Trostchansky Andrés, Lind Summer, Hodara Roberto, Oe Tomoyuki, Blair Ian A, Ischiropoulos Harry, Rubbo Homero, Souza José M
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.
Biochem J. 2006 Jan 1;393(Pt 1):343-9. doi: 10.1042/BJ20051277.
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.
α-突触核蛋白（α-synuclein）的细胞内聚集体是神经退行性疾病（突触核蛋白病）的病理解剖学标志。α-突触核蛋白聚集成丝状内含物的分子机制可能涉及该蛋白的氧化和硝化。虽然已经详细研究了氧化剂和硝化剂对可溶性α-突触核蛋白的影响，但这些活性物质对与脂质相关的α-突触核蛋白的影响仍然未知。在本文中，我们报道与可溶性α-突触核蛋白相比，结合到由磷脂酰胆碱/磷脂酸组成的小单层脂质体上的α-突触核蛋白对氧化和硝化具有抗性。此外，不饱和脂肪酸浓度的增加减少了暴露于过氧亚硝酸盐通量（8 - 20 microM×min(-1)）时α-突触核蛋白的氧化和硝化。为了研究氧化脂质对α-突触核蛋白的影响，将该蛋白与双功能亲电试剂4-羟基壬烯醛（4-HNE）一起孵育。质谱分析显示形成了三种主要产物，分别对应天然蛋白以及与一个或两个4-HNE分子结合的α-突触核蛋白。对修饰后的蛋白进行胰蛋白酶消化，随后进行肽“指纹图谱”分析，结果表明4-HNE修饰了肽段E46GVVHGVATVAEK58。用液相色谱 - 串联质谱对肽段进行进一步分析，确定修饰的残基为His50。数据表明，α-突触核蛋白与生物膜的结合保护该蛋白免受氧化和硝化，从而减少了能够形成聚集体的蛋白质分子的形成。然而，脂质过氧化产物也可以修饰α-突触核蛋白，产生新的蛋白质加合物，这些加合物可作为生物标志物，用于记录人类以及α-突触核蛋白聚集和病理学的动物及细胞模型中的氧化过程。
