The Toll-like Receptors (TLRs) sense the presence of conserved microbial and host """"""""danger"""""""" molecules and elicit primary inflammatory responses that mediate host defense. Excessive TLR signaling, however, often leads to inflammatory disease. Therefore, the ability to control TLR signaling is highly relevant to many pathologic conditions. TLR agonists induce the assembly of a multiprotein, intracellular signaling complex that mediates downstream signaling events. Assembly of this signaling """"""""platform"""""""" is initiated by cooperative interactions of """"""""Toll-IL-1R resistance"""""""" (TIR) domains present in TLR and TLR adapters. This study will identify and provide tools to target the key surface elements of TIR domains that mediate functional TIR-TIR interactions required for TLR2 and TLR4 signaling.
Three Specific Aims are designed.
The first Aim i s to identify positions of the specific TIR """"""""interfaces"""""""" that mediate functional TIR-TIR interactions. To achieve this Aim, for each TIR under study, we will generate a library of """"""""cell-permeable decoy peptides."""""""" Each peptide in a library will be composed of a cell-permeating sequence from Drosophila Antennapedia homeodomain in juxtaposition with a peptide of a TIR that represents a non-fragmented surface area of the TIR, so that each library encompasses the entire surface of the TIR. The peptides will be tested for their ability to inhibit TLR signaling in murine macrophages. The ability of a peptide to block signaling will be interpreted that this peptide represents a functional protein interface. The inhibitory peptides will be tested further in Aim 2 for the ability to bind directly to a TIR by the FRET approach so to confirm specificity of decoy-TIR interaction. Functionality of binary interactions identified by FRET will be further assessed by in silico analysis of the interface positions to identify interfaces that enable a TIR to interact simultaneously with several TIRs of the complex, thus establishing a cooperative, complex-stabilizing interaction.
In Aim 3 we will determine therapeutic potential of targeting these key TIR elements for mitigation of TLR4 signaling elicited in mice by a sub-lethal or lethal LPS challenge. We anticipate that the proposed studies will identify the TIR molecular surfaces that mediate recruitment of adapters to TLR2 and TLR4 signaling complexes, and lay the groundwork for creation of novel anti-inflammatory therapeutics targeting the TLRs specifically. We also expect that the project will result in the development of a more generic approach for studying the formation of diverse signaling complexes.
In this application, we are proposing to identify the parts of proteins that enable them to interact. Once identified, this """"""""interface"""""""" region can be targeted further as a rational way to design drugs that will inhibit interactions of proteins whose malfunctions lead to disease states.
|Javmen, Artur; Szmacinski, Henryk; Lakowicz, Joseph R et al. (2018) Blocking TIR Domain Interactions in TLR9 Signaling. J Immunol 201:995-1006|
|Piao, Wenji; Ru, Lisa W; Toshchakov, Vladimir Y (2016) Differential adapter recruitment by TLR2 co-receptors. Pathog Dis 74:|
|Piao, Wenji; Shirey, Kari Ann; Ru, Lisa W et al. (2015) A Decoy Peptide that Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza. Cell Rep 11:1941-52|
|Szmacinski, Henryk; Toshchakov, Vladimir; Lakowicz, Joseph R (2014) Application of phasor plot and autofluorescence correction for study of heterogeneous cell population. J Biomed Opt 19:046017|
|Piao, Wenji; Vogel, Stefanie N; Toshchakov, Vladimir Y (2013) Inhibition of TLR4 signaling by TRAM-derived decoy peptides in vitro and in vivo. J Immunol 190:2263-72|
|Piao, Wenji; Ru, Lisa W; Piepenbrink, Kurt H et al. (2013) Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain. Proc Natl Acad Sci U S A 110:19036-41|
|Szmacinski, Henryk; Toshchakov, Vladimir; Piao, Wenji et al. (2013) Imaging of Protein Secretion from a Single Cell Using Plasmonic Substrates. Bionanoscience 3:30-36|
|Couture, Leah A; Piao, Wenji; Ru, Lisa W et al. (2012) Targeting Toll-like receptor (TLR) signaling by Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal)-derived decoy peptides. J Biol Chem 287:24641-8|
|Liu, Anguo; Gong, Ping; Hyun, Sang W et al. (2012) TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia. J Biol Chem 287:16132-45|
|Toshchakov, Vladimir Y; Szmacinski, Henryk; Couture, Leah A et al. (2011) Targeting TLR4 signaling by TLR4 Toll/IL-1 receptor domain-derived decoy peptides: identification of the TLR4 Toll/IL-1 receptor domain dimerization interface. J Immunol 186:4819-27|