90 results on '"Inoue, M."'
Search Results
2. Synthesis of a cytochrome c derivative with prolonged in vivo half-life and determination of ascorbyl radicals in the circulation of the rat.
- Author
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Kunitomo, R, primary, Miyauchi, Y, additional, and Inoue, M, additional
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- 1992
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3. Expression of a hybrid Cu/Zn-type superoxide dismutase which has high affinity for heparin-like proteoglycans on vascular endothelial cells.
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Inoue, M., primary, Watanabe, N., additional, Matsuno, K., additional, Sasaki, J., additional, Tanaka, Y., additional, Hatanaka, H., additional, and Amachi, T., additional
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- 1991
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4. Refined x-ray structure of papain.E-64-c complex at 2.1-A resolution
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Yamamoto, D., primary, Matsumoto, K., additional, Ohishi, H., additional, Ishida, T., additional, Inoue, M., additional, Kitamura, K., additional, and Mizuno, H., additional
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- 1991
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5. Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) monoclonal antibody (MLS 128)
- Author
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Nakada, H., primary, Numata, Y., additional, Inoue, M., additional, Tanaka, N., additional, Kitagawa, H., additional, Funakoshi, I., additional, Fukui, S., additional, and Yamashina, I., additional
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- 1991
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6. Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density.
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Terry-Lorenzo, R T, Inoue, M, Connor, J H, Haystead, T A, Armbruster, B N, Gupta, R P, Oliver, C J, and Shenolikar, S
- Abstract
Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.
- Published
- 2000
7. Induction of chromosomal gene mutations in Escherichia coli by direct incorporation of oxidatively damaged nucleotides. New evaluation method for mutagenesis by damaged DNA precursors in vivo.
- Author
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Inoue, M, Kamiya, H, Fujikawa, K, Ootsuyama, Y, Murata-Kamiya, N, Osaki, T, Yasumoto, K, and Kasai, H
- Abstract
We have developed a new strategy for the evaluation of the mutagenicity of a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate) in Escherichia coli. 8-Hydroxydeoxyguanosine triphosphate (8-OH-dGTP) and 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP) were chosen for this study because they appear to be formed abundantly by reactive oxygen species in cells. We introduced the oxidatively damaged nucleotides into competent E. coli and selected mutants of the chromosomal lacI gene. Both damaged nucleotides induced lacI gene mutations in a dose-dependent manner, whereas unmodified dATP and dGTP did not appear to elicit the mutations. The addition of 50 nmol of 8-OH-dGTP and 2-OH-dATP into an E. coli suspension induced 12- and 9-fold more substitution mutations than the spontaneous event, respectively. The 8-OH-dGTP induced A.T --> C.G transversions, and the 2-OH-dATP elicited G.C --> T.A transversions. These results indicate that the two oxidatively damaged nucleotides are mutagenic in vivo and suggest that 8-OH-dGTP and 2-OH-dATP were incorporated opposite A and G residues, respectively, in the E. coli DNA. This new method enables the evaluation and comparison of the mutagenic potentials of damaged DNA precursors in vivo.
- Published
- 1998
8. Probing the ubiquinone reduction site of mitochondrial complex I using novel cationic inhibitors.
- Author
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Miyoshi, H, Inoue, M, Okamoto, S, Ohshima, M, Sakamoto, K, and Iwamura, H
- Abstract
A wide variety of N-methylpyridinium and quinolinium cationic inhibitors of mitochondrial complex I was synthesized to develop potent and specific inhibitors acting selectively at one of the two proposed ubiquinone binding sites of this enzyme (Gluck, M. R., Krueger, M. J., Ramsay, R. R., Sablin, S. O., Singer, T. P., and Nicklas, W. J. (1994) J. Biol. Chem. 269, 3167-3174). N-Methyl-2-n-dodecyl-3-methylquinolinium (MQ18) inhibited electron transfer of complex I at under microM order regardless of whether exogenous or endogenous ubiquinone was used as an electron acceptor. The presence of tetraphenylboron (TPB-) potentiated the inhibition by MQ18 in a different way depending upon the molar ratio of TPB- to MQ18. In the presence of a catalytic amount of TPB-, the inhibitory potency of MQ18 was remarkably enhanced, and the extent of inhibition was almost complete. The presence of equimolar TPB- partially reactivated the enzyme activity, and the inhibition was saturated at an incomplete level (approximately 50%). These results are explained by the proposed dual binding sites model for ubiquinone (cited above). The inhibition behavior of MQ18 for proton pumping activity was similar to that for electron transfer activity. The good correlation of the inhibition behavior for the two activities indicates that both ubiquinone binding sites contribute to redox-driven proton pumping. On the other hand, N-methyl-4-[2-methyl-3-(p-tert-butylphenyl)]propylpyridinium (MP6) without TPB- brought about approximately 50% inhibition at 5 microM, but the inhibition reached a plateau at this level over a wide range of concentrations. Almost complete inhibition was readily obtained at low concentrations of MP6 in the presence of TPB-. Thus MP6 appears to be a selective inhibitor of one of the two ubiquinone binding sites. With a combined use of MP6 and 2,3-diethoxy-5-methyl-6-geranyl-1,4-benzoquinone, we also provided kinetic evidence for the existence of two ubiquinone binding sites.
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- 1997
9. The amino acid residues immediately carboxyl-terminal to the tyrosine phosphorylation site contribute to interleukin 6-specific activation of signal transducer and activator of transcription 3.
- Author
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Inoue, M, Minami, M, Matsumoto, M, Kishimoto, T, and Akira, S
- Abstract
Signal transducers and activators of transcription (Stat) proteins play an important role in signaling through a variety of cytokine and growth factor receptors. Each of the Stat proteins is activated in a ligand-specific manner. Only the Src homology 2 (SH2) domains of Stat1 and Stat2 are critical for the ligand-specific activation of interferon signaling. In this study we determined the domains in Stat3 protein that contribute to interleukin 6 (IL-6)-specific phosphorylation. Based on evidence that Stat3, but not Stat1, is activated in the presence of low levels of IL-6 and soluble IL-6 receptor, we constructed various swap mutants between Stat3 and Stat1 and examined their response to IL-6 after their transient expression into COS7 cells. The region upstream of the SH2 domain was exchangeable between Stat1 and Stat3, whereas the region carboxyl-terminal to the SH2 domain of Stat3 was critical to phosphorylation by IL-6. However, unlike Stat1 and Stat2 in interferon signaling, the swap mutant in which 5 amino acid residues just carboxyl-terminal to the tyrosine phosphorylation site (Tyr705) in Stat3 was replaced by the corresponding region derived from Stat1 was not phosphorylated in response to IL-6. Substituting 1 amino acid (Lys709) at position +4 relative to Tyr705 abolished the tyrosine phosporylation of Stat3 in response to IL-6. Co-immunoprecipitation experiments demonstrated that these mutants were associated with gp130 at an extent similar to wild-type Stat3. Taken together, these results show that the amino acid residues immediately carboxyl-terminal to the tyrosine phosphorylation site are involved in IL-6-specific activation of Stat3.
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- 1997
10. Molecular cloning and genomic analysis of mouse Galbeta1, 3GalNAc-specific GalNAc alpha2,6-sialyltransferase.
- Author
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Kurosawa, N, Inoue, M, Yoshida, Y, and Tsuji, S
- Abstract
cDNA and genomic clones encoding mouse Galbeta1, 3GalNAc-specific GalNAc alpha2,6-sialyltransferase (ST6GalNAc II) were isolated, and the structure organization of the gene was determined. The predicted amino acid sequence is 57.4% identical to the chick ST6GalNAc II sequence but 33.8% identical to the chick ST6GalNA I (GalNAc alpha2, 6-sialyltransferase) sequence. The ST6GalNAc II gene is constitutively expressed in various mouse tissues but highly expressed in lactating mammary gland and adult testis. The gene contains nine exons spanning about 25 kilobases of genomic DNA and encodes a messenger RNA of 1995 nucleotides. Primer extension and S1 nuclease protection analysis of submaxillary gland mRNA showed that the transcription of the ST6GalNAc II gene starts from 68 nucleotides upstream from the translation start site. Characterization of 5'-flanking genomic regions indicated that the Galbeta1,3GalNAc-specific GalNAc alpha2,6-sialyltransferase promoter is embedded in a G+C-rich domain and contains no TATA or CAAT box but has putative binding sites for transcription factors Sp1 and AP-2. Transient transfection experiments involving luciferase reporter genes demonstrated promoter activity in NIH3T3 cells.
