Elucidating mechanisms of amyloid nucleation in vivo

阐明体内淀粉样成核机制

• 批准号: 9886371
• 负责人: Randal Arthur Halfmann
• 金额: $ 31.76万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9886371
• 关键词: Adsorption; Age; Aging; Alzheimer' s Disease; Amyloid; Biochemical; Biophysics; Cells; Cellular biology; Cytology; Data; Dependence; Disease; Event; Flow Cytometry; Fluorescence Resonance Energy Transfer; Genetic Polymorphism; Goals; Grant; Investigation; Kinetics; Laboratories; Lead; Longevity; Memory; Molecular; Molecular Conformation; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Organism; Outcome; Pathologic; Pathologic Processes; Phase; Physical condensation; Physiological; Physiological Processes; Polymers; Polymorph; Population; Probability; Proteins; Research; Role; Signal Transduction; Solubility; Specificity; Structure; Testing; Therapeutic; Thermodynamics; Time; amyloid formation; amyloid structure; analytical tool; density; environmental change; experimental study; fluorophore; in vivo; insight; novel therapeutics; polypeptide; protein function; proteostasis; screening; stress granule; tool

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.

项目概要 淀粉样蛋白发挥多种生理和病理活性。一方面，它们使分子 记忆和转导细胞内信号；另一方面，它们会引发无法治愈的神经退行性病变 与年龄相关的疾病。这些不同的活动的展开时间尺度超过了生物体的寿命。 蛋白质，甚至是含有它们的细胞。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，我们重点关注 淀粉样蛋白形成的关键第一步：成核。成核的概率性质使其研究 使用现有的细胞学工具极其困难。因此我们开发了分布式双氟 FRET (DAmFRET) 来量化成核作为活细胞中给定蛋白质浓度的函数。我们现在有 使用 DAmFRET 分析不同淀粉样蛋白和相关聚合物的成核作用，这些数据使我们得出 提出本次资助的中心假设：低特异性相互作用对比率和 体内淀粉样成核的结构结果。该假设做出以下预测 在我们的具体目标中进行了测试：成核将有利于日益不稳定的淀粉样蛋白，1）增加蛋白质 浓缩或2)蛋白质的浓缩； 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其无序含量而不是其结构。完成这个 研究将揭示活细胞中多种蛋白质成核的关键机制特征，包括 成核势垒的热力学基础和病理性交叉播种网络。它还将 为目前细胞生物学无法企及的基本物理见解开辟了途径。 这些将使人们对与年龄相关的疾病有更深入的了解，并最终获得新的治疗选择。 神经退行性疾病。