Malaria PfCRT Transporter: Structure, Function, Sorting

疟疾 PfCRT 转运蛋白：结构、功能、分类

• 批准号: 7020668
• 负责人: Myles H. Akabas
• 金额: $ 20.38万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7020668
• 关键词: Plasmodium falciparum; analytical ultracentrifugation; cell line; chloroquine; drug resistance; fluorescence microscopy; fluorescence resonance energy transfer; fluorescent dye /probe; gene expression; gene mutation; lysosomes; malaria; membrane proteins; protein binding; protein isoforms; protein localization; protein structure function; protozoal genetics

DESCRIPTION (provided by the applicant): Malaria is a major public health problem in much of the developing world. Until drug resistance developed, chloroquine was the treatment of choice for Plasmodium falciparum malaria because it was inexpensive, relatively safe and effective. Chloroquine resistance in P. falciparum results from mutations in the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT). PfCRT is an integral membrane protein localized in the parasites' digestive vacuole membrane. Sequence analysis predicts that PfCRT has ten membrane-spanning segments. PfCRT's endogenous function and its molecular interactions with chloroquine are unknown. PfCRT is an ideal anti-malarial drug target because there are no close human PfCRT homologues and because PfCRT knockout produces non-viable parasites implying that PfCRT has an essential role for normal parasite physiology. In order to screen for potential new drugs one needs a functional assay for PfCRT. A major goal of this application is to develop such a functional assay. When expressed heterologously in human embryonic kidney HEK-293 cells we showed that PfCRT is targeted to the lysosomal membrane, the digestive vacuole homologue. This application's goals are divided into two groups. The first group, Aims 1 and 2, will provide a basis for future structure-function studies of PfCRT by determining experimentally the transmembrane topology using epitope tag insertion and selective permeabilization (Aim 1) and by determining whether PfCRT is a dimer in the membrane using FRET (Aim 2). Aims 3 to'5 form the second group of experiments. They are a search for functional assays of PfCRT based on its localization in the lysosomal membrane in our heterologous expression system. These experiments are designed to identify a functional assay for PfCRT or a functional effect of PfCRT expression on lysosomal function. It is the current lack of such an assay and the fact that this is a high risk search that has motivated us to apply for an R21 grant, which is exploratory in nature, rather than an RO1.

描述（由申请人提供）：疟疾是发展中国家的主要公共卫生问题。在耐药性发展之前，氯喹是恶性疟原虫疟疾的选择治疗，因为它廉价，相对安全且有效。恶性疟原虫中的氯喹耐药性是由于恶性疟原虫氯喹抗性转运蛋白（PFCRT）突变引起的。 PFCRT是寄生虫消化液泡膜中局部的积分膜蛋白。序列分析预测，PFCRT具有十个膜跨段。 PFCRT的内源性功能及其与氯喹的分子相互作用尚不清楚。 PFCRT是一个理想的抗疟疾药物靶标，因为没有密切的人类PFCRT同源物，并且因为PFCRT敲除产生不可行的寄生虫，这表明PFCRT对于正常的寄生虫生理具有重要作用。为了筛选潜在的新药，需要对PFCRT进行功能测定。该应用程序的主要目标是开发这种功能分析。当在人类胚胎肾脏HEK-293细胞中异源表达时，我们表明PFCRT靶向溶酶体膜，即消化液泡同源物。该应用程序的目标分为两组。 AIMS 1和2的第一组将通过使用表位标签插入和选择性通透性（AIM 1）来确定跨膜拓扑并确定PFCRT是否使用FRET（AIM 2）来确定PFCRT的未来结构 - 功能研究基础。目标3至5构成第二组实验。它们是基于其在异源表达系统中的溶酶体膜中定位的PFCRT的功能测定。这些实验旨在识别PFCRT的功能分析或PFCRT表达对溶酶体功能的功能效应。目前缺乏这样的测定，这是一种高风险搜索，促使我们申请了R21赠款，而R21赠款本质上是探索性的，而不是RO1。

项目成果

Probing the multifactorial basis of Plasmodium falciparum quinine resistance: evidence for a strain-specific contribution of the sodium-proton exchanger PfNHE.

• DOI: 10.1016/j.molbiopara.2009.01.011
• 发表时间: 2009-06
• 期刊: MOLECULAR AND BIOCHEMICAL PARASITOLOGY
• 影响因子: 1.5
• 作者: Nkrumaha, Louis J.;Riegelhaupt, Paul M.;Moura, Pedro;Johnson, David J.;Patel, Jigar;Hayton, Karen;Ferdig, Michael T.;Wellems, Thomas E.;Akabas, Myles H.;Fidock, David A.
• 通讯作者: Fidock, David A.