Project title Proteinase Inhibitors & Crystallin Fragments in Cataract

在老化过程中，越来越多的晶状体总蛋白变成不溶于水的(Wi)。 或者是由于聚集和/或交联性。交联型多聚体的尺寸增大 晶体蛋白变得如此之大，以至于它们最终变得不溶于水，并在 年龄相关性(老年性)白内障的发展。在各种各样的翻译后修饰中， 在人类衰老过程中，晶体蛋白的脱酰胺和截断被认为是最丰富的 隐形眼镜。因此，这些修饰在与年龄相关的聚集和交叉连接中起着重要作用 在老年性白内障的发生发展中，晶状体蛋白和晶状体蛋白是重要的致病因素。我们的 研究表明，~A3-晶状体蛋白是以一种可激活的蛋白水解酶的形式存在于晶状体中的。 酶能降解AA-、AB-、YC-和YD-晶体蛋白。此外，我们的研究表明， ~A3水解酶被AA-和AB-晶状体蛋白抑制。基于这些结果，我们有 假设~A3-蛋白酶活性在体内由AA-和AB-晶体蛋白作为抑制物调节， 激活的~A3-蛋白水解酶分解α-、~-和γ-晶体蛋白。晶体蛋白片段本身 聚合和/或进行翻译后修饰，例如去酰胺化。未修改的和 修饰的晶体蛋白片段与完整的晶体蛋白聚集并交联，首次形成水 可溶性-高分子量(WS-HMW)蛋白质，其中其组分交联并成为 水不溶。为了检验上述假设，拟议的研究将集中于回答 以下两个问题：(1)哪种多肽(氨基酸)形成~A3蛋白酶活性部位， AA-和AB-晶体蛋白是如何抑制该酶活性的？(2)这些蛋白的作用是什么 晶体蛋白片段和/或去酰胺化晶体蛋白在聚集体和交联过程中的作用 体内的晶体蛋白？ 为了回答第一个问题，我们将确定~A3-蛋白酶活性部位在 模体III和IV，蛋白酶诱导的a-、~-和y-晶体蛋白的体内蛋白分解及其抑制 AA和AB-晶状体蛋白对~A3-蛋白酶的作用机制为了回答第二个问题，我们将 确定a-、~-和y-晶体蛋白片段在体内是否被翻译后修饰 在衰老和白内障发展过程中，晶状体蛋白之间形成复合体的机制 AA-和AB-晶体蛋白中ASN的碎片和去胺化及脱酰胺作用(S) 使用转基因小鼠模型研究晶状体透明度。 由于这些研究将使用人类的晶状体，这些发现将与阐明 ~A3-蛋白酶的体内性质及其被AA-和AB-晶体蛋白作为抑制剂的调节 蛋白水解酶诱导晶体蛋白的降解，以及蛋白溶解的晶体蛋白片段和蛋白的潜在作用。 它们在不混浊形成过程中聚集和交联过程中的去酰胺化物种 老化的人类晶状体。 小灵通

During aging, an increasing proportion of total lens proteins becomes water insoluble (WI) either due to aggregation and/or cross-linking. The increased sizes of cross-linked multimers of crystallins become so large that they finally become water insoluble and cause lens opacity during age-related (senile) cataract development. Among the variety of post-translational modifications, deamidation and truncations of crystallins are identified as the most abundant during aging in human lenses. Therefore, these modifications playa major role in age-related aggregation and cross-linking of crystallins, and in tum, are significant causative factors in age-related cataract development. Our studies have shown that ~A3-crystallin exists as an activable proteinase in the lens, and the active enzyme is capable of proteolyzing aA-, aB-, yC- and yD-crystallins. Further, our studies demonstrated that ~A3 proteinase is inhibition by aA- and aB-crystallins. Based on these results, we have hypothesized that ~A3-proteinase activity is regulated in vivo by aA- and aB-crystallins as inhibitors, and the activated ~A3-proteinase proteolyzes a-, ~- and y-crystallins. The crystallin fragments per se aggregate and/or undergo post-translational modifications such as deamidation. The unmodified and modified crystallin fragments aggregate and cross-link with intact crystallins to first form the water soluble-high molecular weight (WS-HMW) proteins, where its components cross-link and become water insoluble. To test the above hypothesis, the proposed studies will be focused to answer the following two questions: (1) Which polypeptide (amino acids) forms the ~A3 proteinase active site, and how is the proteinase activity inhibited by aA- and aB-crystallins? (2) What are the roles of crystallin fragments and/or deamidated crystallins in aggregation and cross-linking processes of crystallins in vivo? To answer the first question, we will determine the ~A3-proteinase active site in the regions of the motifs III and IV, the proteinase-induced proteolysis of a-, ~- and y-crystallins in vivo, and the inhibition mechanism of ~A3-proteinase by aA and aB-crystallins. To answer the second question, we will determine whether the fragments of a-, ~- and y-crystallins are post-translationally modified in vivo during aging and cataract development, the mechanism of complex formation between crystallin fragments and deamidated crystallins, and effects of deamidation of Asn(s) in aA- and aB-crystallins on lens transparency using transgenic mouse models. Because human lenses will be used in these studies, the findings will be relevant in elucidation of in vivo properties of ~A3-proteinase, its regulation by aA- and aB-crystallins as inhibitors, the ~A3- proteinase-induced proteolysis of crystallins, and potential roles of protelyzed crystallin fragments and their deamidated species in aggregation and cross-linking process during development of opacity in aging human lenses. PHS