Conformation and Dynamics of Cataract Mutants of human gammaD crystallin

人γD晶状体蛋白白内障突变体的构象和动力学

• 批准号: 8209151
• 负责人: ANGELA M. GRONENBORN
• 金额: $ 35.05万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8209151
• 关键词: Address; Aging; Animal Model; Behavior; Biological Assay; Biology; Blindness; Cataract; Cell Nucleus; Chemicals; Clinical; Country; Crystallins; Crystallization; Crystallography; Deamination; Defect; Degenerative Disorder; Deposition; Development; Disease; Equilibrium; Eye; Family; Federal Government; Frustration; Future; Gene Mutation; Genes; Goals; Heat shock proteins; Histidine; Human; Impairment; Intervention; Kinetics; Knowledge; Maps; Mass Spectrum Analysis; Measures; Methionine; Methodology; Methods; Modeling; Modification; Molecular; Molecular Biology; Molecular Chaperones; Molecular Conformation; Mus; Mutagenesis; Mutation; NMR Spectroscopy; Nerve Degeneration; Operative Surgical Procedures; Persons; Population; Precipitation; Process; Property; Proteins; Refractory; Relaxation; Research; Resources; Roentgen Rays; Screening procedure; Seeds; Societies; Solutions; Structural Protein; Structure; Structure-Activity Relationship; Thermodynamics; United States; Urea; Variant; Vision; Work; age related; aged; alternative treatment; base; chemical synthesis; congenital cataract; follow-up; inhibitor/antagonist; insight; lens; lens transparency; light scattering; mouse model; mutant; novel; oxidation; polypeptide; protein aggregate; protein aggregation; protein degradation; protein expression; research study; small molecule; small molecule libraries; treatment strategy

Project Summary Cataract is a protein aggregation disease caused by crystallin protein defects in the lens. The congenital form of the disease results from crystallin gene mutations, whereas the age-related degenerative disease results after chemical modification of crystallin proteins. Cataracts are the leading cause of blindness in the world, with approximately 17 million cases per year. Currently, the only available treatment is surgery, which has proven successful. However, a significant fraction of the world population can not access surgery, and, in many cases, problems occur after surgery. Thus, a basic understanding of cataract formation is important to develop novel therapies that delay onset or slow progression. We will investigate the dynamics, structure and folding of cataract-associated ¿D-crystallin mutants. Our aim is to elucidate the structural basis for cataract formation. We hypothesize that not random association of proteins, but specific folding intermediates are involved in aggregation. In addition to providing insight into the process of cataract formation, our studies will explore fundamental questions in protein biology. For example, the interactions that cause frustration of folding, questions about why and how intermediates are stabilized, and the processes that cause a polypeptide chain to misfold and/or aggregate rather than fold into the native state require direct experimental studies to gain new insights. The proposed research will address such outstanding issues through biophysical analyses of wild- type and disease-associated crystallin variants. Crystallins are ideally suited for detailed studies of protein aggregation: they are small; numerous X-ray structures are available; and the folding kinetics for several wild- type proteins to the native state have been investigated. NMR methods will be used to directly investigate folding transitions to obtain novel insights into the energetics of these processes and to elucidate structural details of the intermediates that cannot be obtained by any other methodologies. Our work will involve methods that allow detailed structural and dynamics characterization of proteins, primarily NMR spectroscopy and small angle X-ray scattering. In addition, we will correlate basic biophysical parameters with clinical observations. We plan to determine the three dimensional solution structures of cataract associated human ¿D-crystallins and characterize their dynamic behavior. We will initially focus on two important cataract forming ¿D-crystallin mutants, P23T and V75D. The former is associated with congenital cataracts in humans and the latter is a variant that has been identified to cause cataract in mice and, thus, will lend itself to follow-up studies in an animal model of cataract. We will also characterize the structure and dynamics of ¿D-crystallin folding intermediates. Further, we will investigate whether and how a previously identified, partially folded ¿D-crystallin intermediate causes aggregation. In particular, we will establish whether such partially folded intermediates are seeds for aggregation. This will prepare the basis for discovering small molecule inhibitors of aggregation, an approach that has already yielded some results in a number of neurodegenerative protein deposition diseases.

项目摘要 白内障是由透镜中的晶状体蛋白缺陷引起的蛋白聚集性疾病。的 这种疾病的先天性形式是由晶体蛋白基因突变引起的，而与年龄相关的退行性疾病是由晶体蛋白基因突变引起的。 晶状体蛋白的化学修饰后导致疾病。白内障是导致失明的主要原因 每年大约有1700万例。目前，唯一可用的治疗方法是手术， 这已经证明是成功的。然而，世界人口中有很大一部分无法接受手术， 并且在许多情况下，问题发生在手术之后。因此，对白内障形成的基本理解是 重要的是开发新的治疗方法，延迟发病或减缓进展。我们将调查其动态， 白内障相关的D-晶状体蛋白突变体的结构和折叠。我们的目的是阐明 白内障的形成。我们假设蛋白质不是随机结合，而是特异性折叠 中间体参与聚集。 除了深入了解白内障形成的过程，我们的研究还将探讨 蛋白质生物学的基本问题。例如，导致折叠失败的相互作用， 关于中间体为什么和如何稳定的问题，以及引起多肽链的过程 错误折叠和/或聚集而不是折叠成天然状态需要直接的实验研究以获得新的 见解.拟议的研究将通过对野生动物的生物物理分析来解决这些悬而未决的问题， 类型和疾病相关晶体蛋白变体。晶体蛋白非常适合于蛋白质的详细研究 聚集：它们很小;许多X射线结构是可用的;和几个野生型的折叠动力学。 型蛋白质的天然状态进行了研究。NMR方法将用于直接研究 折叠转变以获得对这些过程能量学的新见解并阐明结构 无法通过任何其他方法获得的中间体的详细信息。 我们的工作将涉及允许详细的蛋白质结构和动力学表征的方法， 主要是NMR光谱和小角X射线散射。此外，我们将把基本的生物物理学 参数与临床观察。我们计划确定的三维解决方案的结构， 白内障与人类D-晶状体蛋白相关，并表征其动态行为。我们将首先关注两个 形成D-晶状体蛋白突变体P23 T和V75 D的重要白内障。前者与先天性 白内障，后者是一种变异体，已被鉴定为引起小鼠白内障，因此， 适用于白内障动物模型的后续研究。我们还将描述结构和 D-晶体蛋白折叠中间体的动力学。此外，我们将调查是否以及如何以前 鉴定的、部分折叠的D-晶状体蛋白中间体引起聚集。特别是，我们将确定是否 这种部分折叠的中间体是用于聚集的种子。这将为发现小 聚集的分子抑制剂，这种方法已经在许多领域取得了一些成果， 神经退行性蛋白质沉积疾病。