Nucleoside/Nucleobase Transporters in Leishmania major

大利什曼原虫中的核苷/核碱基转运蛋白

• 批准号: 7232668
• 负责人: Scott M Landfear
• 金额: $ 32.21万
• 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 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7232668
• 关键词: Adenine; Adenosine; Affinity; Biochemical; Biochemical Genetics; Biochemistry; Biological Process; Cellular biology; Code; Databases; Dissection; Family; Family member; Funding; Genes; Genetic; Genetic Screening; Genome; Guanosine; Immunosuppressive Agents; Inosine; Investigation; Knock-out; Leishmania; Leishmania major; Life Cycle Stages; Ligands; Mammalian Cell; Mediating; Melarsoprol; Membrane; Metabolic; Metabolism; Missense Mutation; Modeling; Molecular; Molecular Biology; Mutation; Nucleoside Transporter; Nucleosides; Nutritional; Oocytes; Open Reading Frames; Parasites; Parasitic Diseases; Pentamidine; Pharmaceutical Preparations; Positioning Attribute; Purine Nucleosides; Purine Nucleotides; Purines; Pyrimidine Nucleosides; Reagent; Role; Saccharomyces cerevisiae; Site-Directed Mutagenesis; Specificity; Structure; Techniques; Testing; Therapeutic; Trypanosoma brucei brucei; Viral; Virulence; Xenopus laevis; Xenopus oocyte; Yeasts; analog; gene replacement; genetic analysis; loss of function; loss of function mutation; mRNA Expression; member; mutant; neoplastic; novel; nucleobase; nucleobase analog; permease; purine; purine permease; research study; tool; uptake

Amalgamating tools of molecular biology, genetics, biochemistry, and cell biology, this collaborative competing continuation application offers an interdisciplinary dissection of the nucleoside and nucleobase transporters from Leishmania. Because protozoan parasites are incapable of synthesizing purine nucleotides or nucleobases de novo, purine transporters provide an important, if not obligatory, nutritional function for the parasite and suggest several therapeutic paradigms. In the course of the previously funded investigations, we have cloned and functionally characterized two equilibrative nucleoside transporter (ENT) genes from L. donovani; LdNT1, the gene encoding the adenosine-pyrimidine nucleoside transporter, and LdNT2, the inosine-guanosine transporter gene. We have also created Aldntl and Aldnt2 knockouts by targeted gene replacement and characterized mutationally derived parasites deficient in either LdNT1 or LdNT2 activity. More recently, two additional ENT family members have been identified within the Leishmania major genome project database; LmaNT3, which recognizes purine nucleobases but not nucleosides, and a previously unidentified open reading frame, LmaNT4, which has recently been shown to possess nucleobase transport activity. These molecular and cellular reagents are the cornerstone of the three Specific Aims of this proposal. The first Specific Aim will examine the functional roles performed by these transporters in intact L. major parasites using targeted gene replacement strategies. We will test LmaNT1, LmaNT2, LmaNT3, and LmaNT4 function in L. major promastigotes, metacyclics, and infectious amastigotes by creating and characterizing deltalmant1, deltamant2, deltamant3, and deltalmant4 knockouts in various permutations. In Specific Aim II, we will implement an unbiased genetic screen for Imant2 loss-of-function mutants. This will enable us to identify in a nonintuitive manner key residues within LmaNT2 that are required for either permeation or ligand recognition. The final Specific Aim will be to functionally characterize the novel LmaNT4 nucleobase transporter in Xenopus laevis oocytes. We will determine the ligand specificity and affinities for LmaNT4 and evaluate LmaNT4 expression throughout the L. major life cycle.

融合分子生物学，遗传学，生物化学和细胞生物学的工具，这种合作竞争的继续应用提供了一个跨学科的解剖利什曼原虫的核苷和核碱基转运蛋白。由于原生动物寄生虫不能从头合成嘌呤核苷酸或核碱基，嘌呤转运蛋白为寄生虫提供了重要的（如果不是强制性的）营养功能，并提出了几种治疗模式。在前期研究中，我们克隆了两个平衡型核苷转运蛋白（ENT）基因，并对其进行了功能鉴定。LdNT 1，编码腺苷-嘧啶核苷转运蛋白的基因，和 LdNT 2，肌苷-鸟苷转运蛋白基因。我们还通过靶向基因置换产生了Aldntl和Aldnt 2敲除，并表征了LdNT 1或LdNT 2活性缺陷的突变衍生的寄生虫。最近，在利什曼原虫主要基因组计划数据库中鉴定了另外两个ENT家族成员; LmaNT 3，其识别嘌呤核碱基但不识别核苷，以及先前未鉴定的开放阅读框，LmaNT 4，其最近被证明具有核碱基转运活性。这些分子和细胞试剂是本提案三个具体目标的基石。第一个具体目标将检查这些转运蛋白在完整的L。主要寄生虫使用有针对性的基因置换策略。我们将测试 LmaNT 1、LmaNT 2、LmaNT 3和LmaNT 4在L.主要前鞭毛体、后循环体和感染性无鞭毛体，通过以各种排列产生和表征deltamant 1、deltamant 2、deltamant 3和deltamant 4敲除。在Specific Aim II中，我们将对Imant 2功能丧失突变体进行无偏遗传筛选。这将使我们能够以非直观的方式识别LmaNT 2内渗透或配体识别所需的关键残基。最后的具体目标将是功能特性的新的LmaNT 4核碱基转运蛋白在非洲爪蟾卵母细胞。我们将确定LmaNT 4的配体特异性和亲和力，并评估LmaNT 4在整个L.主要生命周期