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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目概要白内障是一种晶状体混浊，可导致失明。2010 年，白内障影响了 15% 的人口。到 60 岁，近 70% 的人口到 80 岁。尽管这种疾病普遍存在，但手术替代带有合成植入物的晶状体仍然是当前的护理标准，这种策略阻碍了治疗的获得。对于没有足够医疗保健的人来说，大多数白内障是由晶状体中的蛋白质聚集引起的。它由一类称为晶状体蛋白的蛋白质组成，了解晶状体蛋白的机制。聚集形成白内障对于开发可预防白内障形成的替代疗法至关重要白内障手术中晶状体的成熟伴随着细胞器的损失和蛋白质的降解。机制；因此，眼睛晶状体代谢不活跃，晶状体蛋白必然非常长。对活体蛋白质的生物物理特性的研究证明，晶状体蛋白具有异常可溶性和稳定性。然而，由于遭受数十年的破坏性辐射以及晶状体稳态的丧失。机制中，晶状体蛋白积累翻译后修饰 (PTM)，例如氧化和脱酰胺。这些 PTM 被认为可以改变其溶解度和稳定性，促进白内障相关的聚集脱酰胺作用，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有所增加。该项目的长期目标是降低对聚集和氧化的敏感性以及稳定性。研究脱酰胺如何促进聚集。这项研究将针对具有 3、5、7 和的变体进行。 9 个脱酰胺位点，建立在我们实验室之前开发的见解的基础上。这些变体的聚集程度取决于脱酰胺位点较少的变体的程度。脱酰胺，我预测聚集是通过增加阴离子-p 相互作用形成的。聚集会增加蛋白质动力学改变带来的疏水性暴露。这些可能性将通过溶液态核磁共振和质谱动力学实验来实现。与量化聚集、二聚和氧化程度的生物物理技术相辅相成。然后将用固态核磁共振观察脱酰胺变体聚集体的结构。最后，初步证据。我们的镜头表明 HgS 可以作为最后的氧化还原缓冲剂在镜头中发挥新颖的积极作用。通过监测与此相关的二硫键的蛋白质组学实验来研究这一点角色。