Alpha-Crystallin Function in Lens Biology

α-晶状体蛋白在晶状体生物学中的功能

基本信息

批准号：
7387384
负责人：
USHA P ANDLEY
金额：
$ 46.34万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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/7387384
关键词：
Actins Age Alleles Antibodies Apoptosis Apoptotic Area Arginine Binding Biochemical Biological Biological Models Biology Breeding Bromodeoxyuridine Cataract Cell Cycle Cell Death Cell Nucleus Cell Proliferation Cell Survival Cell division phases Cells Centrosome Complex Condition Consult Crystallins Cysteine Cytokinesis Cytoskeleton Desmin Disease Disruption Ensure Epithelial Epithelial Cells Epithelium Fluorescence Microscopy Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Genes Glycine Goals Growth Heat shock proteins Heat-Shock Proteins 70 Homeostasis Hour Human Immunoblot Analysis Immunofluorescence Immunologic In Situ Nick-End Labeling In Vitro Incidence Individual Injection of therapeutic agent Intermediate Filaments Investigation Knock-in Mouse Knock-out Knockout Mice Label Laboratories Lead Lens Fiber Lens Opacities Letters Life Link MAPK14 gene MAPK8 gene Methods Microtubule-Associated Proteins Microtubule-Organizing Center Microtubules Mitosis Mitotic Mitotic spindle Molecular Chaperones Mus Mutation Myopathy Optics Other Genetics PTK2 gene Phase Phenotype Physiological Play Proliferating Protein Overexpression Protein p53 Proteins Rate Regulation Research Design Research Personnel Role Series Signal Transduction Signaling Protein Staining method Stains System TP53 gene Testing Transgenic Model Tubulin Tubulin Interaction Water Work alpha-Crystallins cell type congenital cataract cytotoxic day early onset fiber cell in vivo insight intracellular protein transport lens lens transparency mouse model movie mutant polypeptide progenitor programs protein localization location research study stoichiometry time use

项目摘要

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-晶状蛋白是两个多肽α-和α的聚集体，它们是小热震蛋白，并充当分子伴侣。 αA和α-晶状体蛋白的突变与早期发作人白内障有关。靶向αA基因的靶向破坏在很小的时候就诱导白内障，这意味着该蛋白质在维持纤维细胞透明度中的关键作用。 αA敲除镜头比野生型镜片小40％。我们发现，衍生自αA基因敲除小鼠的透镜上皮细胞的生长速度慢50％。在体内细胞周期的有丝分裂（细胞分裂）期间，αA基因敲除透镜上皮细胞的一部分死亡，其表型与异常的微管组件相关。 αA-晶状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，其表达是在同步的原代小鼠透镜上皮培养物中依赖于细胞周期相。现在，我们提出了一系列的生化和细胞生物学实验，以进一步检查α-rystallin，增殖和凋亡是否直接在体内透镜上皮中连接。 AIM 1检验了以下假设：α-结晶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。我们将确定来自野生型和α敲除透镜上皮提取物的微管组装，并检查αA和α-晶状体与微管的相互作用。 AIM 2检验了以下假设：αA-和Alphab-晶状体的突变对体内的晶状体上皮细胞是细胞毒性的。我们正在生成敲入小鼠模型，以分析两个与人白内障相关的两个突变（Alphab-crystallin中的R120G和αA-Crystallin中的R49C）的体内效应。我们将确定这些小鼠模型的晶状体上皮的增殖和凋亡，以深入了解这些突变导致白内障形成的机制。 AIM 3检验了以下假设：α-晶状体在调节细胞凋亡和增殖中的功能取决于其与信号蛋白和生存因子的相互作用。我们将解决α-晶状蛋白与晶状体上皮相互作用的细胞存活相关蛋白。这些机械研究可能会深入了解正常α-晶状蛋白功能的丧失如何导致白内障形成。