Mechanisms and functional effects of fibrillar aggregation in the lens alpha-crystallins

晶状体α-晶状体蛋白中纤维聚集的机制和功能效应

• 批准号: 10541819
• 负责人: Russell McFarland
• 金额: $ 4.08万
• 依托单位: PORTLAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10541819
• 关键词: Age; Age of Onset; Amyloid; Amyloid Fibrils; Biochemical; Biological Assay; Biological Models; Biophysics; Blindness; C-terminal; Cardiomyopathies; Cataract; Cells; Chemicals; Chemoresistance; Coupled; Cryoelectron Microscopy; Crystalline Lens; Crystallins; DNA Sequence Alteration; Detection; Development; Disease; Heat shock proteins; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Methods; Modification; Molecular; Molecular Chaperones; Mutation; N-terminal; Nature; Neurodegenerative Disorders; Pathogenesis; Pathogenicity; Pathway interactions; Persons; Physiological; Process; Protein Isoforms; Proteins; Resolution; Role; Structure; System; Techniques; Technology; Therapeutic; Thermodynamics; Time; Variant; Visual impairment; age related; aggregation pathway; alpha-Crystallins; amyloid formation; amyloidogenesis; biophysical techniques; comparative; congenital cataract; early onset; human disease; insight; interdisciplinary approach; lens; lens transparency; light scattering; novel; particle; prevent; protein aggregation; protein function; protein misfolding; response; success

Project Summary Lens transparency is maintained by the abundance of a pair of small heat-shock proteins, αA- and αB-crystallin. Over time, lens proteins (including the α-crystallins) accumulate chemical damage that lead to destabilized states and the nucleation of light-scattering aggregates, which take on a pathogenic state known as cataracts. Understanding how this age-related process of protein aggregation in the lens occurs, or how to prevent/reverse it by therapeutics, has been hindered by our poor understanding of the pathways of α-crystallin aggregation, and the molecular nature (or structures) that define light-scattering opacities in the eye lens. The most common state of protein aggregation is presumably amorphous in nature (i.e., proteinaceous precipitates). However, recent advancement in the detection of amyloids has also supported the hypothesis that one of the other prominent aggregate pathways in the lens may be amyloidogenesis (or the formation of amyloids). In fact, over 5% of the insoluble extracts of cataract lenses have been detected to be amyloid in nature. Furthermore, I have discovered that subtle chemical changes to α-crystallin structure can both promote amyloid formation in the α-crystallins, as well as lead to the formation of a completely novel type of fibrillar aggregation state of αB-crystallin inside living cells, that potentiate formation of light-scattering aggregation in response to other destabilized proteins. This proposal seeks to: 1) characterize a novel and reversible form of fibrillar aggregation in αB-crystallin; and 2) determine how disruption of a conserved motif present in both αA- and αB-crystallin leads to the enhancement of amyloid formation. To accomplish these aims, I will employ a collaborative and multi-disciplinary approach that leverages the enabling technology of single particle CryoEM, together with various biochemical and biophysical methods to characterize these aggregation states of the α-crystallins. Success in these aims are expected to contribute to our understanding of protein misfolding and aggregation in the eye lens – the hallmark of the world's leading cause of vision loss.

项目摘要 透镜透明度是通过一对小热冲蛋白αA和αB-晶状体蛋白的抽象来维持的。 随着时间的流逝，晶状体蛋白（包括α-晶状体）会累积化学损伤，导致不稳定状态 以及光碎片骨料的成核，该聚集体具有称为白内障的致病状态。 了解镜头中与年龄相关的蛋白质聚集过程如何发生，或如何预防/反向 通过治疗，我们对α-晶状体聚集的途径的不良理解和 定义眼镜镜片中光散射的分子性质（或结构）。最常见的状态 蛋白质聚集的本质本质上是无定形的（即蛋白质沉淀）。但是，最近 检测淀粉样蛋白的进步也支持了以下假设 晶状体中的骨料途径可能是淀粉样生成（或淀粉样蛋白的形成）。实际上，超过5％ 已检测到白内障透镜的不溶性提取物本质上是淀粉样蛋白。此外，我发现了 对α-晶状蛋白结构的微妙化学变化都可以促进α-晶状体中的淀粉样蛋白形成，因为 并导致形成一种完全新颖的原纤维聚集状态的αB-晶状体蛋白。 细胞，响应于其他不稳定蛋白的光散射聚集的潜在形成。这 提案寻求：1）表征αB-晶状体中原纤维聚集的一种新颖而可逆的形式；和2） 确定在αA-和αB-晶状体中存在的组成基序的破坏如何导致增强 淀粉样蛋白形成。为了实现这些目标，我将采用一种协作和多学科的方法 这利用了单个粒子冷冻的能力技术，以及各种生化和 生物物理方法来表征α-晶状体的这些聚集态。这些目标的成功是 有望有助于我们对眼镜镜头中蛋白质错误折叠和聚集的理解 - 标志 世界上视力丧失的主要原因。