Comparative Proteomics to Study Age-Related Maculopathy

虽然导致老年性黄斑变性(AMD)的遗传因素正在变得越来越清楚，但这种疾病的病理生理学仍然很难理解。AMD的特征是视网膜色素上皮(RPE)基底层和Bruch膜之间的玻璃体积聚，导致RPE细胞和光感受器的进行性退化。了解玻璃疱疹的细胞和分子起源将有助于寻找新的治疗靶点来预防或减缓疾病的进展。本研究的目的是确定RPE细胞在玻璃体形成中的作用。潜在的假设是RPE细胞参与了玻璃体形成，AMD捐赠者RPE对不同环境应激源的异常反应是导致玻璃体积聚的原因之一。蛋白质表达模式，特别是RPE细胞分泌的蛋白质，可通过蛋白质组学技术检测，有望直接反映RPE细胞和玻璃疣形成之间的关系。利用最先进的蛋白质组学方法(氨基酸稳定同位素标记)，我们发现RPE细胞分泌多种蛋白质，据报道这些蛋白质是玻璃体蛋白的主要成分。此外，我们的数据显示，来自AMD捐赠者的RPE细胞(通过对黄斑的组织学检查诊断，并对Y402H-CFH变体进行基因分型)比年龄匹配的健康捐赠者的RPE分泌的这些蛋白质多2到3倍。在特定的目标I中，我们将使用定量蛋白质组图谱来确定AMD RPE和健康RPE之间的差异表达蛋白质。我们将专注于分泌蛋白，但最终将分析其他亚细胞部分，以进行全面的比较蛋白质组学。在特定的目标II中，AMD RPE细胞与健康RPE细胞相比，在氧化和/或炎症介导的损伤后，蛋白质分泌的变化将被监测。总体而言，这些结果将有助于识别与AMD发病相关的视网膜色素上皮细胞中异常蛋白的表达和运输。我们预计，新出现的蛋白质组学技术的应用将导致对AMD发病机制的更好理解，并将有助于寻找新的治疗靶点来延缓或阻止疾病的进展。PHS 398/2590(版本09/04,2006年4月再次发布)页面延续格式页面首席研究员/项目总监(最后、第一、中间)：Hathout，Yetrib本项目的目标是研究与老年性黄斑变性(AMD)病理相关的细胞和分子机制。我们将实施蛋白质组学方法来评估来自AMD和年龄匹配的健康捐赠者的RPE细胞培养的差异蛋白分泌。

While the genetic factors responsible for age related macular degeneration (AMD) are becoming unraveled, the pathophysiology of the disease remains difficult to understand. AMD is characterized by an accumulation of drusen between the basal lamina of the retinal pigment epithelium (RPE) and Bruch's membrane resulting in progressive degeneration of RPE cells and photoreceptors. Understanding the cellular and molecular origins of drusen will help in finding new therapeutic targets to prevent or slow down the progression of the disease. The objective of this study is to determine the role of RPE cells in drusen formation. The underlying hypothesis is that RPE cells are involved in drusen formation and that an aberrant response of RPE from AMD donors to different environmental stressors is a cause of drusen accumulation. Protein expression patterns and especially proteins secreted by RPE cells, detectable by proteomic techniques, are expected to be a direct reflection of the relationship between RPE cells and drusen formation. Using a cutting edge proteomics approach (stable isotope labeling by amino acids) we show that RPE cells secrete a variety of proteins that have been reported to be major constituent of drusen. Additionally, our data reveals that RPE cells from AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-CFH variant) secreted 2 to 3 fold more of these proteins than RPE from age matched healthy donors. In Specific Aim I, we will determine differentially expressed proteins between AMD RPE and healthy RPE using quantitative proteome profiling. We will focus on secreted proteins but eventually other subcellular fraction will be analyzed for comprehensive comparative proteomics. In Specific Aim II, changes in protein secretion in AMD RPE cells versus healthy RPE cells will be monitored following oxidative and/or inflammatory mediated injury. Overall, the results will help identify abnormal protein expression and trafficking associated with AMD pathogenesis in the retinal pigment epithelial cells. We anticipate that the application of newly emerging proteomic techniques will result in an improved understanding of the pathways involved in AMD pathogenesis and will help in finding new therapeutic targets to delay or stop progression of the disease. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page Principal Investigator/Program Director (Last, First, Middle):Hathout, Yetrib The goal of this project is to study cellular and molecular mechanisms involved in age related macular degeneration (AMD) pathology. We will implement a proteomic approach to assess differential protein secretion in RPE cell cultures derived from AMD and age matched healthy donors.