Abstract
Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102–111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8–MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.
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The authors would like to thank J. Bolen for support and guidance.
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Yang, X., Brownell, J.E., Xu, Q. et al. Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry. Cell Biochem Biophys 67, 139–147 (2013). https://doi.org/10.1007/s12013-013-9625-5
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DOI: https://doi.org/10.1007/s12013-013-9625-5