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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.

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，提供了一个跨学科的解剖恶性疟原虫的核苷转运蛋白。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一个重要的，如果不是强制性的，营养功能的寄生虫，并提出了几个治疗范例。两个核苷转运蛋白基因，PfNT 1和PfNT 2，已被确定存在于恶性疟原虫中数据库，两者都在这个实验室被克隆和测序。PfNT 1活性已被在将PfNT 1 cRNA注射到非洲爪蟾卵母细胞中后，也在核苷转运缺陷型杜氏利什曼原虫中功能性过表达。此外已经在兔中产生了PfNT 1特异性的多克隆抗血清，并用于将PfNT 1定位于寄生虫通过共聚焦和免疫电镜观察质膜。针对PfNT 2的抗体还被生成。这些试剂是本提案中三个具体目标的基石。的多组分具体目标I将包括：i.，PfNT 1的彻底生物化学表征，关于配体特异性和亲和力以及对哺乳动物核苷转运抑制剂的敏感性; 二、PfNT 2是否是功能性核苷转运蛋白的评估，如果是，初步的分子生物学评估。和生物化学表征，包括恶性疟原虫感染的小鼠中蛋白质的免疫定位，红细胞;和iii.，使用以下方法验证PfNT 1和PfNT 2是否是产电转运蛋白：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，以无偏见的方式进行鉴定。 PfNT 1中的时尚关键氨基酸是配体渗透和/或配体选择性所需的。最后一具体目的将探讨PfNT 1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，我们将尝试创建Apfntl淘汰赛，野生型或基因互补的恶性疟原虫，以测试PfNT 1功能是否对完整的寄生虫至关重要然后，我们将表征所得的运输和生长表型。