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Ubiquitin is a versatile post-translational 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 is 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. We then validated the E3 ubiquitin ligase HECTD1 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 knock out 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 which assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.

Original publication

DOI

10.1074/jbc.RA120.015162

Type

Journal article

Journal

J Biol Chem

Publication Date

30/12/2020

Keywords

E3 ubiquitin ligase, HECTD1, K29/K48-linked polyubiquitin chain, TRABID, branched ubiquitin chains, deubiquitylation (deubiquitination), polyubiquitin chain, protein degradation, protein-protein interaction, proteomics, ubiquitin, ubiquitin ligase, ubiquitin thioesterase (OTUB1)