Assembly of the TLR2/6 Transmembrane Domains Is Essential for Activation and Is a Target for Prevention of Sepsis

Abstract

TLR2, together with TLR1 and TLR6, is essential for detecting lipopeptides and bacterial cell wall components such as lipoteichoic acid from Gram-positive bacteria. In this study, we report that transmembrane domain (TMD)–derived peptides from TLR2 and TLR6 specifically inhibit TLR2 activation. Secretion of the cytokines TNF-α and IL-6 by cultured macrophages (RAW264.7 cell line) was inhibited by these peptides in response to TLR2 activation by lipoteichoic acid (TLR2/6 activator) or palmitoyl (3)-Cys-Ser-Lys(4)-OH (TLR2/1 activator) but not by LPS (TLR4 activator). Extensive biophysical and biochemical assays, combined with GALLEX experiments, show that these peptides heterodimerize with their complementary TMDs on their reciprocal protein. These results suggest that TLR2/6/1 TMD assembly is essential for activating this complex. Importantly, when administered to mice inflicted by TLR2, but not TLR4-driven lethal inflammation, a selected peptide rescued 60% of these septic mice, showing potent in vivo inhibition of TNF-α and IL-6 secretion. Furthermore, this peptide also showed high protection in a whole bacteria model. Owing to the importance of TLR2 regulation under a variety of pathological conditions, compounds that can fine-tune this activity are of great importance.

The role of protein–protein interactions in Toll-like receptor function

“…All human TLR TMD constructs exhibited strong homotypic interactions, whilst TLR2 formed stable heterodimers only with their associated subfamily members, TLR1, TLR6 and TLR10 [75], consistent with their biologically relevant roles in ligand recognition discussed above, and with their high sequence identities of ~50-90%. Further biochemical and biophysical assays showed that exogenous synthetic peptides of mouse TLR2 and TLR6 TMDs, expressed in macrophages, affect TLR2 activation by inhibiting TNF-α secretion upon stimulation by lipoteichoic acid, a TLR2/6 activator from Grampositive bacteria [5]. These studies confirmed heterodimerization and strong associated heterotypic interactions between the TLR2 and TLR6 TMDs.…”

“…These studies confirmed heterodimerization and strong associated heterotypic interactions between the TLR2 and TLR6 TMDs. Moreover, in vivo studies have demonstrated the potential for therapeutic targeting by TMDs, by showing that these peptide constructs could inhibit TLR2 activation, reducing lethal inflammation in mice [5].…”

“…Such ligands cause dimerization by facilitating the formation of stable protein-protein interfaces between TLR chains, either directly, or via coreceptor proteins in the case of TLR4. Dimerization is proposed to initiate a signal, either through homodimerization (TLR 3,4,5,8 & 9) or heterodimerization (TLR 1, 2, 6 & 10) [3,4,[20][21][22][23]. The consequent rearrangements resulting from formation of the dimeric ectodomain complex are transferred via an ill-defined mechanism to the now adjacent carboxy-terminal juxtamembrane and TM helices, inducing dimerization of the cytoplasmic TIR domains.…”

“…In 1997 Janeway further identified the family of PRRs called the Toll-like receptors (TLRs) [4], which remain the most extensively studied PRRs today. The importance of TLRs is highlighted by the various infectious and noninfectious disease states that can result from their dysregulation, and by their consequent extensive therapeutic targeting [5][6][7][8][9].…”

An Immunomodulating Motif of the HIV-1 Fusion Protein Is Chirality-independent

“…Therefore, peptides composed of D-amino acids are being used as a tool to study TMD-TMD interactions of membrane-embedded receptors, as in the case of the glycophorin A and the bacterial aspartate receptor (33,46). Furthermore, exogenously added TMD peptides can be used to investigate the assembly of membrane proteins in vivo by interacting with their corresponding segments within the membrane (43,(47)(48)(49)(50).…”