Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis PROTEIN-PROTEIN INTERACTIONS IN LIPID MEMBRANES
Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis PROTEIN-PROTEIN INTERACTIONS IN LIPID MEMBRANES
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DOI：
10.1074/jbc.m109.006064
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发表时间：
2009-11-27
影响因子：
4.8
通讯作者：
Martin, Lisandra L.
中科院分区：
文献类型：
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作者：
Praporski, Slavica;Ng, Su May;Martin, Lisandra L.
越来越多的证据强调了蛋白质-蛋白质相互作用在药物代谢P450功能调控中的重要性，但在膜环境中进行的研究很少，也没有研究P450催化性类固醇合成。在这里，我们报道了全长的人野生型类固醇细胞色素P450(P450，CyP)酶：17α-羟基酶/17，20-裂解酶(P450c17，CyP17)和芳香化酶(P450arom，CyP19)以及它们的电子供体NADPH-细胞色素P450氧化还原酶(CPR)的蛋白质-蛋白质相互作用。用荧光共振能量转移(FRET)3结合石英晶体微天平(QCM)和原子力显微镜(AFM)研究了磷脂酰胆碱+/-胆固醇(哺乳动物)仿生膜与类固醇P450的相互作用。活细胞的FRET结果表明，P450c17和P450arom都是同源二聚体，但不是异二聚体，尽管它们都与CPR发生异二聚体。QCM证实P450c17和P450arom之间没有异源结合，两种酶都不能与饱和的膜结合。相反，CPR很容易结合到P450c17或P450arom饱和的表面上。有趣的是，N-末端修饰的P450arom被稳定地掺入，并给出了与野生型相似的结果，尽管用更少的蛋白质就能达到饱和，这表明假定的跨膜结构域不是膜结合所必需的，而是定位所必需的。事实上，所有的蛋白质在膜中都非常稳定，因此获得了高分辨率的AFM图像，进一步支持了P450c17、P450arom和CPR同源二聚体和低聚物在脂质双层中的形成。这种体内和体外研究的独特组合为P450c17和P450arom的同源二聚化提供了强有力的证据，也许还提供了一些更高级别的相互作用。
Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17 alpha-hydroxylase/17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET) 3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline +/- cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.