Mechanisms of Protein Aging in Normal and Cataractous Lenses

描述(申请人提供)：白内障是世界范围内导致失明的主要原因，其唯一的治疗方法是手术切除，可能会导致并发症，并给我们的经济带来巨大的经济负担。预计到2050年，美国的白内障患病率将增加一倍以上。尽管进行了几十年的研究，但与年龄相关的蛋白质聚集和晶状体混浊的原因仍然不清楚，也没有预防或延缓白内障的治疗方法。我们研究的长期目标是确定晶状体蛋白质在 衰老和白内障发生，以了解蛋白质衰老机制，并开发预防或延缓有害事件的方法。最近，我们发现了一种年龄相关的非酶生化机制，导致大量与年龄相关的晶状体蛋白修饰和与白内障相关的晶状体蛋白交联。这种化学作用发生在磷酸丝氨酸和半胱氨酸残基上，涉及谷胱甘肽(GSH)和晶状体中其他亲核试剂的修饰，包括蛋白质硫醇，导致不可逆的蛋白质交联。或假说，晶状体蛋白质被GSH非二硫硫基化阻止蛋白质-蛋白质交联，但随着年龄的增长，这种保护机制被淹没，导致不可逆转的交联、蛋白质聚集和晶状体混浊。为了验证这一假设，我们将使用最先进的蛋白质组学方法来进一步定义与年龄相关的和特定于白内障的修饰，评估发生化学变化的条件，并验证检测到的修饰和未来治疗用途的动物模型。具体地说，我们建议：1)表征和定量正常人和白内障人晶状体中的蛋白质非二硫修饰和蛋白质交联；2)确定控制非二硫修饰和蛋白质交联的条件；以及3)在人类核性白内障的动物模型中表征晶状体蛋白质的非二硫修饰和蛋白质交联。这些拟议的实验有望提供蛋白质老化的新机制细节，不仅将为新的白内障治疗方法的开发提供信息，还将指导其他衰老和蛋白质聚集性疾病的治疗开发。

DESCRIPTION (provided by applicant): Cataract is the leading cause of blindness worldwide and its only cure, surgical removal, can result in complications and places an enormous financial burden on our economy. The prevalence of cataract in the U.S. is expected to more than double by the year 2050. Despite decades of research, the causes of age-related protein aggregation and lens opacity remain ill- defined and no therapies exist to prevent or delay cataract. The long-term goals of our research are to identify modifications to lens proteins during aging and cataractogenesis in order to understand protein aging mechanisms and to develop ways to prevent or delay deleterious events. Recently, we have discovered an age-dependent, non-enzymatic biochemical mechanism that results in significant amounts of both age-related lens protein modification and cataract-related lens protein crosslinking. This chemistry occurs at phosphoserine and cysteine residues and involves modification by glutathione (GSH) and other nucleophiles in the lens including protein thiols resulting in irreversible protein crosslinking. Or hypothesis is that lens protein non-disulfide thiolation by GSH prevents protein-protein crosslinking, but with age this protective mechanism is overwhelmed resulting in irreversible crosslinking, protein aggregation, and lens opacification. To test this hypothesis we will employ state-of-the-art proteomics methodology to further define age-related and cataract-specific modifications, evaluate conditions upon which the chemistry occurs, and validate an animal model for detected modifications and future therapeutic use. Specifically we propose to: 1) characterize and quantify protein non-disulfide thiol modifications and protein crosslinks in normal and cataractous human lenses, 2) determine conditions controlling non-disulfide thiol modification and protein crosslinking, and 3) characterize lens protein non-disulfide modifications and protein crosslinks in an animal model of human nuclear cataract. The proposed experiments are expected to provide new mechanistic details on protein aging that will not only inform the development of new cataract treatments, but also guide therapeutic development for other aging and protein aggregation diseases.