Nature communications, 2013-10, Vol.4 (1), p.2660, Article 2660
2013
Details
- Autor(en) / Beteiligte
- Titel
- Visualization and targeted disruption of protein interactions in living cells
- Ist Teil von
- Nature communications, 2013-10, Vol.4 (1), p.2660, Article 2660
- Ort / Verlag
- England: Nature Publishing Group
- Erscheinungsjahr
- 2013
- Link zum Volltext
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- 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.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 2041-1723eISSN: 2041-1723DOI: 10.1038/ncomms3660Titel-ID: cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_3826628
- Format
- –
- Schlagworte
- 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, Red Fluorescent Protein, Single-Domain Antibodies - chemistry, Tumor Suppressor Protein p53 - antagonists & inhibitors, Tumor Suppressor Protein p53 - genetics, Tumor Suppressor Protein p53 - metabolism