TIR domains mediate transient interactions of signaling proteins involved in inflammatory signaling and host defense. TIR domains tend to interact with other TIR domains; yet functional TIR-TIR interactions are specific, as exemplified by the recruitment of specific TIR domain-containing adapters in response to activation of a particular receptor. Some TIR domain-containing adapters participate in multiple signaling pathways, whereas others interact with a smaller set of proteins. For example, MyD88 is a necessary adapter for all members of the IL-1R family and all TLRs with the exception of TLR3, whereas TIRAP/Mal participates only in TLR2 and TLR4 signaling. Despite considerable effort, the molecular mechanisms that determine specificity of TIR-TIR interactions are not understood.
Previously, we examined a set of 11 TLR4 TIR-derived decoy peptides for the ability to inhibit TLR4 signaling (1
). The TIRAP peptides tested in this study were designed similarly to the TLR4 peptides so that each peptide represents a region that is structurally homologous to the corresponding TLR4 TIR region () (1
). Five TIRAP-derived peptides, TR3, TR5, TR6, TR9, and TR11, potently inhibited TLR4 signaling. This set is different from the set of inhibitory peptides derived from the TLR4 TIR domain, in which peptides from regions 1, 3, 4 (BB loop), 9, and 11 inhibited. Peptides derived from regions 3, 9, and 11 of both TLR4 and TIRAP TIR domains were inhibitory. It is noteworthy that the sequences of these TIRAP and TLR4 regions are very dissimilar () (1
); for example, the inhibitory peptides derived from region 3 of TIRAP and TLR4 TIR domains have sequences EGSQASLRCF and EEGVPRFHLC, respectively. The absence of local sequence conservancy, especially in the surface-exposed segments of TIR domains, is well documented (e.g.
). Peptides derived from regions 5 and 6 of TIRAP/Mal potently inhibited LPS signaling, whereas peptides from the structurally homologous regions of TLR4 were poor inhibitors. Regions 5 and 6 represent surface-exposed amino acids that are adjacent to strand C, the most central strand of the TIR β-sheet that spans the core of the domain. Therefore, regions 5 and 6 are not contiguous on the TIR surface () and are unlikely to represent one TIRAP interface. Instead of the peptide derived from TR5, the peptide derived from the BB loop of TLR4, which is juxtaposed to region 5, inhibited LPS signaling. The BB loop of TLR4 and several other TIR domains is involved in TIR homodimerization (1
). Poor inhibitory activity of the TIRAP BB peptide may indicate that the TIRAP/Mal homodimerization is not important for the function of this protein.
FIGURE 5. Relative positions of inhibitory peptides on TIRAP TIR surface. Regions represented by peptides that potently inhibited TLR4 signaling are shown in red. Peptides that inhibited both TLR4 and TLR2 signaling are shown blue. This model of the mouse TIRAP (more ...)
Although TIRAP/Mal is necessary for recruitment of MyD88, but not TRIF, to the TLR4 signaling complex, all inhibitory TIRAP peptides blocked both MyD88-dependent and MyD88-independent cytokine genes induced by LPS. This finding agrees with and expands our previous reports that TIR-derived peptides do not preferentially affect MyD88-dependent cytokine production (1
). The ability of TIRAP-derived peptides to inhibit both MyD88-dependent and MyD88-independent cytokines suggests that TLR4 recruits adapters of MyD88-dependent or MyD88-independent pathways either through the same or significantly overlapping sites. This notion is amenable with the concept that the recruitment of adapters of MyD88-dependent and MyD88-independent pathways occurs sequentially at different cellular locations and is controlled by TLR4 trafficking (31
Two TIRAP peptides, TR3 and TR6, inhibited TLR2/TLR1-mediated signaling. Interestingly, TR3 and TR6 inhibited P3C-induced, but not P2C-induced, signaling. This finding suggests that these peptides target TLR1. However, regions 3 and 6 are located on opposite sides of the TIRAP TIR domain (). Therefore, it is unlikely that they target the same molecule. Interestingly, both TR3 and TR6 induced activation of JNK, but not ERK MAPK. This “unspecific” effect of TR3 and TR6, together with findings that both peptides target TLR2 in addition to both MyD88-dependent and TRIF-dependent arms of TLR4 signaling, suggests that TR3 and TR6 may target a wider set of proteins. Further studies will be required to understand the molecular mechanisms that underlie the specificity and strength of inhibition by these decoy peptides.
