Journal of Biological Chemistry
Volume 296, January–June 2021, 100246
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Research Article
The deubiquitinase TRABID stabilizes the K29/K48-specific E3 ubiquitin ligase HECTD1

https://doi.org/10.1074/jbc.RA120.015162Get rights and content
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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.

Keywords

ubiquitin
deubiquitination
polyubiquitin chain
E3 ubiquitin ligase
ubiquitin thioesterase
protein degradation
HECTD1
TRABID
K29/K48-linked polyubiquitin chain

Abbreviations

DUB
deubiquitinase
E1
E1-activating enzyme
E2
E2-conjugating enzyme
E3
E3 ubiquitin ligase
HECT
homologous to the E6-AP carboxyl terminus (HECT) domain family
NZF
Np14 zinc finger
OTU
ovarian tumor
Ub-AQUA
ubiquitin-absolute QUAntification
UBD
ubiquitin binding domain
UbiCREST
ubiquitin chain restriction analysis

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This article contains supporting information.

These authors contributed equally.