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

描述（由申请方提供）：属于锥虫科利什曼原虫属的原生动物寄生虫，导致人类利什曼病，影响全球88个国家的3.5亿人。该寄生虫主要通过携带该生物体感染阶段的雌性白蛉叮咬传播给人类。利什曼病包括三种主要的临床综合征，主要由感染的寄生虫种类决定，它们在地理上各不相同。目前可用的疗法很少，但强烈的副作用和寄生虫抗性限制了它们的有效性。 最近在确定几个锥虫基因组的进展提供了一个宝贵的工具，收集有关这些生物体所使用的分子和生化机制的信息。然而，已经证明很难正确地将特定功能分配给基因编码序列，因为这些寄生虫与它们的高等真核生物对应物之间的序列同源性极低。直到最近，很少有人在人类阶段对利什曼原虫帽结合复合物进行投资，这是启动寄生虫蛋白质合成所必需的。在这个提议中，我们专注于三个翻译启动因子从利什曼原虫在人类阶段：LeishIF 4 E-1，Leish 4 E-IP和LeishIF 3。虽然LeishIF 4 E-1已经成为关键的帽结合蛋白，但假设Leish 4 E-IP和LeishIF 3分别是翻译起始的调节剂和负责在信使RNA的5'端募集小核糖体亚基的支架蛋白。为了研究是否是这种情况，我们将应用生物物理技术的组合，包括NMR光谱学和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的靶点。 这一提议将使我们能够突出人类疟原虫和利什曼原虫翻译起始之间的相似性和差异，并验证利什曼原虫翻译机制作为潜在的药物靶点。这一概念得到了先前在瓦格纳实验室中获得的数据的支持，其中两种人翻译起始因子（eIF 4 E/eIF 4 G）之间的关键相互作用已经在HTP筛选中被靶向，并导致发现具有抗癌活性的化合物。本研究将追求三个具体目标：1。确定LeishIF 4 E-1与其配体cap-4和Leish 4 E-IP复合物的结构。2.表征利什曼原虫帽结合复合物的利什IF 3募集。3.开发HTP筛选试验，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.