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: 10670099
• 负责人: Megan Rocha
• 金额: $ 4.32万
• 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/10670099
• 关键词: Affect; Age; Alzheimer' s Disease; Amyloid Fibrils; Anions; Asparagine; Aspartic Acid; Biological; Blindness; Buffers; Cataract; Cataract Extraction; Cell Nucleus; Charge; Chemicals; 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; Life Cycle Stages; Link; Magic; Mass Spectrum Analysis; Metabolic; Methods; 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; Role; Site; Solubility; Structure; System; Techniques; Technology; Therapeutic; Ultraviolet Rays; Variant; Work; age related; biophysical properties; biophysical techniques; deamidation; design; dimer; disulfide bond; experimental study; 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晶状体混浊。2010年，15%的人口患上了白内障 到60岁时，接近70%的人口到80岁时。尽管这种疾病很普遍，但取代 带有合成植入物的镜片仍然是目前的护理标准。这一战略阻止了获得治疗的机会 对于没有足够医疗保健的人。白内障是由晶状体中的蛋白质聚集引起的，大多数 它由一类名为晶体蛋白的蛋白质组成。了解晶体蛋白的作用机制 凝聚体形成白内障对于开发预防白内障形成的治疗方法至关重要 去做白内障手术。晶状体成熟伴随着细胞器的丧失和蛋白质的降解。 机制；因此，眼晶状体在新陈代谢中处于不活跃状态，晶体蛋白必然非常长- 活蛋白质。对其生物物理性质的研究已经证明，晶状体蛋白具有异常的溶解性和 稳定。然而，由于数十年的破坏性辐射以及晶状体内稳态的丧失， 机制，晶体蛋白积累翻译后修饰(PTM)，如氧化和脱酰亚胺。 这些PTM被认为改变了它们的溶解性和稳定性，从而促进了与白内障相关的聚集 镜头。脱酰胺，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亚胺位点的变体已经增加 易聚集和氧化，稳定性降低。这个项目的长期目标是 研究脱酰胺是如何促进聚集的。这项研究将在3、5、7和 9个脱酰胺部位，并建立在我们实验室之前的洞察力基础上。我假设这一机制 这些变异体的聚集程度将取决于脱酰胺的程度。在具有更少的位置的变体中 脱酰胺，我预测聚集是由增加的阴离子-P相互作用形成的。在高度去胺化的变种中，我 建议聚集增加蛋白质动力学改变后的疏水性暴露。在这里，我将调查 这些可能性与溶液状态核磁共振和质谱学动力学实验。这些将是 辅以生物物理技术，量化聚集、二聚化和氧化的程度。我 然后将用固体核磁共振观察脱酰胺化不同聚集体的结构。最后，初步证据显示 我们的镜头表明，HGS可能在晶状体中扮演着一个新的积极角色，作为最后的氧化还原缓冲区。这就做 用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 角色。