Differential effects of ubiquitin aldehyde on ubiquitin and ATP-dependent protein degradation.
ATP-dependent proteolysis of 125I-labeled human alpha-globin, bovine alpha-lactalbumin, bovine serum albumin, or chicken lysozyme was assessed in a rabbit reticulocyte extract supplemented with ATP, excess ubiquitin, and variable amounts of ubiquitin aldehyde (Ubal), an inhibitor of many ubiquitin-protein isopeptidases. Low concentrations (0.8 microM) of Ubal increased the ATP-dependent degradation of 125I-alpha-globin by approximately 30% after 2 h at 37 degrees C, had little effect on 125I-lysozyme turnover, and decreased 125I-alpha-lactalbumin or 125I-albumin degradation by approximately 20%. The ATP-dependent degradation of all substrates was inhibited by high concentrations (> 3 microM) of Ubal throughout the incubation (15 min to 2 h); after 2 h, this inhibition ranged from 15% for 125I-alpha-globin to approximately 85% for 125I-alpha-lactalbumin and 125I-albumin. Levels of ubiquitin-125I-protein conjugates were increased significantly with Ubal; with > or = 8.0 microM Ubal, high molecular mass multiubiquitinated conjugates were particularly evident for 125I-alpha-globin and 125I-alpha-lactalbumin. These mixtures also accumulated ubiquitin conjugates with sizes expected for di- through pentaubiquitin oligomers. The results are consistent with the following proposed events: The ATP-dependent degradation of 125I-alpha-lactalbumin or 125I-albumin is probably mediated almost exclusively through polyubiquitinated intermediates. High Ubal concentrations inhibit an isopeptidase(s) which normally disassembles "unanchored" polyubiquitin chains that remain after substrate degradation by the 26S proteasome; these chains accumulate to inhibit further conjugate degradation. Much of the ATP-dependent degradation of 125I-alpha-globin and, to a lesser degree, 125I-lysozyme may occur through alternative structures where ubiquitin monomers or short oligomers are ligated to one or more substrate lysines. For 125I-alpha-globin, even low concentrations of Ubal effectively inhibit deubiquitination of these conjugates to enhance alpha-globin degradation.
Shaeffer
J R
JR
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1109, USA.
Cohen
R E
RE
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002645:Chickens; D006801:Humans; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D014452:Ubiquitins / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology
D007457:Iodine Radioisotopes; D011506:Proteins; D014452:Ubiquitins; C051927:ubiquitin-aldehyde; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases