STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

Abstract

STING activation by cyclic dinucleotides induces IRF3- and NF-κB-mediated gene expression in mammals, as well as lipidation of LC3B at Golgi-related membranes. While mechanisms of the IRF3 response are well understood, the mechanisms of NF-κB activation via STING remain unclear. We report here that STING activation induces linear/M1-linked ubiquitin chain (M1-Ub) formation and recruitment of the LUBAC E3 ligase, HOIP, to LC3B-associated Golgi membranes where ubiquitin is also localized. Loss of HOIP prevents formation of M1-Ub chains and reduces STING-induced NF-κB and IRF3 signaling in human THP1 monocytes and mouse bone marrow-derived macrophages, without affecting STING activation. STING-induced LC3B lipidation is not required for M1-Ub chain formation or for immune-related gene expression, but the recently reported STING function in neutralizing Golgi pH may be involved. Thus, LUBAC synthesis of M1-linked ubiquitin chains mediates STING-induced innate immune signaling.

Keywords: Golgi; Innate Immunity; LC3B; LUBAC; NFkB.

Figure 1. STING activation induces M1 and K63-ubiquitin chain formation.

(A) Representative Airyscan-processed confocal images of wild-type (WT) HeLa cells stably expressing BFP-P2A-STING (HeLa^STING) and mEGFP-LC3B (green) treated with 60 µg/mL of cGAMP for 8 h prior to PFA-fixation and immunostaining with antibodies raised against mono- and poly-ubiquitin chains (Ub; magenta) and STING (cyan). Scale bar = 10 and 2 µm (inset). Imaging was replicated in >3 independent experiments. (B) Quantification of the percentage (%) of cells positive for mEGFP-LC3B foci and immunostained Ub foci (left panel), and the percentage (%) of mEGFP-LC3B foci with overlapping immunolabeled signal for Ub, STING, or both (right panel) from experiments represented in Fig. EV1A, and similar conditions to Fig. 1A. Mean ± s.d. from n = 3 replicates analyzed in the same experiment. Imaging was replicated in three independent experiments. (C–F) Representative immunoblots of indicated proteins detected in HeLa^STING cell lysates prepared after treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in three independent experiments. (G) Quantification of immunoblots in (D–F). Values represent the relative intensity of cGAMP treated to untreated lanes. Error bars represent the mean ± s.d. of three independent experiments. (H) Quantification of ubiquitin-GG linked peptides from lysate (left) and Pan-TUBE (tandem-ubiquitin-binding entities) enriched samples (right) identified by targeted LC-MS/MS. HeLa^STING cells stably expressing mEGFP-LC3B were treated with 120 µg/mL of cGAMP for 8 h prior to cell lysis, Pan-TUBE enrichment, and LC-MS/MS analysis. Values represent the relative amount of peptides detected in cGAMP-treated cells over untreated cells. (Lysates) Mean ± s.d. from n = 2 independent experiments with 3–5 technical replicates each; (Pan-TUBEs) Mean ± s.d. from n = 1 of the same cell lysates analyzed in the lysate panel with three technical replicates. (I) Representative Airyscan-processed confocal images of HeLa^STING; mEGFP-LC3B (green) cells treated with 100 µM Monensin for 1 h prior to PFA-fixation and immunostaining with antibodies raised against mono- and poly-ubiquitin chains (Ub; magenta). Scale bar = 10 and 2 µm (inset). Imaging was replicated in two independent experiments. (J, K) Representative immunoblots for indicated linkage-specific ubiquitin chains in HeLa^STING cell lysates prepared after treatment with 100 µM Monensin for 1 h. Immunoblotting was replicated in three independent experiments. (L) Quantification of immunoblots in (J, K). Values represent the relative intensity of Monensin treated to untreated lanes. Error bars represent the mean ± s.d. of three independent experiments. Source data are available online for this figure.

Figure 2. HOIP mediates STING activation-induced M1 ubiquitin chain formation.

