Mechanisms of structural plasticity, client interactions, and co-aggregation of the lens ⍺-crystallins

项目摘要 β-晶体蛋白（β-A-和β-B-亚型）对眼睛的发育和终身透明性至关重要 透镜。作为分子伴侣--小热休克蛋白家族的成员--β-晶体蛋白发挥着重要的作用。 是透镜蛋白质稳态稳定性和防止年龄相关混浊形成的组成部分（即，蛋白 聚集体）导致白内障形成。然而，该系统可能变得不稳定和/或 由于透镜蛋白质的长寿命性质而不堪重负，最终导致疾病 进展尽管在透镜透明度和疾病中有这些基本作用，但在晶状体的透明度和疾病方面仍然存在关键差距。 我们对β-晶体蛋白功能的分子机制的理解，以及这些蛋白质如何 响应于透镜中的异常（或与年龄相关的）条件而聚集。晶体蛋白的一个标志性特征 是一个显着程度的结构塑性，是由子域相互作用，涉及灵活的N-和 C-末端，这是一种被认为可以防止在高浓度环境下结晶的特征。 眼睛透镜。这种分子可塑性也有助于β-晶体蛋白伴侣蛋白功能，使其能够适应并 隔离各种不稳定的蛋白质（aka客户）并防止细胞毒性聚集。然而，在这方面， 内在动力学的这些相同特征阻碍了以前获得详细结构的努力， 特征（原子细节）需要提供这些关键功能的机械见解， 眼睛透镜的组成部分。该提案的目的将利用最新的最先进的 单粒子CryoEM冷冻电子断层扫描（CryoET）方法-结合生物物理和功能 研究-为了获得关于β-晶体蛋白机制的高分辨率结构信息， 结构可塑性/多分散性（目标1）。对伴侣/客户合作途径的其他见解 聚集（目标2）将从NSEM、光散射和交联-MS/MS方法获得。成功 这些目标将填补几十年来一直回避该领域的关键知识空白，并提供详细的 对晶状体蛋白结构可塑性标志的分子基础的机械见解，以及透镜 分子伴侣系统变得不堪重负，导致细胞毒性蛋白质聚集体，如在 白内障

Project Summary The ⍺-crystallins (⍺A- and ⍺B- isoforms) are essential for the development and lifelong transparency of the eye lens. As molecular chaperones – members of the small heat shock protein family – the ⍺-crystallins play an integral part of the stability of lens proteostasis and preventing the formation of age-related opacities (i.e., protein aggregates) responsible for cataract formation. However, this system may become destabilized and/or overwhelmed as a result of the long-lived nature of lens proteins, and ultimately contribute to disease progression. Despite these fundamental roles in lens transparency and disease, there remains a critical gap in our understanding of the molecular mechanisms by which the ⍺-crystallins function, and how these proteins aggregate in response to aberrant (or age-related) conditions in the lens. A hallmark feature of the ⍺-crystallins is a remarkable degree of structural plasticity that is driven by sub-domain interactions involving flexible N- and C-termini, a feature that is thought to prevent crystallization under the highly concentrated environment of the eye lens. This molecular plasticity also contributes to ⍺-crystallin chaperone function, allowing it to adapt to and sequester a diverse range of destabilized proteins (aka clients) and prevent cytotoxic aggregation. However, these same features of intrinsic dynamics have stymied previous efforts to obtain the detailed structural characterizations (atomistic details) required to provide mechanistic insights into the function of these critical components of the eye lens. The aims of this proposal will leverage recent advances in the state-of-the-art methods of single particle CryoEM cryo-electron tomography (CryoET) – coupled with biophysical and functional studies – in order to obtain high-resolution structural information regarding the mechanism of ⍺-crystallin structural plasticity/polydispersity (Aim 1). Additional insights into the pathway(s) of chaperone/client co- aggregation (Aim 2) will be garnered from NSEM, light scattering, and crosslinking-MS/MS methods. Success of these aims will fill critical gaps in knowledge that have evaded the field for decades and provide detailed mechanistic insights into the molecular basis of ⍺-crystallin’s hallmark of structural plasticity and how the lens chaperone system becomes overwhelmed leading to cytotoxic protein aggregates such as those found in cataract.