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。在出现耐药性之前，氯喹是恶性疟原虫疟疾的首选治疗方法，因为它价格低廉，相对安全有效。恶性疟原虫氯喹耐药是由恶性疟原虫氯喹耐药转运体（PfCRT）突变引起的。PfCRT是一种定位于寄生虫消化液泡膜上的完整膜蛋白。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是一种理想的抗疟疾药物靶点，因为PfCRT在人类中没有密切的同源物，而且敲除PfCRT会产生无法存活的寄生虫，这意味着PfCRT对正常的寄生虫生理有重要作用。为了筛选潜在的新药，需要对PfCRT进行功能分析。本应用程序的主要目标是开发这样一种功能分析。当在人胚胎肾HEK-293细胞中异种表达时，我们发现PfCRT靶向溶酶体膜，消化液泡同源物。此应用程序的目标分为两组。第一组，Aims 1和2，通过实验确定表位标签插入和选择性渗透的跨膜拓扑结构（Aim 1），以及通过FRET确定PfCRT是否是膜中的二聚体（Aim 2），将为PfCRT未来的结构-功能研究提供基础。目标3 - 5组成第二组实验。在我们的异源表达系统中，基于PfCRT在溶酶体膜中的定位，他们正在寻找PfCRT的功能测定方法。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。正是由于目前缺乏这样的试验，而且这是一个高风险的研究，促使我们申请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.