Post-translational modifications mediated by carbon dioxide

Signalling by gaseous molecules is fundamental to physiology and disease. Nitric oxide was originally identified as a molecule regulating cardiovascular physiology (Nobel Prize 1998). Oxygen is sensed by the HIF-1 transcription factor to aid cell survival under hypoxia (Nobel Prize 2019). Carbon dioxide is also vital to life processes that include metabolism, cellular homeostasis, chemosensing and pathogenesis. CO2 is of strategic research importance in plants through its role in climate change and physiology. CO2 is of further strategic research importance in mammals through the contribution of elevated CO2 (hypercapnia) to poor prognosis in sleep apnoea, obesity and chronic obstructive pulmonary disorder. Remarkably, we have almost no knowledge of direct CO2 targets in biological systems.

We have developed a chemical proteomics tool for the systematic identification of CO2-binding proteins and demonstrated that CO2 can impact the activity of these proteins (Linthwaite et al. The identification of carbon dioxide mediated protein post-translational modifications. Nature Communications (2018) 9:3092 DOI: 10.1038/s41467-018-05475-z).

A proteomic screen with our chemical genetics tool identified Ubiquitin (Ub) as a CO2-binding protein. Ub is an 8.5 kDa protein found in all eukaryotic cells and regulates protein activity through conjugation to target proteins. It is established that elevated CO2 suppresses NF-kB-mediated transcription. Ub-mediated protein cross-linking regulates NF-kB signalling. We therefore hypothesise that CO2 regulation of Ub cross linking alters NF-kB signalling.

We will investigate how fluctuations in cellular CO2 alter ubiquitin-dependent NF-kB signalling.

In vitro: We will use a combination of 13C-NMR, chemical genetics and ubiquitin cross-linking assays to demonstrate that CO2 inhibits Ub cross-linking specifically at K48 over physiologically relevant concentrations.

In vivo: We will use a combination chemical genetics and mass spectrometry to demonstrate that CO2 inhibits Ub cross-linking specifically at K48 over physiologically relevant concentrations. We will use a combination of Immunoprecipitation and Western Blotting using antibodies specific for components of the NF-kB signalling pathway to elucidate which ubiquitin target is influenced by CO2.