Molecular mechanisms of protein crosslinking in the lens

晶状体中蛋白质交联的分子机制

基本信息

批准号：
8887124
负责人：
Ram H Nagaraj
金额：
$ 38.1万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
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/8887124
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 Health 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 research study

项目摘要

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.

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防止人类白内障。该项目建立在我们以前在衰老和白内生中修饰晶状体蛋白质修饰的工作近25年的工作。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，在人类晶状体中以高达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终产物（AGES）形成色素和交联蛋白。减少的谷胱甘肽（GSH）可以保护此过程，但是老化和白内障透镜中GSH的降低有利于ASC氧化。最近的工作表明，白内障透镜中的许多蛋白质交联是ASC氧化产物介导的。我们知道ASC在衰老和白内障透镜中被氧化，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，但不太可能发生在近乎缺氧的核中。尽管有这一限制，但通过年龄形成的蛋白质交联和聚集在白内科镜头的核中最为突出。 Kynurenine是由吲哚美碱2、3-二加氧酶发起的Kynurenine途径产生的色氨酸氧化产物。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均促进ASC氧化。尽管机制仍然晦涩难懂，但UVA光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氧化是通过UVA光显着加速的，并且在存在和不存在氧气的情况下可以发生这种氧化。基于这些观察结果，我们假设Kynurenine介导的ASC氧化，然后蛋白质修饰在老年性白内障的病因中起重要作用。我们将通过以下三个目标检验这一假设。在AIM 1中，我们将在存在和不存在氧气和UVA光的情况下确定Kynurenine介导的ASC氧化，模仿人透镜的皮质和核的条件。在AIM 2中，我们将确定Kynurenine介导的ASC氧化对晶状体蛋白的共价交联和聚集的影响，在AIM 3中，我们将测试我们对Kyn/Ascy介导的蛋白质修饰和交叉链接的新开发的前药化合物，并评估其对canaract发育的影响。拟议的研究一起将揭示人白内障中晶状体蛋白质修饰中Kynurenines与ASC之间的相互作用，发现可能导致创新的疗法，以预防或延迟人类的白内障。