Ubiquitin is a carbon dioxide–binding protein

Abstract

The identification of CO 2 -binding proteins is crucial to understanding CO 2 -regulated molecular processes. CO 2 can form a reversible posttranslational modification through carbamylation of neutral N-terminal -amino or lysine -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 CO 2 -binding protein. We use 13 C-NMR spectroscopy to demonstrate that CO 2 forms carbamates on the ubiquitin N terminus and -amino groups of lysines 6, 33, 48, and 63. We demonstrate that biologically relevant pCO 2 levels reduce ubiquitin conjugation at lysine-48 and down-regulate ubiquitin-dependent NF-B pathway activation. Our results show that ubiquitin is a CO 2 -binding protein and demonstrates carbamylation as a viable mechanism by which mammalian cells can respond to fluctuating pCO 2 .

Editorial: Elevated Carbon Dioxide Sensing and Physiologic Effects

“…TRAF2 is well known as an E3-ubiquitin ligase capable of facilitating K63 polyubiquitination, and also as a ubiquitin ligase important for NF-kappaB-dependent signaling cascades ( Zhou et al, 2013 ). These findings linking hypercapnia and an E3-ubiquitin ligase are of particular interest given the recent findings by the Cann group in Durham University implicating ubiquitin itself as a direct target for post-translational carboxylation ( Linthwaite et al, 2021 ).…”

Carbon Dioxide and the Carbamate Post-Translational Modification

“…When PreLysCar was applied to a subset of solved protein structures from the PDB (structures greater than 200 residues, solved by X-ray crystallography, containing 14,261 protein chains after 90% redundancy removal), it predicted that at least 1.3% of proteins with more than 200 amino acids in the PDB could potentially be subject to spontaneous lysine carboxylation. As the model has been trained using previously identified stable carbamates, which are predominantly buried, it may only represent a subset of possible KCX sites ( Linthwaite et al, 2020 ).…”

“… Linthwaite et al, 2021 recently explored the potential modification of human Ub by CO 2 . They demonstrated that Ub K33 and K48 could be carbamylated under physiological conditions by CO 2 (25 mM CO 2 /HCO 3 at pH 7.4), using the “trapping” method ( Figure 4 ) outlined in Linthwaite et al, 2018 .…”

“…Ub carbamylation was confirmed independently by 13 C-NMR, identifying K6 and K63 as additional carbamylation sites. Using in vitro conjugation assays, Linthwaite et al (2021) found that di-Ub formation at K48 was significantly decreased when the CO 2 concentration was increased from the normal physiological reference range (1.8–2.3 mM dissolved CO 2 ) to the hypercapnic range (>2.3 mM CO 2 ).…”

“…When cells carrying an NF-κB dependent GFP reporter were exposed to increasing TNFα concentrations under normal (5% (v/v) CO 2 in air) and elevated CO 2 conditions (10% (v/v) CO 2 in air), NF-kB dependent GFP reporter activity was significantly decreased under elevated CO 2 ( Linthwaite et al, 2021 ). Transfection of the same cells with plasmids encoding wild-type Ub, mutant K48R Ub, mutant K63R Ub, or an empty vector was used to address the hypothesis that overexpression of K48R Ub would alter the relative response of the NF-kB pathway to elevated CO 2 .…”