Molecular mechanisms of protein crosslinking in the lens

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。这个项目建立在我们之前近25年的关于晶状体蛋白在衰老和白内障发生中的修饰的工作基础上。蛋白质交联是老化和白内障晶状体的主要修饰。抗坏血酸（ASC）是晶状体的主要成分，其在人体晶状体中的浓度高达2毫米。ASC在老年和白内障晶状体中被氧化，其氧化产物通过形成晚期糖基化终产物（AGEs）与晶状体蛋白迅速反应形成色素和交联蛋白。还原型谷胱甘肽（GSH）对这一过程提供了一定的保护，但衰老和白内障晶状体中GSH水平的降低有利于ASC氧化。最近的研究表明，白内障晶状体中的大部分蛋白质交联是由ASC氧化产物介导的。我们知道ASC在老化和白内障晶状体中被氧化，但我们不知道这种氧化的机制。虽然分子氧介导的氧化很可能发生在皮层，但不太可能发生在缺氧核附近。尽管存在这种局限性，但通过AGE形成的蛋白质交联和聚集在白内障晶状体核中最为突出。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。它们在人体晶状体中的含量相对较高。犬尿氨酸可以自发脱氨并与晶状体蛋白共价结合。我们的初步研究表明，无蛋白和蛋白结合的犬尿氨酸都促进ASC氧化。UVA光被认为是白内障发生的一个重要危险因素，尽管其机制尚不清楚。我们的初步实验表明，UVA光显著加速了犬尿氨酸介导的ASC氧化，并且这种氧化可以在有氧和无氧的情况下发生。基于这些观察结果，我们假设犬尿氨酸介导的ASC氧化随后的蛋白质修饰在老年性白内障的病因中起重要作用。我们将用以下三个目标来检验这一假设。在目标1中，我们将在模拟人类晶状体皮层和晶状体核的条件下，在有和没有氧气和UVA光的情况下，确定犬尿氨酸介导的ASC氧化。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介导的蛋白修饰和交联的影响，并评估它们对白内障发展的影响。总之，拟议的研究将揭示犬尿氨酸和ASC在人类白内障晶状体蛋白修饰中的相互作用，这些发现可能会导致预防或延缓人类白内障的创新疗法。

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, which in the human lens 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 crosslinked proteins through formation of advanced glycation end products (AGEs). Reduced glutathione (GSH) offers some protection against this process, but the decreased levels of GSH in aged and cataractous lenses favor ASC oxidation. Recent work suggests that much of the protein crosslinking in cataractous lenses are ASC oxidation product-mediated. We know that ASC is oxidized in aging and cataractous lenses, but we do not know the mechanisms for such oxidation. Although molecular oxygen- mediated oxidation is likely to occur in the cortex, it is unlikely to occur in the near anoxic nucleus. Despite this limitation, protein crosslinking and aggregation through AGE formation is 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 as 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 protein modification 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. Together, the proposed studies will 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.