An HTS approach to screen for antagonists of the nuclear export machinery using high content cell-based assays
An HTS approach to screen for antagonists of the nuclear export machinery using high content cell-based assays
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DOI：
10.1089/adt.2007.058
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发表时间：
2007-06-01
影响因子：
1.8
通讯作者：
Link, Wolfgang
中科院分区：
文献类型：
--
作者：
Zanella, Fabian;Rosado, Aranzazu;Link, Wolfgang
细胞内定位对于许多与疾病相关途径相关的信号分子的调节活性是必不可少的。高含量筛选是监测小分子或干扰rna对完整细胞内蛋白质易位影响的有力技术。已经开发了几种检测方法来测量核因子κ B、FoxO或活化t细胞核因子等蛋白的核细胞质穿梭，这些蛋白参与不同的信号网络。然而，由于所有这些蛋白质都携带富含亮氨酸的核输出信号（NES），依赖于NES的输出机制的调节剂可能导致分析读数的误解。在这里，我们报道了U2nesRELOC的产生，这是一个基于细胞的系统，用于识别核出口抑制剂和核出口机制的特异性沉默者，以及它对高通量筛选的适应性。该试验是基于稳定表达绿色荧光蛋白（GFP）标记的Rev蛋白的哺乳动物细胞，该蛋白含有很强的外源NES。未经处理的U2nesRELOC细胞的荧光信号仅局限于细胞质。用核输出抑制剂leptomycin B处理后，GFP标记的报告蛋白在细胞核中迅速积累。该分析已适应96个多井格式，并且完全自动化。对50种测试化合物使用三种不同浓度的先导实验产生了非常一致的数据集，具有出色的可重复性，平均Z'值为0.76。总之，U2nesRELOC是一种基于细胞的核输出试验，适用于高通量筛选，为途径反卷积提供反屏蔽。
Intracellular localization is essential for the regulated activity of many signaling molecules associated with disease- relevant pathways. High content screening is a powerful technology to monitor the impact of small molecules or interfering RNAs on translocation of proteins within intact cells. Several assays have been developed to measure the nucleocytoplasmic shuttling of proteins like nuclear factor kappa B, FoxO, or nuclear factor of activated T-cells involved in distinct signaling networks. However, since all these proteins bear a leucine-rich nuclear export signal (NES), modulators of the NES-dependent export machinery can lead to misinterpretation of the assay readout. Here we report the generation of U2nesRELOC, a cell-based system for the identification of nuclear export inhibitors and specific silencers of the nuclear export machinery, and its adaptation to high throughput screening. The assay is based on mammalian cells stably expressing green fluorescent protein (GFP)-labeled Rev protein, which contains a strong heterologous NES. The fluorescent signal of untreated U2nesRELOC cells localizes exclusively to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B the GFP- labeled reporter protein accumulates rapidly in the cell nucleus. The assay has been adapted to 96-multiwell format and fully automated. Pilot experiments with a panel of 50 test compounds using three different concentrations per compound resulted in very consistent data sets with excellent reproducibility and an average Z' value of 0.76. In summary, U2nesRELOC is a cell-based nuclear export assay suitable for high throughput screening, providing counterscreens for pathway deconvolution.