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

• 批准号: 10018021
• 负责人: ANGELA M. GRONENBORN
• 金额: $ 33.51万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10018021
• 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

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.

项目摘要 白内障是世界失明的领导，每年大约有2200万个Cass 疾病是由眼镜中的蛋白质聚集引起的，其主要征菌素是晶体蛋白。 目前，唯一可用的治疗方法是在发达国家中广泛使用的手术 手术不适合大部分世界人口，并且与任何手术一样，并发症 因此，可能会提供对白内障形成的结构理解 治疗方法是 该疾病的先天性形式已映射到结晶蛋白基因突变，即与年龄相关的退行性 疾病曾涉及化学改性的晶体蛋白。 我们解决的与先天性白内障相关的人GD-晶状体突变体的结构和折叠。 NMR和X射线晶体结构的严重程度变体，并通过溶液NMR分析了它们的动态行为 光谱法。 相关的高浓度。 先天性白内障相关突变体。 行为 我们的研究将无法提供到白内障形成过程中 蛋白质科学中的基本问题。 单个结晶蛋白结构和稳定性的图片，需要大量研究来评估解释 在不同的结晶蛋白之间，从而提供有关晶体结构/功能的关键数据 关系。 在正常晶状体中，在没有聚集的情况下相互作用的哪种，原因以及如何直接相互作用 实验研究以获得新的新见解。 据报道发生在晶状体衰老时，影响镜片透明度。 有助于蛋白质稳定性和聚集是未知的。 野生型，疾病相关和化学改性的“老化”，结晶蛋白的生物物理分析的问题 溶液和固态核磁共振（NMR）的结构研究。 角度X射线散射（SAX）和电子显微镜方法将用于直接研究不同的不同 结晶蛋白的混合物以获得对行为的新见解。 相关的蛋白质。