Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes.
Bianca D M van TolBjorn R van DoodewaerdGuinevere S M Lageveen-KammeijerBas C JansenCami M P Talavera OrmeñoPaul J M HekkingAysegul SapmazRobbert Q KimAngeliki MoutsiopoulouDavid KomanderDana L E VergoossenGerbrand J van der Heden van NoortHuib OvaaPaul P GeurinkPublished in: Nature communications (2023)
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.
Keyphrases
- hiv testing
- genome wide
- mass spectrometry
- amino acid
- induced apoptosis
- men who have sex with men
- structural basis
- small molecule
- high resolution
- high throughput
- gene expression
- cell cycle arrest
- transcription factor
- liquid chromatography
- oxidative stress
- signaling pathway
- risk assessment
- adverse drug
- gas chromatography
- human immunodeficiency virus
- hiv infected
- hepatitis c virus
- cell death
- human health