Structural basis of digoxin that antagonizes RORgamma t receptor activity and suppresses Th17 cell differentiation and interleukin (IL)-17 production

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4⁺ T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.     

Heregulin activation of ErbB2/ErbB3 signaling potentiates the integrity of airway epithelial barrier

Background

Members of the ErbB family of the receptor protein tyrosine kinase superfamily mediate heregulin (HRG)-induced cell responses. Here we investigated HRG activation of ErbB receptors, and the role of this activation in the development of the permeability barrier in airway epithelial cells (AECs).

Methods

Two airway epithelial-like cell lines, Calu-3 and 16HBE were exposed to HRG or no stimulus and were evaluated with respect to their paracellular permeability as determined by transepithelial electric resistance (TER) and fluorescein isothiocyanate (FITC)-dextran flux. Tight junctions (TJs) were assessed by immunocytochemical localization of occludin and zonula occludens-1.

Results

HRG promoted the development of the permeability barrier and TJ formation by monolayers of Calu-3 and 16HBE cells. Calu-3 cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4, on their surface. ErbB3 knockdown by small interference RNA (siRNA) blunted the effects of HRG on the permeability barrier. ErbB3 is known as a kinase-dead receptor and relies on other members of the family for its phosphorylation. To identify its heterodimerization partner, we knocked down the expression of other ErbB family receptors. We found that HRG's effect on the permeability barrier could be significantly attenuated by transfecting cells with ErbB2 siRNA but not with EGFR siRNA.

Conclusion

These results indicate that HRG activation of ErbB2/ErbB3 heterodimers is essential for regulation of the permeability barrier in AECs.     

Ambient ozone primes pulmonary innate immunity in mice

Exposure to ozone in air pollution in urban environments is associated with increases in pulmonary-related hospitalizations and mortality. Because ozone also alters clearance of pulmonary bacterial pathogens, we hypothesized that inhalation of ozone modifies innate immunity in the lung. To address our hypothesis, we exposed C57BL/6J mice to either free air or ozone, and then subsequently challenged with an aerosol of Escherichia coli LPS. Pre-exposure to ozone resulted in enhanced airway hyperreactivity, higher concentrations of both total protein and proinflammatory cytokines in lung lavage fluid, enhanced LPS-mediated signaling in lung tissue, and higher concentrations of serum IL-6 following inhalation of LPS. However, pre-exposure to ozone dramatically reduced inflammatory cell accumulation to the lower airways in response to inhaled LPS. The reduced concentration of cells in the lower airways was associated with enhanced apoptosis of both lung macrophages and systemic circulating monocytes. Moreover, both flow cytometry and confocal microscopy indicate that inhaled ozone causes altered distribution of TLR4 on alveolar macrophages and enhanced functional response to endotoxin by macrophages. These observations indicate that ozone exposure increases both the pulmonary and the systemic biologic response to inhaled LPS by priming the innate immune system.     

2-O-alpha-D-glucopyranosyl-L-ascorbic acid scavenges 1,1-diphenyl-2-picrylhydrazyl radicals via a covalent adduct formation

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC-MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable -OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.     

Differentiation of murine B cells induced by chondroitin sulfate B

A two-step culture system was used to investigate the role of chondroitin sulfate (CS) B, which is mitogenic to B cells, in differentiation of B cells. Mouse spleen B cells were incubated for 3 days with CSB in the presence of interleukin (IL)-4 and IL-5. After washing, the cells were replated at 10(5) viable cells/well and recultured without CSB in the presence of IL-4 and IL-5. CSB dose-dependently increased IgM production, the greatest enhancement being 450%. Dextran sulfate had a similar effect, whereas other glycosaminoglycans, CSA, CSC, heparin and hyaluronic acid, were marginally effective. Treatment of B cells with CSB resulted in increases in the number of IgM-secreting cells and numbers of CD138-positive cells and CD45R/B220-negative cells. CSB-induced IgM production was inhibited by the protein kinase C (PKC) inhibitor GF109203X but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. These results demonstrated that CSB promoted differentiation of B cells in the presence of IL-4 and IL-5 and suggested that PKC but not PI3K is crucial for CSB-induced IgM production.     

Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.     

