80 results on '"Nakayama, T."'
Search Results
2. A protein which binds preferentially to single-stranded core sequence of autonomously replicating sequence is essential for respiratory function in mitochondrial of Saccharomyces cerevisiae.
- Author
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Yamazoe, M., primary, Shirahige, K., additional, Rashid, M.B., additional, Kaneko, Y., additional, Nakayama, T., additional, Ogasawara, N., additional, and Yoshikawa, H., additional
- Published
- 1994
- Full Text
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3. Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin
- Author
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Nakayama, T A, primary and Khorana, H G, additional
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- 1991
- Full Text
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4. A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern.
- Author
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Moritz, O L, Tam, B M, Papermaster, D S, and Nakayama, T
- Abstract
To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A(max) at 500 nm) and GFP (A(max) at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the C-terminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.
- Published
- 2001
- Full Text
- View/download PDF
5. Synergism between the calmodulin-binding and autoinhibitory domains on calcineurin is essential for the induction of their phosphatase activity.
- Author
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Tokoyoda, K, Takemoto, Y, Nakayama, T, Arai, T, and Kubo, M
- Abstract
Elevation of the intracellular calcium concentration is necessary for cell growth and the activation of several lymphokine genes. The immunosuppressive drugs cyclosporin A and FK506 profoundly inhibit the calcium-dependent signaling pathway in T lymphocytes by interfering with the activity of the calcium/calmodulin (CaM)-dependent serine/threonine phosphatase, calcineurin (CN). Little is known, however, about how activation of CN enzyme activity or interaction with its substrate, nuclear factor of activated T cell (NF-AT), is regulated. We show here that the binding of CaM to CN may affect the conformation of CN at both the CaM-binding and the autoinhibitory (AI) domains and that this is critical for activation of CN to dephosphorylate NF-AT. Dissociation of the AI domain from the enzyme active site on CN leads to expose a binding site for NF-AT at the N terminus of CN and allows the CN binding to NF-AT. Since the cytoplasmic form of NF-AT can interact with CN mutants lacking enzyme activity, the interaction of the two molecules is independent of CN enzyme activity and occurs prior to the dephosphorylation of NF-AT. Dephosphorylation converts NF-AT from the cytoplasmic to the nuclear form by exposing the DNA-binding domain on NF-AT, and the nuclear form of NF-AT brings CN together into the nuclei. We therefore propose that the activation of CN by the CaM binding independently regulates the interaction with NF-AT and the dephosphorylation of NF-AT.
- Published
- 2000
6. WD repeats of the p48 subunit of chicken chromatin assembly factor-1 required for in vitro interaction with chicken histone deacetylase-2.
- Author
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Ahmad, A, Takami, Y, and Nakayama, T
- Abstract
Chromatin assembly factor-1 (CAF-1) is essential for chromatin assembly in eukaryotes, and comprises three subunits of 150 kDa (p150), 60 kDa (p60), and 48 kDa (p48). We cloned and sequenced cDNA encoding the small subunit of the chicken CAF-1, chCAF-1p48. It consists of 425 amino acid residues including a putative initiation Met, possesses seven WD repeat motifs, and contains only one amino acid change relative to the human and mouse CAF-1p48s. The immunoprecipitation experiment followed by Western blotting revealed that chCAF-1p48 interacts with chicken histone deacetylases (chHDAC-1 and -2) in vivo. The glutathione S-transferase pulldown affinity assay revealed the in vitro interaction of chCAF-1p48 with chHDAC-1, -2, and -3. We showed that the p48 subunit tightly binds to two regions of chHDAC-2, located between amino acid residues 82-180 and 245-314, respectively. We also established that two N-terminal, two C-terminal, or one N-terminal and one C-terminal WD repeat motif of chCAF-1p48 are required for this interaction, using deletion mutants of the respective regions. These results suggest that chCAF-1p48 is involved in many aspects of DNA-utilizing processes, through alterations in the chromatin structure based on both the acetylation and deacetylation of core histones.
- Published
- 1999
7. Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties.
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Maenaka, K, Juji, T, Nakayama, T, Wyer, J R, Gao, G F, Maenaka, T, Zaccai, N R, Kikuchi, A, Yabe, T, Tokunaga, K, Tadokoro, K, Stuart, D I, Jones, E Y, and van der Merwe, P A
- Abstract
Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K(d) approximately 7 microM at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., Wyer, J. R. , Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.
