Chemical Approaches to Understand O-GlcNAc Glycosylation and Its Roles in Neurodegeneration - Project Summary/Abstract Carbohydrates (also known as glycans) comprise one of the largest, most diverse collections of biologically active molecules. However, relative to other biomolecules such as nucleic acids and proteins, carbohydrates remain poorly understood due to challenges in their detection, synthesis, and analysis. The broad objective of this program is to develop chemical approaches to advance a fundamental understanding of the roles of carbohydrates in biology and disease. In the last granting period, we developed a novel Networking of Interactors and SubstratEs (NISE) method to study the biological functions of O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation. O-GlcNAc is an abundant, essential post-translational modification that is emerging as a key regulator of many physiological functions, ranging from epigenetic and transcriptional gene regulation to insulin signaling, cancer cell metabolism, and neurodegeneration. Our NISE approach combines new chemoproteomic tools, genetic engineering, mass spectrometry analysis, and bioinformatics methods to quantitatively profile O- GlcNAc sites and O-GlcNAc transferase (OGT) interactors across the proteome and to determine key interconnections between the interactors and substrates. The resulting networks have revealed novel, unexpected functions for O-GlcNAc and highlighted potential mechanisms to explain the unique specificity of OGT. In the coming granting period, we will expand on this approach and investigate the roles of O- GlcNAcylation in neurons and in the context of neurodegenerative diseases as we continue to tackle the next critical barriers in the field. In Aim 1, we will focus on understanding how O-GlcNAcylation within intrinsically disordered, low-complexity domains of proteins affects their functions and alters biomolecular condensate (BMC) formation, composition, and dynamics. These studies should provide new paradigms and methods for understanding the fundamental mechanisms by which O-GlcNAc regulates proteins and its role in aberrant BMC activity linked to Alzheimer’s disease and related dementias (AD/ADRDs). In Aim 2, we will test specific hypotheses revealed by our NISE networks regarding the regulation of OGT activity at neuronal synapses and specifically toward proteins implicated in AD/ADRDs. In Aim 3, we will apply our NISE method to examine directly how O-GlcNAcylation of specific proteins and pathways becomes dysregulated during AD pathogenesis and with disease-specific mutations by using patient-derived induced pluripotent stem cells (iPSCs) and human AD brain samples. Together, the proposed studies will provide a powerful approach to identify and understand physiologically important and/or disease-causing O-GlcNAcylation events. In turn, this information is expected to provide new potential therapeutic targets and/or strategies to combat progressive neurodegeneration and AD/ADRDs.
