3，5）P2的产生，并确定它们在内吞运输中的作用。最后，我们将使用亲和纯化方法来鉴定PI（3）P和PI（3，5）P2的潜在效应蛋白。这项工作将在T.布鲁氏菌细胞生物学，并可能为利用该途径的未来药物开发奠定基础。

DESCRIPTION (provided by applicant): Trypanosoma brucei ssp. are parasitic protozoa responsible for causing human African trypanosomiasis (HAT, sleeping sickness) in humans and nagana in cattle and other livestock. The parasite is endemic to 36 countries in sub-Saharan Africa, where tsetse fly vector control programs have been the major factors in the recent decline of the disease. Still, the disease is invariably fatal when untreated, and the few drugs available to treat HAT have serious limitations such as prolonged treatment regimen, expense, difficult storage requirements, and serious side effects including death. Understanding the basic cell biology of T. brucei is essential to the development of better drugs that will contribute to the WHO goal of total elimination of HAT as a public health problem. One aspect of T. brucei biology that could be exploited for the development of drugs is the trafficking of endocytic cargo. Bloodstream form trypanosomes have an unusually streamlined endomembrane system that allows for very rapid uptake and recycling or degradation of material. Some aspects of this system have been explored, such as the necessity for small Rab GTPases in regulating different compartments. In other eukaryotes, multiple steps in the endocytic pathway are regulated by signaling lipids called phosphoinositides. Phosphatidylinositol can be phosphorylated on positions 3,4, and 5 of the inositol ring to form seven distinct species. Through the localized action of kinases and phosphatases, individual phosphoinositides are restricted to specific membranes. Enrichment of specific phosphoinositides can therefore mark the identity of functional regions or membrane-bound compartments. Downstream functions arise from the subsequent recruitment of effector proteins, which bind to phosphoinositides through conserved domains including PH, PX, and FYVE domains. PI(3)P and PI(3,5)P2 have particularly been implicated in trafficking between endocytic organelles. Despite their importance in other systems, very little is known about phosphoinositides in T. brucei. This proposal seeks to specifically explore the roles of PI(3)P and PI(3,5)P2 in T. brucei endocytic trafficking. We intend to use biosensors to map the subcellular localization of those phosphoinositides by light and electron microscopy. Next, we will use a knockdown approach to disrupt production of PI(3)P and PI(3,5)P2 independently and define their roles in endocytic trafficking. Finally, we will use an affinity purification method t identify potential effector proteins of PI(3)P and PI(3,5)P2. This work will raise many new questions in T. brucei cell biology and potentially lay the groundwork for future drug development exploiting this pathway.