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Structural characterization of interacting and aggregating cataract-associated crystallins

白内障相关晶状体蛋白相互作用和聚集的结构表征

基本信息

批准号：
10463640
负责人：
ANGELA M. GRONENBORN
金额：
$ 32.51万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-30 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10463640
关键词：
Address Adult Affect Aging American Behavior Biochemical Biophysics Blindness Cataract Cells Chemicals Complex Crystalline Lens Crystallins Crystallization DNA Sequence Alteration Data Degenerative Disorder Deposition Development Diffusion Disease Disease Progression Electron Microscopy Environment Exhibits Eye Gene Family Gene Mutation Genes Heterogeneity Human Individual Knowledge Lens Opacities Light Liquid substance Magic Maps Measures Medical Membrane Lipids Methodology Methods Modification Molecular Molecular Chaperones Molecular Sieve Chromatography Molecular Structure Mutation NMR Spectroscopy Nuclear Magnetic Resonance Operative Surgical Procedures Organelles Pathologic Persons Phase Physiological Population Post-Translational Protein Processing Process Proteins Refractive Indices Reporting Research Resolution Roentgen Rays S-crystallin Sampling Science Series Solubility Structure Structure-Activity Relationship Surface Surgical complication System Testing Variant Visual impairment Work age effect age related aged alpha-Crystallins biophysical analysis biophysical properties congenital cataract deamidation early onset experimental study fiber cell glycation insight intermolecular interaction lens lens transparency light scattering mutant novel novel therapeutic intervention novel therapeutics oxidation protein aggregation protein protein interaction protein structure solid state solid state nuclear magnetic resonance

项目摘要

Project Summary Cataracts are the leading cause of blindness in the world, with approximately 22 million cases per year. The disease is caused by protein aggregation in the eye lens, involving its major constituents, the crystallins. Currently, the only available treatment is surgery, widely used in the developed world. However, access to surgery is not available to a significant fraction of the world population, and, as with any surgery, complications may ensue. Therefore, it is important to provide a structural understanding of cataract formation, if novel therapeutic approaches are to be developed for delaying the onset or slowing the progression of cataracts. While the congenital form of the disease has been mapped to crystallin gene mutations, the age-related degenerative disease is believed to involve chemically-modified crystallin proteins. Previously, we investigated the dynamics, structure and folding of human gD-crystallin mutants that are associated with congenital cataracts. We solved NMR and X-ray crystal structures of several variants and analyzed their dynamic behavior by solution NMR spectroscopy. Our aim now is to elucidate the interactions between different crystallins, under physiologically- relevant high concentrations. We will investigate proteins with modifications that mimic aging and using congenital cataract-associated mutants. We hypothesize that surface changes that impact the liquid phase behavior of the crystallin and/or generate aberrant protein-protein interactions contribute to aggregation. Our studies will not only provide insight into the process of cataract formation but also will shed light on fundamental questions in protein science. Although biochemical and biophysical studies have provided a detailed picture of individual crystallin structures and stability, extensive studies are needed to assess the interplay between different crystallin proteins and, thereby, provide critical data on crystallin structure/function relationships. For example, the interactions that permit high protein concentrations in lens cells and questions about which, why, and how certain crystallins interact without aggregation in the normal lens require direct experimental studies to gain new insights. In addition, several post-translational modifications have been reported to occur upon lens aging, impacting lens transparency. Whether and how such "aged" crystallins contribute to protein stability and aggregation is unknown. The proposed research will address these outstanding issues through biophysical analyses of wild-type, disease-associated, and chemically-modified, “aged”, crystallin variants. Structural studies by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy, small angle x-ray scattering (SAXS), and electron microscopy methods will be used to directly investigate different crystallin mixtures to obtain novel insights into the behavior of normal assemblies involving lens-opacity associated proteins. Structural details of such assemblies cannot be obtained by any other methodologies.

项目概要白内障是世界上导致失明的主要原因，每年约有 2200 万例白内障。这该疾病是由眼睛晶状体中的蛋白质聚集引起的，涉及其主要成分晶状体蛋白。目前，唯一可用的治疗方法是手术，在发达国家广泛使用。然而，访问世界上很大一部分人口无法接受手术，并且与任何手术一样，都会出现并发症可能会发生。因此，如果新颖的话，提供对白内障形成的结构理解非常重要将开发延迟白内障发生或减缓白内障进展的治疗方法。尽管该疾病的先天性形式已被定位为晶状体蛋白基因突变，即与年龄相关的退行性病变据信疾病与化学修饰的晶状体蛋白有关。之前，我们研究了动态，与先天性白内障相关的人类 gD-晶状体蛋白突变体的结构和折叠。我们解决了几种变体的 NMR 和 X 射线晶体结构，并通过溶液 NMR 分析了它们的动态行为光谱学。我们现在的目标是在生理学条件下阐明不同晶状体蛋白之间的相互作用：相关的高浓度。我们将研究经过修饰来模拟衰老的蛋白质，并使用先天性白内障相关突变体。我们假设影响液相的表面变化晶状体蛋白的行为和/或产生异常的蛋白质-蛋白质相互作用有助于聚集。我们的研究不仅将深入了解白内障形成的过程，还将揭示蛋白质科学的基本问题。尽管生物化学和生物物理研究已经提供了详细的单个晶体蛋白结构和稳定性的图片，需要进行广泛的研究来评估相互作用不同晶状体蛋白之间的差异，从而提供有关晶状体蛋白结构/功能的关键数据关系。例如，允许晶状体细胞中蛋白质浓度高的相互作用和问题关于某些晶状体蛋白在正常晶状体中不聚集的情况下相互作用的原因、原因以及如何相互作用，需要直接实验研究以获得新的见解。此外，还进行了一些翻译后修饰据报道，镜片老化时会发生这种情况，影响镜片透明度。晶状体蛋白是否以及如何“老化” 对蛋白质稳定性和聚集的贡献尚不清楚。拟议的研究将解决这些突出的问题通过对野生型、疾病相关的和化学修饰的、“老化的”、晶状体蛋白进行生物物理分析来解决问题变种。通过溶液和固态核磁共振 (NMR) 光谱进行结构研究，小型角X射线散射（SAXS）和电子显微镜方法将用于直接研究不同的晶状体蛋白混合物以获得对涉及晶状体混浊的正常组装行为的新见解相关蛋白质。此类组件的结构细节无法通过任何其他方法获得。