Splicing factor SF3b as a target of the antitumor natural product pladienolide
Splicing factor SF3b as a target of the antitumor natural product pladienolide
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DOI：
10.1038/nchembio.2007.16
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发表时间：
2007-09-01
影响因子：
14.8
通讯作者：
Mizui, Yoshiharu
中科院分区：
文献类型：
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作者：
Kotake, Yoshihiko;Sagane, Koji;Mizui, Yoshiharu
普拉地那肽是一种天然存在的抗肿瘤大环内酯类药物，通过使用由人血管内皮生长因子启动子控制的基于细胞的报告基因表达试验发现（1，2）。尽管普拉二烯内酯B和D在不同的体外和体内系统中具有独特的作用机制（3）和显著的抗肿瘤活性（3），但其靶蛋白仍不清楚。我们使用H-3标记、荧光标记和光亲和/生物素（PB）标记的“化学探针”来鉴定剪接因子SF 3b中的140 kDa蛋白质作为普拉地内酯的结合靶点。SF 3 b亚基3（SAP 130）的增强型绿色荧光蛋白融合蛋白的免疫印迹显示PB探针和SAP 130之间的直接相互作用。普拉地辛衍生物与SF 3b复合物的结合亲和力与其对报告基因表达和细胞增殖的抑制活性高度相关。此外，普拉地那肽B以剂量依赖性方式损害体内剪接。我们的研究结果表明，SF 3b复合物是普拉地那肽的一个剪接相关的蛋白质靶点，并表明该剪接因子是一个潜在的抗肿瘤药物靶点。
Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter(1,2). Despite the unique mechanisms of action(3) and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems(3), their target protein has remained unclear. We used H-3-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.