The deubiquitinase TRABID stabilizes the K29/K48-specific E3 ubiquitin ligase HECTD1.
Amino Acid Sequence
/ genetics
Animals
COS Cells
Chlorocebus aethiops
Dogs
Endopeptidases
/ chemistry
HEK293 Cells
Humans
Madin Darby Canine Kidney Cells
Point Mutation
/ genetics
Proteolysis
Proteomics
Signal Transduction
/ genetics
Substrate Specificity
/ genetics
Ubiquitin
/ chemistry
Ubiquitin-Protein Ligases
/ chemistry
Ubiquitination
/ genetics
E3 ubiquitin ligase
HECTD1
K29/K48-linked polyubiquitin chain
TRABID
deubiquitination
polyubiquitin chain
protein degradation
ubiquitin
ubiquitin thioesterase
Journal
The Journal of biological chemistry
ISSN: 1083-351X
Titre abrégé: J Biol Chem
Pays: United States
ID NLM: 2985121R
Informations de publication
Date de publication:
Historique:
received:
08
07
2020
revised:
23
12
2020
accepted:
30
12
2020
pubmed:
16
4
2021
medline:
25
8
2021
entrez:
15
4
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33853758
pii: S0021-9258(21)00011-9
doi: 10.1074/jbc.RA120.015162
pmc: PMC7948964
pii:
doi:
Substances chimiques
Ubiquitin
0
HectD1 protein, human
EC 2.3.2.26
Ubiquitin-Protein Ligases
EC 2.3.2.27
Endopeptidases
EC 3.4.-
ZRANB1 protein, human
EC 3.4.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
100246Subventions
Organisme : Medical Research Council
ID : MC_U105192713
Pays : United Kingdom
Organisme : Medical Research Council
ID : U105192713
Pays : United Kingdom
Informations de copyright
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
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