Dongqin Yang

Background/Aims: Excessive lipid accumulation in hepatocytes is a critical cause of metabolic dysfunction-associated steatotic liver disease (MASLD) progression. Ankyrin repeat and SOCS box protein 3 (ASB3) is an E3 ubiquitin ligase that mediates diverse disease processes; however, the direct substrates of ASB3 in lipid metabolism and its role in MASLD remain unexplored.Methods: We generated ASB3 knockout mice fed a high-fat diet to induce MASLD. Oxygen consumption and fatty acid oxidation (FAO) were used to assess lipid metabolism. LC-MS/MS and IP were used to verify the ASB3 target protein. Correlation analysis was conducted on the cohort of MASLD patients vs. the control group.Results: Loss of the ASB3 E3 ubiquitin ligase in hepatocytes strengthens mitochondrial FAO, thereby influencing energy consumption to decrease triglyceride storage and lipid accumulation. Quantitative lysine ubiquitination proteomics revealed that ASB3 directly mediated the ubiquitin levels at two sites (K180 and K639) in carnitine palmitoyl transferase 1A (CPT1A), a rate-limiting enzyme of FAO, to induce CPT1A degradation. Moreover, both constitutive and hepatocyte-specific ASB3 knockout enhance FAO and delay lipid accumulation, liver steatosis, and MASLD progression in a CPT1A-dependent manner. Hepatic ASB3 deficiency also delays fibrosis in MASLD. Analysis of public databases and liver tissue samples from MASLD patients revealed that ASB3 was highly expressed in MASLD patients and was negatively correlated with CPT1A.Conclusions: Our study reveals the key roles of ASB3 in the development of MASLD and suggests a novel therapeutic potential for MASLD.

E3 ubiquitin ligases are very important for regulating antiviral immunity during viral infection. Here, we discovered that Ankyrin repeat and SOCS box-containing protein 3 (ASB3), an E3 ligase, are upregulated in the presence of RNA viruses, particularly influenza A virus (IAV). Notably, overexpression of ASB3 inhibits type I IFN (IFN-I) responses induced by Sendai virus (SeV) and IAV, and ablation of ASB3 restores SeV and H9N2 infection-mediated transcription of IFN-β and its downstream interferon-stimulated genes (ISGs). Interestingly, animals lacking ASB3 presented decreased susceptibility to H9N2 and H1N1 infections. Mechanistically, ASB3 interacts with MAVS and directly mediates K48-linked polyubiquitination and degradation of MAVS at K297, thereby inhibiting the phosphorylation of TBK1 and IRF3 and downregulating downstream antiviral signaling. These findings establish ASB3 as a critical negative regulator that controls the activation of antiviral signaling and describe a novel function of ASB3 that has not been previously reported.

E3 ubiquitin ligase (E3) plays a vital role in regulating inflammatory responses by mediating ubiquitination. Previous studies have shown that ankyrin repeat and SOCS box-containing protein 3 (ASB3) is involved in immunomodulatory functions associated with cancer. However, the impact of ASB3 on the dynamic interplay of microbiota and inflammatory responses in inflammatory bowel disease (IBD) is unclear. Here, we systematically identify the E3 ligase ASB3 as a facilitative regulator in the development and progression of IBD. We observed that ASB3 exhibited significant upregulation in the lesions of patients with IBD. ASB3 ^−/− mice are resistant to dextran sodium sulfate-induced colitis. IκBα phosphorylation levels and production of proinflammatory factors IL-1β, IL-6, and TNF-α were reduced in the colonic tissues of ASB3 ^−/− mice compared to WT mice. This colitis-resistant phenotype was suppressed after coprophagic microbial transfer and reversed after combined antibiotics removed the gut commensal microbiome. Mechanistically, ASB3 specifically catalyzes K48-linked polyubiquitination of TRAF6 in intestinal epithelial cells. In contrast, in ASB3-deficient organoids, the integrity of the TRAF6 protein is shielded, consequently decelerating the onset of intestinal inflammation. ASB3 is associated with dysregulation of the colitis microbiota and promotes proinflammatory factors’ production by disrupting TRAF6 stability. Strategies to limit the protein level of ASB3 in intestinal epithelial cells may help in the treatment of colitis. IMPORTANCE: Ubiquitination is a key process that controls protein stability. We determined the ubiquitination of TRAF6 by ASB3 in intestinal epithelial cells during colonic inflammation. Inflammatory bowel disease patients exhibit upregulated ASB3 expression at focal sites, supporting the involvement of degradation of TRAF6, which promotes TLR-Myd88/TRIF-independent NF-κB aberrant activation and intestinal microbiota imbalance. Sustained inflammatory signaling in intestinal epithelial cells and dysregulated protective probiotic immune responses mediated by ASB3 collectively contribute to the exacerbation of inflammatory bowel disease. These findings provide insights into the pathogenesis of inflammatory bowel disease and suggest a novel mechanism by which ASB3 increases the risk of colitis. Our results suggest that future inhibition of ASB3 in intestinal epithelial cells may be a novel clinical strategy.

