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Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes

Author

Listed:
  • 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)

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.

Suggested Citation

  • 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, 2023. "Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-37363-6
    DOI: 10.1038/s41467-023-37363-6
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    References listed on IDEAS

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    1. Kirby N. Swatek & Joanne L. Usher & Anja F. Kueck & Christina Gladkova & Tycho E. T. Mevissen & Jonathan N. Pruneda & Tim Skern & David Komander, 2019. "Insights into ubiquitin chain architecture using Ub-clipping," Nature, Nature, vol. 572(7770), pages 533-537, August.
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