Alpha-Crystallin Function in Lens Biology

α-晶状体蛋白在晶状体生物学中的功能

• 批准号: 6921677
• 负责人: USHA P ANDLEY
• 金额: $ 48.08万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-05-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6921677
• 关键词: cataract; cell biology; cell line; clinical research; crystallins; cytoskeleton; disease /disorder onset; epithelium; fluorimetry; gene mutation; gene targeting; genetically modified animals; heat shock proteins; human genetic material tag; laboratory mouse; lens; microtubules; molecular /cellular imaging; molecular chaperones; molecular pathology; protein protein interaction; protein quantitation /detection; protein signal sequence; protein structure function; tubulin; western blottings

DESCRIPTION: Lens alpha-crystallin is an aggregate of two polypeptides alphaA- and alphaB, which are small heat shock proteins and act as molecular chaperones. Mutations in alphaA- and alphaB-crystallin are associated with early onset human cataract. Targeted disruption of alphaA gene induces cataract at an early age implying a critical role for this protein in maintaining fiber cell transparency. AlphaA knockout lenses are 40% smaller than wild type lenses. We found that lens epithelial cells derived from alphaA knockout mice have 50% slower growth; a fraction of alphaA knockout lens epithelial cells die during mitotic (cell division) phase of the cell cycle in vivo with a phenotype that correlates with abnormal microtubule assembly. AlphaA-crystallin is concentrated in centrosomes and intercellular bridge microtubules of dividing wild type cells, and its expression is cell cycle phase-dependent in synchronized primary mouse lens epithelial cultures. We now propose a series of biochemical and cell biological experiments to further examine whether alphaA-crystallin and proliferation and apoptosis are linked directly in lens epithelium in vivo. Aim 1 tests the hypothesis that one of the functions of alpha-crystallin is to stabilize the tubulin cytoskeleton in lens epithelial cells. We will determine the assembly of microtubules from wild type and alphaA knockout lens epithelial extracts and examine the interaction of alphaA and alphaB-crystallin with microtubules. Aim 2 tests the hypothesis that mutations in alphaA- and alphaB-crystallin are cytotoxic to lens epithelial cells in vivo. We are generating knock-in mouse models to analyze the in vivo effect of two mutations (R120G in alphaB-crystallin and R49C in alphaA-crystallin) associated with human cataract. We will determine proliferation and apoptosis in lens epithelium of these mouse models to gain insight into the mechanism by which these mutations lead to cataract formation. Aim 3 tests the hypothesis that the function of alphaA-crystallin in regulating apoptosis and proliferation is dependent on its interaction with signaling proteins and survival factors. We will resolve the cell survival-related proteins that alpha-crystallin interacts with in the lens epithelium. These mechanistic studies may provide insight into how the loss of normal alpha-crystallin function can lead to cataract formation.

描述：晶状体α-晶状体蛋白是两种多肽αA-和αB的聚集体，它们是小的热休克蛋白并充当分子伴侣。 αA-和αB-晶状体蛋白的突变与早发性人类白内障有关。 αA 基因的靶向破坏会在幼年时诱发白内障，这意味着该蛋白质在维持纤维细胞透明度方面发挥着关键作用。 AlphaA 淘汰镜片比野生型镜片小 40%。我们发现来自αA基因敲除小鼠的晶状体上皮细胞生长速度减慢了50%；一部分 alphaA 敲除晶状体上皮细胞在体内细胞周期的有丝分裂（细胞分裂）阶段死亡，其表型与异常微管组装相关。 AlphaA-晶状体蛋白集中在分裂的野生型细胞的中心体和细胞间桥微管中，其表达在同步原代小鼠晶状体上皮培养物中呈细胞周期阶段依赖性。我们现在提出一系列生化和细胞生物学实验，以进一步检查αA-晶状体蛋白与体内晶状体上皮细胞的增殖和凋亡是否直接相关。目标 1 检验以下假设：α-晶状体蛋白的功能之一是稳定晶状体上皮细胞中的微管蛋白细胞骨架。我们将确定野生型和 αA 敲除晶状体上皮提取物中微管的组装，并检查 αA 和 αB-晶状体蛋白与微管的相互作用。目标 2 检验以下假设：αA-和 αB-晶状体蛋白的突变对体内晶状体上皮细胞具有细胞毒性。我们正在构建敲入小鼠模型来分析与人类白内障相关的两种突变（αB-晶状体蛋白中的 R120G 和 αA-晶状体蛋白中的 R49C）的体内效应。我们将测定这些小鼠模型的晶状体上皮细胞的增殖和凋亡，以深入了解这些突变导致白内障形成的机制。目标 3 检验以下假设：αA-晶状体蛋白调节细胞凋亡和增殖的功能取决于其与信号蛋白和生存因子的相互作用。我们将解析与α-晶状体蛋白在晶状体上皮细胞中相互作用的细胞存活相关蛋白。这些机制研究可能有助于了解正常α-晶状体蛋白功能的丧失如何导致白内障形成。