Gamma crystallin modifications & mechanisms of lens opacity

描述(由申请人提供)：本提案的长期目标是确定晶体蛋白的修饰(遗传的或年龄起病的)导致白内障形成的分子机制。建议对已知与遗传性或老年性白内障有关的修饰晶状体蛋白进行研究。在目标1中，我们将研究修饰晶状体蛋白之间的相互作用(自结合)，而在目标2中，将研究修饰晶状体蛋白与1-晶状体蛋白之间的相互作用。到目前为止，大多数研究都是第一类，并使人们更好地理解了修饰的晶体蛋白是如何自结合并形成“凝聚相”的，这是导致光散射和不透明的原因。第二种类型的相互作用在保持镜头透明度方面最近出现了同样重要的作用。基于现有的知识，我们提出了以下假设：晶状体蛋白的修饰改变了蛋白质-蛋白质的相互作用，并导致(A)突变蛋白的自结合产生各种凝聚相，或(B)突变晶状体蛋白和晶状体蛋白之间的净吸引相互作用的改变。目标1中的实验将比较修饰形式的人？D-、？C-和？S晶体蛋白与野生型蛋白的热力学相图、低分辨率和高分辨率结构(使用紫外可见、圆二色、荧光、傅里叶变换红外光谱、拉曼光谱和核磁共振)以及稳定性。相图为凝聚相的形成提供了边界条件，凝聚相导致光散射和不透明。因此，这些研究和光散射测量(静态和动态)是拟议研究的组成部分。在目标2中，也将使用类似的技术来检查那些不自结合的修饰形式的？？晶体蛋白与天然？-晶体蛋白的相互作用。因此，拟议的实验将确定由于晶状体中的晶状体蛋白修饰而导致光散射和混浊的分子机制，并应提供对两种形式的白内障的全面了解--遗传性白内障以及老年性白内障。 与公共卫生相关：白内障是世界范围内导致失明的主要原因，是卫生保健系统的一项主要费用。这项建议调查了衰老引起的基因突变和蛋白质修饰是如何导致白内障的。研究结果应有助于制定有效的预防或治疗措施。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine the molecular mechanisms by which modifications of the ??crystallins (genetic or age-onset), lead to cataract formation. Proposed here are studies on modified ??crystallins known to be associated with either genetic or age-onset cataract. In Aim 1 interactions between modified ??crystallins alone will be examined (self-association), and in Aim 2 interactions of modified ??crystallins with 1-crystallin will be examined. Most studies to date have been of the first type and have led to a better understanding of how modified ??crystallins self-associate and form "condensed phases" which are responsible for light scattering and opacity. The second type of interaction has recently emerged as being equally important in maintaining lens transparency. Based on current knowledge, we propose the following hypothesis: Modifications of the ??crystallins alter the protein-protein interactions and lead to either (a) self- association of mutant proteins to yield a variety of condensed phases or (b) a change in the net attractive interactions between mutant ??crystallins and 1-crystallin. Experiments in Aim 1 will compare the thermodynamic phase diagrams, the low and high-resolution structures (using u.v.-visible, circular dichroism, fluorescence, FTIR, Raman and NMR), and stabilities of the modified forms of human ?D-, ?C-, and ?S- crystallins, with those of the wild-type protein. Phase diagrams provide the boundary conditions for the formation of condensed phases which lead to light scattering and opacity. Therefore, these studies and light scattering measurements (static and dynamic) are an integral part of the proposed studies. In Aim 2, the interactions of those modified forms of ??crystallins which do not self-associate will be examined with native ?-crystallin, also using similar techniques. Thus, the proposed experiments will identify molecular mechanisms that lead to light scattering and opacity due to ??crystallin modifications in the lens, and should provide a comprehensive understanding of both forms of cataract disease - genetic, as well as age-onset cataract. PUBLIC HEALTH RELEVANCE: Cataracts are the leading cause of blindness worldwide and constitute a major expense to the health care system. This proposal investigates how genetic mutations and protein- modifications due to aging, lead to cataract. The results should help devise effective preventive or therapeutic measures.