Calcium-activated protease in cataract formation

描述(申请人提供)：这项资助的重点是提供一个详细的机制，为钙激活的蛋白水解酶(钙调蛋白)在晶体中的作用。目前正在测试的总体假设是，在正常的晶状体纤维分化和成熟过程中，Calain的蛋白分解对于有限截断、更紧密地堆积被截断的β-晶体蛋白以及细胞骨架元素的重排是必不可少的。在白内障形成过程中，由于钙离子的大量流入，钙蛋白酶的过度激活会导致蛋白质迅速分解和光散射，因为截短的晶体蛋白不再有正确的顺序。在最新的资助周期中，研究产生了几个与这一假说相关的意外发现：晶状体特异的calain Lp82，一种肌肉p94的剪接变体，而不是m-calain的活性，与年轻啮齿动物的白内障形成最密切相关。此外，在人类晶状体中发现了第三种普遍存在的钙蛋白酶，在糖尿病大鼠的白内障中，钙蛋白酶10显著增加。Lp82、Lp85(晶状体中的一种低丰度剪接变异体)和calain 10可能在晶状体成熟或病理性白内障形成所必需的独特位置截断特定的晶状体蛋白质或切割蛋白质。为了检验这些想法，我们将追求三个具体目标： 1.对Lp82的生化性质和底物进行了测定。 2.确定p94变异体是否在人类晶状体中扮演啮齿动物Lp82的角色，并对人类晶状体中不明原因的蛋白质分解负责。 3.确定钙蛋白酶10在人类晶状体中的作用。 这些研究将为使用蛋白酶抑制剂预防白内障提供合理的依据。

DESCRIPTION (provided by applicant): This grant is focused on providing a detailed mechanism for the role of calcium-activated proteases (calpains) in lens. The overall hypothesis being tested is that proteolysis by calpains is essential for limited truncation, tighter packing of truncated beta-crystallins, and rearrangement of the cytoskeletal elements occurring during normal lens fiber differentiation and maturation. During cataract formation, over-activation of calpains by massive influx of calcium produces rapid proteolysis and light scattering because the truncated crystallins are no longer properly ordered. Studies during the latest grant cycle produced several unexpected findings relevant to this hypothesis: Activity of lens-specific calpain Lp82, a splice variant of muscle p94, and not m-calpain, was most strongly associated with cataract formation in young rodents. Further, calpain 10 was discovered as a third ubiquitous calpain in human lens, and calpain 10 was markedly increased in diabetic cataracts in rats. Lp82, Lp85 (a low-abundance splice variant in lens), and calpain 10 may truncate specific lens proteins or cleave proteins at unique sites necessary for proper lens maturation or pathologic cataract formation. To test these ideas, three specific aims will be pursued: 1. Determine the biochemical properties and substrates of Lp82. 2. Determine if p94 variants assume the role of rodent Lp82 in human lenses and are responsible for unexplained proteolysis in human lenses. 3. Define the roles of calpain 10 in human lens. These studies will provide a rational basis for the use of protease inhibitors to prevent cataracts.