Molecular and Cellular Mechanisms of Glaucoma

青光眼的分子和细胞机制

基本信息

批准号：
7176780
负责人：
OLEG G KISSELEV
金额：
$ 21.41万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-01 至 2010-02-28
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The gene, GLC1A, was found to be associated with juvenile onset glaucoma and some adult onset glaucoma. GLC1A encodes myocilin, which is expressed in several key ocular cell types. The structure(s) and function(s) of myocilin remain unknown. Analysis of endogenous human myocilin reveals that several sizes are produced. Because alternative RNA splicing has not been observed in myocilin, the size differences are presumed to be due to use of different start sites and post-translational modifications. I hypothesize that the various sizes ere produced by differential regulation in different tissues and cell types. I want to solve the structure(s) of myocilin and learn its function(s). To do this, I will analyze myocilin synthesis, processing, and secretion in healthy cells from different cell types. Using native secreted myocilin from cultured cells and recombinant myocilin, my specific aims are: (1) To select the richest source of native myocilin. I will analyze the synthesis of the different kinds of native myocilin from different ocular cell types using cultures of human oligodendrocytes, astrocytes, trabecular meshwork cells and vascular endothelial cells. Different forms of myocilin will be purified independently in order to analyze the properties of each. (2) To determine the biochemical and biophysical properties of myocilin synthesized by human ocular cells using purified soluble, native myocilin proteins. (3) To screen crystallization conditions using both native secreted and recombinant myocilin and to obtain good, diffracting crystals. Given the functional importance of ocular cells in determining neuronal survival or degeneration, analysis of the regulation, synthesis and structure of myocilin will provide valuable information to elucidate the functions of myocilin in normal and glaucomatous eyes.

描述（由申请人提供）：发现基因GLC1A与青少年的青光眼和一些成年的青光眼有关。 GLC1A编码肌动蛋白，该肌动蛋白在几种关键的眼细胞类型中表达。肌动蛋白的结构和功能仍然未知。内源性人肌动蛋白的分析表明，产生了几种尺寸。由于在肌动蛋白中尚未观察到替代性RNA剪接，因此假定大小差异是由于使用不同的起始位点和翻译后修饰所致。我假设由不同组织和细胞类型中的差分调节产生的各种尺寸。我想解决肌动蛋白的结构并学习其功能。为此，我将分析来自不同细胞类型的健康细胞中肌动蛋白的合成，加工和分泌。使用来自培养细胞和重组肌动蛋白的本地分泌的肌动蛋白，我的具体目的是：（1）选择天然肌动蛋白的最丰富来源。我将使用人类少突胶质细胞，星形胶质细胞，小梁网状细胞和血管内皮细胞的培养物分析不同眼细胞类型的不同类型肌动蛋白的合成。不同形式的肌动蛋白将独立纯化，以分析每种特性。（2）使用纯化的可溶性，天然的肌动蛋白蛋白来确定由人眼细胞合成的肌动蛋白的生化和生物物理特性。（3）使用天然分泌和重组肌动蛋白筛选结晶条件，并获得良好的衍射晶体。鉴于眼细胞在确定神经元存活或变性方面的功能重要性，对肌动蛋白的调节，合成和结构的分析将提供有价值的信息，以阐明在正常和胶状眼中肌动蛋白的功能。