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

项目摘要 导致年龄相关性白内障的光散射混浊是由白内障颗粒的聚集和沉淀引起的。 透镜晶体蛋白（α、β和γ-晶体蛋白）。α-晶体蛋白（αA和αB亚型）以多分散的形式组装 寡聚复合物并作为ATP非依赖性分子伴侣（即，蛋白质保持酶）。两 这些性质被认为是防止聚集事件，聚集事件会破坏 透镜。众所周知，在我们的一生中， 使透镜晶体蛋白不稳定，并诱导复杂形式的蛋白质-蛋白质相互作用， 聚集（无定形和潜在的原纤维）。然而，理解聚合的一个主要障碍 与白内障相关的途径，一直缺乏关于主要透镜α-晶状体蛋白的结构信息。 这种知识上的差距是由于缺乏有效的方法来表征固有的多分散的 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回避性 在生理条件下鉴定的聚集状态。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，围绕PI在单粒子CryoEM使能技术方面的专业知识， 这将最终使我们能够询问α-晶体蛋白分子可塑性的基础。具体来说，我们的目标是定义 高分辨率结构的α-晶体蛋白在其内在的多分散状态（目的1），解决关键 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纤维形成机制，该机制可用于αB- 晶状体蛋白在细胞条件下的作用（目的3）。结构研究将得到生物物理和 功能表征，与Kirsten Lampi教授（OHSU）合作进行，目的是 阐明了定义α-晶状体蛋白结构、多分散性和稳定性的机械原理， 对于避免在透镜中聚集至关重要，因此对于药物设计策略的未来成功至关重要 目标是控制与年龄有关的白内障（以及一系列其他人类堆叠病）。

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).