A genetically encoded indicator for assaying bioactive chemicals that induce nuclear transport of glucocorticoid receptor
A genetically encoded indicator for assaying bioactive chemicals that induce nuclear transport of glucocorticoid receptor
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DOI：
10.1016/j.ab.2005.09.011
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发表时间：
2005-12-15
影响因子：
2.9
通讯作者：
Umezawa, Y
中科院分区：
文献类型：
--
作者：
Kim, SB;Ozawa, T;Umezawa, Y
糖皮质激素是在压力下分泌的肾上腺类固醇激素，对人体内的稳态和代谢至关重要。由于合成化学物质引起的糖皮质激素受体（GR）功能障碍而导致的糖皮质激素信号传导受损可引起疾病以及体内平衡和代谢的破坏。在这里，我们展示了一种方法的开发，用于筛选内分泌干扰化学品和人类疾病的潜在危险因素的基础上的GR的核贩运。我们构建了一个新的检测使用一对遗传指标与全长的GR，分裂海肾荧光素酶（RLuc），分裂DnaE（蛋白质剪接元件）。具有DnaE和RLuc的C-末端一半的含GR的融合蛋白由于GR的胞质性质而定位在胞质溶胶中，而具有DnaE和RLuc的N-末端一半的融合蛋白由于共融合的核定位信号而停留在核中。在用配体刺激时，GR移位到细胞核中。因此，通过每个DnaE片段的剪接点之间的相互作用，在细胞核中发生蛋白质剪接。来自重构RLuc的酶活性允许配体依赖性发光强度。该方法的可行性进行了评估，通过定量的激素活性的20种不同的类固醇和合成化学品使用的NTH 3 T3细胞携带的对指示剂。根据化学构效关系讨论了受试配体的激素活性。我们发现，雄激素，睾酮和19-去甲睾酮弱诱导GR的核转运。目前的测定允许高通量筛选的风险化学品和候选药物的影响GR的信号转导途径。（c）2005年爱思唯尔公司。All rights reserved.
Glucocorticoids, the adrenal steroid hormones secreted during stress, are essential to homeostasis and metabolism in the human body. An impaired glucocorticoid signaling due to dysfunction of the glucocorticoid receptor (GR) by synthetic chemicals can cause diseases and disruptions of the homeostasis and metabolism. Here we demonstrate the development of a method for screening endocrine-disrupting chemicals and potent risk factors of human diseases based on the nuclear trafficking of the GR. We constructed a new assay using a pair of genetic indicators with the full length of the GR, split Renilla luciferase (RLuc), and split DnaE (a protein splicing element). The GR-containing fusion protein with C-terminal halves of DnaE and RLuc is localized in cytosol due to the cytosolic character of the GR, whereas the fusion protein with N-terminal halves of DnaE and RLuc stays in the nucleus due to the cofused nucleus localization signal. On being stimulated with a ligand, the GR is translocated into the cellular nucleus. Thus, a protein splicing occurs in the nucleus by an interaction between the splicing junctions of each DnaE fragment. The enzymatic activities from the reconstituted RLuc allow the ligand-dependent luminescence intensities. The feasibility of the method was evaluated by quantifying the hormonal activities of 20 different kinds of steroids and synthetic chemicals using the NTH 3T3 cells carrying the pair of indicators. The hormonal activities of tested ligands are discussed based on the chemical structure-activity relationship. We found that androgens, testosterone, and 19-nortestosterone weakly induce the nuclear transport of the GR. The current assay allows high-throughput screening of risk chemicals and drug candidates influential to a signal transduction pathway of the GR. (c) 2005 Elsevier Inc. All rights reserved.