Within higher eukaryotes, hundreds of nucleocytoplasmic proteins are modified by an N-acetylglucosamine (GlcNAc) residue. This O-GlcNAc modification is dynamic, a property imparted by the actions of two enzymes: O-GlcNAc transferase catalyzes the installation of GlcNAc onto specific serine and threonine residues of target proteins via a -glycosidic linkage, while O-GlcNAcase removes the modification. A leading hypothesis for the cellular role(s) of the O-GlcNAc modification is that an interplay between O-GlcNAc and phosphorylation exists to fine-tune cellular signaling pathways. In particular, elevated O-GlcNAc levels are thought to prevent phosphorylation of key signaling molecules in the insulin signaling cascade and cause insulin resistance. A key to many of these studies is the ability to modulate O-GlcNAc levels on proteins using two previously described inhibitors of O-GlcNAcase. These two inhibitors, however, also inhibit other targets and so it was unclear if results obtained using these compounds were an effect of modulating O-GlcNAc levels or, alternatively, an off-target effect of these inhibitors. With the goal of developing a selective O-GlcNAcase inhibitor, a series of biochemical studies provided evidence that O-GlcNAcase uses a catalytic mechanism involving substrate-assisted catalysis. Through these studies, a compound called NAG-thiazoline was found to be a potent inhibitor of O-GlcNAcase. By chemical modification of NAG-thiazoline, an inhibitor termed NButGT was generated that is selective for O-GlcNAcase over functionally-related enzymes yet retains high potency. NButGT functions within cells as well as in vivo within rodents to elevate O-GlcNAc levels. Surprisingly, neither NButGT nor another new inhibitor cause insulin resistance, strongly suggesting that off-target effects of previously used inhibitors are a concern. More surprising is that cultured cells and animals treated long-term with high doses of NButGT show no adverse effects. These results are challenging to reconcile with the leading hypothesis for the role of O-GlcNAc. Therefore, a new hypothesis was formed whereby O-GlcNAc may be acting as a protein chaperone. Preliminary experiments examining the biophysical properties imparted to a protein by O-GlcNAc support this new hypothesis. These new chemical tools and this newly formed hypothesis should enable exciting future research aimed at understanding the functional role of the O-GlcNAc modification.
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Thesis advisor: Vocadlo, David
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