Role of S-nitrosothiols in Akt1 signaling and pneumonia resolution

项目概要/摘要 本提案描述了一个全面的五年培训计划，旨在促进博士的发展。 布莱恩·D Kraft，医学博士（主要研究者）转变为独立的医学科学家。克拉夫特博士完成了他的 在杜克大学内科住院医师和肺、过敏和重症监护医学奖学金 大学医学中心，他现在是一名医学讲师。Kraft博士的长期目标是开发新的 解决危重患者急性肺损伤（ALI）的治疗方法。卡夫博士的研究 重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， 使用抗生素。为了实现他的目标，Kraft博士用了四年的时间， 灵长类动物（NHP）肺炎模型在他的赞助商，克劳德Piantadosi博士，专家的指导下， ALI和线粒体质量控制机制。Piantadosi博士有一个成功地指导更多的记录。 超过40名初级调查员卡夫博士提出的职业发展计划包括实验室 培训、正式教学法、参加科学会议，以及一个咨询委员会， S-nitrosothiol（SNO）生物学和线粒体信号机制方面的专家。在他的奖学金期间 训练后，很明显，ALI后肺修复的关键细胞程序是线粒体 生物发生，新的线粒体质量的产生。该提案中提出的初步研究表明， 线粒体生物合成可以在肺泡II型上皮细胞（AT 2）中被激活， 肺泡区域，通过增加总肺SNO的水平。SNO蛋白具有 经历S-亚硝基化，这是一种翻译后修饰，由此NO被转移到 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-亚硝基化（和 因此失活）使Akt 1去磷酸化（失活）的蛋白磷酸酶PTEN和PHLPP， 线粒体生物合成转录网络的蛋白激酶激活剂。的中心假设 提出以下观点：1）SNOs通过磷酸酶抑制作用激活AT 2细胞线粒体生物合成， 增加Akt 1磷酸化和关键下游修复基因的激活;和2）SNO介导的 线粒体生物发生的诱导将加速细菌性肺炎后的肺修复。这些 将用以下具体目的检验假设：1）确定（a）PTEN的S-亚硝基化是否 和/或（B）PHLPP激活肺AT 2细胞中的Akt 1和线粒体生物发生;和2）确定是否 药理学SNO增加可加速小鼠S.金黄色 肺炎这项工作预计将产生重要的见解，为什么内源性机制可能不会 足以解决严重肺炎与ALI以及新的调节机制，线粒体 生物发生可以被充分利用来加速肺炎的消退。

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.