Copper Coordination and Copper-dioxygen Reactivity of alpha-Synuclein

尽管最近的研究指出帕金森病的遗传成分涉及神经元蛋白α-syn的积累和沉积，但这种散发性疾病要常见得多，可能与促进氧化应激和异常氧化还原活性金属代谢的环境因素有关。例如，帕金森病患者多巴胺能神经元中纤维的选择性积累归因于刺激蛋白质交联的易氧化的儿茶酚的存在，以及帕金森病患者路易小体中铁浓度的增加和脑脊液中铜浓度的增加。此外，在体外观察到了金属增强的氧化齐聚作用，并且，特定的金属-蛋白质相互作用被认为是其他涉及淀粉样变性生物分子的神经退行性疾病的关键，例如淀粉样β-肽(阿尔茨海默氏病)、Prion蛋白(海绵状脑病)和超氧化物歧化酶(肌萎缩侧索硬化症)。一个难以解决的问题是，金属离子是否扰乱了蛋白质的结构从而改变了功能，或者金属-蛋白质复合体是否直接参与了活性氧的产生，或者这两种机制是否都在发挥作用。 铜(II)促进膜结合α-突触核蛋白螺旋的形成 我们研究了铜(II)对α-突触素/脂质相互作用的影响，并测试了磷脂微囊是否能够调节铜(II)的结合性质。已知铜(II)通过N端胺和主链酰胺螯合作用(Jackson MS，Lee JC Inorg)与前四个残基(MDVF)中的可溶性α-syn配位。化学。2009、48、9303-9307)。重要的是，N-末端区域还预计通过初级氨基酸序列中的七个不完全氨基酸重复(KXKEGV)与细胞膜静电相互作用。我们发现，铜(II)与α-SYN的N-末端结合影响其螺旋倾向，此外，我们的结果表明，当蛋白质被膜结合时，F4Wα-SYN与铜(II)的结合亲和力增强。有趣的是，这项工作表明N-末端膜结合是一个动态过程，至少就穿透小泡表面的铜结合部位而言是这样(Lucas HR，Lee JC，Metallology 2011，3,280-283)。 Asp2在α-突触核蛋白初级铜(II)结合部位中的作用 突变的α-突触核蛋白(F4W和F4W/H50S)的色氨酸荧光测量以及与合成的N端肽的比较表明，主要的铜(II)结合位点位于前四个残基。此外，α-氨基末端是铜(II)结合所必需的。利用定点突变(D2N/F4W和D2E/F4W)，我们正在探索铜(II)-D2相互作用的性质，表明D2是一个不太可能的铜(II)配体。通过圆二色谱分析和合成肽模型的使用，对铜配位环境的分子细节也有了更深入的了解。 铜(I)结合α-突触核蛋白的配位性质 在以前的工作中，铜的配位环境的结构信息是通过X射线吸收光谱(Lucas，HR，DeBeer，S，Hong，M-S，Lee JC，J.Am)获得的化学。SoC。2010、132、6636-6637)。特别是，我们用CuK边和扩展的X射线精细结构吸收光谱表征了可溶性和纤维状两种构象的局部构象变化，表明在淀粉样蛋白形成过程中存在铜(I)/氧反应。目前的工作集中在直接研究α-SYN与铜(I)的结合能力，这是生理上相关的金属氧化态。

Although recent work points to a genetic component to PD involving the accumulation and deposit of a neuronal protein, alpha-syn, the sporadic form of the disease is far more common and possibly connected to environmental factors that promote oxidative stress and aberrant redox-active metal metabolism. For example, selective accumulation of fibrils in dopaminergic neurons in PD has been attributed to the presence of easily oxidizable catechols that stimulate protein cross-links, as well as to increased iron concentration in Lewy bodies and copper in cerebrospinal fluid of PD patients. Furthermore, metal-enhanced oxidative oligomerization has been observed for alpha-syn in vitro, and, specific metal-protein interactions have been proposed to be critical in other neurodegenerative diseases involving amyloidogenic biomolecules such as amyloid beta-peptide (Alzheimers disease), prion protein (spongiform encephalopathies), and superoxide dismutase (amyotrophic lateral sclerosis). A difficult issue to resolve is whether metal ions perturb protein structures and thereby alter functions, or whether metal-protein complexes directly participate in the production of reactive oxygen species, or whether both mechanisms are at work. Copper(II) Enhances Membrane-bound alpha-Synuclein Helix Formation We have examined the effect of copper(II) on alpha-syn/lipid interactions and to test whether the phospholipid vesicles can modulate the copper(II) binding properties. Copper(II) is known to coordinate to soluble alpha-syn within the first four residues (MDVF) through the N-terminal amine and backbone amide chelation (Jackson MS, Lee JC Inorg. Chem. 2009, 48, 9303-9307). Importantly, the N-terminal region is also anticipated to interact electrostatically with cellular membranes through seven imperfect amino acid repeats (KXKEGV) in the primary amino acid sequence. We show that copper(II) binding to the N-terminus of alpha-syn affects its helical propensity and moreover, our results indicate that the copper(II) binding affinity of F4W alpha-syn is enhanced when the protein is membrane-bound. Interestingly, this work demonstrates that N-terminal membrane association is a dynamic process at least in regards to the copper-binding site penetrating the vesicle surface (Lucas HR, Lee JC, Metallomics 2011, 3, 280-283). Role of Asp2 in the Primary Copper (II) Binding Site in alpha-Synuclein Tryptophan fluorescence measurements involving mutant alpha-synucleins (F4W and F4W/H50S) and comparisons to synthetic N-terminal peptides suggest that the primary copper(II) binding site is localized in the first four residues. Moreover, the alpha-amino terminus is required for Cu(II) binding. Using site-directed mutagenesis (D2N/F4W and D2E/F4W), we are probing the nature of the copper(II)-D2 interaction showing that D2 is an unlikely copper(II) ligand. Insights on the molecular details of copper coordination environment also are gained through circular dichroism analyses and the use of synthetic peptide models. Coordination Properties of Cu(I) Bound alpha-Synuclein In prior work, structural information on copper coordination environment was obtained through X-ray absorption spectroscopy (Lucas, HR, DeBeer, S, Hong, M-S, Lee JC, J. Am. Chem. Soc. 2010, 132, 6636-6637). Particularly, we have characterized the local conformational changes in both soluble and fibrillar Cu-alpha-syn forms using Cu K-edge and extended X-ray fine structure absorption spectroscopy and have shown that there is Cu(I)/dioxygen reactivity during amyloid formation. Current work is focused on studying directly the ability of alpha-syn to bind copper(I), the physiologically relevant metal oxidation state.