Modified Project Summary/Abstract (The abstract contained no specific references to the in vivo mouse experiments and therefore is unchanged): O-GlcNAc modification is a dynamic protein-modification that is absolutely required for embryonic development in mammals, and is misregulated in diseases, including diabetes, neurodegeneration and cancer. Although approximately 1,000 proteins are modified by O-GlcNAc, the effects of the vast majority of these modifications on protein function are completely unknown. The long-term goal of our research program is to fill in these missing gaps by determining the biochemical consequences of O-GlcNAc on proteins that are key to human disease. To accomplish this goal, we use a combination of carbohydrate and synthetic protein chemistries to build O-GlcNAc modified proteins for subsequent biological experiments. This chemical approach is uniquely enabling, as it is currently the only way to generate homogeneous and site-specifically O-GlcNAc modified proteins. We have been very successful and have used synthetic proteins to determine that O-GlcNAc has a multifaceted role in preventing the amyloid aggregation of proteins in neurodegenerative diseases. Specifically, we have found that O-GlcNAc both directly inhibits the aggregation of amyloid forming proteins and activates the activity of certain small chaperones. In this proposal we will continue to build on these discoveries. In Aim 1, we will determine how O-GlcNAc inhibits the early stages of α-synuclein amyloid formation. In Aim 2, we will test whether O-GlcNAc alters the structure/toxicity relationships of α-synuclein amyloids. In Aim 3, we examine how O-GlcNAc alters the small heat shock protein interactome. Finally, in Aim 4, we will determine if O-GlcNAc can rescue the activity of mutant chaperones that cause Charcot-Marie-Tooth disease. At the conclusion of these independent aims, we will have further unravelled the mechanisms by which O-GlcNAc inhibits protein aggregation and provided critical data to support the ongoing efforts to target O-GlcNAc therapeutically.