Cell signaling through O-linked glycosylation - Project Summary/Abstract Glycosylation – the enzymatic attachment of carbohydrates onto biomolecules – is the most abundant post-translational modification (PTM) of proteins in nature. In mammals, glycosylation influences all aspects of cell biology, including protein quality control and secretion, intracellular signaling, membrane composition and fluidity, cell adhesion and migration, cell-cell communication, and organogenesis. Aberrant glycosylation also underlies a wide range of human diseases, such as developmental defects, obesity and metabolic syndrome, cancer, neurodegeneration, and atherosclerosis. Despite its significance, protein glycosylation remains an under-studied aspect of cell biology, due partly to the unique challenges in characterizing it. Indeed, protein glycosylation is heterogeneous, chemically complex, and created ab initio, without a template molecule, unlike DNA, RNA, or protein biosynthesis. New, interdisciplinary approaches are needed to understand protein glycosylation in physiology and disease. My research program focuses on protein O-glycosylation (i.e., glycans modifying serine and threonine side-chains). In particular, we have a longstanding interest in O-linked β-N-acetylglucosamine (O-GlcNAc), a ubiquitous intracellular PTM in mammals, which decorates thousands of nuclear and cytoplasmic substrates. O- GlcNAc is an essential regulator of myriad aspects of cell physiology and is dysregulated in numerous human diseases, such as cancer, X-linked intellectual disability, and neurodegeneration. However, major aspects of O- GlcNAc signaling are incompletely understood, including the biochemical mechanisms through which O-GlcNAc transduces information. Our work has addressed this challenge by studying O-GlcNAc-mediated protein-protein interactions (PPIs) in fundamental cell biological processes, including intermediate filament structure and dynamics, vesicle trafficking in the early secretory pathway, and regulation of proteostasis through ubiquitin E3 ligase adaptor proteins. We have also systematically identified candidate human O-GlcNAc “reader” proteins that bind this glycan directly and reported the first structures of potential readers with model glycopeptides, illuminating the biophysical basis of these PPIs. Our work is highly interdisciplinary, combining cell biology with structural biology, glycoproteomics, protein biochemistry, and embryonic development. Moreover, as an important complement to conventional techniques, we frequently develop and deploy novel chemical biology approaches to understand O-glycosylation. Building on this foundation, here we propose to study the biochemical mechanisms and downstream functions of O-glycosylation, with a specific focus on O-GlcNAc-mediated PPIs in key cell biological processes. Our work will provide new insight into several pathways dysregulated in human disease and advance our long-term goal of understanding the mechanisms and functions of mammalian O-glycosylation.
