The Really Interesting New Gene (RING) ubiquitin E3 ligase family comprises a large number of members and plays a crucial role in the antiviral process. RING finger protein 115 (RNF115), also known as BCA2, Rabring7, ...The Really Interesting New Gene (RING) ubiquitin E3 ligase family comprises a large number of members and plays a crucial role in the antiviral process. RING finger protein 115 (RNF115), also known as BCA2, Rabring7, or ZNF364, is a novel RING domain protein. In this paper, we cloned the RNF115 homologue from black carp (Mylopharyngodon piceus) and characterized it. The open reading frame of black carp RNF115 contains 933 nucleotides and encodes 310 amino acids. The C-terminal RING domain of RNF115 is highly conserved among various homologous species. Immunofluorescence assays revealed the cytoplasmic and nuclear distribution of RNF115 in the presence or absence of spring viremia of carp virus (SVCV) infection. Overexpression of RNF115 impaired interferon (IFN) and the related interferon-stimulated gene (ISG) mRNA expression, while upregulating SVCV replication. Ex vivo knockdown of RNF115 offered the host cells enhanced antiviral signaling. In vivo knockdown of RNF115 also strengthened black carp's antiviral capacity. Additionally, the results of a dual-luciferase reporter assay, plaque assay, and qRT-PCR assay demonstrated that co-transfection of RNF115 with IRF3/7 reduced IRF3/7-induced IFN transcription and antiviral ability. The association between RNF115 and IRF3/7 was detected by co-immunoprecipitation and immunofluorescence assays. Co-transfection of RNF115 with IRF3/7 also reduced the protein levels of IRF3/7, which were rescued by MG132. The enhanced K48-linked ubiquitination of IRF3/7 under the condition of RNF115 co-transfection implied the ubiquitin/proteasome degradation pathway catalyzed by RNF115. Cysteine 238 and 241 in the RING domain are the main enzyme active sites for RNF115, and the mutant C238/241A lost most of its ability to restrict IRF3/7. In conclusion, black carp RNF115 dampens IRF3/7-mediated IFN signaling through facilitating the ubiquitination and degradation of IRF3/7, which sheds light on the regulation of IFN signaling.展开更多
STING(also known as MITA)is an adaptor protein that mediates cytoplasmic DNA-triggered signaling,and aberrant activation of STING/MITA by cytosolic self-DNA or gain-of-function mutations causes severe inflammation.Her...STING(also known as MITA)is an adaptor protein that mediates cytoplasmic DNA-triggered signaling,and aberrant activation of STING/MITA by cytosolic self-DNA or gain-of-function mutations causes severe inflammation.Here,we show that STING-mediated inflammation and autoimmunity are promoted by RNF115 and alleviated by the RNF115 inhibitor disulfiram(DSF).Knockout of RNF115 or treatment with DSF significantly inhibit systemic inflammation and autoimmune lethality and restore immune cell development in Trex1^(–/–)mice and STING^(N153S/WT) bone marrow chimeric mice.In addition,knockdown or pharmacological inhibition of RNF115 substantially downregulate the expression of IFN-α,IFN-γand proinflammatory cytokines in PBMCs from patients with systemic lupus erythematosus(SLE)who exhibit high concentrations of dsDNA in peripheral blood.Mechanistically,knockout or inhibition of RNF115 impair the oligomerization and Golgi localization of STING in various types of cells transfected with cGAMP and in organs and cells from Trex1^(–/–)mice.Interestingly,knockout of RNF115 inhibits the activation and Golgi localization of STINGN153S as well as the expression of proinflammatory cytokines in myeloid cells but not in endothelial cells or fibroblasts.Taken together,these findings highlight the RNF115-mediated cell type-specific regulation of STING and STINGN153S and provide potential targeted intervention strategies for STING-related autoimmune diseases.展开更多
基金the institutional review board or ethics committee of Hunan Normal University (2024195)。
