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

项目总结 NIH K08职业导师奖的总体目标是发现和研究新的治疗方法 慢性阻塞性肺疾病(COPD)吸烟致上皮损伤的靶点，并促进 发展基本技能，使应聘者成为一名有效和独立的 医生兼科学家。候选人和她的导师已经制定了一份严格的培训计划，将提供 这是一个强大而成功的学术生涯的基础。这项建议探讨了 腺核苷酸转位酶(ANT)如何定位于纤毛呼吸道上皮质膜 以及蚂蚁如何被用于更高级的细胞功能，如纤毛ATP调节影响 呼吸道水化和纤毛跳动。慢性阻塞性肺病是美国吸烟导致死亡的第三大原因 (Cs)是导致疾病的主要侮辱。尽管哺乳动物研究仍在进行，但没有新的生物学 目标已经确定，目前的治疗方法对疾病进展或死亡率的影响有限。我 利用了一种遗传上易驯化的模型系统--盘状网柄蠕虫--的力量， 作为肺部生物学的基因选择发现工具。我鉴定了人蚂蚁(肺中存在ANT1和ANT2) 作为对CS诱导的人支气管上皮细胞死亡的保护作用。蚂蚁是一种线粒体 在线粒体代谢和ATP动态平衡中发挥积极作用的ADP/ATP转运体。而当 为了探索蚂蚁在人类呼吸道组织中的定位，我做了一个有趣的观察，除了 线粒体定位、人ANT1和ANT2定位于人活动纤毛质膜 呼吸道和分离的原代呼吸道细胞(正常人支气管上皮细胞，NHBE)。GFP-标记 腺病毒ANT1和ANT2也进入原发NHBE和ANT1的睫状膜(和线粒体) 经Western分析，ANT2在孤立的人睫状轴丝中存在。此外，ANT2特别指出 在CS的情况下，增强呼吸道表面的水合作用并保留纤毛搏动频率。这个早期的职业生涯 Proposal回答了以下问题：人类ANT1和ANT2如何定位于血浆 呼吸道上皮膜；质膜与线粒体蚂蚁如何调节呼吸道 纤毛呼吸道上皮的代谢、气道水化和纤毛功能；ANT2的影响是什么？ 通过小鼠疾病模型研究呼吸道功能障碍和慢性阻塞性肺疾病发展的功能丧失和获得 鉴于其对呼吸道上皮的保护表型。这将使用以下组合来实现 分子工具、蚂蚁异构体基因修饰、细胞和组织成像、细胞的实时分析 新陈代谢、呼吸道水合作用和睫毛功能的评估以及与烟雾小鼠模型的相关性 曝光。归根结底，ANT在肺部疾病中的作用还没有被描述，治疗操作也没有。 它的生物学可能会让我们把细胞推向更健康的状态。这里探讨的核心生物学主题很可能 具有广泛的适用性，并对其他肺部疾病产生影响。

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.