Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3)

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2–glycans with the conserved Lewis^X binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam₂CSK₄ effectively, yet allows SSL3 to bind to an already formed TLR2–Pam₂CSK₄ complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2–lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.In recent years, Staphylococcus aureus has become a major health threat to both humans and domestic animals. It is found as a commensal bacterium in ∼30% of the human population, but when it becomes infectious it can cause a wide diversity of diseases, ranging from mild skin infections to life-threatening invasive conditions such as pneumonia and sepsis (1). Increased antibiotic resistance and a high amount of virulence factors secreted by S. aureus contribute to its emergence as a pathogen. Among these secreted virulence factors are the staphylococcal superantigen-like proteins (SSLs), a family of 14 proteins located on two genomic clusters (2–4). Recently, we and others identified SSL3 as a potent inhibitor of Toll-like receptor 2 (TLR2) (5, 6), an innate immunity receptor that is a dominant factor in immune recognition of S. aureus (7–10).TLR2 belongs to a family of 10 homologous innate immunity receptors that are activated by pathogen-associated molecular patterns (PAMPs) (11). TLR2 binds bacterial lipopeptides and lipoproteins. Subsequent formation of heterodimers with TLR1 or TLR6 leads to MyD88-dependent activation of the NF-κB pathway (12). TLR2 has dual ligand specificity that is determined by its dimerization partner; stimulation by diacyl lipopeptides from Gram-positive bacteria, including S. aureus, induces the formation of heterodimers with TLR6 (13), whereas triacyl lipopeptides from Gram-negative bacteria initiate formation of TLR2–TLR1 dimers (14). The structural basis for lipopeptide specificity was revealed by crystal structures of TLR2–TLR1 and TLR2–TLR6 complexes with their respective lipopeptide analogs Pam₃CSK₄ and Pam₂CSK₄: TLR2 binds two lipid tails in a large hydrophobic pocket, whereas the third lipid tail of triacyl lipopeptides is accommodated by a smaller pocket present in TLR1, but not in TLR6 (15, 16).The family of SSL proteins, including SSL3, share structural similarities to superantigens, but lack superantigenic activity. Interestingly, the functions that have been discovered for SSLs so far have all been linked to immune evasion. SSL5 inhibits neutrophil extravasation (17, 18) and phagocyte function (19, 20), SSL7 binds IgA and inhibits complement (21), and SSL10 inhibits IgG1-mediated phagocytosis (22, 23), blood coagulation (24), and the chemokine receptor CXCR4 (25). In addition to SSL3, also weak TLR2 inhibitory activity was observed for SSL4 (5), but it remains unknown whether that is its dominant function. This variety of immunomodulatory molecules and functions reflects the importance of the different components of our innate immune system in the defense against S. aureus (26).In this study we determined the crystal structures of SSL3 and the SSL3–TLR2 complex. In combination with mutagenesis and binding studies, our data provide a novel working mechanism of a functional TLR2 antagonist.     

