Objective: Our aim was to study α-syn Nt-acetylation and its impact on the disease-relevant properties of this protein in cell models to shed light on the significance of this modification in disease.

Background: α-synuclein (α-syn) has been widely studied due to its involvement in synucleinopathies, such as Parkinson’s disease (PD), where accumulation of fibril forms of the protein contributes to neuronal degeneration (1, 2). α-syn is N-terminally acetylated and is indicated it to be a suitable substrate for the N-terminal acetyltransferase B (NatB) (3, 4).The effect of Nt-acetylation on α-syn was overlooked for a long time, until in vitro experiments using purified protein suggested that it might play a crucial role in its secondary structure and folding properties, consequently affecting its function and toxicity (5-8).

Method: We here established and characterized an α-syn Nt-acetylation-deficient human cell CRISPR NatB knockout model, through a quantitative mass spectrometry approach. In these cells, we examined the effect of α-syn Nt-acetylation on several properties: protein stability and degradation by quantitative proteomics and western blot, membrane-binding by microscopy, and lastly we optimized an assay to use microscopy and flow cytometry to measure the aggregation propensity in cells by triggering aggregate formation with α-syn overexpression combined with uptake of preformed fibrils.

Results: α-syn protein levels were markedly reduced in the absence of NatB and could be partially restored by proteasomal inhibition. Quantitative N-terminomics revealed how absence of NatB does not completely abolish α-syn Nt-acetylation but rather reduces it. Nonetheless, imaging of KO cells revealed a lower occurrence of cells retaining membrane-bound species of α-syn. When triggering the aggregation of overexpressed α-syn, flow cytometry analysis of NatB KO cells displayed a higher percentage of cells containing intracellular aggregates.

Conclusion: This work presents a cell model with reduced α-syn Nt-acetylation and establishes the effect of this modification on several properties of the protein. Furthermore, our results show that α-syn Nt-acetylation may act as a protective modification hindering aggregation, thus arguing that studying this modification may have clinical and diagnostic value.

References: 1. Goedert M, Spillantini MG, Del Tredici K, Braak H. 100 years of Lewy pathology. Nature Reviews Neurology. 2013;9(1):13-24.
2. Villar-Piqué A, Lopes da Fonseca T, Outeiro TF. Structure, function and toxicity of alpha-synuclein: the Bermuda triangle in synucleinopathies. Journal of Neurochemistry. 2016;139(S1):240-55.
3. Aksnes H, Ree R, Arnesen T. Co-translational, Post-translational, and Non-catalytic Roles of N-Terminal Acetyltransferases. Molecular Cell. 2019;73(6):1097-114.
4. Deng S, Pan B, Gottlieb L, Petersson EJ, Marmorstein R. Molecular basis for N-terminal alpha-synuclein acetylation by human NatB. eLife. 2020;9:e57491.
5. Kang L, Moriarty GM, Woods LA, Ashcroft AE, Radford SE, Baum J. N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer. Protein Science. 2012;21(7):911-7.
6. Kang L, Janowska MK, Moriarty GM, Baum J. Mechanistic Insight into the Relationship between N-Terminal Acetylation of α-Synuclein and Fibril Formation Rates by NMR and Fluorescence. PLOS ONE. 2013;8(9):e75018.
7. Bu B, Tong X, Li D, Hu Y, He W, Zhao C, et al. N-Terminal Acetylation Preserves α-Synuclein from Oligomerization by Blocking Intermolecular Hydrogen Bonds. ACS Chemical Neuroscience. 2017;8(10):2145-51.
8. Watson MD, Lee JC. N-Terminal Acetylation Affects α-Synuclein Fibril Polymorphism. Biochemistry. 2019;58(35):3630-3.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/insight-into-%ce%b1-synuclein-pathogenicity-by-a-human-cell-model-with-suppressed-natb-mediated-n-terminal-acetylation/