Expression and function of ABCA3 in type 2 cells

ABCA3在2型细胞中的表达和功能

• 批准号: 7316827
• 负责人: HENRY SHUMAN
• 金额: $ 29.97万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7316827
• 关键词: ABCA3 gene; ATP phosphohydrolase; Acute; Affect; Alveolar; Animal Model; Antibodies; Binding; Biochemical; Biogenesis; Biotechnology; Birth; Cell Line; Cells; Characteristics; Childhood; Choline; Collaborations; Cultured Cells; Defect; Development; Disease; Epithelial Cells; Funding; Generations; Genetic; Goals; Homologous Gene; Human; In Vitro; Insecta; Interstitial Lung Diseases; Knock-in Mouse; Length; Lipid Binding; Lipids; Location; Lung; Lung diseases; Lysosomes; Measures; Membrane; Membrane Proteins; Methods; Morphology; Mus; Mutation; Newborn Respiratory Distress Syndrome; Organelles; Phospholipids; Proteins; Pulmonary Surfactant-Associated Protein B; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Rate; Rattus; Research; Research Personnel; Role; Secretory Vesicles; Testing; Therapeutic; Transgenic Mice; Vanadates; Work; alveolar lamellar body; base; follow-up; in vitro Model; in vivo; lipid transport; lysosome membrane; mouse model; mutant; novel; programs; small molecule; surfactant; surfactant deficiency; surfactant deficiency in infants; trafficking

DESCRIPTION (provided by applicant): Lamellar bodies (LBs) are the secretory granules of the type 2 alveolar epithelial cell (AT2) through which surfactant packaging and secretion are regulated. Our previous work elucidated how lamellar bodies are formed, maintained, and utilized. An antibody identifying a unique protein of the lamellar body membrane, LBM180, was developed. Mass spectrometric sequencing showed LBM180 is identical to the ATP-Binding Cassette protein, ABCA3. This study was the first to identify ABCA3 as a lamellar body membrane, protein and to suggest its role in lamellar body biogenesis. Subsequently several genetics studies showed that mutations of the ABCA3 gene are associated with fatal surfactant deficiency and pediatric interstitial lung disease (Shulenin, 2004; Bullard, 2005) with markedly abnormal lamellar bodies in AT2 cells (Edwards, 2005). In an animal model we intend to firmly establish a causal relationship between ABCA3 mutations and lung disease and develop methods for testing therapeutics for treating these diseases. During the previous funding period we determined the location and function of ABCA3 in AT2 cells using in vitro methods. We showed: 1) ABCA3 is normally trafficked to the membranes of lamellar bodies and lysosomes and is necessary for converting lysosomes to lamellar bodies, 2) ABCA3 is necessary and sufficient for lipid accumulation, especially of choline phospholipids, in lamellar bodies and lysosomes and 3) mutations that disrupt ABCA3 expression, trafficking or function in cells could, in principal, be rescued with small molecule therapeutics. A recent development allows us to follow up these results with an additional strategy. We purchased five mice from Deltagen, a biotechnology company, and established a colony at Penn. The ABCA3-/- mice die soon after birth and their lungs fail to inflate. Aim 1: We will examine and analyze the morphology of AT2 cells from ABCA3+/+, +/- and -/- mouse lungs and measure the effect that ABCA3 expression has on LB protein and lipid content of lung cells and their secreted products to determine how differences in ABCA3 expression affect lamellar body characteristics. The LB content and PC secretion after acute changes of ABCA3 expression in cell cultures will also be measured. Aim 2: Using biochemical approaches we will determine the (lipid) substrates for ABCA3 ATPase. Aim 3: We will develop cell based screens of small molecule therapeutics for ABCA3 mutations with trafficking and folding defects. Further we will develop animal models of neonatal RDS and interstitial lung disease by generating knock-in mice with ABCA3 mutations and transgenic mice expressing conditionally controlled mutant or wild type ABCA3 in a wild type or ABCA3 KO background.

描述（由申请人提供）：板层体（LB）是2型肺泡上皮细胞（AT 2）的分泌颗粒，通过其调节表面活性剂包装和分泌。我们以前的工作阐明了板层体是如何形成、维持和利用的。开发了识别板层体膜的独特蛋白质LBM 180的抗体。质谱测序显示LBM 180与ATP结合盒蛋白ABCA 3相同。这项研究首次将ABCA 3鉴定为板层体膜蛋白，并表明其在板层体生物发生中的作用。随后的几项遗传学研究表明，ABCA 3基因的突变与致命性表面活性物质缺乏症和儿科间质性肺病（Shulenin，2004; Bullard，2005）以及AT 2细胞中明显异常的板层体（Edwards，2005）相关。在动物模型中，我们打算牢固地建立ABCA 3突变和肺部疾病之间的因果关系，并开发用于测试治疗这些疾病的疗法的方法。在之前的资助期间，我们使用体外方法确定了ABCA 3在AT 2细胞中的位置和功能。我们展示了：1）ABCA 3通常被运输到板层体和溶酶体的膜，并且是将溶酶体转化为板层体所必需的，2）ABCA 3对于板层体和溶酶体中的脂质积累，特别是胆碱磷脂的积累是必需的和足够的，以及3）破坏ABCA 3在细胞中的表达、运输或功能的突变原则上可以用小分子治疗剂拯救。最近的发展使我们能够通过额外的策略来跟进这些结果。我们从生物技术公司Deltagen购买了五只小鼠，并在宾夕法尼亚大学建立了一个种群。ABCA 3-/-小鼠出生后很快死亡，它们的肺无法膨胀。目标1：我们将检查和分析来自ABCA 3 +/+、+/-和-/-小鼠肺的AT 2细胞的形态，并测量ABCA 3表达对肺细胞及其分泌产物的LB蛋白和脂质含量的影响，以确定ABCA 3表达的差异如何影响板层体特征。还将测量细胞培养物中ABCA 3表达急性变化后的LB含量和PC分泌。目的2：利用生物化学方法，我们将确定ABCA 3 ATP酶的（脂质）底物。目标3：我们将开发基于细胞的筛选具有运输和折叠缺陷的ABCA 3突变的小分子治疗剂。此外，我们将通过产生具有ABCA 3突变的基因敲入小鼠和在野生型或ABCA 3 KO背景下表达条件控制突变体或野生型ABCA 3的转基因小鼠来开发新生儿RDS和间质性肺病的动物模型。