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: 10402122
• 负责人: Adeleye J afolayan
• 金额: $ 4.13万
• 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/10402122
• 关键词: AKT1 gene; ATP phosphohydrolase; Binding; Birth; Blood Pressure; Blood Vessels; Blood flow; Cardiopulmonary; Cells; Complex; Disease; Environment; Equilibrium; Event; Failure; Fetal Lung; Heat-Shock Proteins 70; High Pressure Liquid Chromatography; Impairment; In Vitro; Infant Health; Interruption; K-Series Research Career Programs; Lung; Measurement; Mediating; Mentors; Mitochondria; Molecular; Molecular Chaperones; Muscle; Neonatal Intensive Care Units; Newborn Infant; Oxidation-Reduction; Oxidative Stress; Oxygen; Peptides; Permeability; Persistent Fetal Circulation Syndrome; Phosphorylation; Physiological; Play; Premature Infant; Preparation; Process; Proto-Oncogene Proteins c-akt; Public Health; Pulmonary Circulation; Pulmonary artery structure; Rattus; SOD2 gene; Series; Serine; Sprague-Dawley Rats; Stains; Stimulus; Stress; Superoxide Dismutase; Testing; Therapeutic; Ubiquitination; United States; Vascular remodeling; Vasodilation; heart function; improved; in vivo; in vivo Model; inhibitor/antagonist; novel; postnatal; prenatal exposure; protein activation; pulmonary artery endothelial cell; recruit; response; therapeutic target

Project Summary Pulmonary circulation undergoes adaptive processes critical for survival of the newborn at birth. For a successful postnatal transition, several events come to play, including increased pulmonary blood flow and remodeling of muscularized small-medium size pulmonary arteries (PAs). One important mechanism regulating these processes is the activation of adaptive processes that regulate mitochondrial oxidative stress. However, the mechanism is unknown. For the past five years, through the mentored career development award, we have been exploring the molecular processes that maintain the activity of mitochondrial-localized superoxide dismutase (SOD2) appropriate for ROS levels in mitochondria during postnatal transition. Our findings demonstrate that phosphorylation of heat shock protein-70 (hsp70), a major cytosolic molecular chaperone is a critical mechanism regulating mitochondrial oxidative stress during exposure of the fetal lungs to high oxygen environment. We found that elevated ROS induces the activation of protein kinase-B (AKT1), which in turn phosphorylates Hsp70 on Serine-(S631) to promote the import of SOD2 into the mitochondria in response to stress. We also found that when phosphorylated on S631, Hsp70 recruits Obg-like ATPase-1 (OLA1) to Hsp70-SOD2 complexes and the binding of OLA1 drives the mitochondrial SOD2 import by antagonizing CHIP-mediated ubiquitination and proteasomal degradation of Hsp70 and its downstream target, SOD2. Disruption of AKT-mediated phosphorylation of Hsp70S631 increases mitochondrial ROS levels. We also observed that AKT activity is significantly decreased in persistent pulmonary hypertension of the newborn (PPHN) and contributes to impaired vasorelaxation in the disease. However, all these studies were done in vitro, therefore it is important to verify this critical mechanism in vivo. Our working hypothesis is that dynamic phosphorylation of Hsp70S631 by AKT is a novel mechanism promoting postnatal adaptation of pulmonary circulation at birth through mechanisms regulating mitochondrial import of SOD2 and redox balance. To test this hypothesis in an in vivo model, we will test the effect of disrupting OLA1 binding to hsp70 or AKT-mediated phosphorylation of Hsp70 in vivo using Sprague Dawley (SD) rats. We have generated a series of cell-permeable decoy peptide inhibitors of AKT and OLA1. For this, 4 weeks old rats will be treated with GSG (Akt inhibitor), or GLGIV (OLA1 inhibitor) peptide for 4 weeks in normoxic conditions. Echocardiographic measurements of tricuspid regurgitant jets (TR jets) and cardiac function will be performed. We will also quantify mitochondrial ROS levels using mitoSOX staining and HPLC. Vasorelaxation responses of PA in the treated and untreated groups to physiological stimuli in an ex-vivo preparations will be determined. It is anticipated that interruptions of hsp70 interactions with Akt or OLA1 will induce vascular remodeling and PPHN. While CHIP inhibition would increase vasorelaxation response of PAs to stimuli. Thus, increasing AKT activity may be a potential therapeutic option in the treatment of PPHN.