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- 1996
11. Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes.
- Author
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Inoue, M and Morino, Y
- Abstract
Transport of GSH was studied in isolated rat kidney cortical brush-border membrane vesicles in which gamma-glutamyltransferase had been inactivated by a specific affinity labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Transport of intact 2-3H-glycine-labeled GSH occurred into an osmotically active intravesicular space of AT-125-treated membranes. The initial rate of transport followed saturation kinetics with respect to GSH concentrations; an apparent Km of 0.21 mM and Vmax of 0.23 nmol/mg protein X 20 were calculated at 25 degrees C with a 0.1 M NaCl gradient (vesicle inside less than vesicle outside). Sodium chloride in the transport medium could be replaced with KCl without affecting transport activity. The rate of GSH uptake was enhanced by replacing KCl in the transport medium with K2SO4, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. The rate of GSH transport markedly decreased in the absence of a K+ gradient across the vesicular membranes and was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). These results indicate that GSH transport is dependent on membrane potential and involves the transfer of negative charge. The rate of GSH transport was inhibited by S-benzyl glutathione but not by glycine, glutamic acid, and gamma-glutamyl-p-nitroanilide. When incubated with [2-3H]glycine-labeled GSH, intact untreated vesicles also accumulated radioactivity; the rate of uptake was significantly higher in a Na+ gradient than in a K+ gradient. Sodium-dependent transport, but not sodium-independent uptake, was almost completely inhibited by a high concentration of unlabeled glycine. At equilibrium, most of the radioactivity which accumulated in the intravesicular space was accounted for by free glycine. These results suggest that GSH which is secreted into the tubular lumen by a specific translocase in the lumenal membranes or filtered by the glomerulus may be degraded in situ by membranous gamma-glutamyltransferase and peptidase activities which hydrolyze peptide bonds of cysteinylglycine and its derivatives. The resulting free amino acids can be reabsorbed into tubule cells by sodium-dependent transport systems in renal cortical brush-border membranes.
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- 1985
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12. Selective incorporation of polyunsaturated fatty acids into phosphatidylcholine by rat liver microsomes.
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Lands, W E, Inoue, M, Sugiura, Y, and Okuyama, H
- Abstract
The various polyunsaturated fatty acids found in cellular lipids are transferred from their coenzyme A thiol esters to phospholipid acceptors with greatly different maximal velocities. The very low apparent Km values for the thiol esters in acylating 1-acylglycerol-3-phosphocholine could not be directly measured, but their values could be estimated relative to that for arachidonate. The competitive effectiveness of various polyunsaturated acyl-CoAs was estimated by measuring the equivalent concentrations which allow incorporations equal to arachidonyl-CoA. These values help predict the way in which various polyunsaturated acyl-CoAs may be selectively esterified to membrane lipids by the 1-acylglyceol 3-phosphocholine (1-acyl-GPC) acyltransferase system of liver microsomes. The acyl-CoA esters of saturated acids, as well as those for 22:1, 22:2, and 22:3, had negligible ability to compete for the active sites of the 1-acyl-GPC acyltransferase system. The acyl-CoA esters of arachidonate (20:4n-6), eicosatrienoate (20:3n-6), eicosapentaenoate (20:5n-3), and both isomers of linolenate (18:3n-6 and n-3) were handled preferentially by the 1-acyl-GPC acyltransferases. The system from liver has a high selectivity for unsaturated acids but does not appear to discriminate among the polyunsaturated acids of the n-6 and n-3 series that serve as precursors of prostaglandins and leukotrienes.
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- 1982
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13. Intrinsic ADP-ATP exchange activity is a novel function of the molecular chaperone, Hsp70.
- Author
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Hiromura, M, Yano, M, Mori, H, Inoue, M, and Kido, H
- Abstract
Hsp70 is a multifunctional molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis and ADP-ATP exchange. We show here that, in addition to ATPase activity, purified Hsp70 free from nucleoside-diphosphate (NDP) kinase exhibits intrinsic ADP-ATP exchange activity. The rate constants for ATP hydrolysis and ATP synthesis were in a similar range at the optimum pH of 7.5-8.5 in the presence of 5 mM ATP and 0.5 mM ADP. Hsp70 exhibited a considerably strict preference for ATP as a phosphate donor, and a biased substrate specificity, unlike NDP kinase; ADP, UDP, CDP > dTDP, dCDP > GDP, dGDP. During the reaction, Hsp70 formed an acid-labile autophosphorylated intermediate, and nucleoside diphosphate-dependent dephosphorylation of the latter then occurred. These properties of Hsp70 are not identical but similar to those of NDP kinase, but are not similar to those of adenylate kinase and ATP synthase.
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- 1998
14. Bacillus stearothermophilus qcr operon encoding rieske FeS protein, cytochrome b6, and a novel-type cytochrome c1 of quinol-cytochrome c reductase.
- Author
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Sone, N, Tsuchiya, N, Inoue, M, and Noguchi, S
- Abstract
The gcr of Bacillus stearothermophilus K1041 encoding three subunits of the quinol-cytochrome c oxidoreductase (cytochrome reductase, b6c1 complex) was cloned and sequenced. The gene (qcrA) for a Rieske FeS protein of 19,144 Da with 169 amino acid residues, and the gene (qcrC) for cytochrome c1 of 27,342 Da with 250 amino acid residues were found at adjacent upstream and downstream sides of the previously reported qcrB (petB) for cytochrome b6 of subunit 25,425 Da with 224 residues (Sone, N., Sawa, G., Sone, T., and Noguchi, S. (1995) J. Biol. Chem. 270, 10612-10617). The three structural genes for thermophilic Bacillus cytochrome reductase form a transcriptional unit. In the deduced amino acid sequence for the FeS protein, the domain including four cysteines and two histidines binding the 2Fe-2S cluster was conserved. Its N-terminal part more closely resembled the cyanobacteria-plastid type than the proteobacteria-mitochondria type when their sequences were compared. The amino acid sequence of cytochrome c1 was not similar to either type; the thermophilic Bacillus cytochrome c1 is composed of an N-terminal part corresponding to subunit IV with three membrane-spanning segments, and a C-terminal part of cytochrome c reminiscent of cytochrome c-551 of thermophilic Bacillus. The subunit IV in the enzyme of cyanobacteria and plastids is the counterpart of C-terminal part of cytochrome b of proteobacteria and mitochondria. These characteristics indicate that Bacillus cytochrome b6c1 complex is unique.
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- 1996
15. Rat liver canalicular membrane vesicles. Isolation and topological characterization.
- Author
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Inoue, M, Kinne, R, Tran, T, Biempica, L, and Arias, I M
- Abstract
Canalicular plasma membranes were isolated from rat liver homogenates using nitrogen cavitation and calcium precipitation methods. Compared with homogenates, the membranes were enriched 55- to 56-fold in gamma-glutamyltransferase, aminopeptidase M, and alkaline phosphatase activities and showed very low enrichment in markers of other membranes. By electron microscopy, the membrane preparation contained neither junctional complexes nor contaminating organelles and consisted exclusively of vesicles. The presence of vesicles was also evident from the osmotic sensitivity of D-[6-3H]glucose uptake into the membrane preparation. Antisera obtained from rabbits immunized with highly purified rat kidney gamma-glutamyltransferase inhibited the transferase activity of intact or Triton X-100-solubilized membranes by 45-55%. Treatment of vesicles with anti-gamma-glutamyltransferase antisera and anti-rabbit IgG antisera increased the apparent density of the membranes during sucrose density gradient centrifugation. gamma-Glutamyltransferase and aminopeptidase M activities were selectively removed from the vesicles by limited proteolysis with papain without changing the intravesicular space or alkaline phosphatase activity of the membranes. Specific binding of anti-gamma-glutamyltransferase antibody to the outer surface of isolated hepatocytes was observed as measured by the antisera and 125I-labeled protein A; binding followed saturation kinetics with respect to antibody concentration. These data indicate that the isolated canalicular membrane vesicles are exclusively oriented right-side-out and that gamma-glutamyltransferase and aminopeptidase M are located on the luminal side of rat liver canalicular plasma membranes.