Relationship Between Aortic Mineral Elements and Osteodystrophy in Mice with Chronic Kidney Disease

In chronic kidney disease (CKD), osteodystrophy and arterial calcification often coexist. However, arterial alterations have not been addressed in CKD unaccompanied by evidence of calcification. We investigated the association of phosphate (P) and calcium (Ca) accumulation in calcification-free aortas with CKD-induced osteodystrophy. Aortic accumulation of magnesium (Mg), an inhibitor of calcification, was also examined. Male mice aged 26?weeks with CKD characterized by hyperparathyroidism and hyperphosphatemia (Nx, n?=?8) and age-matched healthy male mice (shams, n?=?8) were sampled for blood, and thoracic vertebrae and aortas were harvested. Bone structure and chemicals were analyzed by microcomputed tomography and infrared microspectroscopy, respectively, and aortic accumulation of P, Ca, and Mg was evaluated by plasma-atomic emission spectrometry. Volume fractions of cortical and trabecular bones were smaller in Nx than in sham animals (P?<?0.05), attributed to cortical thinning and reduction in trabecular number, respectively. Bone chemicals were not different between the groups. No calcification was found in either group, but P, Ca, and Mg contents were higher in Nx than in shams (P?<?0.05). The mass ratio of Ca/P was lower in Nx than in shams (P?<?0.05), but that of Mg/Ca and Mg/P was not different between the groups. Aortic P and Ca contents were inversely correlated with the volume fraction of cortical bone (P?<?0.05). In conclusion, the relationship of osteodystrophy with aortic P and Ca accumulation suggests the existence of a bone-vascular axis, even in calcification-free arteries in CKD. The preservation of ratios of Mg/Ca and Mg/P despite CKD development might contribute to calcification resistance.     

Predicting subsite interactions of plasmin with substrates and inhibitors through computational docking analysis

Plasmin plays important roles in various physiological systems. The identification of inhibitors controlling its regulation represents a promising drug-discovery challenge. To develop selective inhibitors of plasmin, structural information of the binding modes is crucial. Here, a computational docking study was conducted to provide structural insight into plasmin subsite interactions with substrates/inhibitors. Predicted binding modes of two peptide-substrates (D/L-Ile-Phe-Lys), and potent and weak inhibitors (YO-2 and PKSI-527) suggested non-prime and prime subsite interactions relevant to recognition by plasmin. Predicted binding modes also correlated well with the experimental structure-activity relationships for plasmin substrates/inhibitors, namely the differences of K(M) values between the D- and L-peptide-substrates and inhibitory potencies of YO-2 and PKSI-527. In particular, interaction observed at a hydrophobic pocket near S2 and at a tunnel-shaped hydrophobic S1' was strongly suggested to be significantly involved in tight binding of inhibitors to plasmin. Our present findings may aid in the design of potent and selective plasmin inhibitors.     

Hypothermic preservation effect on mammalian cells of type III antifreeze proteins from notched-fin eelpout

Antifreeze proteins (AFPs) can bind to the surface of ice crystals and have also been suggested to protect cells from hypothermic damage. The present study reports that type III AFPs from notched-fin eelpout, Zoarces elongatus Kner, can protect cells during hypothermic storage. This fish naturally expresses at least 13 isoforms of type III AFP (denoted NfeAFPs), the primary sequences of which were categorized into SP- and QAE-Sephadex binding groups (SP- and QAE-isoforms). We compared the preservation ability between the extracted isoform mixtures (NfeAFPs) and a recombinant single SP-isoform (RcNfeAFP6). Experiments were performed using cultivated mammalian cells (HepG2) exposed to 4 °C for 24–72 h. The preserved cells were evaluated by measuring LDH released, intracellular ATP, and WST-8 reduction. It appeared that the protective effect of the 2 samples increases dose-dependently at concentrations between 2 and 10 mg/ml. Under highest soluble amount of the protein (10 mg/ml), cell viability significantly improved compared with the ordinary preservation fluid (P < 0.01). This effect was larger with NfeAFPs than with RcNfeAFP6 at the same concentration. The successful hypothermic preservation of cells using natural NfeAFPs may have a wide range of applications for cell engineering and clinical medical care.