Molecular Analysis of Microdissected Human Lenses

描述(申请人提供)：这项建议的中心前提是，晶状体已经进化出几个关键的保护和修复系统，这些系统保持其透明功能，这些系统的功能与年龄相关的功能下降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成蛋白质蛋氨酸亚砜(PMSO)。PMSO在人类晶状体中的水平随着年龄的增长而增加，在人类老年性白内障中，PMSO占晶状体总蛋白的60%。PMSO导致蛋白质功能丧失、蛋白质聚集和细胞死亡。然而，到目前为止，PMSO在晶状体老化和白内障形成中的作用尚未确定。解锁PMSO在晶状体老化和白内障形成中的作用的关键之一是确定晶状体进化来防御PMSO损伤的修复机制，以及那些在蛋氨酸亚砜(MSO)形成时功能丧失的蛋白质。我们发现了一种新的PMSO修复酶，称为蛋氨酸亚砜还原酶A(MSRA)，对于晶状体抵御氧化应激，生存和防止白内障形成是必不可少的。由于PMSO随着年龄的增长在人类晶状体中积累，而且MSRA活性对于抗白内障是必不可少的，很可能是一个或多个晶状体蛋白在衰老时氧化为PMSO的MSRA修复丢失和/或氧化应激导致蛋白质功能丧失和白内障。MSRA修复的一个可能的目标是β-晶体蛋白，它以两个亚单位(？)的寡聚体存在于晶状体中。然后呢？B)每个含有两个保守的蛋氨酸。除了作为结构晶状体蛋白的作用外，β-晶状体蛋白作为分子伴侣保护晶状体蛋白免受年龄相关损伤，对晶状体功能也是必不可少的。这一应用将验证一种假设，即β-晶体蛋白氧化成蛋氨酸亚砜(？-Crystal allin-MSO)会导致伴侣功能的丧失，并且MSRA可以修复和恢复β-晶体蛋白-MSO的伴侣功能。因此，随着年龄的增长，MSRA活性的丧失可能导致β-晶体蛋白-MSO水平的增加，晶状体伴侣功能的丧失，最终导致白内障的形成。这些研究结果将为白内障的发展提供一个新的机制，并提供一个描述关键晶状体保护和修复系统相互依赖功能的创新模型。从这项工作中获得的信息可能会对基于增加MSRA活性从而预防白内障形成的合理治疗方案的设计产生重大影响。 与公共健康相关：晶状体已经进化出几个关键的保护和修复系统，以保持其透明的功能，这些系统的功能与年龄相关的功能下降导致年龄相关性白内障，这是世界范围内视力损害的主要原因。从这项工作中获得的信息将对合理设计预防白内障形成的治疗方法产生重大影响。

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.