Proteinase Inhibitors and Crystallin Fragments in Cataract

描述（由申请人提供）：晶状体相关（老年性）白内障主要是一种晶状体蛋白聚集和随后的沉淀性疾病，发生在数年的时间范围内。在白内障发展过程中，聚集和交联的晶状体蛋白多聚体的增加的尺寸变得如此之大，以至于它们最终变得不溶于水并引起透镜不透明。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），并且被认为是年龄相关性白内障发展的主要致病因素。然而，脱酰胺或截短的晶状体蛋白引起白内障的机制仍不清楚。基于我们的广泛结果，我们假设晶状体蛋白的脱酰胺和截短通过产生聚集体协同引起白内障，所述聚集体存款在透镜膜上并导致纤维细胞变性。聚集过程是由截短的晶体蛋白片段开始的，这些片段形成淀粉样纤维型的 单独的复合物和/或与脱酰胺的晶状体蛋白、filensin和phakinin的片段的复合物。在这个过程中，脱酰胺晶体蛋白的Δ 3-蛋白酶衍生片段起主要作用。我们计划通过以下三个问题来验证上述假设：（1）A-N101 D转基因小鼠模型中白内障发生的分子机制是什么？我们的第一个可用的转基因小鼠模型与<$A-N101 D转基因发展皮质性白内障在约7个月大。由于与野生型小鼠相比，转基因小鼠在7月龄时发生了几种先前的表型变化，因此将使用小鼠模型来阐明脱酰胺AN 101 D诱导的白内障发生的分子机制。(2)在体内，<$A3蛋白酶的活性位点是如何调节和激活以蛋白水解晶体蛋白的？我们将确定活性位点<$A3蛋白酶是如何由其N 通过<$A-和<$B-晶体蛋白作为抑制剂的末端臂和外排，以及<$A3-蛋白酶如何在体外和<$A-N101 D转基因小鼠中相对于未修饰的晶体蛋白优先蛋白水解脱酰胺和/或截短的晶体蛋白。(3)晶体蛋白（通过<$A3蛋白酶的蛋白水解作用获得）、phakinin和filensin片段与脱酰胺的4 kDa <$B片段聚集的分子机制是什么？将确定淀粉样蛋白生成是否是晶状体蛋白片段（通过<$A3-蛋白酶的蛋白水解衍生）、filensin和phakinin片段以及体内存在的脱酰胺4-kDa <$B片段之间聚集的机制。由于这些研究主要使用人类晶状体，因此研究结果将与年龄相关性白内障发生的体内机制相关。该结果对延缓年龄相关性白内障的发生和发展具有重要的治疗价值。 公共卫生相关性：该提案的总体目标是阐明人类年龄相关性白内障发展的分子机制，这是由于晶状体蛋白的两种最普遍但协同的体内翻译后修饰：脱酰胺和截短。这些研究将为脱酰胺和截短的晶状体蛋白如何聚集并导致透镜混浊的机制这一中心问题提供答案。这些发现将用于治疗目的，以延缓人类年龄相关性白内障。

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. PUBLIC HEALTH RELEVANCE: The overall goal of the proposal is to elucidate molecular mechanism of development of human age-related cataract due to the two most prevalent but synergistic in vivo post-translational modifications of crystallins: deamidation and truncation. Th proposed studies will provide answers to the central question about the mechanism of how deamidated and truncated crystallins aggregate and cause lens opacity. The findings would be used for therapeutic purpose to delay human age-related cataract.