Live-cell imaging of p53 interactions using a novel Venus-based bimolecular fluorescence complementation system

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• 作者：Joana Dias Amaral^a ; ^b ; ^{jamaral@ff.ul.pt} ; Federico Herrera^c ; Pedro Miguel Rodrigues^a ; Pedro Antunes Dion&iacute ; sio^a ; Tiago Fleming Outeiro^c ; ^d ; ^e ; Cec&iacute ; lia Maria Pereira Rodrigues^a ; ^b
• 关键词：BiFC assay ; p53&ndash ; Mdm2 interaction ; HCT116 cells ; Drug discovery ; Cancer
• 刊名：Biochemical Pharmacology
• 出版年：2013
• 出版时间：15 March, 2013
• 年：2013
• 卷：85
• 期：6
• 页码：745-752
• 全文大小：1064 K

文摘

p53 plays an important role in regulating a wide variety of cellular processes, such as cell cycle arrest and/or apoptosis. Dysfunction of p53 is frequently associated with several pathologies, such as cancer and neurodegenerative diseases. In recent years substantial progress has been made in developing novel p53-activating molecules. Importantly, modulation of p53 interaction with its main inhibitor, Mdm2, has been highlighted as a promising therapeutic target. In this regard, bimolecular fluorescence complementation (BiFC) analysis, by providing direct visualization of protein interactions in living cells, offers a straightforward method to identify potential modulators of protein interactions. In this study, we developed a simple and robust Venus-based BiFC system to screen for modulators of p53-p53 and p53-Mdm2 interactions in live mammalian cells. We used nutlin-3, a well-known disruptor of p53-Mdm2 interaction, to validate the specificity of the assay. The reduction of BiFC signal mediated by nutlin-3 was correlated with an increase in Puma transactivation, PARP cleavage, and cell death. Finally, this novel BiFC approach was exploited to identify potential modulators of p53-Mdm2 complex formation among a commercially available chemical library of 33 protein phosphatase inhibitors. Our results constitute ¡°proof-of-concept¡± that this model has strong potential as an alternative to traditional target-based drug discovery strategies. Identification of new modulators of p53-p53 and p53-Mdm2 interactions will be useful to achieve synergistic drug efficacy with currently used anti-tumor therapies.