SULFUR-CENTERED CRYSTALLIN MODIFICATIONS & LENS OPACITY

以硫为中心的晶状蛋白修饰

• 批准号: 6661650
• 负责人: Jayanti Pande
• 金额: $ 11.1万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6661650
• 关键词: Raman spectrometry; SDS polyacrylamide gel electrophoresis; animal tissue; autooxidation; cataract; chemical aggregate; circular dichroism; crystallins; cysteine; disulfide bond; electrofocusing; glutathione; high performance liquid chromatography; hydropathy; mass spectrometry; methionine; oxidation; point mutation; posttranslational modifications; site directed mutagenesis; sulfur aminoacid; thiols

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.

描述：以硫为中心的过渡后修饰 晶状体蛋白是许多白内障晶状体中反复出现的特征。 这是 尤其是成熟期开始的人类核白内障，其中 发现了 β 和 γ 晶状体蛋白的半胱氨酸和蛋氨酸残基 进行化学修饰。 一般认为，所有此类 尽管存在巨大的化学差异，但修饰仍会导致白内障 他们之间。 在本提案中，提出了一项战略来确定 各种致白内障的一般化学决定因素 晶状体蛋白对半胱氨酸和蛋氨酸残基进行修饰。 这 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，边际极性和疏水性 修饰增加了蛋白质之间的净吸引力，因此 “导致白内障”。 建议采取以下具体目标来测试这一点 假设。 1. 引入体外晶状体中常见的氧化修饰， 分别位于 β 和 γ 晶状体蛋白的硫中心 混合物，并测量“致白内障”或“抑制白内障” 修饰蛋白质的特性，如上文所定义。 2. 检查 电荷、亲水性和空间效应对 γ晶状体蛋白和的导致白内障或抑制白内障的倾向 半胱氨酸或蛋氨酸修饰的 β-γ 晶状体蛋白混合物 使用选定的化学改性剂去除残留物。 3. 评估角色 α-晶状体蛋白及其亚基α-A和α-B在抑制蛋白中的作用 由于γ晶状体蛋白以硫为中心的修饰而发生聚集 和β-γ晶状体蛋白混合物。 4. 确定个人角色 通过引入点来研究半胱氨酸和蛋氨酸残基在白内障发生中的作用 使用定点诱变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 体内修饰。 目标 2 中拟议的研究预计将 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下式确定 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 浑浊测量以确定 Tph。离子交换 HPLC，低压 色谱、等电聚焦、拉曼和质谱将 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。