The lipopolysaccharide (LPS) of Gram negative bacteria is a well-known inducer of the innate immune response. Toll-like receptor (TLR) 4 and myeloid differentiation factor 2 (MD-2) form a heterodimer that recognizes a common 'pattern' in structurally diverse LPS molecules. To understand the ligand specificity and receptor activation mechanism of the TLR4-MD-2-LPS complex we determined its crystal structure. LPS binding induced the formation of an m-shaped receptor multimer composed of two copies of the TLR4-MD-2-LPS complex arranged symmetrically. LPS interacts with a large hydrophobic pocket in MD-2 and directly bridges the two components of the multimer. Five of the six lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD-2, forming a hydrophobic interaction with the conserved phenylalanines of TLR4. The F126 loop of MD-2 undergoes localized structural change and supports this core hydrophobic interface by making hydrophilic interactions with TLR4. Comparison with the structures of tetra-acylated antagonists bound to MD-2 indicates that two other lipid chains in LPS displace the phosphorylated glucosamine backbone by approximately 5 A towards the solvent area. This structural shift allows phosphate groups of LPS to contribute to receptor multimerization by forming ionic interactions with a cluster of positively charged residues in TLR4 and MD-2. The TLR4-MD-2-LPS structure illustrates the remarkable versatility of the ligand recognition mechanisms employed by the TLR family, which is essential for defence against diverse microbial infection.

Toll-like receptors (TLRs) sense structural patterns in microbial molecules and initiate immune defense mechanisms. The structures of many extracellular and intracellular domains of TLRs have been studied in the last 10 years. These structures reveal the extraordinary diversity of TLR-ligand interactions. Some TLRs use internal hydrophobic pockets to bind bacterial ligands and others use solvent-exposed surfaces to bind hydrophilic ligands. The structures suggest a common activation mechanism for TLRs: ligand binding to extracellular domains induces dimerization of the intracellular domains and so activates intracellular signaling pathways. Recently, the structure of the death domain complex of one of the signaling adapters, myeloid differentiation factor 88 (MyD88), has been determined. This structure shows how aggregation of signaling adapters recruits downstream kinases. However, we are still far from a complete understanding of TLR activation. We need to study the structures of TLR7-10 in complex with their ligands. We also need to determine the structures of TLR-adapter aggregates to understand activation mechanisms and the specificity of the signaling pathways. Ultimately, we will have to study the structures of the complete TLR signaling complexes containing full-length receptors, ligands, signaling, and bridging adapters, and some of the downstream kinases to understand how TLRs sense microbial infections and activate immune responses against them.

CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/ CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser 132 loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38-and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di-or trimerization is necessary but not sufficient for complete activation of CD40.

OBJECTIVEAMP-activated protein kinase (AMPK) and the ATP-sensitive K+ (KATP) channel are metabolic sensors that become activated during metabolic stress. AMPK is an important regulator of metabolism, whereas the KATP channel is a regulator of cellular excitability. Cross talk between these systems is poorly understood.RESEARCH DESIGN AND METHODSRat pancreatic β-cells or INS-1 cells were pretreated for 2 h at various concentrations of glucose. Maximum KATP conductance (Gmax) was monitored by whole-cell measurements after intracellular ATP washout using ATP-free internal solutions. KATP channel activity (NPo) was monitored by inside-out patch recordings in the presence of diazoxide. Distributions of KATP channel proteins (Kir6.2 and SUR1) were examined using immunofluorescence imaging and surface biotinylation studies. Insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay.RESULTSGmax and NPo in cells pretreated with glucose-free or 3 mmol/l glucose solutions were significantly higher than in cells pretreated in 11.1 mmol/l glucose solutions. Immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of Kir6.2 without affecting the total cellular amount. Increases in Gmax and the surface level of Kir6.2 were inhibited by compound C, an AMPK inhibitor, and siAMPK transfection. The effects of glucose deprivation on KATP channels were mimicked by an AMPK activator. Glucose deprivation reduced insulin secretion, but this response was attenuated by compound C.CONCLUSIONSKATP channel trafficking is regulated by energy status via AMPK, and this mechanism may play a key role in inhibiting insulin secretion under low energy status.

An outbreak of febrile respiratory illness associated with human adenovirus (HAdV) occurred in the South Korea military during the 2014–15 influenza season and thereafter. Molecular typing and phylogenetic analysis of patient samples identified HAdV type 55 as the causative agent. Emergence of this novel HAdV necessitates continued surveillance in military and civilian populations.

Pancreatic b-cells are critical in the regulation of glucose homeostasis by controlled secretion of insulin in mammals. Activation of protein kinase A by cAMP is shown to be responsible for enhancing this pathway, which is countered by phosphodiesterase (PDE) that converts cAMP to AMP and turns off the signal. Salt-inducible kinases (SIKs) were also known to inhibit cAMP signaling, mostly by promoting inhibitory phosphorylation on CREBregulated transcription coactivators. Here, we showed that SIK1 regulates insulin secretion in b-cells by modulating PDE4D and cAMP concentrations. Haploinsufficiency of SIK1 led to the improved glucose tolerance due to the increased glucose-stimulated insulin secretion. Depletion of SIK1 promoted higher cAMP concentration and increased insulin secretion from primary islets, suggesting that SIK1 controls insulin secretion through the regulation of cAMP signaling. By using a consensus phosphorylation site of SIK1, we identified PDE4D as a new substrate for this kinase family. In vitro kinase assay as well as mass spectrometry analysis revealed that the predicted Ser 136 and the adjacent Ser 141 of PDE4D are critical in SIK1-mediated phosphorylation. We found that overexpression of either SIK1 or PDE4D in b-cells reduced insulin secretion, while inhibition of PDE4 activity by rolipram or knockdown of PDE4D restored it, showing indeed that SIK1-dependent phosphorylation of PDE4D is critical in reducing cAMP concentration and insulin secretion from b-cells. Taken together, we propose that SIK1 serves as a part of a self-regulatory circuit to modulate insulin secretion from pancreatic b-cells by controlling cAMP concentration through modulation of PDE4D activity.

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The anti-diabetic drug, metformin, exerts its action through AMP-activated protein kinase (AMPK), and Sirtuin (Sirt1) signaling. Insulin-like growth factor (IGF)-binding protein 2 (IGFBP-2) prevents IGF-1 binding to its receptors, thereby contributing to modulate insulin sensitivity. In this study, we demonstrate that metformin upregulates Igfbp-2 expression through the AMPK-Sirt1-PPARα cascade pathway. In the liver of high fat diet, ob/ob, and db/db mice, Igfbp-2 expression was significantly decreased compared to the expression levels in the wild-type mice (p < 0.05). Upregulation of Igfbp-2 expression by metformin administration was disrupted by gene silencing of Ampk and Sirt1, and this phenomenon was not observed in Pparα-null mice. Notably, activation of IGF-1 receptor (IGF-1R)-dependent signaling by IGF-1 was inhibited by metformin. Finally, when compared to untreated type 2 diabetes patients, the metformin-treated diabetic patients showed increased IGFBP-2 levels with diminished serum IGF-1 levels. Taken together, these findings indicate that IGFBP-2 might be a new target of metformin action in diabetes and the metformin-AMPK-Sirt1-PPARα-IGFBP-2 network may provide a novel pathway that could be applied to ameliorate metabolic syndromes by controlling IGF-1 bioavailability.