Functional and pathological interactions of TDP-43

项目摘要 细胞内蛋白质包涵体是绝大多数神经退行性疾病的主要特征。在……里面 常见形式的肌萎缩侧索硬化症(ALS)、阿尔茨海默病相关痴呆(额颞叶 痴呆；以边缘为主的年龄相关性TDP-43脑病(晚期))以及某些形式的 阿尔茨海默病，人类必需的43 kDa TAR DNA结合蛋白(TDP-43)形成神经元内 集合体。重要的是，TDP-43易于聚集的区域中的数十个错义突变已经被 发现于家族性和散发的肌萎缩侧索硬化症和额颞叶痴呆病例。这些数据提供了有力的支持 TDP-43在阿尔茨海默病相关神经变性中的直接致病病理作用 痴呆和运动神经元病。此外，最近调节TDP-43相互作用组的研究 表明TDP-43是治疗这些疾病的一个重要的潜在靶点。然而， 治疗的发展在很大程度上是由于缺乏对正常 TDP-43的功能，导致肌萎缩侧索硬化症和肌萎缩侧索硬化的突变的分子效应 额颞叶痴呆和神经退行性疾病中的TDP-43基因中断。这些差距是由于 这在很大程度上是因为缺乏关于TDP-43及其络合物的原子结构数据，以及它转化为 聚集体，这又是由于难以通过传统的结构观察TDP-43络合物 生物学技术。该项目将利用综合实验和计算结构生物学 与分子和细胞生物学方法相结合的技术，以1)确定原子细节 TDP-43螺旋亚区的组装及其对剪接功能的贡献及其结构转换 疾病聚合，2)确定已知和新的翻译后修饰和疾病相关 突变改变了介导和调节TDP-43液-液相的TDP-43自身和异体蛋白的接触 分离和疾病相关聚集，以及3)绘制TDP-43相互作用的结构基础 用聚(ADP-核糖)和导入机制作为有希望的治疗靶点。具有挑战性的， 动态的、结构的目标使得这种方法有必要强调分子之间的紧密联系 模拟和实验生物物理技术(主要是核磁共振光谱学)。这些方法将 生成相互作用的详细分子模型，这些模型将在细胞内进行功能相关性测试 聚集、细胞剪接和细胞蛋白/RNA结合结构(ICLIP)。这些研究的结果是 TDP-43络合物、相分离、功能和聚集将提供直接的结构和机制 对预防阿尔茨海默病相关痴呆患者TDP-43毒性干扰的策略设计的投入 和运动神经元病。这些洞察力代表着未来治疗ALS的潜力，额颞部 痴呆症和其他与TDP-43相关的疾病，目前还没有治愈或有效的治疗方法。

Project Summary Cellular inclusions of proteins are primary hallmarks of a great majority of neurodegenerative diseases. In common forms of amyotrophic lateral sclerosis (ALS), Alzheimer’s disease related dementias (frontotemporal dementia; limbic-predominant age-related TDP-43 encephalopathy (LATE)) as well as some forms of Alzheimer’s disease, the essential human TAR DNA binding protein of 43 kDa (TDP-43) forms intraneuronal aggregates. Importantly, dozens of missense mutations in an aggregation-prone domain of TDP-43 have been found in familial and sporadic cases of ALS and frontotemporal dementia. These data provide strong support for the direct causative pathological role for TDP-43 in neurodegeneration in Alzheimer’s disease related dementias and motor neuron disease. Additionally, recent research modulating the TDP-43 interactome demonstrates that TDP-43 is an important potential therapeutic targets in these diseases. However, therapeutic development is hampered in large part by an absence of mechanistic understanding of normal TDP-43 function, the molecular effect of the mutations causing amyotrophic lateral sclerosis and frontotemporal dementia, and the TDP-43 disruption in neurodegenerative disease. These gaps are due in large part to a lack of atomic structural data regarding TDP-43, its complexes, and its conversion to aggregates, which in turn is due to the difficulty in observing TDP-43 complexes via traditional structural biology techniques. This project will make use of integrated experimental and computational structural biology techniques combined with molecular and cell biology approaches to 1) determine the atomistic details of the assembly of a helical sub-region of TDP-43, its contribution to splicing function, and its structural conversion in disease aggregates, 2) identify how known and novel post-translational modifications and disease-associated mutations alter TDP-43 self- and hetero-protein contacts that mediate and regulate TDP-43 liquid-liquid phase separation and disease-associated aggregation, and 3) map the structural basis of the interactions of TDP-43 with poly(ADP-ribose) and importin machinery that serve as promising therapeutic targets. The challenging, dynamic, structural targets necessitate the approach highlighting a tight connection between molecular simulation and experimental biophysical techniques (primarily NMR spectroscopy). These approaches will generate detailed molecular models of the interactions that will be tested for functional relevance using in cell aggregation, in cell splicing, and in cell protein/RNA binding structure (iCLIP). The results of these studies on TDP-43 complexes, phase-separation, function, and aggregation will provide direct structural and mechanistic input to the design of strategies to prevent toxic disruption of TDP-43 in Alzheimer’s disease related dementias and motor neuron disease. These insights represent potential for future treatments for ALS, frontotemporal dementia, and other TDP-43-associated diseases that currently have no cure or effective treatments.