RESUMO
HBV infection is challenging to cure due to the persistence of viral covalently closed circular viral DNA (cccDNA). The dedicator of cytokinesis 11 (DOCK11) is recognized as a guanine nucleotide exchange factor (GEF) for CDC42 that has been reported to be required for HBV persistence. DOCK11 is expressed in both the cytoplasm and nucleus of human hepatocytes and is functionally associated with retrograde trafficking proteins Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with the HBV capsid, in the trans-Golgi network (TGN). This opens an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. DOCK11 also facilitates the association of cccDNA with H3K4me3 and RNA Pol II for activating cccDNA transcription. In addition, DOCK11 plays a crucial role in the host DNA repair system, being essential for cccDNA synthesis. This function can be inhibited by 10M-D42AN, a novel DOCK11-binding peptide, leading to the suppression of HBV replication both in vitro and in vivo. Treatment with a combination of 10M-D42AN and entecavir may represent a promising therapeutic strategy for patients with chronic hepatitis B (CHB). Consequently, DOCK11 may be seen as a potential candidate molecule in the development of molecularly targeted drugs against CHB.
Assuntos
Fatores de Troca do Nucleotídeo Guanina , Vírus da Hepatite B , Hepatócitos , Humanos , Vírus da Hepatite B/fisiologia , Vírus da Hepatite B/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Hepatócitos/virologia , Hepatócitos/metabolismo , Internalização do Vírus , Replicação Viral , Hepatite B/virologia , Hepatite B/metabolismo , DNA Viral/metabolismo , DNA Viral/genética , AnimaisRESUMO
Overcoming hepatitis B virus (HBV) is a challenging problem because HBV deceives the host immune system. We have found that DENN domain-containing 2A (DENND2A) was essential for HBV maintenance, although its role remains unclear. In this study, we elucidate its function by screening a novel DENND2A-binding peptide, DENP4-3S. DENP4-3S exhibits homology to SAM and SH3 domain-containing protein 1 (SASH1), a scaffold protein involved in Toll-like receptor signaling that promotes proinflammatory cytokine production. We confirmed that DENND2A interacts with SASH1 specifically. Overexpression and knockdown experiments showed that overexpression of DENND2A suppressed the transcriptional activity of NF-κB, and the knockdown of DENND2A promoted it and the production of cytokines and interferons. Here, we constructed a fusion protein (10M-DEN3SN) consisting of an anti-asialoglycoprotein receptor antibody and DENP4-3S to deliver the peptide to hepatocytes specifically. 10M-DEN3SN inhibited the interaction between DENND2A and SASH1, and rescued SASH1 trapped by DENND2A, leading to the upregulation of NF-κB and its downstream signaling. In addition, 10M-DEN3SN suppressed HBV proliferation in PXB chimeric mice. These results with the DENND2A-binding peptide delivered into hepatocytes suggested the involvement of DENND2A, SASH, and NF-κB signaling pathway in the HBV infection and onset of hepatitis. In conclusion, this study indicates that HBV utilizes DENND2A and SASH1 to evade the immune system.IMPORTANCEHepatitis B virus (HBV) is a serious liver infection with no established cure, causing an abnormal host immune response. Here, we identified a novel peptide that interacts with DENN domain-containing 2A (DENND2A), a host factor essential for HBV maintenance. The resulting peptide showed sequence homology, revealing an interaction between DENND2A and the immune system regulator SASH1. This study suggests that DENND2A contributes to HBV infection by suppressing the cellular immune system by inhibiting SASH1. The DENND2A-binding peptide, incorporated into our hepatocyte-specific peptide delivery system, inhibited the DENND2A-SASH1 interaction and promoted the production of cytokines and interferons in cultured hepatocytes. As a consequence, the peptide suppressed HBV proliferation in humanized mice. We report new insights into the role of DENND2A and SASH1 in HBV maintenance and highlight the importance of the immune system.
Assuntos
Vírus da Hepatite B , Hepatite B , Camundongos , Animais , Vírus da Hepatite B/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , Interferons , Citocinas/metabolismo , Sistema ImunitárioRESUMO
BACKGROUND: Recent reports have unveiled the potential utility of l-carnitine to alleviate metabolic dysfunction-associated steatohepatitis (MASH) by enhancing mitochondrial metabolic function. However, its efficacy at preventing the development of HCC has not been assessed fully. METHODS: l-carnitine (2 g/d) was administered to 11 patients with MASH for 10 weeks, and blood liver function tests were performed. Five patients received a serial liver biopsy, and liver histology and hepatic gene expression were evaluated using this tissue. An atherogenic plus high-fat diet MASH mouse model received long-term l-carnitine administration, and liver histology and liver tumor development were evaluated. RESULTS: Ten-week l-carnitine administration significantly improved serum alanine transaminase and aspartate transaminase levels along with a histological improvement in the NAFLD activity score, while steatosis and fibrosis were not improved. Gene expression profiling revealed a significant improvement in the inflammation and profibrotic gene signature as well as the recovery of lipid metabolism. Long-term l-carnitine administration to atherogenic plus high-fat diet MASH mice substantially improved liver histology (inflammation, steatosis, and fibrosis) and significantly reduced the incidence of liver tumors. l-carnitine directly reduced the expression of the MASH-associated and stress-induced transcriptional factor early growth response 1. Early growth response 1 activated the promoter activity of neural precursor cell expressed, developmentally downregulated protein 9 (NEDD9), an oncogenic protein. Thus, l-carnitine reduced the activation of the NEDD9, focal adhesion kinase 1, and AKT oncogenic signaling pathway. CONCLUSIONS: Short-term l-carnitine administration ameliorated MASH through its anti-inflammatory effects. Long-term l-carnitine administration potentially improved the steatosis and fibrosis of MASH and may eventually reduce the risk of HCC.
Assuntos
Carcinoma Hepatocelular , Fígado Gorduroso , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Neoplasias Hepáticas/prevenção & controle , Carcinoma Hepatocelular/prevenção & controle , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/prevenção & controle , Carnitina/farmacologia , Carnitina/uso terapêutico , Fibrose , Inflamação , Proteínas Adaptadoras de Transdução de SinalRESUMO
BACKGROUND: Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. METHODS: The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. RESULTS: NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRß and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRß. CONCLUSIONS: NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.