The identification of carbon dioxide-binding proteins

二氧化碳对生命至关重要。它在每个生命过程的开始时作为光合作用或化学合成的基本底物，在每个生命过程的结束时作为有氧呼吸和死后腐烂的产物。因此，这种气体调节细胞化学反应、运输、细胞环境的维持、行为和免疫力等多种过程也就不足为奇了。二氧化碳是一个具有重要战略意义的研究目标，与作物对环境变化、虫媒疾病和公共卫生的反应有关。尽管二氧化碳对生物学非常重要，但我们对二氧化碳与细胞的直接相互作用知之甚少。1928年，在血红蛋白上发现了最早的蛋白质翻译后修饰（PTM）。二氧化碳可以直接与选定的蛋白质基团联合收割机结合形成氨基甲酸酯。20世纪20 -80年代的有影响力的研究计划表明，氨基甲酸酯PTM调节血红蛋白中的氧结合并激活二氧化碳固定酶Rubisco。1983年，乔治洛里默（George Lorimer）提出氨基甲酸酯PTM作为调节生物对二氧化碳反应的机制。然而，氨基甲酸酯PTM在细胞外是不稳定的，并且其鉴定提出了显著的分析挑战。已经在蛋白质分子结构中鉴定了几种稳定的氨基甲酸酯，但在其广泛鉴定中的技术困难导致二氧化碳介导的氨甲酰化几乎被遗忘为PTM。例如，在61个已鉴定的PTM中，PTM的维基百科页面没有提到二氧化碳介导的氨甲酰化（不要与异氰酸介导的类似命名的修饰混淆）。我们证明了该工具可以将二氧化碳捕获在蛋白质上，并开发了一个工作流程来识别这些蛋白质。我们进一步研究了泛素作为一种二氧化碳结合蛋白。泛素存在于所有多细胞生物中，并与其他蛋白质结合以调节其活性和命运。二氧化碳调节炎症，我们提供的证据表明，这是由泛素氨基甲酸酯的形成解释。我们假设氨基甲酸酯PTM在细胞中广泛存在，并且氨甲酰化水平响应于二氧化碳的变化。我们假设氨基甲酸酯PTM介导了对二氧化碳的生物相关反应，我们将使用基于质谱的蛋白质组学来解决第一个假设。这些实验将展示细胞中二氧化碳结合蛋白的多样性，以及二氧化碳结合蛋白如何响应波动的气体而变化。这些实验将为二氧化碳调节的分子过程的多样性提供新的见解，并为在其他生物系统中识别此类过程提供工具。我们将结合生物化学，细胞和组织分析来解决第二个假设。具体来说，我们将证明泛素上的氨基甲酸酯PTM介导二氧化碳对炎症转录控制的影响。这些实验将提供新发现的氨基甲酸酯PTM具有生物相关性的原理证明。此外，这些实验将研究二氧化碳和泛素化之间令人惊讶的联系，泛素化是一种蛋白质修饰，其本身对蛋白质活性产生多种影响。由于所产生的工具和见解可以直接应用于各种生物学问题和系统，该提案将改变我们对二氧化碳直接分子反应的理解。

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.