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）：该项目将探测嘌呤核苷和核碱酶转运蛋白（NTS）在利什曼原虫寄生虫生物学中的作用。迄今为止，所有检查的寄生原生动物都无法合成嘌呤，必须从宿主那里挽救这些基本营养素。挽救的第一步是通过NTS摄取嘌呤核苷或核苷酶在寄生虫质膜上的摄取。有三个特定的目标。第一个具体目的是研究核苷转运蛋白的结构和功能，从L. donovani，ldnt1.1，以阐明核苷转运蛋白在利什曼尼亚和其他寄生虫原生动物中的功能。该特定目的的第一个组成部分是分析18个先前确定的LDNT1.1的错义突变体，这些突变体在运输方面非功能或底物特异性改变。这些突变体将对那些非功能性的突变体进行检查，因为它们无法结合底物，并且仍然能够结合底物但无法在质膜上易位的突变体。这些实验将确定在底物识别和结合或底物易位中起着至关重要的作用的氨基酸，从而定义了越过渗透酶的成分，从而促进其两种必不可少的机械功能。特定目标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，在功能上重要的残基分别相互作用，以关闭触及孔的渗透孔和向外方向的渗透孔。第二个特定目的需要创建LMANT4的结构模型，Lmant4是L. Major的独特核碱酶转运蛋白，该转运蛋白在pH 5处最佳起作用，pH 5，巨噬细胞吞噬体的细胞内pH值，寄生虫属于哺乳动物宿主中的巨噬细胞吞噬体，以及可能控制这种不寻常的pH Optimual PH Optimual ph。第三个具体目的是检查L. donovani使用的分子机制适应嘌呤饥饿。初步证据表明，LDNT2蛋白在嘌呤饥饿时显着上调，而其mRNA的水平不受影响。计划的实验将确定该调节是在翻译，蛋白质周转或两者兼而有之的水平。还将检查其他LDNT对其他LDNT的潜在调节。 公共卫生相关性：该提案的总体目的是详细研究寄生虫Donovani中嘌呤核苷转运蛋白的结构，功能和调节，从而导致致命性疾病内脏利什曼病。嘌呤的吸收对于利什曼原虫和所有寄生虫原生动物至关重要，强调了嘌呤核苷/核苷酶转运蛋白在这些微生物中的关键作用，这些转运蛋白还介导了对利什马尼亚或其他帕亚马尼亚质子原生素的几种药物或实验性药物的摄取。嘌呤核苷/核苷酶转运蛋白在寄生虫生存能力中起着的重要作用进一步表明，可以利用对这些转运蛋白的选择性抑制来开发针对寄生虫病的新药物。