Effects of oxidative and nitrative challenges on α-synuclein fibrillogenesis involve distinct mechanisms of protein modifications

Abstract

Filamentous inclusions of α-synuclein protein are hallmarks of neurodegenerative diseases collectively known as synucleinopathies. Previous studies have shown that exposure to oxidative and nitrative species stabilizes α-synuclein filaments in vitro, and this stabilization may be due to dityrosine cross-linking. To test this hypothesis, we mutated tyrosine residues to phenylalanine and generated recombinant wild type and mutant α-synuclein proteins. α-Synuclein proteins lacking some or all tyrosine residues form fibrils to the same extent as the wild type protein. Tyrosine residues are not required for protein cross-linking or filament stabilization resulting from transition metal-mediated oxidation, because higher Mr SDS-resistant oligomers and filaments stable to chaotropic agents are detected using all Tyr → Phe α-synuclein mutants. By contrast, cross-linking resulting from exposure to nitrating agents required the presence of one or more tyrosine residues. Furthermore, tyrosine cross-linking is involved in filament stabilization, because nitrating agent-exposed assembled wild type, but not mutant α-synuclein lacking all tyrosine residues, was stable to chaotropic treatment. In addition, the formation of stable α-synuclein inclusions in intact cells after exposure to oxidizing and nitrating species requires tyrosine residues. These findings demonstrate that nitrative and/or oxidative stress results in distinct mechanisms of α-synuclein protein modifications that can influence the formation of stable α-synuclein fibrils.