TRIB3-TRIM8 complex drives NAFLD progression by regulating HNF4 a stability

Background & Aims: Endoplasmic reticulum (ER) stress of hepatocytes plays a causative role in non-alcoholic fatty liver disease (NAFLD). Reduced expression of hepatic nuclear factor 4a (HNF4a) is a critical event in the pathogenesis of NAFLD and other liver diseases. Whether ER stress regulates HNF4a expression remains unknown. The aim of this study was to delineate the machinery of HNF4a protein degradation and explore a therapeutic strategy based on protecting HNF4a stability during NAFLD progression. Methods: Correlation of HNF4a and tribbles homologue 3 (TRIB3), an ER stress sensor, was evaluated in human and mouse NAFLD tissues. RNA-sequencing, mass spectrometry analysis, co-immunoprecipitation, in vivo and in vitro ubiquitination assays were used to elucidate the mechanisms of TRIB3-mediated HNF4a degradation. Molecular docking and co-immunoprecipitation analyses were performed to identify a cell -penetrating peptide that ablates the TRIB3-HNF4a interaction. Results: TRIB3 directly interacts with HNF4a and mediates ER stress -induced HNF4a degradation. TRIB3 recruits tripartite motif containing 8 (TRIM8) to form an E3 ligase complex that catalyzes K48 -linked polyubiquitination of HNF4a on lysine 470. Abrogating the degradation of HNF4a attenuated the effect of TRIB3 on a diet -induced NAFLD model. Moreover, the TRIB3 gain -offunction variant p.Q84R is associated with NAFLD progression in patients, and induces lower HNF4a levels and more severe hepatic steatosis in mice. Importantly, disrupting the TRIB3-HNF4a interaction using a cell -penetrating peptide restores HNF4a levels and ameliorates NAFLD progression in mice. Conclusions: Our findings unravel the machinery of HNF4a protein degradation and indicate that targeting TRIB3-TRIM8 E3 complex -mediated HNF4a polyubiquitination may be an ideal strategy for NAFLD therapy.