Chapter title |
High Content Screening Biosensor Assay to Identify Disruptors of p53–hDM2 Protein-Protein Interactions
|
---|---|
Chapter number | 37 |
Book title |
Protein-Protein Interactions
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2425-7_37 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2424-0, 978-1-4939-2425-7
|
Authors |
Yun Hua, Christopher J. Strock, Paul A. Johnston, Hua, Yun, Strock, Christopher J., Johnston, Paul A. |
Abstract |
This chapter describes the implementation of the p53-hDM2 protein-protein interaction (PPI) biosensor (PPIB) HCS assay to identify disruptors of p53-hDM2 PPIs. Recombinant adenovirus expression constructs were generated bearing the individual p53-GFP and hDM2-RFP PPI partners. The N-terminal p53 transactivating domain that contains the binding site for hDM2 is expressed as a GFP fusion protein that is targeted and anchored in the nucleolus of infected cells by a nuclear localization (NLS) sequence. The p53-GFP biosensor is localized to the nucleolus to enhance and facilitate the image acquisition and analysis of the PPIs. The N-terminus of hDM2 encodes the domain for binding to the transactivating domain of p53, and is expressed as a RFP fusion protein that includes both an NLS and a nuclear export sequence (NES). In U-2 OS cells co-infected with both adenovirus constructs, the binding interactions between hDM2 and p53 result in both biosensors becoming co-localized within the nucleolus. Upon disruption of the p53-hDM2 PPIs, the p53-GFP biosensor remains in the nucleolus while the shuttling hDM2-RFP biosensor redistributes into the cytoplasm. p53-hDM2 PPIs are measured by acquiring fluorescent images of cells co-infected with both adenovirus biosensors on an automated HCS imaging platform and using an image analysis algorithm to quantify the relative distribution of the hDM2-RFP shuttling component of the biosensor between the cytoplasm and nuclear regions of compound treated cells. |
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