Trinh Dan V, Zhu Norman, Farhang Ghazal, Kim Brian J, Huxford Tom
Structural Biochemistry Laboratory, Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, USA.
J Mol Biol. 2008 May 23;379(1):122-35. doi: 10.1016/j.jmb.2008.03.060. Epub 2008 Apr 3.
Although they share sequence homology to classical cytoplasmic I kappaB inhibitors of transcription factor NF-kappaB, the proteins I kappaB zeta, Bcl-3, and I kappa BNS function in the nucleus as factors that influence NF-kappaB-dependent gene expression profiles. Through the use of purified recombinant proteins and by comparison with the classical I kappaB protein I kappaB alpha, we have discovered mechanistic details of the interaction between I kappaB zeta and functional NF-kappaB dimers. Whereas I kappaB alpha and other classical I kappaB proteins bind tightly to NF-kappaB dimers that possess the p65 subunit, I kappaB zeta binds preferentially to NF-kappaB p50 homodimers. This altered specificity is particularly interesting in light of the fact that both NF-kappaB subunits exhibit high sequence and structural homology, while the I kappaB alpha and I kappaB zeta proteins are also conserved in primary amino acid sequence. We further show that I kappaB zeta is capable of forming a stable ternary complex with the NF-kappaB p50 homodimer and kappaB DNA. Again, this is a stark contrast from I kappaB alpha, which inhibits NF-kappaB p65 homodimer binding to NF-kappaB target DNA sequences. Removal of the DNA sequences flanking the NF-kappaB binding site does not directly affect the interaction of p50 and I kappaB zeta. Rather, we have discovered that the carboxy-terminal glycine-rich region of the NF-kappaB p50 homodimer is involved in mediating high-affinity binding of I kappaB zeta and NF-kappaB p50. We conclude that the NF-kappaB p50 homodimer functions as a legitimate activator of gene expression through formation of a ternary complex between itself, I kappaB zeta, and DNA. The requirement for formation of this complex could explain why the nuclear I kappaB protein I kappaB zeta is absolutely required for expression of the pluripotent pro-inflammatory cytokine interleukin-6 in peritoneal macrophages.
尽管IκBζ、Bcl-3和IκBNS蛋白与转录因子NF-κB的经典细胞质IκB抑制剂具有序列同源性，但它们在细胞核中作为影响NF-κB依赖性基因表达谱的因子发挥作用。通过使用纯化的重组蛋白并与经典的IκB蛋白IκBα进行比较，我们发现了IκBζ与功能性NF-κB二聚体之间相互作用的机制细节。IκBα和其他经典IκB蛋白紧密结合具有p65亚基的NF-κB二聚体，而IκBζ则优先结合NF-κB p50同二聚体。鉴于NF-κB的两个亚基都表现出高度的序列和结构同源性，而IκBα和IκBζ蛋白在一级氨基酸序列上也保守，这种改变的特异性尤其有趣。我们进一步表明，IκBζ能够与NF-κB p50同二聚体和κB DNA形成稳定的三元复合物。同样，这与IκBα形成鲜明对比，IκBα抑制NF-κB p65同二聚体与NF-κB靶DNA序列的结合。去除NF-κB结合位点两侧的DNA序列不会直接影响p50和IκBζ的相互作用。相反，我们发现NF-κB p50同二聚体的羧基末端富含甘氨酸区域参与介导IκBζ与NF-κB p50的高亲和力结合。我们得出结论，NF-κB p50同二聚体通过自身、IκBζ和DNA之间形成三元复合物作为基因表达的合法激活剂发挥作用。形成这种复合物的必要性可以解释为什么核IκB蛋白IκBζ对于腹膜巨噬细胞中多能促炎细胞因子白细胞介素-6的表达是绝对必需的。
