title: Visualization and targeted disruption of protein interactions in living cells.
abstract: Protein-protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein-protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53-HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use 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.
authors.affiliation: 1] Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany [2].
authors.forename: Henry D
authors.initials: HD
authors.lastname: Herce
authors.forename: Wen
authors.initials: W
authors.lastname: Deng
authors.forename: Jonas
authors.initials: J
authors.lastname: Helma
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Cardoso
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001681:Biological Assay; D045744:Cell Line, Tumor; D006224:Cricetinae; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping / Q000379:methods*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D000970:Antineoplastic Agents; D049452:Green Fluorescent Proteins; D007093:Imidazoles; D008164:Luminescent Proteins; D010879:Piperazines; D051736:Proto-Oncogene Proteins c-mdm2; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D016159:Tumor Suppressor Protein p53; D000097573:Red Fluorescent Protein; C482205:nutlin 3; C497206:MDM2 protein, human