- Published
- 1983
- Full Text
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16. Phospholipase A1 acting on phosphatidic acid in porcine platelet membranes.
- Author
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Inoue, M and Okuyama, H
- Abstract
1-[14C]Palmitoyl-2-[3H]arachidonoyl-sn-glycerol 3-phosphate was hydrolyzed to form [14C]palmitic acid and 2-[3H]arachidonoyl-glycerophosphate by porcine platelet membranes. This phospholipase A1 activity was relatively specific for phosphatidic acid; the addition of several other phospholipids in equimolar amounts did not have a significant effect on the hydrolysis of radiolabeled phosphatidic acid, and the specific activity for phosphatidic acid hydrolysis was 20-fold higher than that of the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol under the conditions used. This phospholipase A1 acting on phosphatidic acid has properties different from those reported for other phospholipases and lipases present in platelets.
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- 1984
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17. Biliary transport of glutathione S-conjugate by rat liver canalicular membrane vesicles.
- Author
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Inoue, M, Akerboom, T P, Sies, H, Kinne, R, Thao, T, and Arias, I M
- Abstract
Transport of S-dinitrophenyl glutathione, a model compound of glutathione S-conjugates, was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited time-dependent uptake of [2-3H]glycine-glutathione conjugate into an osmotically sensitive intravesicular space. Inactivation of vesicle-associated gamma-glutamyltransferase by affinity labeling with L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid had no effect on the initial rate of transport. Chemical analysis revealed that the intact glutathione conjugate accounted for most vesicle-associated radioactivity, reflecting the low transferase activity in the liver and membrane vesicles. The initial rate of transport followed saturation kinetics with respect to conjugate concentrations; an apparent Km of 1.0 mM and Vmax of 1.7 nmol/mg of protein X 20 s were calculated. These results indicate that transport of the glutathione S-conjugate across the canalicular membranes is a carrier-mediated process. Sodium chloride in the transport medium could be replaced by KCl, LiCl, or choline chloride without any changes in transport activity. The rate of conjugate transport was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). The rate of conjugate uptake was enhanced by replacing KCl in the transport medium with K gluconate, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. These data indicate that conjugate transport is electrogenic and involves the transfer of negative charge. Transport of S-dinitrophenyl glutathione was inhibited by S-benzyl glutathione, oxidized glutathione, or reduced glutathione. This transport system in canalicular membranes may function in biliary secretion of glutathione S-conjugates of xenobiotics whose synthesis in hepatocytes requires glutathione S-transferases.
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- 1984
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18. Revised Amino Acid Sequence, Crystallization, and Preliminary X-ray Diffraction Analysis of Acidic Phospholipase A2 from the Venom of Agkistrodon Halys blomhoffii
- Author
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Tomoo, K, primary, Ohishi, H, additional, Ishida, T, additional, Inoue, M, additional, Ikeda, K, additional, Aoki, Y, additional, and Samejima, Y, additional
- Published
- 1989
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19. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum.
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Takai, Y., primary, Kishimoto, A., additional, Inoue, M., additional, and Nishizuka, Y., additional
- Published
- 1977
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20. Comparison of mode of activation of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.
- Author
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Takai, Y, primary, Nakaya, S, additional, Inoue, M, additional, Kishimoto, A, additional, Nishiyama, K, additional, Yamamura, H, additional, and Nishizuka, Y, additional
- Published
- 1976
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21. Guanosine 3':5'-monophosphate-dependent protein kinase from silkworm, properties of a catalytic fragment obtained by limited proteolysis.
- Author
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Inoue, M, primary, Kishimoto, A, additional, Takai, Y, additional, and Nishizuka, Y, additional
- Published
- 1976
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22. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. II. Proenzyme and its activation by calcium-dependent protease from rat brain.
- Author
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Inoue, M., primary, Kishimoto, A., additional, Takai, Y., additional, and Nishizuka, Y., additional
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- 1977
- Full Text
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23. Helicobacter pylori generates superoxide radicals and modulates nitric oxide metabolism.
- Author
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Nagata, K, Yu, H, Nishikawa, M, Kashiba, M, Nakamura, A, Sato, E F, Tamura, T, and Inoue, M
- Abstract
During studies of the bactericidal action of nitric oxide (NO), we found that it reversibly inhibited the respiration of Escherichia coli and irreversibly inhibited the respiration of Helicobacter pylori. Peroxynitrite, a reaction product of NO and superoxide, irreversibly inhibited the respiration of both H. pylori and E. coli. H. pylori, but not E. coli, generated substantial amounts of superoxide radicals. These results suggest that NO directly inhibits the respiration of E. coli whereas it rapidly reacts with endogenously generated superoxide radicals in H. pylori. The resulting peroxynitrite inactivates the respiration of H. pylori.
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- 1998
24. Revised Amino Acid Sequence, Crystallization, and Preliminary X-ray Diffraction Analysis of Acidic Phospholipase A2from the Venom of Agkistrodon Halys blomhoffii
- Author
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Tomoo, K, Ohishi, H, Ishida, T, Inoue, M, Ikeda, K, Aoki, Y, and Samejima, Y
- Abstract
The complete amino acid sequence of acidic Agkistrodon halys blomhoffiiphospholipase A2has been redetermined by a combination of manual and automatic Edman degradations. Acidic A. halys blomhoffiphospholipase is a single polypeptide chain consisting of 122 amino acids and is highly homologous in sequence with corresponding regions of phospholipase A2from a variety of sources. Prism crystals of acidic A. halys blomhoffiiphospholipase have been reproducibly grown from 2-methyl-2,4-pentanediol solution adjusted to pH 8.0 with 50 mMTris-HCl buffer in the presence of 10 mMCaCl2. The crystals belong to space group P6122 or P6522 with hexagonal unit cell dimensions of a= b= 76.22 Å and c= 76.56 Å. One molecule occupies the asymmetric unit of the crystal. The diffraction extends to at least 2.5 Å.
- Published
- 1989
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25. RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state.
- Author
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Kukimoto-Niino M, Katsura K, Ishizuka-Katsura Y, Mishima-Tsumagari C, Yonemochi M, Inoue M, Nakagawa R, Kaushik R, Zhang KYJ, and Shirouzu M
- Subjects
- Humans, GTPase-Activating Proteins metabolism, GTPase-Activating Proteins chemistry, GTPase-Activating Proteins genetics, Protein Binding, Protein Conformation, rho GTP-Binding Proteins metabolism, rho GTP-Binding Proteins chemistry, Adaptor Proteins, Signal Transducing metabolism, Adaptor Proteins, Signal Transducing chemistry, Guanine Nucleotide Exchange Factors metabolism, Guanine Nucleotide Exchange Factors chemistry, rac1 GTP-Binding Protein metabolism, rac1 GTP-Binding Protein chemistry
- Abstract
The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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26. Mechanistic characterization of disulfide bond reduction of an ERAD substrate mediated by cooperation between ERdj5 and BiP.
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Cai X, Ito S, Noi K, Inoue M, Ushioda R, Kato Y, Nagata K, and Inaba K
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- Protein Folding, HEK293 Cells, Immunoglobulin J-Chains metabolism, Protein Domains, Endoplasmic Reticulum Chaperone BiP chemistry, Endoplasmic Reticulum Chaperone BiP genetics, Endoplasmic Reticulum Chaperone BiP metabolism, Endoplasmic Reticulum-Associated Degradation, Molecular Chaperones chemistry, Molecular Chaperones genetics, Molecular Chaperones metabolism
- Abstract
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control process that eliminates misfolded proteins from the ER. DnaJ homolog subfamily C member 10 (ERdj5) is a protein disulfide isomerase family member that accelerates ERAD by reducing disulfide bonds of aberrant proteins with the help of an ER-resident chaperone BiP. However, the detailed mechanisms by which ERdj5 acts in concert with BiP are poorly understood. In this study, we reconstituted an in vitro system that monitors ERdj5-mediated reduction of disulfide-linked J-chain oligomers, known to be physiological ERAD substrates. Biochemical analyses using purified proteins revealed that J-chain oligomers were reduced to monomers by ERdj5 in a stepwise manner via trimeric and dimeric intermediates, and BiP synergistically enhanced this action in an ATP-dependent manner. Single-molecule observations of ERdj5-catalyzed J-chain disaggregation using high-speed atomic force microscopy, demonstrated the stochastic release of small J-chain oligomers through repeated actions of ERdj5 on peripheral and flexible regions of large J-chain aggregates. Using systematic mutational analyses, ERAD substrate disaggregation mediated by ERdj5 and BiP was dissected at the molecular level., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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27. RNA editing of AZIN1 coding sites is catalyzed by ADAR1 p150 after splicing.
