Abstract
SummarySixteen 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.
Highlights
Protein ubiquitination is a posttranslational modification of mostly Lys residues that regulates many cellular processes, including protein degradation, intracellular trafficking, cell signaling, autophagy, transcription, translation, and the DNA damage response (Komander and Rape, 2012)
Using Ub chains of all eight linkage types, we reveal that most human ovarian tumor (OTU) enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains
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
Summary
Protein ubiquitination is a posttranslational modification of mostly Lys residues that regulates many cellular processes, including protein degradation, intracellular trafficking, cell signaling, autophagy, transcription, translation, and the DNA damage response (Komander and Rape, 2012). This functional diversity is achieved by the ability of ubiquitin (Ub) to form topologically distinct signals. Within Ub chains, linkages can be formed via seven Ub Lys residues or via the N-terminal Met, generating homotypic (one linkage type per polymer) or heterotypic (multiple linkage types per polymer) Ub chains (Komander and Rape, 2012). Met1-linked chains cooperate with Lys linkages in NF-kB signaling (Iwai, 2011). For the remaining four Ub chain types (Lys, Lys, Lys, and Lys33), cellular roles are elusive (Kulathu and Komander, 2012)
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