项目摘要 肺循环经历对新生儿出生时的存活至关重要的适应性过程。对于一个成功 出生后过渡期，几个事件开始发挥作用，包括增加肺血流量和重建 肌化的中小型肺动脉（PA）。调节这些的一个重要机制是 过程是调节线粒体氧化应激的适应性过程的激活。但 机制不明。在过去的五年里，通过指导职业发展奖，我们一直在 探索维持脑组织局部超氧化物歧化酶活性的分子过程 （SOD 2）适合于出生后过渡期间线粒体中的ROS水平。我们的研究结果表明， 热休克蛋白70（HSP 70）是一种主要的胞质分子伴侣，其磷酸化是一种关键机制 在胎儿肺暴露于高氧环境期间调节线粒体氧化应激。我们 发现升高的ROS诱导蛋白激酶B（AKT 1）的活化，进而磷酸化Hsp 70 丝氨酸-（S631）促进SOD 2进入线粒体以响应应激。我们还发现 当在S631上磷酸化时，Hsp 70募集Obg样ATP酶-1（OLA 1）至Hsp 70-SOD 2复合物， OLA 1的结合通过拮抗CHIP介导的泛素化驱动线粒体SOD 2输入， 蛋白酶体降解Hsp 70及其下游靶标SOD 2。破坏AKT介导的 Hsp 70 S631的磷酸化增加线粒体ROS水平。我们还观察到AKT活性是 显著降低新生儿持续性肺动脉高压（PPHN），并有助于受损 血管舒张的影响然而，所有这些研究都是在体外进行的，因此验证这一点很重要 关键机制。我们的工作假设是AKT对Hsp 70 S631的动态磷酸化是一个过程。 通过机制促进出生时肺循环的出生后适应的新机制 调节线粒体SOD 2输入和氧化还原平衡。为了在体内模型中检验这一假设，我们将 使用以下方法在体内测试破坏OLA 1与Hsp 70的结合或AKT介导的Hsp 70磷酸化的作用 Sprague道利（SD）大鼠。我们已经产生了一系列细胞可渗透的AKT诱饵肽抑制剂， OLA1.为此，将用GSG（Akt抑制剂）或GLGIV（OLA 1抑制剂）肽处理4周龄大鼠4 在正常含氧量的条件下。超声心动图测量三尖瓣反流束（TR射流）， 将进行心脏功能检查。我们还将使用mitoSOX染色定量线粒体ROS水平， 高效液相色谱法处理组和未处理组中PA对离体生理刺激的血管舒张反应 准备工作将确定。可以预期，中断hsp 70与Akt或OLA 1的相互作用将使hsp 70的活性降低。 诱导血管重塑和PPHN。而CHIP抑制则可增强PA的血管舒张反应 刺激。因此，增加AKT活性可能是治疗PPHN的潜在治疗选择。

项目成果

Redox Signaling and Persistent Pulmonary Hypertension of the Newborn.

• DOI: 10.1007/978-3-319-63245-2_16
• 发表时间: 2017
• 期刊: Advances in experimental medicine and biology
• 作者: Megha Sharma;Adeleye J Afolayan

OLA1 Phosphorylation Governs the Mitochondrial Bioenergetic Function of Pulmonary Vascular Cells.

OLA1 磷酸化控制肺血管细胞的线粒体生物能功能。

• DOI: 10.1165/rcmb.2022-0186oc
• 发表时间: 2023
• 期刊: American journal of respiratory cell and molecular biology
• 影响因子: 6.4
• 作者: Sidlowski,Paul;Czerwinski,Amanda;Liu,Yong;Liu,Pengyuan;Teng,Ru-Jeng;Kumar,Suresh;Wells,Clive;PritchardJr,Kirkwood;Konduri,GirijaG;Afolayan,AdeleyeJ
• 通讯作者: Afolayan,AdeleyeJ