Journal of Molecular Biology
Volume 429, Issue 22, 10 November 2017, Pages 3525-3545
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Review
Meddling with Fate: The Proteasomal Deubiquitinating Enzymes

https://doi.org/10.1016/j.jmb.2017.09.015Get rights and content

Highlights

  • The three proteasomal DUBs differ strongly in mechanism, specificity, and function.

  • Rpn11 removes ubiquitin chains from substrates that are committed to degradation.

  • Usp14 and Uch37 can work before commitment to rescue substrates from degradation.

  • Usp14 deubiquitinates substrates with multiple Ub modifications.

  • Uch37 is found in both the proteasome and the INO80 chromatin remodeling complex.

Abstract

Three deubiquitinating enzymes—Rpn11, Usp14, and Uch37—are associated with the proteasome regulatory particle. These enzymes allow proteasomes to remove ubiquitin from substrates before they are translocated into the core particle to be degraded. Although the translocation channel is too narrow for folded proteins, the force of translocation unfolds them mechanically. As translocation proceeds, ubiquitin chains bound to substrate are drawn to the channel's entry port, where they can impede further translocation. Rpn11, situated over the port, can remove these chains without compromising degradation because substrates must be irreversibly committed to degradation before Rpn11 acts. This coupling between deubiquitination and substrate degradation is ensured by the Ins-1 loop of Rpn11, which controls ubiquitin access to its catalytic site. In contrast to Rpn11, Usp14 and Uch37 can rescue substrates from degradation by promoting substrate dissociation from the proteasome prior to the commitment step. Uch37 is unique in being a component of both the proteasome and a second multisubunit assembly, the INO80 complex. However, only recruitment into the proteasome activates Uch37. Recruitment to the proteasome likewise activates Usp14. However, the influence of Usp14 on the proteasome depends on the substrate, due to its marked preference for proteins that carry multiple ubiquitin chains. Usp14 exerts complex control over the proteasome, suppressing proteasome activity even when inactive in deubiquitination. A major challenge for the field will be to elucidate the specificities of Rpn11, Usp14, and Uch37 in greater depth, employing not only model in vitro substrates but also their endogenous targets.

Section snippets

The ubiquitin–proteasome system

The proteasome controls the levels of myriad regulatory proteins and maintains cellular homeostasis by removing misfolded and potentially toxic proteins (reviewed in this issue by Budenholzer et al.; see also Refs. [1], [2], [3]). Proteins are marked for proteasomal degradation with ubiquitin, a highly conserved, 76-residue protein. Ubiquitination of substrate proteins is achieved via the sequential action of E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-ligating)

Substrate recognition and translocation

Ubiquitinated proteins associate with the proteasome through a set of integral RP subunits that serve as substrate receptors: Rpn10, Rpn13, and Rpn1 [1], [19], [20], [21], [22], [23] (Fig. 1). Each of these receptors is capable of recognizing ubiquitin directly as well as indirectly; in ubiquitin recognition, Rpn10 is the strongest. Indirect recognition is mediated by a family of proteins often referred to as shuttling ubiquitin receptors. These shuttle proteins have an N-terminal

Proteasome-associated DUBs

There are three DUBs intrinsic to or associated with mammalian proteasomes: proteasome subunit Rpn11/Poh1/PSMD14, a member of the JAB1/MPN/Mov34 (JAMM) family of DUBs; Usp14 (and its Saccharomyces cerevisiae ortholog Ubp6), of the ubiquitin specific protease (USP/UBP) family; and Uch37/UCHL5, of the ubiquitin C-terminal hydrolase (UCH) family. Because Rpn11, Usp14, and Uch37 belong to different families of enzymes, their association with the proteasome must have arisen independently in

Enzymatic activity and its regulation

All three proteasomal DUBs are highly regulated. They are active preferentially in the context of the proteasome, with a degree of activation as high as 800-fold in the case of Usp14 [47]. However, even when assembled into the proteasome, Usp14 and Rpn11 are not constitutively in an active state. A common theme among these enzymes is the existence of loop segments that restrict the access of ubiquitin to the catalytic site. These loops are displaced to activate the enzyme. The structural

Substrate specificity

The canonical substrate of the proteasome is traditionally considered to be modified by a single polyubiquitin chain containing four or more ubiquitin groups linked via residue K48 of ubiquitin [1]. However, the architecture of ubiquitin modification is now understood to be highly diverse. Each of the seven lysines within ubiquitin can be used for chain formation as well as the N-terminal amino group. Using these target sites, mixed-linkage chains and branched chains of various lengths may be

Usp14

Usp14 and Ubp6 are routinely assayed using artificial substrates such as Ub-AMC. However, with bona fide ubiquitin–protein conjugates as substrates, it has often been difficult to convincingly observe Usp14 and Ubp6 activity [90]. This has been the case even when Usp14/Ubp6 is associated with the proteasome, and thus in an activated state, and even when using established proteasome substrates. As free Usp14 and Ubp6 can hydrolyze free ubiquitin chains, these have been widely used as a measure

Effects of deubiquitination on substrate degradation

As discussed above, deubiquitination by Rpn11 stimulates the degradation of substrates that are already committed to degradation by removing bulky ubiquitin chains that otherwise might stall the proteasome. In contrast, deubiquitination by the ATP-independent DUBs Usp14/Ubp6 and Uch37 has been proposed to suppress degradation through promoting premature substrate dissociation [40], [47], [51], [98]. Most of the work on this problem has concerned Usp14/Ubp6, so we will focus on this enzyme.

Loss

Physiological function

While there has been strong progress in the biochemistry and structural biology of the proteasomal DUBs, our understanding of their physiological roles in mammals is still at an early stage. All three of these enzymes are essential in the mouse [114], [115], [116], suggestive of distinct physiological functions. However, it is not clear for any of these DUBs whether the functional requirement applies to their DUB activity per se. No conditional mutants exist. In cultured cells, there does not

Challenges for the future

Over the last two decades, major progress has been made in our understanding of the proteasome-associated DUBs. However, the picture of proteasomal deubiquitination that emerges from this work remains poorly defined in many respects. Most notably, to date, we have only a preliminary account of the endogenous substrates of Usp14 and Uch37. Solving this problem will require application of proteomic methods in conjunction with careful in vitro biochemistry to show that effects observed in vivo are

Acknowledgments

We thank members of the Finley lab, particularly S. Elsasser, for critical reading of the manuscript. Funding was provided by grants from the National Institutes of Health to D.F. (R01GM043601) and the Dutch Cancer Foundation to S.d.P. (BUIT 2015-7517).

Competing Financial Interests: The Usp14 inhibitor IU1 is under patent, which is held by D.F. and others.

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    Present address: Department of Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.

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