TR3 represents the AB loop of TIRAP/Mal. The highly surface-exposed residues of this loop include two charged amino acids, Glu-128 and Arg-135, and a polar amino acid, Gln-131. Interestingly, the same motif (-ExxQxxxR-) formed by Glu-152, Gln-155, and Arg-159 is also present in TR5 (). Because these TIRAP regions have different secondary structure and the residues are therefore spaced differently on the TIRAP surface, it is unlikely that these regions would have the same docking sites. Nevertheless, the corresponding peptides have higher conformational flexibility and may be predicted to target the same binding site. A counterargument for the statement that the -ExxQxxxR- sequence is solely responsible for inhibition by both TR3 and TR5 is that the TR45 peptide, which is not a strong inhibitor of TLR4 (C), also has this motif. In addition to TLR4, TR3 inhibits TLR2 signaling, whereas TR5 does not. This finding suggests that the -ExxQxxxR- motif does not play a major role in inhibiting the TLR2 pathway.
New data confirm our previous finding that the peptide derived from the BB loop of TIRAP, TR4 (QLRD
VS), is not a particularly strong inhibitor of TLR4 (20
). Interestingly, the peptide derived from the corresponding region of the TLR4 TIR domain, 4BB (LHYRD
AA; here and below, the underlined residues are identical in TLR4 and TIRAP peptides), is a quite potent inhibitor of TLR4 that targets the TLR4 TIR dimerization surface (1
). A comparison of TR4 and 4BB sequences suggests that hydrophobic amino acids of 4BB at positions 6, 7, and 10 might be important for the binding of 4BB to its target.
Region 6 represents the N-terminal part of the third helical region of the TIRAP TIR domain. The peptide derived from region 6 of TIRAP, TR6 (PGFLRDPWCKYQML), inhibited both TLR4 and TLR2 signaling, whereas the peptide derived from the structurally homologous region of TLR4 (RHFIQSRWCIFEYE) did not inhibit TLR4 as strongly. Sequence conservancy between TR6 and the homologous TLR4 region is less than that in the BB loop region, and it has yet to be determined which residues of TR6 are more important for the inhibitory properties of this peptide.
Peptides derived from region 9, the extended fourth helical region, and region 11, the fifth TIR helix, of both TIRAP and TLR4 TIR domains inhibited LPS signaling, yet there is very little sequence similarity in the corresponding regions of TIRAP and TLR4. TR9 represents the region that has been identified as the TRAF6-binding site (33
). Further studies are required to elucidate if TR9 indeed targets TRAF6.
An important and practical addition to the tests we used in the past to characterize TIR-derived decoy peptides (1
) is that, in this study, we investigated in more detail the peptide effects on cytokine secretion after a prolonged 24-h incubation of macrophages with LPS. Every peptide that has been identified as inhibitory based on the manifestations of early TLR4 signaling (i.e.
1-h mRNA induction and MAPK activation) profoundly suppressed secretion of every cytokine measured over the 24-h time course. Importantly, our study included an “immediate-early” and transiently induced cytokine (i.e.
TNF-α), a cytokine that is induced with some delay (i.e.
IL-6), and two cytokines whose secretion significantly increases several hours after LPS stimulation (RANTES and IFN-γ). The ability of decoy peptides to inhibit cytokine secretion over a long period after a single treatment implies the high translational potential of these substances as TLR inhibitors or lead therapeutics.
Finally, an in vivo examination of two inhibitory peptides demonstrated that the cell-permeable decoy peptide inhibitors effectively block systemic manifestations of TLR signaling. Although preliminary, this result is very exciting, as it provides a new avenue for development of novel TLR-targeting therapeutics.
TLR signaling is a significant therapeutic target because uncontrolled TLR signaling is a pathogenic mechanism in many inflammatory diseases, including sepsis. This study has identified several novel TLR inhibitors effective in vitro and in vivo, each of which can be used for further refinement as a lead substance in development of TLR-targeting pharmaceutics.