Investigating Protein-Specific Functions of O-GlcNAc Using Induced Proximity Methods

Abstract

Proteins are molecular machines that control nearly all cellular processes and physiological functions in the body, and their activity is tightly regulated by post-translational modifications (PTMs). O-Linked β-N-acetylglucosamine (O-GlcNAc) is an essential and dynamic, nutrient-responsive monosaccharide PTM that decorates serine and threonine residues on nuclear and cytoplasmic proteins. The addition and removal of O-GlcNAc are mediated by a single pair of enzymes, the writer O-GlcNAc transferase (OGT) and the eraser O-GlcNAcase (OGA). Although O-GlcNAc is broadly involved in diverse cellular processes and disease states, many protein- and site-specific functions of O-GlcNAc remain poorly characterized. Induced proximity strategies that selectively install or remove O-GlcNAc from desired protein targets offer a powerful approach to accelerate functional annotation and uncover therapeutic opportunities for O-GlcNAc. Here, I present work describing the development and application of a generalizable induced-proximity method using a destabilized nanobody-OGT fusion that enables targeted protein O-GlcNAcylation and direct investigation of protein-specific O-GlcNAc functions, uncovering regulatory roles in transcriptional activation and immune signaling. In Chapter 1, I provide a comprehensive review of the O-GlcNAc field. This chapter highlights the discovery of O-GlcNAc, the foundational studies that established its biological significance, and summarizes the biochemical and cellular biology of the O-GlcNAc cycling enzymes, OGT and OGA, and current understanding of protein- and site-specific functions of O-GlcNAc across biological pathways implicated in various human diseases. Efforts to therapeutically modulate O-GlcNAc are also discussed, with particular emphasis on neurodegenerative disorders. Finally, emerging technologies and future opportunities for investigating O-GlcNAc biology and translating mechanistic insights into therapeutic strategies are outlined. Chapter 2 details my efforts to develop a next-generation induced-proximity approach using a destabilized nanobody-OGT fusion (dnGFP-OGT) to selectively install O-GlcNAc on target proteins. This chapter outlines the design principles, optimization, and validation of this method. The generalizability of this strategy is demonstrated across diverse classes of O-GlcNAc-modified proteins, revealing which substrates can be efficiently O-GlcNAcylated through induced proximity. This strategy facilitates precise, target-specific modulation of O-GlcNAc and establishes a platform for investigating the functional consequences of O-GlcNAc across multiple cellular contexts, as explored in subsequent chapters. In Chapter 3, I describe the application of dnGFP-OGT to directly investigate the effects of protein-specific O-GlcNAcylation on seven transcription factors. Coupling this approach with luciferase reporter systems enables quantitative assessment of changes in transcriptional activation as a direct effect of O-GlcNAcylation. Using this strategy, I uncovered a nutrient-sensing inhibitory role for O-GlcNAc on the AP-1 transcriptional complex. This chapter further discusses the mechanistic impact of O-GlcNAc on AP-1 proteins, c-Fos and c-Jun, through protein- and glycosite-level analyses and highlights downstream implications for immune signaling. In Chapter 4, I examine the roles of O-GlcNAc in cellular stress response, transcriptional signaling, and circadian rhythm. Using targeted protein O-GlcNAc approaches in combination with glycosite mapping, this chapter explores how O-GlcNAc modulates these complex biological processes at the protein- and site-levels. Collectively, these studies highlight the contextual complexity of O-GlcNAc regulation and provide a framework for future investigations into how O-GlcNAc regulates dynamic pathways in mammalian systems. In Chapter 5, I investigate the regulation of OGT protein stability and alternative splicing. OGA inhibition and nutrient supplementation were found to trigger proteasome-dependent degradation of OGT, and the potential contributions of previously implicated E3 ligases were assessed. I describe mechanistic studies using systematic OGT tetratricopeptide repeat (TPR) domain truncations to identify determinants of OGT alternative splicing, and quantitative proteomics experiments were performed to identify candidate splicing factors that may be O-GlcNAc-modified to signal OGT splicing. Together, these studies provide further insight into cellular mechanisms controlling OGT abundance and expression.