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Xing Y, Nakahama T, Wu Y, Inoue M, Kim JI, Todo H, Shibuya T, Kato Y, and Kawahara Y
- Subjects
- Animals, Humans, Mice, Catalysis, RNA, Double-Stranded genetics, RNA, Messenger metabolism, HEK293 Cells, Mice, Knockout, RAW 264.7 Cells, Interferons pharmacology, Protein Transport, Adenosine Deaminase metabolism, Carrier Proteins genetics, Carrier Proteins metabolism, RNA Editing drug effects, RNA Editing genetics
- Abstract
Adenosine-to-inosine RNA editing is catalyzed by nuclear adenosine deaminase acting on RNA 1 (ADAR1) p110 and ADAR2, and cytoplasmic ADAR1 p150 in mammals, all of which recognize dsRNAs as targets. RNA editing occurs in some coding regions, which alters protein functions by exchanging amino acid sequences, and is therefore physiologically significant. In general, such coding sites are edited by ADAR1 p110 and ADAR2 before splicing, given that the corresponding exon forms a dsRNA structure with an adjacent intron. We previously found that RNA editing at two coding sites of antizyme inhibitor 1 (AZIN1) is sustained in Adar1 p110/Aadr2 double KO mice. However, the molecular mechanisms underlying RNA editing of AZIN1 remain unknown. Here, we showed that Azin1 editing levels were increased upon type I interferon treatment, which activated Adar1 p150 transcription, in mouse Raw 264.7 cells. Azin1 RNA editing was observed in mature mRNA but not precursor mRNA. Furthermore, we revealed that the two coding sites were editable only by ADAR1 p150 in both mouse Raw 264.7 and human embryonic kidney 293T cells. This unique editing was achieved by forming a dsRNA structure with a downstream exon after splicing, and the intervening intron suppressed RNA editing. Therefore, deletion of a nuclear export signal from ADAR1 p150, shifting its localization to the nucleus, decreased Azin1 editing levels. Finally, we demonstrated that Azin1 RNA editing was completely absent in Adar1 p150 KO mice. Thus, these findings indicate that RNA editing of AZIN1 coding sites is exceptionally catalyzed by ADAR1 p150 after splicing., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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28. Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics.
- Author
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Inoue M, Tsuji Y, Yoshimine C, Enomoto S, Morita Y, Osaki N, Kunishige M, Miki M, Amano S, Yamashita K, Kamada H, Tsutsumi Y, and Tsunoda SI
- Subjects
- Amino Acid Substitution, Animals, Cell Line, Immunoglobulin Fc Fragments genetics, Immunoglobulin G genetics, Mice, Mice, Inbred BALB C, Receptors, Tumor Necrosis Factor, Type I genetics, Receptors, Tumor Necrosis Factor, Type I immunology, Recombinant Fusion Proteins genetics, T-Lymphocytes, Regulatory immunology, Tumor Necrosis Factor-alpha genetics, Immunoglobulin Fc Fragments pharmacology, Immunoglobulin G pharmacology, Mutation, Missense, Receptors, Tumor Necrosis Factor, Type I antagonists & inhibitors, Recombinant Fusion Proteins pharmacology, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t
1/2 in vivo In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Inoue et al.)- Published
- 2020
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29. NMR-based site-resolved profiling of β-amyloid misfolding reveals structural transitions from pathologically relevant spherical oligomer to fibril.
- Author
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Xiao Y, Matsuda I, Inoue M, Sasahara T, Hoshi M, and Ishii Y
- Subjects
- Alzheimer Disease pathology, Amyloid ultrastructure, Humans, Nuclear Magnetic Resonance, Biomolecular, Protein Folding, Protein Structure, Secondary, Amyloid chemistry, Amyloid beta-Peptides chemistry, Peptide Fragments chemistry
- Abstract
Increasing evidence highlights the central role of neurotoxic oligomers of the 42-residue-long β-amyloid (Aβ42) in Alzheimer's disease (AD). However, very limited information is available on the structural transition from oligomer to fibril, particularly for pathologically relevant amyloids. To the best of our knowledge, we present here the first site-specific structural characterization of Aβ42 misfolding, from toxic oligomeric assembly yielding a similar conformation to an AD-associated Aβ42 oligomer, into a fibril. Transmission EM (TEM) analysis revealed that a spherical amyloid assembly (SPA) of Aβ42 with a 15.6 ± 2.1-nm diameter forms in a ∼30-μm Aβ42 solution after a ∼10-h incubation at 4 °C, followed by a slow conversion into fibril at ∼180 h. Immunological analysis suggested that the SPA has a surface structure similar to that of amylospheroid (ASPD), a patient-derived toxic Aβ oligomer, which had a diameter of 10-15 nm in negative-stain TEM. Solid-state NMR analyses indicated that the SPA structure involves a β-loop-β motif, which significantly differed from the triple-β motif observed for the Aβ42 fibril. The comparison of the
13 C chemical shifts of SPA with those of the fibril prepared in the above conditions and interstrand distance measurements suggested a large conformational change involving rearrangements of intermolecular β-sheet into in-register parallel β-sheet during the misfolding. A comparison of the SPA and ASPD13 C chemical shifts indicated that SPA is structurally similar to the ASPD relevant to AD. These observations provide insights into the architecture and key structural transitions of amyloid oligomers relevant for AD pathology., (© 2020 Xiao et al.)- Published
- 2020
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30. The Lon protease-like domain in the bacterial RecA paralog RadA is required for DNA binding and repair.
- Author
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Inoue M, Fukui K, Fujii Y, Nakagawa N, Yano T, Kuramitsu S, and Masui R
- Subjects
- Bacterial Proteins genetics, Binding Sites, Crystallography, X-Ray, DNA, Bacterial genetics, Mutagenesis, Site-Directed, Protein Domains, Protein Structure, Quaternary, Rec A Recombinases genetics, Thermus thermophilus genetics, Bacterial Proteins chemistry, DNA Repair, DNA, Bacterial chemistry, Rec A Recombinases chemistry, Thermus thermophilus enzymology
- Abstract
Homologous recombination (HR) plays an essential role in the maintenance of genome integrity. RecA/Rad51 paralogs have been recognized as an important factor of HR. Among them, only one bacterial RecA/Rad51 paralog, RadA, is involved in HR as an accessory factor of RecA recombinase. RadA has a unique Lon protease-like domain (LonC) at its C terminus, in addition to a RecA-like ATPase domain. Unlike Lon protease, RadA's LonC domain does not show protease activity but is still essential for RadA-mediated DNA repair. Reconciling these two facts has been difficult because RadA's tertiary structure and molecular function are unknown. Here, we describe the hexameric ring structure of RadA's LonC domain, as determined by X-ray crystallography. The structure revealed the two positively charged regions unique to the LonC domain of RadA are located at the intersubunit cleft and the central hole of a hexameric ring. Surprisingly, a functional domain analysis demonstrated the LonC domain of RadA binds DNA, with site-directed mutagenesis showing that the two positively charged regions are critical for this DNA-binding activity. Interestingly, only the intersubunit cleft was required for the DNA-dependent stimulation of ATPase activity of RadA, and at least the central hole was essential for DNA repair function. Our data provide the structural and functional features of the LonC domain and their function in RadA-mediated DNA repair., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
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31. Hypoxia reduces HNF4α/MODY1 protein expression in pancreatic β-cells by activating AMP-activated protein kinase.
