We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 microM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 microM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase.

中文翻译：

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Tryprostatin A，一种微管装配的特异性和新型抑制剂。

我们研究了从烟曲霉纯化的胰蛋白酶抑制素A（TPS-A）的细胞周期抑制机制和主要靶标。TPS-A以剂量和时间依赖性的方式抑制了M相中异步培养的3Y1细胞的细胞周期进程。相反，TPS-B（TPS-A的脱甲氧基类似物）显示出细胞周期对细胞生长的非特异性抑制，即使它在低于TPS-A的浓度下也能抑制细胞生长。通过间接免疫荧光显微镜观察，TPS-A处理可诱导3Y1细胞胞质微管的可逆破坏，其浓度范围可特异性抑制M期进程。TPS-A抑制从牛脑纯化的微管的体外组装（在250 microM时抑制40％）；然而，当谷氨酸诱导聚合反应时，即使在250 microM TPS-A下，对纯化的微管蛋白的自组装几乎没有影响。TPS-A不抑制由紫杉醇或微管蛋白C末端结构域促进的组装。但是，TPS-A阻断了由与C末端域，微管相关蛋白2（MAP2），tau和聚-（1-赖氨酸）相互作用的诱导物诱导的微管蛋白装配。这些结果表明，TPS-A是MAP依赖的微管组装的新型抑制剂，并且通过破坏微管纺锤体，特异性抑制M期的细胞周期进程。TPS-A阻断了由与C末端结构域，微管相关蛋白2（MAP2），tau和聚-（1-赖氨酸）相互作用的诱导物诱导的微管蛋白装配。这些结果表明，TPS-A是MAP依赖的微管组装的新型抑制剂，并且通过破坏微管纺锤体，特异性抑制M期的细胞周期进程。TPS-A阻断了由与C末端结构域，微管相关蛋白2（MAP2），tau和聚-（1-赖氨酸）相互作用的诱导物诱导的微管蛋白装配。这些结果表明，TPS-A是MAP依赖的微管组装的新型抑制剂，并且通过破坏微管纺锤体，特异性抑制M期的细胞周期进程。