International immunopharmacology · 2026
NEDD4L-mediated ALYREF ubiquitination degradation suppresses RUBCNL expression to impair autophagy and promote lipogenesis in alcoholic hepatic fibrosis.
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Hu Xianliang, Wu Jianhua, Huang Yuxin, Guo Qipeng, Xie Zhengyuan
Аннотация
BACKGROUND: Alcoholic hepatic fibrosis (AHF) is a progressive disease with limited therapeutic targets. This study investigated the role and mechanism of RUBCNL in AHF pathogenesis.
METHODS: The AHF mouse model was established using a chronic-plus-binge ethanol feeding protocol combined with CCl4 injection. Acetaldehyde-stimulated LX-2 cells served as the cellular model. RUBCNL, ALYREF and NEDD4L expression were detected via qRT-PCR, Western blot, and immunofluorescence. Gain- and loss-of-function studies were performed using overexpression and siRNA approaches. Mechanisms were explored via Co-IP, CHX chase, and MeRIP assays. AAV-mediated combinatorial gene knockdown/overexpression rescue experiments were performed in AHF mice to verify the linear regulatory axis in vivo.
RESULTS: RUBCNL was significantly downregulated in AHF models. Mechanistically, upregulated NEDD4L directly interacted with and promoted ubiquitination and degradation of the m5C reader ALYREF, reducing its binding to RUBCNL mRNA and leading to decreased RUBCNL mRNA stability. RUBCNL deficiency impaired autophagy (reduced ATG5-ATG12, p-ULK1/ULK1, and downregulated p-mTOR/mTOR), enhanced lipid accumulation (SREBP1c, FASN), and upregulated fibrotic markers (COL1A1, α-SMA). AAV-mediated RUBCNL overexpression in vivo restored autophagic flux, suppressed lipid synthesis and fibrosis, and improved serum ALT, AST, and TG levels. In vivo and in vitro genetic rescue assays further validated that the protective effects of NEDD4L depletion were partially reversed by ALYREF silencing, while the therapeutic benefits of ALYREF overexpression were abolished by RUBCNL knockdown.
CONCLUSION: NEDD4L-mediated ALYREF degradation downregulates RUBCNL, driving AHF progression via autophagy dysfunction and lipid dysregulation, identifying RUBCNL as a potential therapeutic target.
METHODS: The AHF mouse model was established using a chronic-plus-binge ethanol feeding protocol combined with CCl4 injection. Acetaldehyde-stimulated LX-2 cells served as the cellular model. RUBCNL, ALYREF and NEDD4L expression were detected via qRT-PCR, Western blot, and immunofluorescence. Gain- and loss-of-function studies were performed using overexpression and siRNA approaches. Mechanisms were explored via Co-IP, CHX chase, and MeRIP assays. AAV-mediated combinatorial gene knockdown/overexpression rescue experiments were performed in AHF mice to verify the linear regulatory axis in vivo.
RESULTS: RUBCNL was significantly downregulated in AHF models. Mechanistically, upregulated NEDD4L directly interacted with and promoted ubiquitination and degradation of the m5C reader ALYREF, reducing its binding to RUBCNL mRNA and leading to decreased RUBCNL mRNA stability. RUBCNL deficiency impaired autophagy (reduced ATG5-ATG12, p-ULK1/ULK1, and downregulated p-mTOR/mTOR), enhanced lipid accumulation (SREBP1c, FASN), and upregulated fibrotic markers (COL1A1, α-SMA). AAV-mediated RUBCNL overexpression in vivo restored autophagic flux, suppressed lipid synthesis and fibrosis, and improved serum ALT, AST, and TG levels. In vivo and in vitro genetic rescue assays further validated that the protective effects of NEDD4L depletion were partially reversed by ALYREF silencing, while the therapeutic benefits of ALYREF overexpression were abolished by RUBCNL knockdown.
CONCLUSION: NEDD4L-mediated ALYREF degradation downregulates RUBCNL, driving AHF progression via autophagy dysfunction and lipid dysregulation, identifying RUBCNL as a potential therapeutic target.