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Title: An investigation into the effect of N-terminal acetylation upon the human neuronal protein, alpha Synuclein, and influence of NatH on actin function in C. elegans
Author: Eastwood, Tara Ann
ISNI:       0000 0004 8499 7513
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2019
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N-terminal (Nt) acetylation is one of the most common post-translational modifications of proteins, which occurs on 70-90% of human proteins. The effect of Nt-acetylation of the Parkinson's disease associated protein, alpha Synuclein (aS), is poorly understood. Nt- acetylation of aS is generally believed to increase the helicity of the N-terminal 10-12 amino acids of the protein and to effect protein-protein interactions and membrane binding, though previous studies are contradictory. It is difficult to investigate the effect of aS Nt-acetylation in eukaryote cells, as the knockout of either of the genes (naa20 or naa25) which code for the two sub-units of N-terminal acetyltransferase B (NatB), which catalyses the Nt-acetylation of aS, also prevents the Nt-acetylation of many other proteins. In this investigation aS was produced within E. coli, with or without the co-expression of NatB, using a plasmid coding for both naa20 and naa25 from S. pombe, to enable direct comparison of the protein with and without Nt-acetylation, in vivo and in vitro. Differences were consistently seen between the two forms of aS, in protein-protein interactions and in membrane and lipid interactions. Nt- acetylation of aS resulted in a different oligomer conformation, which was more stable and liable to form amyloid in vivo, compared to oligomers produced when aS was not Nt-acetylated. E. coli expressing Nt-acetylated aS form internal structures with a tubular appearance, whereas cells expressing non Nt-acetylated aS, produced large extracellular vesicles. The toxic form of aS has not been identified and differences identified here may have relevance in the disease state. The formation of large aS-induced vesicles, which can be harvested from media, have potential for use in the enhancement of therapeutic protein production. The effect of knockout, of the recently characterised actin specific N-terminal acetyltransferase, NAA80, within C. elegans, was also investigated. No NAA80 knockout-specific phenotype could be identified.
Supervisor: Mulvihill, Daniel Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP506 Molecular biology