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.
Highlights
The ubiquitin/proteasome pathway (UPP)1 is involved in regulating the cell cycle, signal transduction, differentiation, stress response, and DNA repair
The results show that 1) Lys6 was the most readily labeled with sulfo-NHS-biotin; 2) Lys6-biotinylated ubiquitin was used as efficiently as unmodified ubiquitin to form high molecular mass ubiquitin conjugates; 3) Lys6-biotinylated ubiquitin inhibited ubiquitin-dependent degradation because conjugates formed with Lys6-biotinylated ubiquitin were less susceptible to degradation by the 26 S proteasome; 4) K6W mutant ubiquitin had effects similar to those of Lys6-biotinylated ubiquitin; and 5) intracellular expression of K6W mutant ubiquitin resulted in stabilization of substrates for the UPP and enhanced susceptibility to oxidative stress
4) Conjugates formed with Lys6-modified ubiquitin were resistant to degradation by the proteasome, as the conjugates formed with Lys6-modified ubiquitin bound to the proteasome with reduced avidity
Summary
Materials—Tris, acrylamide, N,NЈ-methylenebisacrylamide, N,N,NЈ,NЈtetramethylenediamine, 2-mercaptoethanol, SDS, glycine, and protein molecular mass standards were purchased from Bio-Rad. The assay was conducted in a final volume of 25 l containing (final concentrations) 12 mg/ml RPE cell supernatant or 10 mg/ml fraction II, 50 mM Tris (pH 7.6), 2 mM ATP, 1 mM dithiothreitol, 5 mM MgCl2, 4 M 125I-labeled substrate, and 8 M wild-type or mutant ubiquitin. The assay was conducted in a final volume of 25 l containing (final concentrations) 12 mg/ml RPE cell supernatant, 8 M wild-type or mutant ubiquitin, 50 mM Tris (pH 7.6), 2 mM ATP, 1 mM dithiothreitol, 5 mM MgCl2, 4 mM creatine phosphate, 2 g/ml creatine phosphokinase, and 2– 4 M 125I-labeled substrate. Ubiquitin conjugates of 125I-labeled ␣-lactalbumin were formed in proteasomefree fraction II of rabbit reticulocytes upon addition of wild-type, Lys6biotinylated, K6W, or L8A mutant ubiquitin. The yeast cells were cultured in the presence or absence of 1 M canavanine, an amino acid analog
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