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

项目概要 导致与年龄相关的白内障的光散射混浊是光散射聚集和沉淀的结果。 晶状体蛋白（α、β 和 γ-晶状体蛋白）。 α-晶状体蛋白（αA 和 αB 亚型）以多分散形式组装 寡聚复合物并作为不依赖于 ATP 的分子伴侣（即蛋白质保持酶）发挥作用。两者都 这些特性被认为可以防止聚集事件破坏微妙的蛋白质稳态 镜头。众所周知，在我们的一生中会产生环境压力和化学变化 破坏晶状体晶状体蛋白的稳定性，并诱导复杂形式的蛋白质-蛋白质相互作用，从而导致 聚集（无定形和潜在原纤维）。然而，理解聚合的一个主要障碍 与白内障相关的途径一直缺乏主要晶状体α-晶状体蛋白的结构信息。 这种知识差距是由于缺乏有效的方法来表征固有的多分散性 α-晶状体蛋白的结构、伴侣-客户聚集体形成的异质性以及原纤维的逃避性 在生理条件下鉴定的聚集状态。在本提案中，我们描述了我们的多学科 基于团队的方法，以 PI 在单粒子冷冻电镜支持技术方面的专业知识为中心， 这最终将使我们能够探究 α-晶状体蛋白分子可塑性的基础。具体来说，我们的目标是定义 α-晶状体蛋白在其内在多分散状态下的高分辨率结构（目标 1），解析关键 在饱和客户条件下诱导的结构中间体（又名“预聚集状态”）（目标 2），以及 描述了我们实验室发现的一种新的原纤维形成机制，该机制可通过 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学和 与 Kirsten Lampi 教授（OHSU）合作进行功能表征，目的是 阐明定义 α-晶状蛋白结构、多分散性和稳定性的机制原理 - 这些原理是 对于避免晶状体中的聚集至关重要，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状体蛋白疾病）。

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).