Optimization of chemical reporters of O-GlcNAc for improved specificity and metabolic mapping

2014-09-12Post-translational modifications (PTMs) are ancillary decorations that are transferred onto fully-synthesized proteins. These modifications have been shown to significantly alter the fate and function of their substrates, and they include protein acetylation, phosphorylation, ubquitination, lipidation and glycosylation, among others. PTMs have been shown to be vital for development and are often misregulated in human disease. Throughout my graduate research, I have been particularly focused on the nutrient-senstitive, intracellular-glycosylation PTM N-acetylglucosamine (O-GlcNAc) and, to lesser extents, acetylation and mucin-type O-linked glycosylation. O-GlcNAc modification is a dynamic, signaling PTM that has been implicated in diabetes, neurodegeneration and cancer and is known to be upregulated in response to cell stress as a cytoprotective mechanism. In order to study O-GlcNAc’s role in disease progression, I have developed and optimized chemical reporters of O-GlcNAc that behave similarly to endogenous O-GlcNAc but are equipped with bioorthogonal functionality (azides or alkynes) that can be reacted with corresponding alkyne- or azide-containing tags through Cu(I)-catalyzed Azide-Alkyne Cycloadditon (CuAAC) to visualize the modification and/or identify protein substrates. Importantly, these new chemical reporters of O-GlcNAc are significantly more selective than what was previously available. I employed these probes to identify novel O-GlcNAc substrates as well as characterize the specific biochemical role of O-GlcNAcylation on a particular protein of interest, NEDD₄₋₁. I have also utilized this strategy in developing chemical reporters of protein acetylation and cell surface glycosylation. In addition, I have exploited chemical reporters in order to read-out on metabolic cross-talk between biosynthetic pathways as a way to further understand how cells amend their metabolic machinery in order to adapt to changes in nutrient availability and cellular demand