Proteasomes, the primary mediators of ubiquitin–protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that USP14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin–protein conjugates both in vitro and in cells. A catalytically inactive variant of USP14 has reduced inhibitory activity, indicating that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human USP14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. USP14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of USP14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.

The yin and yang of proteasomal regulation: The ubiquitin-proteasome pathway regulates myriad proteins through their selective proteolysis. The small protein ubiquitin is attached, typically in many copies, to the target protein, which is then recognized and broken down by the proteasome. Shi et al. found a repeat structure in the proteasome for recognizing ubiquitin as well as ubiquitin-like (UBL) proteins. Tandem binding sites allow the proteasome to dock multiple proteins. One of the bound UBL proteins is an enzyme that cleaves ubiquitin-protein conjugates, which antagonizes degradation. Thus, the repetition of related binding sites with distinct specificity achieves a balance of positive and negative regulation of the proteasome. Science , this issue p. 10.1126/science.aad9421

Single-molecule assay of ubiquitylation: Many biological processes in cells are regulated by ubiquitin peptides that are attached to proteins. Measurement of single fluorescent molecules in cell extracts can be used to trace the kinetics of such reactions. Lu et al. refined assay conditions to follow ubiquitination by an E3 ubiquitin ligase (see the Perspective by Komander). They visualized the activity of the anaphase-promoting complex (APC), a ubiquitin ligase critical for control of the cell division cycle. The processive initial reaction catalyzed by the APC was replaced by slower reactions. The results show how small, commonly occurring recognition motifs can guide specific and highly controlled enzymatic events. In a companion paper, Lu et al. explored how the number and arrangement of added ubiquitin chains affected the interaction of ubiquitylated proteins with the proteasome (a protein complex that recognizes ubiquitylated proteins and degrades them). The extent of ubiquitylation determined the strength of interaction of a substrate protein with the proteasome, and the arrangement of the ubiquitin chains determined the movement of the protein into the proteasome and thus the rate of degradation. Science , this issue 10.1126/science.1248737 , 10.1126/science.1250834 ; see also p. 183

The proteasome generally recognizes substrate via its multiubiquitin chain followed by ATP-dependent unfolding and translocation of the substrate from the regulatory particle into the proteolytic core particle to be degraded. Substrate-bound ubiquitin groups are for the most part not delivered to the core particle and broken down together with substrate but instead recovered as intact free ubiquitin and ubiquitin chains. Substrate deubiquitination on the proteasome is mediated by three distinct deubiquitinating enzymes associated with the regulatory particle: RPN11, UCH37, and USP14. RPN11 cleaves at the base of the ubiquitin chain where it is linked to the substrate, whereas UCH37 and apparently USP14 mediate a stepwise removal of ubiquitin from the substrate by disassembling the chain from its distal tip. In contrast to UCH37 and USP14, RPN11 shows degradation-coupled activity; RPN11-mediated deubiquitination is apparently delayed until the proteasome is committed to degrade the substrate. Accordingly, RPN11-mediated deubiquitination promotes substrate degradation. In contrast, removal of ubiquitin prior to commitment could antagonize substrate degradation by promoting substrate dissociation from the proteasome. Emerging evidence suggests that USP14 and UCH37 can both suppress substrate degradation in this way. One line of study has shown that small molecule USP14 inhibitors can enhance proteasome function in cells, which is consistent with this model. Enhancing protein degradation could potentially have therapeutic applications for diseases involving toxic proteins that are proteasome substrates. However, the responsiveness of substrates to inhibition of proteasomal deubiquitinating enzymes may vary substantially. This substrate specificity and its mechanistic basis should be addressed in future studies.

The proteasome-associated enzyme USP14 regulates protein degradation by removing ubiquitin from proteins; here it is shown that USP14 removes ubiquitin chains from in vitro generated cyclin B conjugates en bloc and within milliseconds, before the proteasome has a chance to initiate degradation, and proceeds until a single chain remains. The proteasome machinery degrades proteins that are tagged with the small modifier protein ubiquitin. The proteasome-associated enzyme USP14 is a major regulator of this process, removing ubiquitin tags from only some proteins, thereby suppressing their degradation. Looking at one such proteasome substrate, cyclin B, these authors ask how USP14 selects certain substrates for deubiquitination. They find that USP14 shows preference for ubiquitin–cyclin B conjugates that carry more than one ubiquitin modification or chain. Contrary to what was believed, USP14 seems to remove ubiquitin chains from cyclin B en bloc and within a millisecond time scale — before the proteasome has a chance to initiate degradation. And it proceeds until a single chain remains. This specificity for multi-(poly)ubiquitin chains is apparently unique among the characterized deubiquitinating enzymes. USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes1,2,3,4,5,6,7,8,9. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked preference for ubiquitin–cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14’s catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture.