Exploring Crystallin Deamidation as a Causative Agent of Cataracts

探索晶状体蛋白脱酰胺作用作为白内障的致病因素

• 批准号: 10538422
• 负责人: Megan Rocha
• 金额: $ 4.22万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10538422
• 关键词: Affect; Age; Alzheimer' s Disease; Amyloid Fibrils; Anions; Asparagine; Aspartic Acid; Biological; Blindness; Buffers; Cataract; Cataract Extraction; Cell Nucleus; Charge; Crystalline Lens; Crystallins; Cysteine; Deposition; Development; Dimerization; Disease; Disulfide Linkage; Disulfides; Exposure to; Glutamic Acid; Glutamine; Goals; Health Services Accessibility; Healthcare; Homeostasis; Human; Huntington Disease; Hydrophobicity; Implant; Investigation; Knowledge; Lead; Life Cycle Stages; Link; Magic; Mass Spectrum Analysis; Metabolic; Methods; Modification; Monitor; Operative Surgical Procedures; Organelles; Oxidation-Reduction; Pattern; Persons; Play; Population; Post-Translational Protein Processing; Predisposition; Prevalence; Protein Disulfide Isomerase; Protein Dynamics; Proteins; Proteomics; Radiation induced damage; Regulation; Research; Resolution; Resort; Role; S-crystallin; Site; Solubility; Structure; System; Techniques; Technology; Therapeutic; Translations; Ultraviolet Rays; Variant; Work; age related; alpha-Crystallins; biophysical properties; biophysical techniques; chemical function; deamidation; design; dimer; disulfide bond; experimental study; gamma-Crystallins; inhibitor; innovation; insight; lens; lens cortex; novel; oxidation; prevent; protein aggregation; protein degradation; solid state nuclear magnetic resonance; standard of care; stem

Project Summary Cataract is an opacity of the eye lens that can result in blindness. In 2010, cataract affected 15% of the population by age 60 and nearly 70% of the population by age 80. Despite the disease’s prevalence, surgery that replaces the lens with a synthetic implant remains the current standard of care. This strategy prevents access to treatment for people without adequate healthcare. Cataract is caused by protein aggregation in the lens, the majority of which is comprised of a class of proteins called crystallins. Understanding the mechanisms by which crystallins aggregate to form cataract is critical for developing therapeutics that can prevent their formation as an alternative to cataract surgery. Maturation of the lens is accompanied by loss of organelles and protein degradation mechanisms; therefore, the eye lens is metabolically inactive and crystallins by necessity are extremely long- lived proteins. Research on their biophysical properties have proven that crystallins are unusually soluble and stable. However, due to subjection to decades of damaging radiation as well as the loss of lens homeostasis mechanisms, crystallins accumulate post-translational modifications (PTMs) such as oxidation and deamidation. These PTMs are thought to alter their solubility and stability, thereby promoting cataract-related aggregation in the lens. Deamidation, the conversion of asparagine or glutamine to aspartic or glutamic acid, respectively, is the most common PTM. It has been shown that variants with one or two deamidation sites have increased susceptibility to aggregation and oxidation as well as decreased stability. The long-term goal of this project is investigate how deamidation promotes aggregation. This research will be performed on variants with 3, 5, 7, and 9 sites of deamidation and builds on previous insights developed by our lab. I hypothesize that the mechanism of aggregation of these variants will depend on the extent of deamidation. In variants with fewer sites of deamidation, I predict aggregation is formed by increased anion-p interactions. In highly deamidated variants, I propose aggregation increases hydrophobic exposure from altered protein dynamics. Here, I will investigate these possibilities with solution-state NMR and mass spectrometry dynamics experiments. These will be complimented with biophysical techniques that quantify the extent of aggregation, dimerization, and oxidation. I will then look structure of deamidation variant aggregates with solid-state NMR. Finally, preliminary evidence in our lens suggests the HgS could be performing a novel active role in the lens as a last resort redox buffer. I will investigate this with proteomics experiments that monitor the disulfide linkages that have been implicated in this role.

项目摘要 白内障是一种可导致失明的眼透镜不透明现象。2010年，白内障影响了15%的人口 到60岁，近70%的人口到80岁。尽管这种疾病的流行，手术，取代 具有合成植入物的透镜仍然是当前的护理标准。这一战略阻碍了获得治疗 为没有足够医疗保健的人提供服务。白内障是由蛋白质聚集在透镜中引起的，大多数蛋白质聚集在晶状体中。 它由一类叫做晶体蛋白的蛋白质组成。了解晶体蛋白 聚集形成白内障对于开发可以防止其形成的替代疗法至关重要 白内障手术透镜的成熟伴随着细胞器的丧失和蛋白质的降解 机制;因此，眼睛透镜是代谢不活跃的，晶体蛋白必然是非常长的- 活蛋白质对它们的生物物理性质的研究已经证明，晶体蛋白是异常可溶的， 稳定然而，由于遭受数十年的破坏性辐射以及透镜体内平衡的丧失， 由于晶体蛋白的作用机制，晶体蛋白积累翻译后修饰（PTM），如氧化和脱酰胺。 这些PTM被认为改变了它们的溶解度和稳定性，从而促进了白内障相关的聚集。 透镜。脱酰胺，即天冬酰胺或谷氨酰胺分别转化为天冬氨酸或谷氨酸， 最常见的PTM已经表明，具有一个或两个脱酰胺位点的变体增加了 对聚集和氧化的敏感性以及降低的稳定性。该项目的长期目标是 研究脱酰胺如何促进聚集。这项研究将在具有3、5、7和 9个脱酰胺位点，并建立在我们实验室先前开发的见解基础上。我假设这个机制 这些变体聚集的程度将取决于脱酰胺的程度。在具有较少位点的变体中， 脱酰胺，我预测聚集是由增加阴离子-p相互作用形成的。在高度脱酰胺的变体中， 提出聚集增加来自改变的蛋白质动力学的疏水暴露。在这里，我将调查 这些可能性与溶液状态核磁共振和质谱动力学实验。这些将是 与量化聚集、二聚化和氧化程度的生物物理技术互补。我 然后将用固态NMR观察脱酰胺变体聚集体的结构。最后，初步证据显示， 我们的透镜表明HgS可以在透镜中作为最后手段的氧化还原缓冲剂发挥新的活性作用。我会 通过蛋白质组学实验来研究这一点，这些实验监测了与此有关的二硫键， 作用