Ubiquitination of Rhomboid 5 Homolog 2 by Constitutive Photomorphogenic 1 Alleviates Hepatic Ischemia-reperfusion Injury by Regulating the Transforming Growth Factor-β Activating Kinase 1-C-Jun N-terminal Kinase/p38 Signaling Pathway.

BACKGROUND & AIMS: Hepatic ischemia-reperfusion injury (HIRI) is one of the common complications of liver transplantation. Rhomboid 5 homolog 2 (Rhbdf2) plays a crucial role in apoptosis, inflammation, and liver injury, but its role and regulatory mechanism in HIRI remain unclear. The aim of this study was to investigate the role of Rhbdf2 in HIRI and elucidate its molecular mechanism. METHODS: Rhbdf2 expression levels were detected in pre-ischemia-reperfusion (Pre) and post-ischemia-reperfusion (Post) livers. Western blot analysis, flow cytometry, quantitative real-time polymerase chain reaction, and immunofluorescence staining were used to investigate the effects of Rhbdf2 on hepatic ischemia-reperfusion (HI/R). The potential molecular mechanisms of the effects of Rhbdf2 on HI/R were investigated by combining RNA sequencing and mass spectrometry analysis, as well as co-immunoprecipitation and in vitro ubiquitination assays. RESULTS: The level of Rhbdf2 protein was significantly increased in HI/R. Overexpression of Rhbdf2 in mice exacerbated HI/R-induced liver injury, apoptosis, and the inflammatory response, whereas knockdown of Rhbdf2 produced the opposite results. Mechanistically, overexpression of Rhbdf2 promoted the phosphorylation of mitogen-activated protein kinase kinase kinase 7 (MAP3K7, also known as TAK1), thereby activating the JNK/p38 signaling pathway and ultimately exacerbating HIRI. Mass spectrometry analysis, co-immunoprecipitation, and in vitro ubiquitination assays revealed that the E3 ubiquitin ligase constitutive photomorphogenic 1 (Cop1) interacts with Rhbdf2 and mediates its degradation through K48-linked ubiquitination, thereby inhibiting the TAK1- JNK/p38 axis and reducing HIRI. CONCLUSIONS: This study revealed that Rhbdf2 exacerbates HIRI by activating the TAK1- JNK/p38 axis, whereas Cop1-mediated Rhbdf2 ubiquitination and degradation can significantly inhibit this process. These findings provide potential therapeutic targets and insights for the clinical treatment of HIRI.