SULFUR-CENTERED CRYSTALLIN MODIFICATIONS & LENS OPACITY

描述：以硫为中心的过渡后修饰 晶体蛋白是许多白内障晶状体中的反复出现的特征。 这是 尤其是成熟期发作的人核性白内障，其中 发现β和γ晶体蛋白的半胱氨酸和甲硫氨酸残基 进行化学修饰。 一般认为，所有这些 尽管存在巨大的化学差异， 他们之间 本提案提出了一项战略， 在各种类型的白内障中， 半胱氨酸和甲硫氨酸残基处的晶状体蛋白修饰。 这 该策略基于以下假设：极性或带电修饰 减少蛋白质之间的净吸引力，因此 “抑制白内障” 相反，轻微极性和疏水性 修饰增加了蛋白质之间的净吸引力，因此 白内障“提出以下具体目标来检验这一点 假说. 1. 引入体外氧化修饰，通常在透镜中发现， 分别在β和γ晶体蛋白的硫中心， 混合物，并测量“白内障”或“白内障抑制” 修饰的蛋白质的性质，如上文所定义。 2. 检查 电荷、亲水性和硬脂酸效应对 γ晶状体蛋白的白内障发生或白内障抑制倾向， 在半胱氨酸或甲硫氨酸处修饰的β-γ晶状体蛋白混合物 残留物，使用选定的化学改性剂。 3. 评价的作用 α-晶状体蛋白及其抑制蛋白亚基α-A和α-B 由于γ晶体蛋白的硫中心修饰而导致的聚集 和β-γ晶体蛋白混合物。 4. 确定个人的角色 半胱氨酸和蛋氨酸残基在白内障形成中的作用 使用定点诱变在这些残基处进行突变。 长期目标是制定预防白内障的策略 体内修饰 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。 透镜晶体蛋白之间净吸引力的变化将通过以下方法确定： 测量Tph、相分离温度和蛋白质聚集。 SDS-PAGE、尺寸排阻HPLC、准弹性光散射和蛋白质 使测量变浑浊以确定Tph。离子交换HPLC，低压 色谱，等电聚焦，拉曼和质谱将是 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.