Matthews J R, Wakasugi N, Virelizier J L, Yodoi J, Hay R T
Division of Biochemistry and Molecular Biology, School of Biological and Medical Sciences, University of St Andrews, Fife, UK.
Nucleic Acids Res. 1992 Aug 11;20(15):3821-30. doi: 10.1093/nar/20.15.3821.
A role for redox regulation in activation of the NF-kappa B transcription factor was suggested by the observation that DNA binding activity of free protein, but not preformed DNA-protein complex, is inhibited by -SH modifying agents but enhanced by reducing agents. Mutagenesis of conserved cysteine residues in the p50 subunit identified amino acid 62 as being important for DNA binding, as a serine substitution at this position reduces DNA binding affinity, but renders the protein insensitive to -SH modifying agents. DNA binding activity of the wild type protein but not the amino acid 62 mutant was also stimulated by thioredoxin while detection of disulphide cross linked dimers in p50 but not the amino acid 62 mutant suggests that thioredoxin stimulates DNA binding by reduction of a disulphide bond involving cysteine 62. The physiological relevance of these findings was supported by the observation that cotransfection of a plasmid expressing human thioredoxin and an HIV LTR driven reporter construct resulted in an NF-kappa B dependent increase in expression of the reporter gene. Thus modification of p50 by thioredoxin, a gene induced by stimulation of T-lymphocytes in parallel with NF-kappa B translocation, is a likely step in the cascade of events leading to full NF-kappa B activation.
氧化还原调节在核因子-κB转录因子激活中的作用是基于以下观察结果提出的：游离蛋白的DNA结合活性可被-SH修饰剂抑制，但预先形成的DNA-蛋白复合物的DNA结合活性不受影响，而还原剂可增强游离蛋白的DNA结合活性。对p50亚基中保守半胱氨酸残基进行诱变，确定第62位氨基酸对DNA结合很重要，因为该位置的丝氨酸替代会降低DNA结合亲和力，但使蛋白质对-SH修饰剂不敏感。硫氧还蛋白可刺激野生型蛋白而非第62位氨基酸突变体的DNA结合活性，同时在p50中检测到二硫键交联二聚体，而在第62位氨基酸突变体中未检测到，这表明硫氧还蛋白通过还原涉及半胱氨酸62的二硫键来刺激DNA结合。共转染表达人硫氧还蛋白的质粒和HIV长末端重复序列驱动的报告构建体，导致报告基因的表达在核因子-κB依赖的情况下增加，这一观察结果支持了这些发现的生理相关性。因此，硫氧还蛋白对p50的修饰是导致核因子-κB完全激活的一系列事件中的一个可能步骤，硫氧还蛋白是一种与核因子-κB易位同时由T淋巴细胞刺激诱导的基因。
