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: 8391719
• 负责人: ANGELA M. GRONENBORN
• 金额: $ 33.22万
• 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/8391719
• 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; 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; screening; 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 白内障是由晶状体中的结晶蛋白蛋白缺损引起的蛋白质聚集疾病。这 该疾病的先天性形式是由结晶蛋白基因突变引起的，而与年龄相关的退行性 晶体蛋白化学修饰后的疾病结果。白内障是失明的主要原因 在世界上，每年约有1700万个案件。目前，唯一可用的治疗方法是手术， 事实证明了成功。但是，世界人口中很大一部分无法接受手术， 而且，在许多情况下，手术后出现问题。那是对白内障形成的基本理解是 开发延迟发作或缓慢进展的新型疗法很重要。我们将研究动态， 白内障相关的结构和折叠»d-晶格蛋白突变体。我们的目的是阐明结构基础 为白内障形成。我们假设不是蛋白质的随机关联，而是特定的折叠 中间体参与聚合。 除了洞悉白内障形成过程外，我们的研究还将探索 蛋白质生物学中的基本问题。例如，引起折叠挫败感的相互作用， 有关中间体的稳定以及导致多肽链的过程的问题 要折叠和/或骨料而不是折叠到本地状态需要直接的实验研究才能获得新的 见解。拟议的研究将通过野生的生物物理分析来解决此类杰出问题 类型和疾病相关的晶体变体。结晶蛋白非常适合详细研究蛋白质 聚合：它们很小；有许多X射线结构可用；以及几种野生的折叠动力学 已经研究了原生状态的类型蛋白质。 NMR方法将用于直接调查 折叠过渡以获得对这些过程的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蛋白质的详细结构和动态表征的方法， 初级NMR光谱和小角度X射线散射。此外，我们将相关 具有临床观察的参数。我们计划确定三维解决方案结构 白内障相关的人»d-crystallins并表征其动态行为。我们最初将专注于两个 重要的白内障形成�D-晶状蛋白突变体，P23T和V75D。前者与先天性有关 人类的白内障和后者是一种已鉴定出可引起小鼠白内障的变体，因此会导致白内障 在白内障动物模型中进行后续研究。我们还将表征结构和 »d-rystallin折叠中间体的动力学。此外，我们将调查是否以及如何以前 鉴定出的，部分折叠�D-晶状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聚集的种子。这将为发现小的基础准备 聚集的分子抑制剂，这种方法已经产生了许多结果 神经退行性蛋白沉积疾病。