(A) Representative Airyscan-processed confocal images of HeLa^STING cells stably expressing mScarletI-LC3B (magenta) and mEGFP-HOIP (green) treated with 120 µg/mL of cGAMP for 8 h prior to saponin extraction and PFA-fixation. Scale bar = 10 and 2 µm (inset). Imaging was replicated in three independent experiments. Representative images from non-saponin extracted cells are in Fig. EV2A. (B) Quantification of the percentage (%) of mEGFP-LC3B foci with overlapping immunolabeled signal for HOIP, or HOIP and STING, from experiments represented in Fig. EV2B, and corresponding conditions to Fig. 2A. Mean ± s.d. from n = 3 replicates analyzed in the same experiment. Imaging was replicated in three independent experiments. Representative images are in Fig. EV2B. (C) Representative immunoblots of indicated proteins detected in HeLa^STING cell lysates from WT, HOIPKO, and HOIPKO stably expressing untagged HOIP prepared following treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in three independent experiments. (D) Quantification of M1-Ub in (C). Values represent the relative intensity of corresponding lanes to WT untreated lanes. Error bars represent the mean ± s.d. of three independent experiments. (E) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT, HOIPKO^pool, and HOIPKO^pool stably expressing untagged HOIP prepared following treatment with 1 µM diABZI for 4 h. Immunoblotting was replicated in three independent experiments. (F) Quantification of M1-Ub in (E). Values represent the relative intensity of corresponding lanes to WT untreated lanes. Error bars represent the mean ± s.d. of three independent experiments. (G) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and HOIPKO^pool cells prepared following treatment with 1 µM diABZI for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments. (H) Representative immunoblots of indicated proteins detected in iBMDM cell lysates from shCtrl and shHOIP cells prepared following treatment with 0.2 µM diABZI for 1, 2, and 4 h. Immunoblotting was replicated in three independent experiments. (I) Quantification of M1-Ub in (H). Values represent the relative intensity of corresponding lanes to untreated lanes per cell line. Error bars represent the mean ± s.d. of three independent experiments. Source data are available online for this figure.

Figure 3. Loss of STING-mediated VAIL disrupts the perinuclear localization of ubiquitin and HOIP, but not M1-Ub chain formation.

(A) Representative immunoblots of indicated proteins detected lysates from HeLa^STING WT and HeLa^STING with stable overexpression of mEGFP-SopF prepared after treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in 3 independent experiments. (B) Quantification of M1-Ub in (A). Values represent the relative intensity of lanes to WT cGAMP-treated lanes. Error bars represent the mean ± s.d. of three independent experiments. (C) Representative Airyscan-processed confocal images of WT HeLa^STING cells stably expressing mScarletI-LC3B alone or with stable expression of mEGFP-SopF treated with 120 µg/mL of cGAMP for 8 h prior to PFA-fixation and immunostaining with antibodies raised against mono- and poly-ubiquitin chains (Ub). Scale bar = 20 µm. Imaging was replicated in three independent experiments. (D) Representative immunoblots of indicated proteins detected lysates from HeLa^STING WT and ATG16L1KO cells prepared after treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in three independent experiments. (E) Quantification of M1-Ub in (D). Values represent the relative intensity of lanes to WT cGAMP-treated lanes. Error bars represent the mean ± s.d. of three independent experiments. (F) Representative spinning disk confocal images of HeLa^STING; mEGFP-HOIP (green); mScarletI-LC3B (orange) cells treated with 120 µg/mL of cGAMP for 8 h prior to prior to saponin extraction, PFA-fixation, and immunostaining for ubiquitin (Ub; magenta). Scale bar = 20 µm. (G) Quantification and distribution of the number and size of HOIP (top) and Ubiquitin punctae (bottom) in the experiment represented in (F). Large “punctae” can be interpreted as “foci”. Imaging was replicated in three independent experiments. Source data are available online for this figure.

Figure 4. M1 ubiquitin chains stimulate STING-mediated NFκB-related immune signaling.

