Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic<i>Escherichia coli</i>

Snyder, Greg; Cirl, Christine; Jiang, Jiansheng; Kang, Chul; Waldhuber, Anna; Smith, Peter C.; Römmler, Franziska; Snyder, Nathaniel W.; Fresquez, Theresa; Dürr, Susanne; Tjandra, Nico; Miethke, Thomas; Xiao, Tsan Sam

doi:10.1073/pnas.1215770110

Cited by 79 publications

(113 citation statements)

References 58 publications

(100 reference statements)

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“…The BB loop peptide was identified previously as a moderately inhibitory peptide compared with peptides derived from the BB loop of the four TLR4 adapters (24). The sequence of the newly identified inhibitory TF5 peptide is not similar to any decoy peptide previously identified by us or others as a TLR4 inhibitor (20)(21)(22)(32)(33)(34). One example of dissimilarity of inhibitory sequences is the peptides derived from region 5 of TIRAP (ELCQALSRSHCR) (21) and TRIF (CLQDAIDHSGFT) that have only three amino acids positioned identically.…”

Section: Discussionmentioning

confidence: 97%

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain

Piao¹,

Ru²,

Piepenbrink

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Toll/IL-1R resistance (TIR) domain-containing adapter-inducing IFN-β (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NFκB. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation in wild-type cells. TF5 failed to inhibit LPS-induced cytokine and kinase activation in TRIF-deficient immortalized bone-marrow-derived macrophage, but was fully inhibitory in MyD88 knockout cells. TF5 does not block macrophage activation induced by TLR2, TLR3, TLR9, or retinoic acid-inducible gene 1/melanoma differentiation-associated protein 5 agonists. Immunoprecipitation assays demonstrated that TF4 binds to TLR4 but not TRIF-related adaptor molecule (TRAM), whereas TF5 binds to TRAM strongly and TLR4 to a lesser extent. Although TF5 prevented coimmunoprecipitation of TRIF with both TRAM and TLR4, site-directed mutagenesis of the TRIF B helix residues affected TRIF-TRAM coimmunoprecipitation selectively, as these mutations did not block TRIF-TLR4 association. These results suggest that the folded TRIF TIR domain associates with TRAM through the TRIF B helix region, but uses a different region for TRIF-TLR4 association. The B helix peptide TF5, however, can associate with either TRAM or TLR4. In a mouse model of TLR4-driven inflammation, TF5 decreased plasma cytokine levels and protected mice from a lethal LPS challenge. Our data identify TRIF sites that are important for interaction with TLR4 and TRAM, and demonstrate that TF5 is a potent TLR4 inhibitor with significant potential as a candidate therapeutic for human sepsis.signaling complex assembly | TLR4 targeting | TIR domain recognition site | decoy peptides T oll-like receptors (TLRs) initiate innate immune responses by recognizing specific pathogen-associated molecules; for example, TLR4 recognizes lipopolysaccharides (LPSs) of Gramnegative bacteria (1, 2). Ligand recognition induces dimerization of cytoplasmic Toll/IL-1R resistance (TIR) domains of two receptor molecules and causes recruitment of intracellular TIR domain-containing adapters. Four adapter proteins participate in TLR4 signaling: myeloid differentiation factor 88 (MyD88) (3), TIR domain-containing adapter protein, also known as MyD88-adapterlike (TIRAP-Mal) (4, 5), TIR domain-containing adapter-inducing IFN-β, also known as TLR adaptor molecule 1 (TRIF-TICAM-1) (6, 7), and TRIF-related adaptor molecule also known as TLR adaptor molecule 2 (TRAM-TICAM-2) (8, 9). TIRAP-Mal is important for MyD88 recruitment to the signaling complex located at the plasma membrane to initiate early NF-κB and mitogenactivated protein kinase (MAPK) activation and induce "MyD88-dependent" proinflammatory cytokines, such as TNF-α and IL-1β (4, 5, 10). TRAM is important for TRIF recruitment to the endosomally located TLR4...

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Section: Discussionmentioning

confidence: 97%

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain

Piao¹,

Ru²,

Piepenbrink

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Jiang et al (38) described a murine mutation in MyD88 (I179N), called Pococurante (Poc), that exhibited deficient TLR2/1 signaling but normal TLR2/6 signaling, suggesting that TLR2 interacts with MyD88 in different ways. Using molecular dynamics simulations, Snyder et al (39) demonstrated that in WT MyD88, the BB loop is stabilized, whereas the Poc mutation potentiates its flexibility. A mobile BB loop could potentially result in a greater entropic cost to obtain stable TIR-TIR interactions and lead to deficient signaling (39).…”

Section: C29 Blocks Tlr2 Bacterial Agonist-induced Proinflammatory Genementioning

confidence: 99%

“…Using molecular dynamics simulations, Snyder et al (39) demonstrated that in WT MyD88, the BB loop is stabilized, whereas the Poc mutation potentiates its flexibility. A mobile BB loop could potentially result in a greater entropic cost to obtain stable TIR-TIR interactions and lead to deficient signaling (39). Differences observed in C29-mediated and o-vanillin-mediated inhibition of hTLR2 and mTLR2 signaling and mutagenesis studies of BB loop pocket residues may reflect differential use of the BB loop involving TIR1, TIR2, TIR6, and MyD88 molecular interactions.…”

