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

项目摘要 ⍺-晶体蛋白(⍺A-和⍺B-亚型)对于眼睛的发育和终生透明是必不可少的 镜头。作为分子伴侣-小热休克蛋白家族的成员-⍺-晶体蛋白发挥着 晶状体蛋白稳定和防止与年龄有关的混浊(即蛋白质)的形成的组成部分 凝聚体)导致白内障的形成。但是，该系统可能会变得不稳定和/或 由于晶状体蛋白质的长寿特性而不堪重负，最终导致疾病 进步。尽管在晶状体透明度和疾病方面有这些基本作用，但在以下方面仍然存在严重差距 我们对⍺-晶体蛋白功能的分子机制的理解，以及这些蛋白质是如何 对晶状体中异常(或年龄相关)条件做出反应的聚合。⍺的一个标志性特征--晶体蛋白 是由涉及柔性N-和的子域相互作用驱动的结构塑性的显著程度 C-末端，一种被认为在高度集中的环境下防止结晶的特征 眼镜片。这种分子的可塑性也有助于⍺-晶体蛋白伴侣功能，使其能够适应和 隔离一系列不稳定的蛋白质(又名客户)，防止细胞毒性聚集。然而， 这些内在动力学的相同特征阻碍了先前获得详细结构的努力 提供对这些关键功能的机械性洞察所需的表征(原子细节) 眼镜片的组件。这项提案的目标将利用最新的最先进技术 单粒子冷冻电子断层扫描(CryoET)方法--结合生物物理和功能 研究-为了获得关于⍺机制的高分辨率结构信息-晶状体蛋白 结构可塑性/多分散性(目标1)。对监护人/委托人共同参与的途径(S)的其他见解 聚集(目标2)将通过近扫描电子显微镜、光散射和交联-MS/MS方法获得。取得的成功 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⍺-晶体蛋白结构可塑性标志的分子基础的力学见解以及晶状体如何 伴侣系统变得不堪重负，导致细胞毒性蛋白聚集体，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.