Molecular and Cellular Mechanisms of Glaucoma

描述（由申请人提供）：基因GLC1A被发现与青少年型青光眼和一些成人型青光眼相关。GLC1A编码在几种关键眼细胞类型中表达的心肌蛋白。心肌的结构和功能尚不清楚。内源性人心肌蛋白的分析显示有几种大小。由于在心肌中未观察到选择性RNA剪接，推测其大小差异是由于使用不同的起始位点和翻译后修饰所致。我假设不同的大小是由不同组织和细胞类型的不同调节产生的。我想解决心肌的结构，了解它的功能。为此，我将分析不同细胞类型的健康细胞中心肌素的合成、加工和分泌。利用培养细胞的天然分泌心肌和重组心肌，我的具体目标是：(1)选择最丰富的天然心肌来源。我将分析利用人少突胶质细胞、星形胶质细胞、小梁网细胞和血管内皮细胞的培养物，从不同眼细胞类型合成不同种类的天然心肌。不同形式的心肌素将被单独纯化，以便分析每种心肌素的性质。(2)利用纯化的可溶性天然心肌蛋白，测定人眼细胞合成的心肌蛋白的生化和生物物理特性。(3)利用天然分泌心肌蛋白和重组心肌蛋白筛选结晶条件，获得良好的衍射晶体。鉴于眼细胞在决定神经元存活或退行性变中的功能重要性，分析心肌蛋白的调控、合成和结构将为阐明正常眼和青光眼中心肌蛋白的功能提供有价值的信息。

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.