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 晶状体的透明度是由一对小的热休克蛋白αA-和αB-晶体蛋白的丰富维持的。 随着时间的推移，晶状体蛋白质(包括α-晶体蛋白)会积累化学损伤，从而导致不稳定状态 以及光散射聚集体的成核，呈现出一种称为白内障的致病状态。 了解晶状体中这种与年龄相关的蛋白质聚集过程是如何发生的，或者如何防止/逆转 由于我们对α-晶体蛋白聚集途径的了解不足，它受到了治疗学的阻碍，并且 定义眼睛晶状体中光散射混浊的分子性质(或结构)。最常见的状态是 蛋白质聚集的性质可能是无定形的(即蛋白质类沉淀物)。然而，最近 淀粉样蛋白检测的进步也支持了另一种突出的 晶状体中的聚集途径可能是淀粉样蛋白的形成(或淀粉样蛋白的形成)。事实上，超过5%的 白内障晶状体的不溶性提取物被检测出在性质上是淀粉样蛋白。此外，我还发现 α-晶体蛋白结构的细微化学变化可以促进α-晶体蛋白中淀粉样蛋白的形成，如 并导致αB-晶体蛋白在体内形成一种全新的纤维聚集态 细胞，加强光散射聚集的形成，以响应其他不稳定的蛋白质。这 该提案试图：1)描述αB-晶状体蛋白中一种新的可逆的纤维聚集形式；以及2) 确定αA-和αB-晶体蛋白中存在的保守基序的破坏如何导致增强 淀粉样蛋白的形成。为了实现这些目标，我将采取协作和多学科的方法。 它利用了单粒子低温EM的使能技术，以及各种生化和 生物物理方法来表征α-晶体蛋白的这些聚集状态。实现这些目标的成功是 希望有助于我们理解蛋白质在眼睛晶状体中的错误折叠和聚集--这是一个标志 是世界上导致失明的主要原因。