PROJECT 4 - POLYPHENOLS AND DAMAGE IN THE EYE

眼睛中的蛋白质在维持人类生活质量方面起着至关重要的作用，特别是在衰老方面。其中有几种蛋白质，如透镜蛋白质，合成一次，并保存到生命的其余部分。由于它们暴露在> 320 nm的光下，它们会受到慢性日光诱导的损伤。这种损伤包括透镜蛋白的几种翻译后修饰和交联，其程度尚不清楚。后者导致蛋白质在透镜中沉淀并最终导致白内障形成。通过太阳光的紫外线部分的作用在眼睛中形成的一种重要的化学物质是单线态氧。它与几种蛋白质氨基酸侧链（Cys、His、Met、Trp、Tyr）反应，引入氧原子。它还引起肽骨架在有限位点的裂解。眼房水中的局部化学环境，即浸泡透镜的血浆超滤液，包括重要的抗氧化剂物质，如维生素C和谷胱甘肽。我们的假设是，多酚及其代谢产物来自植物膳食补充剂进入眼的水，从而协同增强其抗氧化环境。这是为了防止UV光诱导的翻译后蛋白质修饰和聚合，从而阻止与年龄相关的眼功能退化。在本提案中，我们将审查以下内容：（1）在大鼠模型中测定所选植物多酚制剂对其在眼睛的透镜和房水中的组成的影响，（2）测定多酚对眼睛特定区域中内源性抗氧化剂谷胱甘肽和外源性抗氧化剂维生素C和E以及类胡萝卜素水平的影响，（3）确定膳食多酚对透镜晶体蛋白（一种具有伴侣活性的热休克蛋白）的翻译后修饰和交联程度的影响，和（4）使用体外模型蛋白和透镜α B-晶体蛋白的特异性突变形式来检查单线态氧诱导的蛋白裂解和翻译后修饰的机制。这些实验将利用微流体分析和蛋白质质谱分析方面的技术和专业知识，这些技术和专业知识是在上一个资助期内在Purdue-UAB植物中心开发的。他们将使用动物模型中的体内实验和体外实验的组合来研究单线态氧诱导的蛋白质损伤的机制以及特定多酚及其代谢物与生理抗氧化剂结合的作用。

The proteins in the eye perform a vital role in sustaining human quality of life, particularly with respect to aging. Several of them, such as the lens proteins, are synthesized one time and preserved for the rest of life. Since they are exposed to light > 320 nm, they are subject to chronic sunlight-induced damage. This damage includes several post-translational modifications and cross-linking of the lens proteins, the extent of which is poorly defined. The latter lead to precipitation of proteins in the lens and ultimately to cataract formation. An important chemical species that is formed in the eye by the action of the ultraviolet portion of sunlight is singlet oxygen. It reacts with several protein amino acid side chains (Cys, His, Met, Trp, Tyr) introducing oxygen atoms. It also causes cleavage of the peptide backbone at limited sites. The local chemical environment in the aqueous of the eye, an ultrafiltrate of plasma that bathes the lens, includes important antioxidant species such a vitamin C and glutathione. Our hypothesis is that polyphenols and their metabolites derived from botanical dietary supplements enter the aqueous of the eye and thereby synergistically enhance its anti-oxidant environment. This is proposed to prevent UV light-induced post-translational protein modification and polymerization and therefore deter age-related degeneration of eye function. In this proposal, we will examine the following: (1) to determine in a rat model the effect of selected botanical polyphenol preparations on their composition in the lens and aqueous humor of the eye, (2) to determine the effects of polyphenols on the levels of the endogenous antioxidant glutathione and the exogenous antioxidants vitamins C and E and carotenoids in specific regions of the eye, (3) to determine the effects of dietary polyphenols on the extent of post-translational modifications and cross-linking of the lens acrystallin (a heat shock protein with chaperone activity), and (4) use specifically mutated forms of a model protein in vitro and lens aB-crystallin to examine the mechanism of singlet oxygen-induced protein cleavage and posttranslational modifications. These experiments will utilize techniques and expertise in microfluidics analysis and protein mass spectrometry that have been developed in the Purdue-UAB Botanicals Center in the previous grant period. They will use a combination of in vivo experiments in animal models and in vitro experiments to examine the mechanisms of singlet oxygen-induced protein damage and the role of specific polyphenols and their metabolites in combination with physiological antioxidants.