TRAM is required for TLR2 endosomal signaling to type I IFN induction.

Detection of microbes by TLRs on the plasma membrane leads to the induction of proinflammatory cytokines such as TNF-α, via activation of NF-κB. Alternatively, activation of endosomal TLRs leads to the induction of type I IFNs via IFN regulatory factors (IRFs). TLR4 signaling from the plasma membrane to NF-κB via the Toll/IL-1R (TIR) adaptor protein MyD88 requires the TIR sorting adaptor Mal, whereas endosomal TLR4 signaling to IRF3 via the TIR domain-containing adaptor-inducing IFN-ß (TRIF) requires the TRIF-related adaptor molecule (TRAM). Similar to TLR4 homodimers, TLR2 heterodimers can also induce both proinflammatory cytokines and type I IFNs. TLR2 plasma membrane signaling to NF-κB is known to require MyD88 and Mal, whereas endosomal IRF activation by TLR2 requires MyD88. However, it was unclear whether TLR2 requires a sorting adaptor for endosomal signaling, like TLR4 does. In this study, we show that TLR2-dependent IRF7 activation at the endosome is both Mal- and TRAM-dependent, and that TRAM is required for the TLR2-dependent movement of MyD88 to endosomes following ligand engagement. TRAM interacted with both TLR2 and MyD88, suggesting that TRAM can act as a bridging adapter between these two molecules. Furthermore, infection of macrophages lacking TRAM with herpes viruses or the bacterium Staphylococcus aureus led to impaired induction of type I IFN, indicating a role for TRAM in TLR2-dependent responses to human pathogens. Our work reveals that TRAM acts as a sorting adaptor not only for TLR4, but also for TLR2, to facilitate signaling to IRF7 at the endosome, which explains how TLR2 is capable of causing type I IFN induction.

Poxviral protein A52 stimulates p38 mitogen-activated protein kinase (MAPK) activation by causing tumor necrosis factor receptor-associated factor 6 (TRAF6) self-association leading to transforming growth factor ß-activated kinase 1 (TAK1) recruitment.

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.

Poxviral protein A46 antagonizes Toll-like receptor 4 signaling by targeting BB loop motifs in Toll-IL-1 receptor adaptor proteins to disrupt receptor:adaptor interactions.

Toll-like receptors (TLRs) have an anti-viral role in that they detect viruses, leading to cytokine and IFN induction, and as such are targeted by viruses for immune evasion. TLR4, although best known for its role in recognizing bacterial LPS, is also strongly implicated in the immune response to viruses. We previously showed that the poxviral protein A46 inhibits TLR4 signaling and interacts with Toll-IL-1 receptor (TIR) domain-containing proteins of the receptor complex. However the exact molecular mechanism whereby A46 disrupts TLR4 signaling remains to be established, and may yield insight into how the TLR4 complex functions, since viruses often optimally target key residues and motifs on host proteins for maximal efficiency. Here we show that A46 targets the BB loop motif of TIR proteins and thereby disrupts receptor:adaptor (TLR4:Mal and TLR4:TRAM), but not receptor:receptor (TLR4:TLR4) nor adaptor:adaptor (Mal:MyD88, TRAM:TRIF, and Mal:Mal) TIR interactions. The requirement for an intact BB loop for TIR adaptor interactions correlated with the protein:protein interfaces antagonized by A46. We previously discovered a peptide fragment derived from A46 termed VIPER (Viral Inhibitory Peptide of TLR4), which specifically inhibits TLR4 responses. Here we demonstrate that the region of A46 from which VIPER is derived represents the TLR4-specific inhibitory motif of the intact protein, and is essential for A46:TRAM interactions. This study provides the molecular basis for pathogen subversion of TLR4 signaling and clarifies the importance of TIR motif BB loops, which have been selected for viral antagonism, in the formation of the TLR4 complex.

Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence.

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence.

Characterisation of viral proteins that inhibit Toll-like receptor signal transduction.

Toll-like receptor (TLR) signalling involves five TIR adapter proteins, which couple to downstream protein kinases that ultimately lead to the activation of transcription factors such as nuclear factor-kappaB (NF-kappaB) and members of the interferon regulatory factor (IRF) family. TLRs play a crucial role in host defence against invading microorganisms, and highlighting their importance in the immune system is the fact that TLRs are targeted by viral immune evasion strategies. Identifying the target host proteins of such viral inhibitors is very important because valuable insights into how host cells respond to infection may be obtained. Also, viral proteins may have potential as therapeutic agents. Luciferase reporter gene assays are a very useful tool for the analysis of TLR signalling pathways, as the effect of a putative viral inhibitor on a large amount of signals can be examined in one experiment. A basic reporter gene assay involves the transfection of cells with a luciferase reporter gene, along with an activating expression plasmid, with or without a plasmid expressing a viral inhibitor. Induction of a signalling pathway leads to luciferase protein expression, which is measured using a luminometer. Results from these assays can be informative for deciding which host proteins to test for interactions with a viral inhibitor. Successful assays for measuring protein-protein interactions include co-immunoprecipitations (Co-IPs) and glutathione-S-transferase (GST)-pulldowns. Co-IP experiments involve precipitating a protein out of a cell lysate using a specific antibody bound to Protein A/G sepharose. Additional molecules complexed to that protein are captured as well and can be detected by Western blot analysis. GST-pulldown experiments are similar in principle to Co-IPs, but a bait GST-fusion protein complexed to glutathione-sepharose (GSH) beads is used to pull down interaction partners instead of an antibody. Again, complexes recovered from the beads are analysed by Western blotting.

