Searching for endogenous molecules that inhibit the Crml-dependent nuclear export system

寻找抑制 Crml 依赖性核输出系统的内源分子

• 批准号: 13672299
• 负责人: NUMAZAWA Satoshi
• 金额: $ 1.47万
• 依托单位: SHOWA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672299/
• 关键词: Leptomycin B; Bufalin; Crml; Nuclear export; PC12

Translocation of macromolecules thorough the nuclear pore is involved in growth, differentiation and functions of eukaryotic cells. Growing evidence indicates that a nuclear exporter Crml plays a central role in the nuclear export system, since Leptomycin B was found to inhibit function of nuclear export signal (NES) in the cytoplasmic protein. The present study was designed to examine a hypothesis that endogenous substances inhibiting Crml function exist, because it is known that LMB binding domain of Crml protein is highly conserved in different species. Based on the fact that the a, β-unsaturated lactone ling of LMB is essential for binding with Crml, we focused on a toad steroid bufalin which possesses similar chemical structure with LMB and has been suggested to exist in human plasma. Bufalin halted THP-1 leukemia cell growth at G2/M boundary as similar to LMB. To examine the effect of bufain on NES, we established HepG2 cells stably express NES-tagged green fluorescent protein (GFP-NES). GFP-NES distributed exclusively in cytoplasm of untreated cells and LMB induced nuclear accumulation of the tagged protein in all cells observed, indicating that this system can properly be applied for searching compounds inhibiting NES. However, bufalin did not induce nuclear accumulation of the fluorescence at any time points or concentrations examined, indicating that this steroid compound does not directly inhibit Crml function. We next screened 82 natural compounds and 32 known kinase inhibitors or anticancer drugs using GFP-NES expressed HepG2 cells. Although there is no compound that inhibits NES as potent as LMB, kinase inhibitors staurosporine and K252a, progesterone, and a ginger component 8-shogaol induced nuclear accumulation of GFP-NES in parallel with cell death. It is, therefore, suggested that these compounds might inhibit the Crml-dependent nuclear export.

大分子通过核孔的转运参与真核细胞的生长、分化和功能。越来越多的证据表明，核输出蛋白Crml在核输出系统中起着核心作用，因为发现Leptomycin B抑制胞质蛋白中的核输出信号（内斯）的功能。本研究旨在验证存在抑制Crml功能的内源性物质的假设，因为已知Crml蛋白的LMB结合结构域在不同物种中高度保守。基于LMB的α，β-不饱和内酯环是与Crml结合所必需的这一事实，我们重点研究了与LMB具有相似化学结构并已被认为存在于人血浆中的蟾蜍甾体蟾毒灵。蟾毒灵在G2/M期终止THP-1白血病细胞生长，与LMB相似。为了检测蟾毒肽对内斯的影响，我们建立了稳定表达NES标记的绿色荧光蛋白（GFP-内斯）的HepG 2细胞。GFP-内斯仅分布于未处理细胞的细胞质中，LMB诱导标记蛋白在所有观察到的细胞中的核积累，表明该系统可以适当地应用于寻找抑制内斯的化合物。然而，蟾蜍灵在任何时间点或检测的浓度下都不诱导荧光的核积累，表明这种类固醇化合物不直接抑制Crml功能。接下来，我们使用GFP-NES表达的HepG 2细胞筛选了82种天然化合物和32种已知的激酶抑制剂或抗癌药物。尽管没有化合物能像LMB一样有效地抑制内斯，但激酶抑制剂星形孢菌素和K252 a、孕酮和生姜组分8-姜烯酚诱导GFP-内斯的核积累，同时细胞死亡。因此，这表明，这些化合物可能会抑制依赖于Crm 1的核输出。

项目成果

S.Numazawa, M.Watabe, S.Nishimura, M.Kurosawa, M.Izuno, T.Yoshida: "Regulation of ERK-mediated signal transduction by p38-MAP kinase in human monocytic THP-1 cells"J. Biochem.. (In press).

S.Numazawa、M.Watabe、S.Nishimura、M.Kurosawa、M.Izuno、T.Yoshida：“人单核细胞 THP-1 细胞中 p38-MAP 激酶调节 ERK 介导的信号转导”J。

S. Numazawa, M. Watabe, S. Nishinmura, M. Kurosawa, M. Izuno, & T. Yoshida: "Regulation of ERK-mediated signal transduction by p38-MAP kinase in human monocytic THP-1 cells"J. Biochem. in press.

S.沼泽、M.渡部、S.西村、M.黑泽、M.伊豆野、

S.Numazawa, M.Watabe, S.Nishinmura, M.Kurosawa, M.Izuno, T.Yoshida: "Regulation of ERK-mediated signal transduction by p38-MAP kinase in human monocytic THP-1 cells"J. Biochem.. (In press).

S.Numazawa、M.Watabe、S.Nishinmura、M.Kurosawa、M.Izuno、T.Yoshida：“人单核 THP-1 细胞中 p38-MAP 激酶调节 ERK 介导的信号转导”J。