Ankyrin‐repeat proteins with a suppressor of cytokine signaling box (ASB) proteins belong to the E3 ubiquitin ligase family. 18 ASB members have been identified whose biological functions are mostly unexplored. Here, we discovered that ASB3 was essential for hepatocellular carcinoma (HCC) development and high ASB3 expression predicted poor clinical outcomes. ASB3 silencing induced HCC cell growth arrest and apoptosis in vitro and in vivo. Liver‐specific deletion of Asb3 gene suppressed diethylnitrosamine (DEN)‐induced liver cancer development. Mechanistically, ASB3 interacted with death receptor 5 (DR5), which promoted ubiquitination and degradation of DR5. We further showed that ASB3 knockdown stabilized DR5 and increased the sensitivity of liver cancer cells to the treatment of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) in a DR5‐dependent manner in cellular and in animal models. In summary, we demonstrated that ASB3 promoted ubiquitination and degradation of DR5 in HCC, suggesting the potential of targeting ASB3 to HCC treatment and overcome TRAIL resistance.

E3 ubiquitin ligases are very important to regulate antiviral immunity during viral infection. Here, we discovered that Ankyrin repeat and SOCS box-containing protein 3 (ASB3), an E3 ligase, are upregulated in the presence of RNA viruses, particularly Influenza A virus (IAV). Notably, overexpression of ASB3 inhibits type I IFN (IFN-I) responses induced by Sendai virus (SeV) and H9N2, and ablation of ASB3 restores SeV and H9N2 infection-mediated transcription of IFN-β and its downstream interferon-stimulated genes (ISGs). Interestingly, animals lacking ASB3 showed a decreased susceptibility to H9N2 and PR8 infections. Mechanistically, ASB3 interacts with MAVS and directly mediates K48-linked polyubiquitination and degradation of MAVS at K297, thereby inhibiting the phosphorylation levels of TBK1 and IRF3, downregulating downstream antiviral signaling. These findings establish ASB3 as a critical negative regulator in controlling the activation of antiviral signaling and describe a novel function of ASB3 that has not been previously reported. IMPORTANCE IAV is a significant zoonotic pathogen that causes infections of the respiratory system. Hosts have evolved multiple strategies to defend against IAV infection. However, not all host proteins play an active defense role. In this study, we found that the E3 ligase ASB3 regulates antiviral immunity by manipulating MAVS stability. Briefly, overexpression of ASB3 degrades MAVS, thereby promoting viral replication. In contrast, ASB3 deletion restores MAVS expression, upregulating IFN-I responses. Additional research revealed that ASB3 mediates the K48-linked polyubiquitination of MAVS at K297, resulting in ASB3 being degraded via the ubiquitin-proteasome pathway. These findings reveal, for the first time, a novel mechanism by which ASB3 negatively regulates antiviral immunity and provides a potential target for anti-IAV drug development.

E3 ubiquitin ligase (E3) plays a vital role in regulating inflammatory responses by mediating ubiquitination. Previous studies have shown that ankyrin repeat and SOCS box-containing protein 3 (ASB3) is involved in immunomodulatory functions associated with cancer. However, the impact of ASB3 on the dynamic interplay of microbiota and inflammatory responses in inflammatory bowel disease (IBD) is unclear. Here we systematically identify the E3 ligase ASB3 as a facilitative regulator in the development and progression of IBD. ASB3 ^-/- mice are resistant to DSS-induced colitis. IκBα phosphorylation levels and production of proinflammatory factors IL-1β, IL-6, and TNF-α were reduced in colonic tissues of ASB3 ^-/- mice compared to WT mice. This colitis-resistant phenotype was suppressed after coprophagic microbial transfer and reversed after combined antibiotic clearance of the microbiota. Mechanistically, ASB3 specifically catalyzes K48-linked polyubiquitination of TRAF6 in IECs. In contrast, in ASB3-deficient organoids, the integrity of the TRAF6 protein is shielded, consequently decelerating the onset of intestinal inflammation. Taken together, Our findings demonstrates that ASB3 is associated with dysregulation of the colitis microbiota and promotes proinflammatory factors production by disrupting the TRAF6 stability. Strategies to limit the protein level of ASB3 in intestinal epithelial cells may help in the treatment of colitis.

