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

晶状体蛋白：ICR/f 大鼠白内障模型中的空间位置和特性

• 批准号: 8117497
• 负责人: STEPHEN BARNES
• 金额: $ 17.91万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8117497
• 关键词: Accounting; Age; Age-Years; Aging; Animal Model; Appearance; Beds; Biochemical; Blindness; Buffers; Cataract; Crystallins; Data; Development; Diabetes Mellitus; Diet; Disulfiram; Drug or chemical Tissue Distribution; Elderly; Event; Eyedrops; Functional disorder; Gel Chromatography; General Population; Genistein; Goals; Image; Individual; Intervention; Ion Exchange; Isoflavones; Isotonic Exercise; Lead; Lesion; Life; Location; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methods; Modeling; Modification; Molecular Chaperones; Molecular Weight; Monitor; Nuclear; Operative Surgical Procedures; Pharmaceutical Preparations; Phosphorylation; Post-Translational Protein Processing; Precipitation; Prevention therapy; Preventive; Process; Property; Proteins; Proteomics; Quality of life; Rattus; Relative (related person); Resolution; Rest; Risk; Risk Factors; Role; Solubility; Solutions; Spatial Distribution; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Staging; Structure; Sunlight; Techniques; Testing; Therapeutic; Time; Tissues; Ultracentrifugation; Urea; Visual impairment; Water; Wistar Rats; age related; aqueous; base; calpain inhibitor; crosslink; deamidation; dietary antioxidant; dietary control; dietary supplements; glycation; grape seed; insight; lens; lens protein; novel therapeutic intervention; oxidation; oxidative damage; prevent; protein misfolding; protein profiling; public health relevance; research study

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.

描述（由申请人提供）：维持镜头的功能？尽管众所周知，a-和b-晶状蛋白在一生中经历了广泛的修饰（交联，糖化，氧化，磷酸化和截断），但何时发生这些修饰或在镜头的哪个区域（核和皮质区域）何时尚不清楚。该应用利用了两种实验方法：（1）ICR/F大鼠是与年龄相关的白内障模型，自发形成白内障10-11周的生命，以及（2）镜头段的质谱成像，可以确定空间分布（以100微分辨率分布（以100微分辨率）（以100微分辨率分辨率）（以个人？a-a-a-a-a-b-crystall？在初步实验中，我们成功地开发了获得大鼠晶状体组织切片的方法，用于用机器人的基质发现并获得晶状体蛋白分布的图像。该应用的目的是检查两个假设：（1）？A-和？B-结晶蛋白经历了修改，尤其是截断，这会导致它们在特定的发展阶段中专门定位在特定阶段的镜头中，这些过程会因仿制和二硫酸和二硫酸的干预而改变的过程，以及（2）在此过程中，请逐步解决问题。在第一个目的中，ICR/F大鼠和质谱成像将用于确定染料在饮食和二硫糖中的作用，并通过眼滴对？将检查来自未处理的ICR/F大鼠和Wistar大鼠（后者没有白内障）的透镜，以确定特定白内障相关的？A-和b-cr​​ystallin片段的外观开始。在第二个目的中，将从透镜（将质谱成像截断为截面？这些将使用色谱和电泳技术进行纯化，然后以确定其准确的分子量，序列和修饰的确定。这些研究将突出显示具有如此延长寿命的蛋白质序列中的溶解度/功能信息，还建立了ICR/F大鼠作为方便的测试床，以探索预防/延迟白内障的干预措施的机制。 公共卫生相关性：达到70岁的普通人群中有50％将需要透镜替代手术，并且以前的视力降低。鉴于美国老年人比例的越来越多以及维持生活质量的重要性，因此有必要采取干预措施来降低白内障风险。 ICR/F大鼠是评估新的治疗干预措施以及饮食补充剂的风险因素的模型。