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

描述（由申请人提供）：维持镜头的功能？A- and ？随着年龄的增长，b晶体蛋白是预防白内障的关键因素。虽然大家都知道？A- and ？b -结晶蛋白在一生中经历广泛的修饰（交联、糖基化、氧化、磷酸化和截断），这些修饰何时发生或发生在晶体的哪个区域（核区和皮质区）尚不清楚。该应用利用了两种实验方法：(1)ICR/f模型，这是一种年龄相关性白内障模型，在10- 11周内自发形成白内障；(2)晶状体部分的质谱成像，可以确定个体的空间分布（100微米分辨率）？A和b晶体蛋白。在初步的实验中，我们已经成功地开发了获得大鼠晶状体组织切片的方法，用于机器人标记基质和获得晶状体蛋白质分布的图像。本应用程序的目标是检验两个假设：(1)？A- and ？b -晶体蛋白经过修饰，特别是截断，导致它们在特定发育阶段特异性地定位在晶状体内，这一过程通过染料木素和双硫仑的干预而改变，并且(2)？A- and ？b晶蛋白降低了它们的溶解度，从而解释了它们的定位。在第一个目标中，将使用ICR/f大鼠和质谱成像来确定饮食中的染料木素和滴眼液中的双硫仑对？A- and ？白内障发育过程中b -晶状体蛋白的截断/修饰及分布。将对未经治疗的ICR/f大鼠和Wistar大鼠（后者没有白内障）的晶状体进行检查，以确定特定白内障相关？A- and ？B-crystallin碎片。在第二个目标中，选择截断？A- and ？b -晶体蛋白（例如，m/z 6409）通过质谱成像显示出明显的区域分布，将从透镜中提取（分为水溶性和水不溶性/尿素溶性部分）。这些将使用色谱和电泳技术纯化，然后通过测定其准确的分子量、序列和修饰来充分表征。这些研究将突出嵌入在具有如此长寿命的蛋白质序列中的溶解度/功能信息，并将ICR/f鼠建立为探索预防/延迟白内障干预机制的方便试验平台。

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.