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，它们使分子永久化 记忆和细胞内信号的传递；另一方面，它们加速了无法治愈的神经退行性变和 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，超出了 蛋白质，甚至是含有蛋白质的细胞。我们的长期目标是确定这些时间是否以及如何 鳞片产生于活细胞中淀粉样蛋白形成的动力学过程。为了实现这一目标，我们把重点放在了 淀粉样蛋白形成的关键第一步：成核。成核的概率性质已经进行了研究。 用现有的细胞学工具是非常困难的。因此，我们开发了分布式两氟FRET (DAmFRET)，以量化成核作为给定蛋白质在活细胞中浓度的函数。我们现在有了 使用DAmFRET分析了各种淀粉样蛋白和相关聚合物的成核作用，这些数据引导我们 提出这项资助的中心假设：低特异性交互作用严重影响比率和 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，即 在我们的特定目标中进行了测试：成核会随着蛋白质的增加而有利于越来越不稳定的淀粉样蛋白 浓缩或2)蛋白质的凝聚；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。完成这项工作 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 成核屏障的热力学基础，以及病理性交叉播种的网络。它还将 已经打开了一条通往目前细胞生物学无法企及的基本物理洞察的管道。 这些将产生对年龄相关和最终新的治疗选择的更深层次的理解 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.