Activation of p53 by scaffold-stabilised expression of Mdm2-binding peptides: visualisation of reporter gene induction at the single-cell level

British Journal of Cancer
G B KarlssonM S Sørensen

Abstract

Small peptides that perturb intracellular signalling pathways are useful tools in the identification and validation of new drug targets. To facilitate the analysis of biologically active peptides, we have developed retroviral vectors expressing an intracellular scaffold protein that significantly enhances the stability of small peptides in mammalian cells. This approach was chosen because retroviral transduction results in efficient and controlled delivery of the gene encoding the effector peptide, while the scaffold protein not only stabilises the peptide but also facilitates the analysis and potential isolation of the target protein. Here, we have adapted a p53-responsive reporter assay to flow cytometry to demonstrate the versatility of this approach by using peptides with known Mdm2-binding activities inserted into a stable scaffold protein that is suitable for intracellular expression in multiple compartments of mammalian cells. This strategy should be generally applicable to the study of small biologically active peptides in diverse functional assays.

References

Apr 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·G P NolanL A Herzenberg
Oct 1, 1995·Trends in Biotechnology·R C Ladner
Jul 1, 1993·Molecular and Cellular Biology·J ChenA J Levine
Aug 1, 1997·Current Opinion in Structural Biology·P A Nygren, M Uhlén
Aug 28, 1998·Current Opinion in Biotechnology·M B ZwickJ K Scott
Oct 27, 1998·Nature Biotechnology·M C Souroujon, D Mochly-Rosen
Jul 27, 1999·Combinatorial Chemistry & High Throughput Screening·G CesareniG Cestra
Mar 14, 2000·Molecular and Cellular Biology·J D WeberC J Sherr
Jul 25, 2000·Chemistry & Biology·T L GururajaD C Anderson
Jan 4, 2001·Nature Genetics·X XuG P Nolan
Apr 18, 2003·Mini Reviews in Medicinal Chemistry·Daniella I ZhelevaPeter M Fischer
Jan 6, 2004·Science·Lyubomir T VassilevEmily A Liu

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Citations

Oct 8, 2005·Nature Biotechnology·H Kaspar BinzAndreas Plückthun
Jul 4, 2006·Nature Reviews. Drug Discovery·Grzegorz NalepaJ Wade Harper
Nov 26, 2009·Nature Reviews. Cancer·Christopher J BrownDavid P Lane
Dec 13, 2005·American Journal of Pharmacogenomics : Genomics-related Research in Drug Development and Clinical Practice·Zhuo ZhangHui Wang
Jul 30, 2009·Expert Opinion on Drug Delivery·Gene L Bidwell, Drazen Raucher

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Methods Mentioned

BETA
phage display
peptide display
FACS
Protein Assay
electrophoresis
FCS

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