Mechanisms of O-GlcNAc signalling . (360G-Wellcome-110061_Z_15_Z)

Glycosylation of serines/threonines with O-linked N-acetylglucosamine (O-GlcNAc, Fig. 1) on nucleocytoplasmic proteins in higher eukaryotes is an abundant and dynamic intracellular post-translational modification that is essential for metazoan life[1,2]. O-GlcNAcylation has been implicated in a wide range of cellular processes, including transcription, the cell cycle, signal transduction networks and protein folding, and shows interplay with regulatory protein phosphorylation[3] (Fig. 1). Dysreg ulation of protein O-GlcNAcylation has been linked to neurodegeneration[4], diabetes[5] and cancer[6]. Although O-GlcNAc has been found on >1000 intracellular proteins, it is not clear how only two single metazoan proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), together build this large, dynamic O-GlcNAc proteome (Fig. 1). Furthermore, it is unknown which O-GlcNAcylated proteins are linked to the severe developmental phenotypes of the ogt gene disruption mutants in mice[7,8], zebrafi sh[9], Xenopus[10] and Drosophila[11]. The central question of this proposal is: How does the OGT enzyme specifically recognise and O-GlcNAcylate proteins that are key for metazoan development. Using Drosophila as a tractable model system, and building on a body of preliminary data, I will use this Investigator Award to: 1. Develop new tools to study O-GlcNAcylation including synthesis of a potent, selective OGT inhibitor and a novel approach for the enrichment and detection of O-GlcNAc pr oteins. 2. Reveal the molecular basis of OGT substrate recognition by applying three parallel strategies to trap stable complexes of OGT with protein substrates and study these by X-ray crystallography and mutagenesis. 3. Genetically and chemically probe the role of O-GlcNAcylation in development by using the OGT inhibitor or carefully designed OGT hypomorphic CRISPR/Cas9 knock-in mutants to throttle OGT activity, and then study the developmental O-GlcNAc proteome to identify the persistin g O-GlcNAc proteins. The results will provide a step-change in our understanding of how OGT specifically recognises and O-GlcNAcylates key developmental proteins and contributes to the regulation of metazoan development.