SULFUR-CENTERED CRYSTALLIN MODIFICATIONS & LENS OPACITY

描述：以硫为中心的过渡后修改 晶状体蛋白是许多白内障晶状体中反复出现的特征。这是 尤其是成熟性人类核性白内障，在这种情况下 在β晶体蛋白和伽马晶体蛋白中发现了半胱氨酸和蛋氨酸残基 进行化学修饰。人们通常认为，所有这些 尽管存在巨大的化学差异，但修饰是导致白内障的 在他们之间。在这项建议中，提出了一种策略来识别 多种疾病致白内障的一般化学决定因素 半胱氨酸和蛋氨酸残基上的晶状体蛋白修饰。这 策略建立在这样的假设基础上：极性修饰或带电修饰 降低蛋白质之间的净吸引力，因此 “抑制白内障”。相反，它具有边缘的极性和疏水性 修饰增加了蛋白质之间的净吸引力，因此 “会导致白内障。”为了测试这一点，提出了以下具体目标 假设。 1.在体外，引入通常在晶状体中发现的氧化修饰， 在β和γ晶体蛋白的硫中心分别和在 混合物，并测量“致白内障”或“抑制白内障” 如上所定义的修饰蛋白质的性质。2.研究 电荷、亲水性和硬脂性对分子筛性能的影响 致白内障或抑制白内障的趋势--γ晶状体蛋白和 以半胱氨酸或蛋氨酸修饰的β-γ晶状体蛋白混合物 残留物，使用选定的化学改性剂。3.评估 抑制蛋白中的α-晶体蛋白及其亚基α-A和α-B 伽马晶体蛋白硫心修饰引起的聚集 和贝塔-伽马晶体蛋白混合物。4.确定个人的角色 穴位导入致白内障中的半胱氨酸和蛋氨酸残留量 使用定点突变技术在这些残基上进行突变。 长期目标是制定预防白内障的策略。 体内的修饰。目标2中的拟议研究预计将 最终指导抗白内障候选药物的开发。 晶状体蛋白之间净吸引力的变化将由以下因素决定 测定TPH、相分离温度和蛋白质聚集性。 十二烷基硫酸钠-PAGE、大小排除高效液相色谱、准弹性光散射和蛋白质 云量测量以确定TPH。离子交换高效液相色谱，低压 层析、等电聚焦、拉曼光谱和质谱学 用作蛋白质表征的分析方法。分子建模 研究将指导试剂的选择。

DESCRIPTION: Sulfur-centered post-transitional modifications of the crystallins are a recurring feature in many cataractous lenses. This is especially true of maturity-onset human nuclear cataract, in which the cysteine and methionine residues of the beta and gamma crystallins are found to be chemically modified. It is generally assumed that all such modifications are cataractogenic, despite the vast chemical differences between them. In this proposal a strategy is presented to identify the general chemical determinants of cataractogenicity in a variety of crystallin modifications at the cysteine and methionine residues. This strategy is based on the hypothesis that: polar or charged modifications decrease the net attraction between proteins, and are therefore "cataract-inhibiting". Conversely, marginally polar and hydrophobic modifications increase the net attraction between proteins and are therefore "cataractogenic." The following Specific Aims are proposed to test this hypothesis. 1. Introduce in vitro, oxidative modifications normally found in the lens, at the sulfur centers of the beta and gamma crystallins individually and in mixtures, and measure the "cataractogenic" or "cataract-inhibiting" properties of the modified proteins, as defined above. 2. Examine the influence of charge, hydrophilicity and stearic effects on the cataractogenic or cataract-inhibiting tendency of the gamma crystallins and beta-gamma crystallin mixtures modified at the cysteine or methionine residues, using selected chemical modifiers. 3. Evaluate the role of alpha-crystallin and its subunits alpha-A and alpha-B in inhibiting protein aggregation due to sulfur-centered modifications of the gamma crystallins and beta gamma crystallin mixtures. 4. Determine the role of individual cysteine and methionine residues in cataractogenesis by introducing point mutations at these residues using site-directed mutagenesis. The long-term objectives are to devise strategies to prevent cataractogenic modifications in vivo. The proposed studies in Aim 2 are expected to eventually guide the development of anticataract drug candidates. Changes in the net attraction between lens crystallins will be determined by measuring Tph, the phase separation temperature and protein aggregation. SDS-PAGE, size exclusion HPLC, quasielastic light scattering and protein clouding measurements to determine Tph. Ion-exchange HPLC, low pressure chromatography, isoelectricfocusing, Raman and mass spectroscopies will be used as analytical methods for protein characterization. Molecular modeling studies will guide the selection of reagents.