SULFUR-CENTERED CRYSTALLIN MODIFICATIONS & LENS OPACITY

以硫为中心的晶状蛋白修饰

• 批准号: 2164450
• 负责人: Jayanti Pande
• 金额: $ 12.9万
• 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 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2164450
• 关键词: Raman spectrometry; animal tissue; autooxidation; cataract; chemical aggregate; circular dichroism; crystallins; cysteine; disulfide bond; glutathione; hydropathy; methionine; oxidation; point mutation; posttranslational modifications; site directed mutagenesis; sulfur aminoacid; thiols

Sulfur-centered post-transnational modifications of the crystallin 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 crystallin are oxidized. However, no direct link has been established between such protein modifications, and the formation of lens opacities. In this proposal, a strategy is presented to establish such a link in vitro, between modifications of the cysteine and methionine residues of the gamma crystallin and the two molecular mechanisms that lead to opacity: protein aggregation and protein phase separation. Our hypothesis is that - not all sulfur-centered modifications are cataractogenic, and that molecular factors such as charge and hydrophilicity are essential elements that determine the role of a particular modification in opacification. To test this hypothesis, the following specific aims are proposed: (1) Introduce in vitro, oxidative modifications normally found in the lens at the sulfur centers of the gamma crystallin, and measure the effect of these modifications on the aggregation and phase separation properties of the protein solutions. (2) Determine the role played by hydrophilicity in sulfur-centered modifications, on lens proteins aggregation and phase separation. (3) Evaluate the role of a crystallin in inhibiting protein aggregation, due to sulfur-centered modifications of the gamma crystallin. (4) Determine the role of individual cysteine and methionine residues in oxidative modifications by introducing point mutations at these residues, using site-directed mutagenesis. These studies are expected to provide the basis for the identification of potentially cataractogenic crystallin modifications in vivo. Since the sulfur centers can be viewed also as anchors for modifying groups, our conclusions should be broadly applicable to all post-transnational protein modifications found in the lens.

以硫为中心的结晶蛋白的翻译后修饰是 许多白内科镜头中的反复出现的特征。 这尤其正确 成熟度发作的人类核白内障，其中半胱氨酸和 β和γ晶体的蛋氨酸残基被氧化。 但是，这种蛋白质之间尚未建立直接联系 修改和晶状体不透明的形成。 在此提案中， 提出了一种在体外建立这种联系的策略 伽马的半胱氨酸和蛋氨酸残基的修饰 结晶蛋白和导致不透明度的两种分子机制：蛋白质 聚集和蛋白相分离。 我们的假设是 - 不是 所有以硫为中心的修饰都是白内生的，该分子 电荷和亲水性等因素是重要因素 确定特定修饰在不拼变中的作用。 到 检验该假设，提出了以下具体目的： （1）通常在体外引入氧化修饰 伽马晶蛋白的硫心中心的镜头，并测量 这些修改对聚集和相分离的影响 蛋白质溶液的性质。 （2）确定亲水性在以硫为中心的作用 修饰，晶状体蛋白聚集和相位分离。 （3）评估晶体在抑制蛋白质聚集中的作用， 由于伽马结晶蛋白的以硫为中心的修饰。 （4）确定单个半胱氨酸和蛋氨酸残基在 通过在这些残基上引入点突变来进行氧化修饰， 使用定向诱变。 这些研究有望为识别提供基础 体内潜在的白内抗性结晶蛋白的修饰。 自从 硫心中心也可以看作是修饰组的锚点， 我们的结论应广泛适用于所有后翻译后 在镜头中发现的蛋白质修饰。