Rabindran S K, Wisniewski J, Li L, Li G C, Wu C
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
Mol Cell Biol. 1994 Oct;14(10):6552-60. doi: 10.1128/mcb.14.10.6552-6560.1994.
The intracellular level of free heat shock proteins, in particular the 70-kDa stress protein family, has been suggested to be the basis of an autoregulatory mechanism by which the cell measures the level of thermal stress and regulates the synthesis of heat shock proteins. It has been proposed that the DNA-binding and oligomeric state of the heat shock transcription factor (HSF) is a principal step in the induction pathway that is responsive to the level of 70-kDa stress protein. To test this hypothesis, we investigated the association between HSF and 70-kDa stress protein by means of a coimmunoprecipitation assay. We found that 70-kDa stress proteins associate to similar extents with both latent and active forms of HSF, although unlike other 70-kDa stress protein substrates, the association with HSF was not significantly disrupted in the presence of ATP. Gel mobility shift assays indicated that active HSF trimers purified from a bacterial expression system could not be substantially deactivated in vitro with purified 70-kDa stress protein and ATP. In addition, elevated concentrations of hsp70 alone could not significantly inhibit induction of the DNA-binding activity of endogenous HSF in cultured rat cells, and the induction was also not inhibited in cultured rat cells or Drosophila cells containing elevated levels of all members of the heat shock protein family. However, the deactivation of HSF to the non-DNA-binding state after prolonged heat stress or during recovery could be accelerated by increased levels of heat shock proteins. Hence, the level of heat shock proteins may affect the rate of disassembly of HSF trimers, but another mechanism, as yet undefined, appears to control the onset of the oligomeric transitions.
细胞内游离热休克蛋白的水平，尤其是70 kDa应激蛋白家族，被认为是一种自动调节机制的基础，通过这种机制细胞可以测量热应激水平并调节热休克蛋白的合成。有人提出，热休克转录因子（HSF）的DNA结合和寡聚状态是诱导途径中的一个主要步骤，该途径对70 kDa应激蛋白的水平有反应。为了验证这一假设，我们通过共免疫沉淀试验研究了HSF与70 kDa应激蛋白之间的关联。我们发现，70 kDa应激蛋白与HSF的潜伏形式和活性形式的结合程度相似，尽管与其他70 kDa应激蛋白底物不同，在ATP存在的情况下，与HSF的结合并未被显著破坏。凝胶迁移率变动分析表明，从细菌表达系统中纯化的活性HSF三聚体在体外不能被纯化的70 kDa应激蛋白和ATP大量失活。此外，单独提高hsp70的浓度并不能显著抑制培养的大鼠细胞中内源性HSF的DNA结合活性的诱导，并且在含有热休克蛋白家族所有成员水平升高的培养大鼠细胞或果蝇细胞中，诱导也未被抑制。然而，长时间热应激后或恢复过程中HSF向非DNA结合状态的失活可因热休克蛋白水平的升高而加速。因此，热休克蛋白的水平可能会影响HSF三聚体的解体速率，但另一种尚未明确的机制似乎控制着寡聚体转变的开始。
