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

项目总结 慢性阻塞性肺疾病(COPD)在美国是一个日益严重的健康问题，没有治愈的方法 治疗。由于难以找到新的治疗方法，新疗法的发展一直停滞不前。 人类肺复杂组织中的生物学和保护途径。因此，罗宾逊实验室 有兴趣使用一种模式生物--社会变形虫盘基网履虫，作为一种发现工具来寻找 新的治疗靶点和途径将防止香烟烟雾(CS)，这是导致 慢性阻塞性肺疾病（慢阻肺）。对于网柄网柄菌，进行了基因筛查以寻找这些目标基因。两个的过度表达 编码腺核苷酸转位酶(ANT)和肌动蛋白相互作用蛋白1(AIP1)的基因提供了最多的 在细胞生长中提供强有力的保护。有趣的是，我们在人类身上看到了这些基因同样的保护作用。 CS暴露对支气管上皮细胞的影响。这项提议的重点将是机械地理解如何 这些蛋白否定了CS损伤的影响。从蚂蚁开始，内部是一个ATP/ADP转运体 线粒体膜，我们预计它的过度表达会增强细胞的新陈代谢。 有趣的是，一些初步数据表明，蚂蚁通过不同的机制发挥保护作用。这个 令人惊讶的是，在纤毛上发现了规范的线粒体蛋白，并调节了纤毛功能，这是已知的 被CS更改。在纤毛原代人支气管上皮细胞(NHBE)中，ANT2(与 蚂蚁)通过增加呼吸道水化和维持正常的睫状体搏动频率来增强睫状体功能 CS的存在。根据这一初步数据和细胞外ATP释放增加的想法 我们假设ANT是细胞外ATP的一种难以捉摸的细胞表面转运体。 这一想法将通过免疫荧光和超分辨率成像在本提案的目标1中进行测试， 表面生物素化分析，以及细胞外ATP对ANT功能NHBE的增减的测量。 由于发现了蚂蚁的保护性表型，还将进行初步的药物筛选，以找到 蚂蚁激活剂。在目标2中，我们将重点介绍AIP1。我们同样会发现它的过度表达是如何防止 CS.研究发现CS影响肌动蛋白动力学和细胞力学，从而导致呼吸道屏障增加。 渗透性。考虑到它作为肌动蛋白解聚的调节因子的作用，我们预计AIP1将否定 CS对肌动蛋白动力学的影响，这将加强细胞与细胞之间的相互作用，增强呼吸道屏障功能。 研究这一点的实验将包括细胞骨架分级，以通过F/G-肌动蛋白比率评估肌动蛋白组装 以及共聚焦成像来评估AIP1是否改变了细胞连接蛋白的表达和定位。 将使用跨上皮耐药(TEER)测量和FITC-葡聚糖通透性分析来评估 上皮屏障密封性。总体而言，这项工作将使我们了解CS是如何扰乱正常细胞的 以及ANT和AIP1如何逆转这些有害影响。这些信息将是至关重要的 为开发可能治疗COPD的药物生成一个框架。

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.