Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes

Bianca D. M. Tol, Bjorn R. Doodewaerd, Guinevere S. M. Lageveen-Kammeijer, Bas C. Jansen, Cami M. P. Talavera Ormeño, Paul J. M. Hekking, Aysegul Sapmaz, Robbert Q. Kim, Angeliki Moutsiopoulou, David Komander, Manfred Wuhrer, Gerbrand J. Heden van Noort, Huib Ovaa and Paul P. Geurink ()
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Bianca D. M. Tol: Leiden University Medical Center
Bjorn R. Doodewaerd: Leiden University Medical Center
Guinevere S. M. Lageveen-Kammeijer: Leiden University Medical Center
Bas C. Jansen: Leiden University Medical Center
Cami M. P. Talavera Ormeño: Leiden University Medical Center
Paul J. M. Hekking: Leiden University Medical Center
Aysegul Sapmaz: Leiden University Medical Center
Robbert Q. Kim: Leiden University Medical Center
Angeliki Moutsiopoulou: Leiden University Medical Center
David Komander: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville
Manfred Wuhrer: Leiden University Medical Center
Gerbrand J. Heden van Noort: Leiden University Medical Center
Huib Ovaa: Leiden University Medical Center
Paul P. Geurink: Leiden University Medical Center

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: Abstract Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.

Date: 2023
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DOI: 10.1038/s41467-023-37363-6

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