The major goal of this project is to define and characterize unknown components of the signaling pathways triggered by Toll-like receptors (TLRs). The function of TLRs on innate immune cells is to recognize pathogens and translate recognition into cell activation and appropriate effector functions, e.g. cytokine production. Ten TLRs are known in humans that specifically recognize diverse pathogen constituents such as lipopolysaccharides (LPS), peptidoglycans (PGN), nucleotides and proteins. This enormous range of specificities enables innate immune cells to respond immediately to virtually all classes of pathogens, including bacteria, fungi, viruses and protozoans. In addition to their physiological role in immune defense, TLRs are also critically involved in the development of immune pathology as elicited by inappropriate TLR stimulation during sepsis and autoimmune diseases. As such, the TLRs and their signal transduction pathways represent important targets for therapeutic intervention strategies. Several hierarchically acting key components of the TLR signal transduction pathways have been defined: TLR activation leads to recruitment and oligomerization of the adaptor protein MyD88, which binds to members of the IRAK family, which in turn recruit TRAF6. TRAF6 oligomerization induces diverse signaling pathways, eventually leading to activation of downstream effector kinases that directly activate transcription factors. Still, it is unknown whether additional proteins exist that act in between MyD88 and TRAF6. Also, little is known about the signaling events between TRAF6 and many of the downstream effector kinases. One reason for this lack of information is that it is technically difficult to explore transiently assembled signaling complexes. We have developed a robust and sensitive technique that allows purification and characterization of such signaling complexes. In this procedure, TLR-mediated dimerization of signaling proteins is mimicked by fusion of these proteins to the bacterial protein Gyrase B, which can inducibly be dimerized by the bivalent antibiotic coumermycin A1. When fused to MyD88 or TRAF6 and equipped with additional epitope tags, the highly selective signaling complexes can be purified and characterized by mass spectrometry (MS). We have demonstrated the proof of principle for this approach by identifying TRAF3 as a critical component of TLR signaling pathways. We have recently optimized the protein analysis using quantitative MS, allowing precise definition of the dynamic composition of signaling complexes. We now propose to pursue two specific aims: First, to define new components of the key TLR signaling complexes marshaled by MyD88 and TRAF6 using our dimerization technique and, second, to define the function of ABIN-1, a novel TLR signaling component recently discovered by our technique. Preliminary analysis of ABIN-1 deficient mice indicates an inflammatory phenotype with increased lymphoid organ size.
Toll-like receptor family members constitute a major pathogen recognition system, which protects us from infectious diseases via partially characterized signaling pathways. The goal of this project is to reveal the molecular composition of these pathways and to define the function of identified molecules in immune responses. This information will be crucial for our understanding of physiological immune responses, and will also be instrumental for future therapeutic approaches in diseases which are controlled by non-physiological activity of these pathways, i.e. autoimmune diseases and sepsis.
