Chemical Tools for Studying O-GlcNAc Glycosylation at the Systems Level

Citation

Thompson, John Warren Lenzi (2020) Chemical Tools for Studying O-GlcNAc Glycosylation at the Systems Level. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/gx3z-k069. https://resolver.caltech.edu/CaltechTHESIS:06112020-001257003

Abstract

The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to intracellular serine and threonine residues is a ubiquitous post-translational modification (PTM) found in all higher eukaryotes. Like other PTMs, it is finely regulated in response to stimuli and dysregulated in multiple diseases. However, unlike other PTMs, methods to detect and profile the dynamics of O-GlcNAc glycosylation are still in their infancy. Herein, we discuss the background, development, and application of new chemical tools that have allowed for some of the first systems-level investigations of O-GlcNAcylation in different cells, organ systems, and disease states. We also significantly advance established techniques for the detection and monitoring of O-GlcNAc on proteins of interest. Using these new techniques, we first uncover a novel O-GlcNAcylation site on Cdk5 and show that this site can dynamically regulate Cdk5 activity in the context of neurodegenerative disease. Next, we apply novel chemical, mass spectrometric, and computational tools to, for the first time, uncover cellular networks engaged by O-GlcNAcylation in vivo. Finally, we undertake the systematic optimization of mass spectrometry based O-GlcNAcomics and use these new insights to significantly advance our understanding of O-GlcNAcylation dynamics in metabolic diseases of the liver. Overall, the techniques developed and data generated herein are closing the methodological and intellectual gaps between the study of O-GlcNAc glycosylation and that of other PTMs.

Thesis Files