Identification of novel proteins that interact with trans-activation domain of transcription factors

我们构建了一个表达vp16融合P53蛋白的表达结构，然后使用哺乳动物双杂交方法（M2H）探索相互作用伙伴（gal4融合蛋白）。从荧光素酶报告活性判断，我们发现了两个重要的候选伴侣；一个是已知的P53结合伙伴mdm2，另一个是17 kDa的蛋白，没有已知的功能。我们尝试使用标记蛋白的共免疫沉淀来证实M2H的结果，尽管我们没有证实后者的关联。由于我们开发了一种新的快速体外下拉测定方法，我们应用该方法进行了验证。然而，我们无法证实后一种相互作用。接下来，我们系统地探索了小鼠转录因子之间的蛋白-蛋白相互作用。为此，我们使用了大约1,700个小鼠cdna作为转录因子/转录调节因子。自激活实验表明，14%的转录因子作为gal4融合蛋白具有反式激活活性，显著高于其他蛋白的2-3%的反式激活活性。我们发现大约有4000种蛋白质与蛋白质之间通过M2H相互作用。结果表明，每720个试验中发现一个相互作用，也显著高于正常情况（千分之一）。通常，双混合数据中存在许多假阳性相互作用。因此，我们使用上述体外下拉试验验证了我们的结果；对于已知的相互作用，验证的成功率非常高（90%），对于未知的相互作用，验证也部分成功（60%）。因此，我们可以尽可能多地提取真阳性蛋白-蛋白相互作用。

We made an expression construct that expresses VP16-fused P53 protein followed by exploration of the interaction partners (Gal4-fusion proteins) using the mammalian two-hybrid methods (M2H). We detected two significant partner candidates judging from luciferase reporter activity ; one was mdm2, a known P53 binding partner, and the other was a 17 kDa protein without known functions. We tried co-immuno-precipitation using the tagged proteins to confirm the M2H result, although we did not confirm the latter association. Because we developed a novel method for rapid in vitro pull-down assay, we applied the method for the confirmation. However, we could not confirm the latter interaction.Next we systematically explored protein-protein interactions among mouse transcription factors. For this purpose, we used approximately 1,700 mouse cDNAs for transcription factors/transcription regulation factors. The self-activation experiment showed that 14% of transcription factors have trans-activation activity as Gal4-fusion proteins, which was significantly higher than 2-3% of trans-activation activity for other proteins. We found approximately 4,000 protein-protein interactions using the M2H. The result showed that one interaction was identified per 720 trials, which was also significantly higher value than normal case (one per thousands). Generally, there are many false-positive interactions in the two-hybrid data. Therefore, we validated our result using in vitro pull-down assay described above ; the validation was successful with very high rate (90%) for the publicly known interactions, and was also partially successful (60%) for the unknown interactions. Thus we could extract the true-positive protein-protein interactions as much as possible.