(A) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and HOIPKO^pool cells prepared following treatment with 1 µM diABZI for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments. (B) Quantification of pIκBα^S32 in (A). Values represent the relative intensity of corresponding bands to untreated bands per cell line. Replicates for each condition from two independent experiments are shown. (C) Quantification of pIκBα^S32 in (D). Values represent the relative intensity of bands to WT-treated bands. Error bars represent the mean ± s.d. of three independent experiments. (D) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT, HOIPKO^pool, and HOIPKO^pool stably expressing untagged HOIP prepared following treatment with 1 µM diABZI for 4 h. Immunoblotting was replicated in three independent experiments. (E, F) Relative expression changes of indicated NFκB- (E) and IRF3/interferon-related (F) genes detected by quantitative RT-PCR in WT and HOIPKO^pool THP1 cells treated with 1 µM diABZI for 1, 2, 4, and 8 h. Quantification of relative expression is from three independent experiments analyzed at the same time. A two-way ANOVA with Sidak’s multiple comparisons test was performed on 2^−ΔΔCt values. Mean ± s.d. n = 3 * < 0.05, ***<0.001, ****<0.0001 (TNF 6 h p = <0.0001, 8 h p = 0.0006; TNFAIP3 6 h p = 0.0001, 8 h p = <0.0001; IL6 6 h p = 0.0117, 8 h p = <0.0001; IFNB1 2 h p = 0.0135, 4 h p = <0.0001, 6 h p = <0.0001). (G, H) Relative expression changes of indicated NFκB- (G) and IRF3/interferon-related (H) genes detected by quantitative RT-PCR in THP1 WT, HOIPKO^pool, and HOIPKO^pool stably expressing untagged HOIP cells treated with 1 µM diABZI for 4 h. Quantification of relative expression is from four independent experiments analyzed at the same time. A two-way ANOVA with Tukey’s multiple comparisons test was performed on 2^−ΔΔCt values. Mean ± s.d. n = 4 *<0.05, **<0.01, ****<0.0001 (TNF WTvHOIPKO^pool p = 0.0019, HOIPKO^poolvHOIPKO^pool + HOIP p = <0.0001; TNFAIP3 WTvHOIPKO^pool p = 0.0333, HOIPKO^poolvHOIPKO^pool + HOIP p = 0.0014; IL6 WTvHOIPKO^pool p = 0.0349, HOIPKO^poolvHOIPKO^pool + HOIP p = <0.0001; IFNB1 WTvHOIPKO^pool p = 0.05, HOIPKO^poolvHOIPKO^pool + HOIP p = <0.0001; IFIT3 WTvHOIPKO^pool p = <0.0001, HOIPKO^poolvHOIPKO^pool + HOIP p = <0.0001; ISG15 HOIPKO^poolvHOIPKO^pool + HOIP p = <0.0001). Source data are available online for this figure.

Figure 5. LC3B lipidation is not required for STING-mediated innate immune responses.

(A) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and ATG16L1KO cells prepared following treatment with 1 µM diABZI for 1, 2, and 4 h. Immunoblotting was replicated in three independent experiments. (B, C) Relative expression changes of indicated NFκB- (B) and IRF3/interferon-related (C) genes detected by quantitative RT-PCR in WT and ATG16L1KO THP1 cells treated with 1 µM diABZI for 2, 4, 6, and 8 h. Quantification of relative expression is from three independent experiments analyzed at the same time. A two-way ANOVA with Sidak’s multiple comparisons test was performed on 2^−ΔΔCt values. Mean ± s.d. n = 3 *<0.05, **<0.01 (TNF 8 h p = 0.0386; TNFAIP3 8 h p = 0.0081). (D) Model for STING-induced ubiquitin chain formation at LC3B-associated Golgi membranes and NFκB signaling. Created in BioRender. Fischer, T. (2024). Source data are available online for this figure.

Figure EV1. Total-Ub and K63-Ub co-localization at LC3B foci following STING activation.

