Journal of Molecular Biology
ReviewMeddling with Fate: The Proteasomal Deubiquitinating Enzymes
Graphical Abstract
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.
References (158)
- et al.
The logic of the 26S proteasome
Cell
(2017) - et al.
Structural basis for the activation and inhibition of the UCH37 deubiquitylase
Mol. Cell
(2015) - et al.
Mechanism of UCH-L5 activation and inhibition by DEUBAD domains in RPN13 and INO80G
Mol. Cell
(2015) - et al.
A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3
Cell
(1998) - et al.
Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry
Mol. Cell
(2007) - et al.
A 26S protease subunit that binds ubiquitin conjugates
J. Biol. Chem.
(1994) - et al.
Structures of Rpn1 T1:Rad23 and hRpn13:hPLIC2 reveal distinct binding mechanisms between substrate receptors and shuttle factors of the proteasome
Structure
(2016) - et al.
Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26S proteasome
J. Biol. Chem.
(1999) - et al.
Structure of the ubiquitin-interacting motif of S5a bound to the ubiquitin-like domain of HR23B
J. Biol. Chem.
(2004) - et al.
Nuclear magnetic resonance studies of the denaturation of ubiquitin
Biochim. Biophys. Acta
(1977)
Multiple associated proteins regulate proteasome structure and function
Mol. Cell
Pleiotropic effects of Ubp6 loss on drug sensitivities and yeast prion are due to depletion of the free ubiquitin pool
J. Biol. Chem.
Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome
J. Biol. Chem.
Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation
Cell
A ubiquitin stress response induces altered proteasome composition
Cell
The deubiquitinating enzyme Usp14 allosterically inhibits multiple proteasomal activities and ubiquitin-independent proteolysis
J. Biol. Chem.
Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening
Mol. Cell
Ubiquitinated proteins activate the proteasomal ATPases by binding to Usp14 or Uch37 homologs
J. Biol. Chem.
Structure of proteasome ubiquitin receptor hRpn13 and its activation by the scaffolding protein hRpn2
Mol. Cell
Distinct modes of regulation of the Uch37 deubiquitinating enzyme in the proteasome and in the Ino80 chromatin-remodeling complex
Mol. Cell
The INO80 chromatin remodeling complex in transcription, replication and repair
Trends Biochem. Sci.
Structural and thermodynamic comparison of the catalytic domain of AMSH and AMSH-LP: nearly identical fold but different stability
J. Mol. Biol.
Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde
Cell
Overexpression of USP14 protease reduces I-κB protein levels and increases cytokine release in lung epithelial cells
J. Biol. Chem.
Structural plasticity allows UCH37 to be primed by RPN13 or locked down by INO80G
Mol. Cell
Protein-linked ubiquitin chain structure restricts activity of deubiquitinating enzymes
J. Biol. Chem.
Concurrent translocation of multiple polypeptide chains through the proteasomal degradation channel
J. Biol. Chem.
Structure of tetraubiquitin shows how multiubiquitin chains can be formed
J. Mol. Biol.
The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26S proteasome
J. Biol. Chem.
Disassembly of Lys 11 and mixed linkage polyubiquitin conjugates provides insights into function of proteasomal deubiquitinases Rpn11 and Ubp6
J. Biol. Chem.
Specificity of the ubiquitin isopeptidase in the PA700 regulatory complex of 26S proteasomes
J. Biol. Chem.
Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast
J. Mol. Biol.
Ubiquitin recognition by the proteasome
J. Biochem.
Recognition of client proteins by the proteasome
Annu. Rev. Biophys.
Deubiquitinases in cancer: new functions and therapeutic options
Oncogene
Mechanisms of deubiquitinase specificity and regulation
Annu. Rev. Biochem.
The increasing complexity of the ubiquitin code
Nat. Cell Biol.
The ubiquitin code
Annu. Rev. Biochem.
Constructing and decoding unconventional ubiquitin chains
Nat. Struct. Mol. Biol.
Structural basis for dynamic regulation of the human 26S proteasome
Proc. Natl. Acad. Sci.
An atomic structure of the human 26S proteasome
Nat. Struct. Mol. Biol.
Structural characterization of the interaction of Ubp6 with the 26S proteasome
Proc. Natl. Acad. Sci.
HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures
BMC Struct. Biol.
A gated channel into the proteasome core particle
Nat. Struct. Biol.
Deubiquitylases from genes to organism
Physiol. Rev.
The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover
Mol. Cell. Biol.
Ubiquitin docking at the proteasome through a novel pleckstrin–homology domain interaction
Nature
Proteasome subunit Rpn13 is a novel ubiquitin receptor
Nature
Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome
Science
Delivery of ubiquitinated substrates to protein-unfolding machines
Nat. Cell Biol.
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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.