Proteasome-Bound UCH37 Debranches Ubiquitin Chains to Promote Degradation

Abstract

1The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables 2 in providing tight control over many biological paradigms. There are clear roles for 3 branched architectures in regulating proteasome-mediated degradation, however the 4 proteins that selectively recognize and process these atypical chains are unknown. Here, 5 using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, 6 we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, exclusively 7 cleaves K48 branched chains. The activity and selectivity toward branched chains is 8 markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using proteasome 9 complexes reconstituted with either active or inactive UCH37 together with protein 10 substrates modified with branched chains, we find that chain debranching promotes 11 degradation under multi-turnover conditions. These results are further supported by 12 proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity 13 impairs global protein turnover. Our work therefore defines UCH37 as a debranching 14 deubiquitinase important for promoting proteasomal degradation.

Deubiquitination Reactions on the Proteasome for Proteasome Versatility

“…Proteasome-bound UCH37 is known to exhibit poor DUB activity for isopeptide-linked ubiquitin–protein conjugates, despite showing predominant activity over USP14 against ubiquitin adducts with small leaving groups, such as ubiquitin amidomethylcoumarin (UbAMC) [ 8 , 37 ]. Similar to USP14, UCH37’s DUB activity can be also markedly increased upon binding to RPN13, a ubiquitin receptor on the proteasome [ 14 , 38 ]. Autoinhibition of the apo form of UCH37 is mainly due to (1) the inhibitory position of UCH37-like (ULD, aka RPN13-binding) domain onto the catalytic domain and (2) the active site crossover loop (ASCL), a flexible segment placed just over the catalytic cysteine ( Figure 1 B).…”

“…This seemingly strict requirement for favored UCH37 substrates may explain why UCH37 preferentially cleaves ubiquitin-adducts with small leaving groups over ubiquitin–protein conjugates. Interestingly, a recent study showed that proteasome-associated UCH37 selectively removes K48-branched chains from a complex mixture of ubiquitin conjugates (see also Section 4 and Figure 2 B) [ 14 ]. The topology of the K48-branching point on bifurcate chains might not be impeded by ASCL-imposed steric hindrance, but this model remains to be further explored.…”

“…A recent study proposed that proteasome-associated UCH37 can debranch ubiquitin chains to promote substrate degradation [ 14 ]. Emerging evidence indicates that branched ubiquitin chains play essential roles in the physiological environment [ 82 , 83 , 84 ], but a branched-chain-specific DUB has so far not been discovered.…”

“…Emerging evidence indicates that branched ubiquitin chains play essential roles in the physiological environment [ 82 , 83 , 84 ], but a branched-chain-specific DUB has so far not been discovered. Strieter’s group found that UCH37 selectively cleaves K48-linked branching points from complex mixture of chains, and its selectivity and activity can be markedly augmented in association with RPN13, at a turnover rate of 3 to 4 s [ 14 ]. This debranching activity essentially promotes the degradation of substrates that are modified with K48-linked branched chains under multi-turnover conditions in vitro and in cells ( Figure 2 B, bottom panel) [ 14 ].…”

“…This indicates that, in spite of having similar cleavage mechanisms, deubiquitination reactions on the proteasome may lead to different proteolytic outcomes. The exact function of UCH37 is less clear, but this enzyme is speculated to edit the ubiquitin chains, yielding repressed or enhanced degradation of substrates depending on the proteasomal demand [ 12 , 13 , 14 ].…”