The Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China.
Autophagy. 2012 Jul 1;8(7):1071-84. doi: 10.4161/auto.20250. Epub 2012 May 11.
Mitoribosome in mammalian cells is responsible for synthesis of 13 mtDNA-encoded proteins, which are integral parts of four mitochondrial respiratory chain complexes (I, III, IV and V). ERAL1 is a nuclear-encoded GTPase important for the formation of the 28S small mitoribosomal subunit. Here, we demonstrate that knockdown of ERAL1 by RNA interference inhibits mitochondrial protein synthesis and promotes reactive oxygen species (ROS) generation, leading to autophagic vacuolization in HeLa cells. Cells that lack ERAL1 expression showed a significant conversion of LC3-I to LC3-II and an enhanced accumulation of autophagic vacuoles carrying the LC3 marker, all of which were blocked by the autophagy inhibitor 3-MA as well as by the ROS scavenger NAC. Inhibition of mitochondrial protein synthesis either by ERAL1 siRNA or chloramphenicol (CAP), a specific inhibitor of mitoribosomes, induced autophagy in HTC-116 TP53 (+/+) cells, but not in HTC-116 TP53 (-/-) cells, indicating that tumor protein 53 (TP53) is essential for the autophagy induction. The ROS elevation resulting from mitochondrial protein synthesis inhibition induced TP53 expression at transcriptional levels by enhancing TP53 promoter activity, and increased TP53 protein stability by suppressing TP53 ubiquitination through MAPK14/p38 MAPK-mediated TP53 phosphorylation. Upregulation of TP53 and its downstream target gene DRAM1, but not CDKN1A/p21, was required for the autophagy induction in ERAL1 siRNA or CAP-treated cells. Altogether, these data indicate that autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.
哺乳动物细胞中的线粒体核糖体负责合成 13 种由 mtDNA 编码的蛋白质，这些蛋白质是线粒体呼吸链复合物（I、III、IV 和 V）的组成部分。ERAL1 是一种核编码的 GTPase，对于 28S 小线粒体核糖体亚基的形成很重要。在这里，我们通过 RNA 干扰证明 ERAL1 的敲低会抑制线粒体蛋白质合成并促进活性氧（ROS）的产生，从而导致 HeLa 细胞发生自噬空泡化。缺乏 ERAL1 表达的细胞中 LC3-I 向 LC3-II 的显著转化和携带 LC3 标记的自噬空泡的积累增强，所有这些都被自噬抑制剂 3-MA 和 ROS 清除剂 NAC 阻断。通过 ERAL1 siRNA 或氯霉素（CAP）抑制线粒体蛋白质合成（CAP 是线粒体核糖体的特异性抑制剂），诱导 HTC-116 TP53（+/+）细胞发生自噬，但在 HTC-116 TP53（-/-）细胞中没有诱导自噬，表明肿瘤蛋白 53（TP53）是自噬诱导所必需的。线粒体蛋白质合成抑制引起的 ROS 升高通过增强 TP53 启动子活性在转录水平上诱导 TP53 表达，并通过 MAPK14/p38 MAPK 介导的 TP53 磷酸化抑制 TP53 泛素化来增加 TP53 蛋白稳定性。上调 TP53 及其下游靶基因 DRAM1，但不是 CDKN1A/p21，是 ERAL1 siRNA 或 CAP 处理的细胞中自噬诱导所必需的。总之，这些数据表明，自噬是通过 ROS-TP53-DRAM1 途径诱导的，以响应线粒体蛋白质合成的抑制。
