Pernelle C, Clerc F F, Dureuil C, Bracco L, Tocque B
Laboratoire Oncologie Moléculaire, Rhone Poulenic Rorer SA, Vitry sur Seine, France.
Biochemistry. 1993 Nov 2;32(43):11682-7. doi: 10.1021/bi00094a026.
The protein products of the jun and fos oncogenes require a functional protein-protein interaction domain, called the "leucine zipper domain", to exert their transcriptional regulatory activity. A scintillation proximity assay was developed in which the biotinylated leucine zipper domain of the Jun protein (275-315) was immobilized on streptavidin-coated microfluorospheres and in which the leucine zipper domain of the Fos protein (160-200) was used as free, labeled ligand. The Fos leucine zipper peptide specifically bound to the Jun leucine zipper peptide, and for the first time, a dissociation constant (Kd = 110 +/- 12 nM in PBS/0.1% Tween) could be determined. Optimal heterodimer formation was reached at neutral pH. Both acidic and alkaline pH decreased the association of the peptides which was, furthermore, completely abolished by 500 mM NaCl, confirming that charged residues are critical for heterodimerization. A commercially obtained recombinant Jun protein competed as efficiently as the Jun leucine zipper peptide for binding to the Fos peptide, confirming the feasibility of using the two leucine zipper peptides to study the interactions between the two transcription factors. We also injected leucine zipper peptides individually into Xenopus oocytes to study whether they would interfere with the activity of the Fos/Jun heterodimer in vivo. Both peptides blocked selectively insulin-mediated oocyte maturation with an IC50 in the range of 15 ng per oocyte. In conclusion, the scintillation proximity assay described here may be used to investigate protein-protein interactions mediated by leucine zipper structures and to identify compounds that inhibit leucine zipper association.
原癌基因jun和fos的蛋白质产物需要一个功能性的蛋白质-蛋白质相互作用结构域，即“亮氨酸拉链结构域”，来发挥其转录调控活性。我们开发了一种闪烁邻近分析方法，其中Jun蛋白（275-315）的生物素化亮氨酸拉链结构域固定在链霉亲和素包被的微荧光球上，而Fos蛋白（160-200）的亮氨酸拉链结构域用作游离的、标记的配体。Fos亮氨酸拉链肽特异性结合Jun亮氨酸拉链肽，并且首次测定了解离常数（在PBS/0.1%吐温中Kd = 110 +/- 12 nM）。在中性pH条件下达到最佳异二聚体形成。酸性和碱性pH均降低了肽的结合，此外，500 mM NaCl完全消除了这种结合，证实带电残基对异二聚化至关重要。一种商业获得的重组Jun蛋白与Jun亮氨酸拉链肽一样有效地竞争与Fos肽的结合，证实了使用这两种亮氨酸拉链肽研究两种转录因子之间相互作用的可行性。我们还将亮氨酸拉链肽分别注射到非洲爪蟾卵母细胞中，以研究它们是否会在体内干扰Fos/Jun异二聚体的活性。两种肽均选择性地阻断胰岛素介导的卵母细胞成熟，IC50在每个卵母细胞15 ng的范围内。总之，本文所述闪烁邻近分析方法可用于研究由亮氨酸拉链结构介导的蛋白质-蛋白质相互作用，并鉴定抑制亮氨酸拉链结合的化合物。
