Fig 1: The CBM complex is linearly ubiquitinated upon TCR stimulation. (A) Parental Jurkat cells were stimulated with 20 ng/ml PMA and 150 ng/ml ionomycin for the indicated time periods. The heat-denatured cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Taking the maximum intensities of linear ubiquitination as 100%, the relative intensities of the linear ubiquitinated CBM complex are indicated. Means ±SD (n = 3). (B) HOIP is required for linear ubiquitination of MALT1. Parental and RNF31-KO Jurkat cells were stimulated with PMA/ionomycin as in (A), immunoprecipitated with anti-MALT1, and immunoblotted with the indicated antibodies. (C) Suppressed linear ubiquitination of NEMO in the TCR-mediated NF-κB activation pathway. Jurkat cells were stimulated with 20 ng/ml PMA and 150 ng/ml ionomycin or 1 µg/ml TNF-α for the indicated time periods, and cell lysates were immunoprecipitated with an anti-NEMO antibody and then immunoblotted with the depicted antibodies. (D) MALT1 is exclusively linearly ubiquitinated upon TCR stimulation. A similar analysis to that in Figure 4C was performed after anti-MALT1 immunoprecipitation. (E) Linear ubiquitination of MALT1 after stimulation with CD3 and CD28. Jurkat cells were stimulated with 5 µg/ml each of anti-CD3 and anti-CD28 antibodies as indicated. The cell lysates and anti-MALT1 immunoprecipitates were immunoblotted by the indicated antibodies. (F) Transient activation of canonical IKK. Jurkat cells were stimulated with 20 ng/ml PMA and 150 ng/ml ionomycin for the indicated time periods. After immunoprecipitation with an anti-NEMO antibody, an in vitro canonical IKK assay was performed using GST-IκBα1–54 as the substrate. Samples were immunoblotted with the indicated antibodies, and taking the maximum intensities of P-IκBα as 100%, the relative intensities are indicated. Means ±SD (n = 3). (A, B, E) *; nonspecific signal.
Fig 2: STAT1-K511/652 mutation enhances IFN-I antiviral activity.a Diagram of bone marrow chimeras using wild-type BALB/c mice as recipients and Stat1−/− (129S6) mice as donors. b The bone marrow was obtained from BALB/c mice that were irradiated and injected (i.v.) with the Stat1−/− bone marrow with lentiviral packaging GFP-STAT1 (WT or K511/652 R), followed by administration with mIFNβ (1500 IU/g, i.p.) for 8 h. Immunoprecipitation and western blot were performed to analyze pY701-STAT1 and GFP-STAT1 levels. c RT-qPCR analysis of the representative ISGs (Ifit1 and Rsad2) mRNA in the bone marrow from recipient mice treated as b. d Linear ubiquitination of STAT1 restricts the interaction between STAT1 and IFNAR2, thus inhibiting STAT1 activation and maintaining homeostasis of IFN signaling. Viral infection rapidly induces production of IFNs, which utilize OTULIN to remove linear ubiquitination of STAT1, thus promoting STAT1 activation. During the late stage of viral infection, HOIP expression is upregulated through the NF-κB signaling pathway, which increases STAT1 linear ubiquitination and inhibits host IFN antiviral response. ***p < 0.001 (two-tailed unpaired Student’s t test). All graphs show the mean ± SEM for six individual mice (c). Data are representative of three independent experiments (b).
Fig 3: STAT1 harbors linear ubiquitination mediated by HOIP.a Mass spectrometry analysis of the potential Flag-STAT1-binding proteins. The number of the identified peptides from the interacting proteins, including HOIP (RNF31), were shown as indicated. CON: control vectors. b Immunoprecipitation (IP) and immunoblotting (IB) analysis of the interaction between Flag-HOIP and Myc-STAT1 in HEK293T cells. c Immunofluorescence analysis of the interaction between Flag-HOIP and Myc-STAT1 in HeLa cells. Scale bar: 1 µm. d Immunoprecipitation analysis of the interaction between endogenous STAT1 and HOIP in mouse primary liver, spleen, and lung cells. e Immunoprecipitation analysis of linear ubiquitination of endogenous STAT1 in HEK293T cells transfected with either control shRNAs (–) or two different shRNAs against HOIP (shHOIP #1, #2). Ub ubiquitination. f Immunoprecipitation analysis of linear ubiquitination of STAT1 in HEK293T cells cotransfected with Flag-HOIL-1L and Flag-Sharpin, together with Flag-HOIP wild-type or its catalytically inactivated mutants (C885A and C916A). g Immunoprecipitation analysis of linear ubiquitination of endogenous STAT1 in HOIP-wild-type (WT) or HOIP-knockout (KO) HEK293T cells. Data are representative of three independent experiments.