Section: C29 Blocks Tlr2 Bacterial Agonist-induced Proinflammatory Genementioning

confidence: 99%

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Mistry¹,

Laird²,

Schwarz³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C 16 H 15 NO 4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.small molecule inhibitor | BB loop | TLR2 pocket | CADD T oll-like receptors (TLRs) are type I transmembrane receptors that detect conserved "pathogen-associated molecular patterns" from microbes, as well as host-derived "danger-associated molecular patterns" (1). TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2, 3), present in gram-positive and gram-negative bacteria (4-9).Ligand engagement of TLR2/1 or TLR2/6 activates the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (i.e., nuclear translocation of NF-κB, activation of MAPKs), resulting in production of proinflammatory cytokines (10). Dysregulated TLR2 signaling has been implicated in numerous diseases (e.g., sepsis, atherosclerosis, tumor metastasis, ischemia/reperfusion injury) (11)(12)(13)(14). Several inhibitors of TLR2 signaling have been developed (15-18), yet none is licensed for human use. A better understanding of the Toll/IL-1 receptor resistance (TIR) domain interactions involved in TLR2 signaling could lead to novel therapeutic agents.Both TLRs and adapter proteins contain a cytoplasmic TIR domain that mediates homotypic and heterotypic interactions ...

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“…11 Three-dimensional structures of TIR domains from MyD88, 11,12 22 Whether and how these results relate to a physiologically relevant process has not been determined so far.…”

Section: Introductionmentioning

confidence: 99%

“…Most TIR domains comprise 5 a helices (A-E) surrounding 5 central b strands (A-E). Three regions that are functionally important and highly conserved in the amino acid sequence are defined as boxes 1 to 3 located at the bA strand, BB loop, and aE helix, respectively.10 Although the hydrophobic core of TIR domains is conserved, the surface residues vary among TIR domains of different signaling proteins, modulating their specificity.11 Three-dimensional structures of TIR domains from MyD88, 11,12 22 Whether and how these results relate to a physiologically relevant process has not been determined so far.Given the importance of the MyD88 TIR domain in cell activation in the context of B-cell malignancies, our study aimed to elucidate the molecular mechanism underlying activation of MyD88. Because lymphoma-associated mutations in the MyD88 TIR domain cause the constitutive activation of MyD88, we surmised that this is primarily a consequence of an enhanced interaction at the level of the MyD88 TIR domain.…”

mentioning

confidence: 99%

Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization

Avbelj

Wolz

Fekonja

et al. 2014

Blood

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Key Points• The hyperactive phenotype of lymphoma-associated mutations is caused by increased oligomerization propensity of the MyD88 TIR domain.• The TIR domain of mutants interacts with wild-type MyD88, explaining why heterozygous mutation could be sufficient as a driver mutation.Myeloid differentiation 88 (MyD88) is the key signaling adapter of Toll-like and interleukin-1 receptors. Recurrent lymphoma-associated mutations, particularly Leu265Pro (L265P), within the MyD88 Toll/interleukin-1 receptor (TIR) domain sustain lymphoma cell survival due to constitutive nuclear factor kB signaling. We found that mutated TIR domains displayed an intrinsic propensity for augmented oligomerization and spontaneous formation of cytosolic Myddosome aggregates in lymphoma cell lines, mimicking the effect of dimerized TIR domains. Blocking of MyD88 oligomerization induced apoptosis. The L265P TIR domain can recruit the endogenous wild-type MyD88 for oligomer formation and hyperactivity. Molecular dynamics simulations and analysis of additional mutations suggest that constitutive activity is caused by allosteric oligomerization. (Blood. 2014;124(26):3896-3904) IntroductionMyeloid differentiation 88 (MyD88) is a pivotal signaling protein in the innate immune system, participating in Toll-like receptor (TLR) signaling pathways during a host's response to infection. 1 Patients with germline MYD88 loss-of-function mutations suffer from severe susceptibility to pyogenic bacterial infection during childhood and only survive into adulthood on a strict therapy of antibiotics.2 Conversely, somatic MyD88 gain-of-function mutations were recently discovered in diffuse large B-cell lymphoma (DBLCL), Waldenström's macroglobulinemia (WM), and chronic lymphocytic leukemia (supplemental Tables 1 and 2 available on the Blood Web site). These data suggest that such mutations, particularly the most prominent Leu265Pro (L265P) mutation, are oncogenic driver mutations that sustain B-cell survival and thus oncogenesis. Indeed, MyD88-mutated cells (cell lines and primary cells) could be selectively killed by ablation of MyD88 downstream signaling using short hairpin RNA or pharmacologic inhibition. 3,4 Although hyperactivation of nuclear factor kB (NF-kB) has been described as a key feature of MyD88-mutated B cells, the molecular mechanism of how the amino acid alterations in lymphomaassociated MyD88 mutations exert this phenotype has to be resolved.MyD88 is composed of an N-terminal death domain (DD) 5 and a highly conserved C-terminal Toll/interleukin-1 receptor (TIR) domain. 6 Whereas the DD and an intermediate linker domain 7 are responsible for downstream signal propagation via kinases of the interleukin-1 receptor-associated kinases (IRAKs), 8 the MyD88 TIR domain integrates signals from upstream TLR and interleukin-1 receptor. Association of TLRs mediated by their agonists triggers the formation of TIR-domain dimers, which results in downstream signaling and expression of genes involved in the host defense system. 1,9 TIR domains comprise 135-...

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Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenicEscherichia coli

Cited by 79 publications

References 58 publications

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization

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