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

描述(申请人提供)：随着年龄的增长，维持晶状体-A-和-B-晶状体蛋白的功能是预防白内障的关键。虽然众所周知，晶状体蛋白在一生中经历了广泛的修饰(交联化、糖基化、氧化、磷酸化和截断)，但尚不清楚这些修饰何时发生或在晶状体的哪个区域(核区和皮质区)。这项应用利用了两种实验方法：(1)ICR/f大鼠，一种年龄相关性白内障的模型，在10-11周内自发形成白内障；(2)对晶状体切片进行质谱仪成像，以确定单个A-和B-晶体蛋白的空间分布(100微米分辨率)。在初步实验中，我们已经成功地开发了获取大鼠晶状体组织切片的方法，用于机器人使用基质进行斑点，并获得晶状体蛋白质分布的图像。本申请的目的是检验两个假说：(1)A-和B-晶体蛋白经历修饰，特别是截断，使它们在特定的发育阶段专门定位于晶状体内，这一过程可通过金雀异黄素和双硫兰的干预而改变，以及(2)？A-和B-晶体蛋白的修饰降低了它们的溶解度，从而解释了它们的定位。在第一个目的中，我们将利用ICR/f大鼠和质谱仪成像来确定饮食中染料木素和滴眼液对晶状体蛋白A和B晶状体蛋白截断/修饰的类型和时间以及在白内障发生发展过程中的分布的影响。来自未经治疗的ICR/f大鼠和Wistar大鼠(后者没有白内障)的晶状体将被检查，以确定特定的与白内障相关的A和B晶状体蛋白片段的出现。在第二个目的中，将从晶状体中提取选定的截短的A-和B-晶体蛋白(例如，m/z 6409)，它们通过质谱学成像显示出明显的区域分布(分为水溶和水不溶/尿素可溶的组分)。这些化合物将使用层析和电泳技术进行纯化，然后通过测定它们的准确分子量、序列和修饰来充分表征它们。这些研究将突出具有如此长寿命的蛋白质序列中嵌入的溶解性/功能信息，并将ICR/f大鼠作为一个方便的试验台，用于探索预防/延缓白内障的干预机制。 公共卫生相关性：50%达到70岁的普通人群将需要接受晶状体置换手术，并有既往视力减退。鉴于美国老年人比例的增加和维持生活质量的重要性，有必要采取干预措施来降低白内障的风险。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.