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

Akt 介导的 hsp70 磷酸化调节出生后过渡期间肺动脉内皮细胞中 SOD2 的线粒体定位和氧化应激

• 批准号: 9981796
• 负责人: Adeleye J afolayan
• 金额: $ 15.62万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2022-01-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9981796
• 关键词: AKT Signaling Pathway; ATP phosphohydrolase; Advisory Committees; Affect; Anions; Antioxidants; Binding; Biochemistry; Biological Assay; Biological Availability; Biology; Birth; Blood Vessels; Cardiopulmonary; Cell Culture Techniques; Cells; Clinical; Cytosol; Data; Development; Development Plans; Disease; Economics; Endothelium; Environment; Enzymes; Exposure to; Failure; Family; Fetal Lung; Free Radicals; Future; Genetic; Goals; Growth; Health; Heat-Shock Response; In Vitro; Infant; Infant Health; Kinetics; Knock-out; Knowledge; Learning Skill; Live Birth; Lung; Measures; Mediating; Medical; Mentors; Mentorship; Mitochondria; Modeling; Molecular; Molecular Chaperones; Mus; NADPH Oxidase; NOS3 gene; Neonatal; Neonatal Intensive Care Units; Neonatology; Neurodevelopmental Impairment; Newborn Infant; Nitric Oxide; Outcome; Oxidation-Reduction; Oxidative Phosphorylation; Oxidative Stress; Oxygen; Oxygen Consumption; Pathogenesis; Pathway interactions; Patients; Perinatal; Persistent Fetal Circulation Syndrome; Phosphorylation; Phosphorylation Site; Play; Positioning Attribute; Post-Translational Protein Processing; Premature Infant; Production; Protein Analysis; Protein Dephosphorylation; Proteins; Proto-Oncogene Proteins c-akt; Public Health; Pulmonary Vascular Resistance; Pulmonary artery structure; Reactive Oxygen Species; Regulation; Research; Research Personnel; Respiration; Respiratory Failure; Role; SOD2 gene; Signal Pathway; Signal Transduction; Source; Superoxide Dismutase; Superoxides; Testing; Training; Training Programs; Transcriptional Regulation; Transgenic Mice; United States; Vasodilation; Wisconsin; base; career development; clinical care; experience; fetal; fetus hypoxia; improved; improved outcome; in utero; in vivo; inhibitor/antagonist; medical schools; member; mortality risk; new therapeutic target; novel; novel therapeutics; postnatal; prenatal exposure; protein protein interaction; pulmonary artery endothelial cell; response; skills; therapeutic target; treatment strategy; ubiquitin ligase

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.

摘要 我的目标是成为一名独立的研究人员，在新生儿疾病为导向的研究与特定的 关注新生儿持续性肺动脉高压中线粒体氧化应激的调节 （PPHN）。PPHN影响2-6/1000活产，是新生儿心肺衰竭的常见原因。 在这些婴儿中，>30%的婴儿无法接受药物治疗，需要有创支持措施（ECMO）。氧化应激 与PPHN的发病机制密切相关。NADPH氧化酶被认为是 肺内皮中的超氧化物（O2 <$）。线粒体呼吸过程中的耗氧量 作为氧化磷酸化的副产品，线粒体中的O2 ′流入。最近的证据表明 线粒体产生的活性氧（ROS）诱导NADPH氧化酶的活化，导致- ROS诱导ROS形成。然而，线粒体O2形成的调节仍然未知。 在暴露于氧-环境中最大限度地减少线粒体O2形成的适应机制的鉴定 出生时的基因可能会确定PPHN的其他治疗靶点。我将开始通过参与以下活动来实现这一目标： 一个职业发展计划，逻辑上允许我扩展我以前的技能，并建立新的技能在鼠标ge- 蛋白质-蛋白质相互作用的分析以及与内源性 上皮生物学该计划将遗传学、生物化学和自由基生物学方面的教学培训与 从我的导师和科学咨询委员会在威斯康星州医学院的技能学习， 结合了内皮生物学、发育血管生物学、自由基和线粒体方面的专业知识 生物学导师和职业发展计划与拟议的研究目标相结合， 验证Akt诱导hsp 70翻译后修饰并调节相互作用的假设 hsp 70与两种最近鉴定的蛋白质，即：一种Obg样ATP酶-1（OLA 1），其促进SOD 2 im-1， port和CHIP，CHIP是一种分子伴侣相关的泛素连接酶，靶向hsp 70进行降解。磷- Akt对hsp 70的磷酸化促进了hsp 70与OLA 1的相互作用，并促进了线粒体免疫。 在产后过渡期间，SOD 2端口减少游离O2。第一个目标是确定捐款额 OLA 1和CHIP对PAECs线粒体氧化还原信号的调节作用。我们将使用转基因小鼠， 体外动力学测定和细胞培养，以确定OLA 1和CHIP对线粒体氧化还原的贡献 PAEC中的信号传导，并确定OLA 1和CHIP如何机制地调节线粒体SOD 2输入， ROS产生。第二个目的是确定PI 3 K/Akt信号通路在调节细胞凋亡中的作用机制。 研究SOD 2的线粒体输入以及这种机制与出生后适应的功能相关性。 成功完成拟议的研究和培训计划将导致未来的研究调查 预防和治疗策略，以改善PPHN患者的结局，目的是减少 PPHN造成的经济和健康负担。