Nucleoside Transporters of Plasmodium falciparum

恶性疟原虫核苷转运蛋白

基本信息

批准号：
7161393
负责人：
BUDDY ULLMAN
金额：
$ 31.25万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-01-01 至 2008-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7161393
关键词：
Adenosine Affinity Amino Acids Antibodies Antiparasitic Agents Antiviral Agents Applications Grants Biochemical Biochemical Genetics Biochemistry Cell membrane Code Complement Databases Disruption Dissection Drug Delivery Systems Erythrocytes Essential Genes Family Gene Targeting Genes Genetic Genetic Screening Goals Growth Guanosine Immune Sera Immunoelectron Microscopy Immunosuppressive Agents Injection of therapeutic agent Inosine Investigation Kinetics Knock-out Laboratories Leishmania donovani Ligands Localized Location Mammalian Cell Mediating Membrane Metabolism Microinjections Missense Mutation Molecular Molecular Biology Mutation Nucleoside Transporter Nucleosides Nutritional Oocytes Oryctolagus cuniculus Parasites Parasitic Diseases Phenotype Physiological Plasmodium falciparum Protein Overexpression Proteins Purine Nucleosides Purine Nucleotides Purines Pyrimidine Nucleosides Reagent Research Research Personnel Role Specificity Staging Structure System Testing Therapeutic Transfection Western Blotting Xenopus laevis Xenopus oocyte base design drug development gene replacement genetic analysis immunocytochemistry inhibitor/antagonist loss of function member mutant nucleoside analog permease programs purine tool

项目摘要

Amalgamating tools of molecular biology, biochemistry, genetics, and immunocytochemistry, this proposal offers an interdisciplinary dissection of the nucleoside transporters of Plasmodium falciparum. As protozoan parasites are incapable of synthesizing purine nucleotides de novo, nucleoside transporters provide an important, if not obligatory, nutritional function for the parasite and present several therapeutic paradigms. Two nucleoside transporter genes, PfNT1 and PfNT2, have been identified within available P. falciparum databases, and both have been cloned and sequenced in this laboratory. PfNT1 activity has been characterized in a preliminary fashion after PfNT1 cRNA injection into Xenopus laevis oocytes, and PfNT1 has also been functionally overexpressed in nucleoside transport-deficient Leishmania donovani. In addition polyclonal antisera specific for PfNT1 have been raised in rabbits and used to localize PfNT1 to the parasite plasma membrane by confocal and immunoelectron microscopy. Antibodies against PfNT2 have also been generated. These reagents are the cornerstone of the three specific aims in this proposal. The multicomponent Specific Aim I will encompass: i., a thorough biochemical characterization of PfNT1 with respect to ligand specificity and affinities and sensitivities to inhibitors of mammalian nucleoside transport; ii., an assessment of whether PfNT2 is a functional nucleoside transporter, and if so, a preliminary molecular and biochemical characterization, including immunolocatization of the protein in P. falciparum-infected erythrocytes; and iii., a verification of whether PfNT1 and PfNT2 are electrogenic transporters using the Xenopus oocyte cRNA expression system. The second Specific Aim initiates a structure-function analysis of PfNTI. We propose to implement a genetic screen for loss-of-function mutants to identify in an unbiased fashion key amino acids in PfNT1 that are required for ligand permeation and/or ligand selectivity. The last Specific Aim will explore the physiological function of PfNT1 within the parasitized erythrocyte using transfection and gene targeting approaches. Specifically, we will attempt to create Apfntl knockouts in either wild type or genetically complemented P. falciparum in order to test whether PfNT1 function is essential to the intact parasite. We will then characterize the resultant transport and growth phenotypes.

该提案融合了分子生物学、生物化学、遗传学和免疫细胞化学的工具对恶性疟原虫核苷转运蛋白进行跨学科剖析。作为原生动物寄生虫不能从头合成嘌呤核苷酸，核苷转运蛋白提供了对于寄生虫来说，即使不是强制性的，也是重要的营养功能，并提出了几种治疗范例。已在可用的恶性疟原虫中鉴定出两个核苷转运蛋白基因 PfNT1 和 PfNT2 数据库，并且两者均已在该实验室中被克隆和测序。 PfNT1 活性将 PfNT1 cRNA 注入非洲爪蟾卵母细胞后初步表征，并且 PfNT1 在核苷转运缺陷的杜氏利什曼原虫中也被功能性过度表达。此外 PfNT1 特异性多克隆抗血清已在兔子中培养并用于将 PfNT1 定位于寄生虫通过共聚焦和免疫电子显微镜观察质膜。针对 PfNT2 的抗体也已已生成。这些试剂是该提案中三个具体目标的基石。这多组分具体目标 I 将包括： i. 对 PfNT1 进行彻底的生化表征关于配体特异性以及对哺乳动物核苷转运抑制剂的亲和力和敏感性； ii. 评估 PfNT2 是否是功能性核苷转运蛋白，如果是，则评估初步分子和生化特征，包括恶性疟原虫感染的蛋白质的免疫定位红细胞； iii. 使用以下方法验证 PfNT1 和 PfNT2 是否是生电转运蛋白非洲爪蟾卵母细胞 cRNA 表达系统。第二个具体目标启动了结构功能分析 PfNTI。我们建议对功能丧失突变体进行遗传筛选，以公正地识别形成 PfNT1 中配体渗透和/或配体选择性所需的关键氨基酸。最后一个具体目标将利用以下方法探索 PfNT1 在寄生红细胞内的生理功能转染和基因靶向方法。具体来说，我们将尝试在以下位置创建 Apfntl 淘汰赛：野生型或基因互补的恶性疟原虫，以测试 PfNT1 功能是否有效对于完整的寄生虫至关重要。然后我们将描述由此产生的运输和生长表型。