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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：
7841106
负责人：
Surafel Mulugeta
金额：
$ 25.5万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7841106
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(申请人提供)：三磷酸腺苷结合盒转运体ABCA3是ABC超家族转运体的成员，其功能是底物跨细胞膜的转运。ABCA3主要定位于肺泡II型细胞板层小体的限制膜，被认为是一种脂类和磷脂转运蛋白。最近，由于ABCA3基因的突变与多种肺部疾病有关，包括新生儿的致死性表面活性物质缺乏和呼吸窘迫综合征(RDS)，以及年龄较大的儿童和成人的间质性肺疾病(ILD)，因此ABCA3受到了相当大的关注。虽然到目前为止，许多研究主要集中在ABCA3作为转运体的功能方面，但由于表达突变的ABCA3亚型，在细胞和肺内稳态中的细胞反应和后果在很大程度上是未知的。本项目的总体目标是使用一种简化论的方法，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蛋白突变异构体表达的后果。具体目标1将检验ABCA3的N-末端结构域由一个短的21个氨基酸组成的假设，该序列含有信号基序，用于将新生蛋白插入内质网膜，并将蛋白靶向高尔基体后远端隔室和细胞表面。利用成熟的体外系统，包括无细胞系统、两个上皮细胞系、人肺泡II型细胞，以及各种分子和生化技术，我们将实验地阐明ABCA3转运蛋白N-末端结构域的跨膜拓扑结构和功能基序。在特定的目标2中，我们将使用体外研究和体内小鼠转基因策略来研究错误折叠的ABCA3突变异构体的表达导致的蛋白质功能障碍和细胞损伤的细胞反应和分子机制。我们将扩大体外研究，包括对化学伴侣的评估。这些分子伴侣有可能通过促进适当的蛋白质折叠和运输以及通过恢复突变转运体的功能来防止或改善细胞损伤。项目简介：最近，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转运体的两个主要方面来了解这些疾病的潜在机制，这两个方面在很大程度上还没有确定。这些包括：1)体外检测ABCA3蛋白的靶向基序和N-末端结构域拓扑结构；2)在体外和体内阐明细胞响应以及对其突变异构体表达的细胞动态平衡的影响。我们相信，从这个项目中获得的知识不仅将被广泛应用于更好地了解肺部的发病机制，而且将为有针对性的治疗干预提供见解。