Phosphoinositides in the T. brucei Endomembrane System

描述（申请人提供）：布鲁氏锥虫。是导致人类非洲人类锥虫病（HAT，昏睡病）和牛和其他牲畜纳迦那病的寄生原生动物。这种寄生虫在撒哈拉以南非洲的36个国家流行，在这些国家，采采蝇病媒控制计划是最近该疾病下降的主要因素。尽管如此，如果不治疗，这种疾病总是致命的，而且治疗HAT的少数药物有严重的局限性，如治疗方案延长、费用高、储存要求困难以及包括死亡在内的严重副作用。了解布鲁氏杆菌的基本细胞生物学对于开发更好的药物至关重要，这将有助于实现世卫组织将彻底消除作为公共卫生问题的艾滋病的目标。可用于药物开发的布鲁氏霉生物学的一个方面是内吞货物的贩运。血流形成的锥虫有一个异常流线型的内膜系统，可以非常迅速地吸收和回收或降解物质。这个系统的一些方面已经被探索，例如小的Rab GTPases在调节不同的区室中的必要性。在其他真核生物中，内吞途径的多个步骤是由称为磷酸肌苷的信号脂质调节的。磷脂酰肌醇可以在肌醇环的3、4、5位被磷酸化，形成7个不同的种类。通过激酶和磷酸酶的局部作用，单个磷酸肌苷被限制在特定的膜上。因此，特定磷酸肌苷的富集可以标记功能区域或膜结合区室的身份。下游功能来自随后募集的效应蛋白，这些效应蛋白通过包括PH、PX和FYVE结构域在内的保守结构域与磷酸肌苷结合。PI(3)P和PI(3,5)P2特别涉及内吞细胞器之间的运输。尽管它们在其他系统中很重要，但对布鲁氏体中的磷酸肌苷知之甚少。本提案旨在具体探讨PI(3)P和PI(3,5)P2在布氏虾内吞贩运中的作用。我们打算利用生物传感器通过光镜和电子显微镜来定位这些磷酸肌苷的亚细胞定位。接下来，我们将使用敲低方法来独立破坏PI(3)P和PI(3,5)P2的产生，并定义它们在内吞贩运中的作用。最后，我们将使用亲和纯化方法鉴定PI(3)P和PI(3,5)P2的潜在效应蛋白。这项工作将为布鲁氏体细胞生物学提出许多新的问题，并可能为未来利用这一途径开发药物奠定基础。

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.