OTU Deubiquitinases Reveal Mechanisms of Linkage Specificity and Enable Ubiquitin Chain Restriction Analysis

Summary

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates.

Graphical Abstract

Highlights

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  The 16 human OTU DUBs cleave distinct sets of ubiquitin chain types

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  Five crystal structures of three human OTU DUBs reveal uncharacterized Ub-binding sites

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  We reveal four distinct mechanisms of linkage specificity in OTU DUBs

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  OTU DUBs can be used to identify the linkage types on a ubiquitinated substrate

Abstract

OTU deubiquitinases use four distinct mechanisms of linkage specificity to hydrolyze ubiquitin, and, due to their specificity, OTU DUBs can be used in ubiquitin chain restriction analysis to characterize the chain types on ubiquitinated proteins.