The aim of this proposal is to identify the mechanism of Toll-like receptor 2 (TLR2) activation by bacterial lipopeptides (BLP) to initiate inflammation. Toll-like receptors (TLR) plays essential role in sensing microbial molecules or pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents. Recently, Dr. Zychlinsky's group identified that bacterial lipopeptides (BLP) are the activators of TLR2. BLPs are made by all prokaryotes and they signal through TLR2 for either the activation of the transcriptional activator NF-kB and for the induction of apoptosis. However, it remains unknown if this effect occurs via direct or indirect interaction of BLP with TLR2. In this study, we will test whether BLP binds directly to TLR2 by binding assays using soluble form of the recombinant TLR2 extra cellular domain in vitro. Synthetic BLP will be used in this study to rule out contaminants bearing others PAMPs motifs. If TLR2 binds directly to BLP, we will identify the BLP-binding motif from the extra cellular domain of TLR2 receptor by site directed mutagenesis. Induced apoptosis and cell activation will be evaluated in the transient mutant cell lines. On the other hand, if BLP fails to bind directly to TLR.2 we hypothesize, that
(1) BLP binds an adaptor protein in the membrane, or
(2) that BLP exhibits high affinity for lipid rafts, based on its acyl moiety. Acylation seems to be a required feature for raft targeting. This aim will be approached by UV cross-linked biotinylated-sBLP (PA-bioBLP) to THP-1 membranes followed by isolation and characterization of PA-bioBLP-containing raft. This approach will provide crucial information about the composition of the sBL/TLR2 signaling platform involved upstream of the TLR2 mediated signaling.