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

晶状体α-晶状体的结构可塑性、客户相互作用和共聚集机制

• 批准号: 10709482
• 负责人: Adam Phillip Miller
• 金额: $ 3.95万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10709482
• 关键词: Alzheimer' s Disease; Binding; Biophysics; Blindness; C-terminal; Cataract; Chemicals; Client; Complex; Coupled; Cryo-electron tomography; Cryoelectron Microscopy; Crystalline Lens; Crystallins; Crystallization; Development; Disease; Disease Progression; Environment; Event; Evolution; Functional disorder; Goals; Health; Heat shock proteins; Heterogeneity; Knowledge; Length; Lens Diseases; Methods; Modeling; Modification; Molecular; Molecular Chaperones; Muramidase; N-terminal; Nature; Parkinson Disease; Pathway interactions; Play; Population; Protein Family; Protein Isoforms; Proteins; Research; Resolution; Role; Solubility; Structure; System; Technology; Therapeutic; Time; Vision; Visualization; Work; age related; aggregation pathway; alpha-Crystallins; biophysical techniques; crosslink; cytotoxic; experimental study; flexibility; insight; lens; lens transparency; light scattering; member; non-Native; novel strategies; particle; prevent; protein aggregation; protein folding; proteostasis; rational design; response; structural biology; structural determinants; success; Alzheimer' s Disease; Binding; Biophysics; Blindness; C-terminal; Cataract; Chemicals; Client; Complex; Coupled; Cryo-electron tomography; Cryoelectron Microscopy; Crystalline Lens; Crystallins; Crystallization; Development; Disease; Disease Progression; Environment; Event; Evolution; Functional disorder; Goals; Health; Heat shock proteins; Heterogeneity; Knowledge; Length; Lens Diseases; Methods; Modeling; Modification; Molecular; Molecular Chaperones; Muramidase; N-terminal; Nature; Parkinson Disease; Pathway interactions; Play; Population; Protein Family; Protein Isoforms; Proteins; Research; Resolution; Role; Solubility; Structure; System; Technology; Therapeutic; Time; Vision; Visualization; Work; age related; aggregation pathway; alpha-Crystallins; biophysical techniques; crosslink; cytotoxic; experimental study; flexibility; insight; lens; lens transparency; light scattering; member; non-Native; novel strategies; particle; prevent; protein aggregation; protein folding; proteostasis; rational design; response; structural biology; structural determinants; success

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.

项目摘要 ⍺-晶状体（⍺a-和⍺b-同工型）对于眼睛的发育和终身透明度至关重要 镜片。作为分子伴侣 - 小热冲击蛋白家族的成员 - ⍺晶状蛋白会发挥 晶状体蛋白抑制剂稳定性的整体部分，并防止与年龄相关的OCACITITS的形成（即蛋白质 聚集体）负责白内障形成。但是，该系统可能变得不稳定和/或 由于晶状体蛋白的长期性质而淹没，并最终导致疾病 进展。尽管在晶状体透明度和疾病中具有这些基本作用，但仍存在关键的差距 我们对⍺-晶状蛋白起作用的分子机制的理解，以及这些蛋白质如何 ⍺-Crystallins的标志性功能 是一个显着程度的结构可塑性，由亚域相互作用驱动，涉及柔性n-和 C-termini，该功能被认为可以防止在高度集中环境下结晶 眼镜镜头。该分子可塑性也有助于⍺-晶状蛋白链烯唯一功能，从而使其适应和适应 隔离一系列不稳定的蛋白质（又称客户端），并防止细胞毒性聚集。然而， 这些内在动力学的相同特征已经阻碍了以前的努力来获得详细的结构 为这些关键的功能提供机械见解所需的字符（原子细节） 眼镜的组成部分。该提案的目的将利用最新的最新进展 单个粒子冷冻冷冻电子层析成像（冷冻）的方法 - 与生物物理和功能 研究 - 为了获得有关⍺-晶状体机制的高分辨率结构信息 结构可塑性/多分散性（AIM 1）。对伴侣/客户端共同的途径的其他见解 聚集（AIM 2）将从NSEM，光散射和交联MS/MS方法中获得。成功 这些目标将填补数十年来逃避该领域并提供详细信息的关键空白 对⍺-晶状体的结构可塑性标志的分子基础的机械洞察力以及镜头的方式 伴侣系统变得不知所措，导致细胞毒性蛋白聚集体，例如在 白内障。