SULFUR-CENTERED CRYSTALLIN MODIFICATIONS & LENS OPACITY

晶体蛋白跨国后的硫中心修饰是 在许多白内障晶状体中反复出现的特征。这一点尤其正确 在成熟性人类核性白内障中，半胱氨酸和 贝塔和伽马晶体蛋白的蛋氨酸残基被氧化。 然而，还没有建立起这种蛋白质之间的直接联系 修改，以及晶状体混浊的形成。在这份提案中， 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 晶体蛋白与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--不是 所有以硫为中心的修饰都会导致白内障，而那个分子 电荷和亲水性等因素是 确定特定修饰在不混浊中的作用。至 验证这一假设时，提出了以下具体目标： (1)在体外引入氧化修饰，通常在 透镜在伽马晶体蛋白的硫中心，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作用 修饰，对晶状体蛋白质聚集和相分离。 (3)评估晶体蛋白在抑制蛋白质聚集中的作用， 由于以硫为中心的伽马晶体蛋白的修饰。 (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进行氧化修饰， 使用定点突变。 这些研究可望为鉴定提供依据。 体内可能导致白内障的晶状体蛋白修饰。自.以来 硫中心也可以被视为修饰基团的锚， 我们的结论应该广泛适用于所有后跨国 在晶状体中发现蛋白质修饰。

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.