Molecular Basis for Lens Transparency

描述（由申请人提供）：晶状体老化的“磨损”是通过其纤维细胞中晶体蛋白的累积修饰在分子水平上记录的。在没有蛋白质翻转的情况下，这种缓慢的环境扰动会导致晶体蛋白稳定性和溶解度的逐渐丧失，并改变蛋白质之间的相互作用，所有这些都会损害晶状体的透明度和折射率。大约35%的纤维细胞重量由分子伴侣a-晶体蛋白组成，a-晶体蛋白是一种蛋白质稳定性传感器，可以结合容易聚集的蛋白质。a-晶体蛋白与不稳定的¿- and ？晶体蛋白是晶状体老化的主要原因。监护人能力的衰竭被认为是老年性白内障的主要催化剂。此外，年龄相关的核性白内障的最早阶段与晶体蛋白的二硫交联聚集体的出现在时间上相关。这项资助的长期目标是了解晶状体老化和白内障发展过程中晶体蛋白稳定性、伴侣结构、亲和力和容量以及分子拥挤之间的相互关系。在下一个资助期，我们将测试关于年龄相关和遗传性白内障的分子机制的两个假设。目标1和目标2将对触发不稳定的¿和？并确定伴侣驱动的相互作用是否通过络合物形成所提供的熵优势导致二硫交联的形成。这项工作的关键是使用一种创新的无标签方法，即后向散射干涉测量法，它可以实时表征这些相互作用。我们还将从a-晶状体容量的滴定和聚集体形成的角度，验证与先天性白内障相关的a-晶状体半胱氨酸突变导致压缩老化过程的假设。根据初步数据，这些突变体的复合物形成是由它们增加的亲和力和容量驱动的，而不是由底物不稳定驱动的，并导致二硫交联的形成。在目标3中，将采用最先进的结构工具来提供伴侣激活状态及其与底物的复合物的快照。这些研究将通过识别与结合相关的序列和结构基序，以及通过小热休克蛋白的低聚物扩张来定义激活的基础，来补充目标1和目标2的机制见解。白内障是全球失明的主要原因之一，在美国影响着2000万成年人。在最常见的核性白内障中，蛋白质聚集和二硫交联是主要的分子事件。对这些分子转化的机制观点的发展具有基本的生化重要性，并可能对干预和治疗策略的发展产生影响。

DESCRIPTION (provided by applicant): The "wear and tear" of lens aging is recorded at the molecular level through accumulated modifications of the crystallins in its fiber cells. This slow environmental perturbation in the absence of protein turn-over leads to a progressive loss in crystallins stability and solubility and alters protein-protein interactions, all of which compromise lens transparency and refractivity. Roughly 35% of the fiber cell weight consists of the molecular chaperone, a-crystallin, a protein-stability sensor that binds aggregation-prone proteins. Attractive interactions between a-crystallin and destabilized ¿- and ?-crystallins are a central facet of lens aging. The exhaustion of chaperone capacity is hypothesized to be a central catalyst for age-related cataract. Furthermore, the earliest stage of age-related nuclear cataract is temporally correlated with the appearance of disulfide cross-linked aggregates of crystallins. The long term goal of this grant is to develop an understanding of the interrelationships between crystallins stability, chaperone structure, affinity and capacity, and molecular crowding in the process of lens aging and the development of cataract. In the next funding period, we will test two hypotheses regarding the molecular mechanisms of age-related and hereditary cataracts. Aims 1 and 2 will undertake a systematic analysis of the energetic threshold required to trigger binding of destabilized ¿- and ?-crystallins to a-crystallin and determine whether chaperone-driven interactions lead to formation of disulfide cross-links through the entropic advantage afforded by complex formation. Critical to this endeavor is the use of an innovative label-free approach, backscattering interferometry, which allows characterization of these interactions in real time. We will also test the hypothesis that A-crystallin cysteine mutants linked to congenital cataract lead to a compressed aging process from the perspective of titration of a-crystallin capacity and formation of aggregates. Based on preliminary data, complex formation by these mutants is driven by their increased affinity and capacity rather than by substrate destabilization and results in the formation of disulfide cross-links. In aim 3, state of the art structural tools will be employed to provide snapshots of a chaperone activated state and its complex with the substrate. These studies will complement the mechanistic insight of aims 1 and 2 by identifying sequence and structural motifs involved in binding, and defining the basis of activation by oligomer expansion of small heat-shock proteins. Cataract formation is a leading cause of blindness worldwide and affects 20 million adults in the United States. In the most common type, human nuclear cataract, protein aggregation and disulfide cross-linking are major molecular events. The development of a mechanistic perspective on these molecular transformations is of fundamental biochemical importance and may well have an impact on the development of intervention and therapeutic strategies.