<Prof. Heinrich Leonhardt>

Visualization and Targeted Disruption of Protein Interactions

Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. I will review the most popular methods to study protein interactions and briefly discuss their technical challenges, advantages and shortcomings. I will then present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions (Herce et al., 2013, Nat Commun 4, 2660). We used a high-affinity nanobody to anchor a GFP-fusion protein of interest at a defined cellular structure and measured the enrichment of red-labelled interacting proteins at these sites. With this approach, we visualized the p53–HDM2 interaction in living cells and directly monitored the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further used this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species.