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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.

项目总结