AGE AND CATARACT RELATED CHANGES IN LENS PROTEINS

晶状体蛋白随着年龄的增长和白内障的发生更容易聚集。增加的易感性可能与它们处于不太稳定的状态有关，要么是内在的，要么是翻译后的修饰。为了验证这一假设，将对未修饰或修饰状态下的主要透镜晶体蛋白的稳定性进行定量分析。稳定性通常用模糊和不精确的构象或构象变化来表示。本课题将通过研究重组晶体蛋白的折叠反应得到标准自由能deltaGH20和活化能deltaGplusplusH20的值，重组晶体蛋白是纯的，未经修饰的。相对稳定性将与它们对聚集的易感性相关，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是否更容易聚集。将对重组α -、β -和γ -晶体蛋白的稳定性进行比较；它们是人体晶状体中的主要晶体蛋白。对于修饰的研究，将使用非酶糖基化，混合二硫化物形成和c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，将进行位点定向突变。定点突变更具特异性；可以研究氨基酸序列的微小变化所产生的影响。影响蛋白质稳定性的另一个因素是小或大的配体与α -结晶蛋白的结合；小分子，如Ca2+和ATP，大分子，如部分展开的-和-结晶蛋白。主要使用的技术包括荧光和圆二色的平衡和动力学分析，以及FPLC液相色谱和光散射的聚集检测。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。从这个建议中获得的知识可能在分子水平上提供对白内障形成的见解，并可能有助于制定开发抗白内障药物的策略。

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.