Structure and conformational dynamics of alpha-synuclein

描述(申请人提供)：帕金森病(PD)是一种常见的神经疾病，从轻微的肌肉协调能力丧失发展到严重的身体和精神残疾，并最终死亡。没有治愈的方法，也没有治疗方法来减缓疾病的进展。目前，美国约有100万帕金森病患者，每年的费用约为250亿美元。随着老年人口的快速增长，预计到2050年，这一数字将增加两倍。因此，迫切需要有效的疾病修正治疗。为了对药物开发工作做出贡献，该项目的目标是确定突触核蛋白(Syn)的结构，以深入了解其在帕金森病发病机制中的功能及其机制，这对合理的药物设计至关重要。？-syn的聚集导致所谓的路易小体(LBS)的出现，作为帕金森氏病的一个明确的病理标志。类似的？-syn聚集体也与其他神经退行性疾病有关，包括多系统萎缩(MSA)、路易体痴呆(DLB)和其他统称为突触核病。？-Syn在大脑中含量非常丰富，似乎对学习和记忆很重要；然而，它的确切功能仍不清楚。它没有已知的酶活性，因此其未知的生物学功能取决于其物理结构；此外，它通过与其结构转变为高度有序的纤维相关的毒性功能而致病。因此，详细的结构信息对于理解其功能和洞察其向致病实体的转变至关重要。然而，目前还不能获得这样的结构细节，主要是因为直到最近，？-syn在溶液中缺乏持久结构，被认为是一种天然的未折叠蛋白质，不适合结构确定。然而，我们最近在溶液中分离到了一种有序的四聚形式的？-syn。我们通过开发一种表达和纯化程序来做到这一点，目的是在整个纯化过程中保持其天然结构。几乎在同一时间，哈佛医学院的Dennis Selkoe团队独立证明了从大鼠大脑和活的人类细胞中分离出来的？-syn也是天然折叠和四聚体的，与我们的重组？-syn惊人地相似。此外，我们还进一步证明了我们制备的四聚体具有抗聚集、不损害脂质体膜、对细胞无毒等特点，从而有力地支持了有序四聚体具有生理学意义。现在我们有了一个服从于传统生物物理工具的有序形式的-syn，这个提议的具体目标是：1)确定？-syn的原子结构；和2)确定其四元结构的动力学。我们将使用X射线结晶学方法、核磁共振方法和单分子荧光方法相结合的方法来实现这一点。 公共卫生相关性：本研究建议确定突触核蛋白的分子结构，并确定其四聚体结构组装的分子动力学，这将为合理设计治疗帕金森病的药物提供分子基础和框架。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a common neurological condition that progresses from subtle loss of muscular coordination to severe physical and mental disability and eventual death. There is no cure and no treatments to slow the disease progression. Currently there are ~1 million PD sufferers in the US, which costs about $25 billion USD per year. These numbers are expected to triple by 2050 as the population of the elderly is increasing rapidly. Thus, effective disease-modifying treatments are desperately needed. To contribute to the drug development effort, the goal of this project is to determine the structure o ¿-synuclein (¿-syn) to gain insights into its function and its mechanisms in the pathogenesis of Parkinson's disease, which are critical for rational drug design. The aggregation of ¿-syn leads to appearance of so-called Lewy bodies (LBs) as a defining pathological hallmark of Parkinson's disease. Similar aggregates of ¿-syn are also implicated in other neurodegenerative diseases, including multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and others collectively known as synucleinopathies. ¿-Syn is highly abundant in the brain and it appears to be important for learning and memory; however, its precise function is still unknown. It has no known enzymatic activity, thus its unknown biological function depends on its physical structure; moreover, it causes disease via a gain-of-toxic function that is associated with its structural transformation into highly ordered fibrils. Thus, detailed structural information of ¿-syn is crucil for understanding its function and to gain insights into its transformation into a disease-causing entity. However, such structural details are currently unavailable mainly because, until recently, ¿-syn lacked a persistent structure in solution and was thought to be a natively unfolded protein, which is not amenable for structure determination. However, we have recently isolated an ordered tetrameric form of ¿-syn in solution. We did so by developing an expression and purification procedure aimed at preserving its native structure throughout the purification process. At about the same time, Dennis Selkoe's group at Harvard Medical School independently demonstrated that ¿-syn isolated from rat brains and living human cells are also natively folded and tetrameric with striking resemblance to our recombinant ¿-syn. Moreover, we further demonstrated that our ¿-syn preparation was resistant to aggregation, did not compromise liposome membranes, and non-toxic to cells, thus strongly supporting that the ordered tetrameric ¿-syn is physiologically relevant. Now that we have an ordered form of ¿-syn that is amenable to traditional biophysical tools, the specific aims of this proposal are; 1) To determine the atomic structure of ¿-syn; and 2) To determine the dynamics of its quaternary structure. We will do so by using a combination of X-ray crystallographic methods, nuclear magnetic resonance (NMR) methods, and single-molecular fluorescence methods. PUBLIC HEALTH RELEVANCE: This study proposes to determine the molecular structure of ¿-synuclein and to determine the molecular kinetics of its tetrameric structure assembly, which will provide the molecular basis and a framework for rational drug design to combat Parkinson's disease.