ABCA3: Biosynthesis, Trafficking, and Cellular Responses in Health and Disease

ABCA3：健康和疾病中的生物合成、贩运和细胞反应

基本信息

批准号：
7799794
负责人：
Surafel Mulugeta
金额：
$ 35.44万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7799794
关键词：
A549 ABCA3 gene ATP-Binding Cassette Transporters Adult Alanine Alveolar Amino Acid Sequence Amino Acids Anabolism Antibodies Apoptotic Attention Binding Biochemical Biological Assay Cell Death Signaling Process Cell Line Cell membrane Cell surface Cell-Free System Cells Characteristics Chemicals Child Chimeric Proteins Chloride Ion Chlorides Cholesterol Coupled Cystic Fibrosis Cytosol Data Defect Disease Distal DsRed Epithelial Epithelial Cells Epitopes Evaluation Exhibits Functional disorder Genes Golgi Apparatus Health Homeostasis Human In Vitro Infant Integral Membrane Protein Interstitial Lung Diseases Knowledge Left Link Lipids Lung Lung diseases Mediating Membrane Metabolism Mitochondria Molecular Molecular Chaperones Mutagenesis Mutation N,N&apos -bis(4-azidobenzoyl)cystine N-terminal Neonatal Newborn Infant Newborn Respiratory Distress Syndrome Organelles Pathogenesis Pathway interactions Peptides Phenotype Phospholipids Process Proteasome Inhibition Protein Isoforms Proteins Publishing Pulmonary Surfactants Pump Quality Control Reporting Role Sagittaria Scanning Sequence Analysis Side Signal Pathway Signal Transduction Site Sorting - Cell Movement Sphingomyelins Stress Structural Models System Techniques Testing Therapeutic Intervention Time Transgenic Mice Transmembrane Domain Walkers alveolar lamellar body alveolar type II cell base cell injury functional restoration glycosylation hydropathy in vivo insight lipid transport mathematical algorithm member mutant prevent protein folding protein misfolding protein transport respiratory distress syndrome response surfactant deficiency trafficking

项目摘要

DESCRIPTION (provided by applicant): The ATP-binding cassette transporter ABCA3 is a member of the ABC superfamily of transporters that function in the translocation of substrates across cell membranes. Predominantly localized in the limiting membrane of the lamellar bodies of lung alveolar type II cells, ABCA3 is believed to function as a lipid and phospholipid transporter. Recently, ABCA3 has received considerable attention because mutations in the gene are associated with various lung disorders including fatal surfactant deficiency and respiratory distress syndrome (RDS) in newborns and interstitial lung disease (ILD) in older children and adults. While many of the studies thus far have focused primarily on the functional aspects of ABCA3 as a transporter, the cellular responses and consequences in cellular and pulmonary homeostasis as a result of expressing mutant isoforms of ABCA3 are largely undefined. The overall objective of this project is to use a reductionist approach aimed at understanding the molecular mechanisms underlying ABCA3 biosynthesis, and to elucidate the consequences of expression of mutant isoforms of ABCA3 proteins associated with RDS and ILD. Specific Aim 1 will test the hypothesis that the N-terminal domain of ABCA3 is comprised of a short 21 amino acid sequence that harbors signal motifs for insertion of the nascent protein into the ER membrane and for targeting of the protein to post-Golgi distal compartments and the cell surface. Using well established in vitro systems including cell free systems, two epithelial cell lines, human alveolar type II cells, and various molecular and biochemical techniques, we will experimentally elucidate the transmembrane topology and functional motifs of the N-terminal domain of the ABCA3 transporter. In Specific Aim 2, we will investigate cellular responses and molecular mechanisms underlying protein dysfunction and cell injury caused by the expression of misfolded mutant isoforms of ABCA3 using both in vitro studies and in vivo mouse transgenic strategies. We will extend the in vitro studies to include the evaluation of chemical chaperones. These chaperones have the potential to prevent or ameliorate cellular damage caused by promoting proper protein folding and trafficking and by restoring function of the mutant transporter. PROJECT NARRATIVE: In recent times, a considerable attention has been given to the ABCA3 transporter because mutation in the gene is believed to cause various lung diseases. We are taking logical steps to understand the mechanisms underlying the cause of these diseases by focusing on two major aspects of the ABCA3 transporter that have been largely undefined. These include: 1) In vitro examination of ABCA3 protein make up in terms of its targeting motifs and N-terminal domain topology; and 2) In vitro and in vivo elucidation of cellular response as well as effects on cellular homeostasis in response to the expression of its mutant isoforms. We believe the knowledge gained from this project will have a broad application not only toward a better understanding of lung pathogenesis but will offer insights for targeted therapeutic intervention.

描述（由申请人提供）：ATP 结合盒转运蛋白 ABCA3 是转运蛋白 ABC 超家族的成员，其功能是使底物跨细胞膜易位。 ABCA3 主要位于 II 型肺泡细胞板层体的限制膜中，据信具有脂质和磷脂转运蛋白的功能。最近，ABCA3 受到了相当多的关注，因为该基因的突变与各种肺部疾病有关，包括致命的表面活性剂缺乏和新生儿呼吸窘迫综合征 (RDS) 以及年龄较大儿童和成人的间质性肺疾病 (ILD)。虽然迄今为止的许多研究主要集中在 ABCA3 作为转运蛋白的功能方面，但由于表达 ABCA3 突变同工型而导致的细胞反应和细胞和肺稳态的后果在很大程度上尚不清楚。该项目的总体目标是使用还原论方法，旨在了解 ABCA3 生物合成的分子机制，并阐明与 RDS 和 ILD 相关的 ABCA3 蛋白突变亚型表达的后果。具体目标 1 将测试这样的假设：ABCA3 的 N 末端结构域由一个短的 21 个氨基酸序列组成，该序列包含用于将新生蛋白插入 ER 膜并将该蛋白靶向至高尔基体后远端区室的信号基序和细胞表面。使用成熟的体外系统，包括无细胞系统、两种上皮细胞系、人肺泡 II 型细胞以及各种分子和生化技术，我们将通过实验阐明 ABCA3 转运蛋白 N 端结构域的跨膜拓扑结构和功能基序。在具体目标 2 中，我们将使用体外研究和体内小鼠转基因策略来研究由 ABCA3 错误折叠突变亚型表达引起的蛋白质功能障碍和细胞损伤的细胞反应和分子机制。我们将扩展体外研究以包括化学伴侣的评估。这些伴侣有可能预防或改善因促进适当的蛋白质折叠和运输以及恢复突变转运蛋白的功能而引起的细胞损伤。项目叙述：近年来，ABCA3 转运蛋白受到了相当大的关注，因为该基因的突变被认为会导致各种肺部疾病。我们正在采取合乎逻辑的步骤，通过关注 ABCA3 转运蛋白的两个主要方面（这两个方面基本上尚未定义）来了解这些疾病的潜在机制。这些包括： 1) 根据其靶向基序和 N 末端结构域拓扑结构对 ABCA3 蛋白的组成进行体外检查； 2) 体外和体内阐明细胞反应以及对其突变亚型表达的细胞稳态影响。我们相信，从该项目中获得的知识将具有广泛的应用，不仅有助于更好地了解肺部发病机制，而且将为有针对性的治疗干预提供见解。