Characterization of Adenine Nucleotide Translocase (ANT) and Actin-Interacting Protein 1 (AIP1) as Protectors Against Cigarette Smoke

腺嘌呤核苷酸转位酶 (ANT) 和肌动蛋白相互作用蛋白 1 (AIP1) 作为香烟烟雾保护剂的表征

• 批准号: 9917578
• 负责人: Jennifer Nguyen
• 金额: $ 4.55万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-16 至 2022-02-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9917578
• 关键词: Actins; Address; Adenine Nucleotide Translocase; Adenoviruses; Affect; Amoeba genus; Animal Model; Biological Assay; Biology; Biotinylation; Cause of Death; Cell Communication; Cell Death; Cell Survival; Cell physiology; Cell surface; Cells; Characteristics; Chronic Obstructive Airway Disease; Cilia; Complex; Cytoskeleton; Data; Defect; Development; Dictyostelium; Dictyostelium discoideum; Disease Progression; Drug Screening; Epithelial; Epithelial Cells; Epithelium; Fractionation; Frequencies; G Actin; Genes; Genetic Screening; Health; Height; Human; Hydration status; Image; Immunofluorescence Immunologic; Inflammation; Injury; Inner mitochondrial membrane; Intercellular Junctions; Lactate Dehydrogenase; Luciferases; Lung diseases; Measurement; Measures; Mechanics; Membrane; Metabolism; Mitochondrial Proteins; Mucous body substance; Pathogenesis; Pathway interactions; Permeability; Pharmaceutical Preparations; Phenocopy; Phenotype; Proteins; Research; Resistance; Resolution; Role; SLC25A5 gene; Signal Transduction; Structure of parenchyma of lung; Surface; System; Testing; Therapeutic; United States; Work; airway epithelium; airway surface liquid; base; bronchial epithelium; cDNA Library; cell growth; cigarette smoke; cigarette smoke-induced; confocal imaging; curative treatments; depolymerization; experimental study; exposure to cigarette smoke; extracellular; fluorescein isothiocyanate dextran; human tissue; insight; interest; knock-down; loss of function; luciferin; lung injury; new therapeutic target; novel therapeutics; overexpression; paralogous gene; persistent symptom; prevent; protective effect; social; therapeutic target; tool

PROJECT SUMMARY Chronic obstructive pulmonary disease (COPD) is a growing health concern in the United States with no curative treatments. The development of new therapeutics has been stagnant due to the difficulty of finding new essential biology and protective pathways in the complex tissue of the human lung. Hence, the Robinson lab was interested in using a model organism, the social amoeba Dictyostelium discoideum, as a discovery tool to find new therapeutic targets and pathways that will protect against cigarette smoke (CS), one of the main causes of COPD. With Dictyostelium, a genetic screen was conducted to find these target genes. Overexpression of two genes encoding for adenine nucleotide translocase (ANT) and actin-interacting protein 1 (AIP1) offered the most robust protection in cell growth. Interestingly, we see the same protective effects from these genes in human bronchial epithelial cells exposed to CS. The focus of this proposal will be to mechanistically understand how these proteins negate the effects of CS injury. Beginning with ANT, an ATP/ADP transporter in the inner membrane of the mitochondria, we expected that its overexpression would enhance cellular metabolism. Interestingly, some preliminary data suggested that ANT was protective through different mechanisms. The canonical mitochondria protein was surprisingly found at cilia and modulated ciliary function, which is known to be altered by CS. In ciliated primary human bronchial epithelial cells (NHBEs), ANT2 (one of the paralogs of ANT) enhanced ciliary function by increasing airway hydration and maintaining normal ciliary beat frequency in the presence of CS. Based on this preliminary data and the idea that extracellular ATP is released to increase airway hydration, we hypothesize that ANT is one of the elusive cell surface transporters of extracellular ATP. This idea will be tested in aim 1 of this proposal through immunofluorescence and super-resolution imaging, surface biotinylation assays, and the measurement of extracellular ATP on ANT gain- or loss- of function NHBEs. Since protective phenotypes of ANT were found, a preliminary drug screen will also be conducted to find activators of ANT. In aim 2, we will focus on AIP1. We will similarly find how its overexpression protects against CS. CS was found to affect actin dynamics and cellular mechanics, which caused increased airway barrier permeability. Considering its role as a regulator of actin depolymerization, we expect that AIP1 will negate the effects of CS on actin dynamics, which will tighten cell-cell interactions and fortify airway barrier function. Experiments to study this will include cytoskeletal fractionation to evaluate actin assembly via F/G-actin ratios and confocal imaging to assess whether AIP1 changes the expression and localization of cell junction proteins. Trans-epithelial resistance (TEER) measurements and a FITC-dextran permeability assay will be used to assess epithelial barrier tightness. Overall, this work will allow us to understand what CS does to disrupt normal cellular functions in the airway, and how ANT and AIP1 can reverse these harmful effects. This information will be critical to generate a framework for developing drugs that can potentially treat COPD.

