A synthetic biology approach for tau post-translational modifications in AD

tau的寡聚体形式是广泛的神经退行性疾病中神经毒性的主要来源。 对从tau蛋白病患者脑中分离的病理性tau聚集体的结构研究已经揭示了一种新的结构。 疾病特异性tau构象和结构多态性的多样性，但它们如何 错误折叠成致病物种的情况仍不清楚。在这里，我们的目标是测试一个长期存在的假设，后- 翻译修饰（PTM），如疾病相关位点的磷酸化和乙酰化，介导 tau寡聚化、结构多态性依赖性增殖和神经毒性。我们建议自下而上 用于直接评估预定义的疾病相关磷酸化和乙酰化的影响的方法 全长的人类tau蛋白。这是我们第一次证明最先进的合成生物学方法 能够产生重组的位点特异性磷酸化的tau（rp-tau），并且具有单一磷酸化的rp-tau能够产生重组的位点特异性磷酸化的tau（rp-tau）， 修饰足以采用病理构象。这一新能力将扩大到包括 tau的位点特异性赖氨酸乙酰化以产生具有确定的PTM的全长tau（包括同时进行的 磷酸-和乙酰基-）参与阿尔茨海默病（AD）和AD相关痴呆（ADRD）。新 本文开发的试剂将用于确定位点特异性tau蛋白修饰修饰对tau蛋白构象的影响 使用冷冻EM、微管结合、寡聚化、神经毒性和tau接种。我们将产生 针对tau的单克隆抗体，具有定义的PTM和AD/ADRD相关构象，通过以下方法进行验证： 冷冻电镜该项目的影响将是提供可转移的资源，以前所未有的方式测试tau PTM。 解析并揭示介导tau寡聚化的分子特征和机制， 构象变化和播种。

Oligomeric forms of tau are the primary source of neurotoxicity in a wide range of neurodegenerative diseases. Structural studies of pathological tau aggregates isolated from tauopathy patient brains have uncovered a diversity of disease-specific tau conformations and structural polymorphs, but the mechanism for how they misfold into pathogenic species remains unknown. Here we aim to test a long-standing hypothesis that post- translational modifications (PTMs), such as phosphorylation and acetylation at disease-associated sites, mediate tau oligomerization, structural polymorph-dependent propagation, and neurotoxicity. We propose a bottom-up approach for directly assessing the impact of pre-defined, disease-associated phosphorylation and acetylation of full-length human tau. For the first time, we demonstrate that a state-of-the-art synthetic biology approach enables the production of recombinant site-specifically phosphorylated tau (rp-tau), and that rp-tau with a single modification is sufficient to adopt pathological conformations. This new capability will be expanded to include site-specific lysine acetylation of tau to produce full-length tau with defined PTMs (including simultaneous phospho- and acetyl-) implicated in Alzheimer’s disease (AD) and AD-related dementias (ADRD). The new reagents developed here will be used to determine the impact of site-specific tau PTMs on tau conformation using cryo-EM, microtubule binding, oligomerization, neurotoxicity, and tau seeding. We will generate monoclonal antibodies against tau with defined PTMs and AD/ADRD relevant conformations validated through cryo-EM. The impact of the project will be to provide transferrable resources to test tau PTMs with unprecedented resolution and uncover the molecular signature and mechanisms that mediate tau oligomerization, conformational change, and seeding.