title: Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation.
abstract: 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.
authors.affiliation: Laboratory for Nutrition and Vision Research, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111, USA. fu.shang@tufts.edu
authors.forename: Fu
authors.initials: F
authors.lastname: Shang
authors.forename: Gejing
authors.initials: G
authors.lastname: Deng
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.forename: Weimin
authors.initials: W
authors.lastname: Guo
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Haas
authors.forename: Bernat
authors.initials: B
authors.lastname: Crosas
authors.forename: Daniel
authors.initials: D
authors.lastname: Finley
authors.forename: Allen
authors.initials: A
authors.lastname: Taylor
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives*; D019921:Biotinylation; D019759:Carbon-Nitrogen Lyases / Q000378:metabolism; D002417:Cattle; D004926:Escherichia coli; D006801:Humans; D007457:Iodine Radioisotopes; D007768:Lactalbumin / Q000378:metabolism; D008239:Lysine / Q000737:chemistry*; D051379:Mice; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D018384:Oxidative Stress; D010447:Peptide Hydrolases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D013388:Succinimides; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*
chemical_list: D007457:Iodine Radioisotopes; D011480:Protease Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; D013388:Succinimides; D025801:Ubiquitin; C053827:biotinyl-N-hydroxysulfosuccinimide ester; D001710:Biotin; D000255:Adenosine Triphosphate; D007768:Lactalbumin; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; D008239:Lysine