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Bicarbonate enhances alpha-synuclein oligomerization and nitration: intermediacy of carbonate radical anion and nitrogen dioxide radical.

ABSTRACT: alpha-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 alpha-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 alpha-synuclein and HCO3- markedly enhanced both nitration and aggregation of alpha-synuclein through dityrosine formation. Bicarbonate-dependent peroxidase activity of Cu,Zn-superoxide dismutase (SOD1) also induced covalent aggregation of alpha-synuclein via a CO3*--dependent mechanism. Nitrone spin traps completely inhibited CO3*--mediated oxidation/nitration and aggregation of alpha-synuclein. Conversely, alpha-synuclein inhibited CO3*--induced spin adduct formation. Independent evidence for CO3*--mediated oxidation and dimerization of alpha-synuclein was obtained from UV photolysis of [(NH3)5CoCO3]+, which generates authentic CO3*-. Irradiation of [(NH3)5CoCO3]+ and NO2- in the presence of alpha-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 alpha-synuclein aggregation and nitration in these systems. We conclude that both CO3*- and NO2* could play a major role in the nitration/aggregation of alpha-synuclein.

SUBMITTER: Andrekopoulos C

PROVIDER: S-EPMC1223984 | biostudies-other | 2004 Mar

REPOSITORIES: biostudies-other

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Project description:Protein tyrosine nitration has been observed in a variety of human diseases associated with oxidative stress, such as inflammatory, neurodegenerative, and cardiovascular conditions. However, the pathways leading to nitration of tyrosine residues are still unclear. Recent studies have shown that peroxynitrite (PN), produced by the reaction of superoxide and nitric oxide, can lead to protein nitration and inactivation. Tyrosine nitration may also be mediated by nitrogen dioxide produced by the oxidation of nitrite by peroxidases. Manganese superoxide dismutase (MnSOD), which plays a critical role in cellular defense against oxidative stress by decomposing superoxide within mitochondria, is nitrated and inactivated under pathological conditions. In this study, MnSOD is shown to catalyze PN-mediated self-nitration. Direct, spectroscopic observation of the kinetics of PN decay and nitrotyrosine formation (k(cat) = 9.3 × 10(2) M(-1) s(-1)) indicates that the mechanism involves redox cycling between Mn(2+) and Mn(3+), similar to that observed with superoxide. Distinctive patterns of tyrosine nitration within MnSOD by various reagents were revealed and quantified by MS/MS analysis of MnSOD trypsin digest peptides. These analyses showed that three of the seven tyrosine residues of MnSOD (Tyr34, Tyr9, and Tyr11) were the most susceptible to nitration and that the relative amounts of nitration of these residues varied widely depending upon the nature of the nitrating agent. Notably, nitration mediated by PN, in both the presence and absence of CO2, resulted in nitration of the active site tyrosine, Tyr34, while nitration by freely diffusing nitrogen dioxide led to surface nitration at Tyr9 and Tyr11. Flux analysis of the nitration of Tyr34 by PN-CO2 showed that the nitration rate coincided with the kinetics of the reaction of PN with CO2. These kinetics and the 20-fold increase in the efficiency of tyrosine nitration in the presence of CO2 suggest a specific role for the carbonate radical anion (•CO3(-)) in MnSOD nitration by PN. We also observed that the nitration of Tyr34 caused inactivation of the enzyme, while nitration of Tyr9 and Tyr11 did not interfere with the superoxide dismutase activity. The loss of MnSOD activity upon Tyr34 nitration implies that the responsible reagent in vivo is peroxynitrite, acting either directly or through the action of •CO3(-).

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Bicarbonate enhances alpha-synuclein oligomerization and nitration: intermediacy of carbonate radical anion and nitrogen dioxide radical.

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