Activation of p53 by scaffold-stabilised expression of Mdm2-binding peptides: visualisation of reporter gene induction at the single-cell level.
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.
Karlsson
G B
GB
Pharmexa A/S, Kogle Allé 6, DK-2970 Hørsholm, Denmark. Nilla.Karlsson@mtc.ki.se
Jensen
A
A
Stevenson
L F
LF
Woods
Y L
YL
Lane
D P
DP
Sørensen
M S
MS
D001859:Bone Neoplasms / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; C484065:NGFR protein, human; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D001616:beta-Galactosidase