The identification of carbon dioxide-binding proteins

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.

Carbon dioxide is vital to life processes. Some physiological effects of carbon dioxide on the cell are known along with the controlling signalling pathways. However, direct carbon dioxide targets are almost completely unknown. Interactions between carbon dioxide and neutral amine groups on proteins to form anionic carbamates have been proposed as a mechanism for biological regulation by carbon dioxide. Here we demonstrate a tool to covalently trap such protein carbamates and make them accessible to proteomic analysis. We use this tool to identify carbon dioxide-binding proteins and demonstrate their relevance in carbon dioxide-responsive processes.

We will use an immunoaffinity enrichment strategy for trapped carbamylated peptides and Liquid Chromatography-Electrospray Ionisation-Mass Spectrometry for their identification. This will enable us to identify further proteins that bind and respond to altered carbon dioxide levels in cells and whole tissues.

We will expand upon our identification of ubiquitin as a new carbon dioxide target to investigate the influence of carbon dioxide on ubiquitin regulation of the transcriptional control of inflammation. We will use a combination of i) Ub cross-linking in vitro, NF-kB reporter assays, Western blotting and immunoprecipitation in cells and ii) analysis of inflammatory responses in the lung.

A consequence of the previous inability to identify carbon dioxide-proteins is a major knowledge gap in our understanding of carbon dioxide-regulated molecular processes. Our proposal will highlight the diversity of molecular processes likely to respond to carbon dioxide. Proof of principle of biological relevance will be demonstrated through an analysis of carbon dioxide regulation of ubiquitin cross-linking in transcriptional control of inflammation. The proposal therefore represents a leap in developing our understanding of molecular processes regulated by carbon dioxide.

We will generate Impact in two broad areas 1) Exploitation of carbon dioxide-directed research as a target for translation to the biopharmaceutical industry with AstraZeneca and 2) Public engagement. These two strands are expanded upon in the Pathways to Impact.

EXPLOITATION OF CARBON DIOXIDE-DIRECTED RESEARCH AS A TARGET FOR TRANSLATION TO THE BIOPHARMACEUTICAL INDUSTRY

Carbon dioxide research is of significant value to AstraZeneca through its contribution to disease states and the possibility of altered drug performance. We will collaborate with AstraZeneca to drive a translational research agenda.

IMPACT IN PUBLIC ENGAGEMENT

Carbon dioxide is of intense public interest and garners much debate. Areas of public interest include 1) the impact of carbon dioxide on crops through climate change, 2) the impact of carbon dioxide on health in pathologies that arise through smoking and obesity and 3) the role of carbon dioxide as a chemoattractant implicated in insect-borne disease. The public will therefore benefit from the knowledge outputs in a highly topical area. We will engage with the public to enhance understanding of the science and issues surrounding carbon dioxide. This will be achieved through lectures at local schools, outreach activities and offering work experience placements for students from a local state-funded sixth form.