The Toll/IL-1 receptor (TIR) domains are crucial signaling modules during innate immune responses involving the Toll-like receptors (TLRs) and IL-1 receptor (IL-1R). Myeloid differential factor 88 (MyD88) is a central TIR domain-containing adapter molecule responsible for nearly all TLR-mediated signaling and is targeted by a TIR domain-containing protein C (TcpC) from virulent uropathogenic Escherichia coli, a common human pathogen. The mechanism of such molecular antagonism has remained elusive. We present the crystal structure of the MyD88 TIR domain with distinct loop conformations that underscore the functional specialization of the adapter, receptor, and microbial TIR domains. Our structural analyses shed light on the genetic mutations at these loops as well as the Poc site. We demonstrate that TcpC directly associates with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE, and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provide a molecular basis for the subversion of TLR signaling by the uropathogenic E. coli virulence factor TcpC and furnish a framework for the design of novel therapeutic agents that modulate immune activation.innate immune cells | bacterial pathogens
Toll-like receptor 4 (TLR4) induces an innate immune response in mammals by recognizing lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria. In this study, we show that tyrosine kinase Syk constitutively associates with TLR4 in THP-1 cells. As previously reported in peripheral blood mononuclear cells, TLR4 gets inducibly tyrosine phosphorylated upon LPS engagement in THP-1 cells. Piceatannol, a pharmacological inhibitor of the tyrosine kinase Syk, abrogates TLR4 tyrosine phosphorylation at low doses. The kinetics of TLR4 tyrosine phosphorylation in THP-1 cells coincides with an early wave of Syk tyrosine phosphorylation. Additionally, serine threonine kinase interleukin-1 (IL1) receptor-associated kinase 1 (IRAK-1) is transiently recruited to the complex containing adaptor molecule MyD88, TLR4 and Syk within 1 min of LPS engagement and dissociates by 30 min. Finally, the inhibition of Syk with piceatannol has no effect on LPS-mediated release of cytokines IL6, IL1b, tumor necrosis factor-a, neither on chemokines macrophage inhibitory protein (MIP)1a, MIP1b, monocyte chemoattractant protein -1, IL8, Groa and RANTES. However, IL10 and IL12p40 releases are significantly inhibited. Our findings implicate Syk as a novel modulator of LPS-mediated TLR4 responses in human monocytic cells and shed insight into the kinetics of early complex formation upon LPS engagement. The early detection of pathogens is critical for protection from infection. 1 Toll-like receptors (TLR) are pathogen recognition receptors that play an important role in sensing conserved molecular signatures of pathogens and inducing antimicrobial immunity in the host. 1,2 Toll-like receptor 4 (TLR4), as the receptor for the Gramnegative bacterial product lipopolysaccharide (LPS), is the prototypical member of the family of type I transmembrane receptors characterized by an extracellular leucine repeat domain and an intracellular Toll/IL1 receptor (TIR) domain. The TIR domain is responsible for signaling and binds to TIR-containing adaptors such as MyD88 and Mal. Receptor engagement through LPS allows subsequent recruitment and activation of IL1 receptor associated kinase 1 (IRAK-1) and 4 to the TLR4-MyD88 complex, and the association of IRAK-1 with tumor necrosis factor (TNF) receptor activating factor 6 (TRAF 6), triggering its phosphorylation. [3][4][5] This cascade culminates in the activation of nuclear factor-kB (NFkB), mitogen-activated protein kinases (MAPK) and interferon regulatory factor 3-mediated gene transcription, and the subsequent production and release of proinflammatory cytokine such as IL1b, TNFa and chemokines such as IL8 and monocyte chemoattractant protein (MCP)-1.Strong evidence indicates that protein tyrosine kinases play critical roles in LPS signaling. Within minutes of LPS stimulation, numerous proteins have been reported to be inducibly tyrosine phosphorylated, in particular, Src family of protein kinases, Hck, Fgr and Lyn, 6 C-terminal src kinase, 7 Bruton's tyrosine kinase (Btk), 8 Pyk2 and Paxi...
Background:The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Results:The crystal structures of the TIR domains from TcpB and TIRAP reveal similar folds and distinct features. Conclusion: TcpB may mimic the function of TIRAP through their similar TIR domain structures. Significance: These findings suggest mechanisms of bacterial mimicry of host signaling adaptor proteins.
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