Structure, function and aggregation of lens α-crystallins by CryoEM

CryoEM 晶状体α-晶状体蛋白的结构、功能和聚集

• 批准号: 10876690
• 负责人: Stephen Loen Reichow
• 金额: $ 37.82万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10876690
• 关键词: Amyloid; Bacteria; Binding; Biophysics; Cataract; Cells; Classification; Client; Collaborations; Complex; Cryoelectron Microscopy; Crystallins; Data; Development; Drug Design; Emerging Technologies; Event; Future; Goals; Heterogeneity; Human; Hybrids; Image; In Vitro; Knowledge; Laboratories; Methods; Modeling; Modification; Molecular; Molecular Chaperones; Molecular Conformation; Muramidase; N-terminal; Pathogenesis; Pathogenicity; Pathway interactions; Physiological; Precipitation; Property; Protein Isoforms; Proteins; Resolution; Role; Stress; Structure; Technology; age related; aggregation pathway; alpha-Crystallins; biophysical techniques; environmental chemical; fibrillogenesis; gamma-Crystallins; lens; lens transparency; light scattering; multidisciplinary; mutant; novel; particle; prevent; protein function; protein protein interaction; proteostasis; structural biology; success; tool

Project Summary Light-scattering opacities responsible for age-related cataracts are a result of aggregation and precipitation of the lens crystallins (α, β, and γ-crystallins). The α-crystallins (αA and αB isoforms) assemble as polydispersed oligomeric complexes and function as ATP-independent molecular chaperones (i.e., protein hold-ases). Both of these properties are thought to guard against aggregation events that would disrupt the delicate proteostasis of the lens. It is known that environmental stress and chemical modifications that accrue over our lifetimes destabilize the lens crystallins, and induce complex forms of protein-protein interactions that lead to aggregation (amorphous and potentially fibril). However, a major hurdle to understanding the aggregation pathways associated with cataracts, has been the lack of structural information on the major lens α-crystallins. This gap in knowledge is due to the lack of effective methods to characterize the inherently polydispersed structure of α-crystallin, the heterogeneity of chaperone-client aggregate formations, and evasiveness of fibril aggregation states identified under physiological conditions. In this proposal, we describe our multidisciplinary team-based approach, centered around the PI's expertise in the enabling technology of single particle CryoEM, that will finally allow us to interrogate the basis of α-crystallin molecular plasticity. Specifically, we aim to define high-resolution structures of the α-crystallins in their intrinsic polydispersed states (Aim 1), resolve key structural intermediates (aka “pre-aggregation states”) induced under saturating client conditions (Aim 2), and characterize a novel mechanism of fibrillogenesis discovered by our laboratory that is accessible to αB- crystallin under cellular conditions (Aim 3). Structural studies will be complimented by biophysical and functional characterization, performed in collaboration with Prof. Kirsten Lampi (OHSU), with the aim of illuminating mechanistic principles that define α-crystallin structure, polydispersity and stability – which are critical to avoidance of aggregation in the lens and therefore key to future success of drug-design strategies targeted at controlling age-related cataracts (and a range of other human crystallin-opathies).

项目摘要 负责年龄相关性白内障的光片散落是汇总和降水的结果 透镜晶体（α，β和γ-晶状蛋白）。 α-晶状体（αA和αb同工型）组装为多分散 寡聚复合物和与ATP无关的分子伴侣（即蛋白质保持）的功能。两个 这些特性被认为可以防止聚集事件破坏会破坏细腻的蛋白质的蛋白质 镜头。众所周知，在我们一生中产生的环境压力和化学修饰 破坏透镜晶体的稳定，并诱导蛋白质 - 蛋白质相互作用的复杂形式，导致 聚集（非洲群岛和潜在的原纤维）。但是，了解聚合的主要障碍 与白内障相关的途径一直缺乏有关主要晶状体α-晶体蛋白的结构信息。 知识的差距是由于缺乏表征固有多分散的有效方法 α-晶状体的结构，伴侣 - 晶状体骨料的异质性以及原纤维的逃避性 在生理条件下确定的聚集状态。在此提案中，我们描述了我们的多学科 基于团队的方法以Pi的专业知识为中心 这最终将使我们能够审问α-晶体蛋白分子可塑性的基础。具体来说，我们旨在定义 α-晶状体在其内部多分散状态中的高分辨率结构（AIM 1），解决键 在饱和的客户条件下（AIM 2）和 表征了我们实验室发现的原纤维发生的新机制，该机制可用于αB- 在细胞条件下结晶蛋白（AIM 3）。结构研究将由生物物理和 功能表征，与Kirsten Lampi教授（OHSU）合作进行，目的是 定义α-晶体结构，多分散性和稳定性的照明机械原理 - 对于避免镜头中的聚集至关重要，因此是药物设计策略未来成功的关键 针对控制年龄相关的白内障（以及其他人类晶体蛋白质）。