α-Synuclein, a neuronal presynaptic protein, has been reported to undergo oligomerization to form toxic Lewy bodies in neurodegenerative disorders. One of the proposed mechanisms for aggregation of α-synuclein involves oxidative and nitrative modifications. In the present study, we show that addition of 3-morpholino-sydnonimine chloride (SIN-1) or slow infusion of pre-formed peroxynitrite (ONOO) to mixtures containing α-synuclein and HCO3 markedly enhanced both nitration and aggregation of α-synuclein through dityrosine formation. Bicarbonate-dependent peroxidase activity of Cu,Zn-superoxide dismutase (SOD1) also induced covalent aggregation of α-synuclein via a CO3•−-dependent mechanism. Nitrone spin traps completely inhibited CO3•−-mediated oxidation/nitration and aggregation of α-synuclein. Conversely, α-synuclein inhibited CO3•−-induced spin adduct formation. Independent evidence for CO3•−-mediated oxidation and dimerization of α-synuclein was obtained from UV photolysis of [(NH3)5CoCO3]+, which generates authentic CO3•−. Irradiation of [(NH3)5CoCO3]+ and NO2 in the presence of α-synuclein yielded nitration and aggregation products that were similar to those obtained from a SIN-1 (or slowly infused ONOO) and HCO3 or a myeloperoxidase/H2O2/NO2 system. Hydrophobic membranes greatly influenced α-synuclein aggregation and nitration in these systems. We conclude that both CO3•− and NO2 could play a major role in the nitration/aggregation of α-synuclein.

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