Abstract
Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl–tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.
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Acknowledgements
This work was supported in part by grant Deutsche Forschungsgemeinschaft AL 484/7-2 to K.A. and grant Sonderforschungsbereich 642 to K.A., D.W., R.S.G. and H.W. U.T.T.N. was supported by the predoctoral fellowship of Fonds der chemischen Industrie. R.S.B. and C. Deraeve thank the Alexander von Humboldt Stiftung for a scholarship. We thank R. Heuann (Ruhr-Universität Bochum) for supplying mouse brains. The authors gratefully acknowledge M. Terbeck, A. Sander, T. Rogowsky, S. Thuns and N. Lupilova for excellent technical assistance. The authors are very grateful to T. Bergbrede and the Dortmund Protein Facility at the Max Planck Institute. The use of beamlines at the Swiss Light Source (Paul Scherrrer Institute) and the help of the X-ray communities at the Max Planck Institute of Molecular Physiology and the Max Planck Insitute of Medical Research with data collection is gratefully acknowledged. We thank A. Barnekow (University of Münster) for the generous gift of Rab6A antibody.
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Nguyen, U., Guo, Z., Delon, C. et al. Analysis of the eukaryotic prenylome by isoprenoid affinity tagging. Nat Chem Biol 5, 227–235 (2009). https://doi.org/10.1038/nchembio.149
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DOI: https://doi.org/10.1038/nchembio.149
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