Comparative Proteomics to Study Age-Related Maculopathy

虽然负责年龄相关性黄斑变性（AMD）的遗传因素正在逐渐解开，但该疾病的病理生理学仍然难以理解。AMD的特征是视网膜色素上皮（RPE）的基底层和Bruch膜之间的蛋白积累，导致RPE细胞和光感受器的进行性变性。了解drusen的细胞和分子起源将有助于找到新的治疗靶点，以预防或减缓疾病的进展。本研究的目的是确定RPE细胞在囊肿形成中的作用。潜在的假设是RPE细胞参与了囊肿的形成，AMD供体RPE对不同环境压力的异常反应是囊肿积累的原因。蛋白质组学技术可检测到的蛋白质表达模式，特别是RPE细胞分泌的蛋白质，有望直接反映RPE细胞与囊肿形成之间的关系。使用尖端的蛋白质组学方法（氨基酸稳定同位素标记），我们发现RPE细胞分泌多种蛋白质，这些蛋白质已被报道为drusen的主要成分。此外，我们的数据显示，来自AMD供者的RPE细胞（通过黄斑的组织学检查诊断，基因分型为Y402H-CFH变体）分泌的这些蛋白质比来自年龄匹配的健康供者的RPE多2至3倍。在Specific Aim I中，我们将使用定量蛋白质组分析来确定AMD RPE和健康RPE之间差异表达的蛋白质。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Specific Aim II中，将监测氧化和/或炎症介导的损伤后AMD RPE细胞与健康RPE细胞中蛋白质分泌的变化。总之，这些结果将有助于识别视网膜色素上皮细胞中与AMD发病机制相关的异常蛋白表达和转运。我们预计，新出现的蛋白质组学技术的应用将导致对AMD发病机制的途径的更好理解，并将有助于找到新的治疗靶点来延缓或阻止疾病的进展。PHS 398/2590（09/04修订版，4/2006修订版）项目负责人/项目主任（最后，第一，中）：Hathout， Yetrib该项目的目标是研究年龄相关性黄斑变性（AMD）病理的细胞和分子机制。我们将实施蛋白质组学方法来评估来自AMD和年龄匹配的健康供者的RPE细胞培养物的差异蛋白分泌。

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.