Abstract
USP37 is a deubiquitinase (DUB) with roles in the regulation of DNA damage repair and the cohesion of sister chromatids during mitosis. USP37 contains a unique insert of three ubiquitin interacting motifs (UIMs) within its catalytic DUB domain. We investigated the role of the three UIMs in the ability of USP37 to cleave di-ubiquitin chains. We found that the third UIM of USP37 recognizes the proximal ubiquitin moiety of K48 di-Ub to potentiate cleavage activity and posit that this mechanism of action may be generalizable to other chain types. In the case of K48-linked ubiquitin chains this potentiation stemmed largely from a dramatic increase in catalytic rate (kcat). We also developed and characterized three ubiquitin variant (UbV) inhibitors that selectively engage distinct binding sites in USP37. In addition to validating the deduced functional roles of the three UIMs in catalysis, the UbVs highlight a novel and effective means to selectively inhibit members of the difficult to drug DUB family.
Highlights
Ubiquitination is the process whereby the small protein ubiquitin (Ub) is covalently attached to a substrate protein via a cascade of three (E1-E2-E3) enzymes
The importance of UIM2 was revealed when UIM2 and UIM3 were disabled together, resulting in an enzyme (USP37mUIM2,3) in which activity was almost completely abolished relative to WT (~4% activity remaining based on V0). Such a drastic effect was not observed for the same double mutant when cleaving K11- or K63-linked chains, where USP37mUIM2,3 retained ~20% or ~18% of WT activity, respectively. These results suggested that UIM1 does not contribute to the activity of USP37 towards K11, K48- and K63-linked chains, whereas UIM2 and UIM3 do contribute to activity towards these chains, but to varying degrees that are dependent on the specific type of chain linkage
Our results are consistent with a model whereby UIM2 and UIM3 of USP37 bind the proximal Ub of K48 di-Ub to increase catalytic efficiency
Summary
Ubiquitination is the process whereby the small protein ubiquitin (Ub) is covalently attached to a substrate protein via a cascade of three (E1-E2-E3) enzymes. Mutation of UIM2 and/or UIM3 perturbed USP37 binding to the cohesin regulator WAPL and to endogenous Ub-protein conjugates[15,26] While these observations made clear that UIM2 and UIM3 play an important functional role in supporting the DUB activity of USP37, several questions remain unresolved, including: 1) do the individual UIMs impact the ability of USP37 to cleave the 6 Ub chain types not previously tested, 2) does mutation of the UIMs in USP37 selectively affect the kcat or KM of the enzyme, and 3) do the UIMs of USP37 act by engaging the proximal or distal position Ub moiety in a Ub chain.
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.
