人热休克转录因子1功能反应与其半胱氨酸巯基氧化还原性能的相关性

Human heat shock transcription factor 1 (hHSF1) may have integrated redox chemistry of cysteine sulfhydryl into its functional responses

目的 评价半胱氨酸巯基氧化还原介导剂对人热休克转录因子 1(hHSF1)的氧化还原状态、结构和功能的作用 ,并试图去解释氧化作用在细胞衰老中对hHSF1转录反应的影响。 方法 通过改良的Westernblot方法检测不同氧化还原介导剂对hHSF1构象的影响 ;通过凝胶电泳迁移率变动试验检测hHSF1的DNA的结合活性。 结果 促进二硫键交联的氧化型介导剂二酰胺(DM )、一氧化氮 (NO+ )载体亚硝基谷胱甘肽 (GSNO)与含hHSF1的HeLa细胞S10 0提取液预培育 ,能在体外形成一个致密的、电泳中移动快速、分子内二硫键交联的氧化型hHSF1(ox hHSF1)单体构象 ;在电泳前加入还原型介导剂二硫苏糖醇 (DTT)能完全逆转。由于ox hHSF1形成 ,使hHSF1在体外不能被热诱导激活形成三体 ,失去和DNA结合的活性 ,而且这个作用仅对含半胱氨酸巯基的转录因子有特异性。 结论 hHSF1功能反应与其半胱氨酸巯基的氧化还原化学性能相关 ,氧化作用和hHSF1分子内二硫键交联有可能是衰老细胞热休克转录反应呈渐减性的原因。

Objective To evaluate the in vitro effects of cysteine-SH-directed reagents on the redox status, structure, and function of human heat shock transcription factor1 (hHSF1). Methods The effects of redox reagents on regulation of the conformation of hHSF1 were examined by modified western blot, and the hHSF1-DNA binding activity was determined by electrophoretic gel mobility assay. Results Preincubation of latent hHSF1 with diamide, an oxidizing reagent known to promotes disulfide cross-link, and S-nitrosoglutathione, a NO + carrier, caused in vitro the formation of a compact, intramolecularly disulfide cross-linked oxidized hHSF1 (ox-hHSF1). The effect of diamide was reversed by dithiothreitol added to the sample prior to gel electrophoresis. The disulfide cross-linked ox-hHSF1 was monomeric and resistant to the in vitro heat-induced trimerization and activation. This effect on hHSF1 and transcription factors with a highly conserved cysteine residue appeared to be specific. Conclusions The results suggest that redox-dependent thiol-disulfide exchange may provide a mechanism regulating the conformation and activity of hHSF1 and it is possible that oxidation and intramolecular disulfide cross-link of hHSF1 may contribute to the attenuated heat shock transcriptional response in aged human cell.