Structural characterization of interacting and aggregating cataract-associated crystallins

项目摘要 白内障是世界上主要的致盲原因，每年约有2200万例。这个 疾病是由眼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。但是，访问 世界上有很大一部分人口不能接受手术，而且，和任何手术一样，手术也有并发症 可能会接踵而至。因此，如果是新的，提供对白内障形成的结构性理解是重要的。 将开发治疗方法来延缓或减缓白内障的发病或进展。而当 这种先天性疾病被认为与晶体蛋白基因突变有关，这是一种与年龄相关的退行性疾病 这种疾病被认为与化学修饰的晶体蛋白有关。在此之前，我们研究了动力学， 与先天性白内障相关的人类GD-晶体蛋白突变体的结构和折叠。我们解决了 几种变种的核磁共振和X射线晶体结构及溶液核磁共振动态行为分析 光谱学。我们现在的目标是阐明不同晶体蛋白之间的相互作用，在生理- 相关的高浓度。我们将研究具有模拟衰老和使用的修饰的蛋白质 先天性白内障相关突变。我们假设表面的变化会影响液体相 晶体蛋白的行为和/或产生异常的蛋白质-蛋白质相互作用有助于聚集。 我们的研究不仅将提供对白内障形成过程的洞察，而且将有助于 蛋白质科学中的基本问题。尽管生化和生物物理研究提供了详细的 单个晶体蛋白结构和稳定性的图片，需要广泛的研究来评估相互作用 不同晶体蛋白之间的关系，从而提供关于晶体蛋白结构/功能的关键数据 两性关系。例如，允许晶状体细胞和问题中的高蛋白浓度的相互作用 关于哪些、为什么以及某些晶体蛋白如何在正常晶状体中无聚集地相互作用需要直接 实验研究，以获得新的见解。此外，还进行了几个翻译后的修改 据报道，发生在晶状体老化，影响晶状体透明度。这种“老化”晶体蛋白是否以及如何“老化” 对蛋白质稳定性和聚集性的贡献尚不清楚。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、老化的结晶进行生物物理分析的问题 变种。用溶液和固体核磁共振波谱进行结构研究，小 角X射线散射(SAXS)和电子显微镜方法将被用来直接研究不同 晶体蛋白混合物，以获得对涉及晶状体混浊的正常组件行为的新见解 相关蛋白质。此类程序集的结构细节无法通过任何其他方法获得。

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.