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Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin

Item Type:Article
Title:Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin
Creators Name:Lectez, B. and Migotti, R. and Lee, S.Y. and Ramirez, J. and Beraza, N. and Mansfield, B. and Sutherland, J. and Martinez-Chantar, M.L. and Dittmar, G. and Mayor, U.
Abstract:Ubiquitination is behind most cellular processes, with ubiquitin substrates being regulated variously according to the number of covalently conjugated ubiquitin molecules and type of chain formed. Here we report the first mammalian system for ubiquitin proteomics allowing direct validation of the MS-identified proteins. We created a transgenic mouse expressing biotinylated ubiquitin and demonstrate its use for the isolation of ubiquitinated proteins from liver and other tissues. The specificity and strength of the biotin-avidin interaction allow very stringent washes so only proteins conjugated to ubiquitin are isolated. In contrast to recently available antibody-based approaches, our strategy allows direct validation by immunoblotting, therefore revealing the type of ubiquitin chains (mono or poly) formed in vivo. We also identify the conjugating E2 enzymes that are ubiquitin-loaded in the mouse tissue. Furthermore, our strategy allows the identification of candidate cysteine-ubiquitinated proteins, providing a strategy to identify those in a proteomic scale. The novel in vivo system described here allows broad access to tissue-specific ubiquitomes and can be combined with established mouse disease models to investigate ubiquitin-dependent therapeutical approaches.
Keywords:Ubiquitin, In Vivo Biotinylation, Proteomics, Liver, Mouse Model, Animals, Mice
Source:Journal of Proteome Research
ISSN:1535-3893
Publisher:American Chemical Society (U.S.A.)
Volume:13
Number:6
Page Range:3016-3026
Date:6 June 2014
Official Publication:https://doi.org/10.1021/pr5001913
PubMed:View item in PubMed

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