MtDNA repair: An isolated pharmacologic target in acute lung injury

描述（由申请人提供）：许多研究探索了抗氧化剂作为急性肺损伤（ALI）的治疗药物，但其临床试验结果令人失望。对这种失败的一种解释是，ALI中产生的氧化剂不仅具有细胞毒性，而且它们还在肺细胞增殖和分化所需的途径中充当第二信使。因此，非选择性抗氧化剂可能会破坏细胞存活和恢复所需的信号传导。一个相关的可能性是，关键分子整合活性氧对肺血管系统的作用尚未确定;抗氧化剂可能无法保护这些假定的哨兵分子的损伤，这些哨兵分子控制细胞死亡程序的激活。基于从细胞培养研究中获得的多条令人信服的证据，本文中我们提出测试线粒体（mt）DNA是决定肺细胞命运的哨兵分子的概念，以响应氧化应激，线粒体DNA修复途径是干预ALI的靶点。特异性目标1将开始将细胞培养的发现转化为实际应用，通过验证以下假设：使用新型融合蛋白构建体增加mtDNA糖基化酶活性可保护和逆转患有铜绿假单胞菌或呼吸机诱导的ALI的啮齿动物的ALI。这些实验是朝着我们开发mtDNA修复融合蛋白作为治疗ALI的平台药物分子的长期目标迈出的重要一步。尽管线粒体DNA修复的增强抑制了ROS介导的细胞死亡，但这种有益作用的机制仍然难以捉摸。传统观念认为，加速的mtDNA修复阻止了氧化性mtDNA损伤的积累，从而减弱了促凋亡ROS的线粒体产生和伴随的细胞死亡驱动的细胞死亡的激活，但不是所有目前可用的数据 一些研究表明mtDNA修复本身并不重要。因此，特异性目标2将使用野生型和突变型Ogg 1蛋白，其修复（但不结合）mtDNA的能力不同，以确定RS诱导的mtDNA损伤的修复是否是预防RS诱导的EC细胞毒性、屏障功能障碍和损伤传播mtDNA片段的细胞外释放的形成所必需的。

DESCRIPTION (provided by applicant): Numerous studies have explored anti-oxidants as therapeutic agents in acute lung injury (ALI), but their outcome in clinical trials has been disappointing. One explanation for this failure is that oxidants generated in ALI are not only cytotoxic, but they also function as second messengers in pathways required for lung cell proliferation and differentiation. Thus, non-selective antioxidants may disrupt signaling required for cell survival and recovery. A related possibility is that key molecule(s) integrating ROS actions on the pulmonary vasculature have yet to be identified; antioxidants may fail to protect against damage to these putative sentinel molecule(s) governing activation of cell death programs. Based on multiple lines of compelling evidence obtained from cell culture studies, herein we propose to test the concept that mitochondrial (mt) DNA is a sentinel molecule dictating lung cell fate in response to oxidant stress and that the mtDNA repair pathway is a target for intervention in ALI. Specific Aim 1 will begin translating findings from cell culture ino practical application by testing the hypothesis that increasing mtDNA glycosylase activity using novel fusion protein constructs protects against, and reverses, ALI in rodents with either Pseudomonas aeruginosa- or ventilator- induced ALI. These experiments comprise an important step towards our longer term goal of developing mtDNA repair fusion proteins as platform drug molecules for treating ALI. Even though enhancement of mtDNA repair suppresses ROS-mediated cell death, the mechanism of this salutary effect has remained elusive. Traditional concepts hold that accelerated mtDNA repair prevents accumulation of oxidative mtDNA damage, thereby attenuating mitochondrial generation of pro-apoptotic ROS and attendant activation of mitochondrially-driven cell death, but not all currently available data support this model and some studies suggest that mtDNA repair, per se, is unimportant. Accordingly, Specific Aim 2 will use wild type and mutant Ogg1 proteins differing in their capacities to repair - but not to bind - mtDNA to determine whether repair of RS-induced mtDNA damage is required for prevention of RS-induced EC cytotoxicity, barrier dysfunction, and formation an extracellular release of injury-propagating mtDNA fragments.