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Site-Specific Incorporation of Post-Translational Modifications in Alpha-Synuclein: Insights into Aggregation, Structure, and Function Marie Shimogawa
- Format:
- Book
- Thesis/Dissertation
- Author/Creator:
- Shimogawa, Marie, author.
- Language:
- English
- Subjects (All):
- Biochemistry.
- Biophysics.
- Analytical chemistry.
- Molecular biology.
- 0487.
- 0786.
- 0486.
- 0307.
- Local Subjects:
- Biochemistry.
- Biophysics.
- Analytical chemistry.
- Molecular biology.
- 0487.
- 0786.
- 0486.
- 0307.
- Physical Description:
- 1 electronic resource (154 pages)
- Contained In:
- Dissertations Abstracts International 86-12B
- Place of Publication:
- Ann Arbor : ProQuest Dissertations and Theses, 2025
- Language Note:
- English
- Summary:
- The misfolding and aggregation of alpha-synuclein (αS) are central to synucleinopathies, including Parkinson's disease. Post-translational modifications (PTMs) regulate αS aggregation and interactions, yet their site-specific effects remain unclear. This dissertation employs genetic code expansion (GCE), enzymatic modification, and protein semi-synthesis to incorporate PTMs and investigate their influence on αS function and pathology. To study lysine acetylation, GCE was used to incorporate acetyl lysine (AcK) at twelve sites, revealing that AcK12, AcK43, and AcK80 slow aggregation in vitro and in neurons, with AcK80 emerging as a promising therapeutic target due to its minimal impact on membrane interactions. While HDAC8 exhibited high deacetylation activity toward AcK80, its lack of specificity over AcK43 suggests that better Lys acetylltransferases or deacetylase targets must be identified. Structural analysis suggests AcK80-mediated aggregation suppression extends beyond simple monomer conformational changes or fibril morphology alterations. Given the frequent use of glutamine as an AcK mimic, its validity was tested, revealing that Gln43 and Gln80 mutants replicated AcK's effects on aggregation, while Gln12 did not, emphasizing the need to validate mimics on a site-by-site basis. Alternative chemical strategies, including thioether acetylation mimic and thioamide acetyl lysine analog, were explored to enhance PTM stability and analytical utility. The effects of phosphorylation were examined using GCE (phosphorylation at Ser87, pS87) and enzymatic modification (phosphorylation at Tyr39, pY39) - pS87 had minimal impact on vesicle binding and pY39 does not dramatically influence fibril structure at physiological stoichiometry (10-25%), unlike when it is present at 100%, as detected by changes in binding of site-specific radioligands. Preliminary work suggests a combinatorial effect between pY39 and AcK43 in aggregation, though future synthetic efforts are needed to efficiently generate doubly modified αS and elucidate this effect. These findings establish methodologies for precise PTM incorporation and highlight site-specific PTM effects on αS aggregation, fibril structure, and function, providing a foundation for future studies on PTM crosstalk and therapeutic targeting
- Notes:
- Source: Dissertations Abstracts International, Volume: 86-12, Section: B.
- Advisors: Petersson, E. James Committee members: Chenoweth, David M.; Marmorstein, Ronen; Burslem, George
- Ph.D. University of Pennsylvania 2025
- Local Notes:
- School code: 0175
- ISBN:
- 9798280759145
- Access Restriction:
- Restricted for use by site license
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