Differential effects of ubiquitin aldehyde on ubiquitin and ATP-dependent protein degradation

ATP-dependent proteolysis of I-125-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 mu M) of Ubal increased the ATP-dependent degradation of I-125-alpha-globin by similar to 30% after 2 h at 37 degrees C, had little effect on I-125-lysozyme turnover, and decreased I-125-alpha-lactalbumin or I-125-albumin degradation by similar to 20%. The ATP-dependent degradation of all substrates was inhibited by high concentrations (>3 mu M) of Ubal throughout the incubation (15 min to 2 h); after 2 h, this inhibition ranged from 15% for I-125-alpha-globin to similar to 85% for I-125-alpha-lactalbumin and I-125-albumin. Levels of ubiquitin-I-125-protein conjugates were increased significantly with Ubal; with greater than or equal to 8.0 mu M Ubal, high molecular mass multiubiquitinated conjugates were particularly evident for I-125-alpha-globin and I-125-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 I-125-alpha-lactalbumin or I-125-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 I-125-alpha-globin and, to a lesser degree, I-125-lysozyme may occur through alternative structures when ubiquitin monomers or short oligomers are ligated to one or more substrate lysines. For I-125-alpha-globin, even low concentrations of Ubal effectively inhibit deubiquitination of these conjugates to enhance alpha-globin degradation.