Function of antimalarial drug resistance proteins

描述(申请人提供)：经过十年的艰苦工作，Wellems和他的同事最近发现了似乎对疟疾寄生虫恶性疟原虫产生氯喹抗药性(CQR)的基因。这个名为pfcrt的基因编码的似乎是一个具有10个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液泡膜。Pfcrt的几种不同的突变模式提供了CQR，但突变蛋白的功能尚不清楚，野生型蛋白催化的内源生理也不清楚。在分子水平上破译Pfcrt功能对于确定CQR、预测额外的耐药途径和设计改进的治疗方法是至关重要的。体内分子水平的功能分析是非常具有挑战性的，因为该蛋白定位于细胞内寄生虫的内膜。因此，在过去的几年里，我们一直致力于在酵母中高效表达疟疾完整膜蛋白，最近报道了突变型和野生型Pfcrt蛋白在酿酒酵母和巴斯德毕赤酵母中的高水平过表达。据我们所知，这是首次成功地在异源系统中过表达疟疾多聚体完整膜蛋白。基于这一成功，我们现在提出一项基于囊泡和蛋白脂质体的详细研究，以破译Pfcrt的功能。我们还建议将这种方法的成功扩展到另一种参与抗疟疾耐药的关键膜蛋白Pf mdr1的分析。所产生的数据对于了解该系统和其他系统的耐药性至关重要，并将为进一步深入研究apicplexan膜转运蛋白提供一个模板。

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.