Implications of In Vitro Multi-Serine Phosphorylation of Alpha-Synuclein in Aggregation and Cytotoxicity

Post-translational modifications guide the functionaldiversityand identity of proteins. Phosphorylation is one such post-translationalmodification that has been reported in pathological proteins relatedto various neurodegenerative disorders such as & alpha;-synuclein (& alpha;-syn)phosphorylation in Parkinson's disease and other synucleinopathies.In & alpha;-syn, the phosphorylation has mostly been observed at S129;however, the occurrence of other serine modifications at S9, S42,and S87 is partially explored. In pathogenic conditions, where & alpha;-synis phosphorylated by complex kinase pathways, multi-site modificationsmay happen and alter the mechanism of & alpha;-syn aggregation. Here,using Polo-like kinase 2 and G-protein coupled receptor kinase 4,the in vitro phosphorylation of & alpha;-syn was performed, which revealedmulti-serine phosphorylation. Mass spectrometry with customized proteolyticdigestion showed prominent phosphorylation at S129 and modificationsat S87 and S42 with PLK2 and S87 with GRK4. The phosphorylation atthe identified serine residues was further validated with NMR andwestern blotting. Multi-serine phosphorylation aggravates the aggregationpotential of monomeric & alpha;-syn, seeding capacity, and cytotoxicityin the SH-SY5Y cell line. This study proposes evidence for in vitromulti-site phosphorylation and its significance in & alpha;-syn aggregation,toxicity, and related pathogenesis.