- Author
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Sato Y, Tsuyama T, Sato C, Karim MF, Yoshizawa T, Inoue M, and Yamagata K
- Subjects
- AMP-Activated Protein Kinases genetics, Animals, Cell Hypoxia drug effects, Cell Hypoxia genetics, Hepatocyte Nuclear Factor 4 genetics, Humans, Insulin genetics, Insulin metabolism, Insulin Secretion, Insulin-Secreting Cells pathology, Metformin pharmacology, Mice, Mice, Obese, Proteasome Inhibitors pharmacology, AMP-Activated Protein Kinases metabolism, Down-Regulation, Hepatocyte Nuclear Factor 4 biosynthesis, Insulin-Secreting Cells metabolism
- Abstract
Hypoxia plays a role in the deterioration of β-cell function. Hepatocyte nuclear factor 4α (HNF4α) has an important role in pancreatic β-cells, and mutations of the human HNF4A gene cause a type of maturity-onset diabetes of the young (MODY1). However, it remains unclear whether hypoxia affects the expression of HNF4α in β-cells. Here, we report that hypoxia reduces HNF4α protein expression in β-cells. Hypoxia-inducible factor was not involved in the down-regulation of HNF4α under hypoxic conditions. The down-regulation of HNF4α was dependent on the activation of AMP-activated protein kinase (AMPK), and the reduction of HNF4α protein expression by metformin, an AMPK activator, and hypoxia was inhibited by the overexpression of a kinase-dead (KD) form of AMPKα2. In addition, hypoxia decreased the stability of the HNF4α protein, and the down-regulation of HNF4α was sensitive to proteasome inhibitors. Adenovirus-mediated overexpression of KD-AMPKα2 improved insulin secretion in metformin-treated islets, hypoxic islets, and ob / ob mouse islets. These results suggest that down-regulation of HNF4α could be of importance in β-cell dysfunction by hypoxia., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
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32. A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification.
- Author
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Inoue M, Ando D, Kamada H, Taki S, Niiyama M, Mukai Y, Tadokoro T, Maenaka K, Nakayama T, Kado Y, Inoue T, Tsutsumi Y, and Tsunoda SI
- Subjects
- Animals, Anti-Inflammatory Agents therapeutic use, Autoimmune Diseases drug therapy, Binding Sites, Calorimetry, Differential Scanning, Cell Line, Tumor, Cytokines metabolism, Drug Design, Female, Fibroblasts metabolism, Humans, Inflammation, Mice, Mice, Inbred BALB C, Polyethylene Glycols chemistry, Protein Conformation, Protein Engineering, Protein Multimerization, Receptors, Tumor Necrosis Factor, Type II antagonists & inhibitors, Recombinant Fusion Proteins chemistry, Mutant Proteins chemistry, Mutation, Receptors, Tumor Necrosis Factor, Type I antagonists & inhibitors, Tumor Necrosis Factor-alpha chemistry, Tumor Necrosis Factor-alpha genetics
- Abstract
Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
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33. Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders.
- Author
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Shibata N, Ohoka N, Sugaki Y, Onodera C, Inoue M, Sakuraba Y, Takakura D, Hashii N, Kawasaki N, Gondo Y, and Naito M
- Subjects
- Animals, CASP8 and FADD-Like Apoptosis Regulating Protein metabolism, Homozygote, Mice, Mice, Mutant Strains, Protein Binding, Ribonucleoproteins metabolism, CASP8 and FADD-Like Apoptosis Regulating Protein genetics, Codon, Terminator, Genetic Diseases, Inborn genetics, Mutation, Proteasome Endopeptidase Complex metabolism, Ubiquitin metabolism
- Abstract
During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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34. MUC1 protein induces urokinase-type plasminogen activator (uPA) by forming a complex with NF-κB p65 transcription factor and binding to the uPA promoter, leading to enhanced invasiveness of cancer cells.
- Author
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Mori Y, Akita K, Tanida S, Ishida A, Toda M, Inoue M, Yashiro M, Sawada T, Hirakawa K, and Nakada H
- Subjects
- Binding Sites genetics, Cell Line, Tumor, Cell Movement drug effects, Dipeptides pharmacology, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, HCT116 Cells, Humans, Immunoblotting, Immunohistochemistry, Matrix Metalloproteinase 2, Matrix Metalloproteinase 9, Mucin-1 metabolism, Neoplasm Invasiveness, Neoplasms genetics, Neoplasms metabolism, Neoplasms pathology, Oligonucleotide Array Sequence Analysis, Phenylenediamines pharmacology, Protein Binding drug effects, RNA Interference, Transcription Factor RelA antagonists & inhibitors, Transcription Factor RelA metabolism, Urokinase-Type Plasminogen Activator metabolism, Cell Movement genetics, Mucin-1 genetics, Promoter Regions, Genetic genetics, Transcription Factor RelA genetics, Urokinase-Type Plasminogen Activator genetics
- Abstract
Mucin 1 (MUC1) is overexpressed in various human malignant tumors and its expression is correlated with a poor prognosis. MUC1 engages in signal transduction by interacting with receptors for growth and differentiation factors, which contributes to the growth and survival of cancer cells. However, the mechanism by which MUC1 promotes cancer cell invasion remains unclear. Microarray analysis revealed that expression of urokinase-type plasminogen activator (uPA) was elevated in MUC1-overexpressing cells. Furthermore, up- and down-modulation of MUC1 expression was clearly correlated with the change of uPA expression. An immunochemical study showed that the distribution of uPA coincided with that of MUC1 in various human cancer tissues. The MUC1 C-terminal domain (MUC1-CD) was associated with nuclear factor-κB (NF-κB) p65 in MUC1-expressing cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that MUC1-CD existed with NF-κB p65 on the uPA promoter. Luciferase assays indicated that the uPA transcriptional activity was correlated with the level of MUC1 expression and that this MUC1-enhancing effect on the uPA transcription was abolished by introduction of mutations into the NF-κB binding sites on the uPA promoter. These results indicate that formation of the MUC1-CD and NF-κB p65 complex enhanced nuclear translocation of NF-κB p65 and subsequent occupancy of NF-κB binding region on the uPA promoter, leading to elevated transcription of uPA. We also demonstrated that uPA induced by MUC1 enhanced the matrix metalloproteinase (MMP)-2 and -9 activities, and consequently promoted cancer cell invasion. Thus, a MUC1 co-operating NF-κB signaling pathway plays a critical role in cancer cell invasion in MUC1-expressing cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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35. Negative regulation of Toll-like receptor-4 signaling through the binding of glycosylphosphatidylinositol-anchored glycoprotein, CD14, with the sialic acid-binding lectin, CD33.
- Author
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Ishida A, Akita K, Mori Y, Tanida S, Toda M, Inoue M, and Nakada H
- Subjects
- Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacology, Blotting, Western, Cell Membrane metabolism, Dendritic Cells drug effects, Dendritic Cells metabolism, HEK293 Cells, Humans, Interleukin-2 metabolism, Ligands, Lipopolysaccharide Receptors genetics, Lipopolysaccharides metabolism, Microscopy, Confocal, Models, Biological, Mutation, NF-kappa B metabolism, Phosphorylation drug effects, Protein Binding, Sialic Acid Binding Ig-like Lectin 3 genetics, Sialic Acid Binding Ig-like Lectin 3 immunology, Toll-Like Receptor 4 genetics, Lipopolysaccharide Receptors metabolism, Sialic Acid Binding Ig-like Lectin 3 metabolism, Signal Transduction, Toll-Like Receptor 4 metabolism
- Abstract
When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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36. Role of activating transcription factor 3 (ATF3) in endoplasmic reticulum (ER) stress-induced sensitization of p53-deficient human colon cancer cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through up-regulation of death receptor 5 (DR5) by zerumbone and celecoxib.
