A CC' loop decoy peptide blocks the interaction between Act1 and IL-17RA to attenuate IL-17- and IL-25-induced inflammation - PubMed (original) (raw)

. 2011 Nov 1;4(197):ra72.

doi: 10.1126/scisignal.2001843.

Shadi Swaidani, Wen Qian, Zizhen Kang, Paige Sun, Yue Han, Chenhui Wang, Muhammet Fatih Gulen, Weiguo Yin, Chunjiang Zhang, Paul L Fox, Mark Aronica, Thomas A Hamilton, Saurav Misra, Junpeng Deng, Xiaoxia Li

Affiliations

A CC' loop decoy peptide blocks the interaction between Act1 and IL-17RA to attenuate IL-17- and IL-25-induced inflammation

Caini Liu et al. Sci Signal. 2011.

Abstract

Interleukin-17 (IL-17) and IL-25 signaling induce the expression of genes encoding inflammatory factors and are implicated in the pathology of various inflammatory diseases. Nuclear factor κB (NF-κB) activator 1 (Act1) is an adaptor protein and E3 ubiquitin ligase that is critical for signaling by either IL-17 or IL-25, and it is recruited to their receptors (IL-17R and IL-25R) through heterotypic interactions between the SEFIR [SEF (similar expression to fibroblast growth factor genes) and IL-17R] domain of Act1 and that of the receptor. SEFIR domains have structural similarity with the Toll-IL-1 receptor (TIR) domains of Toll-like receptors and IL-1R. Whereas the BB' loop of TIR is required for TIR-TIR interactions, we found that deletion of the BB' loop from Act1 or IL-17RA (a common subunit of both IL-17R and IL-25R) did not affect Act1-IL-17RA interactions; rather, deletion of the CC' loop from Act1 or IL-17RA abolished the interaction between both proteins. Surface plasmon resonance measurements showed that a peptide corresponding to the CC' loop of Act1 bound directly to IL-17RA. A cell-permeable decoy peptide based on the CC' loop sequence inhibited IL-17- or IL-25-mediated signaling in vitro, as well as IL-17- and IL-25-induced pulmonary inflammation in mice. Together, these findings provide the molecular basis for the specificity of SEFIR-SEFIR versus TIR-TIR domain interactions and consequent signaling. Moreover, we suggest that the CC' loop motif of SEFIR domains is a promising target for therapeutic strategies against inflammatory diseases associated with IL-17 or IL-25 signaling.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1

Fig. 1

The SEFIR3 region is critical for the interaction between the Act1 SEFIR domain and IL-17RA. (A) Schematic of wild-type (WT) mAct1 and mAct1 SEFIR deletion mutants. (B) HeLa cells were transiently cotransfected with plasmids encoding FLAG-tagged mouse Act1 or its deletion mutants together with plasmid encoding V5-tagged IL-17RA. Lysates of transfected cells were subjected to immunoprecipation (IP) with antibody against the V5 tag, after which they were analyzed by Western blotting (IB) with antibodies against FLAG and V5. WCL, whole-cell lysate. (C) _Act1_−/− MEFs expressing IL-17RB were reconstituted with either empty vector, FLAG-tagged mAct1, or FLAG-tagged deletion mutants of mAct1 by retroviral infection, after which they were treated with IL-17 (50 ng/ml) or IL-25 (100 ng/ml) for 3 hours. The abundances of Cxcl1 and Il13 messenger RNAs (mRNAs) were measured by real-time RT-PCR and the results are expressed as fold-induction calculated as a ratio of the abundance of a given mRNA in the treated sample to that in the untreated sample. The experiment was repeated five times and the data are shown as the mean ± the standard error of the mean (SEM). All other experiments were performed three times, with representative blots shown.

