A new cell-based reporter system for sensitive screening of nuclear export inhibitors
A new cell-based reporter system for sensitive screening of nuclear export inhibitors
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DOI：
10.5582/ddt.2011.v5.6.286
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发表时间：
2011-01-01
影响因子：
3.1
通讯作者：
Kobayashi, Nobuyuki
中科院分区：
文献类型：
--
作者：
Abkallo, Hussein Molu;Kawano, Hiroaki;Kobayashi, Nobuyuki
细胞核定位信号（NLS）和核输出信号（NES）介导的蛋白质跨核孔复合物（NPC）的核质转运是真核细胞中重要的稳态过程，也是肿瘤细胞增殖中丝裂原活化蛋白激酶（MEK）信号分子的一个重要过程。有些病毒，包括流感病毒和艾滋病毒-1，在其生命周期中也采用这种核输出机制。因此，控制蛋白核胞质转运的药物被认为是抗病毒或抗癌药物的候选药物。因此，我们先前开发了GFP/NES-MDCK报告细胞系统，用于筛选新的核输出抑制剂。NES信号偶联的GFP在核输出抑制剂leptomycin B （LMB）存在的情况下在细胞核中积累。本研究建立了一个稳定的表达GFP/NLS/NES融合蛋白的细胞系，并对其作为报告细胞的潜力进行了评估。GFP/NLS/NES-MDCK细胞系在LMB的时间过程中显示出核中GFP积累的改善。此外，剂量效应数据显示GFP/NLS/NES-MDCK比GFP/NES-MDCK细胞更敏感。在GFP/NLS/NES-MDCK细胞中，低至0.01 ng/mL的LMB就足以使GFP融合蛋白在细胞核中积累，而在GFP/NES-MDCK细胞中，GFP融合蛋白在细胞核中积累至少需要1 ng/mL的LMB。这些结果表明，新建立的GFP/NLS/NES-MDCK细胞系是筛选新型核输出抑制剂的潜在有力工具。
Nucleocytoplasmic transport of proteins across the nuclear pore complex (NPC), mediated by the nuclear localization signal (NLS) and the nuclear export signal (NES), is a vital homeostatic process in eukaryotic cells and also in mitogen-activated protein kinase (MEK) signaling molecule in tumor cell proliferation. Some viruses, including the influenza virus and HIV-1, also employ this nuclear export mechanism during their life cycle. Hence, drugs that control nucleocytoplasmic transport of proteins are putative candidate antivirals or anti-cancer agents. Thus, we previously developed a GFP/NES-MDCK reporter cell system for screening novel nuclear export inhibitors. NES signal-conjugated GFP accumulates in the nucleus in the presence of the nuclear export inhibitor leptomycin B (LMB). In this study, a stable GFP/NLS/NES fusion protein-expressing cell line was established, and its potential as a reporter was evaluated. The GFP/NLS/NES-MDCK cell line demonstrates improved nuclear accumulation of GFP in a time-course treatment with LMB. In addition, the dose-response data demonstrated superior sensitivity of GFP/NLS/NES-MDCK over GFP/NES-MDCK cells. As low as 0.01 ng/mL LMB is sufficient to cause accumulation of the GFP fusion protein in the nucleus in GFP/NLS/NES-MDCK cells, while at least 1 ng/mL of LMB is needed for the accumulation of GFP fusion protein in the nucleus of GFP/NES-MDCK cells. These results indicate that the newly established GFP/NLS/NES-MDCK cell line is a potentially powerful tool to screen for novel nuclear export inhibitors.