Department of Life Sciences, Chalmers University of Technology, Gothenburg, Sweden.
Protein Sci. 2024 Apr;33(4):e4956. doi: 10.1002/pro.4956.
Copper ion dys-homeostasis is linked to neurodegenerative diseases involving amyloid formation. Even if many amyloidogenic proteins can bind copper ions as monomers, little is known about copper interactions with the resulting amyloid fibers. Here, we investigate copper interactions with α-synuclein, the amyloid-forming protein in Parkinson's disease. Copper (Cu(II)) binds tightly to monomeric α-synuclein in vitro involving the N-terminal amine and the side chain of His50. Using purified protein and biophysical methods in vitro, we reveal that copper ions are readily incorporated into the formed amyloid fibers when present at the start of aggregation reactions, and the metal ions also bind if added to pre-formed amyloids. Efficient incorporation is observed for α-synuclein variants with perturbation of either one of the high-affinity monomer copper-binding residues (i.e., N-terminus or His50) whereas a variant with both N-terminal acetylation and His50 substituted with Ala does not incorporate any copper into the amyloids. Both the morphology of the resulting α-synuclein amyloids (amyloid fiber pitch, secondary structure, proteinase sensitivity) and the copper chemical properties (redox activity, chemical potential) are altered when copper is incorporated into amyloids. We speculate that copper chelation by α-synuclein amyloids contributes to the observed copper dys-homeostasis (e.g., reduced bioavailable levels) in Parkinson's disease patients. At the same time, amyloid-copper interactions may be protective to neuronal cells as they will shield aberrantly free copper ions from promotion of toxic reactive oxygen species.
铜离子稳态失调与涉及淀粉样形成的神经退行性疾病有关。尽管许多淀粉样蛋白原纤维可以作为单体结合铜离子，但对于铜与形成的淀粉样纤维的相互作用知之甚少。在这里，我们研究了铜与α-突触核蛋白（帕金森病中形成淀粉样的蛋白质）的相互作用。铜（Cu(II)）在体外与单体α-突触核蛋白紧密结合，涉及 N 端胺和 His50 的侧链。使用纯化蛋白和体外生物物理方法，我们揭示了当铜离子存在于聚集反应开始时，铜离子很容易掺入形成的淀粉样纤维中，如果添加到预先形成的淀粉样纤维中，金属离子也会结合。当干扰任一高亲和力单体铜结合残基（即 N 端或 His50）时，α-突触核蛋白变体中观察到有效的掺入，而具有 N 端乙酰化和 His50 被 Ala 取代的变体则不会将任何铜掺入淀粉样纤维中。当铜掺入淀粉样纤维中时，形成的α-突触核蛋白淀粉样纤维的形态（淀粉样纤维间距、二级结构、蛋白酶敏感性）和铜的化学性质（氧化还原活性、化学势）都会发生改变。我们推测，α-突触核蛋白淀粉样纤维中的铜螯合作用导致帕金森病患者观察到的铜稳态失调（例如，生物可利用水平降低）。同时，淀粉样纤维-铜相互作用可能对神经元细胞具有保护作用，因为它们将使异常游离的铜离子免受促进毒性活性氧物质的影响。
