Phosphoinositides in the T. brucei Endomembrane System

描述(申请人提供)：布氏锥虫。是导致人类患非洲锥虫病(HAT、昏睡病)以及牛和其他牲畜感染Nagana的寄生原虫。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，采采蝇媒介控制计划一直是该疾病最近下降的主要因素。尽管如此，这种疾病在不治疗的情况下总是致命的，可用于治疗这种疾病的为数不多的药物具有严重的局限性，如治疗方案延长、费用、难以储存的要求以及包括死亡在内的严重副作用。了解布氏毛滴虫的基本细胞生物学对于开发更好的药物至关重要，这些药物将有助于世卫组织彻底消除HAT这一公共卫生问题的目标。布鲁氏毛滴虫生物学中可用于药物开发的一个方面是内吞货物的贩运。血流形式的锥虫有一个非同寻常的流线型内膜系统，允许非常快速地吸收和回收或降解材料。这个系统的一些方面已经被探索，例如小的Rab GTP酶在调节不同的隔室中的必要性。在其他真核生物中，内吞途径中的多个步骤受到称为磷脂酰肌醇的信号脂类的调节。磷脂酰肌醇可以在肌醇环的第3、4和5位被磷酸化，形成7个不同的物种。通过激酶和磷酸酶的局部作用，个别的肌醇磷脂被限制在特定的膜上。因此，特定的磷脂酰肌醇的浓缩可以标记功能区域或膜结合室的特性。下游功能产生于随后的效应器蛋白的招募，这些效应器蛋白通过包括PH、PX和FYVE结构域的保守结构域与磷脂酰肌醇结合。PI(3)P和PI(3，5)P2尤其与胞内细胞器之间的转运有关。尽管它们在其他系统中很重要，但人们对布氏毛滴虫中的肌醇磷脂知之甚少。本研究旨在探讨PI(3)P和PI(3，5)P2在布氏毛滴虫胞内转运中的作用。我们打算使用生物传感器通过光学和电子显微镜来定位这些磷脂酰肌醇。接下来，我们将使用一种敲除方法来单独干扰PI(3)P和PI(3，5)P2的生产，并确定它们在细胞内转运中的作用。最后，我们将使用亲和纯化的方法来鉴定PI(3)P和PI(3，5)P2的潜在效应蛋白。这项工作将在布鲁氏毛滴虫细胞生物学中提出许多新的问题，并可能为未来利用这一途径进行药物开发奠定基础。

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.