Helicase DDX41 is a cytosolic sensor capable of detecting double-stranded DNA in mammals. However, the function of DDX41 remains poorly understood in invertebrates. In a previous study, we identified the first DDX41 sensor in the black tiger shrimp Penaeus monodon (PmDDX41) and showed that it played a role in anti-viral response. In the present study, we demonstrated that PmDDX41 was localized in the cytoplasm of shrimp hemocytes. However, PmDDX41 was localized in both the cytoplasm and nucleus of hemocytes in the presence of white spot syndrome virus (WSSV) infection or when stimulated by the nucleic acid mimics, poly(dA:dT) and poly(I:C). Similar results were observed when PmDDX41 was transfected into human embryonic kidney 293T (HEK293T) cells. Immunoprecipitation further demonstrated that PmDDX41 bound to biotin-labeled poly(dA:dT) but not poly(I:C). The overexpression of shrimp PmDDX41 and mouse stimulator of interferon gene (MmSTING) in HEK293T cells synergistically promoted IFN-β and NF-κB promoter activity via the DEADc domain. Co-immunoprecipitation (Co-IP) also confirmed that there was an interaction between PmDDX41 and STING after stimulation with poly(dA:dT) but not poly(I:C). Our study is the first to demonstrate that PmDDX41 acts as a cytosolic DNA sensor that interacts with STING via its DEADc domain to trigger the IFN-β and NF-κB signaling pathways, thus activating antiviral innate immune responses.
Previously, a designed ankyrin repeat protein, AnkGAG1D4, was generated for intracellular targeting of the HIV-1 capsid domain. The efficiency was satisfactory in interfering with the HIV assembly process. Consequently, improved AnkGAG1D4 binding affinity was introduced by substituting tyrosine (Y) for serine (S) at position 45. However, the intracellular anti-HIV-1 activity of AnkGAG1D4-S45Y has not yet been validated. In this study, the performance of AnkGAG1D4 and AnkGAG1D4-S45Y in inhibiting wild-type HIV-1 and HIV-1 maturation inhibitor-resistant replication in SupT1 cells was evaluated. HIV-1 p24 and viral load assays were used to verify the biological activity of AnkGAG1D4 and AnkGAG1D4-S45Y as assembly inhibitors. In addition, retardation of syncytium formation in infected SupT1 cells was observed. Of note, the defense mechanism of both ankyrins did not induce the mutation of target amino acids in the capsid domain. The present data show that the potency of AnkGAG1D4-S45Y was superior to AnkGAG1D4 in interrupting either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain.
Interferon regulatory factors (IRFs) are transcription factors found in both vertebrates and invertebrates that were recently identified and found to play an important role in antiviral immunity in black tiger shrimp Penaeus monodon. In this study, we investigated the mechanism by which P. monodon IRF (PmIRF) regulates the immune-related genes downstream of the cytosolic DNA sensing pathway. Depletion of PmIRF by double-stranded RNA-mediated gene silencing significantly reduced the mRNA expression levels of the IFN-like factors PmVago1, PmVago4, and PmVago5 and antilipopolysaccharide factor 6 (ALFPm6) in shrimp. In human embryonic kidney (HEK293T) cells transfected with PmIRF or co-transfected with DEAD-box polypeptide (PmDDX41) and simulator of IFN genes (PmSTING) expression plasmids, the promoter activity of IFN-β, nuclear factor (NF-κB), and ALFPm6 was synergistically enhanced following stimulation with the nucleic acid mimics deoxyadenylic–deoxythymidylic acid sodium salt [poly(dA:dT)] and high molecular weight (HMW) polyinosinic–polycytidylic acid [poly(I:C)]. Both nucleic acid mimics also significantly induced PmSTING, PmIRF, and ALFPm6 gene expression. Co-immunoprecipitation experiments showed that PmIRF interacted with PmSTING in cells stimulated with poly(dA:dT). PmSTING, PmIRF, and PmDDX41 were localized in the cytoplasm of unstimulated HEK293T cells and PmIRF and PmDDX41 were translocated to the nucleus upon stimulation with the nucleic acid mimics while PmSTING remained in the cytoplasm. These results indicate that PmIRF transduces the pathogen signal via the PmDDX41–PmSTING DNA sensing pathway to induce downstream production of interferon-like molecules and antimicrobial peptides.