- Author
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Edagawa M, Kawauchi J, Hirata M, Goshima H, Inoue M, Okamoto T, Murakami A, Maehara Y, and Kitajima S
- Subjects
- Activating Transcription Factor 3 genetics, Apoptosis drug effects, Base Sequence, Celecoxib, Cell Line, Tumor, DNA Primers, Humans, Promoter Regions, Genetic, Pyrazoles pharmacology, Receptors, TNF-Related Apoptosis-Inducing Ligand genetics, Reverse Transcriptase Polymerase Chain Reaction, Sesquiterpenes pharmacology, Sulfonamides pharmacology, Activating Transcription Factor 3 physiology, Apoptosis physiology, Colonic Neoplasms pathology, Endoplasmic Reticulum physiology, Receptors, TNF-Related Apoptosis-Inducing Ligand metabolism, TNF-Related Apoptosis-Inducing Ligand physiology
- Abstract
Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers cell death upon binding to its ligand, TNF-related apoptosis-inducing ligand (TRAIL), and a combination of TRAIL and agents that increase the expression of DR5 is expected to be a novel anticancer therapy. In this report, we demonstrate that the stress response gene ATF3 is required for endoplasmic reticulum stress-mediated DR5 induction upon zerumbone (ZER) and celecoxib (CCB) in human p53-deficient colorectal cancer cells. Both agents activated PERK-eIF2α kinases and induced the expression of activating transcription factor 4 (ATF4)-CCAAT enhancer-binding protein (C/EBP) homologous protein, which were remarkably suppressed by reactive oxygen species scavengers. In the absence of ATF3, the induction of DR5 mRNA and protein was abrogated significantly, and this was associated with reduced cell death by cotreatment of TRAIL with ZER or CCB. By contrast, exogenous expression of ATF3 caused a more rapid and elevated expression of DR5, resulting in enhanced sensitivity to apoptotic cell death by TRAIL/ZER or TRAIL/CCB. A reporter assay demonstrated that at least two ATF/cAMP response element motifs as well as C/EBP homologous protein motif at the proximal region of the human DR5 gene promoter were required for ZER-induced DR5 gene transcription. Taken together, our results provide novel insights into the role of ATF3 as an essential transcription factor for p53-independent DR5 induction upon both ZER and CCB treatment, and this may be a useful biomarker for TRAIL-based anticancer therapy., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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37. Neuroprotective role of the basic leucine zipper transcription factor NFIL3 in models of amyotrophic lateral sclerosis.
- Author
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Tamai S, Imaizumi K, Kurabayashi N, Nguyen MD, Abe T, Inoue M, Fukada Y, and Sanada K
- Subjects
- Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis pathology, Animals, Axons pathology, Basic-Leucine Zipper Transcription Factors genetics, Disease Models, Animal, HEK293 Cells, Humans, Mice, Mice, Transgenic, Motor Neurons pathology, Amyotrophic Lateral Sclerosis metabolism, Axons metabolism, Basic-Leucine Zipper Transcription Factors metabolism, Motor Neurons metabolism, Neuroprotective Agents metabolism
- Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons. Here we show that the basic leucine zipper transcription factor NFIL3 (also called E4BP4) confers neuroprotection in models of ALS. NFIL3 is up-regulated in primary neurons challenged with neurotoxic insults and in a mouse model of ALS. Overexpression of NFIL3 attenuates excitotoxic neuronal damage and protects neurons against neurodegeneration in a cell-based ALS model. Conversely, reduction of NFIL3 exacerbates neuronal demise in adverse conditions. Transgenic neuronal expression of NFIL3 in ALS mice delays disease onset and attenuates motor axon and neuron degeneration. These results suggest that NFIL3 plays a neuroprotective role in neurons and constitutes a potential therapeutic target for neurodegeneration.
- Published
- 2014
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38. Binding of the sialic acid-binding lectin, Siglec-9, to the membrane mucin, MUC1, induces recruitment of β-catenin and subsequent cell growth.
- Author
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Tanida S, Akita K, Ishida A, Mori Y, Toda M, Inoue M, Ohta M, Yashiro M, Sawada T, Hirakawa K, and Nakada H
- Subjects
- Animals, Antigens, CD genetics, Antigens, CD immunology, Antigens, Differentiation, B-Lymphocyte genetics, Antigens, Differentiation, B-Lymphocyte immunology, Cell Line, Tumor, Coculture Techniques, HEK293 Cells, Humans, Mice, Mucin-1 genetics, Mucin-1 immunology, NIH 3T3 Cells, Protein Binding, Protein Structure, Tertiary, Sialic Acid Binding Immunoglobulin-like Lectins genetics, Sialic Acid Binding Immunoglobulin-like Lectins immunology, Stem Cell Niche genetics, Stem Cell Niche immunology, Tumor Microenvironment genetics, Tumor Microenvironment immunology, beta Catenin genetics, beta Catenin immunology, Antigens, CD metabolism, Antigens, Differentiation, B-Lymphocyte metabolism, Mucin-1 metabolism, Sialic Acid Binding Immunoglobulin-like Lectins metabolism, Signal Transduction, beta Catenin metabolism
- Abstract
Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, β-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of β-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited β-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway.
- Published
- 2013
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39. Transcriptional properties of mammalian elongin A and its role in stress response.
- Author
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Kawauchi J, Inoue M, Fukuda M, Uchida Y, Yasukawa T, Conaway RC, Conaway JW, Aso T, and Kitajima S
- Subjects
- Activating Transcription Factor 3 biosynthesis, Activating Transcription Factor 3 genetics, Animals, Dichlororibofuranosylbenzimidazole pharmacology, Elongin, Enzyme Inhibitors pharmacology, HeLa Cells, Humans, Mice, RNA Polymerase II antagonists & inhibitors, RNA Polymerase II genetics, Rats, Stress, Physiological drug effects, Stress, Physiological physiology, Transcription Factors genetics, Transcription Initiation, Genetic drug effects, RNA Polymerase II metabolism, Transcription Factors metabolism, Transcription Initiation, Genetic physiology
- Abstract
Elongin A was shown previously to be capable of potently activating the rate of RNA polymerase II (RNAPII) transcription elongation in vitro by suppressing transient pausing by the enzyme at many sites along DNA templates. The role of Elongin A in RNAPII transcription in mammalian cells, however, has not been clearly established. In this report, we investigate the function of Elongin A in RNAPII transcription. We present evidence that Elongin A associates with the IIO form of RNAPII at sites of newly transcribed RNA and is relocated to dotlike domains distinct from those containing RNAPII when cells are treated with the kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole. Significantly, Elongin A is required for maximal induction of transcription of the stress response genes ATF3 and p21 in response to several stimuli. Evidence from structure-function studies argues that Elongin A transcription elongation activity, but not its ubiquitination activity, is most important for its function in induction of transcription of ATF3 and p21. Taken together, our data provide new insights into the function of Elongin A in RNAPII transcription and bring to light a previously unrecognized role for Elongin A in the regulation of stress response genes.
- Published
- 2013
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40. Generation of N-acylphosphatidylethanolamine by members of the phospholipase A/acyltransferase (PLA/AT) family.
- Author
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Uyama T, Ikematsu N, Inoue M, Shinohara N, Jin XH, Tsuboi K, Tonai T, Tokumura A, and Ueda N
- Subjects
- Animals, Base Sequence, COS Cells, Chlorocebus aethiops, HEK293 Cells, HeLa Cells, Humans, Molecular Sequence Data, Multigene Family, Peroxisomes metabolism, Phospholipase D metabolism, Phospholipids chemistry, RNA Interference, Tandem Mass Spectrometry methods, Acyltransferases chemistry, Gene Expression Regulation, Phosphatidylethanolamines chemistry, Phospholipases A chemistry
- Abstract
Bioactive N-acylethanolamines (NAEs), including N-palmitoylethanolamine, N-oleoylethanolamine, and N-arachidonoylethanolamine (anandamide), are formed from membrane glycerophospholipids in animal tissues. The pathway is initiated by N-acylation of phosphatidylethanolamine to form N-acylphosphatidylethanolamine (NAPE). Despite the physiological importance of this reaction, the enzyme responsible, N-acyltransferase, remains molecularly uncharacterized. We recently demonstrated that all five members of the HRAS-like suppressor tumor family are phospholipid-metabolizing enzymes with N-acyltransferase activity and are renamed HRASLS1-5 as phospholipase A/acyltransferase (PLA/AT)-1-5. However, it was poorly understood whether these proteins were involved in the formation of NAPE in living cells. In the present studies, we first show that COS-7 cells transiently expressing recombinant PLA/AT-1, -2, -4, or -5, and HEK293 cells stably expressing PLA/AT-2 generated significant amounts of [(14)C]NAPE and [(14)C]NAE when cells were metabolically labeled with [(14)C]ethanolamine. Second, as analyzed by liquid chromatography-tandem mass spectrometry, the stable expression of PLA/AT-2 in cells remarkably increased endogenous levels of NAPEs and NAEs with various N-acyl species. Third, when NAPE-hydrolyzing phospholipase D was additionally expressed in PLA/AT-2-expressing cells, accumulating NAPE was efficiently converted to NAE. We also found that PLA/AT-2 was partly responsible for NAPE formation in HeLa cells that endogenously express PLA/AT-2. These results suggest that PLA/AT family proteins may produce NAPEs serving as precursors of bioactive NAEs in vivo.
