Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation

J Biol Chem. 2005 May 27;280(21):20365-74. doi: 10.1074/jbc.M414356200. Epub 2005 Mar 24.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Biotin / analogs & derivatives*
  • Biotinylation
  • Carbon-Nitrogen Lyases / metabolism
  • Cattle
  • Escherichia coli
  • Humans
  • Iodine Radioisotopes
  • Lactalbumin / metabolism
  • Lysine / chemistry*
  • Mice
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Oxidative Stress
  • Peptide Hydrolases / metabolism
  • Polymerase Chain Reaction
  • Protease Inhibitors
  • Proteasome Endopeptidase Complex / metabolism
  • Proteins / metabolism*
  • Recombinant Proteins
  • Saccharomyces cerevisiae
  • Structure-Activity Relationship
  • Succinimides
  • Transfection
  • Ubiquitin / chemistry*
  • Ubiquitin / genetics
  • Ubiquitin / pharmacology*

Substances

  • Iodine Radioisotopes
  • Protease Inhibitors
  • Proteins
  • Recombinant Proteins
  • Succinimides
  • Ubiquitin
  • biotinyl-N-hydroxysulfosuccinimide ester
  • Biotin
  • Adenosine Triphosphate
  • Lactalbumin
  • Peptide Hydrolases
  • Proteasome Endopeptidase Complex
  • Carbon-Nitrogen Lyases
  • isopeptidase
  • Lysine