KCMF1 promotes MASLD progression via K48-linked ubiquitination and degradation of AMPKα

Background/aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) lacks effective pharmacotherapy. Although AMP-activated protein kinase (AMPK) is a central metabolic regulator with hepatoprotective effects, the upstream mechanisms governing its degradation in MASLD remain poorly defined. Here, we identify Potassium Channel modulatory factor 1 (KCMF1) as a previously unrecognized E3 ubiquitin Ligase targeting AMPKα and investigate its role in MASLD pathogenesis.

Methods: Protein interactions were characterized by co-immunoprecipitation and GST pull-down assays. Ubiquitin-linkage specificity and AMPKα stability were assessed biochemically. Hepatocyte-targeted gain-of-function and hepatocyte-specific KCMF1 ablation were evaluated across multiple mouse MASLD models (HFD, GAN, CDAHFD, and ob/ob), with AMPK dependency assessed by pharmacological modulation. An AI-guided virtual screen with biophysical validation was conducted to identify candidate KCMF1 inhibitors.

Results: KCMF1 expression was markedly upregulated in hepatocytes from murine and human MASLD livers. KCMF1 directly interacted with AMPKα and catalyzed its K48-linked polyubiquitination, promoting its degradation and suppressing hepatic AMPK signaling. Functionally, hepatic KCMF1 overexpression exacerbated steatosis, inflammation, and fibrosis, whereas its knockdown or hepatocyte-specific deletion conferred protection across MASLD models. Pharmacological modulation of AMPK in vivo and in primary hepatocytes demonstrated pathway dependency, as AMPK activation attenuated KCMF1-driven pathology. Furthermore, the flavonoid Rhoifolin was identified as a direct KCMF1 binder that stabilized AMPKα and ameliorated MASLD in mice.

Conclusions: KCMF1 drives MASLD pathogenesis by promoting K48-linked ubiquitination and degradation of AMPKα. Targeting the KCMF1-AMPK axis restores hepatic metabolic homeostasis and represents a potential therapeutic strategy for MASLD.