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

Lookup NU author(s): Professor Tiago OuteiroORCiD

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Abstract

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. © 2012 Elsevier Inc.

Publication metadata

Author(s): Amaral JD, Herrera F, Rodrigues PM, Dionisio PA, Outeiro TF, Rodrigues CMP

Publication type: Article

Publication status: Published

Journal: Biochemical Pharmacology

Year: 2013

Volume: 85

Issue: 6

Pages: 745-752

Print publication date: 15/03/2013

Online publication date: 19/12/2012

ISSN (print): 0006-2952

ISSN (electronic): 1873-2968

Publisher: Elsevier

URL: https://doi.org/10.1016/j.bcp.2012.12.009

DOI: 10.1016/j.bcp.2012.12.009

PubMed id: 23261524

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