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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

Smaducin-6 shows bactericidal effects through neutrophil recruitmentSubcutaneous injection of Smaducin-6 into CLP mice reduced bacterial loads in peritoneal fluid. n = 10 mice per group per experiment. Data were statistically analyzed by the Mann–Whitney U-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils.CXCR2-expressing neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram peptide were analyzed by FACS. n = 5 mice per group were used. Data were statistically analyzed by a t-test and show the mean ± SD of at least three independent experiments. **P < 0.005 compared to sham or vehicle controls (CLP + PBS and CLP + Pal-Scram #1).GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram #1 peptide.Data information: Data in (C), (D), and (F) are representative of three independent experiments. Source data are available online for this figure.
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fig06: Smaducin-6 shows bactericidal effects through neutrophil recruitmentSubcutaneous injection of Smaducin-6 into CLP mice reduced bacterial loads in peritoneal fluid. n = 10 mice per group per experiment. Data were statistically analyzed by the Mann–Whitney U-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils.CXCR2-expressing neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram peptide were analyzed by FACS. n = 5 mice per group were used. Data were statistically analyzed by a t-test and show the mean ± SD of at least three independent experiments. **P < 0.005 compared to sham or vehicle controls (CLP + PBS and CLP + Pal-Scram #1).GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram #1 peptide.Data information: Data in (C), (D), and (F) are representative of three independent experiments. Source data are available online for this figure.

Mentions: CLP-induced lethality is correlated with bacterial colony counts in peritoneal fluid (Cohen, 2002). Subcutaneous injection of Smaducin-6 into CLP mice dramatically decreased bacterial colony counts in both peritoneal fluid and blood (Fig6A; Supplementary Fig S13A). However, a direct killing effect of Smaducin-6 against bacteria was not observed (Supplementary Fig S13B). Since bactericidal effects in the sepsis model have been reported to be mediated mainly by neutrophil recruitment (Nathan, 2006), we examined whether Smaducin-6 increases neutrophil recruitment in an LPS-induced endotoxemia model. Smaducin-6 was subcutaneously administered 2 h after LPS injection into the peritoneum and injected again after 12 h. Numbers of neutrophils in collected peritoneal fluid was counted 24 h post-LPS injection. Treatment with Smaducin-6 significantly increased the numbers of recruited neutrophils in the peritoneal fluid, whereas treatment with the scrambled peptide did not (Fig6B).


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)

Smaducin-6 shows bactericidal effects through neutrophil recruitmentSubcutaneous injection of Smaducin-6 into CLP mice reduced bacterial loads in peritoneal fluid. n = 10 mice per group per experiment. Data were statistically analyzed by the Mann–Whitney U-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils.CXCR2-expressing neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram peptide were analyzed by FACS. n = 5 mice per group were used. Data were statistically analyzed by a t-test and show the mean ± SD of at least three independent experiments. **P < 0.005 compared to sham or vehicle controls (CLP + PBS and CLP + Pal-Scram #1).GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram #1 peptide.Data information: Data in (C), (D), and (F) 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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Show All Figures
getmorefigures.php?uid=PMC4492818&req=5

fig06: Smaducin-6 shows bactericidal effects through neutrophil recruitmentSubcutaneous injection of Smaducin-6 into CLP mice reduced bacterial loads in peritoneal fluid. n = 10 mice per group per experiment. Data were statistically analyzed by the Mann–Whitney U-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils.CXCR2-expressing neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram peptide were analyzed by FACS. n = 5 mice per group were used. Data were statistically analyzed by a t-test and show the mean ± SD of at least three independent experiments. **P < 0.005 compared to sham or vehicle controls (CLP + PBS and CLP + Pal-Scram #1).GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram #1 peptide.Data information: Data in (C), (D), and (F) are representative of three independent experiments. Source data are available online for this figure.
Mentions: CLP-induced lethality is correlated with bacterial colony counts in peritoneal fluid (Cohen, 2002). Subcutaneous injection of Smaducin-6 into CLP mice dramatically decreased bacterial colony counts in both peritoneal fluid and blood (Fig6A; Supplementary Fig S13A). However, a direct killing effect of Smaducin-6 against bacteria was not observed (Supplementary Fig S13B). Since bactericidal effects in the sepsis model have been reported to be mediated mainly by neutrophil recruitment (Nathan, 2006), we examined whether Smaducin-6 increases neutrophil recruitment in an LPS-induced endotoxemia model. Smaducin-6 was subcutaneously administered 2 h after LPS injection into the peritoneum and injected again after 12 h. Numbers of neutrophils in collected peritoneal fluid was counted 24 h post-LPS injection. Treatment with Smaducin-6 significantly increased the numbers of recruited neutrophils in the peritoneal fluid, whereas treatment with the scrambled peptide did not (Fig6B).

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