A Bipartite Recombinant Yeast System for the Identification of Subtype-Selective Estrogen Receptor Ligands
A Bipartite Recombinant Yeast System for the Identification of Subtype-Selective Estrogen Receptor Ligands
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用于鉴定亚型选择性雌激素受体配体的二分重组酵母系统
DOI：
10.1007/s12033-008-9097-9
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发表时间：
2009-01-01
影响因子：
2.6
通讯作者：
Huang, Jian
中科院分区：
文献类型：
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作者：
Liang, Kaiwei;Yang, Liuqing;Huang, Jian
由于雌激素受体α（ER α）和β（ER β）被认为介导不同的生物学效应，因此对设计或筛选亚型选择性ER配体有强烈的兴趣。在此，我们构建了一个生物传感器鉴定为二分重组酵母系统（BRYS）筛选和评估亚型选择性ER配体。采用均匀设计和免疫印迹法确定通过铜诱导型金属硫蛋白启动子（CUP 1）控制ER表达的最佳铜剂量。一些化学品，包括天然雌激素（17 β-雌二醇），特异性雌激素受体激动剂（PPT和DPN），和植物雌激素（染料木素）进行了测试，以验证该系统。当这些化学物质被鉴定和表征时，在激动活性之间存在明显的和预期的区分。此外，拮抗剂（ICI 182，780），它可以拮抗雌激素的反式激活，染色质免疫沉淀（ChIP）被用来证实，激动作用是通过ER介导的。比较研究表明，该体系重现性好，灵敏度高。4.7 pM（1.3 ng/l）雌激素是可检测到的ER α和ER β的最低浓度，分别为0.12 nM（33.5 ng/l）。我们的研究结果表明，在体外筛选亚型选择性ER配体可以进行一个简单的酵母系统，是快速，灵敏，可靠，定量。
Since the estrogen receptor alpha (ER alpha) and beta (ER beta) are thought to mediate different biological effects, there is intense interest in designing or screening subtype-selective ER ligands. Here, we constructed a biosensor identified as bipartite recombinant yeast system (BRYS) to screen and evaluate subtype-selective ER ligands. Uniform design and immunoblotting was used to determine an optimal dose of copper which control the expression of ERs through a copper inducible metallothionine promoter (CUP1). Some chemicals including natural estrogen (17 beta-estradiol), specific estrogen receptor agonist (PPT and DPN), and phytoestrogens (genistein) were tested to validate this system. There was obvious and anticipative discrimination between the agonistic activities when these chemicals were identified and characterized. Furthermore, antagonist (ICI 182,780), which could antagonize the transactivation of estrogen, and chromatin immunoprecipitation (ChIP) were used to confirm that the agonistic effects were mediated through ERs. Comparative studies showed that this system was reproducible and sensitive. 4.7 pM (1.3 ng/l) estrogen was the lowest concentration that could be detected to ER alpha and 0.12 nM (33.5 ng/l) for ER beta. The results from our study showed that in vitro screening for subtype-selective ER ligands could be conducted in a simple yeast system that is rapid, sensitive, reliable, and quantitative.