Alpha-Crystallin Function in Lens Biology

晶状体α -晶体蛋白是两个多肽α -和α b的聚集体，它们是小的热休克蛋白，起分子伴侣的作用。α -和α -晶体蛋白突变与早发性白内障有关。α α基因的靶向破坏可在早期诱发白内障，这意味着该蛋白在维持纤维细胞透明度方面起着关键作用。α基因敲除型镜片比野生型镜片小40%。我们发现来自α a基因敲除小鼠的晶状体上皮细胞生长速度减慢50%；在体内，一部分α α敲除的晶状体上皮细胞在细胞周期的有丝分裂（细胞分裂）阶段死亡，其表型与异常的微管组装相关。α -晶体蛋白主要集中在野生型细胞分裂的中心体和细胞间桥微管中，在小鼠晶状体上皮同步原代培养中，α -晶体蛋白的表达与细胞周期相依赖。我们现在提出了一系列的生化和细胞生物学实验来进一步研究α -晶体蛋白是否与晶状体上皮细胞的增殖和凋亡直接相关。目的1验证了α -晶体蛋白的功能之一是稳定晶状体上皮细胞的微管蛋白骨架的假设。我们将从野生型和α α敲除的晶状体上皮提取物中确定微管的组装，并检查α α和α b -晶体蛋白与微管的相互作用。目的2在体内验证α α -和α b -晶体蛋白突变对晶状体上皮细胞具有细胞毒性的假设。我们正在建立敲入小鼠模型，以分析与人类白内障相关的两种突变（α -晶体蛋白中的R120G和α -晶体蛋白中的R49C）的体内效应。我们将检测这些小鼠模型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。目的3验证α -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。我们将解析晶体蛋白与晶状体上皮相互作用的细胞存活相关蛋白。这些机制研究可能为了解正常α -晶体蛋白功能的丧失如何导致白内障的形成提供见解。

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.