Biology of Native Alpha-Synuclein Tetramers in Parkinson's Disease

天然 α-突触核蛋白四聚体在帕金森病中的生物学

• 批准号: 8631204
• 负责人: DENNIS J SELKOE
• 金额: $ 36.82万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8631204
• 关键词: Address; Amyloid beta-Protein; Autophagocytosis; Binding; Biochemical; Biological; Biological Assay; Biology; Brain; Brain Diseases; Cell Line; Cells; Circular Dichroism; Complex; Congo Red; Data; Degradation Pathway; Detergents; Dialysis procedure; Dimethyl Sulfoxide; Disease; Engineering; Enzyme-Linked Immunosorbent Assay; Equilibrium; Erythrocytes; Erythroid Cells; Excision; Health; Heat-Shock Response; Human; Kinetics; Life; Lipids; Liquid substance; Luciferases; Mass Spectrum Analysis; Methods; Missense Mutation; Mitochondria; Molecular; Molecular Chaperones; Molecular Conformation; Monoclonal Antibodies; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organ; Paraquat; Parkinson Disease; Patients; Pharmaceutical Preparations; Physiologic pulse; Physiological; Production; Property; Proteins; Protocols documentation; Publishing; Rattus; Recombinant Proteins; Relative (related person); Reporting; Role; Rotenone; Salts; Scanning Transmission Electron Microscopy Procedures; Series; Solvents; Stress; Structure; Temperature; Thapsigargin; Therapeutic; Tunicamycin; Urea; Venus; Vesicle; Work; age related; aged; alpha synuclein; analytical ultracentrifugation; base; beta pleated sheet; biophysical techniques; brain cell; crosslink; dimer; effective therapy; immunocytochemistry; in vivo; knock-down; meetings; monomer; multicatalytic endopeptidase complex; mutant; neurotoxic; novel; protein folding; public health relevance; reconstitution; research study; sarkosyl; scyllo-inositol; sedimentation equilibrium; small molecule; synuclein; synuclein, alpha (non A4 component of amyloid precursor) protein, human; synucleinopathy; tau Proteins; tool

Pathogenic aggregation of ¿-synuclein (¿Syn) is increasingly implicated in familial and sporadic Parkinson's disease (PD) and other human synucleinopathies. Based largely on studies of the recombinant protein, ¿Syn has long been defined as a "natively unfolded" monomer of 14 kD that is believed to acquire secondary (¿- helical) structure only upon binding to certain lipid vesicles. In contrast, our lab discovered in 2011 that endogenous ¿Syn isolated under non-denaturing conditions from living human cells and neuronal lines occurs principally as a helically-folded tetramer of ~60 kD 1. Multiple methods, including sedimentation equilibrium analysis by analytical ultracentrifugation, scanning transmission electron microscopy, mass spectrometry and circular dichroism, established the existence of an ¿-helically folded tetramer. Not unexpectedly, this discovery engendered controversy, but we have recently completed an extensive analysis of living cells (including neurons) using in vivo crosslinking that has confirmed that the principal form of native ¿Syn in intact cells is a 60 kD tetramer. Also, 3 other labs recently provided evidence for the occurrence of ¿-helical oligomeric structures based on certain methods of preparing ¿Syn 4-6. If this finding that endogenous ¿Syn exists as a helically-folded tetramer can be further extended, it will have major implications for the biology of ¿Syn in health and disease, as the reviewers of our first version of this application recognized. A central concept of human neurodegenerative diseases -- that normally soluble proteins (¿Syn, tau, A¿, etc.) can misfold and aggregate into neurotoxic species - depends on understanding the normal state of the protein and what "misfolding" and "aggregation" actually mean. Accordingly, we propose an integrated series of entirely novel Specific Aims to characterize the dynamic relationship of the metastable ¿Syn tetramers to the unfolded monomer believed to be the native structure of ¿Syn since its description 20 years ago. Aim 1 Raise conformation-specific monoclonal antibodies to purified ¿Syn tetramers as key tools for all Aims. Aim 2 Examine the effects of four PD-causing missense mutations and certain structure-altering artificial mutations on the kinetic equilibrium of tetramers and monomers in intact cells. Aim 3 Study the biochemical mechanism and dynamics of the assembly of freshly synthesized monomers into tetramers (and other oligomers), their stability in the cell, and their subsequent disassembly. Aim 4 Purify endogenous human ¿Syn from normal and diseased (DLB) brains to establish its structure and assembly state in the most disease-relevant organ; then, systematically analyze its biochemical properties. We have made major progress towards these Aims since our first submission (see Preliminary Data). Our work elucidates the dynamic relationship between metastable physiological (¿-helical) oligomers, unfolded monomers, and abnormally folded (¿-sheet-rich) oligomers of ¿Syn in human cells and brain, with attendant mechanistic and therapeutic implications for PD.

¿-突触核蛋白 (¿Syn) 的致病性聚集越来越多地与家族性和散发性帕金森病有关 疾病（PD）和其他人类突触核蛋白病。主要基于重组蛋白的研究，¿Syn 长期以来被定义为 14 kD 的“天然未折叠”单体，据信它会获得二级（¿ 仅在与某些脂质囊泡结合时才形成螺旋）结构。相比之下，我们的实验室在 2011 年发现 在非变性条件下从活的人类细胞和神经元系中分离出内源性 ¿ 主要作为约 60 kD 的螺旋折叠四聚体 1. 多种方法，包括沉降平衡 通过分析超速离心、扫描透射电子显微镜、质谱和 圆二色性，证实了 ¿ 螺旋折叠四聚体的存在。这一发现并不出人意料 引起了争议，但我们最近完成了对活细胞的广泛分析（包括 神经元）使用体内交联，已证实完整细胞中天然 ¿Syn 的主要形式是 60 kD 四聚体。此外，另外 3 个实验室最近提供了 ¿-螺旋寡聚体出现的证据 基于某些制备¿Syn 4-6的方法的结构。如果这一发现表明内源性 ¿Syn 作为 螺旋折叠四聚体可以进一步延伸，这将对 ¿Syn 的生物学产生重大影响 正如我们该应用程序第一个版本的审阅者所认可的那样，健康和疾病。一个中心概念是 人类神经退行性疾病——通常可溶性蛋白质（“Syn、tau、A”等）可能会错误折叠并 聚集成神经毒性物质 - 取决于对蛋白质正常状态的理解以及什么 “错误折叠”和“聚合”实际上是什么意思。因此，我们提出了一个全新的综合系列 具体目标是表征亚稳态 ¿Syn 四聚体与展开的动态关系 自 20 年前被描述以来，单体就被认为是 ¿Syn 的天然结构。目标 1 加注 纯化的 ¿Syn 四聚体的构象特异性单克隆抗体是实现所有目标的关键工具。目标2 检查四种导致 PD 的错义突变和某些改变结构的人工突变的影响 完整细胞中四聚体和单体的动力学平衡。目标3 研究生化机制 以及新合成的单体组装成四聚体（和其他低聚物）的动力学，它们的 细胞中的稳定性及其随后的分解。目标 4 纯化内源性人体 ¿Syn 与正常值 和患病（DLB）大脑，以在与疾病最相关的器官中建立其结构和组装状态； 然后，系统分析其生化特性。我们在实现这些目标方面取得了重大进展 自我们第一次提交以来（参见初步数据）。我们的工作阐明了之间的动态关系 亚稳态生理（¿-螺旋）低聚物、未折叠单体和异常折叠（¿-片层丰富） 人类细胞和大脑中的 ¿Syn 寡聚物，对 PD 具有随之而来的机制和治疗意义。