AGE AND CATARACT RELATED CHANGES IN LENS PROTEINS

随着年龄和白内障的增加，晶状体蛋白质变得更容易聚集。易感性的增加可能与它们处于不太稳定的状态有关，无论是内在的还是翻译后的修饰。为了验证这一假设，将对主晶状体晶体蛋白的稳定性进行定量分析，无论是在未修饰状态还是在修饰状态。稳定性通常用模糊和不精确的术语构象或构象变化来表示。在这个方案中，标准自由能deltaGH20和活化能deltaGplusplusH20将通过研究重组晶体蛋白的折叠反应得到，这些重组晶体蛋白是纯的和未修饰的。相对稳定性将与它们对聚集的敏感性相关，即确定不太稳定的蛋白质或通过修饰变得不太稳定的蛋白质中的哪一种更容易聚集。将研究晶状体蛋白，重组αA-、β-B2-和伽马C-晶状体蛋白之间的稳定性比较；它们是人类晶状体中的主要晶状体蛋白。对于修饰的研究，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，我们将进行定点突变。定点突变更具特异性；氨基酸序列的微小变化的影响可以研究。另一个影响蛋白质稳定性的因素是小的或大的配体与α-晶体蛋白的结合；小分子，如钙离子和三磷酸腺苷，以及大分子，如部分展开的β-和伽马-晶体蛋白。将使用的主要技术包括荧光和圆二色谱的平衡和动力学分析，以及FPLC液相色谱和光散射检测聚集。由于晶状体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。从这一建议中获得的知识可能会在分子水平上为白内障的形成提供洞察力，并可能有助于制定开发抗白内障药物的战略。

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.