Amino-Carbonyl Reactions in the Aging Human Lens

描述：这项研究计划提出一种假说，即还原糖、抗坏血酸和蛋白质之间的氨基-羰基反应(所谓的美拉德反应)对晶状体晶体蛋白的广泛损害是导致人类晶状体老化的原因。我们的关键假设是，这些糖基化产物的积累使晶状体易于发生白内障，并且大部分反应是由抗坏血酸氧化产物催化的。对于这一假设的进展，在前一个资助期已经取得了几项突破。首先，荧光团LM-1的结构被鉴定为Vesperlyine A，一种抗坏血酸催化的蛋白质交联物，在糖尿病人晶状体中特异性增加。其次，我们发现人晶状体中的主要抗坏血酸产物--羧甲基赖氨酸与氧化还原活性Cu2+结合。第三，我们开创了一种新的基于氟标记抗坏血酸(F-ASA)和高分辨率19F-核磁共振光谱的代谢组学方法，发现氧化应激晶状体上皮细胞和白内障积累了F-ASA降解产物。第四，我们发现、克隆并阐明了脱糖酶的催化机制，为今后在转基因动物过度糖基化模型中的应用奠定了基础。最后，也是最近，我们发现了在老化的人类晶状体中，精氨酸在糖基化催化下广泛转化为鸟氨酸的证据。在接下来的几年里，我们提出了一种全新的、我们认为非常有希望的方法，通过建立三种加速晶状体损伤的转基因/条件性基因敲除模型来解决通过抗坏血酸和糖基化来解决晶状体老化的问题。基于啮齿动物晶状体对抗坏血酸摄取不足的发现，我们建议首先通过在晶状体中过表达人类维生素C转运蛋白2(HSVCT2)来建立人类晶状体的小鼠模型。将研究转基因对晶状体动态平衡、蛋白质色素沉着、碎裂和交联性的影响。然后，这只人源化的hSVCT+/+小鼠将与一只谷胱甘肽合成不足的小鼠杂交，该小鼠是通过有条件地敲除晶状体中的谷氨酰半胱氨酸连接酶(？-GCLC)而获得的。基因敲除对晶状体晶状体蛋白变化的影响也有待进一步研究。由于抗坏血酸循环障碍，纯合子hSVCT+/+/？-GCIC-/-杂合小鼠晶状体预计会积累大量的反应性抗坏血酸产物。所采取的方法将使我们能够单独研究多种形式的蛋白质损伤，即抗坏血酸、糖基化和氧化对晶状体伴侣蛋白和其他性质的影响。在这些基因操作的帮助下，人们希望将人类晶状体长达70年的衰老过程浓缩为24个毫秒。这一小鼠模型有望成为开发新的遗传和药物干预措施对抗晶状体衰老的有力工具。

DESCRIPTION: This research program addresses the hypothesis that extensive damage to lens crystallins by amino-carbonyl reactions between reducing sugars, ascorbic acid and proteins (so-called Maillard reaction) contributes to the aging of the human lens. Our key hypothesis is that the accumulation of these glycation products predisposes the lens toward cataractogenesis, and that much of the reaction is catalyzed by ascorbic acid oxidation products. Toward this hypothesis progress several breakthroughs have been achieved in the previous funding period. First, the structure of the fluorophore LM-1 was identified as Vesperlysine A, an ascorbate catalyzed protein crosslink that is specifically increased in diabetic human lenses. Second, we discovered carboxymethyl-lysine, a major ascorbylation product, binds redox active Cu 2+ in the human lens. Third, we have pioneered a novel "metabolomics" approach based on fluorine-labeled ascorbic acid (F-ASA) and high resolution 19F-NMR spectroscopy and found that oxidatively stressed lens epithelial cells and cataracts accumulate F-ASA degradation products. Fourth, we discovered, cloned and elucidated the catalytic mechanism of deglycating enzymes for future use in transgenic animal models of excessive glycation. Finally and most recently, we discovered evidence of extensive glycation-catalyzed conversion of arginine into ornithine in the aging human lens. In the coming years we propose an entirely new and, we believe, highly promising approach to the problem of lenticular aging by ascorbylation and glycation through the creation of three transgenic/conditional knockout models of accelerated damage to the lens. Based on the discovery that rodent lenses are deficient in ascorbic acid uptake, we propose to first create a mouse model of the human lens by overexpressing the human Vitamin C Transporter 2 (hSVCT2) in the lens. The impact of this transgene on lenticular homeostasis, protein pigmentation, fragmentation and crosslinking will be studied. This humanized hSVCT+/+ mouse will be then be crossbred with a mouse deficient in glutathione synthesis obtained by conditionally knocking out ?-glutamyl cysteine ligase (?-Gclc) from the lens. The impact of the knockout on lens crystallin changes with be similarly studied. The homozygous hSVCT+/+/ ?-GcIc-/- hybrid mouse lens is expected to accumulate substantial levels of reactive ascorbylation products due to impairment of ascorbic acid recycling. The approach taken will allow us to study separately the impact of multiple forms of protein damage, i.e. ascorbylation, glycation and oxidation on lens chaperone and other properties. With the help of these genetic manipulations, it is hoped that the 70-yr long aging process of the human lens can be condensed into 24 mos. This mouse model is expected to become a potent tool for the development of novel genetic and pharmacological interventions against lenticular aging.