Geldanamycin-associated inhibition of intracellular trafficking is attributed to a co-purified activity
Geldanamycin-associated inhibition of intracellular trafficking is attributed to a co-purified activity
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DOI：
10.1074/jbc.m312799200
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发表时间：
2004-02-20
影响因子：
4.8
通讯作者：
Neumann, D
中科院分区：
文献类型：
--
作者：
Barzilay, E;Ben-Califa, N;Neumann, D
格尔达霉素是一种特异性抑制热休克蛋白-90 （HSP90）及其内质网同源物葡萄糖调节蛋白-94 （GRP94）的安霉素抗生素，可加速特定细胞蛋白的降解。我们之前的研究表明，格尔达霉素除了加速表皮生长因子受体的降解外，还能抑制其成熟和转运(Supino-Rosin， L., Yoshimura， A., Yarden， Y., Elazar， Z., and Neumann， D. (2000) J. Biol。化学，275,21850-21855)。在这里，我们证明了格尔达霉素对细胞内运输和蛋白质成熟的额外活性与其供应来源有关。通过结合化学分离的吸湿链霉菌（Streptomyces vars . geldanus）提取物和生物筛选，我们发现geldanamycin对细胞内运输和蛋白质成熟的相关作用不是由geldanamycin本身介导的，而是由于存在额外的成分。在硅胶柱上对吸湿葡萄树提取物进行色谱分析，分离了对细胞内运输的抑制作用和对格尔达霉素介导的降解作用。一个不含格尔达霉素的部分阻断了一种可溶性促红细胞生成素受体的分泌，延缓了表皮生长因子受体的成熟，但没有增强其降解，并阻断了早期高尔基池中水疱性口炎病毒G蛋白（VSVGtsO45）的温度敏感突变体的顺行运输。该部分在17-去甲基格尔达霉素中富集（bb0 95%）。然而，由于合成衍生的17-去甲基格尔达霉素不抑制细胞内运输，我们得出结论，17-去甲基格尔达霉素不是活性成分。因此，我们提出一种与苯醌类ansamycin共纯化的化合物可抑制细胞内转运。综上所述，我们的数据表明，先前归因于格尔达霉素的蛋白质成熟和细胞内运输的抑制作用是由另一个不同的片段介导的。
Geldanamycin, an ansamycin antibiotic that specifically inhibits heat-shock protein-90 (HSP90) and its endoplasmic reticulum homologue, glucose-regulated protein-94 (GRP94), accelerates the degradation of selected cellular proteins. We showed previously that geldanamycin inhibits maturation and transport of the epidermal growth factor receptor in addition to accelerating its degradation (Supino-Rosin, L., Yoshimura, A., Yarden, Y., Elazar, Z., and Neumann, D. (2000) J. Biol. Chem. 275, 21850-21855). Here we demonstrate that the additional activities of geldanamycin on intracellular transport and protein maturation are related to its supply source. By combining chemical separation of Streptomyces hygroscopicus var. geldanus extracts and biological screens, we show that the geldanamycin-associated effects on intracellular transport and protein maturation are not mediated by geldanamycin itself but are due to the presence of an additional component(s). Chromatography of S. hygroscopicus var. geldanus extracts on a silica-gel column allowed separation between the inhibition of intracellular trafficking and geldanamycin-mediated degradation. One fraction that was devoid of geldanamycin blocked secretion of a soluble form of the erythropoietin receptor, retarded maturation of the epidermal growth factor receptor without enhancing its degradation, and blocked anterograde transport of a temperature-sensitive mutant of the vesicular stomatitis virus G protein (VSVGtsO45) from the early Golgi cisternae. This fraction was enriched (> 95%) in 17-demethylgeldanamycin. However, as synthetically derived 17-demethylgeldanamycin did not inhibit intracellular trafficking, we concluded that 17-demethylgeldanamycin is not the active component. We thus propose that a compound(s) that co-purifies with benzoquinone ansamycins inhibits intracellular transport. Taken together, our data demonstrate that the inhibitory effects on protein maturation and intracellular trafficking, previously attributed to geldanamycin, are mediated by another distinct moiety.