Akt-mediated phosphorylation of hsp70 regulates mitochondrial localization of SOD2 and oxidative stress in pulmonary artery endothelial cells during postnatal transition

摘要 我的目标是成为一名独立的研究员，在新生儿疾病导向研究中拥有特定的 关注新生儿持续性肺动脉高压时线粒体氧化应激的调节 (PPHN)。PPHN影响2-6/1000活产儿，是新生儿心肺衰竭的常见原因。 在这些婴儿中，30%的婴儿未能通过医疗治疗，需要侵入性支持措施(ECMO)。氧化应激 与PPHN的发病机制密切相关。NADPH氧化酶被认为是 肺内皮细胞中的超氧化物(O2‘)。呼吸产生过程中线粒体的耗氧量 氧作为氧化磷酸化的副产物流入线粒体。最近的证据表明 线粒体产生的活性氧种(ROS)诱导NADPH氧化酶的激活，铅- ING诱导ROS形成。然而，线粒体O2形成的调节仍不清楚。 氧暴露期间最大限度减少线粒体O2生成的适应性机制的鉴定 出生时基因可能会识别PPHN的其他治疗靶点。我将通过从事以下工作开始实现这一目标 一个职业发展计划，从逻辑上允许我扩展我以前的技能，并在鼠标领域建立新的技能- Nestics，蛋白质-蛋白质相互作用的分析和与内切相关的新信号通路的鉴定。 上皮生物学。该计划将遗传学、生物化学和自由基生物学的教学培训与 从我的导师和威斯康星医学院科学顾问委员会那里学习技能 拥有内皮生物学、发育血管生物学、自由基和线粒体方面的专业知识 生物学。指导和职业发展计划与拟议的研究目标相结合，以 检验Akt诱导HSP70翻译后修饰并调节相互作用的假设 与最近发现的两种蛋白HSP70结合，即：促进SOD2免疫的Obg样ATPase-1(OLA1) PORT，和CHIP，这是一种伴侣相关的泛素连接酶，靶向HSP70进行降解。磷酸盐- Akt对HSP70的磷酸化促进HSP70与OLA1的相互作用，促进线粒体免疫。 在出生后过渡期间，SOD2端口可减少游离O2.第一个目标是确定捐款 OLA1和CHIP对肺泡内皮细胞线粒体氧化还原信号的调节作用。我们将使用转基因小鼠， 体外动力学分析和细胞培养确定OLA1和CHIP对线粒体氧化还原的贡献 并确定OLA1和CHIP如何机械地调节线粒体SOD2的输入和释放 ROS生产。第二个目标将确定PI3K/Akt信号通路在调节细胞周期中的机制作用。 探讨线粒体SOD2的输入以及该机制与出生后适应的功能相关性。 拟议的研究和培训方案的成功完成将导致未来的研究调查 改善PPHN患者预后的可预防和治疗策略，目标是减少 PPHN造成的经济和卫生负担。

ABSTRACT My goal is to become an independent investigator in disease-oriented research in the newborn with a specific focus on the regulation of mitochondrial oxidative stress in persistent pulmonary hypertension of the newborn (PPHN). PPHN affects 2-6/1000 live births and is a common cause of cardiopulmonary failure in the newborn. Of these infants, >30% fail medical treatment and need invasive support measures (ECMO). Oxidative stress is strongly implicated in the pathogenesis of PPHN. NADPH oxidases are considered the primary source of superoxide (O2¯) in the pulmonary endothelium. Mitochondrial oxygen consumption during respiration produc- es influx of O2¯ in the mitochondria as a byproduct of oxidative phosphorylation. Recent evidence indicates that reactive oxygen species (ROS) produced by mitochondria induce the activation of NADPH oxidases, lead- ing to ROS induced ROS formation. However, the regulation of mitochondrial O2¯formation remains unknown. Identification of the adaptive mechanisms that minimizes mitochondrial O2¯formation during exposure to oxy- gen at birth may identify additional therapeutic targets in PPHN. I will begin to achieve this goal by engaging in a Career Development plan that logically allows me to expand my prior skills and build new skills in mouse ge- netics, analysis of protein-protein interactions and identification of novel signaling pathways pertinent to endo- thelial biology. This plan integrates didactic training in genetics, biochemistry and free radical biology with learning of skills from my Mentors and Scientific Advisory Committee at the Medical College of Wisconsin who combined have expertise in endothelial biology, developmental vascular biology, free radical and mitochondrial biology. The Mentorship and Career Development plan are integrated with the proposed research objectives to test the hypothesis that Akt induces a post translational modification of hsp70 and modulates the interactions of hsp70 with two recently identified proteins namely: an Obg like ATPase-1 (OLA1) that facilitates SOD2 im- port, and CHIP, which is a chaperone-associated ubiquitin ligase that targets hsp70 for degradation. Phos- phorylation of hsp70 by Akt promotes the interaction of hsp70 with OLA1 and facilitates the mitochondrial im- port of SOD2 to reduce free O2¯during postnatal transition. The first aim seeks to determine the contributions of OLA1 and CHIP to the regulation of mitochondrial redox signaling in PAECs. We will use transgenic mice, in vitro kinetic assays and cell culture to determine the contributions of OLA1 and CHIP to mitochondrial redox signaling in PAECs and identify how OLA1 and CHIP mechanistically regulate mitochondrial SOD2 import and ROS production. The second aim will determine the mechanistic role of PI3K/Akt signaling pathway in regulat- ing the mitochondrial import of SOD2 and the functional relevance of this mechanism to postnatal adaptation. The successful completion of the proposed studies and training program will lead to future studies investigating preventable and treatment strategies to improve outcomes in PPHN patients, with the goal of reducing the economic and health burden due to PPHN.