Fig. 2

Fig. 2

A CC′ loop peptide corresponding to a region from SEFIR3 binds directly to IL-17RA. (A) Computational modeling of the SEFIR domain of mAct1 with I-Tasser in comparison to the TIR domain of TLR2. The SEFIR domain of Act1 consists of five-stranded β-sheets (A to D) and of five roughly parallel α-helices (A′, B′, C′, Cins and C′, D′, and E′) interconnected by loops. The critical structures are colored to highlight secondary structural elements: the BB′ loop is orange; the Cins helix is cyan; and the CC′ loop is pink. The amino acid sequence of the SEFIR domain of mAct1 is shown below the structures together with the modeled secondary structure. (B) SPR analysis of the binding of the mAct1 SEFIR domain to the IL-17RA SEFIR domain. Purified mAct1 SEFIR domain (0.625 to 20 μM) and bovine serum albumin (BSA, 20μM) were injected over the surface to which IL-17RA was immobilized. Binding signal is recorded as resonance units (RU). (C) SPR analysis of the binding of the CC′ loop peptide to the IL-17RA SEFIR domain. The CC′ loop peptide (25 to 400 μM) or a scrambled peptide (Scramble, 400 μM) were injected over the surface to which IL-17RA was immobilized. (D) Comparison of the binding of BB′ loop and the CC′ loop peptides to the IL-17RA SEFIR domain. The BB′ loop, Cins, and CC′ loop peptides (400 μM each) and the Act1 SEFIR domain (10 μM) were injected over the surface to which IL-17RAwas immobilized. Graphs are representative of three experiments.

Fig. 3

Fig. 3

Mutations in the CC′ loop region of the Act1 SEFIR domain disrupt its interaction with IL-17RA and inhibit IL-17 and IL-25 signaling. (A and B) _Act1_−/− MEFs expressing IL-17RB were reconstituted with empty vector, FLAG-tagged mAct1, or (A) FLAG-tagged 3-AA CC′ loop mutants or (B) single-AA CC′ loop mutants, as indicated, by retroviral infection, after which cells were treated with IL-17 or IL-25. The abundances of Cxcl1 and Il13 mRNAs were measured by real-time RT-PCR, and the fold-induction in mRNA amounts was calculated as a ratio of the amount of mRNA in the treated sample compared to that in the untreated sample. Data are the means ± SEMs from three experiments. (C) HeLa cells were transiently cotransfected with plasmids encoding FLAG-tagged mAct1 or the indicated single-AA point mutants of Act1 together with plasmid encoding V5-tagged IL-17RA. Cell lysates were subjected to immunoprecipation with antibody against V5, after which they were analyzed by Western blotting with antibodies against the FLAG and V5 tags. (D) HeLa cells were transiently cotransfected with plasmids encoding FLAG-tagged mAct1 or the indicated single-AA point mutants of Act1 together with plasmid encoding V5-tagged mAct1. Cell lysates were subjected to immunoprecipitation with antibody against the V5 tag, after which they were analyzed by Western blotting with antibodies against the FLAG and V5 tags. Data in (C) and (D) are representative of three experiments.

Fig. 4

Fig. 4

A cell-permeable CC′ loop decoy peptide disrupts the interaction between Act1 and IL-17RA and inhibits IL-17R and IL-25R signaling. (A) Sequences of Antennapedia homeodomain (Antp) and decoy peptides with the Antp sequence underlined. (B) MEFs expressing IL-17RB were incubated for 2 or 24 hours with the indicated FITC-tagged peptides (200 μM) and were then analyzed by flow cytometry. Data show the analysis of FITC-positive cells from untreated (black lines) and FITC-tagged peptide-treated (green lines) samples. The percentages of FITC-positive cells in the treated samples are shown. (C) HeLa cells were cotransfected with plasmids encoding FLAG-tagged mAct1 and V5-tagged IL-17RA and were cultured for 24 hours, after which they were incubated for 24 hours with the indicated peptides (200 μM). IL-17RA was immunoprecipitated from the samples with antibody against the V5 tag, and samples were then subjected to Western blotting analysis with the indicated antibodies. (D) MEFs expressing IL-17RB were incubated with the indicated peptides (200 μM) for 24 hours after which they were treated with IL-17 or IL-25 and then analyzed by Western blotting with the indicated antibodies. (E) MEFs expressing IL-17RB were incubated with the indicated peptides (200 μM) for 24 hours, after which they were treated with IL-17 or IL-25 for 3 hours. The abundances of Cxcl1 and Il13 mRNAs were then measured by real-time RT-PCR. Data shown are means ± SEM from three individual experiments. *P < 0.05 (difference between samples treated with DMSO or the CC′ loop peptide). Representative blots are shown from three individual experiments.

