期刊
EMBO REPORTS
卷 10, 期 11, 页码 1250-1258出版社
WILEY
DOI: 10.1038/embor.2009.192
关键词
purification; ubiquitin; proteasome; UBA; DUBs
资金
- Ramon y Cajal Programme, Ministerio de Educacion y Ciencia (Spain) [BFU 2005-04091]
- Fondo de Investigaciones Sanitarias (FIS)
- CIBERhed
- Department of Industry, Tourism and Trade of the Government of the Autonomous Community
- Innovation Technology Department of the Bizkaia Country
Post-translational modification with ubiquitin is one of the most important mechanisms in the regulation of protein stability and function. However, the high reversibility of this modification is the main obstacle for the isolation and characterization of ubiquitylated proteins. To overcome this problem, we have developed tandem-repeated ubiquitin-binding entities (TUBEs) based on ubiquitin-associated (UBA) domains. TUBEs recognize tetra-ubiquitin with a markedly higher affinity than single UBA domains, allowing poly-ubiquitylated proteins to be efficiently purified from cell extracts in native conditions. More significant is the fact that TUBEs protect poly-ubiquitin-conjugated proteins, such as p53 and I kappa B alpha, both from proteasomal degradation and de-ubiquitylating activity present in cell extracts, as well as from existing proteasome and cysteine protease inhibitors. Therefore, these new 'molecular traps' should become valuable tools for purifying endogenous poly-ubiquitylated proteins, thus contributing to a better characterization of many essential functions regulated by these post-translational modifications.
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