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

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.

Regulation of USP28 Deubiquitinating Activity by SUMO Conjugation

“…A previous report with several USP proteins, which includes the homologous USP25, showed only minor differences against all eight diubiquitin substrates (39). In contrast, the deubiquitinating activity of USP28 against diubiquitin linkages resembles the OTU DUB family, where members are specific for one or a small subset of diubiquitin linkages types (40). It is interesting that in addition to the most common Lys 48 and Lys 63 linkages, Lys 11 is preferred by USP28 in our diubiquitin cleavage analysis (Fig.…”

“…In contrast to the other DUBs families, such as the OTU DUBs (40), most of the members of the USP family have been reported to display a promiscuous deubiquitinating activity preference in an assay against the eight possible diubiquitin linkages (39). An exception to this is the tumor suppressor CYLD, which possesses specific deubiquitinating activity for Lys 63 -linked ubiquitin chains synthesized in response to cytokine-mediated activation of TRAF2 and TRAF3 ubiquitin E3 ligases, thus antagonizing NF-B signaling (13).…”

“…In particular, Lys 11 -linked polyubiquitin chain has been recently described as an alternative degradation signal used to facilitate cell cycle progression (41). It will be of interest to define the structural determinants required for the cleavage of Lys 11 -linked chains by USP28, because they likely depend on specific interactions with the catalytic domain, in contrast to what has been described for CYLD and some OTU DUB members (13,40).…”

PTEN regulates RPA1 and protects DNA replication forks

“…Ubiquitin (Ub)-substrate isopeptide bonds can be cleaved by deubiquitinases (DUBs). Five DUB families have been identified including ovarian tumor proteases (OTUs) [15]. OTUB1 is an OTU family DUB cysteine protease highly specific for cleaving Lys48-linked polyubiquitin chains, which targets proteins for proteasomal degradation [16][17][18][19].…”

Structural Basis for Ovarian Tumor Domain-containing Protein 1 (OTU1) Binding to p97/Valosin-containing Protein (VCP)

“…However, UBXL has low sequence identity of less than 15% to UBX and furthermore lacks the signature motif of UBX. So far, the structure of the OTU domain from yeast complexed with ubiquitin has been produced (18), and recently it has been demonstrated that YOD1, the mammalian homolog of OTU1, has high specificity for Lys 11 -linked ubiquitin chains in a report that shows the complex structure of YOD1 with Lys 11 -linked diubiquitin (19). To understand how OTU1 interacts with VCP on a molecular level and to gain insight into how this interaction regulates the ERAD pathway, we carried out a structural study on N VCP complexed with UBXL OTU1 and performed negative staining electron microscopy (EM) and biochemical studies of the full-length proteins.…”