- Published
- 1999
8. A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7.
- Author
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Nakajima, Y, Yamamoto, T, Nakayama, T, and Nakanishi, S
- Abstract
Metabotropic glutamate receptor subtype 7 (mGluR7) is coupled to the inhibitory cyclic AMP cascade and is selectively activated by a glutamate analogue, L-2-amino-4-phosphonobutyrate. Among L-2-amino-4-phosphonobutyrate-sensitive mGluR subtypes, mGluR7 is highly concentrated at the presynaptic terminals and is thought to play an important role in modulation of glutamatergic synaptic transmission by presynaptic inhibition of glutamate release. To gain further insight into the intracellular signaling mechanisms of mGluR7, with the aid of glutathione S-transferase fusion affinity chromatography, we attempted to identify proteins that interact with the intracellular carboxyl terminus of mGluR7. Here, we report that calmodulin (CaM) directly binds to the carboxyl terminus of mGluR7 in a Ca(2+)-dependent manner. The CaM-binding domain is located immediately following the 7th transmembrane segment. We also show that the CaM-binding domain of mGluR7 is phosphorylated by protein kinase C (PKC). This phosphorylation is inhibited by the binding of Ca(2+)/CaM to the receptor. Conversely, the Ca(2+)/CaM binding is prevented by PKC phosphorylation. Collectively, these results suggest that mGluR7 serves to cross-link the cyclic AMP, Ca(2+), and PKC phosphorylation signal transduction cascades.
- Published
- 1999
9. Molecular cloning, expression, and characterization of novel human SULT1C sulfotransferases that catalyze the sulfonation of N-hydroxy-2-acetylaminofluorene.
- Author
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Sakakibara, Y, Yanagisawa, K, Katafuchi, J, Ringer, D P, Takami, Y, Nakayama, T, Suiko, M, and Liu, M C
- Abstract
Upon sulfonation, carcinogenic hydroxyarylamines such as N-hydroxy-2-acetylaminofluorene (N-OH-2AAF) can be further activated to form ultimate carcinogens in vivo. Previous studies have shown that a SULT1C1 sulfotransferase is primarily responsible for the sulfonation of N-OH-2AAF in rat liver. In the present study, two novel human sulfotransferases shown to be members of the SULT1C sulfotransferase subfamily based on sequence analysis have been cloned, expressed, and characterized. Comparisons of the deduced amino acid sequence encoded by the human SULT1C sulfotransferase cDNA 1 reveal 63.7, 61.6, and 85.1% identity to the amino acid sequences of rat SULT1C1 sulfotransferase, mouse SULT1C1 sulfotransferase, and rabbit SULT1C sulfotransferase. In contrast, the deduced amino acid sequence of the human SULT1C sulfotransferase 2 cDNA displays 62.9, 63.1, 63.1, and 62.5% identity to the amino acid sequences of the human SULT1C sulfotransferase 1, rat SULT1C1 sulfotransferase, mouse SULT1C1 sulfotransferase, and rabbit SULT1C sulfotransferase. Recombinant human SULT1C sulfotransferases 1 and 2, expressed in Escherichia coli and purified to near electrophoretic homogeneity, were shown to cross-react with the antiserum against the rat liver SULT1C1 sulfotransferase and exhibited sulfonating activities with N-OH-2AAF as substrate. Tissue-specific expression of these novel human SULT1C sulfotransferases were examined by employing the Northern blotting technique. The results provide a foundation for the investigation into the functional relevance of these new SULT1C sulfotransferases in different human tissues/organs.
- Published
- 1998
10. Chicken histone deacetylase-2 controls the amount of the IgM H-chain at the steps of both transcription of its gene and alternative processing of its pre-mRNA in the DT40 cell line.
- Author
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Takami, Y, Kikuchi, H, and Nakayama, T
- Abstract
Histone deacetylases (HDACs) are involved in the deacetylation of core histones, which is an important event in transcription regulation in eukaryotes through alterations in the chromatin structure. We cloned cDNAs and genomic DNAs encoding two chicken HDACs (chHDAC-1 and -2), which are preferentially localized in nuclei. Treatment with trichostatin A reduced the HDAC activities in immunoprecipitates obtained with anti-chHDAC-1 and -2 antisera. Using gene targeting techniques, we generated homozygous DT40 mutants, DeltachHDAC-1 and -2, devoid of two alleles of the chHDAC-1 and -2 genes, respectively. The protein patterns on two-dimensional PAGE definitely changed for DeltachHDAC-2, and the amounts of the IgM H- and L-chains increased in it. Of the two IgM H-chain forms, the secreted form mu(s) increased in DeltachHDAC-2, but the membrane-bound form mu(m) decreased. The IgM H-chain gene was transcribed more in DeltachHDAC-2 than in DT40 cells. In the mutant, the alternative processing of IgM H-chain pre-mRNA preferentially occurred, resulting in an increase in the amount of mu(s) mRNA, whereas the stability of the two types of mRNA, mu(s) and mu(m), was unchanged. In DT40 cells, treatment with trichostatin A increased both the amounts of IgM H-chain mRNAs and the switch from mu(m) to mu(s) mRNAs. Based on these results, we propose a model for a role of chHDAC-2 in both the transcription and alternative processing steps, resulting in control of the amount of the mu(s) IgM H-chain in the DT40 cell line.
