The translation apparatus of Leishmania: from basic analysis to pursuit of novel

描述（由申请人提供）：属于锥虫科利什曼原虫属的原生动物寄生虫，在人类中引起利什曼病，影响全球88个国家的3.5亿人。这种寄生虫主要是在雌性白蛉叮咬后传播给人类的，雌性白蛉携带着这种生物的感染阶段。利什曼病包括三种主要的临床综合征，主要由感染寄生虫的种类决定，这些寄生虫在地理上有所不同。目前可用的治疗方法很少，但强烈的副作用和寄生虫耐药性限制了它们的有效性。最近在确定几种锥虫基因组方面取得的进展为收集有关这些生物体使用的分子和生化机制的信息提供了宝贵的工具。然而，由于这些寄生虫与其高等真核生物之间的序列同源性极低，因此很难正确地将特定功能分配给基因编码序列。直到最近，在人类阶段的利什曼原虫帽结合复合体上投入的努力很少，这是启动寄生虫蛋白质合成所必需的。在本文中，我们重点研究了人类阶段主要利什曼原虫的三个翻译起始因子：LeishIF4E-1、Leish4E-IP和LeishIF3。虽然LeishIF4E-1已成为关键的帽结合蛋白，但假设Leish4E-IP和LeishIF3分别是翻译起始的调节因子和负责在信使rna的5'端招募小核糖体亚基的支架蛋白。为了研究这种情况是否属实，我们将结合生物物理技术，包括核磁共振波谱和x射线晶体学，以及分子生物学方法来研究这些因子在体外和寄生虫体内的结构/功能。一旦我们确定了促进寄生虫翻译起始的不可或缺的相互作用，我们将在高通量（HTP）小分子抑制剂筛选中探索这些潜在靶点。这一建议将使我们能够突出人类细胞和利什曼原虫寄生虫翻译起始的异同，并验证利什曼原虫翻译机制作为潜在的药物靶点。这一概念得到了Wagner实验室先前获得的数据的支持，其中两个人类翻译起始因子（eIF4E/eIF4G）之间的关键相互作用已在HTP筛选中被靶向，并导致发现具有抗癌活性的化合物。该研究将追求三个具体目标：1。确定LeishIF4E-1配合物及其配体cap-4和Leish4E-IP的结构。2. 描述利什曼原虫帽结合复合体的LeishIF3招募。3. 开发HTP筛选法以寻找利什曼原虫抑制化合物。

DESCRIPTION (provided by applicant): Protozoan parasites that belong to the genus Leishmania from the Trypanosomadae family, causes Leishmaniasis in humans, which affects 350 million people in 88 countries worldwide. The parasites are principally transmitted to humans following the bite of female phlebotomine sandflies, which carry the infectious stage of the organism. Leishmaniasis comprise three major clinical syndromes principally determined by the species of infecting parasites, which vary geographically. Few therapies are currently available, but strong side effects and parasite resistance limit their effectiveness. The recent advancements in determining several trypanosomatid genomes provided an invaluable tool to gather information regarding the molecular and biochemical mechanisms used by these organisms. However, it has proven difficult to correctly assign specific functions to gene coding sequences because the sequence homology between these parasites and their higher eukaryotic counterparts is extremely low. Until recently, little effort was invested in the Leishmania cap-binding complexes in the human stage, which are required to initiate protein synthesis in parasites. In this proposal, we focus on three translation initiation factors from Leishmania major in the human stage: LeishIF4E-1, Leish4E-IP and LeishIF3. While LeishIF4E-1 has emerged as the crucial cap-binding protein, it is hypothesized that Leish4E-IP and LeishIF3 are, respectively, a regulator of translation initiation and a scaffold protein responsibl for recruiting the small ribosomal subunit at the 5' end of messenger RNAs. To investigate whether this is the case, we will apply a combination of biophysical techniques, including NMR spectroscopy and X-ray crystallography, and molecular biology approaches to study the structure/function of these factors in vitro and within parasites. Once we determine indispensable interactions that promote translation initiation in the parasite, we will explore these potential targets in high-throughput (HTP) small molecule inhibitor screens. This proposal will allow us to highlight similarities and differences between translation initiation in human cels and Leishmania parasites, and validate the Leishmania translation machinery as a potential drug target. This concept is backed by data obtained previously in the Wagner lab where a crucial interaction between two human translation initiation factors (eIF4E/eIF4G) has been targeted in a HTP screen and led to the discovery of compounds with anti-cancer activity. The research will pursue three specific aims: 1. Determine the structure of the LeishIF4E-1 complex with its ligand cap-4 and Leish4E-IP. 2. Characterize the LeishIF3 recruitment to the Leishmania cap-binding complex. 3. Develop assays for HTP screens in search of Leishmania inhibitory compounds.