Research ArticleIMMUNOLOGY

Substrate-specific recognition of IKKs mediated by USP16 facilitates autoimmune inflammation

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Science Advances  13 Jan 2021:
Vol. 7, no. 3, eabc4009
DOI: 10.1126/sciadv.abc4009
  • Fig. 1 Ubiquitination of IKKβ inhibits the phosphorylation of p105.

    (A) NEMO-deficient MEFs were reconstituted with the indicated constructs. The Western blot analysis results of p105 and IκBα phosphorylation and steady-state expression levels in these cells are shown. (B) By using these reconstituted MEFs, IKK kinase activity on different substrates was determined with an in vitro kinase assay in the presence of GST-IκBα or GST-p105. (C) Mouse BMDMs isolated from WT mice were stimulated with LPS. Whole-cell lysate (WL) was subjected to IP using an anti-IKKβ antibody, which was followed by IB analysis of the ubiquitination (Ub) level. (D) HEK293T cells were transfected with NF-κB luciferase reporters along with IKKβWT and ubiquitin expression plasmids. The readouts were normalized to Renilla luciferase activity and are presented as the fold changes relative to the values in untransfected cells. (E) IKKβWT (WT) and multiple site mutants were transfected into HEK293T cells. After 8 hours of TNF-α treatment, the cells were lysed for luciferase assays. ns, not significant. (F) Sequence alignment of Ub sites on IKKβ orthologs of different species. (G) IKKβWT and IKKβK238R were reconstituted into IKKβ−/− cells. WLs were subjected to IP using anti-IKKβ followed by Ub analysis. (H) IKK kinase activity was determined by kinase assays upon IP with an anti-IKKβ antibody. (I) HEK293T cells were transfected with HA-IKKβ and subjected to IP using anti-HA before mass analysis. The associated proteins of IKKβ are presented as indicated. The bars and error bars show the means ± SEMs. The significance of the differences in (E) was determined by the two-tailed Student’s t test. *P < 0.05.

  • Fig. 2 USP16 specifically interacts with IKKβ but not p105 or IκBα.

    (A) HEK293T cells were transfected with USP16-, IKKβ-, p105-, and IκBα-expressing plasmids. IB of HA was performed followed by IP with an anti-FLAG antibody on WLs. (B) The interaction among USP16, IKKβ, and p105 was assessed in WT and USP16-deficient BMDMs activated by LPS. WLs were subjected to IP using an anti-IKKβ or anti-p105 antibody and then to IB analyses of the associated USP16. (C) Confocal microscopy analysis of the colocalization of USP16, IKKβ, and DAPI in WT macrophages stimulated with LPS (100 ng/ml) as indicated. Scale bar, 5 μm. NT, nontreatment. (D) HEK293T cells were transfected with the indicated plasmids. The interaction between USP16 and IKK components was evaluated by co-IP assay. (E and F) The associations between USP16 and various truncation mutants of IKKβ (E) or a USP16 interaction-defective mutant (IKKβ∆NBD) (F) were detected through the indicated IP and IB analyses. (G) USP16 competently bound to IKKβ and inhibited the interaction between IKKβ and NEMO. (H and I) The binding amounts of IKKβ and various truncation mutants of USP16 (H) or an IKKβ interaction-defective mutant (USP16∆IBD) (I) were detected by the indicated IP. The data are representative of at least three independent experiments. aa, amino acids.

  • Fig. 3 IKK-induced p105 phosphorylation requires the participation of USP16.

    (A) The phosphorylation of IKKs and their substrates in WLs of WT and USP16-deficient BMDMs was measured by IB analysis. (B) The amounts of each NF-κB member in cytoplasmic (CE) and nuclear (NE) extracts were detected by IB. (C) Electrophoretic mobility shift assay (EMSA) of NE extracts of WT and USP16-deficient BMDMs stimulated with LPS (1 μg/ml), as assessed with HRP-labeled NF-κB, AP1, or OCT1. (D to F) Similar analyses of the activation of IKKs (D) and transcription factors (E) were performed via IB and EMSA (E) in USP16−/− MEFs as described above. (G and H) IKK kinase assays and IB assays using WLs of LPS-stimulated BMDMs derived from WT and USP16MKO mice (G) or USP16−/− MEFs (H). The data are representative of at least three independent experiments.

  • Fig. 4 USP16-mediated DUB of IKKβ is required for its binding to p105.

