Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.     

CHMetal(II) axial interaction in planar complexes (metal = Cu, Pd) and implications for possible environmental effects of alkyl groups at biological copper sites

Intramolecular M(II)H–C interactions (M(II)=Cu(II), Pd(II)) involving a side chain alkyl group of planar d⁸ and d⁹ metal complexes of the N-alkyl (R) derivatives of N,N-bis(2-pyridylmethyl)amine with an N₃Cl donor set were established by structural and spectroscopic methods. The methyl group from the branched alkyl group (R = 2,2-dimethylpropyl and 2-methylbutyl) axially interacts with the metal ion with the MC and MH distances of 3.056(3)–3.352(9) and 2.317(1)–2.606(1) Å, respectively, and the M–H–C angles of 122.4–162.3°. The Cu(II) complexes showing the interaction have a higher redox potential as compared with those without it, and the ¹H NMR signals of the interacting methyl group in Pd(II) complexes shifted downfield relative to the ligand signals. Dependence of the downshift values on the dielectric constants of the solvents used indicated that the M(II)H–C interaction is mainly electrostatic in nature and may be regarded as a weak hydrogen bond. Implications for possible environmental effects of the leucine alkyl group at the type 1 Cu site of fungal laccase are also discussed.     

Regulation of arsenic trioxide-induced cellular responses by Mnk1 and Mnk2

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As(2)O(3) induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38alpha MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1(-/-) or Mnk2(-/-), or double Mnk1(-/-)Mnk2(-/-) knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.     

Reconstitution of bacteriorhodopsin into cyclic lipid vesicles

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.     

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3′ end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either α-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.     

Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species

For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with ¹²⁵I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with ¹²⁵I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K_d (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R_t (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.     

Antagonistic and agonistic effects of indigoids on the transformation of an aryl hydrocarbon receptor

Halogenated and polycyclic aromatic hydrocarbons, exogenous ligands of the aryl hydrocarbon receptor (AhR), cause various toxicological effects through the transformation of the AhR. In this study, we investigated the antagonistic effects of indigoids on the transformation in addition to their agonistic ones. In a cell-free system, indigoids induced the transformation dose-dependently, but suppressed the transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the binding of 3-methylcholanthrene to the AhR. In mouse hepatoma Hepa-1c1c7 cells, indigoids, especially indirubin, suppressed the transformation and expression of CYP1A1 by inhibiting the translocation of AhR into the nucleus. When orally administered to mice at 10 mg/kg BW/day for three successive days, indigoids did not induce AhR transformation and expression of the CYP1A subfamily in the liver, while indirubin and indigo upregulated quinone reductase activity. These results indicate that indigoids are able to bind to the AhR as ligands and exhibit antagonistic effects at lower concentrations in mammalian cells.     

Antagonizing dopamine D1-like receptor inhibits Th17 cell differentiation: preventive and therapeutic effects on experimental autoimmune encephalomyelitis

Five types of dopamine receptors, termed D1 to D5, have been identified to date. The D1 and D5 receptors form the D1-like group that couples with the Gαs class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Gαi class of G proteins. A D2-like-receptor (D2-like-R) antagonist L750667 induced dendritic cell (DC)-mediated Th17 differentiation. In contrast, a D1-like-R antagonist SCH23390 inhibited DC-mediated Th17 differentiation. The D1-like-Rs were expressed on both DCs and T cells, whereas D2-like-Rs were marginally expressed on CD4⁺CD45RA⁺ naïve T cells. In addition, SCH23390 had the ability to prevent experimental autoimmune encephalomyelitis (EAE) in mice. Spleen cells from EAE mice showed decreased IL-17 production, when SCH23390 was administered. Adoptive transfer of DCs treated with SCH23390 successfully prevented EAE. These findings indicate that antagonizing D1-like-Rs on DCs inhibits Th17 differentiation, thereby leading to an amelioration of EAE.     

Overexpression of yccL (gnsA) and ydfY (gnsB) Increases Levels of Unsaturated Fatty Acids and Suppresses both the Temperature-Sensitive fabA6 Mutation and Cold-Sensitive secG Null Mutation of Escherichia coli

A multicopy suppressor of the cold-sensitive secG null mutation was isolated. The suppressor contained sfa and yccL, the former of which has been reported to be a multicopy suppressor of the fabA6 mutation carried by a temperature-sensitive unsaturated fatty acid auxotroph. Subcloning of the suppressor gene revealed that yccL, renamed gnsA (secG null mutant suppressor), was responsible for the suppression of both the secG null mutation and the fabA6 mutation. In contrast, the sfa gene did not suppress the fabA6 mutation. The ydfY (gnsB) gene, encoding a protein which is highly similar to GnsA, also suppressed both the secG null mutation and the fabA6 mutation. Although both gnsA and gnsB are linked to cold shock genes, the levels of GnsA and GnsB did not exhibit a cold shock response. A gnsA-gnsB double null mutant grew normally under all conditions examined; thus, the in vivo functions of gnsA and gnsB remain unresolved. However, overexpression of gnsA and gnsB stimulated proOmpA translocation of the secG null mutant at low temperature and caused a significant increase in the unsaturated fatty acid content of phospholipids. Taken together, these results suggest that an increase in membrane fluidity due to the increase in unsaturated fatty acids compensates for the absence of the SecG function, especially at low temperature.