N-Homocysteinylation of lysine residues in α-Synuclein enhances aggregation propensity and cytotoxicity in SH-SY5Y cells.

In the current study, the effects of N-homocysteine thiolactone (HcyT) modification on expressed α-synuclein and the SH-SY5Y cell line were investigated. Various fluorometric, cell viability, and flow cytometry assays were employed to analyze the extent and impacts of modification under reducing/non-reducing conditions and different incubation durations. Our results confirmed that under non-reducing conditions, protofibrils exhibited a heightened propensity for aggregation with longer incubation periods. The increased tendency for aggregates following modification could be attributed to intermolecular forces, notably the establishment of π-stacking and hydrophobic interactions, consequent to the disruption of electrostatic charges due to lysine residues. Furthermore, our findings corroborated that N-homocysteinylation of α-synuclein by HcyT induces apoptosis in SH-SY5Y cells, suggesting that such a modification may indeed contribute to the onset and progression of Parkinson's disease (PD) in patients. Deciphering the underlying mechanisms using enhanced resolution techniques at the molecular level can pave the way to unraveling the pathogenesis associated with PD, as well as proposing effective strategies and countermeasures to mitigate the onset and progression of the disease. In light of the discoveries reported herein, N-homocysteinylation can be considered a risk factor and a potential biomarker in structural diseases, such as neurodegenerative disorders. Additionally, we propose that inhibiting N-homocysteinylation may impede protein aggregates and prevent the progression of neurodegeneration associated with these aggregates.