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

描述(申请人提供)：原生动物寄生虫属于锥虫科利什曼原虫属，导致人类患利什曼病，影响全球88个国家的3.5亿人。这种寄生虫主要是在雌性血吸虫沙蝇叮咬后传播给人类的，这种沙蝇携带着生物体的感染期。利什曼病包括三个主要的临床症状，主要由感染寄生虫的种类决定，这些寄生虫在地理上各不相同。目前几乎没有可用的治疗方法，但强烈的副作用和寄生虫耐药性限制了它们的有效性。最近在确定几个锥虫基因组方面的进展为收集关于这些生物使用的分子和生化机制的信息提供了宝贵的工具。然而，由于这些寄生虫与它们的高等真核对应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子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。在这个建议中，我们关注利什曼原虫在人类阶段的三个翻译启动因素：LeishIF4E-1，Leish4E-IP和LeishIF3。虽然LeishIF4E-1已成为关键的帽子结合蛋白，但假设Leish4E-IP和LeishIF3分别是翻译启动的调节器和负责招募信使RNA 5‘端小核糖体亚基的支架蛋白。为了研究这种情况，我们将结合生物物理技术，包括核磁共振光谱和X射线结晶学，以及分子生物学方法，在体外和寄生虫内研究这些因子的结构/功能。一旦我们确定了促进寄生虫翻译启动的不可或缺的相互作用，我们将在高通量(HTP)小分子抑制物筛选中探索这些潜在的靶点。这项建议将使我们能够突出人类CEL和利什曼原虫翻译启动之间的异同，并验证利什曼原虫翻译机制是否为潜在的药物靶标。这一概念得到了瓦格纳实验室先前获得的数据的支持，在该实验室中，两个人类翻译起始因子(eIF4E/eIF4G)之间的关键相互作用已被定位在HTP屏幕上，并导致发现了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LeishIF4E-1及其配体CAP-4和Leish4E-IP的结构。2.鉴定LeishIF3招募到利什曼原虫帽子结合复合体的特征。3.建立HTP筛选方法，寻找抑制利什曼原虫的化合物。

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.