亮氨酸拉链
bZIP域
碱性螺旋-环-螺旋-亮氨酸拉链转录因子
ATF3
化学
转录因子
肽
亮氨酸
生物化学
拉链
爪蟾
分子生物学
生物
DNA结合蛋白
氨基酸
发起人
基因
基因表达
计算机科学
算法
作者
Christine Pernelle,François Clerc,Christine Dureuil,Laurent Bracco,Bruno Tocqué
出处
期刊:Biochemistry
[American Chemical Society]
日期:1993-01-26
卷期号:32 (43): 11682-11687
被引量:32
摘要
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.
科研通智能强力驱动
Strongly Powered by AbleSci AI