Nucleoside Transporters of Plasmodium falciparum

恶性疟原虫核苷转运蛋白

• 批准号: 6843158
• 负责人: BUDDY ULLMAN
• 金额: $ 33.36万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6843158
• 关键词: Nucleoside; Transporters; Plasmodium; falciparum

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================

超过提供的空间。该提案的分子生物学，生物化学，遗传学和免疫细胞化学的合并工具提供了对恶性疟原虫的核苷转运蛋白的跨学科解剖。由于原生动物的寄生虫无法合成从头开始的嘌呤核苷酸，因此核苷转运蛋白为寄生虫提供了重要的，即使不是强制性的营养功能，并提供了几种治疗范式。在可用的恶性疟原虫数据库中已经鉴定出了两个核苷转运蛋白基因PFNT1和PFNT2，并且在该实验室中都被克隆和测序。 PFNT1活性已在PFNT1 CRNA注射到Xenopus laevis卵母细胞后以初步方式进行了表征，并且PFNT1在核苷的运输缺陷利什曼原虫Donovani中也在功能过表达。另外，针对PFNT1的多克隆抗血清已经在兔子中升高，并通过共焦和免疫电子显微镜将PFNT1定位在寄生虫质膜中。还产生了针对PFNT2的抗体。这些试剂是该提案三个特定目标的基石。多组分特定的目的I将包括：i。，PFNT1对配体特异性以及对哺乳动物核苷转运抑制剂的敏感性和敏感性的彻底生化表征； ii。，评估PFNT2是否是功能性核苷转运蛋白，如果是的，则是初步的分子和生化表征，包括在恶性疟原虫感染的红细胞促成的红细胞中蛋白质的免疫定位； iii。，使用爪蟾卵母细胞CRNA表达系统的验证PFNT1和PFNT2是否是电源转运蛋白。第二个特定目的启动了PFNTI的结构功能分析。我们建议实施一个遗传筛选，以实现功能丧失的突变体，以在PFNT1中无偏的时尚键氨基酸中识别配体渗透和/或配体选择性所需的遗传性突变体。最后的特定目的将使用转染和基因靶向方法探索PFNT1在寄生的红细胞中的生理功能。具体而言，我们将尝试在野生型或遗传补充的恶性疟原虫中创建APFNTL敲除，以测试PFNT1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。表演网站=================================== ==================================