Alpha synuclein is a 14 kDa protein that is abundant in the pre-synaptic terminals of neurons. It is best known for its association with Parkinson’s Disease and other synucleinopathies, where it aggregates to form inclusion structures called Lewy bodies – the classic pathological hallmark of these conditions.

Although alpha synuclein has long been regarded as an intrinsically disordered protein, it was found in 2009 to adopt an alpha-helical conformation upon binding to lipid membranes, indicating a possible physiological role for the alpha-helical state in synaptic vesicle cycling^1,2. In contrast, the beta-sheet-rich conformation has been closely linked to disease pathogenesis, demonstrating both neuronal toxicity and the ability to seed alpha synuclein aggregates in vivo³. This distinction between the two conformations is important, not least because enhanced alpha-helical folding can increase resistance to aggregation. Targeting the mechanisms that underlie alpha synuclein conformation promises to be an effective therapeutic strategy for a broad range of neurodegenerative disorders.

Alpha synuclein post-translational modifications

Various post-translational modifications (PTMs) have been proposed to modulate the function of alpha synuclein, with phosphorylation, truncation, and ubiquitination being among the most widely studied to date⁴. These PTMs exert their effects in very different ways.

For example, while mono- and di-ubiquitinated forms of alpha synuclein are found in Lewy bodies, polyubiquitinated alpha synuclein is instead targeted for proteasomal degradation; and while approximately 90% of the alpha synuclein in Lewy bodies is phosphorylated at Serine 129, only around 4% of total alpha synuclein in the normal brain features this modification ^5,6. More recently, N-terminal acetylation of alpha synuclein has been investigated for its therapeutic utility, largely due to the protective function this particular PTM performs.

TEM of N-terminal acetylated alpha synuclein pre-formed fibirls (PFFs) (SPR-332)

How does N-terminal acetylation affect alpha synuclein aggregation?

Although numerous reports have indicated that all alpha synuclein is post-translationally modified in vivo by the addition of an acetyl group at the N-terminus, many in vitro experiments have relied on recombinant forms of the protein lacking this modification. However, studies using N-acetylated alpha synuclein have yielded interesting findings.

Where small vesicles of negatively charged lipids were used as model membranes, it was found that N-terminal acetylation of alpha synuclein enhanced membrane-induced alpha-helical folding and resulted in complete resistance of the folded protein to fibrillar aggregation, a finding which was not replicated by the non-acetylated protein². Additionally, mutant forms of alpha synuclein have shown the N-terminus to determine its neuronal toxicity, most likely through its acetylation. While other studies have revealed N-terminally acetylated alpha synuclein to have significantly reduced sensitivity to thioflavin T, a reagent commonly used to seed aggregation from alpha synuclein monomers^7,8. In combination, these findings highlight the value of using N-acetylated alpha synuclein for research aimed at exploiting this post-translational modification for therapeutic purposes.

Supporting the study of N-terminal alpha synuclein acetylation

We offer a comprehensive portfolio of reagents for researchers studying Parkinson’s Disease and other synucleinopathies and have recently expanded our product offering with Active Human Recombinant N-Terminal Acetylated Alpha Synuclein Monomer Type 1 (SPR-331) and Active Human Recombinant N-Terminal Acetylated Alpha Synuclein Pre-Formed Fibrils (SPR-332). These demonstrate increased fluorescence, indicative of beta-sheet formation, when combined in a Thioflavin T seeding assay.

Thioflavin T assay for N-terminal acetylated alpha synuclein monomers (SPR-331) and pre-formed fibrils (SPR-332). Increased fluorescence, indicative of beta-sheet formation, was seen when 50 ug N-acetylated monomer and 5 ug N-acetylated fibril were combined, compared to monomer or fibril alone.

References

1. Alpha-synuclein binds large unilamellar vesicles as an extended helix, Trexler AJ and Rhoades E, Biochemistry. 2009 Mar 24;48(11):2304-6
2. N-alpha-acetylation of α-synuclein increases its helical folding propensity, GM1 binding specificity and resistance to aggregation, Bartels T et al, PLoS One 2014 Jul 30; 9(7)
3. Endogenous alpha-synuclein monomers, oligomers and resulting pathology: let’s talk about the lipids in the room, Killinger BA et al, NPJ Parkinsons Dis. 2019 Nov 12;5: 23
4. Role of post-translational modifications in modulating the structure, function and toxicity of alpha-synuclein: implications for Parkinson’s disease pathogenesis and therapies, Oueslati A et al, Prog Brain Res. 2010;183:115-45
5. Ubiquitination of α-synuclein by Siah-1 promotes α-synuclein aggregation and apoptotic cell death, Lee JT et al, Hum Mol Genet. 2008 Mar 15;17(6):906-17
6. The role of Ser129 phosphorylation of α-synuclein in neurodegeneration of Parkinson’s disease: a review of in vivo models, Sato H et al, Rev Neurosci. 2013;24(2):115-23
7. N-terminal acetylation mutants affect alpha-synuclein stability, protein levels and neuronal toxicity, Vinueza-Gavilanes R et al, Neurobiol Dis. 2020 Apr;137:104781
8. N-Terminal Acetylation Affects α-Synuclein Fibril Polymorphism, Watson MD and Lee JC, Biochemistry 2019, 58, 35, 3630–3633
9. The influence of N-terminal acetylation on micelle-induced conformational changes and aggregation of α-Synuclein, Ruzafa D et al, PLoS ONE 2017; 12(5)