Visualization and targeted disruption of protein interactions in living cells

Nat Commun. 2013;4:2660. doi: 10.1038/ncomms3660.

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

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology
  • Biological Assay*
  • Cell Line, Tumor
  • Cricetinae
  • Gene Expression
  • Genes, Reporter
  • Genetic Vectors
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Imidazoles / pharmacology
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Microscopy, Fluorescence
  • Molecular Imaging
  • Piperazines / pharmacology
  • Protein Binding / drug effects
  • Protein Interaction Mapping / methods*
  • Proto-Oncogene Proteins c-mdm2 / antagonists & inhibitors
  • Proto-Oncogene Proteins c-mdm2 / genetics
  • Proto-Oncogene Proteins c-mdm2 / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Single-Domain Antibodies / chemistry
  • Tumor Suppressor Protein p53 / antagonists & inhibitors
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • Antineoplastic Agents
  • Imidazoles
  • Luminescent Proteins
  • Piperazines
  • Recombinant Fusion Proteins
  • Single-Domain Antibodies
  • Tumor Suppressor Protein p53
  • red fluorescent protein
  • Green Fluorescent Proteins
  • nutlin 3
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2