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Detecting protein–protein interactions based on kinase-mediated growth induction of mammalian cells

机译:基于激酶介导的哺乳动物细胞生长诱导检测蛋白质之间的相互作用

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摘要

Detection of protein–protein interactions (PPIs) is important for understanding numerous processes in mammalian cells; however, existing PPI detection methods often give significant background signals. Here, we propose a novel PPI-detection method based on kinase-mediated growth induction of mammalian cells. In this method, target proteins are fused to the intracellular domain of c-kit (c-kit ICD) and expressed in interleukin-3-dependent mammalian cells. The PPI induces dimerization and activation of c-kit ICDs, which leads to cell growth in the absence of interleukin-3. Using this system, we successfully detected the ligand-dependent homo-interaction of FKBPF36V and hetero-interaction of FKBP and FRBT2098L, as well as the constitutive interaction between MDM2 and a known peptide inhibitor. Intriguingly, cells expressing high-affinity peptide chimeras are selected from the mixture of the cell populations dominantly expressing low-affinity peptide chimeras. These results indicate that this method can detect PPIs with low background levels and is suitable for peptide inhibitor screening.
机译:蛋白质-蛋白质相互作用(PPI)的检测对于了解哺乳动物细胞中的许多过程很重要。但是,现有的PPI检测方法通常会提供明显的背景信号。在这里,我们提出了一种基于激酶介导的哺乳动物细胞生长诱导的新型PPI检测方法。在这种方法中,靶蛋白与c-kit(c-kit ICD)的胞内域融合,并在依赖白介素3的哺乳动物细胞中表达。 PPI诱导c-kit ICD的二聚化和激活,从而在不存在白介素3的情况下导致细胞生长。使用该系统,我们成功检测了FKBPF36V的配体依赖性均相相互作用以及FKBP和FRBT2098L的异质相互作用,以及MDM2与已知肽抑制剂之间的本构相互作用。有趣的是,表达高亲和力肽嵌合体的细胞选自主要表达低亲和力肽嵌合体的细胞群的混合物。这些结果表明该方法可以检测低背景水平的PPI,适用于肽抑制剂的筛选。

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