Maillard Reactions and Oxidation in Cataractogenesis

描述(申请人提供)：晶状体蛋白质在老化和白内障形成过程中经历了大量的物理化学变化；色素沉着和交联是两个主要的变化。虽然有几个生化途径参与了这些变化，但实际的机制及其相互关系仍不清楚。我们的项目解决了这个问题。二羰基化合物与蛋白质的反应似乎是晶状体蛋白质中色素和交联物的主要贡献者。最近的研究，包括我们自己的研究，指出了产生这些变化的另一种机制，涉及犬尿氨酸，色氨酸氧化产物，与晶状体蛋白反应产生色素和交联结构。我们的初步数据表明，二羰基和犬尿氨酸蛋白的修饰过程是相互关联和相互依赖的。虽然这些途径中的大多数反应都是非酶反应，但形成的反应性化合物可以引发进一步的反应，这些反应是由酶机制调节的。我们将在晶状体生理学的背景下研究这些机制。我们提出的研究旨在实现四个具体目标。在第一个目标中，我们将重点放在晶状体中的甲基乙二醛(MGO)上，这是一种高度活性的二羰基化合物。我们将确定乙醛酶I是如何代谢MGO的，以及乙醛酶I对晶状体上皮细胞在高血糖、氧化或亚硝化应激条件下生存的重要性。在目标2中，我们将确定双脱氧酮(DDO)途径在晶状体蛋白修饰中的作用。我们将确定这一最近发现的糖类和抗坏血酸修饰蛋白质的途径是如何促进晶状体老化和白内障形成的。在目标3中，我们将研究犬尿氨酸介导的蛋白质修饰。我们将主要关注吲哚胺2，3-双加氧酶(IDO)，这是一种催化色氨酸代谢为犬尿氨酸的酶。我们将利用转基因和基因敲除小鼠来研究IDO在晶状体蛋白修饰中的作用，并探讨高血糖和氧化应激对IDO活性的影响。犬尿氨酸对晶状体蛋白的损伤将通过使用高度特异的单抗的层析方法和免疫学分析来评估。最后，在目标4中，我们将确定犬尿氨酸、MGO和DDO在晶状体蛋白修饰中的关系。从这些实验中获得的累积信息将为我们提供关键的见解，了解晶状体老化和白内障形成的生物化学机制。

DESCRIPTION (provided by applicant): Lens proteins undergo numerous physico-chemical changes during aging and cataract formation; pigmentation and crosslinking are the two major ones. Although several biochemical pathways participate in these changes, the actual mechanisms and their interrelationships remain unclear. Our project addresses this very issue. Reactions of dicarbonyl compounds with proteins appear to be major contributors to pigmentation and crosslinking in lens proteins. Recent studies, including our own, point to yet another mechanism for producing these changes, involving kynurenines, tryptophan oxidation products that react with lens proteins to generate pigments and crosslinking structures. Our preliminary data suggest that the dicarbonyl and kynurenine protein modification processes are related and interdependent. Although most reactions in these pathways are non-enzymatic, the reactive compounds formed can initiate further reactions that are regulated by enzymatic mechanisms. We will investigate these mechanisms in the context of lens physiology. Our proposed research is organized to achieve four specific aims. In the first aim, we will focus on methylglyoxal (MGO), a highly reactive dicarbonyl compound in the lens. We will determine how MGO is metabolized by glyoxalase I as well as the importance of glyoxalase I for survival of lens epithelial cells under conditions of hyperglycemic, oxidative or nitrosative stress. In aim 2, we will establish the role of the dideoxyosone (DDO) pathway in lens protein modification. We will determine how this recently discovered pathway, through which sugars and ascorbic acid modify proteins, contributes to lens aging and cataract formation. In aim 3, we will study kynurenine-mediated protein modification. We will focus mainly on indoleamine 2,3-dioxygenase (IDO), an enzyme that catalyzes metabolism of tryptophan to kynurenines. We will use transgenic and knockout mice to investigate the role of IDO in lens protein modification, and explore the effect of hyperglycemia and oxidative stress on IDO activity. Kynurenine-mediated damage to lens proteins will be assessed by chromatographic methods and immunological assays with highly specific monoclonal antibodies. Finally, in aim 4, we will determine the relationship among kynurenines, MGO and DDO in lens protein modifications. Cummulative information gained from these experiments will provide us with critical insights into the biochemical mechanisms that underlie lens aging and cataract formation.