Proteinase Inhbitors & Crystallin Fragments in Cataract

描述(申请人提供)：在老化过程中，由于聚集或交联，越来越多的晶状体总蛋白成为不溶于水的(WL)，它们进一步交联成共价多聚体被认为在年龄相关性(老年性)白内障的发展过程中导致混浊。在文献中，晶状体蛋白的各种翻译后修饰被描述为导致交联机制的因素，但它们的相关作用仍未确定。由于老年性白内障的发展是一个缓慢的过程，有时需要数年时间，因此很少有特定的修饰可能会作为触发因素加速交叉连接机制，并导致晶状体混浊。结果表明，修饰后的晶状体蛋白片段在晶状体蛋白的交联过程中起到了积极的作用。要理解 对于晶体蛋白片段的这种作用，人们必须确定它们的来源、翻译后修饰和交联机制，以便将它们作为致病因素。根据我们的结果，我们假设βA3/Al-晶体蛋白具有蛋白酶活性，由于其处于非活性状态，受到α晶体蛋白的抑制，并且在晶体蛋白底物水平上，其活性在体内受到调节。在激活时，βA3/A1-晶状体蛋白水解酶降解晶状体蛋白，晶状体蛋白片段经过翻译后修饰和本身的交联，并与两个珠状细丝蛋白(pahkinin和filensin)形成共价多聚体。为了验证上述假设，拟议的研究将集中回答以下四个问题：(A)与βA3/A1-晶体蛋白相关的Arg-键水解酶活性激活的分子机制是什么？ (B)βA3/A1-晶体蛋白水解酶活性在体内是如何调节的？(C)在衰老和白内障发展过程中，翻译后修饰的晶状体蛋白片段与珠状细丝蛋白(α激动素和细丝蛋白)的共价交联机制是什么？D)新发现的晶体蛋白片段的翻译后修饰(即N到D残基的脱酰胺，W和M残基的氧化，丝氨酸到脱氢丙氨酸的转化，以及H残基的甲酰化)在与珠状细丝蛋白的交联机制中有什么潜在的作用？ 上述研究将为老年性白内障的发展过程中如何发展混浊这一核心问题提供答案。由于这些研究将使用人类的晶状体，这些发现将有助于阐明βA3/A1-晶体蛋白水解酶在晶体蛋白的蛋白分解中的作用，它们在体内的调节，特别是晶体蛋白片段本身以及与pakinin和filensin的交联机制。

DESCRIPTION (provided by applicant): During aging, an increasing proportion of total lens proteins becomes water insoluble (Wl), either due to aggregation or cross-linking, and their further cross-linking into covalent multimers is believed to cause opacity during age-related (senile) cataract development. Various post-translational modifications of crystallins are described in the literature as causative factors for cross-linking mechanism, but their relative roles remain undefined. Because the senile cataract development is a slow process and sometimes takes years, few specific modifications might act as triggers to accelerate the cross-linking mechanism and cause lens opacity. Our results show that modified crystallin fragments play an active role in the crystallin cross-linking process. To understand such a role of crystallin fragments, one must determine their origin, post-translational modifications and cross-linking mechanism in order to implicate them as a causative factor. Based on our results, we have hypothesized that beta A3/Al-crystallin exhibits proteinase activity and the activity is regulated in vivo due to its existence in an inactive state, inhibition by alpha crystallin, and also at the crystailin substrate level. On activation, the beta A3/A1-crystallin proteinase proteolyses crystallins, and crystallin fragments undergo post-translational modifications and cross-link per se and with two beaded filament proteins (pahkinin and filensin) to form covalent multimers. To test the above hypothesis, the proposed studies will be focused to answer the following four questions: (A) What is the molecular mechanism of activation of an Arg-bond hydrolyzing proteinase activity associated with beta A3/A1-crystallin? (B) How is the beta A3/A1-crystallin proteinase activity regulated in vivo? (C) What is the covalent cross-linking mechanism of post-translationally modified crystallin fragments with beaded filaments proteins (phakinin and filensin) during aging and cataract development? D) What are the potential role of newly discovered posttranslational modifications (i.e., deamidation of N to D residue, oxidation of W and M residues, and conversion of serine to dehydroalanine, and formylation of H residue) in crystallin fragments in the cross-linking mechanism with beaded filament proteins? The above studies will provide answers to the central question of how opacity develops during age-related cataract development. Because human lenses will be used in these studies, the findings will be relevant in elucidating the role of beta A3/A1-crystallin proteinase in the proteolysis of crystallins, their regulation in vivo, and, in particular, the mechanism of cross-linking of crystallin fragments per se and with pakinin and filensin.