Ligand-Mediated Receptor Assembly as an End Point for High-Throughput Chemical Toxicity Screening.
Ligand-Mediated Receptor Assembly as an End Point for High-Throughput Chemical Toxicity Screening.
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DOI：
10.1021/acs.est.7b02882
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发表时间：
2017-08-15
影响因子：
11.4
通讯作者：
LeBlanc GA
中科院分区：
文献类型：
--
作者：
Medlock Kakaley EK;Eytcheson SA;LeBlanc GA
高通量筛选与细胞内靶点相互作用的化学品在环境化学品的毒性评价中越来越突出。我们描述了配体介导的受体组装作为受体信号传导的早期事件及其在筛选与靶向受体相互作用的化学品中的应用。我们利用生物发光共振能量转移（BRET）来检测和量化响应于各种高产量和其他化学品的法尼酸甲酯受体（MfR）的组装。激素法呢酸甲酯与MfR结合，调节甲壳类动物生殖和发育的各个方面。将从蚤状蚤中克隆的MfR蛋白亚基Met和SRC分别与荧光团mAmetrine和光子发生器Rluc 2融合。配体介导的受体组装通过光子从光子供体转移到荧光团导致荧光发射来测量。总体而言，BRET测定与传统的报告基因测定相比具有相当或更高的灵敏度。此外，在BRET测定中筛选阳性的化学物质也刺激了由MfR信号传导引起的水蚤的表型结果。我们得出结论，BRET检测是一种准确、灵敏、成本/时间效率高的传统筛查检测替代方法。
The high throughput screening of chemicals for interaction with intracellular targets is gaining prominence in the toxicity evaluation of environmental chemicals. We describe ligand-mediated receptor assembly as an early event in receptor signaling and its application to the screening of chemicals for interaction with targeted receptors. We utilized bioluminescence resonance energy transfer (BRET) to detect and quantify assembly of the methyl farnesoate receptor (MfR) in response to various high-production volume and other chemicals. The hormone methyl farnesoate binds to the MfR to regulate various aspects of reproduction and development in crustaceans. The MfR protein subunits Met and SRC, cloned from Daphnia pulex, were fused to the fluorophore, mAmetrine and the photon generator, Rluc2, respectively. Ligand-mediated receptor assembly was measured by photon transfer from the photon donor to the fluorophore resulting in fluorescence emission. Overall, the BRET assay had comparable or greater sensitivity as compared to a traditional reporter gene assay. Further, chemicals that screened positive in the BRET assay also stimulated phenotypic outcomes in daphnids that result from MfR signaling. We concluded the BRET assay is an accurate, sensitive, and cost/time efficient alternative to traditional screening assays.
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DOI：
10.1668/0003-1569(2001)041
发表时间：
2001-04-01
期刊：
AMERICAN ZOOLOGIST
影响因子：
--
作者：
Kramer, DL;McLaughlin, RL
通讯作者：
McLaughlin, RL
影响因子：
10.4
作者：
Olmstead, AW;LeBlanc, GA
通讯作者：
LeBlanc, GA
影响因子：
8.8
作者：
Oda, S;Tatarazako, N;Iguchi, T
通讯作者：
Iguchi, T
影响因子：
3.4
作者：
Breton, Billy;Sauvageau, Etienne;Bouvier, Michel
通讯作者：
Bouvier, Michel
DOI：
10.1073/pnas.0609568104
发表时间：
2007-05-22
影响因子：
11.1
作者：
Kidd, Karen A.;Blanchfield, Paul J.;Flick, Robert W.
通讯作者：
Flick, Robert W.