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: 8021928
• 负责人: ANGELA M. GRONENBORN
• 金额: $ 35.13万
• 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/8021928
• 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

DESCRIPTION (provided by applicant): 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 cannot 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 3D-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 3D-crystallins and characterize their dynamic behavior. We will initially focus on two important cataract forming 3D-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 3D-crystallin folding intermediates. Further, we will investigate whether and how a previously identified, partially folded 3D-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. PUBLIC HEALTH RELEVANCE: Cataracts are the leading cause of blindness, with approximately 17 million cases worldwide per year. At present, the only available treatment is surgery; however, a significant fraction of the population in the US and elsewhere is unable to access surgery for various reasons. Understanding the mechanisms of cataract formation will open the way for the development of new therapies that delay onset or slow progression.

描述（由申请人提供）：白内障是由晶状体蛋白缺损引起的蛋白质聚集疾病。该疾病的先天性形式是由结晶蛋白基因突变引起的，而化学修饰结晶蛋白蛋白后与年龄相关的退行性疾病。白内障是世界失明的主要原因，每年约有1700万例病例。目前，唯一可用的治疗方法是手术，事实证明是成功的。但是，世界人口中很大一部分无法接受手术，在许多情况下，手术后出现问题。因此，对白内障形成的基本理解对于开发延迟发作或缓慢进展的新型疗法很重要。我们将研究与白内障相关的3D-晶格蛋白突变体的动力学，结构和折叠。我们的目的是阐明白内障形成的结构基础。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集。 除了提供有关白内障形成过程的见解，我们的研究还将探讨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，有关中间体的稳定原因和如何稳定的问题，以及导致多肽链错误折叠和/或聚集的过程，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。拟议的研究将通过对野生型和疾病相关的结晶蛋白变体的生物物理分析来解决此类杰出问题。结晶蛋白非常适合详细研究蛋白质聚集：它们很小；有许多X射线结构可用；并且已经研究了几种野生型蛋白质的折叠动力学。 NMR方法将用于直接研究折叠转变，以获得对这些过程的能量学的新见解，并阐明任何其他方法无法获得的中间体的结构细节。 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，主要是NMR光谱和小角度X射线散射。此外，我们将将基本生物物理参数与临床观察相关联。我们计划确定白内障相关的人类3D-晶体的三维溶液结构，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3D-晶状蛋白突变体P23T和V75D。前者与人类的先天性白内障有关，后者是一种已鉴定出可引起小鼠白内障的变体，因此，在白内障动物模型中会自行进行后续研究。我们还将表征3D-晶状折叠中间体的结构和动力学。此外，我们将研究是否以及如何以及如何部分鉴定，部分折叠的3D-晶状蛋白中间体导致聚集。特别是，我们将确定这种部分折叠的中间体是否是聚集的种子。这将为发现小分子抑制剂的基础准备基础，这种方法已经在许多神经退行性蛋白沉积疾病中产生了一些结果。 公共卫生相关性：白内障是失明的主要原因，全球约有1700万例病例。目前，唯一可用的治疗方法是手术。但是，由于各种原因，美国和其他地方的人口中很大一部分无法接受手术。了解白内障形成的机制将为开发延迟发作或缓慢进展的新疗法开辟道路。