Basis of a high-throughput method for nuclear receptor ligands.
Basis of a high-throughput method for nuclear receptor ligands.
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DOI：
10.1093/jb/mvg101
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发表时间：
2003-06
影响因子：
2.7
通讯作者：
Tomohiko Kanayama;S. Mamiya;T. Nishihara;J. Nishikawa
中科院分区：
文献类型：
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作者：
Tomohiko Kanayama;S. Mamiya;T. Nishihara;J. Nishikawa
评估人类接触与激素受体结合的人造化学品的风险已成为一个重大的公共卫生问题。在激素受体中，核受体往往是外源药物的靶标，因为它们的内源性配体是小的脂溶性分子。核受体是配体诱导的转录因子，调控多种靶基因的转录活性。在转录起始阶段，核受体以激动剂依赖的方式与共激活因子（TIF2、SRC1、ACTR、CBP/p300等）相互作用。利用核受体与辅激活剂的相互作用，我们开发了一种易于使用且灵敏度高的新型快速配体体外筛选方法。该方法被我们称为CoA-BAP系统，适用于大多数核受体，并且由于整个实验操作可以在微孔板上进行，因此适合于高通量筛选。我们使用人类TIF2作为共激活因子，包括在大肠杆菌中作为BAP融合蛋白表达的LXXLL基序和在大肠杆菌中作为GST融合蛋白表达的核受体LBD。在gsh偶联的微孔板上，将这些蛋白与化学物质孵育，以碱性磷酸酶活性检测蛋白质与蛋白质的相互作用。到目前为止，我们已经检测了七种核受体（er α / β, trα, rar α / γ， rxr α和VDR），并证实该方法效果良好。
Assessment of the risk of human exposure to man-made chemicals that bind to hormone receptors has emerged as a major public health issue. Among hormone receptors, nuclear receptors tend to be targets of xenobiotics because their endogenous ligands are small, fat-soluble molecules. Nuclear receptors are ligand-inducible transcriptional factors and regulate the transcriptional activity of various target genes. At the start of the initiation step of transcription, nuclear receptors interact with coactivators (TIF2, SRC1, ACTR, CBP/p300, etc.) in an agonist-dependent manner. Using the interaction of the nuclear receptor with a coactivator, we have developed a novel rapid ligand in vitro screening method that is easy to use and has high sensitivity. This method, called by us the CoA-BAP system, is applicable to most nuclear receptors and is suitable for high-throughput screening because the entire experimental operation can be carried out on a microplate. We used human TIF2 as a coactivator including LXXLL motifs expressed in Escherichia coli as a fusion protein with BAP and nuclear receptor LBD expressed in E. coli as a fusion protein with GST. On a GSH-coupled microplate these proteins were incubated with chemicals and the protein-protein interactions were detected as alkaline phosphatase activity. To date we have examined seven nuclear receptors (ERalpha/beta, TRalpha, RARalpha/gamma, RXRalpha,and VDR) and confirmed that the method works well.