title: High content screening biosensor assay to identify disruptors of p53-hDM2 protein-protein interactions.
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.
authors: Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Room 1014 Salk Hall, 3501 Terrace Street, Pittsburgh, PA, 15261, USA.
authors: Yun
authors: Y
authors: Hua
authors: Christopher J
authors: CJ
authors: Strock
authors: Paul A
authors: PA
authors: Johnston
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: C433227:CNBP protein, human; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins