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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antineoplastic Agents / pharmacology
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Biological Assay*
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Cell Line, Tumor
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Cricetinae
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Gene Expression
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Genes, Reporter
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Genetic Vectors
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Humans
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Imidazoles / pharmacology
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Mice
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Microscopy, Fluorescence
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Molecular Imaging
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Piperazines / pharmacology
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Protein Binding / drug effects
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Protein Interaction Mapping / methods*
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Proto-Oncogene Proteins c-mdm2 / antagonists & inhibitors
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Proto-Oncogene Proteins c-mdm2 / genetics
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Proto-Oncogene Proteins c-mdm2 / metabolism*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism*
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Red Fluorescent Protein
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Single-Domain Antibodies / chemistry
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Tumor Suppressor Protein p53 / antagonists & inhibitors
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Tumor Suppressor Protein p53 / genetics
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Tumor Suppressor Protein p53 / metabolism*
Substances
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Antineoplastic Agents
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Imidazoles
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Luminescent Proteins
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Piperazines
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Recombinant Fusion Proteins
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Single-Domain Antibodies
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Tumor Suppressor Protein p53
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Green Fluorescent Proteins
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nutlin 3
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MDM2 protein, human
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Proto-Oncogene Proteins c-mdm2