CALCIUM ACTIVATED PROTEASE IN CATARACT FORMATION

这项研究的长期目标是了解钙的作用。 正常晶状体和晶状体中激活的中性蛋白水解酶，钙蛋白酶 正在经历白内障形成。在上一笔拨款中进行的研究 循环显示：1)钙蛋白及其内源性抑制物以一种 人和啮齿动物晶状体中的主要蛋白酶系统。2)钙蛋白酶弥漫分布 分布在中央上皮的上皮细胞中，在那里它可能 与细胞骨架有关。相反，钙调蛋白在分化过程中 上皮细胞和外皮质勾勒出纤维的边缘 如果它与质膜有关。3)初步结果 显示钙蛋白酶抑制剂E64能够预防白内障和 培养的大鼠晶状体中的蛋白质分解。 这些数据支持晶状体中钙蛋白逃避抑制的假说。 通过结合到纤维膜上，胞浆中的钙蛋白酶是 激活了。在正常的晶状体中，这会导致细胞骨架的降解。 参与终末纤维细胞分化。在白内障的发生中， 晶状体蛋白和膜蛋白的过度作用 钙蛋白酶会导致不溶解和浑浊。 为了进一步验证这一假说，我们提出了三个实验：1) 检查钙蛋白与晶状体膜结合的性质，并确定 如果钙蛋白酶结合到晶状体膜上，可以逃脱钙调蛋白和 激活。2)研究钙蛋白与亚细胞的关系 用透射电子显微镜观察晶状体中的结构。3)确定 新的、更具特异性的细胞穿透抑制剂的有效性 钙蛋白酶预防白内障及相关生化变化的临床研究 培养的啮齿动物晶状体。 建议的研究应能更详细地了解 钙蛋白在晶状体中的活性调控及其作用。他们还将提供 未来可能将钙蛋白酶抑制剂作为药物进行测试的数据 改善或预防人类的白内障。

The long-term goal of this research is to understand the role of calcium activated neutral protease, calpain, in normal lenses and lenses undergoing cataract formation. Studies conducted in the preceding grant cycle showed: 1) Calpain and its endogenous inhibitor are present as a major protease system in human and rodent lenses. 2) Calpain is diffusely distributed across epithelial cells in central epithelium, where it may be associated with cytoskeleton. In contrast, calpain in differentiating epithelial cells and outer cortex outlined the periphery of the fibers as if it were associated with the plasma membrane. 3) Preliminary results showed that calpain inhibitor E64 was able to prevent cataract and proteolysis in cultured rat lenses. These data support the hypothesis that calpain in lens escapes inhibition of cytosolic calpastatin by binding to fiber membranes, where calpain is activated. In normal lenses, this leads to hydrolysis of cytoskeleton involved in terminal fiber cell differentiation. In cataractogenesis, breakdown of crystallins and membrane proteins by excessive action of calpain leads to insolubilization and opacity. Three experiments are proposed to further test this hypothesis: 1) Examine the nature of calpain binding to the lens membrane, and determine if binding of calpain to lens membrane allows escape from calpastatin and activation. 2) Examine the association of calpain with subcellular structures in lens using transmission electron microscopy. 3) Determine the effectiveness of new, more specific, cell penetrating inhibitors of calpain in preventing cataract and associated biochemical changes in cultured rodent lenses. The studies proposed should provide a more detailed understanding of the regulation and role of calpain activity in lens. They will also provide data for possible future testing of calpain inhibitors as drugs to ameliorate or prevent cataract in man.