Functional Characterization of Tau Mutation and Post-translational Modifications

项目概要/摘要 蛋白质聚集是神经退行性疾病的标志，包括阿尔茨海默病，并且 这些疾病缺乏有效的治疗方法。我们目前缺乏对分子和 细胞机制控制蛋白质聚集在人脑中，这将使新的 治疗策略 tau蛋白在许多称为tau蛋白病的神经退行性疾病中聚集在大脑中， 包括老年痴呆症在疾病的早期阶段，tau蛋白仅聚集在特定的神经元中， 在大脑中的每一个神经元中都有表达，这意味着细胞环境中的特定因素 使tau易于聚集。类似地，tau突变仅与特定的 tau蛋白病总之，这些疾病特征意味着tau蛋白对它的两个序列非常敏感， 特性及其细胞环境。 翻译后修饰（PTM）是细胞环境可以作用于细胞的一种机制。 蛋白质类似于突变。Tau蛋白在转录后被大量修饰，并且Tau蛋白PTM的变化是相关的。 伴随着疾病的发展事实上，tau蛋白磷酸化和蛋白水解被认为是核心 tau蛋白病的发作和进展中的事件。类似地，突变与早期疾病发作相关 并且已知加速体外tau聚集。突变也可以通过改变tau蛋白来引起PTM的变化 互动伙伴。然而，tau序列的这些变化的功能和因果关系尚不清楚。我 假设PTM和突变允许tau进入特定构象以形成聚集体。 本提案的目标是全面鉴定（1）tau PTM的生物学基础 变化和（2）tau突变和PTM如何引起聚集。我已经证明了线粒体电子 转运链功能障碍引起tau蛋白修饰修饰的重塑，包括tau蛋白水解酶的积累， 碎片在目标1中，我将获得基于质谱（MS）的蛋白质组学的新培训，以确定tau蛋白 由于ETC功能障碍而发生的PTM变化以及这些变化如何控制tau聚集。在目标2中，我将使用深度 突变扫描（DMS）来全面探测tau的序列-结构关系。作为目标2的一部分，我 将使用交联MS直接比较体外和体内tau状态，以揭示结构机制 识别的PTM和序列变化。我非常适合完成拟议的研究，因为 这个建议补充了我在蛋白质生物物理学和iPSC衍生神经元的功能基因组学方面的培训 新的MS技术。完成该提案将确定哪些PTM和突变控制 tau聚集，以及开发新技术来探测体外和体内的tau结构。 完成这份提案将为我提供必要的技能和培训，成为一个成功的独立 调查员

PROJECT SUMMARY/ ABSTRACT Protein aggregation is a hallmark of neurodegenerative diseases, including Alzheimer's Disease, and these diseases lack effective therapeutics. We currently lack an understanding of the molecular and cellular mechanisms controlling protein aggregation in the human brain, which would enable new therapeutic strategies. The protein tau aggregates in the brain in a number of neurodegenerative diseases called tauopathies, including Alzheimer's Disease. In early stages of disease, tau aggregates only in specific neurons despite being expressed in every neuron in the brain, implying that specific factors in the cellular environment predispose tau to aggregation. Similarly, tau mutations are associated with the onset of only specific tauopathies. Together, these disease features imply that tau is exquisitely sensitive to both its sequence properties and its cellular environment. Post-translational modifications (PTM) are a mechanism by which the cellular environment can act on a protein similarly to mutation. Tau is heavily post-translationally modified and changes to tau PTMs are correlated with progression of disease. Indeed, tau phosphorylation and proteolysis have been proposed to be central events in the onset and progression of tauopathies. Similarly, mutations are correlated with early disease onset and are known to hasten in vitro tau aggregation. Mutations can also cause changes in PTMs by changing tau interaction partners. The function and causality of these changes to tau sequence, however, is unknown. I hypothesize that PTMs and mutations license tau to access specific conformations to form aggregates. The goal of this proposal is to comprehensively identify (1) the biological basis of tau PTM changes and (2) how tau mutation and PTMs cause aggregation. I have shown that mitochondrial electron transport chain dysfunction causes a remodeling of tau PTMs, including the accumulation of a tau proteolytic fragment. In Aim 1, I will acquire new training in mass spectrometry (MS)-based proteomics to determine the tau PTM changes that occur due to ETC dysfunction and how those control tau aggregation. In Aim 2, I will use deep mutational scanning (DMS) to comprehensively probe tau's sequence-structure relationship. As part of Aim 2, I will use cross-link MS to directly compare in vitro and in vivo tau states to will reveal the structural mechanisms for the identified PTM and sequence changes. I am ideally positioned to complete the proposed research, as this proposal complements my training in protein biophysics and functional genomics in iPSC-derived neurons with novel MS-based techniques. Completion of this proposal will identify which PTMs and mutations control tau aggregation, as well as the development of new technologies to probe tau structure in vitro and in vivo. Completion of this proposal will provide me with essential skills and training to be a successful independent investigator.