文摘The Really Interesting New Gene (RING) ubiquitin E3 ligase family comprises a large number of members and plays a crucial role in the antiviral process. RING finger protein 115 (RNF115), also known as BCA2, Rabring7, or ZNF364, is a novel RING domain protein. In this paper, we cloned the RNF115 homologue from black carp (Mylopharyngodon piceus) and characterized it. The open reading frame of black carp RNF115 contains 933 nucleotides and encodes 310 amino acids. The C-terminal RING domain of RNF115 is highly conserved among various homologous species. Immunofluorescence assays revealed the cytoplasmic and nuclear distribution of RNF115 in the presence or absence of spring viremia of carp virus (SVCV) infection. Overexpression of RNF115 impaired interferon (IFN) and the related interferon-stimulated gene (ISG) mRNA expression, while upregulating SVCV replication. Ex vivo knockdown of RNF115 offered the host cells enhanced antiviral signaling. In vivo knockdown of RNF115 also strengthened black carp's antiviral capacity. Additionally, the results of a dual-luciferase reporter assay, plaque assay, and qRT-PCR assay demonstrated that co-transfection of RNF115 with IRF3/7 reduced IRF3/7-induced IFN transcription and antiviral ability. The association between RNF115 and IRF3/7 was detected by co-immunoprecipitation and immunofluorescence assays. Co-transfection of RNF115 with IRF3/7 also reduced the protein levels of IRF3/7, which were rescued by MG132. The enhanced K48-linked ubiquitination of IRF3/7 under the condition of RNF115 co-transfection implied the ubiquitin/proteasome degradation pathway catalyzed by RNF115. Cysteine 238 and 241 in the RING domain are the main enzyme active sites for RNF115, and the mutant C238/241A lost most of its ability to restrict IRF3/7. In conclusion, black carp RNF115 dampens IRF3/7-mediated IFN signaling through facilitating the ubiquitination and degradation of IRF3/7, which sheds light on the regulation of IFN signaling.
基金supported by grants from the National Key Research and Development Program of China(Grant Nos.2022YFC3401500 and 2023YFC2306100)the Natural Science Foundation of China(Grant Nos.31930040,32070900,82000670,32270951,32200710,and 823B1006)+3 种基金the Fundamental Research Funds for the Central Universities(Grant Nos.2042022kf1187,2042022kf1123 and 2042022dx0003)the Major Scientific and Technological Project of Hubei Province(Grant No.2022ACA005)the Translational Medicine and Interdisciplinary Research Joint Found of Zhongnan Hospital of Wuhan University(Grant.No.ZNJC202218)the Non-Profit Central Research Institute Fund of the Chinese Academy of Medical Sciences(Grant No.2020PT320-004).
文摘STING(also known as MITA)is an adaptor protein that mediates cytoplasmic DNA-triggered signaling,and aberrant activation of STING/MITA by cytosolic self-DNA or gain-of-function mutations causes severe inflammation.Here,we show that STING-mediated inflammation and autoimmunity are promoted by RNF115 and alleviated by the RNF115 inhibitor disulfiram(DSF).Knockout of RNF115 or treatment with DSF significantly inhibit systemic inflammation and autoimmune lethality and restore immune cell development in Trex1^(–/–)mice and STING^(N153S/WT) bone marrow chimeric mice.In addition,knockdown or pharmacological inhibition of RNF115 substantially downregulate the expression of IFN-α,IFN-γand proinflammatory cytokines in PBMCs from patients with systemic lupus erythematosus(SLE)who exhibit high concentrations of dsDNA in peripheral blood.Mechanistically,knockout or inhibition of RNF115 impair the oligomerization and Golgi localization of STING in various types of cells transfected with cGAMP and in organs and cells from Trex1^(–/–)mice.Interestingly,knockout of RNF115 inhibits the activation and Golgi localization of STINGN153S as well as the expression of proinflammatory cytokines in myeloid cells but not in endothelial cells or fibroblasts.Taken together,these findings highlight the RNF115-mediated cell type-specific regulation of STING and STINGN153S and provide potential targeted intervention strategies for STING-related autoimmune diseases.
