Lipotoxic hepatocyte-derived <i>UBQLN1</i> -enriched small extracellular vesicles activate hepatic stellate cells to promote hepatic fibrosis

Metabolic dysfunction-associated steatohepatitis (MASH) serves as a primary contributor to liver fibrosis, cirrhosis, and hepatocellular carcinoma, yet specific diagnostic markers and therapeutic targets remain unavailable. This study elucidates the molecular mechanism by which UBQLN1 (ubiquilin 1) promotes MASH-related liver fibrosis by regulating small extracellular vesicles (sEVs) secretion and the functionality of the lysosome-mitochondria axis, as well as its clinical significance. Analysis of a multicenter cohort (<i>n</i> = 150) demonstrated significantly elevated UBQLN1 levels in both serum and serum-derived sEV from MASH patients, exhibiting diagnostic accuracies of 0.89 and 0.95, respectively. Furthermore, increased UBQLN1 was observed in mouse models of MASH, hiPSCs-derived human liver organoids, and oleic acid and palmitic acid injured hepatocytes (lipotoxic hepatocytes). Mechanistically, lipotoxic stress induces O-GlcNAcylation at the T277 site of UBQLN1 <i>via</i> OGT (O-GlcNAc transferase), which competitively inhibits its phosphorylation, consequently reducing ubiquitin-mediated degradation. Hepatocyte UBQLN1 facilitates the secretion of sEVs by regulating LAMP1-mediated fusion of multivesicular bodies (MVBs) with lysosomes. Subsequently, sEVs containing UBQLN1 regulate the activation of hepatic stellate cells by degrading the V-ATPase subunit ATP6V1B2 through E54D-dependent ubiquitin ligase activity, thereby inhibiting lysosomal acidification and mitophagy. Moreover, hepatic-specific knockdown of <i>Ubqln1</i> or hepatocyte-specific knockdown of <i>Ogt</i> significantly alleviates fibrosis and metabolic disorders in MASH mice. This study elucidates the critical role of the post-translational modification regulatory network of UBQLN1 in the progression of MASH and proposes its translational potential as an integrated therapeutic target, providing a theoretical basis for the development of sEV-based intervention strategies.<b>Abbreviations:</b> ATP6V1B2 ATPase H+ transporting V1 subunit B2; Co-IP: co-immunoprecipitation; CCL4: carbon tetrachloride; ELISA: enzyme linked immunosorbent assay; GOT1/AST: glutamic-oxaloacetic transaminase; GPT/ALT: glutamic-pyruvic transaminase; hiPSCs: human induced pluripotent stem cells; HFD: high-fat diet; HFHC: high-fat and high-cholesterol diet; HSCs: hepatic stellate cells; LAMP1: lysosomal associated membrane protein 1; LTH-sEV: lipotoxic hepatocyte-derived small extracellular vesicles; LSECs: liver sinusoidal endothelial cells; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MVBs: multivesicular bodies; MASH: metabolic dysfunction-associated steatohepatitis; N-sEV: normal hepatocyte-derived sEV; OGT: O-linked N-acetylglucosamine (GlcNAc) transferase; O-GlcNAc: O-linked-β-D-N-acetylglucosamine; PAOA: oleic acid and palmitic acid; sEV: small extracellular vesicle; UBQLN1: ubiquilin 1.