Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions
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Evaluation Summary:
This manuscript addresses the role of the deubiquitylating enzyme UCH37 in facilitating proteasomal clearance of branched polyubiquitylated substrates. Using a wide-range of chemical biological, biophysical and cell biological techniques, the authors have convincingly demonstrated that UCH37 binds to branched ubiquitin trimers, with at least one K48 linkage, by binding to both distal ubiquitins attached to the proximal, or central, ubiquitin. They further demonstrate that mutations of UCH37 lead to the formation of proteasomal foci in cells and that these foci are rich in polyubiquitinated species, presumably due to the lack of debranching by UCH37. Overall, this excellent study adds to our understanding of UCH37 function, especially with regard to the newly observed phenomenon of reversible proteasome aggregation in cells. Readers will benefit from the large array of ubiquitin-centric tools that are described to study key aspects of UCH37 function and from knowledge of the specific role of UCH37.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome for the next round of substrate processing. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique Ub chain architecture is aided by a DUB.
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Evaluation Summary:
This manuscript addresses the role of the deubiquitylating enzyme UCH37 in facilitating proteasomal clearance of branched polyubiquitylated substrates. Using a wide-range of chemical biological, biophysical and cell biological techniques, the authors have convincingly demonstrated that UCH37 binds to branched ubiquitin trimers, with at least one K48 linkage, by binding to both distal ubiquitins attached to the proximal, or central, ubiquitin. They further demonstrate that mutations of UCH37 lead to the formation of proteasomal foci in cells and that these foci are rich in polyubiquitinated species, presumably due to the lack of debranching by UCH37. Overall, this excellent study adds to our understanding of UCH37 function, especially with regard to the newly observed phenomenon of reversible proteasome aggregation in …
Evaluation Summary:
This manuscript addresses the role of the deubiquitylating enzyme UCH37 in facilitating proteasomal clearance of branched polyubiquitylated substrates. Using a wide-range of chemical biological, biophysical and cell biological techniques, the authors have convincingly demonstrated that UCH37 binds to branched ubiquitin trimers, with at least one K48 linkage, by binding to both distal ubiquitins attached to the proximal, or central, ubiquitin. They further demonstrate that mutations of UCH37 lead to the formation of proteasomal foci in cells and that these foci are rich in polyubiquitinated species, presumably due to the lack of debranching by UCH37. Overall, this excellent study adds to our understanding of UCH37 function, especially with regard to the newly observed phenomenon of reversible proteasome aggregation in cells. Readers will benefit from the large array of ubiquitin-centric tools that are described to study key aspects of UCH37 function and from knowledge of the specific role of UCH37.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Joint Public Review:
The proteasome governs the fate of hundreds of proteins in the cell and is the most prominent destination for ubiquitin-protein conjugates. One of the persistently enigmatic aspects of the proteasome function is how it is coupled to deubiquitination of the substrate. Uch37 is one of three deubiquitinating enzymes, each very different, that effect these steps in substrate processing. Undoubtedly, the specificity of Uch37 for branched ubiquitin chains is a significant issue in the field. But it is a challenging problem technically and linking the debranching activity to specific biology is at an early stage at best. This study explores these issues at a high technical level. There are considerable structural data on Uch37's interaction with ubiquitin, but they do not provide a concrete model for how the branch is …
Joint Public Review:
The proteasome governs the fate of hundreds of proteins in the cell and is the most prominent destination for ubiquitin-protein conjugates. One of the persistently enigmatic aspects of the proteasome function is how it is coupled to deubiquitination of the substrate. Uch37 is one of three deubiquitinating enzymes, each very different, that effect these steps in substrate processing. Undoubtedly, the specificity of Uch37 for branched ubiquitin chains is a significant issue in the field. But it is a challenging problem technically and linking the debranching activity to specific biology is at an early stage at best. This study explores these issues at a high technical level. There are considerable structural data on Uch37's interaction with ubiquitin, but they do not provide a concrete model for how the branch is recognized. It is an intuitively reasonable model that, to resolve a branch, two ubiquitins must be recognized, and that the "first" recognition site is simply the active site of Uch37. Through exemplary experiments involving among other things NMR, enzyme kinetics, microscale thermophoresis, and tailor-made mutations in specific ubiquitin groups within the three-part branched structure, the authors construct a convincing argument that specific hydrophobic patches within two of the three ubiquitin are recognized by Uch37, the two ubiquitin groups being the substrate-distal groups. Uch37 functions as part of a complex with Rpn13, a ubiquitin receptor, but it is excluded that the ubiquitin recognition site in Rpn13 provides the "second" site. Interestingly, another domain in Rpn13 does contribute to the specificity of Uch37, by helping to position an active site loop within Uch37 so as to suppress hydrolysis of some non-branched chains.
