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  • Deubiquitinating enzymes and the proteasome regulate preferential sets of ubiquitin substrates.

Deubiquitinating enzymes and the proteasome regulate preferential sets of ubiquitin substrates.

Nature communications (2022-05-19)
Fredrik Trulsson, Vyacheslav Akimov, Mihaela Robu, Nila van Overbeek, David Aureliano Pérez Berrocal, Rashmi G Shah, Jürgen Cox, Girish M Shah, Blagoy Blagoev, Alfred C O Vertegaal
ABSTRACT

The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Here, we compare the contributions of the proteasome and DUBs on the global ubiquitinome, using UbiSite technology, inhibitors and mass spectrometry. We uncover large dynamic ubiquitin signalling networks with substrates and sites preferentially regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitination. DUBs regulate substrates via at least 40,000 unique sites. Regulated networks of ubiquitin substrates are involved in autophagy, apoptosis, genome integrity, telomere integrity, cell cycle progression, mitochondrial function, vesicle transport, signal transduction, transcription, pre-mRNA splicing and many other cellular processes. Moreover, we show that ubiquitin conjugated to SUMO2/3 forms a strong proteasomal degradation signal. Interestingly, PARP1 is hyper-ubiquitinated in response to DUB inhibition, which increases its enzymatic activity. Our study uncovers key regulatory roles of DUBs and provides a resource of endogenous ubiquitination sites to aid the analysis of substrate specific ubiquitin signalling.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
Sigma-Aldrich
PARG human, recombinant, expressed in Sf21 cells, His tagged, >95% (SDS-PAGE)
Sigma-Aldrich
Cycloheximide, from microbial, ≥94% (TLC)
Sigma-Aldrich
Z-Leu-Leu-Leu-al, ≥90% (HPLC)
Sigma-Aldrich
Anti-Ubiquitin (pan) Antibody, clone 2G7B8, clone 2G7B8, from mouse