Interaction with phospholipids modulates alpha-synuclein nitration and lipid-protein adduct formation.
Intracellular aggregates of alpha-syn (alpha-synuclein) represent pathoanatomical hallmarks of neurodegenerative disorders (synucleinopathies). The molecular mechanisms underlying alpha-syn aggregation into filamentous inclusions may involve oxidation and nitration of the protein. Whereas the effects of oxidants and nitrating species on soluble alpha-syn have been studied in detail, the effect of these reactive species on alpha-syn associated with lipids is still unknown. In the present paper, we report that alpha-syn bound to small unilamellar liposomes composed of phosphatidylcholine/phosphatidic acid is resistant to oxidation and nitration when compared with soluble alpha-syn. Additionally, increasing concentrations of unsaturated fatty acids diminished the oxidation and nitration of alpha-syn upon exposure to fluxes of peroxynitrite (8-20 microM x min(-1)). To investigate the effect of oxidized lipids on alpha-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 alpha-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 alpha-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 alpha-syn, generating novel protein adducts that could serve as biomarkers for documenting oxidative processes in human as well as animal and cellular models of alpha-syn aggregation and pathology.
Trostchansky
Andrés
A
Center for Free Radical and Biomedical Research, Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avenida General Flores 2125, CP 11800, Montevideo, Uruguay.
Lind
Summer
S
Hodara
Roberto
R
Oe
Tomoyuki
T
Blair
Ian A
IA
Ischiropoulos
Harry
H
Rubbo
Homero
H
Souza
José M
JM
D000447:Aldehydes / Q000737:chemistry; D005786:Gene Expression Regulation; D006801:Humans; D015227:Lipid Peroxidation; D009566:Nitrates / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism*; D051844:alpha-Synuclein / Q000737:chemistry* / Q000378:metabolism*
D000447:Aldehydes; D009566:Nitrates; D010743:Phospholipids; D051844:alpha-Synuclein; D030421:Peroxynitrous Acid; C027576:4-hydroxy-2-nonenal