项目摘要 慢性阻塞性肺疾病（COPD）是美国日益严重的健康问题， 治疗。由于很难找到新的必需品， 生物学和人类肺部复杂组织中的保护途径。因此，罗宾逊实验室 感兴趣的是使用一种模式生物，社会性阿米巴Dictyosteelium discoideum，作为发现工具， 新的治疗靶点和途径，将防止香烟烟雾（CS），其中一个主要原因， 慢性阻塞性肺病对于网囊藻，进行遗传筛选以找到这些靶基因。两个过度表达 编码腺嘌呤核苷酸移位酶（ANT）和肌动蛋白相互作用蛋白1（AIP 1）的基因提供了最 在细胞生长中提供强大的保护。有趣的是，我们在人类身上也看到了这些基因的保护作用。 暴露于CS的支气管上皮细胞。本提案的重点将是从机制上理解如何 这些蛋白质抵消了CS损伤的影响。从ANT开始，ANT是一种位于内皮层的ATP/ADP转运蛋白， 由于它是线粒体膜上的一个蛋白质，我们预期它的过表达将增强细胞代谢。 有趣的是，一些初步的数据表明，ANT是通过不同的机制保护。的 令人惊讶的是，在纤毛中发现了典型的线粒体蛋白，并调节纤毛功能，这是已知的， 被CS改变。在纤毛原代人支气管上皮细胞（NHBE）中，ANT 2（ANT 2的旁系同源物之一）表达于细胞内。 ANT）通过增加气道水化和维持正常纤毛搏动频率增强纤毛功能， CS的存在。基于这一初步数据以及细胞外ATP释放增加的想法， 气道水化，我们假设ANT是细胞外ATP的难以捉摸的细胞表面转运蛋白之一。 这一想法将在本提案的目标1中通过免疫荧光和超分辨率成像进行测试， 表面生物素化测定，以及ANT功能获得或丧失NHBE的细胞外ATP测量。 由于发现了ANT的保护性表型，因此还将进行初步药物筛选，以发现 ANT的激活剂。在目标2中，我们将重点关注AIP 1。我们将同样发现它的过度表达如何保护 CS. CS可影响肌动蛋白动力学和细胞力学，导致气道屏障增加 磁导率考虑到它作为肌动蛋白解聚调节剂的作用，我们预计AIP 1将否定肌动蛋白解聚的作用。 CS对肌动蛋白动力学的影响，这将加强细胞-细胞相互作用并强化气道屏障功能。 研究这一点的实验将包括通过F/G-肌动蛋白比率来评估肌动蛋白组装的细胞骨架分离 和共聚焦成像以评估AIP 1是否改变细胞连接蛋白的表达和定位。 将使用跨上皮抵抗力（TEER）测量和FITC-葡聚糖渗透性测定来评估 上皮屏障紧密性总的来说，这项工作将使我们了解CS是如何破坏正常细胞的， 在气道中的功能，以及ANT和AIP 1如何逆转这些有害影响。这些信息将至关重要 为开发可能治疗COPD的药物提供了一个框架。