RIP-seq of DDX49 related RNAs in HHL5 cells transfected Flag-DDX49 following OA/PA treatment for 24 hours
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE299232 | GEO | 2025/06/17
REPOSITORIES: GEO
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Project description:RIP-seq of DDX49 related RNAs in HHL5 cells transfected Flag-DDX49 following OA/PA treatment for 24 hours
| PRJNA1273118 | ENA
Project description:To explore d the proteins that associated with DDX49 in metabolic associated fatty liver disease (MAFLD) and metabolic dysfunction-associated steatohepatitis (MASH), We overexpressed DDX49 with Flag tag in HHL5 cells, then treated with OA/PA for 24 hours, and performed immunoprecipitation and mass spectrometry to search for the proteins that bind to DDX49.
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Project description:During the processes of MAFLD and MASH, DDX49 undergoes liquid-liquid phase separation. Subsequently, we used OA/PA (2:1) to simulate the MASH model in vitro. After treating HHL5 cells with OA/PA for 24 hours, we separated the lipid-organized particles of DDX49 by differential centrifugation. To investigate the composition of these particles and study their functions, we conducted a 10-fold proteomic analysis.
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Project description:Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are characterized by excessive triglyceride accumulation in the liver. However, due to an incomplete understanding of its pathogenesis, more efforts are still needed to identify specific and effective treatments. N4-acetylcytidine (ac4C) is a newly discovered RNA modification to regulate mRNA stability post-transcriptionally. N-acetyltransferase 10 (NAT10), the sole enzyme catalyzing mRNA acetylation, has not been fully explored in human diseases, especially in MASLD and MASH. In the current study, abundant RNA acetylation was found in lipid metabolism-related genes in the livers of leptin receptor-deficient (db/db) mice. Besides, hepatic NAT10 expression is significantly increased in multiple mouse models of MASLD and MASH. NAT10 expression is also elevated in patients with MASLD and positively correlated with clinical characteristics. Genetic NAT10 knockdown protects against diet-induced hepatic steatosis and steatohepatitis in mice, while its overexpression exacerbates steatosis. Mechanistically, NAT10 could bind to Srebp-1c mRNA to promote its stability and expression, thereby upregulating lipogenic enzymes. In addition, the translational significance of our findings is that treatment of Remodelin, an NAT10 inhibitor, could improve liver steatosis and dyslipidemia in a preclinical mouse model. Together, these findings highlight the significance of ac4C modification and NAT10 in MASLD and MASH, offering a potential therapeutic target for disease treatment.
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Project description:Metabolic dysfunction-associated steatotic liver disease (MASLD) is estimated to affect approximately 30% of adults worldwide. The progressive form of MASLD, namely metabolic-dysfunction steatohepatitis (MASH), is a leading cause of chronic liver disease. MASH is marked by hepatocellular fat accumulation (steatosis), ballooning, and inflammation. Although many in vitro and in vivo models replicate MASH pathophysiology, no in vitro hepatocyte MASH model has been evaluated for its ability to reflect clinically observed changes in drug metabolizing enzyme (DME) and transporter characteristics such as abundance and activity. This study addressed this knowledge gap by developing a model using sandwich-cultured human hepatocytes (SCHH) that mimics both MASH pathophysiology and alterations in DME and transporter concentrations and function. Lipid-cytokine treatments were first optimized using differentiated HuH-7 cells based on cellular toxicity profiles and their ability to induce a MASH-like phenotype. Three final treatments, all including TNF-α (1 ng/mL) and IL-6 (1.2 ng/mL), were selected for evaluation in SCHH: (1) oleic acid (OA):palmitic acid (PA) (1:2, 0.5 mM), (2) a lipid mix (lysophospholipids mixture + OA:PA), and (3) lipid mix+0.01 mM cholesterol. Treatments were incubated for 72 h with SCHH from three donors. Quantitative targeted absolute proteomics (QTAP), employing stable isotope labeled (SIL) peptide standards and microLC-MS/MS measurement, was used to quantify transporter and DME concentrations in the SCHH total membrane fraction. Concentrations of ten transporters, ten CYP450s, seven UGTs and five other DMEs could be measured above the lower limit of quantification of 0.1 pmol/mg membrane protein. B-CLEAR® technology was used, also, to evaluate transporter function with [3H]-taurocholate (TCA) and [3H]-estradiol-17β-glucuronide (E217G) employed as probe substrates of function. All three treatments significantly increased lipid droplet formation and peroxidation, characteristics of MASH, in SCHH with minimal toxicity. The treatments also altered DME and transporter concentrations in a similar manner to changes observed in liver tissue from patients with MASH. Across treatments, concentrations of bile salt export pump (BSEP), sodium taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and multidrug resistance-associated protein (MRP) 2 were reduced by 0.66–0.57-fold, 0.71–0.52-fold, 0.74–0.63-fold, 0.82–0.80-fold, and 0.71–0.48-fold, respectively. Correspondingly, TCA apparent uptake clearance and biliary clearance were reduced by 0.70–0.26-fold and 0.61–0.27-fold, respectively. E217G apparent uptake clearance was reduced by 0.67–0.35-fold while biliary excretion index values for E217G were reduced to negligible levels. Overall, the findings demonstrate that lipid–cytokine treatments induce MASH-like changes in SCHH, including clinically relevant reductions in DME and transporter concentrations and transporter function. This model may serve as a valuable tool for predicting altered hepatobiliary drug disposition in MASH.
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Project description:Lipotoxicity-induced hepatic DNA damage responses (DDRs) contribute to malignant progression of MASH-related HCC, but the underlying mechanism is unclear. Using DSB marker phosphorylation of the Ser139 residue of the histone variant H2AX (γH2AX) antiby-mediated CUT&Tag-seq, a global increase of DSBs induced by PA+OA was observed.
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Project description:Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are two common liver disorders characterized by abnormal lipid accumulation. Our study found reduced levels of GTPase-activating protein-binding protein1（G3BP1）in patients with MASLD and MASH, suggesting its involvement in these liver disorders. Hepatocyte-specific G3BP1 knockout (G3BP1 HKO) mice had more severe MASLD and MASH than their corresponding controls. Intriguingly, the G3BP1 HKO MASLD model mice exhibit dysregulated autophagy, and biochemical analyses demonstrated that G3BP1 promotes autophagosome-lysosome fusion through direct interactions with the SNARE proteins STX17 and VAMP8. We also show that hepatic knockout of G3BP1 promotes de novo lipogenesis, and ultimately found that G3BP1 is required for the nuclear translocation of the well-known liver-lipid-regulating transcription factor TFE3. Taken together, our results suggest that G3BP1 should be investigated as a potential target for developing medical interventions to treat MASLD and MASH.
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