Cellular metabolite sensing

Overview: Cells have an incredible ability to adapt to both internal and external changes, which is essential for life. One way they do this is by sensing changing metabolite levels through metabolite-responsive post-translational modification (PTM) of proteins. These PTMs allow cells to detect and respond to metabolic changes across all domains of life.

At the King lab, we study two key areas of cellular metabolite sensing: the modification of proteins by CO₂ and the reversible addition of a sugar molecule called O-GlcNAc to proteins. Our aim is to develop sustainable biotechnologies to combat climate change and to provide new insights that could lead to important advances in human therapeutics.

Area 1: Protein carboxylation as a CO₂ sensing mechanism

bacteria carbon dioxide

CO₂ is a vital biological gas that triggers adaptive responses in organisms across all domains of life. For instance, in humans, CO₂ regulates breathing rate and depth, and in certain pathogens, CO₂ exposure prompts the expression of virulence factors within the human host. Additionally, some photosynthetic organisms use CO₂ as their sole carbon source. Understanding how organisms sense CO₂ levels and adjust their metabolism could lead to significant advancements in human therapeutics and biological carbon capture technologies.

Despite its importance, the fundamental biochemical mechanisms through which organisms sense CO₂ remain largely unknown. One direct way CO₂ can impact proteins is by modifying the primary amine of lysine residues to form a carbamate (Lys-CO₂). This modification drastically alters the structure and charge state of these sites in a reversible manner.

At the King lab, we are pioneering chemoproteomic methods to identify Lys-CO₂ sites across the proteome and investigating its potential role as a biochemical regulatory mechanism for CO₂ sensing. Our research aims to uncover the fundamental processes of CO₂ sensing and their applications in science and medicine.

Proteome-wide identification of Lys-CO₂

protein binding CO2

King D.T., Zhu S., Hardie D.B., Serrano-Negrón J.E., Madden Z., Kolappan S., Vocadlo D.J. Nature Chemical Biology. 18(7): 782-791(2022)

We describe the first quantitative proteomic method to identify Lys-CO₂ sites in proteins. Using this method, we identify 20 high-confidence Lys-CO₂ sites. From among our panel of modified proteins, we characterize a novel Lys-CO₂ site within the effector pocket of metabolic sensor PII from cyanobacteria. We uncover a unique negative biochemical control mechanism whereby Lys-CO₂ formation antagonizes binding of PII to its regulatory effector ligand ATP over a physiological range of CO₂. To our knowledge, this represents the first example of CO₂ acting as a negative biochemical regulator of proteins.

 

Area 2: Exploring the fundamental biochemical roles of O-GlcNAc in human health and disease

Mammalian cells exist in a complex soup of metabolites that reflect their internal state and local surroundings, serving as crucial indicators of cellular and organismal health. These metabolites can activate biochemical pathways that help maintain cellular balance. One important way metabolites influence these pathways is through post-translational modifications (PTMs).

A key metabolite-sensitive PTM involves the addition of a single O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues on proteins. Dysregulation of O-GlcNAc is associated with chronic human diseases such as neurodegeneration and cancer. However, the biochemical mechanisms by which O-GlcNAc affects protein function remain largely unknown.

At the King lab, we investigate the fundamental biochemical roles of O-GlcNAc using proteome-wide methods. We then delve into the detailed molecular mechanisms using targeted in-vitro biochemical and cellular experiments. Our research aims to uncover how O-GlcNAc impacts health and disease, potentially leading to new therapeutic strategies.

Uncovering the O-GlcNAc dependent protein meltome

King D.T., Serrano-Negrón J.E., Zhu Y., Moore C.L., Shoulders M.D., Foster L.J., Vocadlo D.J. J. Am. Chem. Soc. 144(9): 3833-3842 (2022)

We developed a proteome-wide approach to monitor the thermostability effect of PTMs and used it to uncover the O-GlcNAc dependent meltome. Surprisingly, O-GlcNAc was predominantly destabilizing, a finding that challenged the predominant view of O-GlcNAc as being stabilizing. This method serves as a blueprint to study the thermostability effect of, in principle, any protein modification.