Copper Coordination and Copper-dioxygen Reactivity of alpha-Synuclein

虽然最近的研究指出PD的遗传成分涉及神经元蛋白α -syn的积累和沉积，但散发形式的疾病更为常见，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，PD患者多巴胺能神经元中原纤维的选择性积累归因于易氧化儿茶酚的存在，儿茶酚刺激蛋白质交联，以及路易小体中铁浓度和PD患者脑脊液中铜浓度的增加。此外，已经在体外观察到α -syn的金属增强氧化寡聚化，并且已经提出了特定的金属-蛋白质相互作用在其他涉及淀粉样蛋白生物分子的神经退行性疾病中至关重要，例如淀粉样β肽（阿尔茨海默病），朊蛋白（海海绵状脑病）和超氧化物歧化酶（肌萎缩性侧索硬化症）。一个难以解决的问题是金属离子是否扰乱蛋白质结构从而改变功能，或者金属-蛋白质复合物是否直接参与活性氧的产生，或者这两种机制是否都在起作用。

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.