A cryptic K48 ubiquitin chain binding site on UCH37 is required for its role in proteasomal degradation
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Evaluation Summary:
This study identifies a cryptic ubiquitin-binding site on the proteasome-associated deubiquitinase UCH37 and reveals that branched ubiquitin chains are bound and processed differently from mono-ubiquitin modifications. The authors use a variety of elegant biochemical and biophysical approaches to characterize this new binding site, and the conclusions are very well supported by the experimental data. These findings provide important new insights into the cleavage of branched ubiquitin chains during protein degradation by the 26S proteasome, therefore represent a critical advance to the ubiquitin-proteasome field and our understanding of how ubiquitin signaling regulates protein turnover in the cell, and will be of interest to a broad audience.
(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. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Degradation by the 26 S proteasome is an intricately regulated process fine tuned by the precise nature of ubiquitin modifications attached to a protein substrate. By debranching ubiquitin chains composed of K48 linkages, the proteasome-associated ubiquitin C-terminal hydrolase UCHL5/UCH37 serves as a positive regulator of protein degradation. How UCH37 achieves specificity for K48 chains is unclear. Here, we use a combination of hydrogen-deuterium mass spectrometry, chemical crosslinking, small-angle X-ray scattering, nuclear magnetic resonance (NMR), molecular docking, and targeted mutagenesis to uncover a cryptic K48 ubiquitin (Ub) chain-specific binding site on the opposite face of UCH37 relative to the canonical S1 (cS1) ubiquitin-binding site. Biochemical assays demonstrate the K48 chain-specific binding site is required for chain debranching and proteasome-mediated degradation of proteins modified with branched chains. Using quantitative proteomics, translation shutoff experiments, and linkage-specific affinity tools, we then identify specific proteins whose degradation depends on the debranching activity of UCH37. Our findings suggest that UCH37 and potentially other DUBs could use more than one S1 site to perform different biochemical functions.
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Evaluation Summary:
This study identifies a cryptic ubiquitin-binding site on the proteasome-associated deubiquitinase UCH37 and reveals that branched ubiquitin chains are bound and processed differently from mono-ubiquitin modifications. The authors use a variety of elegant biochemical and biophysical approaches to characterize this new binding site, and the conclusions are very well supported by the experimental data. These findings provide important new insights into the cleavage of branched ubiquitin chains during protein degradation by the 26S proteasome, therefore represent a critical advance to the ubiquitin-proteasome field and our understanding of how ubiquitin signaling regulates protein turnover in the cell, and will be of interest to a broad audience.
(This preprint has been reviewed by eLife. We include the public reviews …
Evaluation Summary:
This study identifies a cryptic ubiquitin-binding site on the proteasome-associated deubiquitinase UCH37 and reveals that branched ubiquitin chains are bound and processed differently from mono-ubiquitin modifications. The authors use a variety of elegant biochemical and biophysical approaches to characterize this new binding site, and the conclusions are very well supported by the experimental data. These findings provide important new insights into the cleavage of branched ubiquitin chains during protein degradation by the 26S proteasome, therefore represent a critical advance to the ubiquitin-proteasome field and our understanding of how ubiquitin signaling regulates protein turnover in the cell, and will be of interest to a broad audience.
(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. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In the present manuscript, Strieter and coworkers reveal a cryptic, K48-linkage specific ubiquitin binding site on the backside of the proteasome-associated deubiquitinase UCH37, providing important new insights into the debranching activity of UCH37 and its role in proteasomal degradation of substrates marked with branched ubiquitin chains. Other groups had previously postulated the existence of an additional binding site based on functional assays, yet experimental evidence had been missing. Here, the authors use a variety of well-suited biochemical and biophysical approaches, including hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS), chemical crosslinking, NMR, small angle X-ray scattering (SAXS), molecular dynamics simulations, and site-specific mutagenesis, to identify and …
Reviewer #1 (Public Review):
In the present manuscript, Strieter and coworkers reveal a cryptic, K48-linkage specific ubiquitin binding site on the backside of the proteasome-associated deubiquitinase UCH37, providing important new insights into the debranching activity of UCH37 and its role in proteasomal degradation of substrates marked with branched ubiquitin chains. Other groups had previously postulated the existence of an additional binding site based on functional assays, yet experimental evidence had been missing. Here, the authors use a variety of well-suited biochemical and biophysical approaches, including hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS), chemical crosslinking, NMR, small angle X-ray scattering (SAXS), molecular dynamics simulations, and site-specific mutagenesis, to identify and characterize this new site regarding linkage specificity, ubiquitin binding modes and critical motifs, as well as its effects on substrate turnover by the proteasome. Revealing the involved regions and residues in UCH37 enabled the authors to place specific mutations that disrupt ubiquitin binding to this site and inhibit UCH37's debranching activity, allowing the identification of cellular substrates that depend on this activity for degradation by the proteasome.
