Purine Nucleoside/Nucleobase Transporters in Leishmania

描述(申请人提供)：这个项目将探索嘌呤核苷和碱基转运蛋白(NTS)在利什曼原虫生物学中的作用。到目前为止，所有被检查的寄生原虫都无法合成嘌呤，必须从宿主那里抢救这些必要的营养物质。挽救的第一步是NTS通过寄生虫质膜摄取嘌呤核苷或核苷酸碱基。有三个具体目标。第一个特异性目的是研究杜氏利什曼原虫核苷转运蛋白LdNT1.1的结构和功能，以阐明核苷转运蛋白在利什曼原虫和其他寄生原虫中的功能。这个特定目标的第一个组成部分是分析18个先前发现的LdNT1.1错义突变，这些突变在运输中没有功能或改变了底物特异性。这些突变体将被检测那些因为它们不能结合底物而没有功能的突变体，以及那些仍然能够结合底物但不能将其转移到质膜上的突变体。这些实验将确定在底物识别和结合或底物转运中发挥关键作用的氨基酸，从而确定促进其两个基本机制功能的渗透酶成分。特定目的1的第二个组成部分是定义LdNT1.1的胞外和胞内门控残基，LdNT1.1是功能上重要的残基，它们相互作用，分别关闭向内和向外的渗透孔。第二个特定目标需要创建LmaNT4的结构模型，LmaNT4是一种来自重大乳杆菌的独特的碱基转运蛋白，在pH值为5时功能最佳，寄生虫驻留在哺乳动物宿主中的巨噬细胞吞噬酶体的细胞内pH，以及可能控制这一不寻常的最适pH的关键残基的遗传解剖。第三个具体目标是研究L.donovani用来适应嘌呤饥饿的分子机制。初步证据表明，在嘌呤饥饿时，LdNT2蛋白显著上调，而其mRNA水平不受影响。计划中的实验将确定这一调控是否在翻译、蛋白质周转或两者兼而有之的水平上起作用。我们还将研究嘌呤限制对其他LdNTs的潜在调节作用。 公共卫生相关性：这项建议的总体目标是详细研究引起致命内脏利什曼病的寄生虫杜氏利什曼原虫中嘌呤核苷转运体的结构、功能和调节。嘌呤的摄取对利什曼原虫和所有寄生原虫都是必不可少的，这突显了嘌呤核苷/碱基转运体在这些微生物中的关键作用，这些转运体还介导了几种药物或实验药物对利什曼原虫或其他寄生原虫的摄取。嘌呤核苷/碱基转运体在寄生虫存活中的重要作用进一步表明，选择性地抑制这些转运体可以被用来开发治疗寄生虫病的新药。

DESCRIPTION (provided by applicant): This project will probe the role of purine nucleoside and nucleobase transporters (NTs) in the biology of Leishmania parasites. All parasitic protozoa examined to date are unable to synthesize purines and must salvage these essential nutrients from their hosts. The first step in salvage is the uptake of purine nucleosides or nucleobases across the parasite plasma membrane by NTs. There are three Specific Aims. The first Specific Aim is to study the structure and function of a model nucleoside transporter from L. donovani, LdNT1.1, to elucidate how nucleoside transporters function in Leishmania and in other parasitic protozoa. The first component of this Specific Aim is to analyze 18 previously identified missense mutants of LdNT1.1 that are non-functional in transport or have altered substrate specificity. These mutants will be examined for those that are non-functional because they fail to bind substrate and those that are still able to bind substrate but unable to translocate it across the plasma membrane. These experiments will identify amino acids that play crucial roles in substrate recognition and binding or in substrate translocation and will thus define components of the permease that promote its two essential mechanistic functions. The second component of Specific Aim 1 is to define extracellular and intracellular gating residues for LdNT1.1, functionally important residues that interact to close off the permeation pore in the inward-facing and outward-facing orientation respectively. The second Specific Aim entails the creation of a structural model of LmaNT4, a unique nucleobase transporter from L. major that functions optimally at pH 5, the intracellular pH of the macrophage phagolysosome where the parasite resides in the mammalian host, and the genetic dissection of key residues that likely control this unusual pH optimum. The third Specific Aim is to examine the molecular mechanisms L. donovani employs to adapt to purine starvation. Preliminary evidence reveals that the LdNT2 protein is dramatically upregulated upon purine starvation whereas the level of its mRNA is unaffected. Planned experiments will determine whether this regulation operates at the level of translation, protein turnover, or both. The potential regulation of the other LdNTs by purine limitation will also be examined. PUBLIC HEALTH RELEVANCE: The overall objective of this proposal is to study in detail the structure, function, and regulation of purine nucleoside transporters in the parasite Leishmania donovani that causes the fatal disease visceral leishmaniasis. Uptake of purines is essential for Leishmania and all parasitic protozoa, underscoring the crucial role of purine nucleoside/nucleobase transporters in these microorganisms, and these transporters also mediate the uptake of several drugs or experimental drugs employed against Leishmania or other parasitic protozoa. The essential roles that purine nucleoside/nucleobase transporters play in parasite viability further suggests selective inhibition of these transporters could be exploited to develop novel drugs against parasitic diseases.