TY - JOUR
T1 - Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes
AU - van Tol, Bianca D M
AU - van Doodewaerd, Bjorn R
AU - Lageveen-Kammeijer, Guinevere S M
AU - Jansen, Bas C
AU - Talavera Ormeño, Cami M P
AU - Hekking, Paul J M
AU - Sapmaz, Aysegul
AU - Kim, Robbert Q
AU - Moutsiopoulou, Angeliki
AU - Komander, David
AU - Wuhrer, Manfred
AU - van der Heden van Noort, Gerbrand J
AU - Ovaa, Huib
AU - Geurink, Paul P
N1 - © 2023. The Author(s).
PY - 2023/3/25
Y1 - 2023/3/25
N2 - 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.
AB - 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.
KW - Ubiquitination
KW - Polyubiquitin/metabolism
KW - Deubiquitinating Enzymes/metabolism
KW - Ubiquitin/metabolism
KW - Ubiquitins/metabolism
U2 - 10.1038/s41467-023-37363-6
DO - 10.1038/s41467-023-37363-6
M3 - Article
C2 - 36966155
SN - 2041-1723
VL - 14
SP - 1661
JO - Nature Communications
JF - Nature Communications
IS - 1
ER -