Journal: Life Science Alliance

Article Title: FAT10 is phosphorylated by IKKβ to inhibit the antiviral type-I interferon response

doi: 10.26508/lsa.202101282

Figure Lengend Snippet: (A) HEK293 cells were transiently transfected with a His-3xFLAG-FAT10 (FLAG-FAT10) expression construct and stimulated for 24 h with TNF. Lysates were subjected to immunoprecipitation using FLAG-reactive antibodies, coupled to sepharose beads, and subsequently analyzed by Phos-tag/SDS–PAGE/IB analysis. Where indicated, cells were pretreated before TNF stimulation with the displayed inhibitors for a total of 3 h (10 µM each). (B) HEK293 cells were transiently transfected with expression plasmids for the different kinases. Cells were harvested, lysed, and subjected to immunoprecipitation using anti-FLAG or anti-HA antibodies, coupled to sepharose beads. Subsequently, the immunoprecipitated kinases were incubated with recombinant FAT10 (rFAT10) and an in vitro reaction was performed in the kinase buffer. The phosphorylation status of FAT10 was analyzed by Phos-tag/SDS–PAGE and IB. Asterisks mark unspecific background bands. (C) FLAG-FAT10 and the indicated kinases were transiently overexpressed in HEK293 cells followed by TNF stimulation. After 24 h, cells were lysed and subjected to immunoprecipitation against the FLAG-tag, combined with Phos-tag/SDS–PAGE and IB analysis. (D) Recombinant FAT10 (rFAT10) was incubated with recombinant kinases IKKβ, IKKε or MK3 for 45 min at 30°C. Subsequently, proteins were separated on a Phos-tag/SDS–PAGE followed by immunoblot analysis using the antibodies indicated. (E) HEK293 cells were prepared as described in (A). Where specified, cells were pretreated with the inhibitors indicated (10 µM each) for a total of 3 h before stimulation with TNF. One representative example out of three independent experiments with the same outcomes is shown. Source data are available for this figure.

Article Snippet: The following cDNA constructs were obtained from Addgene: FLAG-JNK1 (plasmid #13798, [ ]), JNK2 (plasmid #13755, [ ]), JNK3 (plasmid #13758, [ ]), HA-IKKα (plasmid #15469, [ ]), FLAG-IKKβ (plasmid #15470, [ ]), HA-IKKγ (plasmid #13512, [ ]), FLAG-IKKε (plasmid #26201, [ ]), pM2.G (plasmid #12259; kind gift from Didier Trono), and psPAX2 (plasmid #12260; kind gift from Didier Trono).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page, Incubation, Recombinant, In Vitro, FLAG-tag, Western Blot

Journal: Life Science Alliance

Article Title: FAT10 is phosphorylated by IKKβ to inhibit the antiviral type-I interferon response

doi: 10.26508/lsa.202101282

Figure Lengend Snippet: (A) FAT10 mRNA levels were analyzed and quantified by real-time PCR in A549 cells 24 h after TNF/IFNγ treatment or 24 h after IAV infection. (B) A549 cells were infected with IAV (MOI:1) for 0, 6, 24 or 48 h, as indicated. Cell lysates were subjected to SDS–PAGE combined with immunoblot analysis with the antibodies indicated. (C) Immunoblot showing endogenous FAT10 expression in A549 cells treated for 24 h with TNF/IFNγ, or infected for 24 h with IAV. FAT10 was immunoprecipitated with a monoclonal FAT10-reactive antibody (clone 4F1) and an immunoblot was performed with the antibodies indicated. (D) A549 cells stably expressing His-3xFLAG-FAT10 (FLAG-FAT10) were lysed after 24 h of TNF stimulation or after 24 h of IAV infection. The lysates were subjected to immunoprecipitation using FLAG-reactive antibodies coupled to sepharose beads. Subsequently, a Phos-tag/SDS–PAGE and immunoblot analysis with the indicated antibodies was performed. Asterisk marks an unspecific background band. (E) Recombinant 6His-SUMO-FAT10 was purified via Ni-NTA and left bound to the beads. 400 units λ phosphatase was added and incubated twice for 30 min at 30°C. Beads were extensively washed and FAT10 was eluted by treatment with the ULP1 enzyme to receive untagged and non-phosphorylated FAT10. Lysates from untreated or IAV-infected A549 cells were prepared and incubated with the purified FAT10, as indicated, for 30 min at 30°C. Proteins were separated on a Phos-tag/SDS–PAGE and analyzed by immunoblotting with a FAT10-reactive, polyclonal antibody. (F) A549 cells stably expressing FLAG-FAT10 were infected with IAV for 24 h. Cell lysates were subjected to a FLAG-immunoprecipitation and analyzed by Phos-tag/SDS–PAGE, followed by immunoblotting using the antibodies indicated. Where indicated, cells were pretreated with the indicated inhibitors (5 mM IKKβ inhibitor, 10 μM IKKε inhibitor) for 3 h, before IAV infection. For each panel, one representative example out of three independent experiments with similar outcomes is shown. Source data are available for this figure.

