Elucidating mechanisms of amyloid nucleation in vivo

项目摘要 淀粉样蛋白具有多种生理和病理活性。一方面，它们使分子 记忆和细胞内信号;另一方面，他们沉淀不可治愈的神经退行性疾病， 与年龄相关的疾病。这些不同的活动随着时间的推移而展开， 蛋白质甚至是携带它们的细胞。我们的长期目标是确定这些时间是否以及如何 鳞片是由活细胞中淀粉样蛋白形成的动力学产生的。为了实现这一目标，我们把重点放在 淀粉样蛋白形成的关键第一步：成核。成核的概率性质使它的研究 用现有的细胞学工具非常困难。因此，我们开发了分布式Amphiphibrium FRET （DAmFRET）来量化成核作用作为活细胞中给定蛋白质浓度的函数。我们现在已经 使用DAmFRET分析不同淀粉样蛋白和相关聚合物的成核，这些数据使我们 我提出了这个补助金的中心假设：低特异性相互作用严重影响了利率， 体内淀粉样蛋白成核的结构结果。这一假设做出了以下预测， 在我们的特定目标中测试：随着1）蛋白质含量的增加，成核作用将有利于越来越不稳定的淀粉样蛋白 浓缩或2）蛋白质的缩合;和3）一种蛋白质的淀粉样蛋白成核的能力 不同蛋白质的淀粉样蛋白取决于它们的无序含量而不是它们的结构。完成本 研究将揭示活细胞中不同蛋白质成核的关键机制特征，包括 成核障碍的热力学基础和病理交叉播种的网络。它还将 已经打开了一个管道，基本的物理见解，目前超出了细胞生物学的范围。 这些将产生更深入的理解，并最终新的治疗选择，年龄相关， 神经退行性疾病

Project Summary Amyloids exert numerous physiological and pathological activities. On the one hand, they perpetuate molecular memories and transduce intracellular signals; on the other, they precipitate incurable neurodegenerative and age-associated diseases. These disparate activities unfold over time scales that exceed the lifespan of the proteins or even the cells that harbor them. Our long-term goal is to determine whether, and how, these time scales emerge from the kinetics of amyloid formation in living cells. Toward this goal, we have focused on the critical first step of amyloid formation: nucleation. The probabilistic nature of nucleation has made its study exceedingly difficult with established cytological tools. We therefore developed Distributed Amphifluoric FRET (DAmFRET) to quantify nucleation as a function of a given protein’s concentration in living cells. We have now used DAmFRET to analyze nucleation of diverse amyloids and related polymers, and these data lead us to propose the central hypothesis of this grant: low-specificity interactions critically influence the rates and structural outcomes of amyloid nucleation in vivo. This hypothesis makes the following predictions that will be tested in our Specific Aims: that nucleation will favor increasingly labile amyloids with 1) increasing protein concentration or 2) condensation of the protein; and 3) that the ability of amyloids of one protein to nucleate amyloids of a different protein depends on their disordered content rather than their structure. Completing this investigation will reveal critical mechanistic features of nucleation by diverse proteins in living cells, including the thermodynamic underpinnings of nucleation barriers, and networks of pathological cross-seeding. It will also have opened a conduit to fundamental physical insights that are presently beyond the reach of cell biology. These will yield a deeper understanding of, and ultimately new therapeutic options for, age- associated and neurodegenerative diseases.