Molecular Mechanism Underlying Hypoxic Preconditioning-Promoted Mitochondrial Translocation of DJ-1 in Hypoxia/Reoxygenation H9c2 Cells
Molecular Mechanism Underlying Hypoxic Preconditioning-Promoted Mitochondrial Translocation of DJ-1 in Hypoxia/Reoxygenation H9c2 Cells
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DOI：
10.3390/molecules25010071
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发表时间：
2020-01-01
期刊：
影响因子：
4.6
通讯作者：
Chen, He-Ping
中科院分区：
文献类型：
--
作者：
Deng, Yi-Zhang;Xiao, Lin;Chen, He-Ping
DJ-1通过保护线粒体复合物I的活性，抑制线粒体活性氧（ROS）的产生，参与缺氧预处理（HPC）对缺氧/复氧（H/R）诱导的氧化应激损伤的心肌保护作用。然而，HPC使DJ-1的线粒体易位，其中没有靶向序列，以保护线粒体复合物I的分子机制，在很大程度上是未知的。在这项研究中，免疫共沉淀数据显示DJ-1与葡萄糖调节蛋白75（Grp 75）相关，并且这种关联在HPC后显著增强。免疫荧光成像和Western blot分析表明，HPC显著增强了DJ-1从H/R的H9 c2细胞的胞浆到线粒体的移位，这是由pFlag-DJ-1转染诱导的DJ-1过表达模拟的。更重要的是，Grp 75的敲低显著降低了HPC和pFlag-DJ-1转染诱导的DJ-1的线粒体易位。HPC可促进DJ-1与线粒体复合物I亚基ND 1和NDUFA 4的结合，提高复合物I活性，抑制缺氧/复氧后线粒体ROS的产生和随后的氧化应激损伤，pFlag-DJ-1转染也可模拟HPC的作用。有趣的是，HPC和pFlag-DJ-1转染的这些作用也被Grp 75敲低所阻止。综上所述，这些结果表明，HPC促进DJ-1从细胞质中的线粒体的转运Grp 75依赖的方式和Grp 75是必需的DJ-1介导的HPC对H/R诱导的线粒体复合物I缺陷和随后的氧化应激损伤的保护。
DJ-1 was recently reported to be involved in the cardioprotection of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R)-induced oxidative stress damage, by preserving mitochondrial complex I activity and, subsequently, inhibiting mitochondrial reactive oxygen species (ROS) generation. However, the molecular mechanism by which HPC enables mitochondrial translocation of DJ-1, which has no mitochondria-targeting sequence, to preserve mitochondrial complex I, is largely unknown. In this study, co-immunoprecipitation data showed that DJ-1 was associated with glucose-regulated protein 75 (Grp75), and this association was significantly enhanced after HPC. Immunofluorescence imaging and Western blot analysis showed that HPC substantially enhanced the translocation of DJ-1 from cytosol to mitochondria in H9c2 cells subjected to H/R, which was mimicked by DJ-1 overexpression induced by pFlag-DJ-1 transfection. Importantly, knockdown of Grp75 markedly reduced the mitochondrial translocation of DJ-1 induced by HPC and pFlag-DJ-1 transfection. Moreover, HPC promoted the association of DJ-1 with mitochondrial complex I subunits ND1 and NDUFA4, improved complex I activity, and inhibited mitochondria-derived ROS production and subsequent oxidative stress damage after H/R, which was also mimicked by pFlag-DJ-1 transfection. Intriguingly, these effects of HPC and pFlag-DJ-1 transfection were also prevented by Grp75 knockdown. In conclusion, these results indicated that HPC promotes the translocation of DJ-1 from cytosol to mitochondria in a Grp75-dependent manner and Grp75 is required for DJ-1-mediated protection of HPC on H/R-induced mitochondrial complex I defect and subsequent oxidative stress damage.