Abstract
Toll/IL-1R (TIR) domain-containing adapter-inducing IFN-β (TRIF)-related adapter molecule (TRAM) serves as a bridging adapter that enables recruitment of TRIF to activated TLR4 and thereby mediates the induction of TRIF-dependent cytokines. A library of cell-permeating decoy peptides derived from TRAM TIR domain has been screened for the ability of individual peptides to inhibit TLR4 signaling in primary murine macrophages. Peptides derived from TRAM TIR BB loop (TM4) and C helix (TM6) inhibited the LPS-induced activation of MyD88-dependent and TRIF-dependent cytokines, as well as MAPK activation. TM4 and TM6 did not block macrophage activation induced by TLR2, TLR9, or retinoic acid-inducible gene 1-like receptor agonists. Both TM4 and TM6 blocked coimmunoprecipitation of TRAM and TLR4 ectopically expressed in HEK293T cells. Both peptides also blocked the LPS-induced recruitment of MyD88 to TLR4 in primary murine macrophages. In vivo examination of TRAM-derived peptides demonstrated that all peptides that were inhibitory in vitro profoundly suppressed systemic inflammatory response elicited in mice by a sublethal LPS dose, and protected mice against a lethal LPS challenge. This research identifies novel TLR inhibitors effective in vitro and in vivo and validates the approach taken in this study as a rational way for development of signaling inhibitors and lead therapeutics.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Adaptor Proteins, Vesicular Transport / genetics
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Adaptor Proteins, Vesicular Transport / immunology
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Amino Acid Sequence
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Animals
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Gene Expression / drug effects
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HEK293 Cells
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Humans
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Inflammation / chemically induced
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Inflammation / drug therapy*
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Inflammation / genetics
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Inflammation / immunology
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Lipopolysaccharides
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Macrophages, Peritoneal / drug effects*
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Macrophages, Peritoneal / immunology
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Macrophages, Peritoneal / pathology
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Mice
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Mice, Inbred C57BL
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Mitogen-Activated Protein Kinases / genetics
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Mitogen-Activated Protein Kinases / immunology
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Molecular Sequence Data
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Myeloid Differentiation Factor 88 / antagonists & inhibitors
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Myeloid Differentiation Factor 88 / genetics
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Myeloid Differentiation Factor 88 / immunology
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Peptide Library
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Peptides / chemical synthesis
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Peptides / pharmacology*
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Primary Cell Culture
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Receptors, Interleukin / chemistry
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Receptors, Interleukin / genetics*
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Receptors, Interleukin / immunology
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Signal Transduction / drug effects
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Toll-Like Receptor 4 / antagonists & inhibitors*
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Toll-Like Receptor 4 / genetics
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Toll-Like Receptor 4 / immunology
Substances
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Adaptor Proteins, Vesicular Transport
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Lipopolysaccharides
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Myd88 protein, mouse
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Myeloid Differentiation Factor 88
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Peptide Library
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Peptides
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Receptors, Interleukin
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TICAM-1 protein, mouse
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Ticam2 protein, mouse
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Tlr4 protein, mouse
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Toll-Like Receptor 4
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Mitogen-Activated Protein Kinases