Thousands of intracellular proteins are dynamically modified by monosaccharides of O-linked N- acetylglucosamine (O-GlcNAc). The cycling of O-GlcNAc is regulated by the concerted actions of enzymes encoded by just two genes: the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) that add and remove O-GlcNAc, respectively (1-4). Dysregulation of O-GlcNAc cycling, or levels of the nucleotide sugar used by OGT (UDP-GlcNAc), exacerbates the pathophysiology of a host of diseases including type II diabetes, cancer, neurodegeneration, heart failure, hypertension and aging (5-7). While technological and methodological innovations have improved our ability to detect, modulate, and site-map O-GlcNAc (8-13), many of these techniques have not been adopted by the broader scientific community thus inhibiting a mechanistic understanding of the roles that O-GlcNAc plays in potentiating disease. Often, the aforementioned approaches require specialized equipment and reagents, lack the specificity required to study a modification that is cycled by just two enzymes, or lead to significant off-target effects (10, 14-20). Thus, the goal of the studies proposed herein is to generate facile, inexpensive, chemical genetic tools that probe the roles of O-GlcNAc in vitro and in vivo. Specifically, we will address the following aims:
Specific Aim #1 : Enabling tunable, reversible chemical-genetic regulation of O-GlcNAcylation in an organelle-specific manner. These tools will combine destabilization domain (21-23) and nanotrap (24) technology to enable researchers to modulate the expression of OGT and OGA, as well as key enzymes within the hexosamine biosynthetic pathway, in a spatial, temporal, and dose-dependent manner. These tools will be applied to understanding the role of O-GlcNAc in prostate cancer.
Specific Aim 2 : Enabling tissue- and organelle-specific tagging of O-GlcNAc-modified proteins. This tool will harness the power of proximity Biotin ligation (25, 26) to enable tagging and enrichment of O- GlcNAcylated proteins in a cell-specific or organelle-specific manner. Together, the tools described in this proposal will overcome current experimental limitations associated with studying O-GlcNAc and facilitate studies focused on determining the role of O-GlcNAc at a mechanistic level in a broad range of models.
The sugar O-GlcNAc is essential for life, regulates numerous cellular processes, and in implicated in disease that include heart failture, the development of type II diabetes, neurodegenerative disease and cancer. The goal of this proposal is to devlop tools that will enable researchers to modulate the levels of O-GlcNAc in cell lines and enrich O-GlcNAc- modified proteins. Collectively, these tools should accelerate the rate at which the mechanisms by which O-GlcNAc pariticpates in the pathophysiological of disease.
|Zachara, Natasha E (2018) Critical observations that shaped our understanding of the function(s) of intracellular glycosylation (O-GlcNAc). FEBS Lett 592:3950-3975|