Proteinase Inhibitors and Crystallin Fragments in Cataract

描述（由申请人提供）：年龄相关性（老年性）白内障主要是一种晶体蛋白聚集和随后沉淀的疾病，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非常大，以至于它们最终变得不溶于水，导致晶状体混浊。晶体蛋白的脱酰胺和截断被认为是最丰富的翻译后修饰（PTMs），被认为是年龄相关性白内障发展的主要原因。然而，脱酰胺或截短晶体蛋白对白内障发展的作用机制尚不清楚。基于我们广泛的结果，我们假设晶状体蛋白的脱酰胺和截短通过产生沉积在晶状体膜上的聚集体并导致纤维细胞变性而协同导致白内障。聚集过程由截短的晶体蛋白片段开始，形成淀粉样原纤维型

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.