Background: Ankyrin (Ank) is a protein family with crucial roles in retaining normal cellular physiology. In addition, ankyrin offers the potential as a non-antibody binder against various biomolecules. The designed ankyrin repeat protein (DARPin) selected from phage display libraries is useful for molecular detection and therapy. Monoclonal antibodies (mAbs) specific to the common epitope of DARPin are required to detect protein-protein interaction. Objectives: This study aimed to establish mAbs against common antigenic determinant of ankyrins for further application in immunological techniques. Materials and methods: Ank1D4 monomer and dimer were generated in the Escherichia coli expression system for immunogen preparation and validation of established mAbs. The binding activity of anti-Ank mAb obtained from different hybridoma clones was characterized using Ank1D4 by indirect ELISA. Candidate anti-Ank mAbs were validated for their cross-reactivity against irrelevant ankyrin (Ank2D3). The binding kinetic of mAbs from three candidate hybridoma clones (Ank-54, Ank-59, and Ank-94) was evaluated using bio-layer interferometry (BLI). The highest affinity clone (Ank-94 mAb ) was further validated for its specificity against Ank1D4 and dimeric Ank1D4 using indirect ELISA. The interaction of three anti-Ank mAbs and ankyrins was compared by western immunoblotting analysis. The specificity of Ank-94 mAb was determined using a closely related scaffold, i.e., alpha-helicoidal HEAT-like repeat protein scaffold (αRep) by indirect ELISA. Ankyrins were detected by sandwich ELISA using Ank-94 mAb. Results: The culture supernatant from hybridoma clones were characterized for their anti-ankyrin binding properties. Using indirect ELISA, three clones exhibited positive reactivity against the immunized ankyrin antigen (Ank1D4). The interactive epitope was found to rely on common antigenic determinants found in Ank1D4, dimeric Ank1D4, and an irrelevant ankyrin, Ank2D3. The immunoblotting results suggest that all mAbs interact with the sequential epitope of ankyrins. The cross-reactivity of Ank-94 mAb was not observed with αRep. Ank-94 mAb was selected for further purification and evaluation of binding properties due to its highest degree of binding affinity against Ank1D4. Conclusion: The establishment of a novel Ank-94 mAb could be a valuable research tool in tracing the target of DARPins or developing immunoassays. Ank-94 mAb is superior over formerly produced Ank mAbs since it recognizes a common epitope on DARPins and relies on sequential epitope. Ank-94 mAb has no cross-reactivity with another scaffold, αRep.
Domain 1 of CD147 participates in matrix metalloproteinase (MMP) production and is a candidate for targeted therapy to prevent cancer invasion and metastasis. A functional mouse anti-CD147 monoclonal antibody, M6-1B9, was found to recognize domain 1 of CD147, and its respective mouse single-chain variable fragment (ScFvM61B9) was subsequently generated. The EDLGS epitope candidate for M6-1B9 was identified using the phage display peptide technique in this study. For future clinical applications, humanized ScFv specific to domain 1 of CD147 (HuScFvM61B9) was partially adopted from the hypervariable sequences of parental mouse ScFvM61B9 and grafted onto suitable human immunoglobulin frameworks. Molecular modelling and simulation were performed in silico to generate the conformational structure of HuScFvM61B9. These results elucidated the amino acid residues that contributed to the interactions between CDRs and the epitope motif. The expressed HuScFvM61B9 specifically interacted with CD147 at the same epitope as the original mAb, M6-1B9, and retained immunoreactivity against CD147 in SupT1 cells. Binding affinity of HuScFvM61B9 by biolayer interferometry was higher than the predicted one. should be considered a potential therapeutic antibody. As domain 1 is responsible for cancer invasion and metastasis, HuScFvM61B9 would be a candidate for cancer targeted therapy in the future.
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