Ceramide(d18:1/18:1)-NDUFA6 interaction inactivates respiratory complex I to attenuate oxidative-stress-driven pathogenesis in liver ischemia/reperfusion injury
Kai Wang, … , Yiyi Li, Chuanjiang Li
Kai Wang, … , Yiyi Li, Chuanjiang Li
Published April 17, 2025
Citation Information: JCI Insight. 2025;10(10):e187083. https://doi.org/10.1172/jci.insight.187083.
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Research Article Hepatology Metabolism

Ceramide(d18:1/18:1)-NDUFA6 interaction inactivates respiratory complex I to attenuate oxidative-stress-driven pathogenesis in liver ischemia/reperfusion injury

Abstract

Oxidative stress driven by malfunctioning respiratory complex I (RC-I) is a crucial pathogenic factor in liver ischemia/reperfusion (I/R) injury. This study investigated the role of alkaline ceramidase 3 (ACER3) and its unsaturated long-chain ceramide (CER) substrates in regulating liver I/R injury through RC-I. Our findings demonstrated that I/R upregulated ACER3 and decreased unsaturated long-chain CER levels in human and mouse livers. Both global and hepatocyte-specific Acer3 ablation, as well as treatment with CER(d18:1/18:1), led to a significant increase in CER(d18:1/18:1) levels in the liver, which mitigated the I/R-induced hepatocyte damage and inflammation in mice. Mechanistically, ACER3 modulated CER(d18:1/18:1) levels in mitochondria-associated membranes and the endoplasmic reticulum (ER), thereby influencing the transport of CER(d18:1/18:1) from the ER to mitochondria. Acer3 ablation and CER(d18:1/18:1) treatment elevated CER(d18:1/18:1) in mitochondria, where CER(d18:1/18:1) bound to the RC-I subunit NDUFA6 to inactivate RC-I and reduced reactive oxygen species production in the I/R-injured mouse liver. These findings underscore the role of the CER(d18:1/18:1)-NDUFA6 interaction in suppressing RC-I–mediated oxidative-stress-driven pathogenesis in liver I/R injury.

Authors

Kai Wang, Leyi Liao, Hanbiao Liang, Pengxiang Huang, Qingping Li, Baoxiong Zhuang, Chen Xie, Xiangyue Mo, Xuesong Deng, Jieyuan Li, Yang Lei, Minghui Zeng, Cungui Mao, Ruijuan Xu, Cuiting Liu, Xianqiu Wu, Jie Zhou, Biao Wang, Yiyi Li, Chuanjiang Li

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Figure 4

CER(d18:1/18:1) interacts with RC-I subunit NDUFA6.

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CER(d18:1/18:1) interacts with RC-I subunit NDUFA6. (A) Schematic of the...
(A) Schematic of the experimental workflow for identifying CER(d18:1/18:1)-binding proteins using 3D carbene chip SPR, proteomics, and protein-lipid docking analysis with AutoDock Vina (see Supplemental Methods). (B) SPR analysis showing the binding response of CER(d18:1/18:1)-binding proteins in positive protein samples, inactive protein control, and chip control. (C) Predicted binding energy of CER(d18:1/18:1) with captured proteins, highlighting the highest binding affinity for Ndufa6. (D–F) Model of the CER(d18:1/18:1)–human NDUFA6 complex (D), interaction details (E), and hydrophobicity analysis (F). vdW, van der Waals; es, electrostatic. (G) Immunoblot analysis showing the overexpression of FLAG-tagged NDUFA6 in HepG2 cells treated with CER(d18:1/18:1). (H and I) Levels of CER(d18:1/18:1) (H) and other CER species in HepG2 cells overexpressing NDUFA6-FLAG (I). (J and K) Immunoprecipitation of FLAG-tagged NDUFA6 in HepG2 cells (J) and levels of CER(d18:1/18:1) in immunoprecipitated NDUFA6 protein from HepG2 cells (K). Images in G–K represent the results of 3 independent experiments. Data in H, I, and K are expressed as mean ± SD. Statistical significance was determined using 1-way ANOVA followed by Tukey’s test for multiple comparisons. ***P < 0.001.