Tau protein is associated with many neurodegenerative disorders known as tauopathies, such as Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and Picks’ disease (PiD). Depending on the alternative splicing of the microtubule-associated protein tau (MAPT) gene in the central nervous system, tau protein may carry either three or four microtubule-binding repeats (3R vs 4R) along with either two, one, or no N-terminal acidic repeats (2N, 1N, 0N) giving rise to 6 primary tau isoforms. Under disease conditions, tau protein aggregates into intracellular inclusions that feature distinct profiles of tau isoforms (e.g. 4R/3R in AD, 4R in PSP, and 3R in PiD). Aggregates of tau were traditionally thought of as the main driver of neurodegeneration in tauopathies. Increasingly, evidence points to earlier, soluble conformations of abnormally modified monomers and multimeric tau as toxic forms of tau. The biological processes driving tau from physiological species to pathological conformations remain incompletely understood; however, the functional consequences of tau mutations, protein-protein interactions, and post-translational modifications (PTMs) are likely contributors. Phosphorylation and acetylation have been the most commonly and extensively studied PTMs of tau. Nonetheless, tau is subject to other PTMs that have not gained as much attention, such as carbamylation, prolyl-isomerization, polyamination, O-linked-N-acetyl β-d-N-glucosaminylation (O-GlcNAcylation), and SUMOylation among others. Moreover, our understanding of the role played by PTMs in regulating the adoption of soluble toxic conformations by tau is in its early days. Therefore, it is of paramount importance to fill those gaps by shedding more light on the understudied PTMs of tau and investigate their role in regulating the formation of pathological tau conformations. In this dissertation, I set out to investigate three of the understudied PTMs of tau: polyamination, O-GlcNAcylation, and SUMOylation. First, I produced a set of recombinant tau proteins individually modified with the three PTMs using different purification approaches. The modified sites on recombinant tau isoforms were identified using mass spectrometry. Then, I assessed the impact of the three PTMs on the interactions of tau with microtubules (MTs) in terms of MT binding and tubulin polymerization. In addition, the unmodified and modified tau proteins were subjected to a set of in vitro biophysical and biochemical assays to determine the contribution of the three PTMs to tau’s transition into the known pathological conformations, including formation of filamentous aggregates, oligomerization, exposure of the phosphatase-activating domain, misfolding, and in-cell seeding capability. Several effects of polyamination, O-GlcNAcylation, and SUMOylation on the interaction of tau with microtubules and the adoption of pathological conformations by tau were identified. Integrating the findings from this work with the current literature can provide insights on the potential role played by PTMs in disease. I show that this framework to study PTMs using recombinant proteins is useful in investigating the regulation of tau pathobiology even by understudied PTMs. Furthermore, integrating the findings from this work with the current literature can provide insights on the potential role played by PTMs in disease. Taken together, this approach facilitates the advancement of our understanding of the relationships between PTMs and tau conformations in health and disease.
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