Lens crystallins: spatial location and properties in the ICR/f rat cataract model

描述（由申请人提供）：保持透镜的功能？然后呢？B-晶状体蛋白与衰老是预防白内障的关键事件。尽管众所周知，？然后呢？B-晶状体蛋白在一生中经历广泛的修饰（交联、糖化、氧化、磷酸化和截短），这些修饰何时发生或在透镜的哪个区（核和皮质区）中不太清楚。该应用利用两种实验方法：（1）ICR/f大鼠，年龄相关性白内障的模型，其在10- 11周的生命中自发形成白内障，和（2）透镜的部分的质谱成像，其允许确定个体的空间分布（在100微米分辨率）。然后呢？B-晶体蛋白在初步的实验中，我们已经成功地开发了用于获得大鼠透镜的组织切片的方法，用于用基质自动点样并获得透镜蛋白质分布的图像。本申请的目的是检查两个假设：（1）？然后呢？B-晶体蛋白进行修改，特别是截断，使他们在特定的发展阶段，在透镜内的具体本地化，通过干预与染料木素和双硫仑改变的过程，和（2），修改？然后呢？B-晶体蛋白降低其溶解度，从而解释其定位。在第一个目标，ICR/f大鼠和质谱成像将被用来确定染料木黄酮在饮食和双硫仑滴眼液的类型和时间的影响？然后呢？白内障发生过程中B-晶状体蛋白的截短/修饰和分布将检查未经治疗的ICR/f大鼠和Wistar大鼠（后者没有白内障）的晶状体，以确定特定白内障相关的出现的开始时间？然后呢？B-晶体蛋白片段。在第二个目标，选择截断？然后呢？B-晶体蛋白（例如，m/z 6409），通过质谱成像显示出显著的区域分布，将从透镜中提取（分成水溶性和水不溶性/尿素溶性部分）。这些将使用色谱和电泳技术进行纯化，然后通过测定其准确分子量、序列和修饰进行充分表征。这些研究将突出嵌入在具有如此延长的寿命的蛋白质序列中的溶解度/功能信息，并且还将ICR/f大鼠建立为探索预防/延迟白内障的干预机制的方便试验床。 公共卫生相关性：50%达到70岁的普通人群将需要透镜置换手术以及先前的视力下降。鉴于美国老年人比例的增加以及维持生活质量的重要性，有必要采取干预措施降低白内障的风险。ICR/f大鼠是评估新的治疗干预措施以及膳食补充剂中的风险因素的模型。

DESCRIPTION (provided by applicant): Maintaining the function of the lens ?A- and ?B-crystallins with aging is the key event in preventing cataracts. Although it is well known that ?A- and ?B-crystallins undergo extensive modifications (crosslinking, glycation, oxidation, phosphorylation and truncation) over a lifetime, it is less clear when these modifications occur or in which zone of the lens (nuclear and cortical regions). This application takes advantage of two experimental approaches: (1) the ICR/f rat, a model of age-related cataracts, that spontaneously forms cataracts witihin 10- 11 weeks of life, and (2) mass spectrometry imaging of sections of the lens that allows determination of the spatial distribution (at 100 micron resolution) of individual ?A- and ?B-crystallins. In preliminary experiments we have successfully developed methods for obtaining tissue sections of the rat lens, for spotting with matrix robotically and obtaining images of the distribution of lens proteins. The goal of this application is to examine two hypotheses: (1) ?A- and ?B-crystallins undergo modifications, particularly truncation, that cause them to be specifically localized within the lens at particular stages of development, processes that are altered by intervention with genistein and disulfiram, and (2) that the modifications of the ?A- and ?B-crystallins decrease their solubility, thereby accounting for their localization. In the first aim, ICR/f rats and mass spectrometry imaging will be used to determine the effect of genistein in the diet and disulfiram by eye drops on the type and timing of ?A- and ?B-crystallins' truncation/modifications and distribution during cataract development. Lenses from untreated ICR/f rats and Wistar rats (the latter do not have cataracts) will be examined to determine the onset of the appearance of specific cataract-associated ?A- and ?B-crystallin fragments. In the second aim, selected truncated ?A- and ?B-crystallins (e.g., m/z 6409), showing marked regional distribution by mass spectrometry imaging, will be extracted from the lens (into water-soluble and water-insoluble/urea- soluble fractions). These will be purified using chromatographic and electrophoretic techniques and then fully characterized by determination of their accurate molecular weights, sequence and modifications. These studies will highlight the solubility/function information embedded in the sequence of a protein that has such an extended lifetime and also establish the ICR/f rat as a convenient test bed for exploring the mechanism of interventions to prevent/delay cataracts. PUBLIC HEALTH RELEVANCE: 50% of the general population who reach 70 years of age will have required lens replacement surgery as well as having previous diminished vision. Given the increasing proportion of the elderly in the USA and the importance of sustaining quality of life, interventions to reduce the risk of cataracts are warranted. The ICR/f rat is a model to evaluate both new therapeutic interventions as well as risk factors in dietary supplements.