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.

分子生物学，生物化学，遗传学和免疫细胞化学的合并工具，该建议提供了恶性疟原虫的核苷转运蛋白的跨学科解剖。作为原生动物寄生虫无法合成从头开始的嘌呤核苷酸，核苷转运蛋白提供重要的是，即使不是强制性的寄生虫营养功能，并提出了几种治疗范例。在可用的恶性疟原虫中已经鉴定出两个核苷转运蛋白基因PFNT1和PFNT2 数据库，并且都在该实验室中克隆和测序。 PFNT1活性已经 PFNT1 CRNA注入Xenopus laevis卵母细胞和PFNT1之后以初步方式进行特征在缺乏核苷转运型利什曼原虫的核苷转运率中也过表达。此外针对PFNT1的多克隆抗血清已在兔子中升高，用于将PFNT1定位于寄生虫共聚焦和免疫电子显微镜的质膜。针对PFNT2的抗体也有被生成。这些试剂是该提案三个特定目标的基石。这多组分特定目的我将包括：i。，PFNT1的彻底生化表征对配体的特异性和亲和力和对哺乳动物核苷转运抑制剂的敏感性； ii。，评估PFNT2是否是功能性核苷转运蛋白，如果是的，则是初步分子和生化表征，包括对恶性疟原虫感染的蛋白质的免疫定位红细胞； iii。，验证PFNT1和PFNT2是否是使用电源转运蛋白爪蟾卵母细胞CRNA表达系统。第二个特定目的启动了结构功能分析 pfnti。我们建议实施一个遗传筛选，以识别功能丧失的突变体以无偏见 PFNT1中的时尚键氨基酸是配体渗透和/或配体选择性所需的。最后具体目标将探索使用寄生的红细胞中PFNT1的生理功能转染和基因靶向方法。具体来说，我们将尝试在为了测试PFNT1功能是否为对于完整的寄生虫必不可少的。然后，我们将表征所得的运输和生长表型。