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 导致老年性白内障的光散射浑浊是由聚集和沉淀的 晶状体中的晶体蛋白(α，β和γ-晶体蛋白)。α-晶状体蛋白(αA和αB亚型)以多分散形式聚集在一起 低聚复合体，并作为不依赖于ATP的分子伴侣(即蛋白质保持酶)发挥作用。两个都是 这些特性被认为可以防止聚集事件，这些聚集事件会扰乱微妙的蛋白质平衡 镜头。众所周知，在我们的一生中积累的环境压力和化学修饰 破坏晶状体晶体蛋白的稳定性，并诱导复杂形式的蛋白质-蛋白质相互作用，从而导致 聚集(无定形和潜在的纤维状)。然而，理解聚合的一个主要障碍是 与白内障相关的通路，一直缺乏关于晶状体α的主要结构信息--晶状体蛋白。 这种认识上的差距是由于缺乏有效的方法来描述固有的多分散的 α-晶体蛋白的结构、伴侣-客户聚集体形成的异质性和原纤维的规避 在生理条件下确定的聚集状态。在这份提案中，我们描述了我们的多学科 以团队为基础的方法，以PI在单粒子低温EM使能技术方面的专业知识为中心， 这将最终使我们能够探究α的基础--晶体蛋白的分子可塑性。具体地说，我们的目标是定义 α晶体蛋白在其固有多分散状态下的高分辨结构(目标1)，解析关键 在饱和客户条件(目标2)下诱导的结构中间体(也称为“预聚合态”)，以及 描述我们实验室发现的一种新的纤维形成机制，该机制可用于αB- 细胞条件下的晶状体蛋白(目标3)。结构研究将得到生物物理和生物科学的赞扬 与Kirsten Lampi教授(OHSU)合作进行的功能表征，目的是 阐明了定义α的机制原理-晶体蛋白结构、多分散性和稳定性-它们是 对避免晶体内聚集至关重要，因此也是药物设计策略未来成功的关键 旨在控制与年龄相关的白内障(以及一系列其他人类晶体蛋白疾病)。