BIOCHEMICAL AND BIOPHYSICAL TECHNIQUES TO INTERROGATE TAU AND ALPHA-SYNUCLEIN INTERACTIONS

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Degree type
Doctor of Philosophy (PhD)
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Biochemistry and Molecular Biophysics
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Biochemistry, Biophysics, and Structural Biology
Biochemistry, Biophysics, and Structural Biology
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2024
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Ramirez, Jennifer
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Abstract

Age-related neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by amyloid deposits in the brain, with tau aggregates linked to AD and α-synuclein (αS) aggregates to PD. Emerging evidence suggests these pathologies overlap, with tau and αS potentially influencing each other’s aggregation. This study employs biochemical and biophysical techniques to investigate tau’s interactions with soluble and fibrillar αS. Fluorescence correlation spectroscopy (FCS) revealed that tau domains interact weakly with monomeric αS but show significant interactions with αS aggregates, which extend tau aggregation slightly without altering its final extent. We observed that specific tau regions have different binding affinities to αS aggregates, and mutations may affect these interactions. In addition, both tau and αS are subject to a myriad of post-translational modifications (PTMs) such as phosphorylation and acetylation, yet our ability to understand how PTMs regulate tau functions in health and disease has been hindered by the absence of tools for generating site specific PTMs on tau. Genetic code expansion (GCE) stands at the forefront of protein PTM research, enabling precise and customizable integration of modified amino acids into proteins expressed through recombinant methods. We synthesized full-length αS with glycosylation or phosphorylation at a key position, achieving nearly complete conversion but with low final yields due to purification losses. Modified αS proteins exhibited weaker binding to vesicles compared to wild-type αS, with similar effects observed for phosphorylation and glycosylation. Additionally, we explored lysine acetylation sites on αS using non-canonical amino acid (ncAA) mutagenesis, providing insights into the role of acetylation in αS aggregation and highlighting key sites for further research. These findings enhance our understanding of tau and αS interactions and the impact of PTMs, suggesting directions for future research into their roles in neurodegenerative diseases.

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Rhoades, Elizabeth
Petersson, E. James
Date of degree
2024
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