Department of Chemistry, University of Copenhagen, Copenhagen, Denmark.
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
Free Radic Biol Med. 2018 Jun;121:38-50. doi: 10.1016/j.freeradbiomed.2018.04.559. Epub 2018 Apr 22.
Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used conditions that result in extensive modification precluding an understanding of the early events in this process. In this study, we have examined time-dependent oxidative events related to α-synuclein modification using six different molar ratios of Cu/HO/protein and Cu/HO/ascorbate/protein resulting in mild to moderate extents of oxidation. For a Cu/HO/protein molar ratio of 2.3:7.8:1 only low levels of carbonyls were detected (0.078 carbonyls per protein), whereas a molar ratio of 4.7:15.6:1 gave 0.22 carbonyls per α-synuclein within 15 min. With the latter conditions, rapid conversion of 3 out of 4 methionines (Met) to methionine sulfoxide, and 2 out of 4 tyrosines (Tyr) were converted to products including inter- and intra-molecular dityrosine cross-links and protein oligomers, as determined by SDS-PAGE and Western blot analysis. Limited histidine (His) modification was observed. The rapid formation of dityrosine cross-links was confirmed by fluorescence and mass-spectrometry. These data indicate that Met and Tyr oxidation are early events in Cu/HO-mediated damage, with carbonyl formation being a minor process. With the Cu/HO/ascorbate system, rapid protein carbonyl formation was detected with the first 5 min, but after this time point, little additional carbonyl formation was detected. With this system, lower levels of Met and Tyr oxidation were detected (2 Met and 1 Tyr modified with a Cu/HO/ascorbate/protein ratio of 2.3:7.8:7.8:1), but greater His oxidation. Only low levels of intra- dityrosine cross-links and no inter- dityrosine oligomers were detected under these conditions, suggesting that ascorbate limits Cu/HO-induced α-synuclein modification.
先前关于金属离子催化的α-突触核蛋白氧化的研究大多使用导致广泛修饰的条件，这使得人们无法了解该过程中的早期事件。在这项研究中，我们使用六种不同的铜/双氧水/蛋白质摩尔比（Cu/HO/protein）和铜/双氧水/抗坏血酸/蛋白质摩尔比（Cu/HO/ascorbate/protein），考察了与α-突触核蛋白修饰相关的时间依赖性氧化事件，这些条件导致的氧化程度从轻到中度不等。对于 Cu/HO/protein 摩尔比为 2.3:7.8:1，仅检测到低水平的羰基（每个蛋白质 0.078 个羰基），而摩尔比为 4.7:15.6:1 时，在 15 分钟内每个α-突触核蛋白就有 0.22 个羰基。在后一种条件下，迅速将 4 个甲硫氨酸（Met）中的 3 个转化为甲硫氨酸亚砜，将 4 个酪氨酸（Tyr）中的 2 个转化为包括二酪氨酸内和内交联以及蛋白质寡聚物在内的产物，这通过 SDS-PAGE 和 Western blot 分析得到证实。观察到有限的组氨酸（His）修饰。二酪氨酸交联的快速形成通过荧光和质谱得到证实。这些数据表明，Met 和 Tyr 的氧化是 Cu/HO 介导的损伤的早期事件，而羰基的形成是一个次要过程。在 Cu/HO/抗坏血酸系统中，在最初的 5 分钟内就检测到了快速的蛋白质羰基形成，但在此时间点之后，几乎没有检测到额外的羰基形成。在该系统中，检测到较低水平的 Met 和 Tyr 氧化（用 Cu/HO/ascorbate/protein 比为 2.3:7.8:7.8:1 修饰 2 个 Met 和 1 个 Tyr），但 His 氧化程度更高。在这些条件下，仅检测到低水平的内二酪氨酸交联，没有检测到内二酪氨酸寡聚物，这表明抗坏血酸限制了 Cu/HO 诱导的α-突触核蛋白修饰。
