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Inhibition of lethal inflammatory responses through the targeting of membrane-associated Toll-like receptor 4 signaling complexes with a Smad6-derived peptide.

Lee YS, Park JS, Jung SM, Kim SD, Kim JH, Lee JY, Jung KC, Mamura M, Lee S, Kim SJ, Bae YS, Park SH - EMBO Mol Med (2015)

Bottom Line: Here, we developed Smaducin-6, a novel membrane-tethered palmitic acid-conjugated Smad6-derived peptide composed of amino acids 422-441 of Smad6.Smaducin-6 interacted with Pellino-1, located in the inner membrane, thereby disrupting the formation of IRAK1-, RIP1-, IKKε-mediated TLR4 signaling complexes.Our findings provide clues to develop new peptide-based drugs to target Pellino-1 protein in TLR4 signaling pathway for the treatment of sepsis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon, Korea.

No MeSH data available.


Related in: MedlinePlus

Membrane-tethered Smaducin-6 inhibits TLR4 signalingA Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by quantitative real-time RT–PCR.B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression and (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.D Peli1 knockdown or wild-type human THP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by quantitative real-time RT–PCR. Data show the mean ± SD of three independent experiments.E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI.F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin–agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.G–I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.Data information: Data in (A) and (B) were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. ***P < 0.001, **P < 0.005, *P < 0.05 compared to no LPS treatment or vehicle control (Pal-Scram #1).Source data are available online for this figure.
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fig03: Membrane-tethered Smaducin-6 inhibits TLR4 signalingA Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by quantitative real-time RT–PCR.B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression and (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.D Peli1 knockdown or wild-type human THP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by quantitative real-time RT–PCR. Data show the mean ± SD of three independent experiments.E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI.F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin–agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.G–I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.Data information: Data in (A) and (B) were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. ***P < 0.001, **P < 0.005, *P < 0.05 compared to no LPS treatment or vehicle control (Pal-Scram #1).Source data are available online for this figure.

Mentions: Palmitoylated peptides have been reported to act as modulators, which target proteins at the intracellular surface of the membrane through a “flip” process (Covic et al, 2002; Tressel et al, 2011). We conjugated palmitic acid to the N-terminus of a commercially synthesized Smad6 (422–441) peptide, named Smaducin-6, to target Pellino-1 (Fig3A). Smaducin-6 was synthesized at a purity of more than 95% to be used in this study (Supplementary Fig S4A). Pre-treatment with Smaducin-6 reduced LPS-induced Il6 gene expression in LPS-treated RAW264.7 cells in a dose-dependent manner (Fig3A), reduced NF-κB-mediated luciferase gene expression (Fig3B), and reduced IκBα degradation and IKKα/β phosphorylation (Fig3C). A scrambled palmitoylated peptide (Pal-Scram #1) was used as a negative control. In contrast, pre-treatment with Smaducin-6 did not affect LPS-induced phosphorylation of MAP kinases such as ERK, JNK, and p38 MPAK, compared to the control scrambled peptide (Supplementary Fig S4B). Also, the reduction in LPS-induced Il6 gene expression by Smaducin-6 was similar to that of Pellino-1 knockdown RAW264.7 cells (Fig3D).


Inhibition of lethal inflammatory responses through the targeting of membrane-associated Toll-like receptor 4 signaling complexes with a Smad6-derived peptide.

Lee YS, Park JS, Jung SM, Kim SD, Kim JH, Lee JY, Jung KC, Mamura M, Lee S, Kim SJ, Bae YS, Park SH - EMBO Mol Med (2015)

Membrane-tethered Smaducin-6 inhibits TLR4 signalingA Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by quantitative real-time RT–PCR.B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression and (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.D Peli1 knockdown or wild-type human THP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by quantitative real-time RT–PCR. Data show the mean ± SD of three independent experiments.E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI.F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin–agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.G–I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.Data information: Data in (A) and (B) were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. ***P < 0.001, **P < 0.005, *P < 0.05 compared to no LPS treatment or vehicle control (Pal-Scram #1).Source data are available online for this figure.
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fig03: Membrane-tethered Smaducin-6 inhibits TLR4 signalingA Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by quantitative real-time RT–PCR.B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression and (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.D Peli1 knockdown or wild-type human THP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by quantitative real-time RT–PCR. Data show the mean ± SD of three independent experiments.E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI.F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin–agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.G–I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.Data information: Data in (A) and (B) were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. ***P < 0.001, **P < 0.005, *P < 0.05 compared to no LPS treatment or vehicle control (Pal-Scram #1).Source data are available online for this figure.
Mentions: Palmitoylated peptides have been reported to act as modulators, which target proteins at the intracellular surface of the membrane through a “flip” process (Covic et al, 2002; Tressel et al, 2011). We conjugated palmitic acid to the N-terminus of a commercially synthesized Smad6 (422–441) peptide, named Smaducin-6, to target Pellino-1 (Fig3A). Smaducin-6 was synthesized at a purity of more than 95% to be used in this study (Supplementary Fig S4A). Pre-treatment with Smaducin-6 reduced LPS-induced Il6 gene expression in LPS-treated RAW264.7 cells in a dose-dependent manner (Fig3A), reduced NF-κB-mediated luciferase gene expression (Fig3B), and reduced IκBα degradation and IKKα/β phosphorylation (Fig3C). A scrambled palmitoylated peptide (Pal-Scram #1) was used as a negative control. In contrast, pre-treatment with Smaducin-6 did not affect LPS-induced phosphorylation of MAP kinases such as ERK, JNK, and p38 MPAK, compared to the control scrambled peptide (Supplementary Fig S4B). Also, the reduction in LPS-induced Il6 gene expression by Smaducin-6 was similar to that of Pellino-1 knockdown RAW264.7 cells (Fig3D).

Bottom Line: Here, we developed Smaducin-6, a novel membrane-tethered palmitic acid-conjugated Smad6-derived peptide composed of amino acids 422-441 of Smad6.Smaducin-6 interacted with Pellino-1, located in the inner membrane, thereby disrupting the formation of IRAK1-, RIP1-, IKKε-mediated TLR4 signaling complexes.Our findings provide clues to develop new peptide-based drugs to target Pellino-1 protein in TLR4 signaling pathway for the treatment of sepsis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon, Korea.

No MeSH data available.


Related in: MedlinePlus