Chemical and molecular biological approaches to elucidate the biochemical and biological functions of polyubiquitin chains
Final Report Abstract
Modification of proteins by the covalent attachment of ubiquitin plays a fundamental role in the control of many, if not all, fundamental biological processes. Substrate proteins are often modified by the attachment of ubiquitin chains consisting of several ubiquitin moieties linked to each other via isopeptide bond formation. Ubiquitin contains 7 lysine residues and each of these lysines can be used for ubiquitin chain formation. Importantly, it appears that the actual lysine residue of ubiquitin used for ubiquitin-ubiquitin conjugation determines the biochemical/biological function of the respective ubiquitin chain. For example, K48-linked ubiquitin chains serve as signal to target modified proteins for degradation by the proteasome, while K63-linked ubiquitin chains have been linked to non-proteolytic processes. An attractive possibility is that the different function(s) of different ubiquitin chains are mediated by proteins that selectively interact with the respective chain. However, with the exception of proteins interacting with K48-linked ubiquitin chains, little was known about the identity of such proteins at the beginning of this project. In this study, we established two methods for the synthesis of defined ubiquitinubiquitin conjugates. One method relies on a thioether ligation approach, while the other is based on click reaction between two non-natural amino acids (azidohomoalanine, pyrrolysine analog) incorporated into respective bacterially expressed ubiquitin moieties. Using the latter method, all seven naturally occurring ubiquitin connectivities could be synthesized (i.e. all possible di-ubiquitins). Furthermore, preliminary evidence indicates that the synthetic diubiquitins are structurally not distorted suggesting that synthetic di-ubiquitins are well suited to serve as affinity matrices to identify proteins that selectively interact with defined ubiquitin-ubiquitin conjugates. To characterize the polyubiquitin chain formation activity of the ubiquitin-protein ligase Mdm2 within cells and to determine the effect of different ubiquitin chains on the turnover and the cellular localization of Mdm2 and its substrate p53, wild-type ubiquitin or defined lysine mutants of ubiquitin were overexpressed in cell culture experiments. Surprisingly, this revealed that at least under the conditions used, ectopic expression of ubiquitin results in stabilization of p53 as well as of other known substrate proteins of the ubiquitin-proteasome system. Based on additional observations, we propose that the inhibitory effect of ectopically expressed ubiquitin is due to squelching of ubiquitin binding proteins required for shuttling p53 or other proteins to the proteasome for degradation (i.e., the amount of ubiquitin binding proteins available for degradation of ubiquitinated proteins within cells becomes rate-limiting upon even mild increases in ubiquitin levels indicating that ubiquitin expression levels have to be tightly controlled).
Publications
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(2005). The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation. Cell 123, 409-421
Adhikary S, Marinoni F, Hock A, Hulleman E, Popov N, Beier R, Bernard S, Quarto M, Capra M, Goettig S, Kogel U, Scheffner M, Helin K, Eilers
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(2006). Regulation of p27 degradation and S-phase progression by Ro52 RING finger protein. Mol Cell Biol 26, 5994-6004
Sabile A, Meyer AM, Wirbelauer C, Hess D, Kogel U, Scheffner M, Krek W
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(2009). Functional ubiquitin conjugates with lysine-ε-amino-specific linkage by thioether ligation of cysteinyl-ubiquitin peptide building blocks. Bioconjug Chem 20, 1152-1162
Jung JE, Wollscheid HP, Marquardt A, Manea M, Scheffner M, Przybylski M
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(2010). Synthesis of defined ubiquitin dimers. J Am Chem Soc 132, 16337-16339
Eger S, Scheffner M, Marx A, Rubini M
