Mechanisms of Protein Aging in Normal and Cataractous Lenses

摘要 白内障是世界范围内导致失明的主要原因。白内障晶状体摘除手术 给我们的经济带来了巨大的财政负担，并不是没有副作用。此外， 预计到2050年，美国的白内障患病率将增加一倍以上。 虽然正在开发新的治疗方法来预防或逆转早期白内障，但我们的 对白内障发生的潜在机制的了解仍然不清楚。长的- 我们研究的学期目标是确定晶状体蛋白质在衰老和 白内障的发生为了确定蛋白质老化机制并开发预防的方法， 延迟，或反向混浊。最近，我们发现了多个年龄相关的、非 酶生化过程导致与年龄相关的晶状体蛋白修饰和 白内障相关晶状体蛋白的交联物。涉及的晶状体蛋白质和氨基酸有 已经确定；然而，机械性细节仍未解决。此外，我们还观察到 在老化的晶状体组织中，可溶性晶状体蛋白戏剧性地重新分布到膜部分。 同样，这种蛋白质转移的操作机制(S)仍然未知。我们的假设是 特定的晶状体蛋白-蛋白质交联物、肽/脂的改变和晶体蛋白与晶状体的结合 膜是导致白内障的。为了验证这一假设，我们将使用最先进的成像技术 质谱学和蛋白质组学/脂质组学方法学，以进一步定义年龄相关和 白内障的特定修饰，并定义发生化学反应的条件 确定化学机理。具体地说，我们建议：1)定义蛋白质位点和 白内障人晶状体中蛋白质交联物的化学和，2)定义白内障特异性 人类晶状体中的多肽和脂质的变化，以及3)阐明导致 晶状体蛋白膜结合力随着年龄的增长和白内障的形成而增加。建议数 预计实验将提供有关蛋白质老化的新机制细节，不仅 告知新的白内障治疗方法的发展，同时也指导治疗的发展 其他衰老和蛋白质聚集性疾病。

ABSTRACT Cataract is the leading cause of blindness worldwide. Surgical removal of cataractous lenses places an enormous financial burden on our economy and is not without side effects. Moreover, the prevalence of cataract in the U.S. is expected to more than double by the year 2050. Although new therapies are being developed to prevent or reverse early cataract, our understanding of the underlying mechanisms of cataractogenesis remains unclear. The long- term goals of our research are to identify modifications to lens proteins during aging and cataractogenesis in order to define protein aging mechanisms and to develop ways to prevent, delay, or reverse opacification. Recently, we have identified multiple age-dependent, non- enzymatic biochemical processes that result in both age-related lens protein modification and cataract-related lens protein crosslinking. The lens proteins and amino acids involved have been identified; however, mechanistic details remain unresolved. In addition, we have observed a dramatic re-distribution of soluble lens proteins to the membrane fraction in aged lens tissue. Again, the operative mechanism(s) of this protein shift remains unknown. Our hypothesis is that specific lens protein-protein crosslinks, peptide/lipid alterations, and crystallin binding to lens membranes are cataractogenic. To test this hypothesis we will employ state-of-the-art imaging mass spectrometry and proteomics/lipidomics methodology to further define age-related and cataract-specific modifications and define conditions upon which the chemistry occurs to determine chemical mechanism. Specifically we propose to: 1) define the protein sites and chemistry of protein crosslinks in cataractous human lenses and, 2) define cataract-specific peptide and lipid changes in human lenses, and 3) elucidate the molecular events that lead to increased lens protein membrane binding with age and cataract formation. 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.