CALCIUM ACTIVATED PROTEASE IN CATARACT FORMATION

白内障形成中的钙激活蛋白酶

• 批准号: 3261303
• 负责人: THOMAS R SHEARER
• 金额: $ 11.65万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 1994-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3261303
• 关键词: X ray; aging; binding proteins; calcium; calpain; cataract; disease /disorder prevention /control; electron microscopy; enzyme linked immunosorbent assay; enzyme mechanism; enzyme substrate; eye disorder chemotherapy; high performance liquid chromatography; histochemistry /cytochemistry; human tissue; immunochemistry; laboratory mouse; laboratory rabbit; laboratory rat; lens; lens proteins; membrane fusion; organ culture; protease inhibitor; protein structure; proteolysis; radiobiology

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.

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含有激活的中性蛋白酶、钙蛋白酶 正在形成白内障。在之前的资助中进行的研究 循环显示：1）钙蛋白酶及其内源性抑制剂作为 人类和啮齿动物晶状体中的主要蛋白酶系统。 2) 钙蛋白酶呈弥漫性 分布在中央上皮的上皮细胞中， 与细胞骨架有关。相反，钙蛋白酶在分化 上皮细胞和外皮层勾画出纤维的外围： 如果它与质膜有关。 3）初步结果 表明钙蛋白酶抑制剂 E64 能够预防白内障 培养大鼠晶状体中的蛋白水解。 这些数据支持晶状体中的钙蛋白酶逃脱抑制的假设 通过与纤维膜结合来产生胞质钙蛋白酶抑制剂，其中钙蛋白酶是 活性。在正常晶状体中，这会导致细胞骨架水解 参与终末纤维细胞分化。在白内障发生过程中， 过度作用导致晶状体蛋白和膜蛋白的分解 钙蛋白酶导致不溶解和混浊。 提出了三个实验来进一步检验这一假设：1） 检查钙蛋白酶与晶状体膜结合的性质，并确定 如果钙蛋白酶与晶状体膜的结合允许钙蛋白酶抑制剂逸出并且 激活。 2) 检查钙蛋白酶与亚细胞的关联 使用透射电子显微镜观察透镜中的结构。 3）确定 新的、更特异的细胞穿透抑制剂的有效性 钙蛋白酶预防白内障和相关生化变化 培养的啮齿动物镜片。 拟议的研究应提供对 晶状体中钙蛋白酶活性的调节和作用。他们还将提供 未来可能测试钙蛋白酶抑制剂作为药物的数据 改善或预防人类白内障。