Inflammation-induced TRIM21 represses hepatic steatosis by promoting the ubiquitination of lipogenic regulators
Kostas C. Nikolaou, Svenja Godbersen, Muthiah Manoharan, Stefan Wieland, Markus H. Heim, Markus Stoffel
Kostas C. Nikolaou, Svenja Godbersen, Muthiah Manoharan, Stefan Wieland, Markus H. Heim, Markus Stoffel
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Research Article Hepatology

Inflammation-induced TRIM21 represses hepatic steatosis by promoting the ubiquitination of lipogenic regulators

Abstract

Nonalcoholic steatohepatitis (NASH) is a leading cause for chronic liver diseases. Current therapeutic options are limited due to an incomplete mechanistic understanding of how steatosis transitions to NASH. Here we show that the TRIM21 E3 ubiquitin ligase is induced by the synergistic actions of proinflammatory TNF-α and fatty acids in livers of humans and mice with NASH. TRIM21 ubiquitinates and degrades ChREBP, SREBP1, ACC1, and FASN, key regulators of de novo lipogenesis, and A1CF, an alternative splicing regulator of the high-activity ketohexokinase-C (KHK-C) isoform and rate-limiting enzyme of fructose metabolism. TRIM21-mediated degradation of these lipogenic activators improved steatosis and hyperglycemia as well as fructose and glucose tolerance. Our study identifies TRIM21 as a negative regulator of liver steatosis in NASH and provides mechanistic insights into an immunometabolic crosstalk that limits fatty acid synthesis and fructose metabolism during metabolic stress. Thus, enhancing this natural counteracting force of steatosis through inhibition of key lipogenic activators via TRIM21-mediated ubiquitination may provide a therapeutic opportunity to treat NASH.

Authors

Kostas C. Nikolaou, Svenja Godbersen, Muthiah Manoharan, Stefan Wieland, Markus H. Heim, Markus Stoffel

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Figure 1

TRIM21 interacts, ubiquitinates, and degrades A1CF in hepatocytes.

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TRIM21 interacts, ubiquitinates, and degrades A1CF in hepatocytes. (A) V...
(A) Volcano plot of proteomics data, depicting protein data P values versus fold change (FC). Normalized total ion current (TIC) was used as a quantitative method to plot the volcano. Proteomics data were produced from 3 liver replicates, 1 mouse/replicate. All data points above threshold (red line) are calculated with t test significance P < 0.05 and Benjamini-Hochberg multiple-test correction. All significant proteins associated with these points are listed in Supplemental Table 1. Highlighted are TRIM21 and RBM47, a known A1CF interactor. (B) Coimmunoprecipitation (Co-IP) experiments to validate the endogenous A1CF and TRIM21 interaction in mouse livers. (C and D) Ubiquitination (Ub) assay determining either K63-linked Ub-specific or total Ub of endogenous A1CF in mouse liver (C) or HepG2 cells (D); ubiquitination assay shown in D was performed in HepG2 cells. (E) Total A1CF ubiquitination levels in Hepa 1–6 cells cotransfected with A1CF and either TRIM21 WT or its ligase-dead (LD) form after MG132 (0.5 μM) treatment for 6 hours. Input levels indicate A1CF and TRIM21 expression. (F) Endogenous A1CF ubiquitination levels in HepG2 cells transfected with si-control or siTrim21. (G and H) Half-life measurements of endogenous A1CF in HepG2 cells (G), or overexpressed A1CF in Hepa 1–6 cells (H), cotransfected with TRIM21 WT or its LD form and treated with cycloheximide (CHX, 100 μg/mL) for the indicated time. (I and J) A1CF ubiquitination assays to identify the A1CF ubiquitination site in Hepa 1–6 cells cotransfected with either TRIM21 and A1CF (WT), single mutants (I), or double ubiquitination mutant (J), that were MG132 treated for 6 hours. (K) A1CF expression in Hepa 1–6 cells cotransfected with TRIM21 and A1CF-WT or indicated mutants. (L) Half-life measurements of A1CF in Hepa 1–6 cells cotransfected with TRIM21 and A1CF-WT or K302R mutant, treated with CHX for the indicated time intervals. ACTIN was used as a loading control.