Conformation and Dynamics of Cataract Mutants of human gammaD crystallin

描述(申请人提供)：白内障是一种由晶状体蛋白缺陷引起的蛋白质聚集性疾病。先天性疾病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的化学修饰引起的。白内障是世界上主要的致盲原因，每年约有1700万例。目前，唯一可用的治疗方法是手术，事实证明手术是成功的。然而，世界上有很大一部分人口无法接受手术，而且在许多情况下，手术后会出现问题。因此，对白内障形成的基本了解对于开发延缓发病或延缓进展的新疗法非常重要。我们将研究与白内障相关的3D-晶体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。我们假设聚集不是蛋白质的随机结合，而是特定的折叠中间产物参与聚集。除了提供对白内障形成过程的洞察外，我们的研究还将探索蛋白质生物学的基本问题。例如，导致折叠受挫的相互作用，中间体为什么以及如何稳定的问题，以及导致多肽链错误折叠和/或聚集而不是折叠到自然状态的过程，需要直接的实验研究来获得新的见解。拟议的研究将通过对野生型和与疾病相关的晶体蛋白变体的生物物理分析来解决这些悬而未决的问题。晶体蛋白非常适合于蛋白质聚集的详细研究：它们很小；可以获得大量的X射线结构；并且已经研究了几种野生型蛋白质到天然状态的折叠动力学。核磁共振方法将被用来直接研究折叠转变，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。我们的工作将涉及到能够对蛋白质进行详细的结构和动力学表征的方法，主要是核磁共振光谱和小角X射线散射。此外，我们将把基本的生物物理参数与临床观察联系起来。我们计划确定与白内障相关的人类3D-晶体蛋白的三维溶液结构，并表征它们的动态行为。我们将首先关注形成3D-晶体蛋白突变体的两个重要的白内障，P23T和V75D。前者与人类先天性白内障有关，后者是一种已被确认可导致小鼠白内障的变异，因此，将有助于在白内障动物模型中进行后续研究。我们还将表征3D-晶体蛋白折叠中间体的结构和动力学。此外，我们将调查以前确定的、部分折叠的3D-晶体蛋白中间体是否以及如何导致聚集。特别是，我们将确定这种部分折叠的中间体是否为聚集的种子。这将为发现聚集的小分子抑制剂奠定基础，这种方法已经在一些神经退行性蛋白质沉积疾病中取得了一些结果。 与公共卫生相关：白内障是导致失明的主要原因，全世界每年约有1700万例。目前，唯一可用的治疗方法是手术；然而，在美国和其他地方，有相当一部分人口由于各种原因无法接受手术。了解白内障的形成机制将为延缓发病或延缓进展的新疗法的开发开辟道路。

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.