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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

Amino acids 400–441 of Smad6 specifically bind to Pellino-1 and inhibit NF-κB signalingA Schematic representation of truncated mutants of the Smad6 MH2 domain and binding to Pellino-1N. HA-tagged Pellino-1N encodes amino acids 1–137 of Pellino-1.B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates.C, D The SBE-Luc or 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400–441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h or LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.E CMT-93 cell lines stably expressing Smad6 amino acids 400–441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.Source data are available online for this figure.
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fig01: Amino acids 400–441 of Smad6 specifically bind to Pellino-1 and inhibit NF-κB signalingA Schematic representation of truncated mutants of the Smad6 MH2 domain and binding to Pellino-1N. HA-tagged Pellino-1N encodes amino acids 1–137 of Pellino-1.B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates.C, D The SBE-Luc or 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400–441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h or LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.E CMT-93 cell lines stably expressing Smad6 amino acids 400–441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.Source data are available online for this figure.

Mentions: We previously showed that the Smad6 MH2 domain (amino acids 332–496) is sufficient for interaction with Pellino-1N (amino acids 1–137) (Choi et al, 2006). To narrow down the subregion of the Smad6 MH2 domain responsible for binding to Pellino-1, serial truncations of the Smad6 MH2 domain were tested for interactions with Pellino-1 by coimmunoprecipitation assays (Fig1A and B). We found that Smad6 (amino acids 400–441) is sufficient for interaction with Pellino-1N, whereas Smad6 constructs with truncations of amino acids 410–441 failed to bind to the Pellino-1 N domain, suggesting that Smad6 (amino acids 410–441) is necessary for interaction with Pellino-1 (Fig1A and B). Interestingly, a subregion of the Smad6 MH2 domain (amino acids 400–441) was predicted to correspond to a β-sheet structure by homology modeling (Arnold et al, 2006) (Supplementary Fig S2).


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)

Amino acids 400–441 of Smad6 specifically bind to Pellino-1 and inhibit NF-κB signalingA Schematic representation of truncated mutants of the Smad6 MH2 domain and binding to Pellino-1N. HA-tagged Pellino-1N encodes amino acids 1–137 of Pellino-1.B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates.C, D The SBE-Luc or 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400–441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h or LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.E CMT-93 cell lines stably expressing Smad6 amino acids 400–441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.Source data are available online for this figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: Amino acids 400–441 of Smad6 specifically bind to Pellino-1 and inhibit NF-κB signalingA Schematic representation of truncated mutants of the Smad6 MH2 domain and binding to Pellino-1N. HA-tagged Pellino-1N encodes amino acids 1–137 of Pellino-1.B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates.C, D The SBE-Luc or 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400–441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h or LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.E CMT-93 cell lines stably expressing Smad6 amino acids 400–441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.Source data are available online for this figure.
Mentions: We previously showed that the Smad6 MH2 domain (amino acids 332–496) is sufficient for interaction with Pellino-1N (amino acids 1–137) (Choi et al, 2006). To narrow down the subregion of the Smad6 MH2 domain responsible for binding to Pellino-1, serial truncations of the Smad6 MH2 domain were tested for interactions with Pellino-1 by coimmunoprecipitation assays (Fig1A and B). We found that Smad6 (amino acids 400–441) is sufficient for interaction with Pellino-1N, whereas Smad6 constructs with truncations of amino acids 410–441 failed to bind to the Pellino-1 N domain, suggesting that Smad6 (amino acids 410–441) is necessary for interaction with Pellino-1 (Fig1A and B). Interestingly, a subregion of the Smad6 MH2 domain (amino acids 400–441) was predicted to correspond to a β-sheet structure by homology modeling (Arnold et al, 2006) (Supplementary Fig S2).

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