Molecular mechanisms of protein crosslinking in the lens

晶状体中蛋白质交联的分子机制

• 批准号: 8999881
• 负责人: Ram H Nagaraj
• 金额: $ 33.01万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-27 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8999881
• 关键词: Molecular; mechanisms; protein; crosslinking; lens

DESCRIPTION (provided by applicant): Our long-term goal is to prevent human cataracts by understanding the molecular mechanisms involved. This project builds on our previous work of nearly 25 years on lens protein modifications in aging and cataractogenesis. Protein crosslinking is a major modification in aged and cataractous lenses. Ascorbate (ASC) is a major constituent of the lens and in humans is present at concentrations up to 2 mM. ASC is oxidized in aged and cataractous lenses, and its oxidation products react rapidly with lens proteins to form pigmented and cross-linked proteins through formation of advanced glycation end products (AGEs). Recent work suggests that ASC oxidation product-mediated reactions contribute significantly to protein crosslinking in cataractous lenses. Reduced glutathione (GSH) offers some protection against this process, but the decreased levels of GSH in aged and cataractous lenses favor ASC oxidation. Mechanisms for such oxidation are unclear. Although molecular oxygen-mediated oxidation is likely to occur in the cortex, it is unlikely to occur in the nearly anoxic nucleus. Despite this limitation, ASC oxidation, along with protein crosslinking, aggregation and AGE-modification of proteins are most prominent in the nucleus of cataractous lenses. Kynurenines are tryptophan oxidation products produced by the kynurenine pathway initiated by indoleamine 2, 3-dioxygenase. They are present in relatively high levels in human lenses. Kynurenines undergo spontaneous deamination and bind covalently to lens proteins. Our preliminary studies show that both protein-free and protein-bound kynurenines promote ASC oxidation. UVA light has been considered to be an important risk factor for cataractogenesis, although the mechanisms are still obscure. Our preliminary experiments suggest that kynurenine-mediated ASC oxidation is significantly accelerated by UVA light, and that such oxidation can occur both in the presence and absence of oxygen. Based on these observations, we hypothesize that kynurenine- mediated ASC oxidation followed by AGE-modification of proteins plays an important role in the etiology of senile cataracts. We will test this hypothesis with the following three aims. In aim 1 we will determine kynurenine-mediated ASC oxidation in the presence and absence of oxygen and UVA light, conditions that emulate cortex and nucleus of the human lens. In aim 2 we will determine the impact of kynurenine-mediated ASC oxidation on covalent crosslinking and aggregation of lens proteins, and in aim 3, we will test our newly developed prodrug compounds on Kyn/ASC-mediated protein modification and crosslinking, and evaluate their effects on cataract development. The proposed studies should unravel the interplay between kynurenines and ASC in lens protein modification in human cataracts, and the findings could lead to innovative therapies to prevent or delay cataracts in humans.

描述（由申请人提供）：我们的长期目标是通过了解所涉及的分子机制来预防人类白内障。该项目建立在我们之前近 25 年的晶状体蛋白修饰与衰老和白内障发生相关的研究基础上。蛋白质交联是老化和白内障镜片的主要修饰。抗坏血酸 (ASC) 是晶状体的主要成分，在人体中的浓度高达 2 mM。 ASC 在老化和白内障晶状体中被氧化，其氧化产物与晶状体蛋白快速反应，通过形成晚期糖基化终产物 (AGE) 形成色素和交联蛋白。最近的研究表明，ASC 氧化产物介导的反应对白内障晶状体中的蛋白质交联有显着贡献。还原型谷胱甘肽 (GSH) 对这一过程提供了一些保护，但老化和白内障晶状体中 GSH 水平的降低有利于 ASC 氧化。这种氧化的机制尚不清楚。虽然分子氧介导的氧化很可能发生在皮质中，但不太可能发生在近缺氧的核中。尽管存在这一限制，ASC 氧化以及蛋白质交联、聚集和蛋白质 AGE 修饰在白内障晶状体的核中最为突出。犬尿氨酸是由吲哚胺 2, 3-双加氧酶启动的犬尿氨酸途径产生的色氨酸氧化产物。它们在人类晶状体中的含量相对较高。犬尿氨酸发生自发脱氨基作用并与晶状体蛋白共价结合。我们的初步研究表明，无蛋白质和蛋白质结合的犬尿氨酸都会促进 ASC 氧化。 UVA 光已被认为是白内障发生的重要危险因素，尽管其机制仍不清楚。我们的初步实验表明，UVA 光显着加速了犬尿氨酸介导的 ASC 氧化，并且这种氧化在有氧和无氧的情况下都可能发生。基于这些观察，我们假设犬尿氨酸介导的 ASC 氧化以及随后的蛋白质 AGE 修饰在老年性白内障的病因学中发挥着重要作用。我们将通过以下三个目标来检验这一假设。在目标 1 中，我们将确定在存在和不存在氧气和 UVA 光的情况下犬尿氨酸介导的 ASC 氧化，这些条件模拟人类晶状体的皮质和核。在目标 2 中，我们将确定犬尿氨酸介导的 ASC 氧化对晶状体蛋白共价交联和聚集的影响，在目标 3 中，我们将测试我们新开发的前药化合物对 Kyn/ASC 介导的蛋白质修饰和交联的影响，并评估它们对白内障发生的影响。拟议的研究应该揭示犬尿氨酸和 ASC 在人类白内障晶状体蛋白修饰中的相互作用，并且这些发现可能会带来预防或延迟人类白内障的创新疗法。