Although the study does not uncover the molecular mechanism of UCH37's backside-mediated ubiquitin cleavage and the details of why certain substrates depend on it for proteasomal turnover, it represents an important advance to our understanding of ubiquitin-chain cleavage and editing at the 26S proteasome, and it sets the stage for future mechanistic investigations.
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Reviewer #2 (Public Review):
Du et al apply an arsenal of biophysical methods to evaluate the mechanistic origin of UCH37 preference for K48-linked Ub chains. They find a new Ub binding site formed by UCH37 residues in helix5-6 and that this region is essential for K48 chain binding and debranching as well as for UCH37-dependent substrate degradation by the proteasome. This new site for Ub binding is distinct from and in addition to that established by previous crystal structures of UCH37 with monoubiquitin. The original site is found to be the most germane for Ub-AMC cleavage, but not important for K48 chain activity. They further find proteins that are dependent on UCH37 for degradation following H2O2 treatment and thereby link UCH37 activity to cellular pathways.
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Reviewer #3 (Public Review):
In this manuscript the Strieter lab studies branched ubiquitin chain cleavage by UCH37/RPN13, using a series of biochemical and structural approaches. Interestingly they find that branched chains bind to a completely different site in the enzyme than the canonical mono-ubiquitin binding site. This allows them to identify a set of very convincing separation of function mutations, and initiate the analysis of the specific roles of UCH37 in chain debranching and mono-ubiquitin clean up. This is highly exciting development that elucidates an unexpected and beautiful biological phenomenon. There is no doubt that this manuscript will have profound impact in the future.
The experiments are creative, very well done and overall well presented. However, there are very many of those, and the following suggestions may …
Reviewer #3 (Public Review):
In this manuscript the Strieter lab studies branched ubiquitin chain cleavage by UCH37/RPN13, using a series of biochemical and structural approaches. Interestingly they find that branched chains bind to a completely different site in the enzyme than the canonical mono-ubiquitin binding site. This allows them to identify a set of very convincing separation of function mutations, and initiate the analysis of the specific roles of UCH37 in chain debranching and mono-ubiquitin clean up. This is highly exciting development that elucidates an unexpected and beautiful biological phenomenon. There is no doubt that this manuscript will have profound impact in the future.
The experiments are creative, very well done and overall well presented. However, there are very many of those, and the following suggestions may help to explain the findings to researchers that do not have a background in UCH catalysis to clarify the big picture
A) The introduction could use some clarification of concepts
a. The existing structure of UCHL37:RPN13:Ub (4WLR) is the base for the definition of the "canonical" S1 site. Yet, no figure exists that shows that structure, and there is only an incidental description of how the ubiquitin positioning in this structure relates to the message of the paper and to the backside (lines 168-169). Please show the structure as a figure, and define clearly where the S1 site is.
b. The cross-over loop (abbreviated as CL, although the abbreviation is never made explicit in the text) is clearly important in (1) determining substrate specificity and size of UCHL DUBs and (2) in the current model of RPN13-mediated regulation of UCHL37. Please add a paragraph in the introduction to discuss the CL.
c. A reminder of what is the definition of proximal and distal ubiquitin (donating the C-terminus) in this particular context should also be in the text.B) It would be very helpful if the main finding of the paper, the fact that branched chains use a novel S1 binding site, could be illustrated in a schematic figure. Ideally such a figure compares the previous S1 and the new one (could be useful to find a consistent name for it: e.g. S1*? ) for branched chains side-by-side. Of course if such a figure could reflect the various interaction sites mentioned, as well as the location of the various named parts of the ub-substrate (distal, proximal) and linkages.
There are some minor points in the writing that could be strengthened.
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