Inhibition of lethal inflammatory responses through the targeting of membrane-associated Toll-like receptor 4 signaling complexes with a Smad6-derived peptide.
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.
Affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon, Korea.
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fig02: The minimal Pellino-1-binding region of Smad6 selectively inhibits NF-κB signalingA Schematic representation of truncated mutants of the Smad6 MH2 domain.B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422–441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) was co-transfected with the HA-tagged full-length Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-Myc or anti-HA antibody, and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, CS3MTBXA-6xMyc, was transfected as a negative control. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates. Data are representative of at least three independent experiments.C The Myc-Smad6(422-441) plasmid was co-transfected with full-length Flag-IRAK1, Flag-TRAF6, Flag-MyD88, or HA-Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-Myc antibody. IgG was added as a negative control for IP. Data are representative of at least three independent experiments.D, E The SBE-Luc or 5xNF-κB-Luc reporter plasmids were co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 6 h or LPS for 2 h, and luciferase activities were measured and normalized.F The BRE-Luc reporter plasmid was co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid or full-length Smad6 into RAW264.7 cells, respectively. After 24 h, cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized.G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422–441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) or full-length Smad6 (Myc-Smad6) was co-transfected with HA-tagged full-length Smad4 or HA-tagged full-length Pellino-1 plasmid into HEK293 cells, respectively. Cell lysates were immunoprecipitated (IP) with anti-Myc and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, pCS3MTBXA-6xMyc, was co-transfected with HA-Smad4 or HA-Pellino-1 as a negative control. Data are representative of at least three independent experiments.Data information: All data in (D–F) were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.005, *P < 0.05 compared to the control (without LPS, TGF-β1, or BMP4).Source data are available online for this figure.
We sought to further narrow down this subregion of Smad6, composed of 42 amino acids, because a peptide of this length is limited in therapeutic usage in vivo. Co-immunoprecipitation assays showed that a minimal region of Smad6 amino acids 422–441 is sufficient for binding to HA-tagged Pellino-1 (Fig2A and B). This minimal region bound only to Pellino-1, but not to other proteins involved in the TLR4 signaling pathway (Fig2C). Expression of this minimal region of Smad6 (amino acids 422–441) inhibited NF-κB-mediated reporter activity, but not TGF-β or BMP-mediated reporter activity (Fig2D–F; Supplementary Fig S3). We next examined the binding of this minimal region to Smad4, because Smad6 MH2 domain interacts with Smad4 (Morén et al, 2005). Smad6 MH2 domain and full-length Smad6 bound to Smad4 and Pellino-1, respectively, whereas the minimal region of Smad6 (422–441) specifically bound to Pellino-1, but not to Smad4 (Fig2G). Thus, this minimal region of Smad6 (422–441) interacting with Pellino-1 specifically inhibits NF-κB activation.