Mechanistic insights into alpha-synuclein neuronal toxicity: Misfolding, serine phoshorylation, and interactions with Rab GTPases.

Alpha-Synuclein(aS), a protein abundant in presynaptic neurons, has been found as the major component of Lewy bodies, the neuronal inclusion identified as the hallmark of Parkinson’s disease (PD). aS adopts intrinsically disordered conformation under physiological conditions and possesses a high propensity for assembling into oligomers and fibrils during the pathological process of PD. Although the pathology of PD is still elusive, several lines of evidence with respect to the neuronal toxicity, cellular physiology, and protein structure indicate that aS plays an important role in the development of PD and affects the normal cellular functions. In this dissertation we study the molecular mechanisms of serine phosphorylation, the interaction with Rab-GTPases and the aggregates structure of aS. Phosphorylation is a major posttranslational modification of aS. Approximately 90% of aS in LB is phosphoylated at Ser129, but the pathological role of phosphorylation has still not been determined. Ser87 is also an important serine phosphorylation site located at NAC region. To understand the mechanisms of phosphorylation, we investigate phosphorylation kinetics and the effects of phosphorylation on aS aggregation. Real-time NMR spectroscopy is employed to study the phosphorylation kinetics of the Ser87 and Ser129 of aS. With the aid of the residue-specific resolution provided by NMR spectroscopy we reveal that the three familial mutations A30P, E46K, and A53T do not change the phosphorylation kinetics of Ser87 but slightly modify the kinetics of Ser129. At the same time, under the conditions of our assay, we also identify that PLK2 and PLK3 are capable of phosphorylating aS at Ser129 with high selectivity and efficiency. Furthermore, we find that phosphorylation of Ser129 by PLK3 and the phosphorylation mimicking mutants (P128E/S129E and M127E/P128E/S129E) do not affect the aggregation propensity of aS. Rab GTPases are important for the regulation of vesicle trafficking in eukaryotic cells. Rab1, Rab3a, and Rab8a were suggested to protect against aS-induced cellular toxicity in different model systems. Focusing on molecular mechanisms involved in the interplay between Rab proteins and aS, we firstly identify that both the GDP- and GppNHp-bound Rab8a bind to the C-terminus of aS. However, aS has a higher affinity to Rab8a(GDP) than Rab8a(GppNHp). Secondly, the positively charged C-terminal peptide of Rab8a is found to reproduce the binding of full-length Rab8a to aS, suggesting that a charge complementation between the C-terminal of both proteins is important for the interaction. Furthermore, we have assigned Rab8a in the GDP-bound state and find that in addition to the C-terminus the Switch I region of Rab8a serves as a binding site for the aS C-terminus. Since the conformation of switch regions is highly dependent on nucleotide binding, this finding elucidates the binding preference for the GDP-bound state. Furthermore, Rab8a enhances the aggregation of aS in both oligomerization and fibril formation. Rab1b and Rab3a do not bind to aS but they can also significantly enhance the fibril formation of aS. Taken together, our results demonstrate a tight interplay between aS and Rab proteins at the molecular level. Furthermore, aS aggregates, namely oligomers and fibrils, are highly associated with PD pathology. We perform solution NMR based hydrogen/deuterium (H/D) exchange to detect the fibril core of E46K and A53T aS fibrils. These two mutations do not cause drastic changes to the fibril core structures in comparison to wt-aS and a conserved core region has been suggested for wt-aS and the three familial mutants. Furthermore, we optimize the H/D exchange method to detect the conformation of compounds-stabilized aS oligomers. Our results suggest that the compounds-stabilized oligomers do not contain strongly hydrogen-bonded regions.