Function of antimalarial drug resistance proteins

描述（由申请人提供）：经过十年的艰苦工作，Wellems及其同事最近确定了似乎负责赋予恶性疟原虫氯喹抗性（CQR）的基因。这个基因，pfcrt，编码什么似乎是一个多位整合膜蛋白与10个假定的跨膜螺旋。Pfcrt蛋白定位于红细胞内寄生虫的消化泡膜。Pfcrt中几种不同的突变模式赋予了CQR，但突变蛋白的功能尚不清楚，也不了解野生型蛋白催化的内源性生理学。在分子水平上破译Pfcrt功能对于定义CQR、预测其他耐药途径以及设计改进的治疗是至关重要的。体内分子水平功能的分析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，在过去的几年里，我们致力于在酵母中高水平过表达疟疾整合膜蛋白，最近报道了突变型和野生型Pfcrt蛋白在S.酿酒酵母和巴斯德毕赤酵母。据我们所知，这代表了第一个成功的过表达疟疾多面体的整体膜蛋白在异源系统。 基于这一成功，我们现在提出了一个详细的囊泡和蛋白脂质体为基础的研究，以破译Pfcrt的功能。我们还建议将我们的成功与这种方法的另一个关键的膜蛋白参与抗疟药物耐药性，Pfmdr 1的分析。要生成的数据是至关重要的了解在这个和其他系统的耐药性，并将提供一个模板，设计额外的深入研究apicomplexan膜转运蛋白。

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.