Purine Nucleoside/Nucleobase Transporters in Leishmania

利什曼原虫中的嘌呤核苷/核碱基转运蛋白

• 批准号: 8056669
• 负责人: Scott M Landfear
• 金额: $ 38.12万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8056669
• 关键词: Acids; Adenosine; Affect; African Trypanosomiasis; Allopurinol; Amino Acids; Antibodies; Binding; Biology; Carrier Proteins; Cell membrane; Complement; Computer Simulation; Disease; Dissection; Environment; Family; Gene Combinations; Genes; Genetic; Histidine; Label; Laboratories; Leishmania; Leishmania donovani; Leishmaniasis; Location; Lysosomes; Mediating; Melarsoprol; Membrane; Messenger RNA; Modeling; Molecular; Molecular Probes; Monitor; Mutate; NTF3 gene; Nucleoside Transporter; Nutrient; Nutritional; Parasites; Parasitic Diseases; Pentamidine; Phagolysosome; Pharmaceutical Preparations; Pharmacology; Physiologic pulse; Pichia; Play; Polyribosomes; Property; Proteins; Protozoa; Purine Nucleosides; Purines; Pyrimidine Nucleosides; Reagent; Regulation; Research Personnel; Role; Saccharomyces cerevisiae; Starvation; Structural Models; Structure; Substrate Specificity; System; Therapeutic; Translations; Visceral Leishmaniasis; Xenopus oocyte; Yeasts; base; expression vector; extracellular; inhibitor/antagonist; macrophage; member; microorganism; mutant; novel; nucleobase; nucleobase analog; nucleoside analog; nutrition; permease; protein degradation; public health relevance; purine; purine analog; research study; sensor; uptake

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.

描述（由申请人提供）：本项目将探讨嘌呤核苷和核碱基转运蛋白（NT）在利什曼原虫生物学中的作用。迄今为止，所有的寄生原生动物都不能合成嘌呤，必须从它们的宿主那里抢救这些必需的营养物质。挽救的第一步是通过NT摄取嘌呤核苷或核碱基穿过寄生虫质膜。有三个具体目标。第一个具体目标是研究L. donovani，LdNT 1.1，阐明核苷转运蛋白在利什曼原虫和其他寄生原生动物中的功能。该特定目标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，这些突变体在转运中无功能或具有改变的底物特异性。将检查这些突变体中因不能结合底物而无功能的突变体和仍能结合底物但不能将底物转运穿过质膜的突变体。这些实验将确定在底物识别和结合或底物易位中发挥关键作用的氨基酸，并因此确定促进其两个基本机械功能的通透酶的组分。具体目标1的第二个组成部分是定义LdNT 1.1的细胞外和细胞内门控残基，这些残基在功能上是重要的，它们相互作用以分别关闭向内和向外方向的渗透孔。第二个特异性目的需要创建LmaNT 4的结构模型，LmaNT 4是来自L.主要的功能最佳的pH值为5，细胞内的pH值的巨噬细胞吞噬溶酶体寄生虫驻留在哺乳动物宿主，和遗传解剖的关键残基，可能控制这种不寻常的pH值最佳。第三个具体目标是研究分子机制L。多诺万尼雇用来适应嘌呤饥饿。初步证据显示，LdNT 2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否在翻译水平、蛋白质周转水平或两者兼而有之。还将研究嘌呤限制对其他LdNT的潜在调节。 公共卫生相关性：本提案的总体目标是详细研究导致致命性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。嘌呤的摄取对于利什曼原虫和所有寄生原虫是必不可少的，这强调了嘌呤核苷/核碱基转运蛋白在这些微生物中的关键作用，并且这些转运蛋白还介导针对利什曼原虫或其他寄生原虫的几种药物或实验药物的摄取。嘌呤核苷/核碱基转运蛋白在寄生虫生存能力中发挥的重要作用进一步表明，选择性抑制这些转运蛋白可用于开发抗寄生虫病的新药。