BIOCHEMICAL INVESTIGATION OF P-GLYCOPROTEIN

多药耐药是癌症患者遇到的一种情况，在这种情况下，肿瘤对各种细胞毒性抗癌化疗药物产生抗药性。它通常涉及质膜蛋白P-糖蛋白(Pgp)的表达增强。Pgp参与了抗艾滋病药物的耐药性，这也是强有力的证据。PGP由1280个氨基酸组成，排列成两个重复的部分，每个部分包含六个预测的跨膜螺旋和一个ATP结合位点。它以一种ATP依赖的方式发挥作用，将药物和广泛的其他疏水化合物从细胞中排除，显示出大量的药物刺激的ATPase活性，现在被广泛认为是ATP驱动的药物外排泵。我们实验室提出的包含交替催化位置的催化循环和ATP-水解与药物传输的耦合机制已被广泛采用作为工作模型。我们最近取得了一项突破，即发展了一种大规模的方法，利用毕赤酵母制备纯的、洗涤剂可溶的、小鼠和人的PGP。现在不仅可以大量获得野生型Pgp，而且还可以获得突变型Pgp，从而促进了更广泛的结构、生物物理和生化方法。这项建议的目的是描述PGP的结构和功能。结构将通过电子显微镜和X射线结晶学来确定。催化机理将通过插入特定的荧光探针来监测核苷酸结合参数和催化位点的占有率，以及通过突变关键催化位点残基来研究。将研究三磷酸腺苷水解与药物转运的耦合。PGP的两个部分将被分别纯化和重组，以便于了解催化位点和膜结构域之间的相互作用。这方面的基础知识对于设计禁用P-糖蛋白和克服患者耐药性的方法将是非常宝贵的。

Multidrug-resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotherapeutic agents. It often involves enhanced expression of P- glycoprotein (Pgp), a plasma membrane protein. Involvement of Pgp in resistance to anti-AIDS drugs is also strongly-indicated. Pgp consists of 1280 amino acids, arranged in two repeated halves, each of which contains six predicted transmembrane helices and one ATP-binding site. It acts in an ATP-dependent manner to exclude drugs and a wide range of other hydrophobic compounds from cells, displays substantial drug- stimulated ATPase activity, and is now widely-believed to act as an ATP- driven drug-efflux pump. A catalytic cycle involving alternating catalytic sites and a mechanism for coupling of ATP-hydrolysis to drug-transport, presented by our laboratory, has become widely-adopted as a working model. We recently made a breakthrough, namely the development of a large- scale method for preparation of pure, detergent-soluble, mouse and human Pgp, using Pichia. Not only wild-type but also mutant Pgp may now be obtained in quantity, facilitating a broader range of structural, biophysical and biochemical approaches. The aim of this proposal is to characterize structure and function of Pgp. Structure will be determined by electron-microscopy and X-ray crystallography. Catalytic mechanism will be studied by specific insertion of fluorescent probes to monitor nucleotide binding parameters and occupancy of catalytic sites, and by mutagenesis of critical catalytic site residues. Coupling of ATP hydrolysis to drug transport will be investigated. The two halves of Pgp will be purified separately and reconstituted, to facilitate understanding of interactions between catalytic sites and membrane domains. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein and overcome drug-resistance in patients.