Article Snippet: The following cDNA constructs were obtained from Addgene: FLAG-JNK1 (plasmid #13798, [ ]), JNK2 (plasmid #13755, [ ]), JNK3 (plasmid #13758, [ ]), HA-IKKα (plasmid #15469, [ ]), FLAG-IKKβ (plasmid #15470, [ ]), HA-IKKγ (plasmid #13512, [ ]), FLAG-IKKε (plasmid #26201, [ ]), pM2.G (plasmid #12259; kind gift from Didier Trono), and psPAX2 (plasmid #12260; kind gift from Didier Trono).

Techniques: Real-time Polymerase Chain Reaction, Infection, SDS Page, Western Blot, Expressing, Immunoprecipitation, Stable Transfection, Recombinant, Purification, Incubation

Journal: The Journal of biological chemistry

Article Title: SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection.

doi: 10.1016/j.jbc.2023.104925

Figure Lengend Snippet: Figure 3. SAMHD1 suppresses MAVS aggregation and disrupts MAVS interaction with IKKε. A, THP-1 Ctrl and SAMHD1 KO cells were mock-infected or infected with SeV (MOI of 10) for 8 h. Crude mitochondria were isolated and subjected to SDD-AGE (top). Whole cell lysates were analyzed by SDS-PAGE (bottom). Quantification of pTBK1 (S172) and pIRF3 (S396) levels was performed by densitometry and normalized relative to total TBK1 and IRF3, respectively. B, HEK293T cells were transfected with expression plasmids encoding the indicated proteins (FLAG-MAVS, myc-IKKε, and increasing amounts of HA-SAMHD1). Cells were lysed 36 h posttransfection, and IP was performed using an anti-FLAG antibody. Indicated proteins were detected by IB. C, THP-1 Ctrl and SAMHD1 cells were mock-infected (M) or infected with SeV (MOI of 10) and harvested at the indicated time points. Cell lysates were analyzed by IB with the indicated antibodies. Quantification of pIKKε (S172) levels was performed by densitometry and expressed relative to total IKKε. A–C, representative results from three independent experiments are shown. IB, immunoblot; IKKε, inhibitor of nuclear factor kappa-B kinase epsilon; IRF, IFN regulatory factor; M, mock infection; MAVS, mitochondrial antiviral-signaling protein; MOI, multiplicity of infection; pIKKε, phospho-IKKε; SAMHD, sterile alpha motif and HD domain-containing protein; SDD-AGE, semidenaturing detergent agarose gel electrophoresis; SeV, Sendai virus; SeV NP, nucleoprotein of SeV; TBK1, TANK binding kinase 1.

Article Snippet: FLAG-tagged kinaseinactive IKKε K38A was a gift from Tom Maniatis (Addgene plasmid #26202). plentiCRISPR v2-Blasticidin was a gift from Mohan Babu (Addgene plasmid # 83480).

Techniques: Infection, Isolation, SDS Page, Transfection, Expressing, Western Blot, Sterility, Agarose Gel Electrophoresis, Virus, Binding Assay

Journal: The Journal of biological chemistry

Article Title: SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection.

doi: 10.1016/j.jbc.2023.104925

Figure Lengend Snippet: Figure 4. SAMHD1 inhibits IKKε-mediated IFN-I signaling and disrupts IRF7 binding to the IKKε kinase domain. A, recombinant SAMHD1 and IKKε purified from Escherichia coli and HEK293T cells, respectively, were pulled down with an anti-SAMHD1 antibody and analyzed by IB. Representative data from two independent experiments are shown. B, HEK293T cells were cotransfected with increasing amounts of plasmid encoding HA-SAMHD1, IFN-β- luciferase reporter, renilla-TK, and myc-tagged IKKε. Dual luciferase assay was performed at 24 h posttransfection, and cell lysates were harvested for IB. Error bars represent mean ± SD. Statistical significance was determined using one-way ANOVA; ***p < 0.001 compared with the vector control in the same group. C, schematic representation of full-length (FL) human IKKε (top). Five myc-tagged C-terminal and N-terminal deletion mutants are represented by the solid bars below the FL IKKε. The aa numbers of IKKε are shown. D–F, HEK293T cells were cotransfected with plasmids encoding HA-SAMHD1 and myc- tagged IKKε FL or C-terminal deletion mutants (D), IKKε N-terminal deletion lacking the kinase domain (ΔKD) (E), or IKKε kinase-inactive mutant K38A (F). Cells were lysed, and IP was performed using anti-HA antibody at 36 h posttransfection. Nonspecific IgG was used as a negative control in IP, and the indicated proteins were detected by IB. G, HEK293T cells were cotransfected with myc-IKKε KD, FLAG-IRF7, and HA-SAMHD1. Cells were lysed, and IP was performed using anti-myc antibody at 36 h posttransfection. The indicated proteins were detected by IB. A and B and D–G, representative data from three independent experiments are shown. HLH, helix-loop-helix domain; IB, immunoblot; IFN, interferon; IKKε, inhibitor of nuclear factor kappa-B kinase epsilon; IRF, IFN regulatory factor; KD, kinase domain; LZ, leucine zipper domain; SAMHD, sterile alpha motif and HD domain-containing protein; ULD, ubiquitin-like domain; V, vector controls; WT, wild-type.