Fig 4: Linear ubiquitination sustains STAT1 signaling homeostasis.a Western blot analysis of endogenous STAT1 in HEK293T cells transfected with Flag-LUBAC. b Western blot analysis of endogenous STAT1 in HEK293T cells transfected with control shRNAs (–) or shHOIP (#1, #2). c Western blot analysis of pY701-STAT1 (pY-STAT1) levels in HEK293T cells transfected with increasing amounts of Flag-LUBAC. d Western blot analysis of pY-STAT1 in HOIP-WT or HOIP-KO HEK293T cells treated with IFNα (1000 IU/ml). e Western blot analysis of pY-STAT1 in HEK293T cells cotransfected with Flag-Sharpin and Flag-HOIL-1L, together with Flag-HOIP wild-type or its catalytically inactivated mutants (C885A and C916A), and then treated with IFNα (1000 IU/ml) for 30 min. f Dual-luciferase reporter assay of the ISRE activity in HEK293T cells cotransfected with control shRNAs (shCON) or shHOIP (#1, #2), together with ISRE luciferase and Renilla, and then stimulated with IFNα (1000 IU/ml) for 20 h. g RT-qPCR analysis of the representative ISG (Ifit1) mRNA in HEK293T cells transfected with either shCON or shHOIP (#1, #2), and then stimulated with IFNα or IFNβ (1000 IU/ml) for 4 h. h RT-qPCR analysis of Ifit1 mRNA in HOIP-WT or HOIP-KO HEK293T cells treated with IFNβ (1000 IU/ml) for 4 h. i RT-qPCR analysis of VSV viral RNA in 2fTGH cells transfected with shHOIP and then stimulated with IFNα (60 IU/ml) for 20 h, followed by infection with VSV (MOI = 0.1) for 24 h. j Western blot analysis of VSV-encoded proteins VSV-G in HeLa cells transfected with Flag-LUBAC and then treated as i. k RT-qPCR analysis of IFN (Ifnɑ, Ifnγ, and Ifnλ) mRNA in HEK293T cells transfected with LUBAC. l Immunoprecipitation analysis of pY-STAT1 in HEK293T cells transfected with shCON (–) or shHOIP (#1, #2). m RT-qPCR analysis of a representative ISG (Isg54) mRNA in HEK293T cells transfected with shHOIP (#1, #2). N.S., not significant (p > 0.05) and *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t test). Data are shown as mean and s.d. of three biological replicates (f–i, k, m), or are representative of three independent experiments (a–e, j, l).
Fig 5: Viruses upregulate HOIP and STAT1 linear ubiquitination.a RT-qPCR analysis of HOIP mRNA in HT1080 cells challenged with SeV (MOI = 1.0) for the indicated times. b RT-qPCR analysis of HOIP mRNA in HT1080 cells infected with SeV (MOI = 1.0) and then treated with a NF-κB inhibitor PDTC (10 µM) as indicated. c RT-qPCR analysis of HOIP mRNA in HT1080 cells stimulated with IFNα (1000 IU/ml) as indicated. d Western blot analysis of HOIP proteins in HEK293T cells infected with VSV (MOI = 0.1) as indicated. e Immunoprecipitation analysis of the interaction between HOIP and STAT1 in 2fTGH cells infected with VSV (MOI = 0.1) as indicated. f Immunoprecipitation analysis of linear ubiquitination of STAT1 in 2fTGH cells infected with VSV (MOI = 0.1 and 0.5) for 9 h. g Immunoprecipitation analysis of linear ubiquitination of STAT1 in HT1080 cells infected with SeV (MOI = 1.0) as indicated. h Immunoprecipitation analysis of linear ubiquitination of STAT1 in HOIP-WT or HOIP-KO HEK293T cells infected with or without VSV (MOI = 0.1) for 9 h. i Immunoprecipitation analysis of linear ubiquitination of STAT1 in HEK293T cells transfected with Myc-STAT1 (WT or DM) and then infected with VSV (MOI = 0.1) as indicated. j Immunoprecipitation analysis of linear ubiquitination of STAT1 and RT-qPCR analysis of Ifnβ mRNA in 2fTGH cells infected with SeV for the indicated times. k Western blot analysis of pY-STAT1 in HOIP-WT or HOIP-KO HEK293T cells infected with VSV (MOI = 0.1) for 12 h. l RT-qPCR analysis of Ifit1 mRNA in U3A cells transfected with Myc-STAT1 (WT or DM) and then infected with SeV (MOI = 1.0) as indicated. N.S., not significant (p > 0.05) and *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t test). Data are shown as mean and s.d. of three biological replicates (a–c, l), or are representative of three independent experiments (d–k).
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