The Role of Adenine Nucleotide Translocase in the Protection of Airway Epithelial Cells in Chronic Obstructive Pulmonary Disease (COPD)

腺嘌呤核苷酸转位酶在保护慢性阻塞性肺疾病 (COPD) 气道上皮细胞中的作用

• 批准号: 10226893
• 负责人: Corrine R Kliment
• 金额: $ 16.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-16 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226893
• 关键词: Adenine Nucleotide Translocase; Animal Model; Apical; Award; Biological; Biological Models; Biology; Cause of Death; Cell Death; Cell Survival; Cell membrane; Cell physiology; Cell surface; Cells; Cellular Metabolic Process; Chronic Bronchitis; Chronic Obstructive Airway Disease; Chronic lung disease; Cilia; Data; Development; Dictyostelium; Dictyostelium discoideum; Disease; Disease Progression; Disease model; Epithelial Cells; Foundations; Frequencies; Functional disorder; Gene Expression; Genetic; Golgi Apparatus; Health Care Costs; Homeostasis; Human; Hydration status; Immunoblotting; Immunofluorescence Immunologic; Impairment; Inner mitochondrial membrane; Knockout Mice; Lung; Lung diseases; Membrane; Mentors; Metabolism; Mitochondria; Mitochondrial Membrane Protein; Modeling; Modification; Molecular; Morbidity - disease rate; Mucociliary Clearance; Mus; Obstructive Lung Diseases; Pathogenesis; Pathway interactions; Patients; Peptide Signal Sequences; Phenotype; Physicians; Plasma; Play; Process; Production; Protein Isoforms; Proteins; Regulation; Research; Respiration; Risk Factors; Role; SLC25A4 gene; SLC25A5 gene; Scheme; Scientist; Smoke; Solid; Surface; Testing; Therapeutic; Time; Tissue imaging; Tissues; Training; United States National Institutes of Health; Work; airway epithelium; airway surface liquid; body system; bronchial epithelium; career; career development; cellular imaging; cigarette smoke; cigarette smoke-induced; cilium motility; effective therapy; epithelial injury; exposure to cigarette smoke; fascinate; gain of function; genetic selection; human model; mitochondrial metabolism; mortality; mouse model; new therapeutic target; novel; overexpression; preservation; prevent; programs; protective effect; reconstitution; skills; therapeutic target; tool; trafficking

PROJECT SUMMARY The overall objectives of this NIH K08 Career Mentored Award are to identify and investigate new therapeutic targets for epithelial injury due to cigarette smoke in chronic obstructive lung disease (COPD), and to facilitate the development of essential skills that will allow the candidate to become an effective and independent physician-scientist. The candidate and her mentors have constructed a rigorous training plan that will provide the foundation for a strong and successful academic career. This proposal explores the core mechanisms of how adenine nucleotide translocase (ANT) is localized to the plasma membrane of ciliated airway epithelium and further how ANT is utilized for more advanced cellular functions, such as ciliary ATP regulation impacting airway hydration and ciliary beating. COPD is the 3rd leading cause of death in the US with cigarette smoke (CS) being the primary insult leading to disease. Despite ongoing mammalian research, no new biologic targets have been identified, and current therapies have limited effect on disease progression or mortality. I have leveraged the power of a genetically tractable model system, the amoebozoan Dictyostelium discoideum, as a genetic selection discovery tool for lung biology. I identified human ANT (ANT1 and ANT2 present in lung) as protective against CS-induced cell death in human bronchial epithelial cells. ANT is a mitochondrial ADP/ATP transporter that plays an active role in mitochondrial metabolism and ATP homeostasis. While exploring the localization of ANTs in human airway tissue, I made a fascinating observation that in addition to mitochondrial localization, human ANT1 and ANT2 localize to the plasma membrane of motile cilia in human airway and isolated primary airway cells (Normal Human Bronchial Epithelial cells, NHBE). GFP-tagged adenoviral ANT1 and ANT2 also go to the ciliary membrane (and mitochondria) in primary NHBEs and ANT1 and ANT2 are present in isolated human ciliary axonemes by western analysis. In addition, ANT2 specifically enhances airway surface hydration and preserves ciliary beat frequency in the context of CS. This early career proposal answers the following questions: How does human ANT1 and ANT2 localize to the plasma membrane of airway epithelium; how do plasma membrane versus mitochondrial ANTs regulate airway metabolism, airway hydration and ciliary function in ciliated airway epithelium; and what is the impact of ANT2 loss and gain of function on airway dysfunction and COPD development through mouse models of disease given its protective phenotype on airway epithelium. This will be accomplished using a combination of molecular tools, ANT isoform genetic modification, cell and tissue imaging, real-time analysis of cellular metabolism, assessment of airway hydration and ciliary function, and correlation with a mouse model of smoke exposure. Ultimately, the role of ANT in lung disease has yet to be described and therapeutic manipulation of its biology may allow us to drive cells to a healthier state. The core biology themes explored here will likely have broad applicability and impact other lung diseases.

