Summary
Ubiquitination involves the tagging of proteins with one (mono-) or more (poly-) ubiquitin molecules. Primarily the role of ubiquitination involves mainly short-lived and regulatory proteins being tagged with a poly-ubiquitin tail, thus introducing a hydrophobic patch that allows the protein to be identified and degraded by the 26S proteasome. Transfer of ubiquitin to the lysine residue of a target protein is a multi-step ATP-dependent process. The functions of ubiquitination have been extended in recent years to all areas of biology, many of them proteasome independent. As a small fraction of any protein may potentially be ubiquitinated, this may explain the wide range and large number of proteins that have been identified as being tagged with ubiquitin in the literature. This chapter outlines a general method for an indication of ubiquitination levels and identification of ubiquitinated proteins by two-dimensional electrophoresis in combination with immunoblotting.
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References
Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annu. Rev. Biochem. 67, 427–479
Mukhopadhyay, D., and Riezman, H. (2007). Proteasome-independent functions of ubiquitin in endocytosis and signalling. Science 315, 201–205
Kaiser, P., and Huang, L. (2005). Global approaches to understanding ubiquitination. Genome Biol. 6, Article 233
Shringarpure, R., Grune, T., Mehlase, J., and Davies, K. J. A. (2003). Ubiquitin conjugation is not required for the degradation of oxidised proteins by proteasome. J. Biol. Chem. 278, 311–318
Shang, F., Gong, X., and Taylor, A. (1997). Activity of ubiquitin-dependent pathway in response to oxidative stress. Ubiquitin activating enzyme is transiently up-regulated. J. Biol. Chem. 268, 15405–15411
Peng, J., Schwartz, D., Elias, J.E., Thoreen, C.C., Cheng, D., Marsischky, G., et al (2003). A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926
Sato, K., Hayami, R., Wu, W., Nishikawa, T., Nishikawa, H., Okuda, Y., et al. Nuclophosmin/B23 is a Candidate substrate for the BRCA1-BARD1 ligase. J. Biol. Chem. 279, 30919–30922
McDonagh, B., and Sheehan, D. (2006). Redox proteomics in the blue mussel Mytilus edulis: carbonylation is not a prerequisite for ubiquitination in acute free radical mediated oxidative stress. Aquat. Toxicol. 79, 325–333
Powell, S.R., Wang, P., Divald, A., Teichberg, S., Haridas, V., McCloskey, T. W., et al (2005). Aggregates of oxidised proteins (lipofuscin) induce apoptosis through proteasome inhibition and dysregulation of proapoptotic proteins. Free Radic. Biol. Med. 38, 1093–1101
Acknowledgments
I acknowledge an Embark fellowship from the Irish Research Council for Science, Engineering and Technology.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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McDonagh, B. (2009). Detection of Ubiquitination in 2DE. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_24
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DOI: https://doi.org/10.1007/978-1-59745-281-6_24
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