Role of EMC3/TMEM111 in Alveolar Epithelial Cell Function

EMC3/TMEM111 在肺泡上皮细胞功能中的作用

基本信息

批准号：
9918951
负责人：
XINHUA LIN
金额：
$ 53.94万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9918951
关键词：
ABCA3 gene Acute respiratory failure Adult Adult Respiratory Distress Syndrome Alveolar Alveolar Cell Antibodies Binding Biochemical Birth Carrier Proteins Cell physiology Cells Child Chronic Client Clinical Research Complex Complex Mixtures Confocal Microscopy Data Defect Degradation Pathway Disease Drosophila genus Endoplasmic Reticulum Environmental Risk Factor Enzymes Epithelial Cells Estrogen receptor positive Functional disorder Genes Genetic Genetic study Homeostasis Human Immunofluorescence Microscopy In Vitro Individual Infant Injury Interstitial Lung Diseases Knock-in Mouse Knowledge Life Lipids Lung Lung Transplantation Lung diseases Mammalian Cell Mass Spectrum Analysis Mediating Modeling Molecular Molecular Chaperones Morbidity - disease rate Mus Mutate Mutation Neonatal Newborn Infant Pathogenesis Pathway interactions Perinatal Phospholipids Physiological Play Premature Infant Process Production Protein Biosynthesis Proteins Proteomics Pulmonary Fibrosis Pulmonary Surfactant-Associated Protein C Pulmonary Surfactants Respiratory Failure Respiratory physiology Role Route Site Structure Surface Tension Telomerase Testing Tomatoes Toxic effect Vascular remodeling Walkers Yeasts airway epithelium alveolar epithelium alveolar lamellar body base cell injury disease diagnosis disease-causing mutation effective therapy endoplasmic reticulum stress experimental study genetic disorder diagnosis in vivo in vivo Model lipid transport loss of function member misfolded protein mortality mouse model mutant neonate novel postnatal prevent protein complex protein folding protein protein interaction protein transport respiratory distress syndrome response surfactant surfactant deficiency surfactant function surfactant production ventilation

项目摘要

PROJECT SUMMARY: Role of Emc3/Tmem111 in Alveolar Type 2 (AT2) Cell Function. Overview: Pulmonary surfactant is a complex mixture of lipids and proteins produced by AT2 cells that is secreted into the alveolar spaces to reduce surface tension and prevent alveolar collapse during ventilation. Lack of pulmonary surfactant leads to respiratory failure in 1) preterm infants and adults with respiratory distress syndrome (RDS/ARDS) and 2) chronic interstitial lung diseases (ILD) caused by mutations in genes encoding surfactant proteins (e.g. ABCA3, SFTPA, SFTPB, and SFTPC). ABCA3, a phospholipid transporter, and SP-B are critical for the formation of lamellar bodies and for surfactant function. Misrouting of mutant SP- C or loss of ABCA3 cause AT2 cell injury leading to ILD. While genetic diagnoses for diseases of surfactant homeostasis are now possible in newborn infants, there are no effective therapies other than lung transplantation for these usually fatal disorders. Lacking is knowledge regarding the specific AT2 cell machinery that integrates the routing and processing of surfactant lipids and proteins in the AT2 cells, and the molecular mechanisms by which disruption of these pathways causes AT2 cell injury, surfactant deficiency and alveolar remodeling. We have identified Emc3/Tmem111 as a critical ER component of the cellular machinery that regulates the processing, routing, and function of SP-B, SP-C, ABCA3, and lipids in AT2 cells. In this project, we will identify the intracellular sites and functions of EMC3 in vivo and in vitro. The effects of loss of function of EMC3 on lung structure, surfactant homeostasis, and lung function will be determined. Its role in a proposed cell-specific ER/EMC3 complex, its protein cargoes, and its requirement for surfactant homeostasis in AT2 cell function will be identified in mouse models in vivo, in primary AT2 cells isolated from the mice, and immortalized airway epithelial cells, representing models of both human and mouse AT2 cell function. Immunofluorescence and confocal microscopy will be used to precisely identify the intracellular sites of action, and an Emc3-flag-tomato construct will be used in conjunction with antibodies for Co-IP experiments to identify protein cargoes by proteomic analysis. Lipidomic analyses and ultrastructural studies will identify its role in lamellar body formation and surfactant lipid homeostasis. The role of EMC3 in ER stress and the recognition, stabilization, and chaperoning of mutant surfactant proteins and ABCA3 produced by defects in SFTPC and ABCA3 genes will be identified. We will determine whether EMC3/EMC complex is regulated by and influences ER stress and endoplasmic reticulum associated degradation (ERAD) pathways that causes AT2 cell toxicity.

项目摘要：Emc3/Tmem111 在肺泡 2 型 (AT2) 细胞功能中的作用。概述：肺表面活性物质是 AT2 细胞产生的脂质和蛋白质的复杂混合物，分泌到肺泡腔中以降低表面张力并防止通气期间肺泡塌陷。缺乏肺表面活性物质会导致 1) 早产儿和患有呼吸道疾病的成人呼吸衰竭窘迫综合征 (RDS/ARDS) 和 2) 由基因突变引起的慢性间质性肺疾病 (ILD) 编码表面活性蛋白（例如 ABCA3、SFTPA、SFTPB 和 SFTPC）。 ABCA3，一种磷脂转运蛋白，和 SP-B 对于层状体的形成和表面活性剂功能至关重要。突变体 SP- 的错误路由 C 或 ABCA3 缺失会导致 AT2 细胞损伤，导致 ILD。表面活性剂疾病的基因诊断现在新生婴儿的体内平衡已成为可能，但除了肺部治疗外，没有有效的治疗方法移植治疗这些通常致命的疾病。缺乏关于特定 AT2 细胞的知识整合 AT2 细胞中表面活性剂脂质和蛋白质的路由和处理的机器，以及这些途径的破坏导致 AT2 细胞损伤、表面活性剂缺乏和肺泡重塑。我们已确定 Emc3/Tmem111 是细胞内质网的关键 ER 成分调节 SP-B、SP-C、ABCA3 和脂质的加工、路由和功能的机制 AT2细胞。在这个项目中，我们将在体内和体外鉴定 EMC3 的细胞内位点和功能。这 EMC3 功能丧失对肺结构、表面活性物质稳态和肺功能的影响决定。它在拟议的细胞特异性 ER/EMC3 复合物中的作用、其蛋白质货物以及其对 AT2细胞功能中的表面活性剂稳态将在小鼠模型体内的原代AT2细胞中得到鉴定从小鼠中分离出来，以及永生化的气道上皮细胞，代表了人类和人类的模型小鼠 AT2 细胞功能。免疫荧光和共聚焦显微镜将用于精确识别胞内作用位点，并且 Emc3-flag-tomato 构建体将与抗体结合使用通过蛋白质组分析鉴定蛋白质货物的 Co-IP 实验。脂质组学分析和超微结构研究将确定其在层状体形成和表面活性剂脂质稳态中的作用。 EMC3的作用内质网应激与突变型表面活性剂蛋白和 ABCA3 的识别、稳定和陪伴由 SFTPC 和 ABCA3 基因缺陷产生的结果将被识别。我们将确定是否为EMC3/EMC 复合物受 ER 应激和内质网相关降解 (ERAD) 调节并影响导致 AT2 细胞毒性的途径。