Copper Coordination and Copper-dioxygen Reactivity of alpha-Synuclein

α-突触核蛋白的铜配位和铜-双氧反应性

基本信息

批准号：
8557988
负责人：
Jennifer Lee
金额：
$ 1.18万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8557988
关键词：
Affect Affinity Alzheimer&apos s Disease Amides Amines Amino Acid Sequence Amino Acids Amyloid beta-Protein Amyotrophic Lateral Sclerosis Binding Binding Sites Catechols Cellular Membrane Cerebrospinal Fluid Chemistry Circular Dichroism Copper Deposition Development Dioxygen Disease Disease Progression Environment Environmental Risk Factor Fluorescence Genetic In Vitro Intervention Ions Iron Lewy Bodies Ligands Lipids Measurement Membrane Metals Modeling Molecular N-terminal Nature Neurodegenerative Disorders Neurons Oxidation-Reduction Oxidative Stress Pathology Patients Peptides Phospholipids Play Prion Diseases Prions Process Production Property Proteins Reactive Oxygen Species Reagent Roentgen Rays Role Site-Directed Mutagenesis Spectrum Analysis Staging Structure Superoxide Dismutase Surface Testing Tryptophan Vertebral column Vesicle Work absorption alpha synuclein amyloid formation chelation crosslink dopaminergic neuron insight metal metabolism mutant novel strategies oxidation protein complex protein structure synthetic peptide

项目摘要

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.

尽管最近的工作表明了PD的遗传成分，涉及神经元蛋白的积累和沉积Alpha-syn，但该疾病的零星形式更为普遍，并且可能与促进氧化应激和异常的氧化还原活性金属代谢的环境因素有关。例如，PD中多巴胺能神经元中原纤维的选择性积累已归因于易于氧化的儿茶酚的存在，这些儿茶素刺激了蛋白质交联，以及在PD患者的脑脊液中的Lewy体和铁浓度的提高。此外，已经观察到在体外α-syn的金属增强氧化寡聚化，并且已经提出，特定的金属蛋白质相互作用在其他涉及淀粉样生物分子的神经退行性疾病中至关重要歧化酶（肌萎缩性侧索硬化症）。很难解决的问题是金属离子是否扰动蛋白质结构，从而改变功能，或者金属蛋白络合物是否直接参与活性氧的产生，或者两种机制是否在起作用。铜（ii）增强膜结合的α-突触核蛋白螺旋形成我们已经检查了铜（II）对α-syn/脂质相互作用的影响，并测试磷脂囊泡是否可以调节铜（II）结合特性。已知铜（II）通过N末端胺和骨链酰胺螯合在前四个残基（MDVF）内与可溶性α-Syn进行协调（Jackson MS，Lee JC Inorg。Chem。2009，48，9303-9307）。重要的是，还预计N末端区域将通过原代氨基酸序列中的七个不完善的氨基酸重复序列（KXKEGV）与细胞膜相互作用。我们表明，铜（II）与α-Syn的N末端结合会影响其螺旋倾向，而且我们的结果表明，当蛋白是膜结合时，F4Wα-Syn的铜（II）结合亲和力会增强。有趣的是，这项工作表明，N末端膜关联至少在穿透囊泡表面的铜结合位点上是一个动态过程（Lucas HR，Lee JC，Meltallomics 2011，3，280-283）。 ASP2在α-突触核蛋白的主要铜（II）结合位点中的作用色氨酸荧光测量涉及突变α-突触核蛋白（F4W和F4W/H50S）和合成N末端肽的比较表明，原代铜（II）结合位点位于前四个残基中。此外，Cu（II）结合需要α-氨基末端。使用位置定向的诱变（D2N/F4W和D2E/F4W），我们正在探测铜（II）-D2相互作用的性质，表明D2是不太可能的铜（II）配体。关于铜配位环境分子细节的见解也可以通过圆形二色性分析和使用合成肽模型来获得。 Cu（i）结合α-核蛋白的协调性能在先前的工作中，通过X射线吸收光谱获得了有关铜协调环境的结构信息（Lucas，Hr，Debeer，S，S，Hong，M-S，Lee JC，J.Am。J.Am。Chem。Soc。2010，132，6636-6637）。特别是，我们使用Cu k-边缘和扩展的X射线良好的结构吸收光谱谱图表征了可溶性和原纤维Cu-Alpha-syn形式的局部构象变化，并表明在淀粉样蛋白形成过程中存在CU（I）/二氧氧的反应性。当前的工作集中在直接研究α-Syn结合铜（I）的能力，即生理相关的金属氧化态。