Norris Erin H, Giasson Benoit I, Ischiropoulos Harry, Lee Virginia M-Y
Center for Neurodegenerative Disease Research and the Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2003 Jul 18;278(29):27230-40. doi: 10.1074/jbc.M212436200. Epub 2003 May 8.
Filamentous inclusions of alpha-synuclein protein are hallmarks of neurodegenerative diseases collectively known as synucleinopathies. Previous studies have shown that exposure to oxidative and nitrative species stabilizes alpha-synuclein filaments in vitro, and this stabilization may be due to dityrosine cross-linking. To test this hypothesis, we mutated tyrosine residues to phenylalanine and generated recombinant wild type and mutant alpha-synuclein proteins. alpha-Synuclein proteins lacking some or all tyrosine residues form fibrils to the same extent as the wild type protein. Tyrosine residues are not required for protein cross-linking or filament stabilization resulting from transition metal-mediated oxidation, because higher Mr SDS-resistant oligomers and filaments stable to chaotropic agents are detected using all Tyr --> Phe alpha-synuclein mutants. By contrast, cross-linking resulting from exposure to nitrating agents required the presence of one or more tyrosine residues. Furthermore, tyrosine cross-linking is involved in filament stabilization, because nitrating agent-exposed assembled wild type, but not mutant alpha-synuclein lacking all tyrosine residues, was stable to chaotropic treatment. In addition, the formation of stable alpha-synuclein inclusions in intact cells after exposure to oxidizing and nitrating species requires tyrosine residues. These findings demonstrate that nitrative and/or oxidative stress results in distinct mechanisms of alpha-synuclein protein modifications that can influence the formation of stable alpha-synuclein fibrils.
α-突触核蛋白的丝状内含物是统称为突触核蛋白病的神经退行性疾病的标志。先前的研究表明，在体外，暴露于氧化和硝化物质会使α-突触核蛋白丝稳定，这种稳定可能是由于二酪氨酸交联所致。为了验证这一假设，我们将酪氨酸残基突变为苯丙氨酸，并生成了重组野生型和突变型α-突触核蛋白。缺乏部分或全部酪氨酸残基的α-突触核蛋白形成原纤维的程度与野生型蛋白相同。酪氨酸残基对于过渡金属介导的氧化所导致的蛋白质交联或丝稳定并非必需，因为使用所有酪氨酸突变为苯丙氨酸的α-突触核蛋白突变体均检测到了更高分子量的耐SDS低聚物和对离液剂稳定的丝。相比之下，暴露于硝化剂所导致的交联需要一个或多个酪氨酸残基的存在。此外，酪氨酸交联参与丝的稳定，因为暴露于硝化剂的组装野生型α-突触核蛋白（而非缺乏所有酪氨酸残基的突变型α-突触核蛋白）对离液处理稳定。此外，暴露于氧化和硝化物质后，完整细胞中稳定的α-突触核蛋白内含物的形成需要酪氨酸残基。这些发现表明，硝化和/或氧化应激导致α-突触核蛋白修饰的不同机制，这些机制可影响稳定的α-突触核蛋白原纤维的形成。
