Alpha-Crystallin Function in Lens Biology

产品说明：透镜α-晶状体蛋白是两种多肽α A-和α B的聚集体，它们是小的热休克蛋白并充当分子伴侣。α A-和α B-晶状体蛋白的突变与早发性人类白内障相关有针对性地破坏alphaA基因在早期诱导白内障，这意味着这种蛋白质在维持纤维细胞透明度中起关键作用。AlphaA敲除镜片比野生型镜片小40%。我们发现，源自alphaA基因敲除小鼠的透镜上皮细胞生长减慢50%;一部分alphaA基因敲除的透镜上皮细胞在体内细胞周期的有丝分裂（细胞分裂）期死亡，其表型与异常微管组装相关。α A-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，并且其表达在同步化的原代小鼠透镜上皮培养物中是细胞周期阶段依赖性的。我们现在提出了一系列的生化和细胞生物学实验，以进一步研究是否α A-晶体蛋白和增殖和凋亡直接联系在体内透镜上皮细胞。目的1验证α-晶状体蛋白的功能之一是稳定透镜上皮细胞中微管蛋白细胞骨架的假设。我们将确定野生型和α A敲除透镜上皮提取物的微管组装，并检查α A和α B-晶体蛋白与微管的相互作用。目的2检验α A-和α B-晶状体蛋白突变对体内透镜上皮细胞具有细胞毒性的假设。我们正在构建基因敲入小鼠模型，以分析与人类白内障相关的两种突变（α B-晶状体蛋白中的R120 G和α A-晶状体蛋白中的R49 C）的体内效应。我们将确定这些小鼠模型的透镜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。目的3验证α A-晶体蛋白调节细胞凋亡和增殖的功能依赖于其与信号蛋白和存活因子的相互作用的假设。我们将解析与透镜上皮细胞中α-晶状体蛋白相互作用的细胞存活相关蛋白。这些机制研究可能会提供洞察正常α-晶状体蛋白功能的丧失如何导致白内障形成。

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.