| Post translational modifications (PTMs) are covalent additions appended to the side chains of amino acids that afford an extra level of biological and chemical complexity to the genome. These modifications can range from the addition of a single methyl group to the installation of a large oligosaccharide. Nevertheless, PTMs have been shown to have the ability to change the function, activity or localization of their target proteins. Furthermore, PTMs can be dynamically regulated and can be added enzymatically or chemically thus allowing them to act as signaling mechanisms or as metabolic sensors. In order to interrogate the consequences of PTMs, many methods have been developed to visualize and identify substrate proteins. One of these methods involves the treatment of live cells with a small molecule memetic of a metabolic precursor, containing an abiotic azide or alkyne, for its enzymatic incorporation onto target proteins and subsequent analysis using copper catalyzed azide alkyne cycloaddition (CuAAC), or click chemistry. These small molecules are termed metabolic chemical reporters (MCRs) and have been extensively utilized in the study of protein glycosylation. Described here is the development and optimization of the first MCRs to be specific for a single type of protein glycosylation called O-GlcNAc. O-GlcNAc modification is defined by the enzymatic addition of a single monosaccharide termed N-acetyl-glucosamine (GlcNAc) to serine and threonine residues of intracellular proteins. With this novel MCR in hand, the modification of the apoptotic regulator proteins called the caspases was revealed and was subsequently shown to affect the cleavage and activation of the protein during cell death. In addition, described here are three N-propargyloxycarbamate MCRs for the investigation of carbohydrate salvage pathways, an MCR for visualizing the metabolic cross-talk between carbohydrate metabolism and protein acetylation, and finally the small molecule 2-azido-2-deoxy-glucose, a reporter that revealed the unexpected promiscuity of O-GlcNAc transferase, the enzyme responsible for the addition of O-GlcNAc. |