Conformational coupling between the active site and residues within the KC-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K^C-channel is a conserved glutamate in subunit III. However, the majority of the K^C-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K^C-channel, was found to depend on the conformation of Y241^Vc, located in subunit I at the interface with subunit III. Mutations of Y241^Vc (to A/F/H/S) in the Vibrio cholerae cbb₃ eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b₃. The data suggest a linkage between residues lining the K^C-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241^Vc and the active site cross-linked Y255^Vc, as well as two Cu_B histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.Nearly all aerobic organisms rely on one or more heme-copper oxygen reductases (HCOs) as the terminal enzyme in their respiratory chains. These enzymes are virtually all proton pumps, coupling the generation of a proton motive force to the chemistry of reducing O₂ to water (1–3). Critical components of these enzymes include proton-conducting channels, which are conserved structures within each major family of HCOs (A, B, and C families) (4–6). These proton channels are defined by conserved polar residues, as well as internal water molecules that provide a hydrogen-bonded pathway for proton diffusion within the protein by a Grotthus-type mechanism (7–9). The channels are required to provide pathways both for chemical protons, which are consumed at the enzyme active site to form water, and for pumped protons, which are transported across the membrane. For prokaryotic A-family HCOs, there are two well-defined proton-input channels leading from the bacterial cytoplasm to the vicinity of the active site, designated the D-channel and the K-channel, respectively (10, 11).For each O₂ consumed, four chemical protons are taken from the bacterial cytoplasm and an additional four protons are taken from the cytoplasm and delivered to the periplasm, for Gram-negative organisms. Previous studies have demonstrated that all four of the pumped protons along with two chemical protons are taken up by the D-channel, whereas the K-channel is required for delivering two chemical protons to the active site during the portion of the catalytic cycle before the binding of O₂ (11). An H-channel has also been proposed for the mammalian cytochrome c oxidase (3), but with no equivalent in the prokaryotic or fungal HCOs.Surprisingly, the B- and C-families of oxygen reductases, which are found only in prokaryotes, each uses only one proton-input channel, located in the homologous part of the protein as the K-channel of the A-family oxygen reductases (6, 12, 13). We refer to these proton channels as the K^B- and K^C-channels for the B- and C-family enzymes, respectively. The patterns of conserved polar residues that define the K^B- and K^C-channels are unique to each family of enzymes, and both differ from the polar residues conserved within the K-channel of the A-family oxygen reductases. Because there is no equivalent of the D-channel in the B- and C-family oxygen reductases, all four of the chemical protons, as well as all pumped protons, must use the K^B- and K^C-channels. The present study explored the properties of mutations within the K^C-channel of cytochrome cbb₃ from Vibrio cholerae.The X-ray structure of the cytochrome cbb₃ (C-family oxygen reductase) from Pseudomonas stutzeri has confirmed the presence of only a single proton-conducting channel, located mostly within subunit I (CcoN) (13). Although the resolution is insufficient to define internal water molecules within the K^C-channel, molecular dynamics (MD) simulations have demonstrated that highly dynamic water wires can form within the K^C-channel and provide a pathway for proton translocation (14). Furthermore, these simulations suggest that the chemical and pumped protons share the same track but diverge after Y317^Ps (P. stutzeri)^*, at which point two pathways are possible, one for chemical protons leading to the active site and another for pumped protons leading to the propionates of heme b₃, which is near the periplasmic bulk aqueous phase.Although the bulk of the K^C-channel is located in subunit I (CcoN), mutagenesis studies of the cytochrome cbb₃ from Rhodobacter sphaeroides have demonstrated that the channel entrance is a highly conserved glutamate located on the cytoplasmic surface of subunit III (CcoP; E49^IIIPs) (15) (Fig. 1). The proton pathway from E49^IIIPs to the portion of the channel within subunit I (CcoN) is not evident. Here we used MD simulations to identify a hydrated pathway from the cytoplasmic solution and mediating direct contacts between E49^III and CcoN portion of the K^C-channel, supporting the role of E49^IIIPs in proton transfer. Site-directed mutagenesis studies of cytochrome cbb₃ from V. cholerae confirm the importance of the residues implicated as being part of the K^C-channel. An important finding is that a number of mutations within the K^C-channel also perturb the active site of the enzyme, suggesting a conformational coupling between the channel and the active site of the enzyme.Open in a separate windowFig. 1.Structure of cytochrome cbb₃ from P. stutzeri. (A) Ribbon structure with the CcoN in pink, CcoO in green, and CcoP in blue. Heme b and heme b₃ are highlighted along with the residues in the K^C-channel. (B) Enlargement of the region showing residues in the K^C-channel examined in the present study. The structure is that of PDB ID code 3MK7 from P. stutzeri (13), but the residue numbering is that from V. cholerae. Displayed water molecules were modeled using the DOWSER program (22).     

Grb-2-associated binder 1 (Gab1) regulates postnatal ischemic and VEGF-induced angiogenesis through the protein kinase A-endothelial NOS pathway

The intracellular signaling mechanisms underlying postnatal angiogenesis are incompletely understood. Herein we show that Grb-2-associated binder 1 (Gab1) plays a critical role in ischemic and VEGF-induced angiogenesis. Endothelium-specific Gab1 KO (EGKO) mice displayed impaired angiogenesis in the ischemic hindlimb despite normal induction of VEGF expression. Matrigel plugs with VEGF implanted in EGKO mice induced fewer capillaries than those in control mice. The vessels and endothelial cells (ECs) derived from EGKO mice were defective in vascular sprouting and tube formation induced by VEGF. Biochemical analyses revealed a substantial reduction of endothelial NOS (eNOS) activation in Gab1-deficient vessels and ECs following VEGF stimulation. Interestingly, the phosphorylation of Akt, an enzyme known to promote VEGF-induced eNOS activation, was increased in Gab1-deficient vessels and ECs whereas protein kinase A (PKA) activity was significantly decreased. Introduction of an active form of PKA rescued VEGF-induced eNOS activation and tube formation in EGKO ECs. Reexpression of WT or mutant Gab1 molecules in EGKO ECs revealed requirement of Gab1/Shp2 association for the activation of PKA and eNOS. Taken together, these results identify Gab1 as a critical upstream signaling component in VEGF-induced eNOS activation and tube formation, which is dependent on PKA. Of note, this pathway is conserved in primary human ECs for VEGF-induced eNOS activation and tube formation, suggesting considerable potential in treatment of human ischemic diseases.     

