Elucidating mechanisms of amyloid nucleation in vivo

阐明体内淀粉样成核机制

• 批准号: 10531912
• 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 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10531912
• 关键词: Acceleration; Adsorption; Age; Aging; Alzheimer' s Disease; Amyloid; Biochemical; Biophysics; Cells; Cellular biology; Cytology; Data; Dependence; Disease; Disparate; Event; Flow Cytometry; Fluorescence Resonance Energy Transfer; Genetic Polymorphism; Goals; Grant; Investigation; Kinetics; Laboratories; 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.

项目摘要 淀粉样蛋白发挥了许多物理和病理活动。一方面，它们使分子永存 记忆和传递细胞内信号；另一方面，它们宝贵的神经退行性和 与年龄相关的疾病。这些不同的活动随时间尺度而展开，超过了寿命 蛋白质甚至藏有其细胞。我们的长期目标是确定这些时间是否以及如何 鳞片从活细胞中淀粉样蛋白形成的动力学中出现。为了实现这一目标，我们专注于 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 具有既定的细胞学工具非常困难。因此，我们开发了分布式的二氟此 （DAMFRET）将成核作为给定蛋白在活细胞中的浓度的函数。我们现在有 使用Damfret分析潜水淀粉样蛋白和相关聚合物的成核，这些数据使我们进入 提议这笔赠款的核心假设：低特异性相互作用严重影响了速率和 体内淀粉样蛋白成核的结构结果。该假设使以下预测将是 在我们的具体目的中测试：成核将有利于越来越不稳定的淀粉样蛋白，而蛋白质增加 浓度或2）蛋白质的缩合； 3）一种蛋白质的淀粉样蛋白具有核的能力 不同蛋白质的淀粉样蛋白取决于其无序含量而不是结构。完成此操作 研究将揭示活细胞中各种蛋白质成核的关键机理特征，包括 成核屏障的热力学基础和病理交叉种子网络。它也会 已经为目前超出细胞生物学范围的基本物理见解开了一个渠道。 这些将对与年龄相关的新治疗选择有更深入的了解，并最终对 神经退行性疾病。

项目成果

Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal.

• DOI: 10.7554/elife.86939
• 发表时间: 2023-11-03
• 期刊: eLife
• 影响因子: 7.7
• 作者: Kandola T;Venkatesan S;Zhang J;Lerbakken BT;Von Schulze A;Blanck JF;Wu J;Unruh JR;Berry P;Lange JJ;Box AC;Cook M;Sagui C;Halfmann R
• 通讯作者: Halfmann R