The identification of carbon dioxide-binding proteins

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Carbon dioxide is essential for life. It is at the beginning of every life process as a fundamental substrate of photosynthesis or chemosynthesis and is at the end of every life process as the product of aerobic respiration and post-mortem decay. As such, it is not a surprise that this gas regulates such diverse processes as cellular chemical reactions, transport, maintenance of the cellular environment, behaviour and immunity. Carbon dioxide is a strategically important research target with relevance to crop responses to environmental change, insect-borne disease and public health. However, we know very little of the direct interactions of carbon dioxide with the cell, despite the importance of the gas to biology.Carbon dioxide mediates the earliest known example of a protein post-translational modification (PTM), identified on haemoglobin in 1928. Carbon dioxide can directly combine with select protein groups to form carbamates. Influential research programmes from the 1920-80's demonstrated that the carbamate PTM regulates oxygen-binding in haemoglobin and activates the carbon dioxide-fixing enzyme Rubisco. George Lorimer proposed carbamate PTMs as a mechanism for regulating biological responses to carbon dioxide in 1983. However, the carbamate PTM is unstable outside the cell and its identification presents significant analytical challenges. Several stable carbamates have been identified in protein molecular structures, but the technical difficulties in their widespread identification has resulted in carbon dioxide-mediated carbamylation being all but forgotten as a PTM. For example, the Wikipedia page for PTM does not mention carbon dioxide-mediated carbamylation (not to be confused with the similarly named modification mediated by isocyanic acid) among 61 identified PTMs.Here we develop a new tool to investigate carbamate PTMs as a widespread mechanism enabling cells to sense carbon dioxide. We demonstrate that the tool can trap carbon dioxide on proteins and we develop a work flow to identify those proteins. We further characterise ubiquitin as a carbon dioxide-binding protein. Ubiquitin is found in all multicellular organisms and is attached to other proteins to regulate their activity and fate. Carbon dioxide regulates inflammation and we provide evidence that this is explained by carbamate formation on ubiquitin.This proposal has two hypotheses in light of these preliminary data:1. We hypothesise that the carbamate PTM is widespread in the cell and that the level of carbamylation responds to changes in carbon dioxide.2. We hypothesise that the carbamate PTM mediates mediates biologically relevant responses to carbon dioxide.We will use mass spectrometry-based proteomics to address the first hypothesis. These experiments will demonstrate the diversity of carbon dioxide-binding proteins in the cell and how carbon dioxide bound to protein changes in response to fluctuating gas. The experiments will provide new insight into the diversity of molecular processes regulated by carbon dioxide and provide tools to identify such processes in other biological systems.We will use a combination of biochemical, cellular and tissue-based analysis to address the second hypothesis. Specifically, we will demonstrate that the carbamate PTM on ubiquitin mediates the influence of carbon dioxide on transcriptional control of inflammation. The experiments will provide proof of principle that newly discovered carbamate PTMs have biological relevance. Furthermore, the experiments will investigate a surprising link between carbon dioxide and ubiquitination, a protein modification that itself exerts a diverse influence on protein activity.As the tools and insights that arise can be directly applied to diverse biological problems and systems, the proposal will transform our understanding of direct molecular responses to carbon dioxide.