Structure and conformational dynamics of alpha-synuclein

描述（由申请人提供）：帕金森病（PD）是一种常见的神经系统疾病，可从轻微的肌肉协调丧失发展到严重的身体和精神残疾，最终导致死亡。目前还没有治愈和治疗方法来减缓疾病的进展。目前，美国约有100万PD患者，每年花费约250亿美元。随着老年人口的迅速增长，预计到2050年这些数字将增加两倍。因此，迫切需要有效的疾病改善治疗。为了促进药物开发，本项目的目标是确定¿-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和机制，这对合理的药物设计至关重要。¿-syn的聚集导致所谓的路易体（LBs）的出现，这是帕金森病的典型病理标志。类似的¿-syn聚集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蛋白病的疾病。-Syn在大脑中含量很高，对学习和记忆很重要；然而，它的确切功能仍然未知。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，它通过与其结构转化为高度有序的原纤维相关的毒性获得功能引起疾病。因此，详细的结构信息对于理解¿-syn的功能和洞察其向致病实体的转变至关重要。然而，这些结构细节目前无法获得，主要是因为直到最近，¿-syn在溶液中缺乏持久的结构，被认为是一种天然未折叠的蛋白质，无法进行结构测定。然而，我们最近在溶液中分离出一种有序的四聚体形式的¿-syn。为此，我们开发了一种表达和纯化程序，旨在在整个纯化过程中保留其天然结构。大约在同一时间，哈佛医学院的Dennis Selkoe小组独立地证明，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，我们进一步证明了我们的¿-syn制备具有抗聚集性，不损害脂质体膜，对细胞无毒，从而有力地支持了有序四聚体¿-syn在生理上的相关性。现在我们有了一个有序的¿-syn形式，可以适用于传统的生物物理工具，这个建议的具体目标是；1)确定¿-syn的原子结构；2)确定其第四纪结构动力学。我们将使用x射线晶体学方法，核磁共振（NMR）方法和单分子荧光方法的组合来做到这一点。

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.