Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, Republic of Korea.
KoBio Labs, Seongnam, 13488, Republic of Korea.
Exp Mol Med. 2023 Jun;55(6):1218-1231. doi: 10.1038/s12276-023-01009-w. Epub 2023 Jun 1.
The signaling pathways governing acetaminophen (APAP)-induced liver injury have been extensively studied. However, little is known about the ubiquitin-modifying enzymes needed for the regulation of APAP-induced liver injury. Here, we examined whether the Pellino3 protein, which has E3 ligase activity, is needed for APAP-induced liver injury and subsequently explored its molecular mechanism. Whole-body Peli3 knockout (KO) and adenovirus-mediated Peli3 knockdown (KD) mice showed reduced levels of centrilobular cell death, infiltration of immune cells, and biomarkers of liver injury, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), upon APAP treatment compared to wild-type (WT) mice. Peli3 deficiency in primary hepatocytes decreased mitochondrial and lysosomal damage and reduced the mitochondrial reactive oxygen species (ROS) levels. In addition, the levels of phosphorylation at serine 9 in the cytoplasm and mitochondrial translocation of GSK3β were decreased in primary hepatocytes obtained from Peli3 KO mice, and these reductions were accompanied by decreases in JNK phosphorylation and mitochondrial translocation. Pellino3 bound more strongly to GSK3β compared with JNK1 and JNK2 and induced the lysine 63 (K63)-mediated polyubiquitination of GSK3β. In rescue experiments, the ectopic expression of wild-type Pellino3 in Peli3 KO hepatocytes restored the mitochondrial translocation of GSK3β, but this restoration was not obtained with expression of a catalytically inactive mutant of Pellino3. These findings are the first to suggest a mechanistic link between Pellino3 and APAP-induced liver injury through the modulation of GSK3β polyubiquitination.
调控对乙酰氨基酚（APAP）诱导肝损伤的信号通路已得到广泛研究。然而，对于调控 APAP 诱导肝损伤所需的泛素修饰酶知之甚少。在这里，我们研究了具有 E3 连接酶活性的 Pellino3 蛋白是否需要参与 APAP 诱导的肝损伤，并随后探讨了其分子机制。与野生型（WT）小鼠相比，全身 Peli3 敲除（KO）和腺病毒介导的 Peli3 敲低（KD）小鼠在 APAP 处理后，中央小叶细胞死亡、免疫细胞浸润以及丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）等肝损伤生物标志物的水平降低。原代肝细胞中 Peli3 的缺失减少了线粒体和溶酶体损伤，并降低了线粒体活性氧（ROS）水平。此外，从 Peli3 KO 小鼠获得的原代肝细胞中细胞质中丝氨酸 9 的磷酸化和 GSK3β 的线粒体易位减少，并且这些减少伴随着 JNK 磷酸化和线粒体易位的减少。与 JNK1 和 JNK2 相比，Pellino3 与 GSK3β 结合更强，并诱导 GSK3β 的赖氨酸 63（K63）介导的多泛素化。在挽救实验中，WT Pellino3 在 Peli3 KO 肝细胞中的异位表达恢复了 GSK3β 的线粒体易位，但在用催化失活突变体 Pellino3 的表达进行挽救时，未获得这种恢复。这些发现首次表明，通过调节 GSK3β 的多泛素化，Pellino3 与 APAP 诱导的肝损伤之间存在机制联系。
