BIOCHEMICAL INVESTIGATION OF P-GLYCOPROTEIN

多药耐药性是癌症患者中遇到的一种情况，其中肿瘤对多种细胞毒性抗癌化疗剂产生耐药性。它通常涉及到P-糖蛋白（Pgp）（一种质膜蛋白）的表达增强. Pgp参与对抗艾滋病药物的耐药性也是强有力的。Pgp由1280个氨基酸组成，排列成两个重复的半部分，每个半部分包含六个预测的跨膜螺旋和一个ATP结合位点。它以ATP依赖性方式起作用以从细胞中排除药物和广泛的其他疏水化合物，显示出显著的药物刺激的ATP酶活性，并且现在被广泛认为充当ATP驱动的药物外排泵。我们实验室提出的一种催化循环，包括交替的催化位点和ATP水解与药物转运的耦合机制，已被广泛采用作为工作模型。我们最近取得了突破性进展，即利用毕赤酵母开发了制备纯的、洗涤剂可溶的、小鼠和人Pgp的大规模方法。不仅野生型，而且突变体Pgp现在可以获得的数量，促进更广泛的结构，生物物理和生物化学的方法。该提案的目的是表征Pgp的结构和功能。将通过电子显微镜和X射线晶体学确定结构。将通过特异性插入荧光探针来监测核苷酸结合参数和催化位点的占用率，并通过关键催化位点残基的诱变来研究催化机制。将研究ATP水解与药物转运的偶联。Pgp的两半将被分别纯化和重构，以促进对催化位点和膜结构域之间相互作用的理解。这类基本知识将是非常宝贵的，在设计方法，以禁用P-糖蛋白和克服耐药性的病人。

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.