Trimming of Ubiquitin Chains by Proteasome-associated Deubiquitinating Enzymes

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 eukaryotic proteasome is dedicated primarily to the degradation of proteins tagged by ubiquitin (1). Proteasomes strongly prefer multiubiquitinated protein substrates. The successive addition of ubiquitin groups to the substrate by ubiquitin ligases is usually accomplished through the formation of ubiquitin chains. The proteasome has much in common with the simple ATP-dependent proteases of prokaryotes and mitochondria (2, 3), although only the proteasome recognizes the ubiquitin modification. In all cases, the ATPases form a hexameric ring complex. These rings are homomeric in the case of the prokaryotic and mitochondrial proteases, whereas in eukaryotic proteasomes, the ATPase ring is heteromeric. Proteasomes and the simple ATP-dependent proteases are fundamentally similar in that they all have an ATPase ring (found within the regulatory particle RP]1 in proteasomes, also known as the 19S particle and PA700) abutting a proteolytic complex (the core particle CP] in proteasomes, also known as the 20S particle), although in some cases, the ATPase and protease domains are present on the same polypeptide chain (Fig. 1). Furthermore, this ancient organization of ATP-dependent proteases involves stacked ring complexes. Substrates are translocated from one ring to the next via the central pore within each ring. For most substrates, movement from ring to ring is driven by ATP hydrolysis. Thus, the substrate is captured by the ATPase ring of the RP and then translocated into the central cavity of the CP where it is hydrolyzed.Open in a separate windowFig. 1.Deubiquitinating enzymes of proteasome. In metazoans, three DUBs associate with the proteasome as shown. Each is associated with the 19-subunit RP. The detailed positioning of these enzymes on the RP is not known and is represented here schematically. RPN11 cuts at the base of the chain to release the chain en bloc. As shown, this is coupled (by an unknown mechanism) to translocation of the substrate from the RP to the CP to be degraded. In contrast, the action of USP14 and UCH37 is thought to promote substrate release from the proteasome rather than degradation. However, it should be noted that the attack of these enzymes on a substrate does not guarantee release, especially as their action on the chain is gradual, proceeding stepwise over time from the distal tip of the ubiquitin chain. Some substrates may carry more than one ubiquitin chain and thus be processed in a more complex manner. Moreover, more than one DUB might act on a given chain. The proteasome icon, adapted from Ref. 30 with permission, is based on cryo-EM imaging.The pathway of translocation contains a series of narrow constrictions through which folded proteins cannot pass. The inability of a typical folded protein to pass through these “filters” defines in part the selectivity of such proteases. However, the ATPases can exert a pulling force on the substrate that is strong enough to unfold the protein, which allows for passage through the series of constrictions. This force is exerted within the central channel of the ATPase complex. Thus, translocation and unfolding of the substrate are generally coupled events (1–3).Although not departing from this paradigm, the eukaryotic proteasome interacts with substrate in a more complex manner as a result of interactions involving the ubiquitin tag. Thus, many of the 13 subunits that were added to the evolutionarily ancient ATPase complex to form the RP in the eukaryotic lineage participate in recognition and processing of the ubiquitin tag (1). For example, the yeast proteasome has five and probably more distinct ubiquitin receptors, two that are integral subunits and three that are reversibly proteasome-associated (4). In addition, proteasomes of mammals have three distinct deubiquitinating enzymes (DUBs). The multiplicity of DUBs points to a surprisingly complex role of deubiquitination in proteasome function.