Activating internal tandem duplication mutations of the fms-like tyrosine kinase-3 (FLT3-ITD) at complete response and relapse in patients with acute myeloid leukemia

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are among the most frequent molecular aberrations in patients with acute myeloid leukemia. We retrospectively analyzed 324 patients with acute myeloid leukemia treated with front-line induction chemotherapy between October 2004 and March 2010. Fifty-six patients had FLT3-ITD mutation at diagnosis. Fifty-one (91%) patients with FLT3-ITD achieved complete remission. Thirteen patients had FLT3 analysis at complete remission. None had FLT3-ITD. Twenty-five (49%) patients with FLT3-ITD relapsed. Of these, 13 (52%) had FLT3-ITD at relapse (3 negative and 9 not done). Among the 201 patients without FLT3-ITD at diagnosis who achieved complete remission, 77 (38%) relapsed among whom 8 (10%) patients acquired FLT3-ITD clone. We conclude that FLT3-ITD mutations are unstable at follow up and may occur for the first time at relapse. Therefore, FLT3-ITD is not a reliable marker for minimal residual disease in acute myeloid leukemia.     

NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL

Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (P=0.003) and +12 as a sole cytogenetic abnormality (P=0.008). NOTCH1 mutations in +12 CLL associated with an approximately 2.4 fold increase in the risk of death, a significant shortening of survival (P<0.01) and proved to be an independent predictor of survival in multivariate analysis. Analogous to +12 CLL with TP53 disruption or del(11q), NOTCH1 mutations in +12 CLL conferred a significantly worse survival compared to that of +12 CLL with del(13q) or +12 only. The overrepresentation of cell cycle/proliferation related genes of +12 CLL with NOTCH1 mutations suggests the biological contribution of NOTCH1 mutations to determine a poor outcome. NOTCH1 mutations refine the intermediate prognosis of +12 CLL.     

The Dielectric Constant of Ba6−3x(Sm1−yNdy)8+2xTi18O54 (x = 2/3) Ceramics for Microwave Communication by Linear Regression Analysis

The electronics related to the fifth generation mobile communication technology (5G) are projected to possess significant market potential. High dielectric constant microwave ceramics used as filters and resonators in 5G have thus attracted great attention. The Ba_6−3x(Sm_1−yNd_y)_8+2xTi₁₈O₅₄ (x = 2/3) ceramic system has aroused people’s interest due to its underlying excellent microwave dielectric properties. In this paper, the relationships between the dielectric constant, Nd-doped content, sintering temperature and the density of Ba_6−3x(Sm_1−yNd_y)_8+2xTi₁₈O₅₄ (x = 2/3) ceramics were studied. The linear regression equation was established by statistical product and service solution (SPSS) data analysis software, and the factors affecting the dielectric constant have been analyzed by using the enter and stepwise methods, respectively. It is found that the model established by the stepwise method is practically significant with Y = −71.168 + 6.946x₁ + 25.799x₃, where Y, x₁ and x₃ represent the dielectric constant, Nd content and the density, respectively. According to this model, the influence of density on the dielectric constant is greater than that of Nd doping concentration. We bring the linear regression analysis method into the research field of microwave dielectric ceramics, hoping to provide an instructive for the optimization of ceramic technology.     

Modulation of Structure and Optical Property of Nitrogen-Incorporated VO2 (M1) Thin Films by Polyvinyl Pyrrolidone

