Proteinase Inhbitors & Crystallin Fragments in Cataract

蛋白酶抑制剂

• 批准号: 7115412
• 负责人: Om Prakash Srivastava
• 金额: $ 2.5万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7115412
• 关键词: SDS polyacrylamide gel electrophoresis; aging; cataract; crosslink; crystallins; endopeptidases; enzyme substrate; gel filtration chromatography; glycation; high performance liquid chromatography; human tissue; immunoprecipitation; membrane proteins; pathologic process; posttranslational modifications; protease inhibitor; protein sequence; proteolysis; radioimmunoassay; western blottings

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.

描述（由申请人提供）：在老化过程中，由于聚集或交联，越来越多比例的总透镜蛋白质变成水不溶性（WI），并且据信它们进一步交联成共价多聚体会在年龄相关性（老年性）白内障发展过程中引起不透明。晶体蛋白的各种翻译后修饰在文献中被描述为交联机制的致病因素，但它们的相对作用仍不明确。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，很少有特定的修改可能会作为触发器，加速交联机制，并导致透镜混浊。我们的研究结果表明，修饰的晶体蛋白片段在晶体蛋白交联过程中起着积极的作用。了解 晶体蛋白片段的这种作用，必须确定它们的来源、翻译后修饰和交联机制，以便将它们牵连为致病因素。基于我们的结果，我们假设β A3/Al-晶状体蛋白表现出蛋白酶活性，并且由于其以非活性状态存在、被α晶状体蛋白抑制以及在晶状体蛋白底物水平，活性在体内受到调节。在活化时，β A3/A1-晶状体蛋白蛋白酶蛋白水解晶状体蛋白，晶状体蛋白片段经历翻译后修饰和本身交联，并与两个珠状丝蛋白（帕激肽和filensin）形成共价多聚体。为了验证上述假设，所提出的研究将集中于回答以下四个问题：（A）与β A3/A1-晶状体蛋白相关的Arg键水解蛋白酶活性的激活的分子机制是什么？ (B)β A3/A1-晶状体蛋白酶活性在体内是如何调节的？(C)在老化和白内障发展过程中，后修饰的晶状体蛋白片段与珠状丝蛋白（phakinin和filensin）的共价交联机制是什么？D）新发现的翻译后修饰的潜在作用是什么（即，N残基脱酰胺为D残基，W残基和M残基氧化，丝氨酸转化为脱氢丙氨酸，H残基脱酰胺）在晶体蛋白片段与珠状丝蛋白的交联机制中？ 上述研究将为年龄相关性白内障发展过程中混浊如何发展这一核心问题提供答案。由于这些研究将使用人类晶状体，因此研究结果将与阐明β A3/A1-晶状体蛋白酶在晶状体蛋白的蛋白水解中的作用、晶状体蛋白在体内的调节以及晶状体蛋白片段本身以及与巴激肽和filensin的交联机制有关。