Function of antimalarial drug resistance proteins

描述（由申请人提供）：经过十年的艰苦工作，wellms及其同事最近确定了一种基因，该基因似乎是赋予疟疾寄生虫恶性疟原虫氯喹抗性（CQR）的原因。这个基因，pfcrt，编码似乎是一个具有10个假定的跨膜螺旋的多面体完整膜蛋白。Pfcrt蛋白定位于红细胞内寄生虫的消化空泡膜。Pfcrt中几种不同的突变模式赋予CQR，但突变蛋白的功能尚不清楚，野生型蛋白催化的内源性生理也不清楚。在分子水平上破译Pfcrt的功能对于定义CQR、预测额外的耐药途径以及设计改进的治疗方法至关重要。分析分子水平的功能在体内是非常具有挑战性的，因为蛋白质定位于细胞内寄生虫的内膜。因此，在过去的几年中，我们努力在酵母中设计疟疾整体膜蛋白的高水平过表达，并且最近报道了突变型和野生型Pfcrt蛋白在酿酒酵母和帕斯德酵母中的高水平过表达。据我们所知，这是第一次在异源系统中成功过表达疟疾多面体整体膜蛋白。基于这一成功，我们现在提出了一个详细的基于囊泡和蛋白脂质体的研究来破译Pfcrt的功能。我们还建议将这种方法的成功扩展到分析另一种与抗疟药耐药性有关的关键膜蛋白pfmdr 1。生成的数据对于了解该系统和其他系统的耐药性至关重要，并将为设计顶复合体膜转运蛋白的其他深入研究提供模板。

DESCRIPTION (provided by applicant): After a decade of painstaking work, Wellems and colleagues recently identified the gene that appears to be responsible for conferring chloroquine resistance (CQR) to the malarial parasite Plasmodium falciparum. This gene, pfcrt, encodes what appears to be a polytopic integral membrane protein with 10 putative transmembraneous helices. Pfcrt protein is localized to the digestive vacuolar membrane of the intraerythrocytic parasite. Several distinct patterns of mutations in Pfcrt confer CQR, but the function of mutant proteins is not yet known, nor is the endogeneous physiology catalyzed by the wild type protein understood. Deciphering Pfcrt function at the molecular level is central to defining CQR, to anticipating additional drug resistance pathways, and for devising improved therapy. Analysis of molecular level function in vivo is extraordinarily challenging since the protein is localized to an endo membrane of an intracellular parasite. Thus, over the past several years we have endeavored to engineer high level overexpression of malarial integral membrane proteins in yeast, and have recently reported the high level overexpression of mutant and wild type Pfcrt proteins in both S. cerevisiae and P. pastoris. To our knowledge, this represents the first successful overexpression of malarial polytopic integral membrane proteins in a heterologous system. Based upon this success, we now propose a detailed vesicle and proteoliposome based study to decipher the function of Pfcrt. We also propose to extend our success with this approach to analysis of another key membrane protein involved in antimalarial drug resistance, Pf mdr 1. The data to be generated are vital for understanding drug resistance in this and other systems, and will provide a template for design of additional in depth studies of apicomplexan membrane transport proteins.