Molecular and Cellular Mechanisms of Glaucoma

描述(由申请人提供)：GLC1A基因被发现与青少年青光眼和一些成人青光眼有关。GLC1a编码Myoclin，它在几种关键的眼细胞类型中表达。霉菌素的结构(S)和功能(S)尚不清楚。对内源性人肌球蛋白的分析表明，产生了几种大小的蛋白。由于在myocin中没有观察到可供选择的RNA剪接，因此推测大小的差异是由于使用了不同的起始点和翻译后修饰。我推测，不同的大小是由不同组织和细胞类型的不同调控所产生的。我想解开霉菌素的结构(S)，学习它的功能(S)。为了做到这一点，我将分析不同细胞类型的健康细胞中肌红素的合成、加工和分泌。利用培养细胞分泌的天然肌球蛋白和重组肌球蛋白，我的具体目的是：(1)筛选最丰富的天然肌球蛋白来源。我将使用人少突胶质细胞、星形胶质细胞、小梁细胞和血管内皮细胞的培养，分析不同类型的眼细胞合成不同种类的天然肌球蛋白。不同形式的霉菌素将被单独提纯，以分析每种形式的性质。(2)用纯化的天然可溶性Myocin蛋白测定人眼细胞合成的myoclin的生化和生物物理性质。(3)筛选天然分泌型和重组型麦角菌素的结晶条件，获得良好的衍射型晶体。鉴于眼细胞在决定神经元存活或退化中的功能重要性，分析myoclin的调节、合成和结构将为阐明myoclin在正常和青光眼中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.