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

项目摘要 该NIH K 08职业指导奖的总体目标是确定和研究新的治疗方法， 慢性阻塞性肺疾病（COPD）中香烟烟雾引起的上皮损伤的靶点， 基本技能的发展，使候选人成为一个有效的和独立的 物理学家兼科学家候选人和她的导师已经制定了严格的培训计划， 一个强大的和成功的学术生涯的基础。该提案探讨了 腺嘌呤核苷酸移位酶（ANT）如何定位于纤毛气道上皮细胞的质膜 以及ANT如何用于更高级的细胞功能，如纤毛ATP调节， 呼吸道水合和纤毛跳动。COPD是美国第三大死亡原因，与吸烟有关 (CS)是导致疾病的主要原因尽管哺乳动物研究正在进行中，但没有新的生物 已经确定了靶点，目前的治疗对疾病进展或死亡率的影响有限。我 利用了一个遗传上易于处理的模型系统，即变形虫盘基网骨藻， 作为肺部生物学的遗传选择发现工具。我鉴定了人ANT（ANT 1和ANT 2存在于肺中） 作为对CS诱导的人支气管上皮细胞的细胞死亡的保护。ANT是一种线粒体 在线粒体代谢和ATP稳态中起积极作用的ADP/ATP转运体。而 在探索ANTs在人类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ANT 1和ANT 2定位于人活动纤毛的质膜 气道和分离的原代气道细胞（正常人支气管上皮细胞，NHBE）。gfp标记 腺病毒ANT 1和ANT 2也进入原发性NHBE和ANT 1中的睫状膜（和线粒体 和ANT 2存在于分离的人睫状体轴突中。此外，ANT 2特别是 增强气道表面水合作用并在CS的情况下保持纤毛搏动频率。早期的职业生涯 该提案回答了以下问题：人类ANT 1和ANT 2如何定位于血浆 气道上皮细胞膜;质膜与线粒体ANT如何调节气道 纤毛气道上皮细胞的代谢、气道水化和纤毛功能;以及ANT 2的影响 通过小鼠疾病模型研究气道功能障碍和COPD发展的功能丧失和获得 考虑到其对气道上皮的保护表型。这将通过以下组合来实现： 分子工具，ANT异构体遗传修饰，细胞和组织成像，实时分析细胞 代谢，评估气道水合作用和纤毛功能，以及与吸烟小鼠模型的相关性 exposure.最后，ANT在肺部疾病中的作用还有待描述， 它的生物学特性可以让我们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。

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.