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: 8482333
• 负责人: Ram H Nagaraj
• 金额: $ 39.63万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8482333
• 关键词: Advanced Glycosylation End Products; Aging; Animal Model; Arginine; Ascorbic Acid; Binding; Cataract; Cell Nucleus; Cells; Chemicals; Crystallins; Data; Deamination; Detection; Development; Environment; Etiology; Eye; Goals; Human; In Vitro; Incubated; Innovative Therapy; Kynurenine; Laboratories; Lead; Lysine; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Methods; Modification; Molecular; Nuclear; Organ Culture Techniques; Oxygen; Oxygen measurement, partial pressure, arterial; Paper; Pathway interactions; Pattern; Physiological; Pigmentation physiologic function; Pigments; Play; Post-Translational Protein Processing; Process; Prodrugs; Protein Binding; Proteins; Reaction; Reduced Glutathione; Risk Factors; Role; Seminal; Senile Cataract; Structure; Surface; Testing; Transgenic Animals; Transgenic Mice; Tryptophan; Tryptophan 2,3 Dioxygenase; Ultraviolet A radiation; Water; Work; adduct; aged; analytical method; ascorbate; base; crosslink; efficacy testing; inhibitor/antagonist; interest; lens; lens cortex; lens protein; molecular mass; novel; oxidation; pentosidine; prevent; protein aggregate; protein aggregation; protein crosslink; public health relevance; research study

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.

我们的长期目标是通过了解所涉及的分子机制来防止人白内障。这 项目基于我们以前在近25年的镜头蛋白质修饰和衰老和 白内生生成。蛋白质交联是老化和白内障透镜的重大修饰。抗坏血酸 （ASC）是晶状体的主要组成部分，在人类镜头中以高达2 mm的浓度存在。 ASC 在老年和白内障透镜中被氧化，其氧化产物与晶状体蛋白迅速反应 通过形成晚期糖基化最终产物（年龄）的色素和交联蛋白。减少 谷胱甘肽（GSH）为这一过程提供了一些保护，但老化的GSH水平降低 白内科镜头有利于ASC氧化。最近的工作表明，大部分蛋白质交联 白内障透镜是ASC氧化产物介导的。我们知道ASC在衰老和 白内科镜头，但我们不知道这种氧化的机制。尽管分子氧 介导的氧化可能发生在皮质中，不太可能发生在近乎缺氧的核中。尽管如此 通过年龄形成的限制，蛋白质交联和聚集在 白内科镜头。 kynurenine是由Kynurenine途径产生的色氨酸氧化产物 由吲哚胺2,3-二氧酶发起。它们存在于人类镜片中相对较高的水平。 Kynurenine会自发脱氨基，并共价与透镜蛋白结合。我们的初步研究 表明无蛋白质和蛋白质结合的kynurenines促进了ASC氧化。 Uva Light一直是 尽管机制仍然晦涩难懂，但被认为是白内生生成的重要危险因素。我们的 初步实验表明，kynurenine介导的ASC氧化被UVA显着加速 光，这种氧化可以在存在和不存在氧气的情况下发生。基于这些 观察结果，我们假设Kynurenine介导的ASC氧化，然后进行蛋白质修饰。 在老年白内障的病因中的重要作用。我们将通过以下三个目标检验这一假设。在 AIM 1我们将在存在和不存在氧气和UVA的情况下确定Kynurenine介导的ASC氧化 光，模仿皮层的条件和人晶状体的核。在AIM 2中，我们将确定 Kynurenine介导的ASC氧化在共价交联和聚集的晶状体蛋白上，在AIM 3中， 我们将在Kyn/ASC介导的蛋白质修饰上测试我们新开发的前药化合物 交联，评估它们对白内障发育的影响。拟议的研究将共同​​解散 kynurenines与ASC之间的相互作用在人白内障中的镜头蛋白质修饰和发现 可能导致创新的疗法，以预防或延迟人类白内障。