谷胱甘肽合成抑制对人热激因子1结合DNA功能的影响

The Effect of Inhibition of GSH Synthesis on hHSF1-DNA Binding Activity

【目的】研究细胞内具抗氧化作用的谷胱甘肽合成受抑制对人热激因子1结合DNA功能的影响。【方法】用谷胱甘肽合成酶抑制剂丁硫堇处理培养中的HeLa细胞,降低细胞内的谷胱甘肽水平,通过非变性聚丙烯酰胺凝胶电泳和Westernblot检测细胞谷胱甘肽缺乏时经热激处理后人热激因子1的氧化还原构象改变;通过凝胶电泳迁移率试验检测其与DNA结合的活性变化。【结果】丁硫堇处理后的HeLa细胞,经热激后出现一种在电泳中移动快速、结构致密、分子内二硫键交联的氧化型热激因子1;凝胶电泳迁移率试验结果显示热激因子1与DNA结合活性降低。【结论】谷胱甘肽在维持细胞的氧化还原状态中起重要作用,谷胱甘肽缺乏导致细胞内氧化型热激因子1蓄积,从而降低热激因子1与DNA结合的功能。

To evaluate the effect of inhibition of glutathione synthesis on hHSF1 DNA binding activity.HeLa cells were incubated with 1 mmol/L buthionine sulfoximine to block γ glutathione synthesis and deplete intracellular glutathine. Redox conformers of hHSF1 were detected by native gel electrophoresis and immuno Western blot probing for hHSF1 and the hHSF1 DNA binding activity was detected by electrophoretic gel mobility assay.Inhibition of glutathione synthesis in HeLa cells by buthionine sulfoximine(BSO) promoted the formation of ox hHSF1 and blocked the heat induced activation of HSE binding activity.[Conclusion]The results suggest that intracellular glutathione may support an important role in the cellular redox and the accumulation of ox hHSF1 in the HeLa cells.