Proteinase Inhibitors and Crystallin Fragments in Cataract

描述(由申请人提供)：老年性(老年性)白内障主要是一种晶体蛋白聚集和随后的沉淀性疾病，发生在几年的时间框架内。在白内障的发展过程中，聚集的和交联的晶体蛋白多聚体的尺寸变得如此之大，以至于它们最终变得不溶于水，导致晶状体混浊。晶状体蛋白的脱酰胺和截短被认为是最丰富的翻译后修饰(PTM)，被认为是老年性白内障发生的主要原因。然而，去酰胺化或截短型晶体蛋白导致白内障发生的机制仍不清楚。基于我们广泛的结果，我们假设晶体蛋白的去酰胺化和截断通过产生沉积在晶状体膜上的聚集体而协同导致白内障，并导致纤维细胞退化。聚集过程是由截断的晶体蛋白片段启动的，这些晶体蛋白片段形成了淀粉样原纤维类型的 单独和/或与去酰胺化的晶体蛋白、细丝蛋白和α激肽的片段形成复合体。在这一过程中，A3-蛋白酶衍生的脱胺化晶体蛋白片段发挥了主要作用。我们计划通过寻求以下三个问题的答案来检验上述假设：(1)A-N101D转基因小鼠模型白内障发生的分子机制是什么？我们有史以来第一个转基因A-N101D转基因小鼠在大约7个月大的时候患上了皮质性白内障。由于与野生型小鼠相比，上述几个表型变化会在7个月大的转基因小鼠中引发白内障的发生，因此将利用小鼠模型来阐明去胺化AN101D诱导白内障发生的分子机制。(2)A3蛋白水解酶的活性部位是如何在体内调节和激活以降解晶体蛋白的？我们将确定活性部位A3蛋白酶是如何由其N进行内在调节的 在体外和在A-N101D转基因小鼠中，与未修饰的晶体蛋白相比，A3-蛋白酶如何优先降解去酰胺化和/或截断的晶体蛋白。(3)4-kDa B片段与晶状体蛋白(经A3-蛋白水解酶分解)、蛋白激动素和细丝蛋白片段聚集的分子机制是什么？将确定淀粉样变是否是晶体蛋白片段(通过A3-蛋白酶降解而来)、细丝蛋白和蛋白激肽片段以及体内存在的脱胺4-kDa？B片段之间的聚集机制。由于这些研究将主要使用人类晶状体，因此这些发现将与年龄相关性白内障发生的体内机制相关。本研究结果对延缓老年性白内障的发生发展具有重要的治疗价值。

DESCRIPTION (provided by applicant): Age-related (senile) cataract is principally a crystallin protein aggregation and subsequent precipitation disease that occurs over the time frame of several years. During cataract development, the increased sizes of aggregated and cross-linked crystallin multimers become so large that they finally become water insoluble and cause lens opacity. Deamidation and truncations of crystallins are identified as the most abundant post-translational modifications (PTMs), and are believed to be the major causative factors in age-related cataract development. However, the mechanisms of cataract development by either deamidated- or truncated-crystallins are still unknown. Based on our extensive results, we have hypothesized that deamidated and truncation of crystallins synergistically cause cataract by producing aggregates that deposit on lens membranes and lead to fiber cells degeneration. The aggregation process is initiated by truncated crystallin fragments that form amyloid fibril-type of complexes alone and/or with fragments of deamidated crystallins, filensin and phakinin. In this process, ¿A3-protease-derived fragments of deamidated crystallins play a major role. We plan to test the above hypothesis by seeking answers to the following three questions: (1) What is molecular mechanism of cataractogenesis in ¿A-N101D transgenic mouse model? Our first ever available transgenic mouse model with ¿A-N101D transgene develops cortical cataract at about 7-month of age. Because several preceding phenotypic changes trigger cataract development at 7-months of age in transgenic mice relative to wild type mice, the mouse model will be used to elucidate the molecular mechanism of deamidated ¿AN101D-induced cataract development. (2) How is the active site of ¿A3 protease regulated and activated in vivo to proteolyze crystallins? We will determine how the active site ¿A3 protease is regulated intrinsically by its N terminal arm and extrinsically by ¿A- and ¿B-crystallins as inhibitors, and how ¿A3-protease preferentially proteolyzes deamidated and/or truncated crystallins relative to unmodified crystallins in vitro and in ¿A-N101D transgenic mice. (3) What is the molecular mechanism of aggregation of fragments of crystallins (derived via proteolysis by ¿A3-protease) and phakinin and filensin with a deamidated 4-kDa ¿B fragment? It will be determined whether amyloidogenesis is the mechanism of aggregation among crystallin fragments (derived via proteolysis by ¿A3-protease), fragments of filensin and phakinin, and an in vivo existing deamidated 4-kDa ¿B fragment. Because mainly human lenses will be used in these studies, the findings will be relevant to in vivo mechanism of age-related cataract development. The results will be of significant therapeutic value to delay the development and progression of age-related cataract.