| Summary: | O-GlcNAcylation is an abundant post-translational modification found on serine and threonine hydroxyl groups of nucleocytoplasmic proteins. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are the sole enzymes responsible for addition and removal of all GlcNAc moieties, respectively. Although O-GlcNAcylation is involved in many diverse cellular processes and has been linked to many diseases, the molecular mechanisms by which it can modulate the activities of proteins remain largely unknown. As such, the primary goal of my thesis is to gain a better structural and functional understanding of the consequences of O-GlcNAcylation of tau, an intrinsically disordered microtubule-binding protein. Upon hyperphosphorylation, tau aggregates, forming neurofibrillary tangles that are a hallmark of Alzheimer’s disease. In contrast, O-GlcNAc has a reciprocal relationship with phosphorylation and reduces tau aggregation. To address my goals, I used NMR spectroscopy to probe local structural and dynamic effects of O-GlcNAc on a fragment of tau spanning residues 353 to 408 which encompasses a microtubule-binding repeat and Ser400, a key O-GlcNAc acceptor. Although chemical shift perturbations were observed near the site of O-GlcNAc-modification, based on main-chain chemical shifts, ³J-coupling and NOE interactions, there were no significant local structural changes compared to the wild-type tau polypeptide. However, there was a small decrease in the nsec-psec time scale mobility on the main-chain of tau around residue 400. In order to investigate the functional impact of O-GlcNAc on tau, I compared the heparin-induced aggregation of the wild-type tau peptide, the O-GlcNAc form, and three serine to aspartate “phosphomimic” mutants. Importantly, the O-GlcNAc modification significantly decreased the amount of aggregation, whereas two of the phosphomimic mutants increased aggregation relative to wild-type tau. I postulate that the O-GlcNAc modification increases the solubility of tau, thereby stabilizing the monomer in solution, and reducing the stability of aggregates.
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