VO₂, as a promising material for smart windows, has attracted much attention, and researchers have been continuously striving to optimize the performance of VO₂-based materials. Herein, nitrogen-incorporated VO₂ (M1) thin films, using a polyvinylpyrrolidone (PVP)-assisted sol–gel method followed by heat treatment in NH₃ atmosphere, were synthesized, which exhibited a good solar modulation efficiency (ΔT_sol) of 4.99% and modulation efficiency of 37.6% at 2000 nm (ΔT_{2000 nm}), while their visible integrated transmittance (T_lum) ranged from 52.19% to 56.79% after the phase transition. The crystallization, microstructure, and thickness of the film could be regulated by varying PVP concentrations. XPS results showed that, in addition to the NH₃ atmosphere-N doped into VO₂ lattice, the pyrrolidone-N introduced N-containing groups with N–N, N–O, or N–H bonds into the vicinity of the surface or void of the film in the form of molecular adsorption or atom (N, O, and H) filling. According to the Tauc plot, the estimated bandgap of N-incorporated VO₂ thin films related to metal-to-insulator transition (E_g1) was 0.16–0.26 eV, while that associated with the visible transparency (E_g2) was 1.31–1.45 eV. The calculated E_g1 and E_g2 from the first-principles theory were 0.1–0.5 eV and 1.4–1.6 eV, respectively. The Tauc plot estimation and theoretical calculations suggested that the combined effect of N-doping and N-adsorption with the extra atom (H, N, and O) decreased the critical temperature (τ_c) due to the reduction in E_g1.     

TGF-β from the Porcine Intestinal Cell Line IPEC-J2 Induced by Porcine Circovirus 2 Increases the Frequency of Treg Cells via the Activation of ERK (in CD4+ T Cells) and NF-κB (in IPEC-J2)

Porcine circovirus 2 (PCV2) causes immunosuppression. Piglets infected with PCV2 can develop enteritis. Given that the gut is the largest immune organ, however, the response of the gut’s immune system to PCV2 is still unclear. Here, IPEC-J2 cells with different treatments were co-cultured with PBMC or CD4⁺ T cells (Transwell). Flow cytometry and Western blotting revealed that PCV2-infected IPEC-J2 increased the frequency of CD4⁺ T cells among piglets’ peripheral blood mononuclear cells (PBMCs) and caused CD4⁺ T cells to undergo a transformation into Foxp3⁺ regulatory T cells (Treg cells) via activating CD4⁺ T ERK. Cytokines production and an inhibitor assay showed that the induction of Tregs by PCV2-infected IPEC-J2 was dependent on TGF-β induced by PCV2 in IPEC-J2, which was associated with the activation of NF-κB. Taken together, PCV2-infected IPEC-J2 activated NF-κB to stimulate the synthesis of TGF-β, which enhanced the differentiation of CD4⁺ T cells into Treg cells through the activation of ERK in CD4⁺ T cells. This information sheds light on PCV2′s function in the intestinal immune system and suggests a potential immunosuppressive mechanism for PCV2 infection.     

Investigation on Crystallization and Magnetic Properties of (Nd,Pr, Ce)2Fe14B/α-Fe Nanocomposite Magnets by Microwave Annealing Treatment

In the present work, the structures and magnetic properties of (Nd, Pr, Ce) ₂Fe₁₄B/α-Fe nanocomposite magnets were thoroughly investigated. The microwave annealing was applied to achieve a uniform heating effect and uniform grains. Microwave annealing is more favorable to obtain α-Fe phase than conventional annealing, which leads to the enhanced coercivity of hysteresis loops. The coercivity of nanocomposite magnets was 245 kA/m after annealing at 2000 W for 10 min.     

Evidence of sp2-like Hybridization of Silicon Valence Orbitals in Thin and Thick Si Grown on α-Phase Si(111)√3 × √3R30°-Bi

One-monolayer (ML) (thin) and 5-ML (thick) Si films were grown on the α-phase Si(111)√3 × √3R30°-Bi at a low substrate temperature of 200 °C. Si films have been studied in situ by reflection electron energy loss spectroscopy (REELS) and Auger electron spectroscopy, as a function of the electron beam incidence angle α and low-energy electron diffraction (LEED), as well as ex situ by grazing incidence X-ray diffraction (GIXRD). Scanning tunneling microscopy (STM), and scanning tunneling spectroscopy (STS) were also reported. The REELS spectra, taken at the Si K absorption edge (~1.840 KeV), reveal the presence of two distinct loss structures attributed to transitions 1s→π* and 1s→σ* according to their intensity dependence on α, attesting to the sp²-like hybridization of the silicon valence orbitals in both thin and thick Si films. The synthesis of a silicon allotrope on the α-phase of Si(111)√3 × √3R30°-Bi substrate was demonstrated by LEED patterns and GIXRD that discloses the presence of a Si stack of 3.099 (3) Å and a √3 × √3 unit cell of 6.474 Å, typically seen for multilayer silicene. STM and STS measurements corroborated the findings. These measurements provided a platform for the new √3 × √3R30° Si allotrope on a Si(111)√3 × √3 R30°-Bi template, paving the way for realizing topological insulator heterostructures from different two-dimensional materials, Bi and Si.