- Published
- 2012
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41. Identification of novel γ-secretase-associated proteins in detergent-resistant membranes from brain.
- Author
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Hur JY, Teranishi Y, Kihara T, Yamamoto NG, Inoue M, Hosia W, Hashimoto M, Winblad B, Frykman S, and Tjernberg LO
- Subjects
- Amino Acid Sequence, Amyloid Precursor Protein Secretases isolation & purification, Amyloid beta-Peptides biosynthesis, Animals, Brain enzymology, Cell Adhesion Molecules, Neuronal genetics, Chromatography, Affinity, Gene Knockdown Techniques, HEK293 Cells, Humans, Male, Membrane Glycoproteins metabolism, Membrane Microdomains ultrastructure, Membrane Proteins chemistry, Membrane Proteins metabolism, Molecular Sequence Data, Peptide Fragments chemistry, Presenilin-1 genetics, Presenilin-1 metabolism, Protein Binding, Rats, Rats, Sprague-Dawley, Receptors, Notch metabolism, Syntaxin 1 chemistry, Syntaxin 1 metabolism, Tandem Mass Spectrometry, Voltage-Dependent Anion Channel 1 genetics, Amyloid Precursor Protein Secretases metabolism, Brain metabolism, Cell Adhesion Molecules, Neuronal metabolism, Membrane Microdomains metabolism, Voltage-Dependent Anion Channel 1 metabolism
- Abstract
In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.
- Published
- 2012
- Full Text
- View/download PDF
42. Identification and structure determination of novel anti-inflammatory mediator resolvin E3, 17,18-dihydroxyeicosapentaenoic acid.
- Author
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Isobe Y, Arita M, Matsueda S, Iwamoto R, Fujihara T, Nakanishi H, Taguchi R, Masuda K, Sasaki K, Urabe D, Inoue M, and Arai H
- Subjects
- Female, HEK293 Cells, Humans, Magnetic Resonance Spectroscopy, Male, Molecular Structure, Peritonitis chemically induced, Zymosan toxicity, Arachidonate 12-Lipoxygenase metabolism, Arachidonate 15-Lipoxygenase metabolism, Docosahexaenoic Acids metabolism, Eosinophils metabolism, Inflammation Mediators metabolism, Neutrophils metabolism, Peritonitis metabolism
- Abstract
Bioactive mediators derived from omega-3 eicosapentaenoic acid (EPA) elicit potent anti-inflammatory actions. Here, we identified novel EPA metabolites, including 8,18-dihydroxyeicosapentaenoic acid (8,18-diHEPE), 11,18-diHEPE, 12,18-diHEPE, and 17,18-diHEPE from 18-HEPE. Unlike resolvins E1 and E2, both of which are biosynthesized by neutrophils via the 5-lipoxygenase pathway, these metabolites are biosynthesized by eosinophils via the 12/15-lipoxygenase pathway. Among them, two stereoisomers of 17,18-diHEPE, collectively termed resolvin E3 (RvE3), displayed a potent anti-inflammatory action by limiting neutrophil infiltration in zymosan-induced peritonitis. The planar structure of RvE3 was unambiguously determined to be 17,18-dihydroxy-5Z,8Z,11Z,13E,15E-EPE by high resolution NMR, and the two stereoisomers were assigned to have 17,18R- and 17,18S-dihydroxy groups, respectively, using chemically synthesized 18R- and 18S-HEPE as precursors. Both 18R- and 18S-RvE3 inhibited neutrophil chemotaxis in vitro at low nanomolar concentrations. These findings suggest that RvE3 contributes to the beneficial actions of EPA in controlling inflammation and related diseases.
- Published
- 2012
- Full Text
- View/download PDF
43. Structural basis for sequence-specific DNA recognition by an Arabidopsis WRKY transcription factor.
- Author
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Yamasaki K, Kigawa T, Watanabe S, Inoue M, Yamasaki T, Seki M, Shinozaki K, and Yokoyama S
- Subjects
- Amino Acid Motifs, Arabidopsis metabolism, Arabidopsis Proteins metabolism, DNA, Plant metabolism, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Tertiary, Surface Plasmon Resonance, Transcription Factors metabolism, Arabidopsis chemistry, Arabidopsis Proteins chemistry, DNA, Plant chemistry, Transcription Factors chemistry
- Abstract
The WRKY family transcription factors regulate plant-specific reactions that are mostly related to biotic and abiotic stresses. They share the WRKY domain, which recognizes a DNA element (TTGAC(C/T)) termed the W-box, in target genes. Here, we determined the solution structure of the C-terminal WRKY domain of Arabidopsis WRKY4 in complex with the W-box DNA by NMR. A four-stranded β-sheet enters the major groove of DNA in an atypical mode termed the β-wedge, where the sheet is nearly perpendicular to the DNA helical axis. Residues in the conserved WRKYGQK motif contact DNA bases mainly through extensive apolar contacts with thymine methyl groups. The importance of these contacts was verified by substituting the relevant T bases with U and by surface plasmon resonance analyses of DNA binding.
- Published
- 2012
- Full Text
- View/download PDF
44. Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro.
- Author
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Sato Y, Endo H, Okuyama H, Takeda T, Iwahashi H, Imagawa A, Yamagata K, Shimomura I, and Inoue M
- Subjects
- Animals, Blotting, Western, Cell Line, Tumor, Female, Flow Cytometry, Immunohistochemistry, In Vitro Techniques, Insulin Secretion, Mice, Mice, Inbred C57BL, Cell Hypoxia physiology, Insulin metabolism, Insulin-Secreting Cells metabolism, Oxygen Consumption physiology
- Abstract
Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic β-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic β-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated β-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.
- Published
- 2011
- Full Text
- View/download PDF
45. Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.
- Author
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Matsumura S, Shinoda K, Yamada M, Yokojima S, Inoue M, Ohnishi T, Shimada T, Kikuchi K, Masui D, Hashimoto S, Sato M, Ito A, Akioka M, Takagi S, Nakamura Y, Nemoto K, Hasegawa Y, Takamoto H, Inoue H, Nakamura S, Nabeshima Y, Teplow DB, Kinjo M, and Hoshi M
- Subjects
- Alzheimer Disease metabolism, Amyloid metabolism, Amyloid pharmacology, Amyloid ultrastructure, Amyloid beta-Peptides metabolism, Amyloid beta-Peptides pharmacology, Animals, Cells, Cultured, Humans, Peptides metabolism, Peptides pharmacology, Rats, Amyloid chemistry, Amyloid beta-Peptides chemistry, Peptides chemistry, Protein Multimerization
- Abstract
Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.
