Molecular and Cellular Mechanisms of Glaucoma

描述（申请人提供）：发现该基因GLC1A与青少年型青光眼和某些成人型青光眼相关。GLC1A编码myocilin，其在几种关键的眼部细胞类型中表达。myocilin的结构和功能仍不清楚。内源性人肌细胞素的分析揭示了产生几种大小。由于在肌球蛋白中未观察到RNA剪接，因此推测大小差异是由于使用不同的起始位点和翻译后修饰所致。我推测不同的大小是由不同组织和细胞类型的差异调节产生的。我想解决myocilin的结构（S），并了解其功能（S）。为了做到这一点，我将分析来自不同细胞类型的健康细胞中的myocilin合成，加工和分泌。本研究利用培养细胞分泌的天然myocilin和重组myocilin，具体目的是：（1）筛选天然myocilin最丰富的来源。我将使用人少突胶质细胞、星形胶质细胞、小梁网细胞和血管内皮细胞的培养物分析来自不同眼细胞类型的不同种类的天然肌钙蛋白的合成。不同形式的myocilin将被单独纯化，以分析每种形式的特性。(2)确定由人眼细胞使用纯化的可溶性天然肌球蛋白合成的肌球蛋白的生物化学和生物物理特性。(3)使用天然分泌和重组myocilin筛选结晶条件并获得良好的衍射晶体。鉴于眼细胞在决定神经元存活或变性方面的重要功能，对肌钙蛋白的调节、合成和结构的分析将为阐明肌钙蛋白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.