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 结晶蛋白是许多白内科镜头中的反复出现的特征。 这是 尤其是成熟的人类核白内障，其中 发现β和伽马晶蛋白的半胱氨酸和蛋氨酸残基 进行化学修饰。 通常认为所有这些 尽管化学差异很大，但修饰是白内生的 他们之间。 在此提案中，提出了一个策略来确定 各种白内障的一般化学决定因素 半胱氨酸和蛋氨酸残基的结晶蛋白修饰。 这 策略基于以下假设：极性或充电修改 减少蛋白质之间的净吸引力，因此 “抑制白内障”。 相反，极性和疏水性 修改会增加蛋白质之间的净吸引力，因此 “白内生。” 提出了以下特定目标来测试 假设。 1。引入体外，氧化修饰，通常在镜头中发现， 在β和伽马结晶蛋白的硫心中心分别和 混合物，测量“白内障”或“抑制白内障” 如上所述，修饰蛋白的特性。 2。检查 电荷，亲水性和硬性影响对 伽马晶蛋白的白内生或白内障抑制趋势 在半胱氨酸或蛋氨酸修饰的β-伽马结晶蛋白混合物 残留物，使用选定的化学修饰符。 3。评估角色 α-晶状体及其亚基α-A和α-B抑制蛋白 由于伽马结晶蛋白的以硫为中心的修饰引起的聚集 和β伽玛结晶蛋白混合物。 4。确定个人的作用 通过引入点的白内术中的半胱氨酸和蛋氨酸残基 这些残基的突变使用定位的诱变。 长期目标是制定防止白内障的策略 体内修饰。 AIM 2中提出的研究预计将 最终指导了抗核药物候选者的发展。 透镜结晶蛋白之间的净吸引力的变化将由 测量TPH，相分离温度和蛋白质聚集。 SDS-PAGE，尺寸排除HPLC，准灯光散射和蛋白质 确定TPH的云测量。离子交换HPLC，低压 色谱法，等电源，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。