- Published
- 2011
- Full Text
- View/download PDF
46. Uncoating of human immunodeficiency virus type 1 requires prolyl isomerase Pin1.
- Author
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Misumi S, Inoue M, Dochi T, Kishimoto N, Hasegawa N, Takamune N, and Shoji S
- Subjects
- Capsid Proteins genetics, Cell Line, Cell Line, Tumor, HIV-1 genetics, Humans, Jurkat Cells, Leukemia, T-Cell, NIMA-Interacting Peptidylprolyl Isomerase, Peptidylprolyl Isomerase genetics, Phosphorylation genetics, Phosphorylation physiology, RNA, Small Interfering genetics, RNA, Small Interfering physiology, Serine metabolism, Threonine metabolism, Capsid Proteins metabolism, HIV-1 metabolism, Peptidylprolyl Isomerase metabolism
- Abstract
The process by which the human immunodeficiency virus type 1 (HIV-1) conical core dissociates is called uncoating, but not much is known about this process. Here, we show that the uncoating process requires the interaction of the capsid (CA) protein with the peptidyl-prolyl isomerase Pin1 that specifically recognizes the phosphorylated serine/threonine residue followed by proline. We found that the HIV-1 core is composed of some isoforms of the CA protein with different isoelectric points, and one isoform is preferentially phosphorylated in the Ser(16)-Pro(17) motif. The mutant virus S16A/P17A shows a severely attenuated HIV-1 replication and an impaired reverse transcription. The S16A/P17A change increased the amount of particulate CA cores in the cytosol of target cells and correlated with the restriction of HIV-1 infection. Glutathione S-transferase pulldown assays demonstrated a direct interaction between Pin1 and the HIV-1 core via the Ser(16)-Pro(17) motif. Suppression of Pin1 expression by RNA interference in a target cell results in an attenuated HIV-1 replication and increases the amount of particulate CA cores in the cytosol of target cells. Furthermore, heat-inactivated, inhibitor-treated, or W34A/K63A Pin1 causes an attenuated in vitro uncoating of the HIV-1 core. The Pin1-dependent uncoating is inhibited by antisera raised against a CA peptide phosphorylated at Ser(16) or treatment of the HIV-1 core with alkaline phosphatase. These findings provide insights into this obscure uncoating process in the HIV-1 life cycle and a new cellular target for HIV-1 drug development.
- Published
- 2010
- Full Text
- View/download PDF
47. Dynein dysfunction induces endocytic pathology accompanied by an increase in Rab GTPases: a potential mechanism underlying age-dependent endocytic dysfunction.
- Author
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Kimura N, Inoue M, Okabayashi S, Ono F, and Negishi T
- Subjects
- Age Factors, Animals, Brain metabolism, Brain pathology, Disease Models, Animal, Dynactin Complex, Dyneins genetics, Endosomes metabolism, Humans, Macaca fascicularis, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, Protein Binding, rab GTP-Binding Proteins genetics, Alzheimer Disease metabolism, Alzheimer Disease pathology, Dyneins metabolism, Endocytosis, rab GTP-Binding Proteins metabolism
- Abstract
Growing evidence suggests that endocytic dysfunction is intimately involved in early stage Alzheimer disease pathology, such as the accumulation of beta-amyloid precursor protein in enlarged early endosomes. However, it remains unclear how endocytic dysfunction is induced in an age-dependent manner. Cytoplasmic dynein, a microtubule-based motor protein, interacts with another microtubule-associated protein, dynactin. The resulting dynein-dynactin complex mediates minus end-directed vesicle transport, including endosome trafficking. We have previously shown that the interaction between dynein-dynactin complexes is clearly attenuated in aged monkey brains, suggesting that dynein-mediated transport dysfunction exists in aged brains. Our immunohistochemical analyses revealed that age-dependent endocytic pathology was accompanied by an increase in Rab GTPases in aged monkey brains. Here, we demonstrated that siRNA-induced dynein dysfunction reproduced the endocytic pathology accompanied by increased Rab GTPases seen in aged monkey brains and significantly disrupted exosome release. Moreover, it also resulted in endosomal beta-amyloid precursor protein accumulation characterized by increased beta-site cleavage. These findings suggest that dynein dysfunction may underlie age-dependent endocytic dysfunction via the up-regulation of Rab GTPases. In addition, this vicious circle may worsen endocytic dysfunction, ultimately leading to Alzheimer disease pathology.
- Published
- 2009
- Full Text
- View/download PDF
48. Four-electron reduction of dioxygen by a multicopper oxidase, CueO, and roles of Asp112 and Glu506 located adjacent to the trinuclear copper center.
- Author
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Kataoka K, Sugiyama R, Hirota S, Inoue M, Urata K, Minagawa Y, Seo D, and Sakurai T
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Circular Dichroism, Electron Spin Resonance Spectroscopy, Escherichia coli Proteins chemistry, Molecular Sequence Data, Mutant Proteins chemistry, Mutant Proteins metabolism, Oxidation-Reduction, Oxidoreductases chemistry, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Structure-Activity Relationship, Aspartic Acid metabolism, Copper metabolism, Electrons, Escherichia coli enzymology, Escherichia coli Proteins metabolism, Glutamic Acid metabolism, Oxidoreductases metabolism, Oxygen metabolism
- Abstract
The mechanism of the four-electron reduction of dioxygen by a multicopper oxidase, CueO, was studied based on reactions of single and double mutants with Cys(500), a type I copper ligand, and the noncoordinating Asp(112) and Glu(506), which form hydrogen bonds with the trinuclear copper center directly and indirectly via a water molecule. The reaction of C500S containing a vacant type I copper center produced intermediate I in an EPR-silent peroxide-bound form. The formation of intermediate I from C500S/D112N was restricted due to a reduction in the affinity of the trinuclear copper center for dioxygen. The state of intermediate I was realized to be the resting form of C500S/E506Q and C500S of the truncated mutant Deltaalpha5-7CueO, in which the 50 amino acids covering the substrate-binding site were removed. Reactions of the recombinant CueO and E506Q afforded intermediate II, a fully oxidized form different from the resting one, with a very broad EPR signal, g < 2, detectable only at cryogenic temperatures and unsaturated with high power microwaves. The lifetime of intermediate II was prolonged by the mutation at Glu(506) involved in the donation of protons. The structure of intermediates I and II and the mechanism of the four-electron reduction of dioxygen driven by Asp(112) and Glu(506) are discussed.
- Published
- 2009
- Full Text
- View/download PDF
49. Synthetic ciguatoxins selectively activate Nav1.8-derived chimeric sodium channels expressed in HEK293 cells.
- Author
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Yamaoka K, Inoue M, Miyazaki K, Hirama M, Kondo C, Kinoshita E, Miyoshi H, and Seyama I
- Subjects
- Animals, Base Sequence, Cell Line, Ciguatera Poisoning genetics, Gene Expression, Humans, Molecular Sequence Data, Muscle Proteins genetics, NAV1.4 Voltage-Gated Sodium Channel, NAV1.8 Voltage-Gated Sodium Channel, Nerve Tissue Proteins genetics, Peptide Mapping, Protein Structure, Tertiary genetics, Rats, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sodium Channels genetics, Ciguatera Poisoning metabolism, Ciguatoxins pharmacology, Muscle Proteins metabolism, Nerve Tissue Proteins metabolism, Sodium Channels metabolism
- Abstract
The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.
- Published
- 2009
- Full Text
- View/download PDF
50. Structure of the C-terminal phosphotyrosine interaction domain of Fe65L1 complexed with the cytoplasmic tail of amyloid precursor protein reveals a novel peptide binding mode.
- Author
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Li H, Koshiba S, Hayashi F, Tochio N, Tomizawa T, Kasai T, Yabuki T, Motoda Y, Harada T, Watanabe S, Inoue M, Hayashizaki Y, Tanaka A, Kigawa T, and Yokoyama S
- Subjects
- Amino Acid Motifs physiology, Amyloid beta-Protein Precursor genetics, Amyloid beta-Protein Precursor metabolism, Animals, Mice, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Nuclear Magnetic Resonance, Biomolecular, Nuclear Proteins genetics, Nuclear Proteins metabolism, Peptides genetics, Peptides metabolism, Protein Binding physiology, Protein Folding, Protein Structure, Quaternary physiology, Protein Structure, Tertiary physiology, Amyloid beta-Protein Precursor chemistry, Nerve Tissue Proteins chemistry, Nuclear Proteins chemistry, Peptides chemistry
- Abstract
Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I beta-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel beta-sheet with the beta5 strand of the PID domain, the binding mode typically observed in the PID/PTB.peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn alpha-helix and interacts extensively with the C-terminal alpha-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB.peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins.
- Published
- 2008
- Full Text
- View/download PDF
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