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.
Highlights
UCH37 is a highly conserved deubiquitinating enzyme (DUB) found from fission yeast to human, but absent in the budding yeast S. cerevisiae
(3) Whereas RPN13C enhances UCH37 hydrolysis of most polyUb substrates, it strongly inhibits activity with linear K48 Ub3 (Figure 1C). (4) Consistently, we found that Ub2 and Ub1 products were always produced in a 2:1 ratio quantified by mass (Figure 1C), which is equivalent to a 1:1 molar ratio
In the crystal structures of 88 UCH37 in complex with Ub and RPN13C, we and others have observed that RPN13C interacts with two distinct regions of UCH3717,18: in addition to binding the UCH37CTD, RPN13C makes 90 contacts with UCH37 active site crossover loop (ASCL) residues M148 and F149
Summary
UCH37 ( known as UCHL5) is a highly conserved DUB found from fission yeast to human, but absent in the budding yeast S. cerevisiae It has a catalytic domain characteristic of the UCH family of DUBs and a unique C-terminal domain (CTD) that mediates interactions with two binding partners, RPN1313-15 and NFRKB. These structures revealed that, whereas RPN13 stabilizes UCH37 in a conformation that promotes Ub binding, NFRKB represses UCH37 by occluding the Ub binding site Despite that these structures provided clear understanding of how UCH37 activity is controlled in different contexts, they did not reveal how UCH37 contributes to the functions of the proteasome or INO80 complex. PolyUb conjugates that accumulate upon proteolytic stresses are greatly enhanced by the loss of UCH37 activity We show that both binding and deubiquitination of branched polyUb by UCH37 facilitate proteasome-dependent clearance of stress-induced inclusions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
