Quantitative analysis of the interaction of constitutive androstane receptor with chemicals and steroid receptor coactivator 1 using surface Plasmon resonance biosensor systems : A case study of the Baikal seal (Pusa sibirica) and mouse.
Quantitative analysis of the interaction of constitutive androstane receptor with chemicals and steroid receptor coactivator 1 using surface Plasmon resonance biosensor systems : A case study of the Baikal seal (Pusa sibirica) and mouse.
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使用表面等离子共振生物传感器系统对组成型雄甾烷受体与化学物质和类固醇受体辅激活剂 1 的相互作用进行定量分析：贝加尔海豹（Pusa sibirica）和小鼠的案例研究。
DOI：
10.1093/toxsci/kfs288
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
H.
中科院分区：
文献类型：
--
作者：
Dau;P.T.;Sakai;H.;Hirano. M.;Ishibashi;H.;Tanaka;Y.;Kameda;K.;Fujino;T.;Kim;E,Y. and Iwata;H.
组成型雄甾烷受体（CAR）不仅具有高的基础转录活性，而且是一种配体依赖性转录因子。已知CAR在物种间表现出不同的配体谱。然而，化学物质激活CAR的机制和物种特异性反应尚未完全了解。本研究的目的是建立一个高通量的工具来筛选CAR配体，并阐明来自贝加尔海豹（bsCAR）和小鼠（mCAR）的CAR蛋白如何与化学物质和类固醇受体辅激活因子1（SRC 1）相互作用。我们开发了表面等离子体共振（SPR）系统来定量评估CAR与潜在配体和SRC 1的相互作用。bsCAR和mCAR的配体结合结构域（LBD）是在麦胚无细胞系统中合成的。然后将纯化的CAR LBD固定在传感器芯片上用于SPR测定，并测量汽车与配体候选物的直接相互作用的动力学。雄甾烷醇和雄烯醇、雌酮、17β-雌二醇、TCPOBOP和CITCO显示出对两种汽车的化合物特异性但相似的亲和力。CAR-SRC 1相互作用是配体依赖性的，但在海豹和小鼠之间表现出不同的配体特征。SRC 1相互作用实验的结果与我们先前的体外CAR介导的反式激活实验的结果一致，计算机模拟分析也支持CAR-SRC 1相互作用的结果;在bsCAR和mCAR的配体结合口袋中几乎没有结构差异，但是在这些受体的螺旋11和12中存在明显的区别，这表明配体结合的CAR和SRC 1的相互作用对于确定物种特异性和配体依赖性反式激活超过基础活性是关键的。SPR分析显示了作为CAR配体的高通量筛选工具的潜力。
The constitutive androstane receptor (CAR) not only displays a high basal transcriptional activity but also acts as a ligand-dependent transcriptional factor. It is known that CAR exhibits different ligand profiles across species. However, the mechanisms underlying CAR activation by chemicals and the species-specific responses are not fully understood. The objectives of this study are to establish a high-throughput tool to screen CAR ligands and to clarify how CAR proteins from the Baikal seal (bsCAR) and the mouse (mCAR) interact with chemicals and steroid receptor coactivator 1 (SRC1). We developed the surface plasmon resonance (SPR) system to assess quantitatively the interaction of CAR with potential ligands and SRC1. The ligand-binding domain (LBD) of bsCAR and mCAR was synthesized in a wheat germ cell–free system. The purified CAR LBD was then immobilized on the sensor chip for the SPR assay, and the kinetics of direct interaction of CARs with ligand candidates was measured. Androstanol and androstenol, estrone, 17β-estradiol, TCPOBOP, and CITCO showed compound-specific but similar affinities for both CARs. The CAR-SRC1 interaction was ligand dependent but exhibited a different ligand profile between the seal and the mouse. The results of SRC1 interaction assay accounted for those of our previousin vitroCAR-mediated transactivation assay.In silicoanalyses also supported the results of CAR-SRC1 interaction; there is little structural difference in the ligand-binding pocket of bsCAR and mCAR, but there is a distinct discrimination in the helix 11 and 12 of these receptors, suggesting that the interaction of ligand-bound CAR and SRC1 is critical for determining species-specific and ligand-dependent transactivation over the basal activity. The SPR assays demonstrated a potential as a high-throughput screening tool of CAR ligands.