Development and Maintenance of Lens Transparency

镜头透明度的开发和维护

基本信息

批准号：
7269860
负责人：
JOHN Irwin CLARK
金额：
$ 56.92万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-06-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7269860
关键词：
Affinity Animal Model Biological Assay Cardiomyopathies Cataract Cells Complex Confocal Microscopy Crystallins Cytoskeletal Filaments Cytoskeletal Modeling Cytoskeletal Proteins Cytoskeleton Desmin Detergents Development Disease Economic Development Electron Microscope Electron Microscopy Electroretinography Elements Evaluation Filament Gene Expression Heat shock proteins Histology Human Imagery In Vitro Lens Fiber Ligands Link Maintenance Measures Methods Microscopic Modeling Molecular Chaperones Mus Mutation Myopathy Nerve Degeneration Pattern Peptides Preparation Protein Array Proteins Proteolysis Rattus Retina Retinal Retinal Degeneration Selenite Site Site-Directed Mutagenesis Staging Structural Protein Structure Surface Plasmon Resonance System Technology Tertiary Protein Structure Testing Thick Transgenic Mice Transgenic Organisms Work base digital immunocytochemistry in vivo interest lens lens transparency mouse model mutant new technology protein aggregation protein aminoacid sequence scaffold

项目摘要

DESCRIPTION (provided by applicant): Human alphaBeta crystallin is the archetype for small heat shock proteins, sHSP, that are involved in protein aggregation and filament assembly diseases including cataracts, neurodegeneration, cardiomyopathy and desmin related myopathy. Interactions between alphaBeta crystallin are necessary for normal filament assembly and organization of crystallins in lens cells. In aim 1, characterization of the interactive sites for subunit assembly, for cytoskeletal proteins and for target peptides on human alphaBeta crystallin, the peptide sequences of the interactive domains on human alphaBeta crystallin will be identified using a protein multipin arrays. The affinities between the interactive domains will be quantified using surface plasmon resonance (SPR) and characterized functionally using in vitro and in vivo assays for chaperone activity. The results are expected to provide new information on the structural basis for the assembly of sHSP subunits to functional complexes and for their interaction with chaperone target proteins and with cellular filaments and cytoskeletal elements. In aim 2, in vivo evaluation of retina - lens relationships that may influence development and maintenance of lens transparency in transgenic mice, the historical hypothesis that a lens - retina relationship is important for normal development of lens cell transparency will be studied. Electroretinograms (ERG) and digital slit lamp recordings of opacity in selected animal models will quantify transparency with retinal function during the development of the lens and during loss of transparency in models for cataract formation. Lastly, the hypothesis that lens cytoskeleton provides a scaffold for development and maintenance of transparent lens fiber structure will be investigated in aim 3, observe the cellular organization of major structural proteins in differentiating lens fibers during development of lens transparency and during loss of transparency in the selenite rat and in selected transgenic mouse models using confocal microscopy and electron microscopy (EM). The patterns and distribution of the cytoskeleton and crystallins during differentiation of transparent lens fibers will be investigated using electron microscopy and confocal immunocytochemistry.

描述（由申请人提供）：人字母晶体蛋白是小型热休克蛋白，SHSP的原型，与蛋白质聚集和细丝组件疾病有关，包括白内障，神经变性，心肌病和脱木蛋白相关。字母结晶蛋白之间的相互作用对于正常细丝组装和晶状体细胞中晶体蛋白的组织是必需的。在AIM 1中，将使用蛋白质多个阵列鉴定出人字母结晶蛋白上的相互源组装，细胞骨架蛋白和针对人字母晶体上靶肽的靶肽的交互性位点的表征。交互式结构域之间的亲和力将使用表面等离子体共振（SPR）进行量化，并使用体外和体内分析进行伴侣活性的功能表征。预计结果将提供有关将SHSP亚基组装到功能复合物以及与伴侣靶靶蛋白以及细胞丝和细胞骨架元素的相互作用的结构基础上的新信息。在AIM 2中，对转基因小鼠的透镜透明度的发展和维持的视网膜 - 晶状体关系的体内评估，将研究晶状体 - 视网膜关系对于透镜细胞透明度的正常发展很重要的历史假设。选定动物模型中不透明度的电子图（ERG）和数字缝隙记录将在镜头的发展过程中用视网膜功能和白内障形成模型的透明度丧失来量化透明度。最后，将在AIM 3中研究透镜细胞骨架为透明镜头纤维结构的开发和维持提供的脚手架的假说，在AIM 3中研究了透明镜头在透明透明度和使用转质小鼠模型中透明度损失过程中透明度和透明度损失期间分化透镜纤维中主要结构蛋白的细胞组织。将使用电子显微镜和共聚焦免疫细胞化学研究研究透明晶状体纤维分化过程中细胞骨架和结晶蛋白的模式和分布。