Div. of Neonatology, Dept. of Pediatrics, Suite C460, Children's Corporate Ctr., 999 N. 92nd St., Wauwatosa, WI.
Am J Physiol Lung Cell Mol Physiol. 2014 Feb 15;306(4):L351-60. doi: 10.1152/ajplung.00264.2013. Epub 2013 Dec 27.
Superoxide dismutase 2 (SOD-2) is synthesized in the cytosol and imported into the mitochondrial matrix, where it is activated and functions as the primary antioxidant for cellular respiration. The specific mechanisms that target SOD-2 to the mitochondria remain unclear. We hypothesize that inducible heat shock protein 70 (iHSP70) targets SOD-2 to the mitochondria via a mechanism facilitated by ATP, and this process is impaired in persistent pulmonary hypertension of the newborn (PPHN). We observed that iHSP70 interacts with SOD-2 and targets SOD-2 to the mitochondria. Interruption of iHSP70-SOD-2 interaction with 2-phenylethylenesulfonamide-μ (PFT-μ, a specific inhibitor of substrate binding to iHSP70 COOH terminus) and siRNA-mediated knockdown of iHSP70 expression disrupted SOD-2 transport to mitochondria. Increasing intracellular ATP levels by stimulation of respiration with CaCl2 facilitated the mitochondrial import of SOD-2, increased SOD-2 activity, and decreased the mitochondrial superoxide (O2(·-)) levels in PPHN pulmonary artery endothelial cells (PAEC) by promoting iHSP70-SOD-2 dissociation at the outer mitochondrial membrane. In contrast, oligomycin, an inhibitor of mitochondrial ATPase, decreased SOD-2 expression and activity and increased O2(·-) levels in the mitochondria of control PAEC. The basal ATP levels and degree of iHSP70-SOD-2 dissociation were lower in PPHN PAEC and lead to increased SOD-2 degradation in cytosol. In normal pulmonary arteries (PA), PFT-μ impaired the relaxation response of PA rings in response to nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine. Pretreatment with Mito-Q, a mitochondrial targeted O2(·-) scavenger, restored the relaxation response in PA rings pretreated with PFT-μ. Our observations suggest that iHSP70 chaperones SOD-2 to the mitochondria. Impaired SOD-2-iHSP70 dissociation decreases SOD-2 import and contributes to mitochondrial oxidative stress in PPHN.
超氧化物歧化酶 2（SOD-2）在细胞质中合成，并被导入线粒体基质，在那里它被激活并作为细胞呼吸的主要抗氧化剂发挥作用。将 SOD-2 靶向线粒体的具体机制尚不清楚。我们假设诱导型热休克蛋白 70（iHSP70）通过一种由 ATP 介导的机制将 SOD-2 靶向线粒体，而这一过程在新生儿持续性肺动脉高压（PPHN）中受到损害。我们观察到 iHSP70 与 SOD-2 相互作用，并将 SOD-2 靶向线粒体。用 2-苯乙磺酰胺-μ（PFT-μ，一种特异性抑制 iHSP70 COOH 末端底物结合的抑制剂）中断 iHSP70-SOD-2 相互作用和 siRNA 介导的 iHSP70 表达敲低破坏了 SOD-2 向线粒体的转运。通过用 CaCl2 刺激呼吸增加细胞内 ATP 水平，促进 SOD-2 向线粒体的输入，增加 SOD-2 活性，并减少 PPHN 肺动脉内皮细胞（PAEC）中线粒体中超氧自由基（O2（·-））的水平，通过促进 iHSP70-SOD-2 在 外 线 粒 体 膜 上 的 解 离 。 相 反 ， 抑 制 线 粒 体 ATP 酶 的 寡 霉 素 降 低 了 控 制 组 PAEC 中 SOD-2 的表达和活性，并增加了线粒体中的 O2（·-）水平。PPHN PAEC 中的基础 ATP 水平和 iHSP70-SOD-2 解离程度较低，导致细胞质中 SOD-2 降解增加。在正常肺动脉（PA）中，PFT-μ 损害了对一氧化氮（NO）供体 S-亚硝基-N-乙酰青霉胺反应的 PA 环的松弛反应。用线粒体靶向 O2（·-）清除剂 Mito-Q 预处理可恢复用 PFT-μ 预处理的 PA 环的松弛反应。我们的观察表明，iHSP70 将 SOD-2 伴侣到线粒体。SOD-2-iHSP70 解离受损会降低 SOD-2 的导入，并导致 PPHN 中线粒体氧化应激。
