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- Title
Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8-MLN4924 Adduct by Mass Spectrometry.
- Authors
Yang, Xiaofeng; Brownell, James; Xu, Qing; Zhu, Fengying; Ma, Jingya; Loke, Huay-Keng; Rollins, Neil; Soucy, Teresa; Minissale, James; Thomas, Michael; Mallender, William; Dick, Lawrence; Li, Ping; Liao, Hua
- 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, ), 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.
- Subjects
UBIQUITIN; MASS spectrometry; BIOCONJUGATES; ENZYME inhibitors; BIOCHEMISTRY; PHARMACODYNAMICS
- Publication
Cell Biochemistry & Biophysics, 2013, Vol 67, Issue 1, p139
- ISSN
1085-9195
- Publication type
Article
- DOI
10.1007/s12013-013-9625-5