MLN4924 is a selective neddylation inhibitor that has shown great potential in treating several cancer and metabolic diseases, including obesity. However, it remains largely unknown whether MLN4924 has similar effect on non-alcoholic liver disease (NAFLD), which is closely associated with metabolic disorders. Here, we investigated the role of MLN4924 in NAFLD treatment and the underlying mechanism of the action using primary hepatocytes stimulated with free fatty acid, as well as high-fat diet (HFD)-induced NAFLD mouse models. We found that MLN4924 can inhibit the accumulation of lipid and reduce the expression of peroxisome proliferator-activated receptor γ (PPARγ), a key player in adipocyte differentiation and function in both in vivo and in vitro models. Moreover, we verified its important role in decreasing the synthesis and accumulation of fat in the liver, thus mitigating the development of NAFLD in the mouse model. The body weight and fat mass in MLN4924-treated animals were significantly reduced compared to the control group, while the metabolic activity, including O₂ consumption, CO₂ and heat production, also increased in these animals. Importantly, we demonstrated for the first time that MLN4924 can markedly boost mitochondrial fat acid oxidation (FAO) to alter liver lipid metabolism. Finally, we compared the metabolites between MLN4924-treated and untreated Huh7 cells after fatty acid induction using lipidomics methods and techniques. We found induction of several metabolites in the treated cells, including Beta-guanidinopropionic acid (b-GPA) and Fluphenazine, which was in accordance with the increase of FAO and metabolism. Together, our study provided a link between neddylation modification and energy metabolism, as well as evidence for targeting neddylation as an emerging therapeutic approach to tackle NAFLD.

Hepatocellular carcinoma (HCC) is one of the largest causes of cancer-related deaths worldwide owing to the limitation of effective treatment options. The ubiquitin-proteasome system has been rapidly recognized as a frequent target of deregulation leading to cancers. Enhanced DNA damage response (DDR) promotes HCC growth and prevents chemosensitivity, and ubiquitin E3 ligases are key modulators in DDR. Therefore, a better understanding of how E3 ligases regulate cell growth and DNA damage may provide novel insights in understanding the oncogenic mechanism and improving the efficacy of DNA damage therapeutic agents. Here, we performed a high-content RNAi screening targeting 52 DDR-related E3 ligases in HCC and found that ring finger protein 4 (RNF4) was essential for HCC growth. RNF4 was highly expressed in HCC tissues, and the expression levels of RNF4 were associated with poor outcomes. RNF4 silencing significantly suppressed the cell growth, and subsequently induced G2/M arrest and apoptosis of HCC cells in vitro; RNF4 silencing also demonstrated the tumor-suppressive efficacy on HCC in vivo. Moreover, RNF4 silencing increased DNA damage, and rendered HCC cells more sensitive to DNA damage drugs and radiation. We found RNF4 functionally interacts with p62, and mechanistic analyses indicated that RNF4 silencing triggered the nuclear enrichment of p62. Moreover, the p62 nuclear targeting was required for increased DNA damage and growth suppression mediated by RNF4 silencing. Thus, our findings suggest RNF4 is essential for HCC proliferation via preventing nuclear translocation of p62. RNF4 silencing promotes DNA damage and may serve as a novel strategy to suppress cell growth and increase the sensitivity of DNA damage therapeutic agents in HCC.

Background: Increasing numbers of studies reported platelet (PLT)- related measures could play a creative role in many malignancies, while the prognostic impact of these measures in hepatocellular carcinoma (HCC) remains limited and controversial. It is worth exploring the predictive value of PLT-related measures in HCC. Methods: A total of 279 HCC patients with hepatectomy were analyzed in the retrospective cohort study. The optimal cut-off points of these PLT-related indices were obtained by the receiver operating characteristic (ROC) curve. The associations of these indices with clinical characteristics and overall survival (OS) were evaluated by Kaplan–Meier curves and Cox proportional hazards models. Results: High PLT count and low prognostic nutritional index (low-PNI) were significantly associated with larger tumor size. The low gamma-glutamyl transpeptidase-to-platelet ratio (low-GPR) group was inclined to more hepatitis infections. Survival curves indicated that preoperative high-PLT, low-GPR, and low-PNI had a worse prognosis after surgery in the cohort. In addition, PLT≥220 × 10⁹/L (HR, 2.274; 95% CI, 1.061-4.876; P = .035), PNI≥51.9 (HR, 0.503; 95% CI, 0.265-0.954; P = .035), and GPR≥0.2 (HR, 0.432; 95% CI, 0.204-0.912; P = .028) were identified as independent prognostic factors for survival outcomes in the multivariable analysis. Conclusion: High-PLT, low-GPR, and low-PNI as the preoperative predictors were associated with poor OS in HCC patients with hepatectomy. Our data reveal that they could be simple, easily obtained, and effective predictors for evaluation of survival outcomes in patients.