Rhodopsin Trafficking and Retinal Degenerations

视紫红质贩运和视网膜变性

• 批准号: 8138432
• 负责人: Alecia K Gross
• 金额: $ 34.8万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8138432
• 关键词: Acute; Affinity; Animals; Binding; Biogenesis; C-terminal; Carrier Proteins; Cells; Cellular biology; Chimeric Proteins; Co-Immunoprecipitations; Cultured Cells; Defect; Development; Disease; Dominant-Negative Mutation; Epitopes; Future; Genes; Goals; Green Fluorescent Proteins; Health; Homozygote; Human; Image; Immunoblotting; In Vitro; Inborn Genetic Diseases; Knock-in Mouse; Lead; Light; Location; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Membrane Proteins; Modeling; Molecular; Monitor; Movement; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurosciences; Pathway interactions; Photoreceptors; Process; Protein Binding; Proteins; Regulatory Element; Research; Retinal; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Outer Segments; Role; Signal Transduction; Signaling Protein; Sorting - Cell Movement; Staging; Structure; Testing; Time; Work; in vivo; insight; membrane assembly; mutant; photoactivation; photoreceptor disc; polarized cell; protein protein interaction; receptor; research study; retinal rods; rho; tool; trafficking

DESCRIPTION (provided by applicant): One of the most fundamental problems in molecular neuroscience and cell biology is the proper assembly of signal-transducing membranes including the transport and sorting of protein components. A major cause of neurodegenerative and other inherited disorders is the improper localization of receptors and other signaling or transport proteins. The goal of this study is to identify proteins that interact with rhodopsin during transport and those involved in the biogenesis of disk membranes in the outer segment of rod cells, and then determine the molecular mechanisms by which the molecular interactions of rhodopsin with other proteins lead to formation of healthy photoreceptor disk membranes. This work will further the understanding of the mechanisms of neurodegenerative disorders caused by improper trafficking of receptors and other membrane proteins. The focus of the proposed research is to understand protein-protein interactions that are defective when rhodopsin lacks the proper structure at its carboxy-terminus, as is the case in several of the most severe forms of autosomal dominant retinitis pigmentosa. We will use powerful mouse knock-in models that my co-workers and I have developed, as well as new models proposed herein. In Aim 1, we will identify proteins that interact with rhodopsin's carboxy-terminus to mediate proper transport and disk membrane assembly through affinity-capture experiments using retinal extracts from homozygote rhodopsin mutants with defective carboxyl-termini knock-in animals. In Aim 2, we will characterize, first in vitro, then in vivo, a mutant rhodopsin, Ter349Glu, containing a carboxyl-terminal extension that causes one of the most severe forms of rhodopsin-mediated autosomal dominant retinitis pigmentosa. In Aim 3, we will develop a new tool, human rhodopsin fused to photoactivatable green fluorescent protein that is followed by a repeat of rhodopsin's carboxyl terminus (rho-paGFP- 1D4). This construct will be used in two distinct ways: first, we will test the hypothesis that an unobstructed rhodopsin carboxy-terminus is sufficient to form proper outer segments in healthy rods in knock-in animals. Second, we will study the role of specific protein-protein interactions in rhodopsin trafficking after photoactivation of GFP, enabling us to track the movement of subpopulations of rhodopsin in cells for the first time. This sets the stage for in vivo trafficking studies in the future. PUBLIC HEALTH RELEVANCE: The focus of this study is to understand protein-protein interactions that are defective when the dim light photoreceptor rhodopsin lacks the proper structure at its carboxy- terminus, as is the case in several of the most severe forms of autosomal dominant retinitis pigmentosa. We will study the role of rhodopsin in proper rod cell formation and degeneration, and monitor its trafficking to better understand these processes.

描述（由申请人提供）：分子神经科学和细胞生物学中最基本的问题之一是信号转导膜的正确组装，包括蛋白质组分的运输和分选。神经退行性疾病和其他遗传性疾病的主要原因是受体和其他信号传导或转运蛋白的不适当定位。本研究的目的是确定在运输过程中与视紫红质相互作用的蛋白质，以及参与视杆细胞外段盘膜生物发生的蛋白质，然后确定视紫红质与其他蛋白质的分子相互作用导致形成健康感光盘膜的分子机制。这项工作将进一步了解神经退行性疾病的机制所造成的不适当的贩运受体和其他膜蛋白。拟议研究的重点是了解当视紫红质在其羧基末端缺乏适当结构时，蛋白质与蛋白质之间的相互作用会出现缺陷，就像几种最严重的常染色体显性视网膜色素变性的情况一样。我们将使用我的同事和我开发的强大的小鼠敲入模型，以及本文提出的新模型。在目的1中，我们将确定与视紫红质的羧基末端相互作用的蛋白质，以介导适当的运输和磁盘膜组装通过亲和捕获实验，使用视网膜提取物从纯合子视紫红质突变体与缺陷的羧基末端敲入动物。在目标2中，我们将首先在体外，然后在体内，一个突变的视紫红质，Ter 349 Glu，含有一个羧基末端的延伸，导致最严重的形式之一的视紫红质介导的常染色体显性视网膜色素变性。在目标3中，我们将开发一种新的工具，人视紫红质融合到可光活化的绿色荧光蛋白，随后是一个重复的视紫红质的羧基末端（rho-paGFP-1D 4）。该构建体将以两种不同的方式使用：首先，我们将测试以下假设：无障碍的视紫红质羧基末端足以在敲入动物的健康视杆中形成适当的外节段。第二，我们将研究特定的蛋白质-蛋白质相互作用的作用，在视紫红质运输后，光活化的绿色荧光蛋白，使我们能够跟踪移动的视紫红质亚群在细胞中的第一次。这为今后的体内贩运研究奠定了基础。公共卫生关系：本研究的重点是了解当昏暗光感受器视紫红质在其羧基末端缺乏适当结构时有缺陷的蛋白质-蛋白质相互作用，如在几种最严重形式的常染色体显性视网膜色素变性中的情况。我们将研究视紫红质在视杆细胞形成和变性中的作用，并监测其运输，以更好地了解这些过程。