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Visualization and targeted disruption of protein interactions in living cells.

Herce, Henry D. ; Deng, Wen ; Helma, Jonas ; Leonhardt, Heinrich ; Cardoso, M. Cristina (2013):
Visualization and targeted disruption of protein interactions in living cells.
In: Nature communications, 4, p. 2660. ISSN 2041-1723,
[Article]

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.

Item Type: Article
Erschienen: 2013
Creators: Herce, Henry D. ; Deng, Wen ; Helma, Jonas ; Leonhardt, Heinrich ; Cardoso, M. Cristina
Title: Visualization and targeted disruption of protein interactions in living cells.
Language: English
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.

Journal or Publication Title: Nature communications
Volume of the journal: 4
Divisions: 10 Department of Biology
10 Department of Biology > Cell Biology and Epigenetics
Date Deposited: 29 Oct 2013 12:27
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