Tau structure and dynamics in Alzheimer's disease

项目摘要-尽管对阿尔茨海默病(AD)进行了数十年的研究，但疾病修改 治疗阿尔茨海默病的方法仍然难以捉摸。这在很大程度上是由于在理解分子和 AD的结构基础。阿尔茨海默病的两个特征之一是由神经纤维缠结形成的 本质上无序的微管相关蛋白tau。Tau细丝在大脑中的扩散是 阿尔茨海默病神经病理分期的基础。Ad tau被过度磷酸化，截断，并用 其他翻译后修饰(PTM)。然而，这些PTM如何导致tau与MTS解离 并错误折叠成-Sheet淀粉样蛋白，以及tau是如何穿过类脂膜传播其病理的，这不是 为人所知。AD成对螺旋长丝(PHF)tau纤维具有C形-Sheet芯，它包含部分 MT结合重复。但包含大多数与疾病相关的PTM的大部分蛋白质， 在低温电子显微镜数据中看不到它的无序。在这里，我们建议使用固态核磁共振 核磁共振波谱、电子显微镜、小鼠神经元毒性分析和其他生化 了解膜结合的AD tau丝的分子结构和动力学的方法 Tau和MT结合的tau。我们假设特定的电荷-电荷相互作用是变化的基础 自聚集和与细胞结合时tau的构象、动力学和性质 合伙人。在过去的四年里，我们论证了应用单核磁共振研究结构的可行性。 和全长的tau纤维在体外形成和种植AD PHF tau。我们现在将应用这一点 回答三个问题的专业知识。在目标1中，我们将研究磷酸化和截断是如何导致 通过测定不含阴离子辅助因子的磷酸化tau(p-tau)的结构； 寻找复制AD PHF tau结构和性质的最低结构；以及 表征tau的半移动性富含脯氨酸区域的动态结构。在目标2中，我们将 通过测量tau的构象、动力学和膜来研究tau与脂膜的相互作用 与小的和大的单层囊泡结合的单体tau插入。我们将确定结构 膜诱导的tau聚集体，并探索磷酸化和截断如何影响结构 和膜结合tau的动力学。这些实验应该会揭示脂膜是如何 有核的tau聚集体以及聚集态tau如何跨膜。在目标3中，我们将调查 MT结合的tau的结构与磷酸化的关系，并探讨精氨酸-磷酸 R‘结构域的相互作用影响tau与MTS的结合。原纤维、膜结合性和结缔组织的联合研究 与MT结合的tau对于理解tau如何从其固有的无序结构转化为 以类普里子的方式传播的聚集结构。这一理解应该为未来提供信息 干扰这一过程的药物的设计。

Project Summary - Despite decades of research into Alzheimer's disease (AD), disease-modifying treatments for AD remain elusive. This is significantly due to challenges in understanding the molecular and structural basis of AD. One of the two hallmarks of AD is the neurofibrillary tangles formed by the intrinsically disordered microtubule (MT)-associated protein tau. Spreading of tau filaments in the brain is the basis of neuropathological staging of AD. AD tau is hyperphosphorylated, truncated, and decorated with other posttranslational modifications (PTMs). However, how these PTMs cause tau to dissociate from MTs and misfold into -sheet amyloids, and how tau crosses the lipid membrane to spread its pathology, is not known. AD paired helical filament (PHF) tau fibrils have a C-shaped -sheet core that encompasses part of the MT-binding repeats. But the majority of the protein, which contains most of the disease-relevant PTMs, is too disordered to be seen in cryo-electron microscopy data. Here we propose to employ solid-state NMR (ssNMR) spectroscopy, electron microscopy, mouse neuron toxicity assays, and other biochemical approaches to understand the molecular structures and dynamics of AD tau filaments, membrane-bound tau, and MT-bound tau. We hypothesize that specific charge-charge interactions underlie the varying conformations, dynamics and properties of tau when self-aggregated and when bound to its cellular partners. In the last four years, we demonstrated the feasibility of applying ssNMR to study the structures and dynamics of full-length tau fibrils formed in vitro and seeded by AD PHF tau. We will now apply this expertise to answer three questions. In Aim 1, we will investigate how phosphorylation and truncation cause AD PHF tau by determining the structures of phosphorylated tau (p-tau) fibrilized without anionic cofactors; searching for minimum constructs that replicate the AD PHF tau structure and properties; and characterizing the dynamic structures of the semi-mobile proline-rich region of tau. In Aim 2, we will investigate tau interactions with lipid membranes by measuring the conformation, dynamics and membrane insertion of monomeric tau bound to small and large unilamellar vesicles. We will determine the structures of membrane-induced tau aggregates, and probe how phosphorylation and truncation affect the structure and dynamics of membrane-bound tau. These experiments should shine light on how lipid membranes nucleate tau aggregates and how aggregated tau crosses the membrane. In Aim 3, we will investigate the structures of MT-bound tau as a function of phosphorylation, and probe how arginine-phosphate interactions in the R' domain affect tau binding to MTs. A joint study of the fibrillar, membrane-bound and MT-bound tau is crucial for understanding how tau converts from its intrinsically disordered structure to an aggregated structure that propagates in a prion-like manner. This understanding should inform the future design of drugs that interfere with this process.