(A) Representative spinning disk confocal images of HeLa^STING; mEGFP-LC3B (green) cells treated with 120 µg/mL of cGAMP for 8 h prior to PFA-fixation and immunostaining for mono- and poly-ubiquitin chains (Ub; magenta), and STING (cyan). Scale bar = 20 µm. Corresponding to quantification in Fig. 1B. (B) Representative spinning disk confocal images of HeLa^STING; mEGFP-LC3B (green) cells treated with 1 µM diABZI for 4 h prior to PFA-fixation and immunostaining for mono- and poly-ubiquitin chains (Ub; magenta), and STING (cyan). Scale bar = 20 µm. Corresponding to quantification in Fig. EV1C. (C) Quantification of the percentage (%) of cells positive for mEGFP-LC3B foci and immunostained Ub foci (left panel), and the percentage (%) of mEGFP-LC3B foci with overlapping immunolabeled signal for Ub, STING, or both (right panel) from experiments represented in Fig. EV1B. Error bars represent ±s.d. from three replicates analyzed in the same experiment. Imaging was replicated in three independent experiments. (D, E) Representative spinning disk confocal images of HeLa^STING; mEGFP-LC3B cells treated with 120 µg/mL of cGAMP for 8 h prior to PFA-fixation and immunostaining for K48- and K63-ubiquitin chains. Scale bar = 20 µm. (F) Quantification of the percentage (%) of mEGFP-LC3B foci with overlapping signal for immunolabeled K48- and K63-ubiquitin chain from experiments represented in Fig. EV1D, E. Mean ± s.d. from n = 3 replicates analyzed in the same experiment. Imaging was replicated in three independent experiments. (G) Pearson’s correlation coefficient of mScarletI-LC3B and Vx3-EGFP over time in the live imaging experiment represented in Movie EV1. FRT/TREX HeLa cells stably expressing FRT/TO-DD-Vx3-EGFP, BFP-P2A-STING, and mScarletI-LC3B were incubated with 1 µg/mL Doxycycline and 500 nM Shield1 for 24 h prior to treatment with either 120 µg/mL cGAMP or 1 µM diABZI and imaging every 30 min for 12 h on a spinning disk confocal microscope. Error bars represent ± s.d. from three replicates analyzed in the same experiment. Imaging was replicated in three independent experiments.

Figure EV2. STING activation by multiple agonists induces HOIP-mediated M1 ubiquitin chain formation, which is not required for STING degradation or LC3B lipidation in HeLa or THP1 cells.

(A) Representative Airyscan-processed confocal images of HeLa^STING cells stably expressing mScarletI-LC3B and mEGFP-HOIP treated with 120 µg/mL of cGAMP for 8 h with no saponin extraction prior to PFA-fixation. Scale bar = 10 µm. Corresponding to representative images in Fig. 2A. Imaging was replicated in three independent experiments. (B) Representative spinning disk confocal images of HeLa^STING; mEGFP-HOIP (green); mScarletI-LC3B (magenta) cells treated with 120 µg/mL of cGAMP for 8 h prior to prior to saponin extraction and PFA-fixation. Scale bar = 20 µm. Corresponding to quantification in Fig. 2B. (C, D) Representative immunoblots of indicated proteins detected in cell lysates from HeLa^STING WT and HOIPKO cells (C) or HeLa^STING and HeLa^STING stably expressing mEGFP-OTULIN (D) prepared following treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in three independent experiments. (E, F) Representative immunoblots of indicated proteins detected in HeLa^STING cell lysates from WT and HOIPKO cells prepared following treatment with 120 µg/mL cGAMP (E) or 1 µM diABZI (F) for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments. (G) Representative immunoblots of endogenous STING were detected in cell lysates from WT and HOIPKO HeLa cells without stable overexpression of STING. Cells were treated with 15 µg/mL cGAMP for 8 h. Immunoblotting was replicated in three independent experiments. (H) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and HOIPKO^pool cells prepared following treatment with 120 µg/mL cGAMP for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments.

Figure EV3. Overexpression of LC3B affects STING activation-induced ubiquitylation, which may involve the Golgi neutralizing function of STING.

