Role of S-nitrosothiols in Akt1 signaling and pneumonia resolution

项目摘要/摘要 这份提案描述了一项旨在促进博士发展的综合五年培训计划。 布莱恩·D·克拉夫特，医学博士(首席研究员)，成为一名独立的内科科学家。克拉夫特博士完成了他的 杜克大学内科住院医师和肺部、过敏和重症监护医学研究员 他现在是大学医学中心的一名医学讲师。克拉夫特博士的长期目标是开发新的 危重患者急性肺损伤(ALI)的治疗方法。克拉夫特博士的研究 焦点是细菌性肺炎后的肺修复机制，这是全球范围内的主要死亡原因，尽管 抗生素的使用。为了追求他的目标，克拉夫特博士已经用老鼠和非人类进行了四年的研究 灵长类(NHP)肺炎模型在他的赞助商克劳德·皮安塔多西博士的指导下建立，克劳德·皮安塔多西博士是 ALI和线粒体质量控制机制。Piantadosi博士有成功指导更多 而不是40名初级调查员。克拉夫特博士提出的职业发展计划结合了实验室 培训，正式教学，出席科学会议，以及由以下人员组成的咨询委员会 S-亚硝硫醇(SNO)生物学和线粒体信号机制方面的专家。在他的团契期间 训练表明，ALI后肺修复的一个关键细胞程序是线粒体 生物发生，产生新的线粒体质量。这项提案中提出的初步研究表明 线粒体的生物发生可以在肺泡II型上皮(AT2)细胞中被激活，AT2细胞是 在肺泡区，通过药理增加总的肺SNO水平。SnO蛋白有 经过S亚硝化，翻译后修饰，NO被转移到硫的部分 半胱氨酸硫醇。初步数据表明，这些发现的一个潜在机制是S-亚硝化(和 因此使Akt1去磷酸化(去激活)的蛋白磷酸酶PTEN和PHLPP失活)， 线粒体生物发生转录网络的蛋白激酶激活剂。的中心假设 提出如下观点：1)SNO通过抑制磷酸酶途径激活AT2细胞线粒体的生物合成 增加Akt1的磷酸化和关键下游修复基因的激活；以及2)SNO介导的 诱导线粒体生物发生将加速细菌性肺炎后的肺修复。这些 假设将以以下具体目的进行检验：1)确定(A)PTEN的S亚硝化反应 和/或(B)PHLPP激活肺AT2细胞Akt1和线粒体生物发生；以及2)确定 药物性SNO增强可加速小鼠金黄色葡萄球菌所致ALI的消退 肺炎。这项工作有望对为什么内生机制可能不是 足以解决ALI的重症肺炎以及线粒体的新调节机制 可通过药物作用加速肺炎消退的生物发生。

PROJECT SUMMARY/ABSTRACT This proposal describes a comprehensive five-year training program designed to foster the development of Dr. Bryan D. Kraft, M.D. (Principal Investigator) into an independent physician-scientist. Dr. Kraft completed his residency in Internal Medicine and fellowship in Pulmonary, Allergy, and Critical Care Medicine at Duke University Medical Center where he is now a Medical Instructor. Dr. Kraft’s long term goal is to develop novel therapeutic approaches to the resolution of acute lung injury (ALI) in critically-ill patients. Dr. Kraft’s research focus is mechanisms of lung repair following bacterial pneumonia, a leading cause of death worldwide despite the use of antibiotics. In pursuit of his goal, Dr. Kraft has worked for four years using murine and nonhuman primate (NHP) pneumonia models under the mentorship of his sponsor, Dr. Claude Piantadosi, an expert in ALI and mitochondrial quality control mechanisms. Dr. Piantadosi has a record of successfully mentoring more than 40 junior investigators. The career development program proposed by Dr. Kraft incorporates laboratory training, formal didactics, attendance at scientific conferences, and an advisory committee composed of experts in S-nitrosothiol (SNO) biology and mitochondrial signaling mechanisms. During his Fellowship training, it became apparent that a critical cellular program for lung repair following ALI is mitochondrial biogenesis, the generation of new mitochondrial mass. Preliminary studies presented in this proposal indicate that mitochondrial biogenesis can be activated in lung alveolar type II epithelial (AT2) cells, the progenitor cells of the alveolar region, by pharmacologically augmenting the levels of total lung SNOs. SNO proteins have undergone S-nitrosylation, a post-translational modification whereby NO is transferred to the sulfur moieties of cysteine thiols. Preliminary data indicate that a potential mechanism for these findings is S-nitrosylation (and therefore inactivation) of the protein phosphatases PTEN and PHLPP that de-phosphorylate (de-activate) Akt1, a protein kinase activator of the transcriptional network for mitochondrial biogenesis. The central hypotheses of the proposal are that 1) SNOs activate mitochondrial biogenesis in AT2 cells via phosphatase inhibition leading to increased Akt1 phosphorylation and activation of key downstream repair genes; and 2) SNO-mediated induction of mitochondrial biogenesis will accelerate lung repair following bacterial pneumonia. These hypotheses will be tested with the following Specific Aims: 1) Determine if S-nitrosylation of (a) PTEN and/or (b) PHLPP activate Akt1 and mitochondrial biogenesis in lung AT2 cells; and 2) Determine if pharmacologic SNO augmentation can accelerate resolution of ALI following murine S. aureus pneumonia. This work is expected to yield important insight into why endogenous mechanisms may not be sufficient to resolve severe pneumonia with ALI as well as novel regulatory mechanisms of mitochondrial biogenesis that could be pharmacologically exploited to accelerate pneumonia resolution.