Department of Anatomy and Cell Biology, College of Medicine, 107 Wiggins Rd., University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
Biochem Cell Biol. 2009 Dec;87(6):845-51. doi: 10.1139/o09-049.
Since Hsp90 is a known modulator of HSF1 activity, we examined the effects of two pharmacological inhibitors of Hsp90, novobiocin and geldanamycin, on HSF1 DNA-binding activity in the Xenopus oocyte model system. Novobiocin exhibits antiproliferative activity in culture cells and interacts with a C-terminal ATP-binding pocket on Hsp90, inhibiting Hsp90 autophosphorylation. Treatment of oocytes with novobiocin followed by heat shock results in a dose-dependent decrease in HSF1 DNA-binding and transcriptional activity. Immunoprecipitation experiments demonstrate novobiocin does not alter HSF1 activity through dissociation of Hsp90 from either monomeric or trimerized HSF1, suggesting that the effect of novobiocin on HSF1 is mediated through alterations in Hsp90 autophosphorylation. Geldanamycin binds the N-terminal ATPase site of Hsp90 and inhibits chaperone activity. Geldanamycin treatment of oocytes resulted in a dose-dependent increase in stability of active HSF1 trimers during submaximal heat shock and a delay in disassembly of trimers during recovery. The results suggest that Hsp90 chaperone activity is required for disassembly of HSF1 trimers. The data obtained with novobiocin suggests the C-terminal ATP-binding activity of Hsp90 is required for the initial steps of HSF1 trimerization, whereas the effects of geldanamycin suggest N-terminal ATPase and chaperone activities are required for disassembly of activated trimers. These data provide important insight into the molecular mechanisms by which pharmacological inhibitors of Hsp90 affect the heat shock response.
由于 Hsp90 是 HSF1 活性的已知调节剂，我们在非洲爪蟾卵母细胞模型系统中检查了两种 Hsp90 的药理学抑制剂（新生霉素和格尔德霉素）对 HSF1 DNA 结合活性的影响。新生霉素在培养细胞中表现出抗增殖活性，并与 Hsp90 的 C 端 ATP 结合口袋相互作用，抑制 Hsp90 自身磷酸化。用新生霉素处理卵母细胞，然后进行热休克，导致 HSF1 DNA 结合和转录活性呈剂量依赖性下降。免疫沉淀实验表明，新生霉素不会通过 Hsp90 从单体或三聚化 HSF1 解离来改变 HSF1 活性，这表明新生霉素对 HSF1 的影响是通过改变 Hsp90 自身磷酸化介导的。格尔德霉素结合 Hsp90 的 N 端 ATPase 位点并抑制伴侣活性。格尔德霉素处理卵母细胞导致在亚最大热休克期间活性 HSF1 三聚体的稳定性呈剂量依赖性增加，并且在恢复期间三聚体的解组装延迟。结果表明，Hsp90 伴侣活性是 HSF1 三聚体解组装所必需的。用新生霉素获得的数据表明 Hsp90 的 C 端 ATP 结合活性是 HSF1 三聚化初始步骤所必需的，而格尔德霉素的作用表明 N 端 ATPase 和伴侣活性是激活的三聚体解组装所必需的。这些数据为药理学抑制剂 Hsp90 影响热休克反应的分子机制提供了重要的见解。
