Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol
Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol
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DOI：
10.1042/bj20060632
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发表时间：
2007-01-15
影响因子：
4.1
通讯作者：
Ueda, Kazumitsu
中科院分区：
文献类型：
--
作者：
Kimura, Yasuhisa;Kioka, Noriyuki;Ueda, Kazumitsu
MDR1（多药耐药性 1）/P-糖蛋白是一种 ATP 驱动的转运蛋白，可从细胞中分泌多种结构不相关的疏水性化合物。研究表明，药物直接从脂质双层与 MDR1 结合，并且双层中的胆固醇也与 MDR1 相互作用。然而，胆固醇对药物-MDR1 相互作用的影响仍不清楚。为了检查这些影响，在昆虫细胞中表达并纯化了人类 MDR1。将纯化的 MDR1 蛋白在含有不同浓度胆固醇的脂蛋白体中重建，并比较药物刺激的 ATP 酶的酶学参数。胆固醇以洗涤剂可溶形式直接与纯化的 MDR1 结合，并且胆固醇对药物刺激的 ATP 酶活性的影响因药物而异。胆固醇对药物刺激的 ATP 酶活性 Km 值的影响与该药物的分子质量密切相关。胆固醇增加小药物（分子量<500Da）的结合亲和力，但不影响分子质量800-900Da药物的结合亲和力，并抑制缬氨霉素（分子量>1000Da）的结合亲和力。胆固醇也会增加罗丹明 B 和紫杉醇的 V-max 值，表明胆固醇影响周转和药物结合。紫杉醇刺激的 MDR1 ATP 酶活性在豆甾醇、谷甾醇和菜油甾醇以及胆固醇（但不存在麦角甾醇）存在的情况下增强。这些结果表明，MDR1的药物结合位点可能最适合分子质量在800至900 Da之间的药物，而胆固醇可能通过调整药物结合位点来支持较小药物的识别，并在MDR1的功能中发挥重要作用。
MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on Km values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs(molecular mass < 500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass > 1000 Da). V-max values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.