项目概要 NIH K08 职业指导奖的总体目标是识别和研究新的治疗方法 慢性阻塞性肺病（COPD）中香烟烟雾引起的上皮损伤的目标，并促进 发展基本技能，使候选人成为有效和独立的人 医生科学家。候选人和她的导师制定了严格的培训计划，将提供 为强大而成功的学术生涯奠定基础。该提案探讨了核心机制 腺嘌呤核苷酸转位酶 (ANT) 如何定位于纤毛气道上皮的质膜 以及如何进一步利用 ANT 来实现更高级的细胞功能，例如影响纤毛 ATP 调节 气道水合作用和纤毛跳动。慢性阻塞性肺病是美国因吸烟而死亡的第三大原因 (CS) 是导致疾病的主要损伤。尽管哺乳动物研究正在进行，但没有新的生物制剂 目标已经确定，目前的疗法对疾病进展或死亡率的影响有限。我 利用了遗传易处理模型系统——变形虫盘基网柄菌（Dictyostelium discoideum）的力量， 作为肺生物学的基因选择发现工具。我发现了人类 ANT（ANT1 和 ANT2 存在于肺部） 作为对 CS 诱导的人支气管上皮细胞细胞死亡的保护作用。 ANT是线粒体 ADP/ATP 转运蛋白在线粒体代谢和 ATP 稳态中发挥积极作用。尽管 在探索 ANT 在人体气道组织中的定位时，我做了一个有趣的观察，除了 线粒体定位，人类 ANT1 和 ANT2 定位于人类活动纤毛的质膜 气道和分离的初级气道细胞（正常人​​支气管上皮细胞，NHBE）。 GFP标记的 腺病毒 ANT1 和 ANT2 也进入原发性 NHBE 和 ANT1 中的睫状膜（和线粒体） 根据蛋白质印迹分析，ANT2 和 ANT2 存在于分离的人纤毛轴丝中。此外，ANT2特别 在 CS 的情况下增强气道表面水合作用并保持纤毛跳动频率。这早期的职业生涯 该提案回答了以下问题：人类 ANT1 和 ANT2 如何定位到血浆 气道上皮膜；质膜与线粒体 ANT 如何调节气道 纤毛气道上皮的新陈代谢、气道水合作用和纤毛功能； ANT2有什么影响 通过小鼠疾病模型，气道功能障碍和慢性阻塞性肺病发展的功能丧失和获得 鉴于其对气道上皮的保护表型。这将通过组合来完成 分子工具、ANT亚型遗传修饰、细胞和组织成像、细胞实时分析 新陈代谢、气道水合和纤毛功能的评估以及与烟雾小鼠模型的相关性 接触。最终，ANT 在肺部疾病中的作用尚未得到描述，治疗操作也尚未得到证实。 它的生物学特性可能使我们能够将细胞推向更健康的状态。这里探讨的核心生物学主题可能会 具有广泛的适用性并影响其他肺部疾病。