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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals
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Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals

机译：萤火虫荧光素酶互补显像揭示细胞和活体动物中调节的蛋白-蛋白相互作用的动力学

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Signaling pathways regulating proliferation, differentiation, and apoptosis are commonly mediated through protein-protein interactions as well as reversible phosphorylation of proteins. To facilitate the study of regulated protein-protein interactions in cells and living animals, we optimized firefly luciferase protein fragment complementation by screening incremental truncation libraries of N- and C-terminal fragments of luciferase. Fused to the rapamycin-binding domain (FRB) of the kinase mammalian target of rapamycin and FK506-bincling protein 12 (FKBP), respectively, the optimized FRB-N-terminal luciferase fragment (NLuc)/C-terminal luciferase fragment (CLuc)-FKBP luciferase complementation imaging (LCI) pair reconstituted luciferase activity in cells upon single-site binding of rapamycin in an FK506-competitive manner. LCI was used in three independent applications. In mice bearing implants of cells expressing the FRB-NLuc/CLuc-FKBP LCI pair, dose-and time-dependent luciferase activity allowed target-specific pharmacodynamic analysis of rapamycin-induced protein-protein interactions in vivo. In cells expressing a Cdc25C-NLuc/CLuc-14-3-3epsilon LCI pair, drug-mediated disruption of cell cycle regulated protein-protein interactions was demonstrated with the protein kinase inhibitor UCN-01 in a phosphoserine-dependent manner. When applied to IFN-gamma-dependent activation of Janus kinase/signal transducer and activator of transcription 1 (STAT1), LCI revealed, in the absence of ligand-induced phosphorylation, STAT1 proteins existing in live cells as preformed dimers. Thus, optimized LCI provides a platform for near real-time detection and characterization of regulated and small molecule-induced protein-protein interactions in intact cells and living animals and should enable a wide range of novel applications in drug discovery, chemical genetics, and proteomics research.
机译：调节增殖，分化和凋亡的信号通路通常是通过蛋白质-蛋白质相互作用以及蛋白质的可逆磷酸化介导的。为了便于研究细胞和活体动物中调节的蛋白质-蛋白质相互作用，我们通过筛选萤光素酶N和C末端片段的增量截断文库来优化萤火虫萤光素酶蛋白质片段的互补性。优化后的FRB-N末端荧光素酶片段（NLuc）/ C末端荧光素酶片段（CLuc）分别与雷帕霉素激酶哺乳动物靶标的雷帕霉素结合域（FRB）和FK506结合蛋白12（FKBP）融合-FKBP荧光素酶互补成像（LCI）对在雷帕霉素单点结合后以FK506竞争方式重建了细胞中的荧光素酶活性。 LCI被用于三个独立的应用程序。在带有表达FRB-NLuc / CLuc-FKBP LCI对的细胞植入物的小鼠中，剂量和时间依赖性的荧光素酶活性可以在体内对雷帕霉素诱导的蛋白质-蛋白质相互作用进行靶标特异性药效分析。在表达Cdc25C-NLuc / CLuc-14-3-3epsilon LCI对的细胞中，用蛋白丝氨酸抑制剂UCN-01以磷酸丝氨酸依赖性方式证明了药物介导的细胞周期调节蛋白-蛋白相互作用的破坏。当应用于干扰素-γ-依赖性的Janus激酶/信号转导子和转录激活子1（STAT1）的激活时，LCI显示，在没有配体诱导的磷酸化的情况下，活细胞中存在的STAT1蛋白是预先形成的二聚体。因此，优化的LCI提供了一个平台，用于在完整细胞和活体动物中近实时检测和表征受调节的和小分子诱导的蛋白质-蛋白质相互作用，并应能在药物发现，化学遗传学和蛋白质组学中广泛应用研究。

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