AGE AND CATARACT RELATED CHANGES IN LENS PROTEINS

透镜蛋白质随着年龄的增长和白内障的发生而变得更容易聚集。 增加的易感性可能与它们处于不稳定状态的事实有关，无论是内在的还是通过翻译后修饰。 为了检验该假设，将对未修饰或修饰状态下的主要透镜晶体蛋白进行稳定性的定量分析。 稳定性通常用模糊不清的术语构象或构象变化来表示。 在该提议中，标准自由能Δ GH 20和活化能Δ GplusplusH 20的值将通过使用纯的和未修饰的重组晶体蛋白研究折叠反应来获得。 相对稳定性将与它们对聚集的敏感性相关，即，以确定较不稳定的蛋白质或通过修饰变得较不稳定的蛋白质是否更易于聚集。 将研究晶状体蛋白、重组α A-、β B2-和γ C-晶状体蛋白之间的稳定性比较;它们是人透镜中的主要晶状体蛋白。 对于修饰研究，将使用非酶糖化、混合二硫键形成和C末端降解作为模型。 为了进一步了解修饰对构象和动力学稳定性的影响，将进行定点突变。定点突变更具有特异性;可以研究氨基酸序列的微小变化的影响。 影响蛋白质稳定性的另一个因素是小或大配体与α-晶状体蛋白的结合;小分子，如Ca 2+和ATP，以及大分子，如部分未折叠的β-和γ-晶状体蛋白。 将使用的主要技术包括通过荧光和圆二色性的平衡和动力学分析，以及通过FPLC液相色谱和光散射检测聚集。由于透镜混浊是由蛋白质聚集引起的，因此诱导蛋白质聚集的潜在机制在白内障研究中至关重要。 从这一建议获得的知识可能提供洞察白内障形成的分子水平上，并可能有助于制定一项战略，开发抗白内障药物。

The lens proteins become more susceptible to aggregation with age and cataract. The increased susceptibility may be related to the fact that they are in less stable states, either intrinsically or by posttranslational modifications. To test the hypothesis, a quantitative analysis of the stability will be made on the main lens crystallins, either in unmodified or modified states. The stability is usually expressed by the vague and imprecise term conformation or conformational change. In this proposal, the values of standard free energy, deltaGH20, and activation energy, deltaGplusplusH20, will be obtained by studying the folding reactions using recombinant crystallins, which are pure and unmodified. The relative stability will be correlated to their susceptibility to aggregation, i.e., to determine whether a less stable protein or a protein that becomes less stable by modification will be more susceptible to aggregation. A comparison of stability among crystallins, recombinant alphaA-, betaB2-, and gammaC-crystallins, will be studied; they are the major crystallins in the human lens. For the study of modifications, nonenzymatic glycation, mixed disulfide formation, and C-terminal degradation will be used as models. To further understand the effect of modification on conformational and kinetic stability, site-directed mutation will be performed. Site-directed mutation is more specific; the effect of a small change in amino acid sequence can be studied. Another factor that affects protein stability is the binding of a small or large ligand to alpha-crystallin; small molecules, such as Ca2+ and ATP, and large molecules, such as partially unfolded beta- and gamma-crystallins. The main techniques that will be used include equilibrium and kinetic analysis by fluorescence and circular dichroism, and detection of aggregation by FPLC liquid chromatography and light scattering. Since lens opacity is caused by protein aggregation, the underlying mechanisms that induce protein aggregation are fundamentally important in cataract research. The knowledge obtained from this proposal may provide insight on cataract formation on a molecular level and may help in formulating a strategy for developing anticataract agents.