Calcium-activated protease in cataract formation

描述（由申请人提供）：该资助的重点是提供钙活化蛋白酶（calpain）在晶状体中的作用的详细机制。总的假设是，在正常晶态纤维分化和成熟过程中，钙蛋白酶的蛋白质水解对有限的截短、截短的β -结晶蛋白的紧密堆积和细胞骨架元素的重排是必不可少的。在白内障形成过程中，由于大量钙流入导致的钙蛋白酶过度激活会产生快速的蛋白水解和光散射，因为截短的晶体蛋白不再有序。在最近的资助周期中，研究产生了一些与这一假设相关的意想不到的发现：晶状体特异性calpain Lp82（肌肉p94的一种剪切变体）的活性，而不是m-calpain，与幼龄啮齿动物的白内障形成最密切相关。此外，还发现calpain10是人体晶状体中普遍存在的第三种calpain10，并且在大鼠糖尿病性白内障中calpain10明显升高。Lp82, Lp85（晶状体中的低丰度剪接变体）和calpain10可以截断特定的晶状体蛋白或在晶状体成熟或病理性白内障形成所必需的独特位置切割蛋白质。为了测试这些想法，将追求三个具体目标：

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.