EFEMPI and Drusen Formation in the Retina

视网膜中的 EFEMPI 和玻璃疣形成

• 批准号: 6792189
• 负责人: LIHUA Y MARMORSTEIN
• 金额: $ 37.63万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6792189
• 关键词: animal tissue; autosomal dominant trait; basement membrane; clinical research; gene mutation; genetic recombination; human tissue; immunocytochemistry; immunoelectron microscopy; immunoprecipitation; laboratory mouse; laboratory rat; macular drusen; mass spectrometry; messenger RNA; pathologic process; protein biosynthesis; protein localization; protein protein interaction; protein purification; protein structure function; protein transport; retinal pigment epithelium; transcytosis

DESCRIPTION (provided by applicant): The long-term objective of this application is to understand how EFEMP1 is involved in the pathogenesis of Doyne honeycomb retinal dystrophy (DHRD) and Malattia Leventinese (ML). A characteristic feature of DHRD/ML is the presence of drusen beneath the retinal pigment epithelium (RPE). Little is known about the composition or formation of drusen. A single mutation (R345W) in the gene EFEMP1 was found to be responsible for DHRD/ML. EFEMP1 is an uncharacterized protein of unknown function. The preliminary results of this application indicate that EFEMPI is a secreted protein and that EFEMP1R345W accumulates within cells and is secreted less efficiently than wild type EFEMP1. In the retina of a ML patient homozygous for the R345W mutation, EFEMP1 was found to accumulate in the RPE and the RPE basement membrane region immediately overlaying drusenoid deposits. No such accumulation was noted in the eyes of healthy human donors. Thus the hypothesis of this application is that EFEMP1R345W is involved in drusen formation through the impairment of trafficking by EFEMP1R345W aggregates between Bruch's membrane and RPE cells and/or within RPE cells. Guided by this hypothesis, the specific aims focus on: expression of the EFEMP1 protein in normal and pathological tissues, determine how the R345W mutation results in the accumulation of EFEMP1 within and beneath RPE cells, and identification of molecules interacting with EFEMP1. The studies outlined in this application should aid in understanding how this gene defect leads to the retinal pathology associated with DHRD/ML and developing effective intervention and beneficial medical treatment protocols for DHRD/ML. The expression of EFEMP1 protein in normal and pathological retina tissues will be determined by immunohistochemistry and immunoelectron microscopy. The effect of the R345W mutation on the fate of EFEMP1 protein will be determined through the comparison of the trafficking of EFEMP1 and EFEMP1R345W in RPE cells and the study on the mutation's effect on the proteasome degradation system and turnover process of EFEMP1 in RPE cells. Proteins interacting with EFEMP1 will be identified by immunoaffinity purification of EFEMP1 complexes followed by mass spectrometry. The effect of the R345W mutation on these interactions will be tested using recombinant EFEMP1R345W.

描述（由申请人提供）：本申请的长期目标是了解EFEMP1如何参与Doyne Honeycomb视网膜营养不良（DHRD）和Malattia Leventinese（ML）的发病机理。 DHRD/ML的一个特征是视网膜色素上皮（RPE）下的drusen。关于drusen的组成或形成知之甚少。发现基因EFEMP1中的单个突变（R345W）负责DHRD/ML。 EFEMP1是一种未知功能的未表征的蛋白质。该应用的初步结果表明EFEMPI是一种分泌的蛋白质，EFEMP1R345W在细胞内积累，并且比野生型EFEMP1效率较低。在ML患者的视网膜中，纯合为R345W突变，发现EFEMP1积聚在RPE中，RPE基底膜区域立即覆盖了drusenoid沉积物。在健康的人类捐助者的眼中，没有发现这样的积累。因此，该应用的假设是EFEMP1R345W通过EFEMP1R345W的运输损害与BRUCH的膜和RPE细胞和RPE细胞内的EFEMP1R345W骨料相关。在这一假设的指导下，具体的目的是：在正常和病理组织中EFEMP1蛋白的表达，确定R345W突变如何导致RPE细胞内和下方EFEMP1的积累，以及与EFEMP1相互作用的分子的鉴定。本应用中概述的研究应有助于了解该基因缺陷如何导致与DHRD/ML相关的视网膜病理，并为DHRD/ML开发有效的干预和有益的医疗治疗方案。 EFEMP1蛋白在正常和病理视网膜组织中的表达将由免疫组织化学和免疫电子显微镜确定。 R345W突变对EFEMP1蛋白命运的影响将通过比较RPE细胞中EFEMP1和EFEMP1和EFEMP1R345W的运输以及对RPE细胞中EFEMP1的蛋白酶体降解系统和EFEMP1的影响的研究的研究。与EFEMP1相互作用的蛋白质将通过EFEMP1复合物的免疫亲和力纯化，然后是质谱法。 R345W突变对这些相互作用的影响将使用重组EFEMP1R345W进行测试。