Malaria PfCRT Transporter: Structure, Function, Sorting

描述(由申请人提供)：疟疾是许多发展中国家的一个主要公共卫生问题。在产生抗药性之前，氯喹是治疗恶性疟疾的首选药物，因为它便宜、相对安全和有效。恶性疟原虫氯喹抗性转运蛋白(PfCRT)基因突变是恶性疟原虫氯喹耐药性产生的原因。PfCRT是一种完整的膜蛋白，定位于寄生虫的消化液泡膜。序列分析预测，PfCRT有10个跨膜片段。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点，因为没有接近的人类PfCRT同源物，而且因为PfCRT基因敲除会产生不活的寄生虫，这意味着PfCRT对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进行功能分析。这一应用的一个主要目标是开发这样一种功能检测。当PfCRT在人胚胎肾HEK-293细胞中异源表达时，我们发现PfCRT靶向于消化液泡同系物--溶酶体膜。这个应用程序的目标分为两组。第一组，目标1和2，将通过使用表位标签插入和选择性通透性(目标1)通过实验确定跨膜拓扑，并通过使用FRET确定PfCRT是否是膜中的二聚体(目标2)，为未来的PfCRT结构功能研究提供基础。第二组实验的目标是3到‘5。它们是基于我们的异源表达系统中PfCRT在溶酶体膜上的定位来寻找其功能分析。这些实验旨在确定PfCRT的功能测定或PfCRT表达对溶酶体功能的功能影响。正是目前缺乏这样的检测方法，以及这是一项高风险的研究，促使我们申请了R21拨款，这是探索性的，而不是RO1。

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.