Shang, FuDeng, GejingLiu, QingGuo, WeiminHaas, Arthur L.Crosas, BernatFinley, DanielTaylor, Allen2019-10-052005Shang, Fu, Gejing Deng, Qing Liu, Weimin Guo, Arthur L. Haas, Bernat Crosas, Daniel Finley, and Allen Taylor. 2005. “Lys6-Modified Ubiquitin Inhibits Ubiquitin-Dependent Protein Degradation.” Journal of Biological Chemistry 280 (21): 20365–74. https://doi.org/10.1074/jbc.m414356200.0021-92581083-351Xhttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41483254Ubiquitin 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, Lys6 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.en-USJournal Article2019-10-0510.1074/jbc.M414356200