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.

晶体蛋白的以硫为中心的跨国后修饰是 这是许多白内障晶状体中反复出现的特征。 尤其如此 成熟期发作的人核性白内障，其中半胱氨酸和 β和γ晶状体蛋白的甲硫氨酸残基被氧化。 然而，这种蛋白质之间没有直接的联系。 变形和透镜混浊的形成。 在这一提议中， 提出了一种在体外建立这种联系的策略， γ-半胱氨酸和蛋氨酸残基的修饰 晶状体蛋白和导致混浊的两种分子机制：蛋白质 聚集和蛋白质相分离。 我们的假设是， 所有以硫为中心的修饰都是致白内障的， 诸如电荷和亲水性的因素是 确定特定修饰在乳浊中的作用。 到 为了检验这一假设，提出了以下具体目标： (1)引入体外氧化修饰，通常在 透镜在硫中心的γ晶体蛋白，并测量 这些修饰对聚集和相分离的影响 蛋白质溶液的性质。 (2)确定亲水性在硫中心的 修饰，对透镜蛋白聚集和相分离的影响。 (3)评价晶状体蛋白在抑制蛋白质聚集中的作用， 这是由于γ晶体蛋白的硫中心修饰。 (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进行氧化修饰， 使用定点诱变。 预计这些研究将为确定 潜在的白内障晶体蛋白的修改在体内。 以来 硫中心也可被视为改性基团的锚， 我们的结论应该广泛适用于所有后跨国 在透镜中发现的蛋白质修饰。