Differential Effects of Ubiquitin Aldehyde on Ubiquitin and ATP-Dependent Protein Degradation

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Abstract
ATP-dependent proteolysis of 125I-labeled human α-globin, bovine α-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 μM) of Ubal increased the ATP-dependent degradation of 125I-α-globin by ∼30% after 2 h at 37 °C, had little effect on 125I-lysozyme turnover, and decreased 125I-α-lactalbumin or 125I-albumin degradation by ∼20%. The ATP-dependent degradation of all substrates was inhibited by high concentrations (>3 μM) of Ubal throughout the incubation (15 min to 2 h); after 2 h, this inhibition ranged from 15% for 125I-α-globin to ∼85% for 125I-α-lactalbumin and 125I-albumin. Levels of ubiquitin−125I-protein conjugates were increased significantly with Ubal; with ≥8.0 μM Ubal, high molecular mass multiubiquitinated conjugates were particularly evident for 125I-α-globin and 125I-α-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-α-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-α-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-α-globin, even low concentrations of Ubal effectively inhibit deubiquitination of these conjugates to enhance α-globin degradation.