Fig. 5

Fig. 5

A cell-permeable CC′ loop decoy peptide inhibits IL-17- and IL-25-induced pulmonary inflammation. Wild-type (WT) female BALB/c mice (n = 4 mice per group) were pretreated with DMSO, scrambled peptide, or the CC′ loop decoy peptide for 4 hours, after which they were administered saline, IL-17, or IL-25 intranasally. Mice were sacrificed and analyzed 16 hours after injection with IL-17 or 4 days after injection with IL-25. (A) Mice pretreated with the CC′ peptide showed reduced accumulation of neutrophils in BAL fluid after intranasal administration of IL-17 compared to that in BAL fluid from mice pretreated with the scrambled peptide. (B) The IL-17-associated cytokines MCP-1 and GM-CSF were measured by enzyme-linked immunosorbent assay (ELISA) analysis of the BAL from IL-17-treated mice. (C) H&E staining of lung tissue from IL-17-treated mice showed that IL-17-induced airway recruitment of granulocytes, was reduced in mice pretreated with the CC′ loop decoy peptide compared to that in mice pretreated with the scrambled peptide or DMSO. (D) Real-time RT-PCR analysis of gene expression in lung tissue from IL-17-treated mice. (E) Mice pretreated with the CC′ decoy peptide showed reduced accumulation of eosinophils in the BAL after intranasal administration of IL-25 compared to that in the BAL from mice pretreated with the scrambled peptide. (F) ELISA analysis of the IL-25-associated cytokines IL-5 and IL-13 in the BAL of IL-25-treated mice. (G) H&E and PAS staining of lung tissue from IL-25-treated mice showed that IL-25-induced airway recruitment of granulocytes, predominantly eosinophils, as well as mucin production, as determined by PAS staining, were reduced in mice pretreated with the CC′ loop decoy peptide compared to that in mice pretreated with the scrambled peptide or DMSO. (H) Real-time RT-PCR analysis of gene expression in lung tissue from IL-25-treated mice. All experiments were performed three times. *P < 0.05.

Fig. 6

Fig. 6

The CC′ loop motif is a common motif among SEFIR family members and is critical for SEFIR-SEFIR interactions. (A) Computational modeling of the SEFIR domains of IL-17RA, IL-17RB, and IL-17RC in comparison with the SEFIR domain of Act1. The critical structures are colored to highlight secondary structure elements: the BB′ loop is orange; the Cins helix is cyan; and the CC′ loop is pink. (B) HeLa cells were transiently cotransfected with plasmids encoding V5-tagged IL-17RA or its D-BB′ or D-CC′ mutants together with plasmid encoding FLAG-tagged mAct1. Cell lysates were subjected to immunoprecipitation with antibody against the V5 tag, after which they were analyzed by Western blotting with antibodies against the FLAG and V5 tags. (C) HeLa cells were transiently cotransfected with plasmid encoding V5-tagged IL-17RB or its D-BB′ or D-CC′ mutants together with plasmid encoding FLAG-tagged mAct1. Cell lysates were subjected to immunopreciptation with antibody against the V5 tag, after which they were analyzed by Western blotting with antibodies against the FLAG and V5 tags. Blots shown are representative of three independent experiments.

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