1. Toll-Like Receptors (TLR)

2. Toll-Like Receptor Signaling Toll receptor initially discovered in Drosophila as important receptor in dorso-ventral embryonic pattern –Toll mutants refers to the fact that these mutants could not establish a proper dorsal-ventral axis –Toll in German means ‘great’, apparently this was one of the words describing the scientists’ enthusiasm after observing the mutant flies Hoffman and colleagues showed that Toll-mutant flies susceptible to fungal infections Mammalian homologues discovered and designated as Toll-Like Receptors (TLR) TLRs recognize specific patterns in pathogens which are not observed in mammals

4. Toll-Like Receptors

8. Large molecules found in outer membrane of Gram- negative bacteria Comprised of a lipid and saccharide component Highly immunogenic Recognized by TLR4 Can cause septic shock and lead to death Often referred to as Endotoxin since it is not secreted but is a byproduct of bacterial lysis Great variability among different bacterial strains LipoPolySaccharides (LPS)

10. TLR3, TLR7 and TLR8 detect viral nucleic acids Found in intracellular membranes since viral nucleic acids are endogenously generated Note the TIR domains of these TLRs face the cytosol of the cell Poly I:C is an agonist for TLR3 that mimics binding of dsRNA to TLR3 Viral Nucleic Acids

11.  Cytoplasmic tails of TLRs show similarities to IL-1 receptor (TIR)  Common adaptor to TLRs is MyD88  Crucial proline residue in all TLR TIR domains, except TLR3  If substituted with histidine, no signaling occurs  All TLRs likely have a MyD88 pathway (TLR3 is an exception)  TLR4 has a MyD88 independent pathway as well Cytoplasmic TIR domain TLRs and TIR Domain

12.  MyD88 knockout mice have no response to LPS  MyD88 is essential to all inflammatory signaling pathways  MyD88s, a splice variant of MyD88 down regulates the inflammatory response  MyD88 interacts with TIR domain of TLR and recruits IRAK-4, IRAK- 1 and TRAF-6 MyD88 Adaptor

13. 4 IRAKs known, IRAK1, IRAK-2, IRAK-M and IRAK-4 IRAK are serine/threonine kinases IRAK-4 phosphorylates IRAK-1 IRAK-M plays an inhibitory role in TLR signaling IRAK-4 phosphorylates IRAK-1 TRAF6 is a member of the TNF receptor associated factor (TRAF) family TRAF6 interacts with IRAK-1 and gets activated Release of TRAF6/IRAK-1 ensues IRAK and TRAF6

14. TRAF6/IRAK-1 complex associates with 3 proteins –TAK1 (TGF-B activated kinase) –TAB1 (TAK1 binding proteins) –TAB2 (TAK1 binding proteins) Large complex associates with membrane Eventually IRAK-1 stays in membrane while TRAF6/TAK1/TAB1/TAB2 move to cytosol E2 Ligases such as Ubc13 and Uev1A join further enlarging complex IRAK and TRAF6 Release

15. TAK-1 Activation The enlarged complex that includes –TRAF6, TAK1, TAB1, TAB2, Ubc13, Uev1A activate TAK1 Activated TAK1 phosphorylates IKK complex Activated TAK1 can also phosphorylate MAP Kinases IKK complex consists of IKK ,  and  /NEMO I  B phosphorylation results in NF-  B translocation to nucleus

21. MyD88 Independent Pathway  MyD88 Knock out mice do not produce inflammatory cytokines such as TNF-   However with TLR4 stimulation NF-  B and JNK delayed activity occurs  This strongly suggests the existence of 2 pathways in TLR signaling  a MyD88 dependent pathway (early)  a MyD88 independent pathway (late)  TLR3 stimulation also exchibits a MyD88 independent pathway TLR4

22. Figures obtained from: Takeda and Akira, 2004 Wikipedia Kuby, 6 th edition Acknowledgements