(A) Representative Airyscan-processed confocal images of HeLa^STING and HeLa^STING cells with stable overexpression of mEGFP-LC3B (green) treated with 120 µg/mL of cGAMP for 8 h prior to PFA-fixation and immunostaining with antibodies raised against mono- and poly-ubiquitin chains (Ub; magenta) and STING (cyan). Scale bar = 20 and 2 µm (inset). Imaging was replicated in two independent experiments. (B) Representative immunoblots of indicated proteins detected lysates from HeLa^STING WT and HeLa^STING with stable overexpression of mEGFP-LC3B prepared after treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in 3 independent experiments. (C) Representative immunoblots of indicated proteins detected in HeLa^STING cell lysates prepared after treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Immunoblotting was replicated in three independent experiments. (D, E) Representative spinning disk confocal images of FRT/TREX HeLa cells stably expressing FRT/TO-DD-Vx3-EGFP, BFP-P2A-STING, and mScarletI-LC3B at the 6-hour timepoint following treatment (D) and quantification of the percentage (%) of cells positive for Vx3-EGFP foci over time (E). Cells were incubated with 1 µg/mL Doxycycline and 500 nM Shield1 for 24 h prior to treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI, and imaging every 30 min for 12 h on a spinning disk confocal microscope. Scale bar = 50 µm. Quantification is from three wells analyzed in the same experiment. Imaging was replicated in two independent experiments.

Figure EV4. HOIP is important for NFκB signaling following STING activation by cGAMP and in mouse bone marrow-derived macrophages.

(A) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and HOIPKO^pool cells prepared following treatment with 120 µg/mL cGAMP for 1, 2, 4, and 8 h. Immunoblotting was replicated in three independent experiments. (B, C) Relative expression changes of indicated NFκB- (B) and IRF3/interferon-related (C) genes detected by quantitative RT-PCR in WT and HOIPKO^pool THP1 cells treated with 120 µg/mL cGAMP for 1, 2, 4, and 8 h. Quantification of relative expression is from three independent experiments analyzed at the same time. A two-way ANOVA with Sidak’s multiple comparisons test was performed on 2^−ΔΔCt values. Mean ± s.d. n = 3 *<0.05, **<0.01, ***<0.001, ****<0.0001 (TNF 6 h p = <0.0001, 8 h p = <0.0001; TNFAIP3 4 h p = 0.0084, 6 h p = <0.0001, 8 h p = <0.0001; IL6 6 h p = <0.0001, 8 h p = <0.0001; IFNB1 2 h p = 0.0019, 4 h p = 0.0005, 8 h p = 0.0281). (D, E) Relative expression changes of indicated NFκB- (D) and IRF3/interferon-related (E) genes detected by quantitative RT-PCR in WT and shCtrl and shHOIP iBMDM cells treated with 0.2 µM diABZI for 1, 2, and 4 h. Quantification of relative expression is from three independent experiments analyzed at the same time. A two-way ANOVA with Sidak’s multiple comparisons test was performed on 2^−ΔΔCt values. Mean ± s.d. n = 3 **<0.01, ***<0.001, ****<0.0001 (Tnf 2 h p = <0.0001, 4 h p = 0.0005; Tnfaip3 2 h p = <0.0001, 4 h p = <0.0001; Il6 4 h p = <0.0001; Ifnb1 2 h p = 0.0011, 4 h p = 0.0031; Ifit3 4 h p = <0.0001; Isg15 4 h p = <0.0001).

Figure EV5. ATG16L1 is not required for IRF3- and NFκB-related signaling induced by STING activation, however the upstream Golgi neutralizing function of STING may affect immune-related gene expression.

(A) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and ATG16L1KO cells prepared following treatment with 1 µM diABZI for 1, 2, and 4 h. Immunoblotting was replicated in three independent experiments. (B) Representative immunoblots of indicated proteins detected in lysates from WT THP1 cells prepared following treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Immunoblotting was replicated in three independent experiments. (C, D) Relative expression changes of indicated NFκB-related genes (C) and interferon-related genes (D) detected by quantitative RT-PCR in THP1 cells treated with DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Quantification of relative expression is from four independent experiments analyzed at the same time. A one-way ANOVA with Tukey’s multiple comparisons test was performed on 2^−ΔΔCt values. Mean ± s.d. n = 4 *<0.05, **<0.01, ****<0.0001 (TNF p = 0.0123; TNFAIP3 p = 0.005; IL6 p = 0.0027; IFNB1 p = <0.0001; ISG15 p = 0.0351).