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

大分子通过核孔的移位参与了真核细胞的生长、分化和功能。越来越多的证据表明，核输出蛋白Crm1在核输出系统中起着核心作用，因为Leptomycin B被发现抑制细胞质蛋白中核输出信号(NES)的功能。本研究旨在验证一种假说，即存在抑制CrML功能的内源性物质，因为已知CrML蛋白的LMB结合域在不同物种中高度保守。根据LMB的α，β-不饱和内酯是与CrML结合所必需的事实，我们重点研究了一种与LMB具有相似化学结构的蟾酥类固醇蟾酥，并推测它存在于人血浆中。与LMB类似，蟾酥能抑制THP-1白血病细胞在G2/M交界处的生长。为了检测蟾酥对NES的影响，我们建立了稳定表达NES标记的绿色荧光蛋白(GFP-NES)的HepG2细胞。GFP-NES仅分布在未处理细胞的胞浆中，LMB可诱导标记蛋白在所有细胞中聚集，表明该体系可用于寻找抑制NES的化合物。然而，在所考察的任何时间点或浓度下，蟾酥灵都没有诱导细胞核内的荧光聚集，表明这种类固醇化合物并不直接抑制CrML的功能。接下来，我们使用GFP-NES表达的HepG2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。虽然没有一种化合物能像LMB那样有效地抑制NES，但激酶抑制剂星形孢子素和K252a、孕酮和生姜成分8-shogaol诱导GFP-NES的核积聚与细胞死亡平行。因此，我们认为这些化合物可能抑制了crml依赖的核输出。

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.