Rhodopsin Trafficking and Retinal Degenerations

视紫红质贩运和视网膜变性

基本信息

批准号：
8324689
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
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-30 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8324689
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 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 abstracting in vivo insight membrane assembly mutant photoactivation photoreceptor disc polarized cell protein protein interaction receptor research study retinal rods rho tool trafficking

项目摘要

Project Summary/Abstract 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. Project Narrative 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.

项目概要/摘要分子神经科学和细胞生物学中最基本的问题之一是正确的信号转导膜的组装，包括蛋白质成分的运输和分类。神经退行性疾病和其他遗传性疾病的一个主要原因是神经元定位不当受体和其他信号或转运蛋白。本研究的目的是鉴定在运输过程中与视紫红质相互作用的蛋白质以及那些参与视杆细胞外节盘膜的生物发生，然后确定视紫红质与其他蛋白质的分子相互作用导致的分子机制形成健康的感光盘膜。这项工作将进一步加深对由受体和其他物质的不当运输引起的神经退行性疾病的机制膜蛋白。拟议研究的重点是了解蛋白质-蛋白质相互作用当视紫红质在其羧基末端缺乏适当的结构时，它们就会有缺陷，就像在几种最严重的常染色体显性遗传性色素性视网膜炎。我们将用强大的我和我的同事开发的小鼠敲入模型，以及提出的新模型在此处。在目标 1 中，我们将鉴定与视紫红质羧基末端相互作用以介导适当的蛋白质使用视网膜提取物通过亲和捕获实验进行运输和盘膜组装具有缺陷的羧基末端敲入动物的纯合视紫红质突变体。在目标 2 中，我们将首先在体外，然后在体内表征含有羧基末端的突变视紫红质 Ter349Glu 延伸导致视紫红质介导的常染色体显性遗传的最严重形式之一视网膜色素变性。在目标 3 中，我们将开发一种新工具，将人类视紫红质与光激活融合绿色荧光蛋白，后面是视紫红质羧基末端的重复 (rho-paGFP- 1D4）。该构造将以两种不同的方式使用：首先，我们将测试以下假设：畅通无阻的视紫红质羧基末端足以在健康的视杆细胞中形成适当的外部片段敲入动物。其次，我们将研究视紫红质中特定蛋白质-蛋白质相互作用的作用 GFP 光激活后的贩运，使我们能够追踪 GFP 亚群的移动视紫红质首次进入细胞。这为未来体内贩运研究奠定了基础。项目叙述这项研究的重点是了解有缺陷的蛋白质-蛋白质相互作用当弱光感光体视紫红质在其羧基处缺乏正确的结构时终点，就像几种最严重的常染色体显性遗传形式的情况一样视网膜色素变性。我们将研究视紫红质在适当的视杆细胞形成中的作用和退化，并监测其贩运以更好地了解这些过程。