    (A and B) The interaction between IKKβ and p105 was assessed in WT and USP16-deficient BMDMs stimulated by LPS (1 μg/ml) (A) or in TNF-α–treated USP16−/− MEFs (B) via IP with an anti-IKKβ antibody. (C) USP16-deficient MEFs were reconstituted with WT or catalytically inactive USP16. IB analysis of the interaction between IKKβ and p105 under TNF-α (50 ng/ml) stimulation was performed as indicated. (D) HEK293T cells were transfected with an NF-κB–luciferase reporter plasmid in the presence (+) or absence (−) of the indicated empty vector or expression plasmids. Luciferase assays were performed, and the results are presented as fold changes based on the empty vector group 36 hours after transfection. (E) IKKβ was isolated by IP (under denaturing conditions) from WLs of WT and USP16-deficient BMDMs and subjected to IB assays using anti-ubiquitin (top). Protein lysates were also subjected to direct IB (bottom). (F) HEK293T cells were transfected with HA-tagged ubiquitin along with the indicated expression plasmids. The ubiquitination levels of IKKβ and IKKα were examined by IB. Cell lysates were also subjected to direct IB (bottom three). (G) In vitro assays were used to evaluate USP16-mediated IKKβ DUB. Recombinant WT or inactive USP16 (USP16mut) was incubated with ubiquitinated IKKβ isolated from transfected HEK293T cells. Ubiquitination was detected by IB. (H) HEK293T cells were transfected with multiple ubiquitin mutants (mutations at K6, K11, K48, K63, K29, and K33) and the indicated expression plasmids. HA-tagged IKKβ was isolated by IP, and the ubiquitination level was then detected by IB. The data are representative of at least three independent experiments.

  • Fig. 5 USP16 is required for the induction of various NF-κB–targeted genes.

    (A) Venn diagram illustrating the overlap of DEGs between WT and USP16-deficient BMDMs under nontreatment or LPS-stimulated conditions for 6 hours. The KEGG analysis results of the enriched biological processes for these DEGs are shown. (B) Heatmap showing basal LPS-responsive (right) NF-κB–targeted genes among the DEGs of WT and USP16-deficient BMDMs. (C) Flow cytometry of the expression of CD40, CD80, and CD86 in WT and USP16-deficient BMDMs in response to LPS stimulation. MFI, mean fluorescence intensity. (D and E) qRT-PCR analysis of mRNA (vertical axes) in WT or USP16-deficient BMDMs unstimulated (0 hour) or stimulated for 2 or 6 hours with LPS (100 ng/ml) (D) or CpG (25 nM) (E). (F) ELISA results for the indicated cytokines in the supernatants of WT or USP16-deficient BMDMs stimulated with LPS for 12 and 24 hours. (G) qRT-PCR analysis of the indicated genes in USP16-deficient BMDMs reconstituted with USP16WT and USP16CI and subjected to LPS stimulation. (H) qRT-PCR analysis of proinflammatory cytokine production in WT and USP16-deficient BMDMs reconstituted with p50. All qRT-PCR data are presented as the fold induction relative to the Actb mRNA level. The data are presented as the means ± SEMs and are representative of at least three independent experiments. The statistical analysis results show the variations among experimental replicates. Two-tailed unpaired t tests were performed. *P < 0.05.

  • Fig. 6 USP16 deficiency in macrophages alleviates experimental colitis and inflammation-mediated colon carcinogenesis.

    (A) CD11b- and CD68-positive colon macrophages were isolated from patients with UC or CD or from healthy donors. qRT-PCR was performed to analyze the USP16 mRNA levels. (B) Immunohistochemical examination of USP16 protein in inflammatory tissue samples of patients with IBD and in healthy samples. Scale bar, 50 μm. (C) Immunofluorescence images of USP16 and CD68 staining in human colon tissue sections. Scale bar, 50 μm. WT and USP16MKO mice were treated with 3% [(D) and (F) to (I)] or 3.5% (E) DSS (in drinking water) for five continuous days and then supplied with normal drinking water. (D and E) Body weight loss (D) and survival rates (E) of DSS-treated WT and USP16MKO mice. (F to H) Colon length (F), proinflammatory cytokine production (G), and hematoxylin and eosin (H&E) histological staining (H) results for day 8 DSS-treated WT and USP16MKO mice (scale bar, 100 mm). (I) FACS analysis results for the total immune cells (CD45+), macrophages (CD11b+F4/80+), and neutrophils (CD11b+Ly6G+), presented in a representative plot for multiple mice (n = 4). (J) Schematic of mouse treatment with AOM/DSS. The colons of WT and USP16MKO mice were photographed. The numbers of tumors of different sizes were measured. (K) Representative images of Ki-67 staining of colon tumors of WT and USP16MKO mice (scale bar, 50 μM). (L) qRT-PCR assay of cytokine levels in colon tissue of AOM/DSS-treated WT and USP16MKO mice. All qRT-PCR data are presented as the fold induction relative to the Actb mRNA level. Photo credits of (B), (C), (F), (H), (J), and (K): Yu Zhang (Zhejiang University). The data are presented as the means ± SEMs and are representative of at least three independent experiments. The statistical analysis results show the variations among experimental replicates. Two-tailed unpaired t tests were performed. *P < 0.05 and **P < 0.01.

Supplementary Materials

  • Supplementary Materials

    Substrate-specific recognition of IKKs mediated by USP16 facilitates autoimmune inflammation

    Jian-shuai Yu, Tao Huang, Yu Zhang, Xin-tao Mao, Ling-jie Huang, Yi-ning Li, Ting-ting Wu, Jiang-yan Zhong, Qian Cao, Yi-yuan Li, Jin Jin

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