Purine Nucleoside/Nucleobase Transporters in Leishmania

项目描述（申请人提供）：本项目将探讨嘌呤核苷和核碱基转运体（nt）在利什曼原虫生物学中的作用。到目前为止，所有的寄生原生动物都不能合成嘌呤，必须从宿主那里获取这些必需的营养物质。救助的第一步是嘌呤核苷或核碱基通过NTs摄取寄生虫的质膜。有三个具体目标。第一个特定目的是研究多诺瓦氏乳杆菌核苷转运体LdNT1.1的结构和功能，以阐明核苷转运体在利什曼原虫和其他寄生原生动物中的作用。特异性目的的第一个组成部分是分析18个先前鉴定的LdNT1.1错义突变体，这些突变体在运输中无功能或改变了底物特异性。这些突变体将被检查为那些没有功能的，因为它们不能结合底物，那些仍然能够结合底物，但不能在质膜上转移。这些实验将确定在底物识别和结合或底物易位中起关键作用的氨基酸，从而确定促进其两种基本机制功能的渗透酶成分。Specific Aim 1的第二个组成部分是定义LdNT1.1的细胞外和细胞内门控残基，这两个功能重要的残基分别在向内和向外方向相互作用以关闭渗透孔。第二个特定目标需要创建LmaNT4的结构模型，LmaNT4是一种来自L. major的独特核碱基转运体，在pH 5，寄生在哺乳动物宿主的巨噬细胞吞噬溶酶体的细胞内pH值下发挥最佳作用，并对可能控制这种不寻常的pH最佳的关键残基进行遗传解剖。第三个具体目的是研究多诺瓦氏乳杆菌适应嘌呤饥饿的分子机制。初步证据表明，LdNT2蛋白在嘌呤饥饿时显著上调，而其mRNA水平不受影响。计划中的实验将确定这种调节是在翻译水平上起作用，还是在蛋白质周转水平上起作用，或者两者都起作用。嘌呤限制对其他ldnt的潜在调节也将被研究。

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.