Carbamylation Promotes Sequence-Specific Amyloidogenesis in the α-Synuclein KTKEGV Repeat Motifs

KTKEGV repeat motifs play a crucial role in regulating the folding of α-synuclein structure and function, including tetramer formation and interaction with membranes. In α-syn, nine KTKEGV imperfect repeats have been reported. Post-translational modifications (PTMs) that could neutralize the positive charge on lysine may disturb the electrostatic interaction of α-syn with the cellular membrane, thereby affecting physiological function. Carbamylation is one such nonenzymatic age-dependent charge-neutralizing PTM which is reported to be elevated in aged people. While some reports have suggested that carbamylation is a proaggregation factor, others have assigned it a protective role. In the present study, we explored the aggregation propensities of KTKEGV repeat motifs and full-length α-syn protein upon carbamylation. We observed distinct aggregation kinetics among various KTKEGV motifs of α-syn, including disease-specific mutated versions, which was confirmed by multiple biophysical techniques such as ThT assay, turbidity measurement, Congo red staining, AFM, and SEM. Notably, the repeat motifs 3 (³²KTKEGVLYV⁴⁰), 5 (⁵⁸KTKEQVTNV⁶⁶), and the core motif (⁷⁷VAQKTV⁸²) exhibited robust fibrillar amyloid formation when carbamylated. Also, repeat motif 4 (⁴³KTKEGVVH⁵⁰), when mutated (E46K, H50Q, and both), becomes an aggregation hotspot, even though the native sequence does not aggregate upon carbamylation. Carbamylation of full-length α-syn protein appears to lead to aggregation with higher fibrillar amyloid content as indicated by a several-fold enhancement of ThT plateau fluorescence. Such carbamylated α-syn aggregates could recruit unmodified α-syn and again led to the formation of well-organized amyloid fibrils. These findings highlight the site-specific role of charge-neutralizing PTMs such as carbamylation α-syn aggregation and provide novel insights into the molecular mechanisms related to synucleinopathies and dementia with Lewy bodies.