Alpha Synuclein : An Intriguing Little Protein (Mini Review)

In this mini review we discuss Alpha Synuclein (αSyn), a small, yet curious 140 amino acid long protein that was originally named because of its localisation on synaptic vesicles from the electric lobe of the Pacific Electric Ray, Torpedo californica [1]. Shortly afterwards, αSyn was reported as the non-amyloid-β-component (NAC) of amyloid plaques of Alzheimer’s patients [2] and subsequently, missense mutations in αSyn’s SNCA (aka PARK1) gene have been linked to “synucleinopathy” diseases that include Parkinson’s disease, Dementia with Lewy bodies and Alzheimer’s disease (reviewed in [3]–[5]).

Indeed, Parkinson’s disease is the second most common neurodegenerative disorder that affects >10 million people worldwide, but unfortunately, to date no disease modifying therapies are available. The economic impact of Parkinson’s disease in just the USA alone is estimated to be around $52 billion which, coupled with the devastating effects of these diseases, means that αSyn is the subject of significant research efforts aiming to develop novel therapeutics, along with methods that will allow an early diagnosis of the conditions.

αSyn is predominantly expressed in the brain [6] and is found at high levels in the presynaptic boutons of axons [7]. It has also been detected in erythroid cells [8] and at low levels in other tissues but, in spite of intense study, the precise physiological roles and function of αSyn still remain unclear (see [4] for a review on the current status of what is known about the function of αSyn). One theory that is gaining support is that αSyn functions to promote membrane curvature, thereby contributing to synaptic trafficking and vesicle budding [9], [10].

Structurally, αSyn is an intriguing protein. Its N-terminal sequence is divided into seven highly conserved 11-mer repeats with a KTKGEV consensus sequence (residues 1–95), which, similar to apolipoproteins, form an amphipathic alpha-helix with 3 turns, and mediate association of αSyn with lipid membranes. This region also contains the NAC domain (residues 60–95), an area believed to be responsible for αSyn aggregation [2] and sensing of lipid properties [11]. Of particular note is that all identified mutations that are associated with synucleinopathies are located in this region: A30P, A30G, E46K, H50Q, G51D, A53E, A53V and A53T [12]–[20] six of which cluster within eight residues, suggesting that lipid binding or lack thereof may be linked to αSyn pathology. The C-terminus of αSyn (residues 96–140) is highly acidic and largely unstructured [21]–[23] and is the target of various post-translational modifications [24]. The C- terminus has been implicated in modulating αSyns interaction with proteins, metal ions, polycation and polyamine, regulating both its membrane binding and nuclear localisation and protecting αSyn from aggregation (see [3] and references therein). Acetylation of the N-terminal methionine has been shown to be important for oligomerisation of αSyn in diseased states [25].

In Parkinson’s disease, there is mounting evidence that tiny amounts of misfolded αSyn species can spread between cells (perhaps in a prion-like manner) and seed the aggregation of the normal, functional αSyn to form amyloidal plaques [27]. The fibrils of aggregated αSyn comprise a major part of the amyloid aggregates, present in so-called Lewy bodies. These, and the intermediate oligomeric aggregates present during the course of the aggregation process, are toxic to dopaminergic neurons and thus contribute to degeneration in Parkinson’s.

There are many NMR, X-Ray and Cryo-EM structures of αSyn monomer and fibrils in the PDB. (e.g. 1XQ8 [23], 2N0A [28] and 7C1D [29] . However, the picture emerging from these studies is complex. For example, the Cryo-EM studies have identified four distinct types of full length αSyn fibril to date, known as type 1a ‘rod’, type 1b ‘twister’, type 2a and type 2b polymorphs [30].

For the majority of these structural programmes the αSyn was expressed in and purified from E. coli with anion exchange chromatography often being included, due to the low 4.67 pI of αSyn. Stephens et al [31] provide an excellent review of the different methods that have been used to produce monomeric αSyn with comment on the impact of various steps (such as low pH) on its subsequent functionality.

Within the Protein Science and Structural Biology department at Sygnature Discovery we have been purifying various forms of αSyn and other fibril forming proteins for several years. The feedback that we have had is that the monomeric human wild type αSyn we make is of the highest quality with excellent batch to batch consistency and stability, that displays no self-seeding when used as a substrate within seed aggregation and RT-QuIC assays.

If this is of interest to your project please dont hesitate to get in touch with us to discuss your particular needs.

References

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