Abstract
The identification of CO2-binding proteins is crucial to understanding CO2-regulated molecular processes. CO2 can form a reversible posttranslational modification through carbamylation of neutral N-terminal α-amino or lysine ε-amino groups. We have previously developed triethyloxonium (TEO) ion as a chemical proteomics tool for covalent trapping of carbamates, and here, we deploy TEO to identify ubiquitin as a mammalian CO2-binding protein. We use 13C-NMR spectroscopy to demonstrate that CO2 forms carbamates on the ubiquitin N terminus and ε-amino groups of lysines 6, 33, 48, and 63. We demonstrate that biologically relevant pCO2 levels reduce ubiquitin conjugation at lysine-48 and down-regulate ubiquitin-dependent NF-κB pathway activation. Our results show that ubiquitin is a CO2-binding protein and demonstrates carbamylation as a viable mechanism by which mammalian cells can respond to fluctuating pCO2.