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

突触核蛋白致病聚集性(Syn)与家族性和散发性帕金森病的关系日益密切 疾病(PD)和其他人类共核病。主要基于对重组蛋白？Syn的研究 长期以来一直被定义为一种14kD的自然展开的单体，被认为获得了二次(？) 螺旋状)仅在与某些脂泡结合时才有结构。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细胞系分离的内源性Syn发生 主要作为~60kD的螺旋折叠四聚体1.多种方法，包括沉淀平衡 用分析超速离心法、扫描电子显微镜、质谱仪和X射线衍射仪对样品进行了分析 圆二色谱证实了螺旋折叠四聚体的存在。不出所料，这一发现 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，已经证实在完整细胞中天然Syn的主要形式是 60kD的四聚体。另外，最近还有另外3个实验室提供了出现螺旋低聚物的证据 基于某些制备方法的结构？Syn 4-6。如果这一发现内源性Syn以 螺旋折叠的四聚体可以进一步延伸，这将对Syn的生物学研究具有重要意义 健康和疾病，正如我们第一个版本的应用程序的审查者所认识到的那样。一个核心概念是 人类神经退行性疾病--通常可溶的蛋白质(Syn、tau、A等)可能会出现折叠错误和 聚合成神经毒性物种-取决于了解蛋白质的正常状态以及 “错误折叠”和“聚合”实际上是指。据此，我们提出了一套完整的系列全新的 具体目的是表征亚稳态四聚体与未折叠的动力学关系 自20年前被描述以来，单体被认为是？Syn的天然结构。目标1加薪 针对纯化的Syn四聚体的构象特异性单抗是实现所有目标的关键工具。目标2 检查四种导致帕金森氏症的错义突变和某些结构改变的人工突变的影响 关于完整细胞中四聚体和单体的动态平衡。目的3研究其生化机制。 和新合成的单体组装成四聚体(和其他低聚物)的动力学，他们的 细胞中的稳定性，以及它们随后的分解。目的4从正常人中分离纯化内源性人Syn 以及患病(DLB)脑，以建立其在与疾病最相关的器官中的结构和组装状态； 然后，对其生化特性进行系统分析。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(见初步数据)。我们的工作阐明了两者之间的动态关系 亚稳的生理(螺旋)低聚物、未折叠的单体和异常折叠(富含片状) Syn在人类细胞和大脑中的寡聚体，以及伴随而来的帕金森病的机制和治疗意义。

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.