Article Snippet: FLAG-tagged kinaseinactive IKKε K38A was a gift from Tom Maniatis (Addgene plasmid #26202). plentiCRISPR v2-Blasticidin was a gift from Mohan Babu (Addgene plasmid # 83480).

Techniques: Binding Assay, Recombinant, Plasmid Preparation, Luciferase, Control, Mutagenesis, Negative Control, Western Blot, Sterility, Ubiquitin Proteomics

Journal: The Journal of biological chemistry

Article Title: SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection.

doi: 10.1016/j.jbc.2023.104925

Figure Lengend Snippet: Figure 10. SAMHD1 impairs IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection. SAMHD1 inhibits MAVS- and IKKε-mediated IFN-I induction in monocytic cells. Mechanistically, SAMHD1 interacts with the CARD of MAVS and suppresses MAVS aggre- gation, resulting in reduced IKKε recruitment to MAVS and IKKε phos- phorylation (indicated with a letter P). SAMHD1 also binds to the kinase domain of IKKε and disrupts binding of IRF7 to IKKε KD. SAMHD1 sup- pression of the IRF7-mediated antiviral response depends on its interaction with the inhibitory domain of IRF7. Our results also indicated that endog- enous IRF7 in THP-1 cells is required for efficient HIV-1 infection and viral gene expression. The red T-shaped symbols indicate inhibitory functions confirmed in experiments, while the black T-shaped symbols indicate pro- posed inhibitory mechanisms. This figure was created with BioRender.com. CARD, caspase-recruitment domain; HIV, human immunodeficiency virus; IFN, interferon; IKKε, inhibitor of nuclear factor kappa-B kinase epsilon; IRF, IFN regulatory factor; KD, kinase domain; MAVS, mitochondrial antiviral- signaling protein; SAMHD, sterile alpha motif and HD domain-containing protein.

Article Snippet: FLAG-tagged kinaseinactive IKKε K38A was a gift from Tom Maniatis (Addgene plasmid #26202). plentiCRISPR v2-Blasticidin was a gift from Mohan Babu (Addgene plasmid # 83480).

Techniques: Infection, Binding Assay, Gene Expression, Virus, Sterility

Journal: The Journal of Biological Chemistry

Article Title: SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection

doi: 10.1016/j.jbc.2023.104925

Figure Lengend Snippet: SAMHD1 inhibits IKKε-mediated IFN-I signaling and disrupts IRF7 binding to the IKKε kinase domain. A , recombinant SAMHD1 and IKKε purified from Escherichia coli and HEK293T cells, respectively, were pulled down with an anti-SAMHD1 antibody and analyzed by IB. Representative data from two independent experiments are shown. B , HEK293T cells were cotransfected with increasing amounts of plasmid encoding HA-SAMHD1, IFN-β-luciferase reporter, renilla-TK, and myc-tagged IKKε. Dual luciferase assay was performed at 24 h posttransfection, and cell lysates were harvested for IB. Error bars represent mean ± SD. Statistical significance was determined using one-way ANOVA; ∗∗∗ p < 0.001 compared with the vector control in the same group. C , schematic representation of full-length (FL) human IKKε ( top ). Five myc-tagged C-terminal and N-terminal deletion mutants are represented by the solid bars below the FL IKKε. The aa numbers of IKKε are shown. D – F , HEK293T cells were cotransfected with plasmids encoding HA-SAMHD1 and myc-tagged IKKε FL or C-terminal deletion mutants ( D ), IKKε N-terminal deletion lacking the kinase domain (ΔKD) ( E ), or IKKε kinase-inactive mutant K38A ( F ). Cells were lysed, and IP was performed using anti-HA antibody at 36 h posttransfection. Nonspecific IgG was used as a negative control in IP, and the indicated proteins were detected by IB. G , HEK293T cells were cotransfected with myc-IKKε KD, FLAG-IRF7, and HA-SAMHD1. Cells were lysed, and IP was performed using anti-myc antibody at 36 h posttransfection. The indicated proteins were detected by IB. A and B and D – G , representative data from three independent experiments are shown. HLH, helix-loop-helix domain; IB, immunoblot; IFN, interferon; IKKε, inhibitor of nuclear factor kappa-B kinase epsilon; IRF, IFN regulatory factor; KD, kinase domain; LZ, leucine zipper domain; SAMHD, sterile alpha motif and HD domain-containing protein; ULD, ubiquitin-like domain; V, vector controls; WT, wild-type.

Article Snippet: FLAG-tagged kinase-inactive IKKε K38A was a gift from Tom Maniatis (Addgene plasmid #26202). plentiCRISPR v2-Blasticidin was a gift from Mohan Babu (Addgene plasmid # 83480).

Techniques: Binding Assay, Recombinant, Purification, Plasmid Preparation, Luciferase, Mutagenesis, Negative Control, Western Blot