Molecular Analysis of Microdissected Human Lenses

描述（由申请人提供）：本提案的中心前提是晶状体已经进化出几个关键的保护和修复系统，以保持其透明功能，并且这些系统功能的年龄相关下降会导致年龄相关性白内障。人类晶状体老化和白内障形成的一个标志是蛋白质蛋氨酸氧化为蛋白质蛋氨酸亚砜（PMSO）。人体晶状体中的PMSO水平随着年龄的增长而增加，在与年龄有关的白内障中，晶状体总蛋白的60%为PMSO。PMSO导致蛋白质功能丧失、蛋白质聚集和细胞死亡。然而，迄今为止，PMSO在晶状体老化和白内障形成中的作用尚未确定。解开PMSO在晶状体老化和白内障形成中的作用的一个关键是确定晶状体已经进化到防御PMSO损伤的修复机制和那些在蛋氨酸亚砜（MSO）形成时功能丧失的蛋白质。我们已经发现了一种新的PMSO修复酶，称为蛋氨酸亚砜还原酶a (MsrA)，对晶状体防御氧化应激，生存能力和防御白内障形成至关重要。由于PMSO会随着年龄的增长而在人体晶状体中积累，并且MsrA的活性对白内障的抵抗至关重要，因此在衰老和/或氧化应激时，一种或多种被氧化为PMSO的晶状体蛋白的MsrA修复功能的丧失可能会导致蛋白质功能的丧失和白内障的发生。MsrA修复的可能目标是什么？晶状体中以两个亚基(？A和？B)每个含有两个保守的蛋氨酸。除了作为晶状体结构结晶蛋白的作用外，？-晶状体蛋白是晶状体功能的重要组成部分，它可以作为分子伴侣保护晶状体蛋白免受年龄相关损伤。这个应用程序将测试氧化？-结晶蛋白转化为蛋氨酸亚砜(？- crystin - mso)导致伴侣功能丧失，MsrA可以修复和恢复？-crystallin-MSO。因此，随着年龄的增长，MsrA活性的丧失可能导致？-晶状体蛋白- mso，晶状体伴侣功能丧失，最终形成白内障。这些研究结果将提供白内障发展的新机制和描述关键晶状体保护和修复系统相互依存功能的创新模型。从这项工作中获得的信息可能对基于增加MsrA活性从而预防白内障形成的治疗方法的合理设计产生重大影响。

DESCRIPTION (provided by applicant): The central premise of this proposal is that the lens has evolved several key protective and repair systems that maintain its transparent function and that an age-related decrease in the function of these systems contributes to age-related cataract. A hallmark of human lens aging and cataract formation is oxidation of protein methionines to protein methionine sulfoxide (PMSO). PMSO levels increase in the human lens with age and in human age-related cataract, 60% of total lens protein is found as PMSO. PMSO causes loss of protein function, protein aggregation and cell death. However, to date, the role of PMSO in lens aging and cataract formation has not been established. One key to unlocking the role of PMSO in lens aging and cataract formation is to identify those repair mechanisms that the lens has evolved to defend against PMSO damage and those proteins whose functions are lost upon methionine sulfoxide (MSO) formation. We have discovered that a novel PMSO repair enzyme called methionine sulfoxide reductase A (MsrA) is essential for lens defense against oxidative stress, viability, and defense against cataract formation. Since PMSO accumulates in the human lens with increasing age and since MsrA activity is essential for cataract resistance, it is likely that loss of MsrA repair of one or more lens proteins oxidized to PMSO upon aging and/or oxidative stress contributes to loss of protein function and cataract. A likely target for MsrA repair is ?-crystallin, which exists in the lens as an oligomer of two subunits (? A and ? B) each containing two conserved methionines. In addition to its role as a structural lens crystallin, ? -crystallin is essential for lens function through its ability to act as a molecular chaperone that protects lens proteins against age-related damage. This application will test the hypothesis that oxidation of ? -crystallin to methionine sulfoxide (? -crystallin-MSO) causes loss of chaperone function and that MsrA can repair and restore the chaperone function of ? -crystallin-MSO. Thus, loss of MsrA activity upon aging could result in increased levels of ? -crystallin-MSO, loss of lens chaperone function and ultimately cataract formation. The results of these studies will provide a novel mechanism for cataract development and an innovative model describing the interdependent functions of key lens protective and repair systems. The information gained from this work could have a major impact on the rational design of therapeutics based on increasing the activity of MsrA thereby preventing cataract formation. PUBLIC HEALTH RELEVANCE: The lens has evolved several key protective and repair systems that maintain its transparent function and an age-related decrease in the function of these systems contributes to age-related cataract the leading cause of visual impairment world-wide. The information gained from this work will have a major impact on the rational design of therapeutics that could prevent cataract formation.