Huang C F, Wang Y C, Tsao D A, Tung S F, Lin Y S, Wu C W
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan.
J Biol Chem. 2000 Apr 21;275(16):12313-20. doi: 10.1074/jbc.275.16.12313.
The HCMV IE2 protein negatively autoregulates its own expression as well as represses the transactivation activity of p53. Using the repression domain of IE2 as bait in the yeast two-hybrid system, Nrf1 and Nrf2, members of the CNC-bZIP family, were found to be IE2-interacting proteins. Residues 331-448 encompassing the DNA-binding and the dimerization domains of Nrf1 are sufficient for the interaction. The interaction was further confirmed in vitro by a glutathione S-transferase pull-down assay and in vivo by co-immunoprecipitation. In transient transfection studies, transcription driven by six copies of an NF-E2 site or by chimeric proteins between the DNA-binding domain of LexA and members of the CNC-bZIP family is repressed by IE2. Importantly, the DNA binding activity of the Nrf1/MafK heterodimer is not impeded by IE2. In a parallel study, CNC-bZIP factors attenuate the negative autoregulation of IE2. The attenuation could be explained by the finding that Nrf1 functions alone and synergistically with its heterodimerization partner, MafK, in inhibiting the DNA binding activity of IE2. Taken together, these results demonstrate the existence of antagonism between members of the CNC-bZIP family and IE2.
人巨细胞病毒IE2蛋白对其自身表达具有负向自调控作用，同时还能抑制p53的反式激活活性。在酵母双杂交系统中，以IE2的抑制结构域为诱饵，发现CNC-bZIP家族成员Nrf1和Nrf2是与IE2相互作用的蛋白。包含Nrf1的DNA结合结构域和二聚化结构域的331 - 448位残基足以介导这种相互作用。通过谷胱甘肽S-转移酶下拉实验在体外进一步证实了这种相互作用，并通过共免疫沉淀在体内进行了验证。在瞬时转染研究中，由六个拷贝的NF-E2位点驱动的转录或由LexA的DNA结合结构域与CNC-bZIP家族成员之间的嵌合蛋白驱动的转录均受到IE2的抑制。重要的是，Nrf1/MafK异二聚体的DNA结合活性不受IE2的影响。在一项平行研究中，CNC-bZIP因子减弱了IE2的负向自调控作用。这种减弱作用可以通过以下发现来解释：Nrf1单独发挥作用，并与其异二聚化伴侣MafK协同抑制IE2的DNA结合活性。综上所述，这些结果证明了CNC-bZIP家族成员与IE2之间存在拮抗作用。
