High-content assay to study protein prenylation.
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Simonen, Marjo, Ibig-Rehm, Yvonne, Hofmann, Gabriele, Zimmermann, Johann, Albrecht, Genevieve, Magnier, Maxime, Heidinger-Millot, Valerie and Gabriel, Daniela (2008) High-content assay to study protein prenylation. Journal of Biomolecular Screening : the official journal of the Society for Biomolecular Screening, 13 (6). pp. 456-467. ISSN 1087-0571
Abstract
The mevalonate pathway leads to synthesis of cholesterol and isoprenoid lipids. Prenyltransferases attach the isoprenoid lipids to the C-terminus of several small guanosine triphosphate-binding proteins. The prenyl groups are essential for the biological activity of these proteins. The prenyltransferases and other components of the mevalonate pathway are either present or potential drug targets for cancer, osteoporosis, restenosis, or high serum cholesterol level. Until recently, cellular assays to study protein prenylation have been tedious, low-throughput assays. The authors have developed a high-content imaging-based assay to study protein prenylation. The assay is based on a green fluorescent protein (GFP) reporter, which is tagged with the prenylation motif of human H-Ras. The C-terminus of H-Ras targets GFP to the plasma membrane. When protein prenylation is inhibited, the tagged GFP cannot be localized to plasma membrane but is soluble in the cells. The localization of the GFP reporter can be analyzed in the 96- or 384-well format using automated microscopy and automated image analysis. Information about cell number and nuclear intensity can be obtained from the same images. In compound screening, these readouts provide valuable information about the toxicity of the compounds. The authors have validated their assay using several inhibitors of the mevalonate pathway as well as siRNA against farnesyl pyrophosphate synthase, a critical enzyme in the synthesis of the isoprenoid lipids.
| Item Type: | Article |
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| Additional Information: | author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used |
| Keywords: | mevalonate pathway; protein prenylation; high-content analysis; green fluorescent protein (GFP); plasma membrane localization |
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| Date Deposited: | 14 Dec 2009 13:53 |
| Last Modified: | 31 Jan 2013 01:05 |
| URI: | https://oak.novartis.com/id/eprint/818 |