Abstract
Ubiquitin is a versatile posttranslational modification, which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains, which have been extensively studied, the regulation and function of most atypical ubiquitin chains are only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. The E3 ubiquitin ligase HECTD1 was then validated as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knockout of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase that assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.
Highlights
Through the sequential activity of E1-activating enzymes, E2conjugating enzymes, and E3 ubiquitin ligases, as a single moiety or as a ubiquitin chain [4, 5]
We analyzed the interactome of two catalytic dead TRABID constructs following transient expression in HEK293ET cells (Fig. 1, A–C)
DUBs often exist as part of protein complexes, which can include other DUBs as well as E3 ubiquitin ligases, and we found both classes of enzymes in our candidate substratome list (Fig. 1, B and C; Table S1) [40]
Summary
Through the sequential activity of E1-activating enzymes, E2conjugating enzymes, and E3 ubiquitin ligases, as a single moiety or as a ubiquitin chain [4, 5]. The discovery of the AnkUBD as a novel UBD abutting the N-terminus of TRABID OTU domain revealed that it is required for full DUB activity and to some extent specificity too These findings as well as recent studies, which identified TRABID’s NZF 1 as the minimal UBD required for the recognition of K29- and K33-linked diubiquitin, establish TRABID as a unique DUB highly tuned for the recognition and processing of these atypical ubiquitin chains [29,30,31]. TRABID was shown to process K29 and K33-linked chains on UVRAG, a Beclin 1 complex component, thereby inhibiting autophagy and increasing hepatocellular cellular carcinoma growth [36] Another interesting finding came through phenotypic studies of Trabid KO mice, which revealed that in dendrocytes, Trabid loss of function led to proteasomal degradation of the histone demethylase Jmjd2 [37]. These studies highlight a role for TRABID in transcriptional regulation as well as novel cross talk between protein ubiquitination and epigenetics mechanisms the types and composition of ubiquitin chains involved remain to be determined
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