College of Life Sciences, Lamprey Research Center, Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Liaoning Normal University, Dalian, 116081, China.
College of Medicine and Biological Information Engineering, Northeastern University, Shenyang, 110169, China.
Fish Shellfish Immunol. 2020 Sep;104:613-621. doi: 10.1016/j.fsi.2020.06.037. Epub 2020 Jun 24.
Before we have reported lamprey PHB2 could enhance the cellular oxidative-stressed tolerance, here the aim was to explore its mechanisms. We used flow cytometry analysis to identify a Lampetra morii homologue of PHB2 (Lm-PHB2) that could significantly decrease the levels of ROS generation in HEK293T cells. According to confocal microscopy observations, Lm-PHB2 contributed to maintain the mitochondrial morphology of HEK293T cells, and then both cellular nuclear location and translocation from the nucleus to mitochondria of Lm-PHB2 were also examined in HEK293T cells under oxidative stress. We also examined the expressions and locations of various Lm-PHB2 deletion mutants and the amino acid mutant by confocal microscopy and the results showed that the translocation of Lm-PHB2 into mitochondria was dependent on the Lm-PHB2 region and the 17th, 48th and 57th three arginines (R) of N-terminal were very critical. In addition, the analyses of QRT-PCR and Western blot demonstrated that Lm-PHB2 increased the expression levels of OPA1 and HAX1 in HEK293T cells treated with HO. The analyses of immunofluorescence and immunoprecipitation showed that Lm-PHB2 could interact with OPA1 and HAX1, respectively. The above mentioned results indicate that Lm-PHB2 could assist OPA1 and HAX1 to maintain mitochondrial morphology and decrease ROS levels by the translocation from the nucleus to mitochondria under oxidative stress.
在我们报道七鳃鳗 PHB2 可以增强细胞氧化应激耐受性之前，本研究旨在探索其机制。我们使用流式细胞术分析鉴定了七鳃鳗 PHB2 的同源物（Lm-PHB2），它可以显著降低 HEK293T 细胞中 ROS 生成的水平。根据共聚焦显微镜观察，Lm-PHB2 有助于维持 HEK293T 细胞的线粒体形态，然后还在氧化应激下检查了 Lm-PHB2 在 HEK293T 细胞中的核定位和从核到线粒体的易位。我们还通过共聚焦显微镜检查了各种 Lm-PHB2 缺失突变体和氨基酸突变体的表达和定位，结果表明 Lm-PHB2 向线粒体的易位依赖于 Lm-PHB2 区域以及 N 端的第 17、48 和 57 位三个精氨酸（R）。此外，QRT-PCR 和 Western blot 分析表明，Lm-PHB2 增加了 HO 处理的 HEK293T 细胞中 OPA1 和 HAX1 的表达水平。免疫荧光和免疫沉淀分析表明，Lm-PHB2 可以分别与 OPA1 和 HAX1 相互作用。上述结果表明，Lm-PHB2 可以通过核到线粒体的易位，在氧化应激下协助 OPA1 和 HAX1 维持线粒体形态并降低 ROS 水平。