Peptide identification from a Porphyra dioica protein hydrolysate with antioxidant, angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities.

A Porphyra dioica protein extract was enzymatically hydrolysed and then fractionated using semi-preparative reverse-phase high performance chromatography. The hydrolysate and its fractions were tested for their oxygen radical absorbance capacity (ORAC) along with their angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. The most potent fraction was analysed by liquid chromatography mass spectrometry. Eight peptide sequences were selected for synthesis based on their structure-activity criteria for bioactivity. Asp-Tyr-Tyr-Lys-Arg showed the highest ORAC activity (4.27 ± 0.15 µmol Trolox equivalent per µM). Thr-Tyr-Ile-Ala had the highest ACE inhibitory activity (IC50: 89.7 ± 7.10 µM). Tyr-Leu-Val-Ala was the only peptide showing DPP-IV inhibitory activity (IC50: 439 ± 44 µM). Apart from Asp-Tyr-Tyr-Lys-Arg and Thr-Tyr-Ile-Ala, which displayed increased ORAC activity, the bioactivities of the peptides were either maintained or decreased following in vitro simulated gastrointestinal digestion. The results indicate that P. dioica-derived peptides may have potential applications as health enhancing ingredients.

The TLR signaling adaptor TRAM interacts with TRAF6 to mediate activation of the inflammatory response by TLR4.

TLRs act as sentinels in professional immune cells to detect and initiate the innate immune response to pathogen challenge. TLR4 is a widely expressed TLR, responsible for initiating potent immune responses to LPS. TRAM acts to bridge TLR4 with TRIF, orchestrating the inflammatory response to pathogen challenge. We have identified a putative TRAF6-binding motif in TRAM that could mediate a novel signaling function for TRAM in TLR4 signaling. TRAM and TRAF6 association was confirmed by immunoprecipitation of endogenous, ectopically expressed and recombinant proteins, which was ablated upon mutation of a key Glu residue in TRAM (TRAM E183A). TRAF6 and TRAM were observed colocalizing using confocal microscopy following ectopic expression in cells and the ability of TRAM and TRAM E183A to activate luciferase-linked reporter assays was determined in HEK293 and TRAF6-deficient cells. Importantly, TRAM-deficient macrophages reconstituted with TRAM E183A display significantly reduced inflammatory TNF-α, IL-6, and RANTES protein production compared with WT TRAM. These results demonstrate a novel role for TRAM in TLR4-mediated signaling in regulating inflammatory responses via its interaction with TRAF6, distinct from its role as a bridging adaptor between TLR4 and TRIF.

c-Jun N-terminal kinase activity supports multiple phases of 3D-mammary epithelial acinus formation.

Primary murine mammary epithelial cells cultured on a laminin-rich-extracellular matrix (ECM) require c-Jun N-terminal kinase (JNK) activity for acinus formation. Inhibition of JNK (using SP600125) or small interfering RNA-mediated knockdown of JNK1 blocked acinus formation, impaired cell polarisation and lumen clearance and allowed sustained extracellular signal-regulated kinase (ERK) phosphorylation, cell proliferation, adhesion-independent cell survival and expression of epithelial-mesenchymal transition markers. ERK inhibition abolished the effects of JNK blockade. Interestingly, inhibition of JNK from the time of cell seeding blocked cell polarisation and lumen clearance; later inhibition (≥ 6 h) only affected lumen clearance. ERK inhibition effectively protected cell polarisation but less so, lumen clearance. SP600125-treatment similarly affected acinus formation by the 'normal' human mammary epithelial MCF10A cell line. Expression of dominant-negative JNK1 in MCF10A cells also undermined acinus formation, generating large 'multi-acinar spheres' whose formation is probably driven by excessive luminal cell proliferation and cell survival. As JNK activity must be suppressed from the time of cell seeding to block cell polarisation, we studied the behaviour of MCF10A cells immediately after seeding in laminin rich matrix: we detected engagement of cells with the matrix, early polarisation, movement of cells into clusters and 'epithelial-cell- like' behaviour of clustered cells. Inhibition of JNK activity or expression of dominant-negative JNK1 allowed cell engagement to the matrix, but blocked cell polarisation and all subsequent 'behaviours'. While integrin activation occurred, tyrosine-phosphorylation of paxillin, Fak and Src was significantly damped by JNK inhibition. These results emphasise the multi-phase dependency of the organisation of mammary cells in 3D on JNK activity and suggest a 'permissive' support of ECM-integrin 'outside-in' signalling and a 'damping' of growth-factor ERK signalling as its two key cell physiological effects.

The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling.

Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll-interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction 'downstream' of TRIF but not of MyD88. SARM associated with TRIF, and 'knockdown' of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling.

Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors.

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.