title: Live-cell imaging of p53 interactions using a novel Venus-based bimolecular fluorescence complementation system.
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.
authors.affiliation: Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Portugal. jamaral@ff.ul.pt
authors.forename: Joana Dias
authors.initials: JD
authors.lastname: Amaral
authors.forename: Federico
authors.initials: F
authors.lastname: Herrera
authors.forename: Pedro Miguel
authors.initials: PM
authors.lastname: Rodrigues
authors.forename: Pedro Antunes
authors.initials: PA
authors.lastname: Dionísio
authors.forename: Tiago Fleming
authors.initials: TF
authors.lastname: Outeiro
authors.forename: Cecília Maria Pereira
authors.initials: CM
authors.lastname: Rodrigues
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D016923:Cell Death; D045744:Cell Line, Tumor; D017931:DNA Primers; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2