Employing neutron-encoded monoUbs to study E2/E3 ligase activity and selectivity for assembling Ub chains

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Abstract While protein ubiquitination is an extensively studied post-translational modification, many aspects of this process remain unclear. Ubiquitin conjugation involves the action of three different types of enzymes working in concert to install ubiquitin onto substrate proteins. Despite efforts, an in vitro mid/high-throughput screen to quickly determine which enzymes work together to build ubiquitin chains and directly analyze the type(s) of chains formed does not exist. In this study, we developed a new multiplexed mass spectrometry-based E1-E2-E3 assay that enables the analysis of whether E2/E3 pairs work together to form ubiquitin chains and concomitantly reports on the nature of the formed ubiquitin chain type(s). The assay employs synthetic modified neutron-encoded monoUb substrates with a distinct molecular weight, enabling the simultaneous analysis of these substrates. Overall, various E2-E3 pairs were screened for their ability to build Ub chains, which furnished a three-dimensional overview of linkage selectivity over time and enzyme concentration. Graphical abstract

Authors 10

1. Bianca D. M. van Tol Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC);

   Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands

2. Bjorn R. van Doodewaerd Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC);

   Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands

3. Guinevere S. M. Lageveen‐Kammeijer Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC);

   Center for Proteomics and Metabolomics, Leiden University Medical Center, 333 ZA Leiden, The Netherlands

4. Bas C. Jansen Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC);

   Center for Proteomics and Metabolomics, Leiden University Medical Center, 333 ZA Leiden, The Netherlands

5. Joanna Liwocha

   Max Planck Institute of Biochemistry

   Affiliation as printed

   Max Planck Institute of Biochemistry, Martinsried, Germany;

   Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany

6. Rishov Mukhopadhyay Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC);

   Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands

7. Manfred Wuhrer Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center;

   Center for Proteomics and Metabolomics, Leiden University Medical Center, 333 ZA Leiden, The Netherlands

8. Gerbrand J. van der Heden van Noort Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology , Division of Chemical Biology and Drug Discovery , Leiden University Medical Center , 2333 ZC Leiden , The Netherlands

   Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands

9. Brenda A. Schulman

   Max Planck Institute of Biochemistry

   Affiliation as printed

   Max Planck Institute of Biochemistry

   Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany

10. Paul P. Geurink corresponding Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

    Leiden University · Leiden University Medical Center

    Affiliation as printed

    Leiden University Medical Center;

    Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands