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The HOIL-1L ligase modulates immune signalling and cell death via monoubiquitination of LUBAC

Abstract

The linear ubiquitin chain assembly complex (LUBAC), which consists of HOIP, SHARPIN and HOIL-1L, promotes NF-κB activation and protects against cell death by generating linear ubiquitin chains. LUBAC contains two RING-IBR-RING (RBR) ubiquitin ligases (E3), and the HOIP RBR is responsible for catalysing linear ubiquitination. We found that HOIL-1L RBR plays a crucial role in regulating LUBAC. HOIL-1L RBR conjugates monoubiquitin onto all LUBAC subunits, followed by HOIP-mediated conjugation of linear chains onto monoubiquitin, and these linear chains attenuate the functions of LUBAC. The introduction of E3-defective HOIL-1L mutants into cells augmented linear ubiquitination, which protected the cells against Salmonella infection and cured dermatitis caused by reduced LUBAC levels due to SHARPIN loss. Our results reveal a regulatory mode of E3 ligases in which the accessory E3 in LUBAC downregulates the main E3 by providing preferred substrates for autolinear ubiquitination. Thus, inhibition of HOIL-1L E3 represents a promising strategy for treating severe infections or immunodeficiency.

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Fig. 1: HOIL-1L E3 activity negatively regulates LUBAC functions, including suppression of apoptosis and NF-κB activation.
Fig. 2: HOIL-1L E3 activity negatively regulates the generation of linear ubiquitin chains by LUBAC.
Fig. 3: Autoubiquitination of HOIL-1L inhibits LUBAC functions by suppressing the generation of linear ubiquitin chains.
Fig. 4: HOIL-1L E3 activity negatively regulates LUBAC functions in vitro.
Fig. 5: HOIL-1L ubiquitinates all LUBAC subunits followed by HOIP-mediated linear ubiquitination onto monoubiquitin, and OTULIN counteracts this effect.
Fig. 6: Defective HOIL-1L E3 activity protects against S. typhimurium infections by activating LUBAC.
Fig. 7: Generation of HOIL-1L-E3-defective ΔRING1 mice.
Fig. 8: Pathophysiological roles of HOIL-1L-E3-defective mice.

Data availability

Source data for Figs. 18 and Extended Data Figs. 15, 7 and 8 have been provided as statistical source data or unprocessed blots. MS raw data have been deposited in the ProteomeXchange database with the accession code PXD018038. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Y. Takeda, T. Jo and Y. Sasaki for insightful discussion, T. Nakagawa and J. Matsuhiro for protein purification, Y. Sugahara and Y. Hayamizu for animal care, and N. Ueno for technical assistance. This study was supported by the following: JSPS KAKENHI grant numbers 24112002, 25253019, JP17H06174 and JP18H05499 (to K.I.); JSPS KAKENHI grant number JP17K08786, a Grant for Joint Research Project of the Institute of Medical Science from the University of Tokyo, and the Hakubi-project grant, Kyoto University (to M.K.); and JSPS KAKENHI grant number JP18H05498 (to F.O.).

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Contributions

Y.F., H.F. and K.I. conceived and designed the project. Y.F. performed most of the experiments. M.K. and A.N. performed the bacterial infection experiments. F.O., Y.S. and K.T. performed the MS experiments. K.S. performed immunostaining of lungs of mice and generation of M1-TUBE. R.T. provided advice on the project. Y.F. and K.I. wrote the manuscript with contributions from all other authors.

Corresponding author

Correspondence to Kazuhiro Iwai.

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Extended data

Extended Data Fig. 1 The highly conserved HOIL-1L RBR ubiquitin ligase negatively regulates LUBAC functions.

a, Conserved residues in the RING1-IBR-RING2 domains of HOIL-1L in the indicated species. Arrow indicates the catalytic cysteine of HOIL-1L. b, Schematic representation of mouse HOIL-1L and its mutants. Cleavage of caspase-3 in LUBAC TKO MEFs stably reconstituted with HOIP and the indicated HOIL-1L protein, stimulated with TNF-α (3 ng ml-1) and CHX (20 μg ml-1), was assessed by immunoblotting. The experiments were repeated twice, independently, with similar results. c, Viability of LUBAC TKO MEFs stably reconstituted with HOIP, SHARPIN, and the indicated HOIL-1L protein, stimulated with TNF-α (10 ng ml-1), was measured using the iCELLigence system. The experiments were repeated three times, independently, with similar results. d, NF-κB activation in HEK293T cells transfected with the indicated expression plasmids and 5× NF-κB luciferase reporters was measured by luciferase assay. Mean is shown; n=3 independent experiments; P-values are from a two-tailed Student’s t-test. e, Quantitative PCR (qPCR) analyses of expression levels of NF-κB target genes in LUBAC TKO MEFs stably reconstituted with HOIP, SHARPIN, and the indicated HOIL-1L protein. Mean ± S.D. is shown; n=5 independent experiments; P-values are from a two-tailed Student’s t-test. f, Phosphorylation and degradation of IκBα in LUBAC TKO MEFs stably reconstituted with HOIP and SHARPIN. The experiments were repeated three times, independently, with similar results. Statistical source data are provided in Source Data Extended Data Fig. 1 and unprocessed immunoblots are provided in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 Loss of HOIL-1L E3 does not overtly affect K48, K63, or other types of chains.

a, Amounts of the indicated types of ubiquitin chains in LUBAC TKO MEFs stably reconstituted with HOIP and SHARPIN, and the indicated HOIL-1L protein. Lysates from TKO MEFs expressing the indicated HOIL-1L proteins were probed with the indicated antibodies. The experiments were repeated three times, independently, with similar results. b, Assessment of ubiquitin linkages in lysates of LUBAC TKO MEFs stably reconstituted with HOIP, SHARPIN, and the indicated HOIL-1L protein using MS analyses. Mean is shown; n=3 independent experiments; P-values were obtained by one-way ANOVA followed by Tukey’s multiple comparison test. Statistical source data are provided in Source Data Extended Data Fig. 2 and unprocessed immunoblots are provided in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 Auto-ubiquitination of HOIL-1L inhibits LUBAC functions.

a, Lysates of LUBAC TKO MEFs stably reconstituted with HOIP C879A, SHARPIN, and the indicated HOIL-1L protein were probed as depicted. Arrow shows the upper band of HOIL-1L. b, In vitro ubiquitination reactions were performed at 37°C for 24 h with His-UBCH7 C/S to generate an oxy-ester bond between ubiquitin and UBCH7, followed by digestion of ubiquitinated UBCH7 with NH2OH or USP2cc at 37°C for 1 h. c,h,l Lysates of indicated MEFs were probed as depicted. d,i Digestion of the upper (modified) HOIL-1L signal with USP2cc or NH2OH at 37°C for 30 min anti-FLAG immunoprecipitates obtained under denaturing conditions from LUBAC TKO MEFs expressing 3×FLAG–HOIL-1L WT (d) or the All-K/R mutant (i). e,j,k, LUBAC TKO MEFs stably reconstituted with HOIP, SHARPIN and 3×FLAG-HOIL WT (e) or the All-K/R mutant (j,k) were subjected to mass spectrometry to assess ubiquitination sites in LUBAC subunits. S107 of hHOIP (e), S915 of mHOIP (j), and T955 of mHOIP (k) were identified as ubiquitination sites. f, Lysates of HEK293T cells transfected with the indicated expression plasmids were probed as indicated. g, NF-kB activation in HEK293T cells transfected with the indicated expression plasmids and 5×NF-kB luciferase reporters was assessed by luciferase assay. Mean is shown; n=3 independent experiments; P-values are from a two-tailed Student’s t-test. The data shown were repeated twice, independently, with similar results. Statistical source data are provided in Source Data Extended Data Fig. 3 and unprocessed immunoblots are provided in Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 Loss of HOIL-1L E3 activity increases LUBAC activity in vitro.

a, b, Linear ubiquitin (a) or modification of indicated HOIL-1L proteins (b) in in vitro ubiquitination assays with Petit-SHARPIN and HOIL-1L WT or the indicated mutants, as assessed by immunoblotting. The data shown were repeated three times, independently, with similar results. Unprocessed immunoblots are provided in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 HOIL-1L ubiquitinates all LUBAC subunits, HOIP conjugates linear ubiquitin onto the ubiquitin, and OTULIN counteracts this effect.

a, Modification of LUBAC subunits in WT or OTULIN KO MEFs treated or not treated with lysates containing MBP-OTULIN at 37°C for 1 h. * shows non-specific bands. The experiments were repeated three times, independently, with similar results. b, Schematic representation of HOIL-1L WT and ΔRING1 (lacking aa 251–341). c, Cleavage of caspase-8 and caspase-3 induced by TNF-a (1 ng ml-1) plus CHX (20 μg ml-1) in WT or OTULIN KO MEFs expressing the indicated HOIL-1L proteins. The experiments were repeated twice, independently, with similar results. Unprocessed immunoblots are provided in Source Data Extended Data Fig. 5. Source data

Extended Data Fig. 6 Loss of HOIL-1L E3 protects against Salmonella typhimurium infection, and infecting S. typhimurium are covered with high levels of linear ubiquitin in MEFs expressing ligase-defective HOIL-1L.

a-d, S. typhimurium infections of LUBAC TKO MEFS stably expressing HOIP, SHARPIN, and 3×FLAG–HOIL-1L WT or C458A. Microscopic images of indicated MEFs were collected 24 hours after infection. Scale bars, 500 μm (a). Confocal micrographs of LUBAC TKO MEFs stably expressing HOIP, SHARPIN, and 3×FLAG–HOIL-1L WT or C458A were infected with S. typhimurium and stained for linear ubiquitin (1E3), FLAG (M2), or Hoechst 33342 at 2 h (b), 4 h (c), and 6 h (d) post-infection. Scale bars, 5 μm (b–d). The data shown were repeated three times, independently, with similar results.

Extended Data Fig. 7 Generation of HOIL-1L ΔRING1 mice as a model for ligase-defective HOIL-1L mice.

a, LUBAC TKO MEFs stably reconstituted with the indicated proteins were probed as shown. b, NF-kB activation in HEK293T cells transfected with the indicated expression plasmids and 5× NF-kB luciferase reporters was assessed by luciferase assay. Mean ± S.D. is shown; n=6 independent experiments; P-values are from one-way ANOVA followed by Tukey’s multiple comparison test. c, Cleavage caspase-3 in LUBAC TKO MEFs stably reconstituted with the indicated proteins and stimulated with TNF-a (2.5 ng ml-1) plus CHX (20 μg ml-1) was assessed by immunoblotting. d, Level of linear ubiquitin in LUBAC TKO MEFs stably reconstituted with the indicated proteins. e, Schematic representation of conditional and deleted loci of HOIL-1L conditional ΔRING1 mice. f, Cell lysates of primary MEFs from WT and HOIL-1L ΔRING1 mice were probed as indicated. g, Indicated MEFs were immunoprecipitated with anti-SHARPIN antibody, and bound and unbound fractions were probed as indicated. h, Lysates of the indicated organs from 8-week-old littermate mice of the indicated genotypes were probed as depicted. i, Splenocytes of 15-week-old littermate mice of the indicated genotypes were examined by flow cytometry. Splenocytes were analyzed for surface expression of CD3e and CD19 (upper panels). B cells were defined as CD19+CD3e- cells and T cells were defined as CD19−CD3e+ cells. T cell subpopulations of splenocytes were analyzed for surface expression of CD4 and CD8 (lower panels). Figures exemplifying the gating strategy for flow cytometry experiments were provided in Supplementary Figure 1c. Experiments were repeated independently with similar results twice (a,c,g,h,i) or at least three times (d,f). Statistical source data are provided in Source Data Extended Data Fig. 7 and unprocessed immunoblots are provided in Source Data Extended Data Fig. 7. Source data

Extended Data Fig. 8 Pathophysiological roles of HOIL-1L ΔRING1 in mice.

a, Primary MEFs from WT and HOIL-1L ΔRING1 mice were probed as depicted after stimulation with TNF-a (5 ng ml-1) plus CHX (20 μg ml-1) for the indicated periods. b, Lysates of primary hepatocytes from 12-week-old WT or HOIL-1L ΔRING1 mice were probed as indicated. c, Cleavage of caspase-3 in primary hepatocytes from 12-week-old WT or HOIL-1L ΔRING1 mice after stimulation with TNF-a (10 ng ml-1) plus actinomycin D (Act-D) (100 ng ml-1) was assessed by immunoblotting. d, Macroscopic appearance of livers of the indicated mice at 18 weeks old. Scale bar, 1 cm. e,f, Mice of the indicated genotypes were injected intraperitoneally with LPS/D-GalN or PBS. Seven hours after injection, DNA ladder (e) or cleaved caspase-8 (f) in liver lysates of the indicated mice was probed as depicted. g, Primary keratinocytes from 7-week-old WT or HOIL-1L ΔRING1 mice were probed as indicated. h, Cleavage of caspase-3 in primary keratinocytes from 7-week-old WT or HOIL-1L ΔRING1 mice after stimulation with TNF-a (10 ng ml-1) plus CHX (20 μg ml-1) was assessed by immunoblotting. Experiments were repeated independently with similar results twice (a,c,e-h) or at least three times (b,d). Unprocessed immunoblots are provided in Source Data Extended Data Fig. 8. Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Tables

Supplementary Table 1: List of antibodies used; Supplementary Table 2: List of primers using for qRT–PCR gene expression analysis.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

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Statistical source data.

Source Data Fig. 7

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Source Data Fig. 7

Unprocessed western blots.

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Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots.

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Statistical source data.

Source Data Extended Data Fig. 2

Unprocessed western blots.

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Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed western blots.

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Unprocessed western blots.

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Unprocessed western blots.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig. 8

Unprocessed western blots.

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Fuseya, Y., Fujita, H., Kim, M. et al. The HOIL-1L ligase modulates immune signalling and cell death via monoubiquitination of LUBAC. Nat Cell Biol 22, 663–673 (2020). https://doi.org/10.1038/s41556-020-0517-9

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