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

项目摘要 肺循环经历了对新生儿出生时存活至关重要的适应性过程。为了一个成功的 出生后过渡，几个事件开始发挥作用，包括肺血流增加和 肌化的中小型肺动脉(PAS)。一个重要的机制就是调节这些 过程是调节线粒体氧化应激的适应性过程的激活。然而， 机制尚不清楚。在过去的五年里，通过导师职业发展奖，我们一直 探讨维持线粒体定位超氧化物歧化酶活性的分子过程 (Sod2)与出生后过渡期间线粒体中的ROS水平相适应。我们的发现表明 热休克蛋白-70(HSP70)是一种主要的胞质分子伴侣，它的磷酸化是一个重要的机制 高氧环境下胎肺线粒体氧化应激的调控。我们 发现ROS升高诱导蛋白激酶B(AKT1)激活，进而使Hsp70磷酸化 丝氨酸-(S631)促进SOD2进入线粒体以应对压力。我们还发现， 当S631上的磷酸化时，Hsp70招募Obg-like ATPase-1(OLA1)形成Hsp70-SOD2复合体，并 OLA1的结合通过拮抗CHIP介导的泛素化和 Hsp70及其下游靶标SOD2的蛋白酶体降解。AKT介导的干扰 Hsp70S631的磷酸化增加了线粒体的ROS水平。我们还观察到AKT活性是 新生儿持续性肺动脉高压(PPHN)显著降低，并有助于 血管松弛在疾病中的作用。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 体内的关键机制。我们的工作假设是AKT对Hsp70S631的动态磷酸化是一种 通过机制促进出生后肺循环适应的新机制 调节线粒体SOD2的输入和氧化还原平衡。为了在活体模型中检验这一假设，我们将 在体法检测OLA1对HSP70或AKT介导的HSP70磷酸化的影响 SD大鼠。我们已经产生了一系列AKT和AKT的细胞渗透性诱骗多肽抑制剂 OLA1.为此，4周龄大鼠将用GSG(Akt抑制剂)或GLGIV(OLA1抑制剂)肽治疗4周 在常氧条件下工作数周。三尖瓣返流的超声心动图测量 将进行心脏功能检查。我们还将使用mitoSOX染色和 高效液相色谱柱。治疗组和未治疗组PA对生理刺激的血管松弛反应 准备工作将会确定。预计HSP70与Akt或OLA1相互作用的中断将 诱导血管重塑和PPHN。而抑制ChIP可增强PAS的血管松弛反应 去刺激。因此，增加AKT活性可能是治疗PPHN的一种潜在的治疗选择。

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.