Abstract
Abstract Dysregulation of protein-protein interactions between the tumor suppressor p53 and its binding partners is implicated in the pathogenesis of various cancers. Here we describe several novel assays for analysis of p53 interactions and their inhibitors both in live mammalian cells and in vitro. To evaluate putative inhibitors of protein-protein interactions between p53 and its negative regulators Mdm2 and Mdm4, we recently developed two comparative live-cell Fluorescent-Two Hybrid (F2H) assays. The F2H principle is based on a tethering strategy: the GFP-tagged protein (here p53) is enriched at the protein interaction platform of the engineered F2H-BHK cells and serves as bait, whereas the RFP-tagged protein serves as a prey (here Mdm2 or Mdm4). By performing p53:Mdm2 and p53:Mdm4 F2H assays side-by-side, we could evaluate the dual inhibitory activity of the previously published stapled peptides. Furthermore, since F2H allows visualization of the dynamics of protein-protein interactions, we could compare the compound's kinetics with real-time imaging. We performed a mutant analysis with F2H and showed that several Nutlin-resistant mutants of Mdm2 are sensitive to inhibition with stapled peptides sMTide-02 and sMTide-02a. For in vitro validation of p53 interactions, we developed novel p53 immunoprecipitation reagents. We employed the single-domain antibody technology in conjunction with phage display to isolate two specific anti-p53 VHHs (also termed nanobodies) from immunized alpacas. When conjugated to agarose beads, these VHHs serve as highly efficient pull-down reagents (p53-Traps), specific exclusively against N- and C-terminus of p53 respectively. Using both p53-Traps, we could confirm our F2H results in immunoprecipitation followed by Western blotting. Furthermore, p53-Traps enabled us to extend our analysis of the p53 interaction network and evaluate interactions between the p53 isoforms. Taken together, we developed a toolbox for analysis of p53 interactions both biochemically and by fluorescence microscopy. Our fully reversible live-cell F2H assays can be applied for side-by-side profiling of new inhibitors of p53:Mdm2 and p53:Mdm4 interactions with respect to their intracellular activity, cell penetration and kinetics. Our N- and C-terminal p53-Traps complement p53 interaction analysis and enable highly efficient biochemical investigation of the p53 network and discovery of new interaction partners of p53. Citation Format: Larisa Yurlova, Andrea Buchfellner, Benjamin Ruf, Sebastien Gabriel Michel Jo, Jean-Christophe Bourdon, Farid J. Ghadessy, Christopher J. Brown, David P. Lane, Tina Romer. Live cell and in vitro analysis of p53 interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1231. doi:10.1158/1538-7445.AM2015-1231
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