Interaction with phospholipids modulates α-synuclein nitration and lipid–protein adduct formation

Intracellular aggregates of α-syn (α-synuclein) represent pathoanatomical hallmarks of neurodegenerative disorders (synucleinopathies). The molecular mechanisms underlying α-syn aggregation into filamentous inclusions may involve oxidation and nitration of the protein. Whereas the effects of oxidants and nitrating species on soluble α-syn have been studied in detail, the effect of these reactive species on α-syn associated with lipids is still unknown. In the present paper, we report that α-syn bound to small unilamellar liposomes composed of phosphatidylcholine/phosphatidic acid is resistant to oxidation and nitration when compared with soluble α-syn. Additionally, increasing concentrations of unsaturated fatty acids diminished the oxidation and nitration of α-syn upon exposure to fluxes of peroxynitrite (8–20 μM·min⁻¹). To investigate the effect of oxidized lipids on α-syn, the protein was incubated with the bifunctional electrophile 4-HNE [4-hydroxy-2(E)-nonenal]. MS analysis showed the formation of three major products corresponding to the native protein and α-syn plus one or two 4-HNE molecules. Trypsin digestion of the modified protein followed by peptide ‘finger-printing’ revealed that 4-HNE modified the peptide E⁴⁶GVVHGVATVAEK⁵⁸. Further analysis of the peptides with liquid chromatography–tandem MS identified the modified residue as His⁵⁰. The data indicate that the association of α-syn with biological membranes protects the protein from oxidation and nitration and thus diminishes the formation of protein molecules capable of forming aggregates. However, products of lipid peroxidation can also modify α-syn, generating novel protein adducts that could serve as biomarkers for documenting oxidative processes in human as well as animal and cellular models of α-syn aggregation and pathology.