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Updated: May 15, 2026
Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation
Published on: March 5, 2018
Live-cell imaging of p53 interactions using a novel Venus-based bimolecular fluorescence complementation system
Joana Dias Amaral1, Federico Herrera, Pedro Miguel Rodrigues
1Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Portugal. jamaral@ff.ul.pt
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.
Insights
Researchers developed a novel Venus-based bimolecular fluorescence complementation (BiFC) system to screen for modulators of p53 interactions. This assay successfully identified potential drug candidates targeting the p53-Mdm2 complex for cancer therapy.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The tumor suppressor protein p53 regulates critical cellular processes like cell cycle arrest and apoptosis.
- Dysfunctional p53 is implicated in various diseases, including cancer and neurodegenerative disorders.
- Targeting the interaction between p53 and its inhibitor Mdm2 is a promising therapeutic strategy.
Purpose of the Study:
- To develop and validate a Venus-based bimolecular fluorescence complementation (BiFC) system for screening p53 interaction modulators.
- To identify novel compounds that modulate p53-p53 and p53-Mdm2 interactions in live mammalian cells.
Main Methods:
- Development of a Venus-based BiFC assay for visualizing protein-protein interactions in real-time.
- Validation of the BiFC system using nutlin-3, a known inhibitor of p53-Mdm2 interaction.
- Screening a library of 33 protein phosphatase inhibitors for modulators of p53-Mdm2 complex formation.
Main Results:
- The BiFC assay demonstrated specificity, with nutlin-3 reducing the BiFC signal, correlating with increased Puma transactivation, PARP cleavage, and cell death.
- The study successfully identified potential modulators of p53-Mdm2 complex formation from the screened chemical library.
- The developed BiFC system provides a robust and direct method for identifying novel therapeutic targets.
Conclusions:
- The Venus-based BiFC system serves as a powerful tool for discovering modulators of p53 interactions.
- This approach offers a viable alternative to traditional drug discovery methods for p53-related pathologies.
- Identification of new modulators can enhance the efficacy of existing anti-tumor therapies.
