DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

A. Barbara Dirac-Svejstrup; Jane Walker; Peter Faull; Vesela Encheva; Vyacheslav Akimov; Michele Puglia; David Perkins; Sandra Kümper; Suchete S. Hunjan; Blagoy Blagoev; Ambrosius P. Snijders; David J. Powell; Jesper Q. Svejstrup

doi:10.1016/j.molcel.2020.05.035

DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

A. Barbara Dirac-Svejstrup, Jane Walker, Peter Faull, Vesela Encheva, Vyacheslav Akimov, Michele Puglia, David Perkins, Sandra Kümper, Suchete S. Hunjan, Blagoy Blagoev, Ambrosius P. Snijders, David J. Powell, Jesper Q. Svejstrup^*

^*Corresponding author for this work

PCE - Proteomics Center of Excellence

Research output: Contribution to journal › Article › peer-review

50 Scopus citations

Abstract

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.

Original language	English (US)
Pages (from-to)	332-341.e7
Journal	Molecular cell
Volume	79
Issue number	2
DOIs	https://doi.org/10.1016/j.molcel.2020.05.035
State	Published - Jul 16 2020

Funding

This work was supported by the Francis Crick Institute (FCI), which receives its core funding from Cancer Research UK ( FC001166 ), the UK Medical Research Council ( FC001166 ), and the Wellcome Trust ( FC001166 ), and by a grant from the European Research Council, Agreements 693327 (TRANSDAM) to JQS, while work in the Blagoev lab was funded by the Danish National Research Foundation (ATLAS, DNRF grant No. 141 ) and the Novo Nordisk Foundation ( NNF18OC0052768 ). We thank Cell Services, the Crick Flow Cytometry Platform, and the High Throughput Screening Facility of the FCI for their support and time spent on this project and Mao Xiang Chen at GSK who designed the lentivirus constructs expressing human DDI2 and generated the infected HEK293 cell pools.

Keywords

Bortezomib
DDI2
Ddi1
NFE2L1
NRF1
proteasome
proteasome inhibition
ubiquitin
ubiquitin protease

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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10.1016/j.molcel.2020.05.035

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Dirac-Svejstrup, A. B., Walker, J., Faull, P., Encheva, V., Akimov, V., Puglia, M., Perkins, D., Kümper, S., Hunjan, S. S., Blagoev, B., Snijders, A. P., Powell, D. J., & Svejstrup, J. Q. (2020). DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1. Molecular cell, 79(2), 332-341.e7. https://doi.org/10.1016/j.molcel.2020.05.035

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abstract = "The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.",

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AU - Faull, Peter

AU - Encheva, Vesela

AU - Akimov, Vyacheslav

AU - Puglia, Michele

AU - Perkins, David

AU - Kümper, Sandra

AU - Hunjan, Suchete S.

AU - Blagoev, Blagoy

AU - Snijders, Ambrosius P.

AU - Powell, David J.

AU - Svejstrup, Jesper Q.

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AB - The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.

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DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

Abstract

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Keywords

ASJC Scopus subject areas

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