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The deubiquitinase TRABID stabilises the K29/K48-specific E3 ubiquitin ligase HECTD1

ABSTRACT: The OTU deubiquitinase TRABID is highly tuned for the recognition and cleavage of K29 and K33-linked ubiquitin chains. Yet the cellular functions of these atypical ubiquitin signals remain unclear. Here, we compared the interactome of two different catalytically-dead TRABID constructs, one that lacks the OTU domain and the other a single point mutant that is catalytically inactive. Since both constructs also act as ubiquitin trapping mutants, this approach was used to differentiate between interactors and substrates. The E3 ubiquitin ligase HECTD1 was confirmed as a TRABID substrate, and using UbiCREST and Ubiquitin-AQUA proteomics, we determined that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that in contrast to UBE3C, HECTD1 requires the formation of branched K29/K48-linked chains for its full activity. Finally, we used transient knockdown and genetic knock out of TRABID in mammalian cells to show that TRABID stabilises HECTD1 protein levels. This study identifies TRABID-HECTD1 as a K29-specific DUB/E3 pair and provides novel insights on the assembly of branched ubiquitin signals.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast

DISEASE(S): Disease Free

SUBMITTER: Mark Skehel

LAB HEAD: Julien D F Licchesi

PROVIDER: PXD022703 | Pride | 2021-06-23

REPOSITORIES: Pride

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			Action	DRS
	MMW273_P1_JL_A1.raw	Raw
	MMW273_P1_JL_A10.raw	Raw
	MMW273_P1_JL_A2.raw	Raw
	MMW273_P1_JL_A3.raw	Raw
	MMW273_P1_JL_A4.raw	Raw

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