Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2005; 280(21):20365-74. DOI: 10.1074/jbc.M414356200
Source: PubMed


Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys(6) of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys(6) was the most readily labeled with sulfosuccinimidobiotin. Lys(6)-biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys(6)-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys(6)-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys(6) had similar phenotypes as Lys(6)-biotinylated ubiquitin. Lys(6) mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys(6)-modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys(6)-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

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    • "K6-sulfosuccinimidobiotin-labled ubiquitin is able to form polyubiquitin chains, but conjugates show a lesser susceptibility to proteasomal degradation. Mutation of the lysine residue (K6W) also gives a similar effect (Shang et al., 2005). This suggests that K6, or the surrounding surface environment, is required for proteasomal degradation through recognition and binding to the 26S proteasome. "
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    • ", compare lanes 10-12 vs. 4-6). Additional support for cross talk between the pathways is indicated by formation of the autophagy indicator, LC3-II, when ubiquitin conjugates accumulate due to inhibition of the UPS by expression of conjugationcompetent but degradation-incompetent ubiquitin (Figure S9B) (Shang et al., 2005). "
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