Mechanisms of Protein Aging in Normal and Cataractous Lenses

描述（由申请人提供）：白内障是全世界导致失明的主要原因，其唯一的治疗方法是手术切除，但可能会导致并发症，并给我们的经济带来巨大的经济负担。预计到 2050 年，美国白内障的患病率将增加一倍以上。尽管经过数十年的研究，与年龄相关的蛋白质聚集和晶状体混浊的原因仍然不明确，并且没有预防或延迟白内障的治疗方法。我们研究的长期目标是确定晶状体蛋白的修饰 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迟有害事件的方法。最近，我们发现了一种年龄依赖性、非酶生化机制，可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。这种化学反应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（包括蛋白质硫醇）的修饰，从而导致不可逆的蛋白质交联。或者假设谷胱甘肽的晶状体蛋白非二硫键硫醇化作用可防止蛋白质与蛋白质交联，但随着年龄的增长，这种保护机制被压垮，导致不可逆的交联、蛋白质聚集和晶状体混浊。为了检验这一假设，我们将采用最先进的蛋白质组学方法来进一步定义与年龄相关和白内障特异性的修饰，评估化学发生的条件，并验证动物模型以检测到的修饰和未来的治疗用途。具体来说，我们建议：1）表征和量化正常和白内障人类晶状体中蛋白质非二硫键修饰和蛋白质交联，2）确定控制非二硫键修饰和蛋白质交联的条件，以及3）表征人类核白内障动物模型中晶状体蛋白质非二硫键修饰和蛋白质交联。拟议的实验预计将提供有关蛋白质衰老的新机制细节，这不仅将为新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.