Synergistic involvement of the NZF domains of the LUBAC accessory subunits HOIL-1L and SHARPIN in the regulation of LUBAC function

Figures

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  Fig. 1. The ubiquitin-binding activity of HOIL-1L NZF preferentially contributes to NF-κB activation. A Schematic illustration of the domains of LUBAC subunits. B Immunoblot analyses of lysates of WT MEFs and HOIL-1L-null MEFs stably reconstituted with the indicated proteins. Data are representative of three independent experiments. C, E WT MEFs and HOIL-1L-null MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (1 ng/mL) (C) or TNF-α (2.5 ng/mL) plus CHX (20 μg/mL) (E) for the indicated times, and analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments. D HOIL-1L-null MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (1 ng/mL), and expression of NF-κB target genes was measured by quantitative PCR. All gene expression levels are normalized against the corresponding levels of ACTB and expressed as fold changes relative to cells with an empty vector at 0 h. Data are shown as the mean ± s.d. of three independent experiments. P values were calculated by a one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. F HOIL-1L-null MEFs…

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  Fig. 2. The NZF domains in HOIL-1L and SHARPIN cooperatively regulate NF-κB activation mainly by binding to linear ubiquitin chains. A, F Immunoblot analyses of lysates of WT MEFs and HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins. Data are representative of three independent experiments. B, G HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (5 ng/mL) for the indicated times and analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments. C, H NF-κB activation in HEK293T cells transiently transfected with the indicated combinations of HOIP, HOIL-1L, and SHARPIN was measured using a luciferase assay. Data are shown as the mean ± s.d. of four (C) or three (H) independent experiments. P values were calculated by one-way ANOVA with Tukey’s post hoc test. D HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (5 ng/mL), and expression of NF-κB target genes was measured by quantitative PCR. All gene expression levels are normalized against the corresponding levels of ACTB and expressed as fold changes relative to…

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  Fig. 3. The NZF domain of HOIL-1L contributes to LUBAC-mediated cell death protection in concert with the NZF domain of SHARPIN. A, F HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (2.5 ng/mL) and CHX (20 μg/mL) for the indicated times and analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments. B HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (2.5 ng/mL) and CHX (20 μg/mL) for the indicated times after pretreatment with Z-VAD-FMK (10 μM) for 1 h, and analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments. C, D, G, H HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (2.5 ng/mL) and CHX (20 μg/mL), and cell viability was continuously measured using the xCELLigence system. A representative image (C, G) and statistical analyses of the normalized cell index at 6 h (D, H). Data are shown as the mean ± s.d. of five independent experiments. P values were calculated by a one-way ANOVA with Tukey’s post hoc test. E…

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  Fig. 4. Screening of HOIL-1L NZF inhibitors. A, B Schematic illustration of the TR-FRET assay for detection of GST-tagged HOIL-1L NZF and His-tagged linear di-ubiquitin interactions (A). Counter TR-FRET assay with 6His-tagged GST protein (B). C, D, E Microscale thermophoresis (MST) assay of binding between HOIL-1L NZF and linear di-ubiquitin in the presence of compounds (C) and between compounds and linear di-ubiquitin (D). Correlation between C and D (E). F, G JR-GFP cells were treated with DMSO or the indicated compounds (10 μM) for 1 h and then stimulated with TNF-α (5 ng/mL) for 4 h (F) or TNF-α (5 ng/mL) plus CHX (20 μg/mL) for 6 h (G). Samples were analyzed by flow cytometry after Annexin V staining. Data are displayed as the mean fluorescence intensity (MFI) of GFP expression (F) and the frequency of Annexin V-positive cells (G). Data are shown as the mean ± s.d. of four independent experiments. P values are from a two-tailed Student’s t test.

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  Fig. 5. FSL0717 suppressed NF-κB activation by inhibiting the HOIL-1L NZF, whereas FSL0720 increased cell death by inhibiting both the HOIL-1L NZF and SHARPIN NZF. A Chemical structures of the identified compounds. B, D, E WT MEFs were stimulated with TNF-α (1 ng/mL) (B) or TNF-α (2.5 ng/mL) plus CHX (20 μg/mL) (D) for the indicated times after treatment with DMSO or the indicated compounds (10 μM) for 1 h and analyzed by immunoblotting with the indicated antibodies. Quantitative analysis of cleaved caspase-3 shown in D was performed at 6 h after stimulation (E). Data are representative of three (B) or five (D) independent experiments and are shown as the mean ± s.d. (E). P values were obtained using a one-sample t test. C HOIL-1L/SHARPIN DKO MEFs stably reconstituted with the indicated proteins were stimulated with TNF-α (1 ng/mL) for the indicated time after treatment with DMSO or the indicated compounds (10 μM), and expression of NF-κB target genes was measured by quantitative PCR. All gene expression levels are normalized against the corresponding levels of ACTB and expressed as fold changes relative to DMSO-treated cells at 0 h. Data are shown as the mean ± s.d. of three independent…