Morel Y, Barouki R
INSERM U490, Université Paris V-René Descartes, Centre Universitaire des Saints-Pères, 45, rue des Saints-Pères, 75006 Paris, France.
Biochem J. 2000 May 15;348 Pt 1(Pt 1):235-40.
The activity of the nuclear factor I/CCAAT transcription factor (NFI/CTF) is negatively regulated by oxidative stress. The addition of relatively high (millimolar) H(2)O(2) concentrations inactivates cellular NFI DNA-binding activity whereas lower concentrations can repress NFI/CTF transactivating function. We have investigated the mechanism of this regulation using Gal4 fusion proteins and transfection assays. We show that micromolar H(2)O(2) concentrations repress the transactivating domain of NFI/CTF in a dose-dependent manner and are less or not active on other transcription factors' transactivating domains. Studies using deletions and point mutations pointed to the critical role of Cys-427. Indeed, when this cysteine is mutated into a serine, the repression by H(2)O(2) is totally blunted. Mutation of other cysteine, serine and tyrosine residues within the transactivating domain had no clear effect on the repression by H(2)O(2). Finally, treatment of cells with the thiol-alkylating reagent N-ethylmaleimide leads to a decrease in the transactivating function, which is dependent on Cys-427. This study shows that transactivating domains of transcription factors can constitute very sensitive targets of oxidative stress and highlights the critical role of these domains.
核因子I/CCAAT转录因子（NFI/CTF）的活性受到氧化应激的负调控。添加相对较高（毫摩尔）浓度的H₂O₂会使细胞NFI DNA结合活性失活，而较低浓度则可抑制NFI/CTF的反式激活功能。我们使用Gal4融合蛋白和转染试验研究了这种调控机制。我们发现微摩尔浓度的H₂O₂以剂量依赖的方式抑制NFI/CTF的反式激活结构域，而对其他转录因子的反式激活结构域作用较小或无作用。使用缺失和点突变的研究指出了Cys-427的关键作用。实际上，当这个半胱氨酸突变为丝氨酸时，H₂O₂的抑制作用完全消失。反式激活结构域内其他半胱氨酸、丝氨酸和酪氨酸残基的突变对H₂O₂的抑制作用没有明显影响。最后，用硫醇烷基化试剂N-乙基马来酰亚胺处理细胞会导致反式激活功能降低，这依赖于Cys-427。这项研究表明转录因子的反式激活结构域可能是氧化应激非常敏感的靶点，并突出了这些结构域的关键作用。