The latter half of the paper consists mainly of studies in cells that explore the biological significance of debranching. When ubiquitin chain levels were examined (Fig 6A), the strengths of the effects of various mutants varied in arguably surprising ways, as did the relative strengths of the effects when assessed by different anti-ubiquitin antibodies (where the exact same samples were being examined). Thus, the EWI mutant had a modest effect with FK2 antibodies, much less than C88A, but was spot on with C88A with the K11/K48 anti-branch antibody. Should this be interpreted as the K11/K48 antibody reporting on deubiquitination of true Uch37 substrates whereas the FK2 antibody is reporting as well (perhaps mainly) on some more indirect perturbation of proteasome function (where ubiquitin conjugates may be unproductively retained at Uch37, not only not deubiquitinated). It would also help to show the extent of the enzymatic defect in the EWI mutant using the standard in vitro assays in the paper.
This paper is technically excellent and is a significant contribution to our understanding of deubiquitination, focusing on a very interesting member of this enzyme family. The manuscript follows up on a previous publication that UCH37 catalyzes deubiquitination of branched ubiquitin chains, cleaving the K48 linkage. The authors find preference for K6/K48 branches over K11/K48 and K48/K63 branches and use NMR to identify interactions with the hydrophobic patches of both distal ubiquitins. Amino acid substitutions demonstrate the importance of the K6 distal Ub hydrophobic patch and not the proximal Ub for debranching of the chain. UCH37 is further found to localize to proteasome and K48 ubiquitin containing stress granules, which increase in abundance and K11/K48 ubiquitin chains when UCH37 is knocked out of cells. Altogether this manuscript provides new advances in characterizing UCH37 activity in cells.
This manuscript reports a careful mechanistic study of the deubiquitylating enzyme UCH37 that was shown to be involved in hydrolyzing K48-linked branched ubiquitin structures. Using a clever combination of chemical biology techniques, the authors synthesized homogenous branched ubiquitin substrates for assays with UCH37. This led to the discovery that K11/K48 linkage is turned over the fastest and that UCH37 binds both ubiquitin proteins attached to the central ubiquitin in K6/K48 linked triubiquitin. Furthermore, the authors demonstrated that mutation of UCH37 leads to the formation of proteasomal foci due to the accumulation of polyubiquitylated species. Thus, the authors have associated the debranching function of UCH37 with efficient protein turnover by 26S proteasomes. As such, the manuscript is an excellent addition to our knowledge of UCH37 from a previous study by Strieter and colleagues in Mol. Cell 2020 that also demonstrated that debranching activity of UCH37 was associated with efficient proteasomal turnover of ubiquitylated proteins. The novel aspect of the current study lies in the demonstration that UCH-37 inactivation leads to enhanced proteasomal foci formation, and these foci are also enriched in the shuttle protein RAD23B. Thus, the authors provide a comprehensive picture of UCH37 function that confirms the Molecular Cell study and extends it to specific binding modes and cellular phenomena such as foci formation.
Overall, this is an excellent story with well thought out experiments. The diversity of chemistry applied was marvellous.
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