- Published
- 1999
11. The affinity maturation of anti-4-hydroxy-3-nitrophenylacetyl mouse monoclonal antibody. A calorimetric study of the antigen-antibody interaction.
- Author
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Torigoe, H, Nakayama, T, Imazato, M, Shimada, I, Arata, Y, and Sarai, A
- Abstract
To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45 degrees C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.
- Published
- 1995
12. Physical and functional interaction of murine and Xenopus Smad7 with bone morphogenetic protein receptors and transforming growth factor-beta receptors.
- Author
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Souchelnytskyi, S, Nakayama, T, Nakao, A, Morén, A, Heldin, C H, Christian, J L, and ten Dijke, P
- Abstract
Members of the transforming growth factor-beta (TGF-beta) family transmit signals from membrane to nucleus via intracellular proteins known as Smads. A subclass of Smad proteins has recently been identified that antagonize, rather than transduce, TGF-beta family signals. Smad7, for example, binds to and inhibits signaling downstream of TGF-beta receptors. Here we report that the C-terminal MAD homology domain of murine Smad7 (mSmad7) is sufficient for both of these activities. In addition, we show that mSmad7 interacts with activated bone morphogenetic protein (BMP) type I receptors (BMPR-Is), inhibits BMPR-I-mediated Smad phosphorylation, and phenocopies the effect of known BMP antagonists when overexpressed in ventral cells of Xenopus embryos. Xenopus Smad7 (XSmad7, previously termed Smad8) and mSmad7 are nearly identical within their bioactive C-domain, but have quite distinct N-domains. We found that XSmad7, similar to mSmad7, interacted with BMP and TGF-beta type I receptors and inhibited receptor-mediated phosphorylation of downstream signal-transducing Smads. However, XSmad7 is a less efficient inhibitor of TbetaR-I-mediated responses in mammalian cells than is mSmad7. Furthermore, overexpression of XSmad7 in Xenopus embryos produces patterning defects that are not observed following overexpression of mSmad7, suggesting that mSmad7 and XSmad7 may preferentially target distinct signaling pathways. Our results are consistent with the possibility that the C-domain of antagonistic Smads is an effector domain whereas the N-domain may confer specificity for distinct signaling pathways.
- Published
- 1998
13. Localization and functional analysis of the substrate specificity/catalytic domains of human M-form and P-form phenol sulfotransferases.
- Author
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Sakakibara, Y, Takami, Y, Nakayama, T, Suiko, M, and Liu, M C
- Abstract
Human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases (PSTs), which are greater than 93% identical in their primary sequences, were used as models for investigating the structural determinants responsible for their distinct substrate specificity and other enzymatic properties. A series of chimeric PSTs were constructed by reciprocal exchanges of DNA segments between cDNAs encoding M-form and P-form PSTs. Functional characterization of the recombinant wild-type M-form, P-form, and chimeric PSTs expressed in Escherichia coli and purified to homogeneity revealed that internal domain-spanning amino acid residues 84-148 contain the structural determinants for the substrate specificity of either M-form or P-form PST. Data on the kinetic constants (Km, Vmax, and Vmax/Km) further showed the differential roles of the two highly variable regions (Region I spanning amino acid residues 84-89 and Region II spanning amino acid residues 143-148) in substrate binding, catalysis, and sensitivity to the inhibition by 2,6-dichloro-4-nitrophenol. In contrast to the differential sulfotransferase activities of M-form and P-form PSTs toward dopamine and p-nitrophenol, the Dopa/tyrosine sulfotransferase activities were found to be restricted to M-form, but not P-form, PST. Furthermore, the variable Region II of M-form PST appeared to play a predominant role in determining the Dopa/tyrosine sulfotransferase activities of chimeric PSTs. Kinetic studies indicated the role of manganese ions in dramatically enhancing the binding of D-p-tyrosine to wild-type M-form PST. Taken together, these results pinpoint unequivocally the sequence encompassing amino acid residues 84-148 to be the substrate specificity/catalytic domain of both M-form and P-form PSTs and indicate the importance of the variable Regions I and II in determining their distinct enzymatic properties.
- Published
- 1998
14. Altering substrate specificity of Bacillus sp. SAM1606 alpha-glucosidase by comparative site-specific mutagenesis.
- Author
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Inohara-Ochiai, M, Nakayama, T, Goto, R, Nakao, M, Ueda, T, and Shibano, Y
- Abstract
The Bacillus sp. SAM1606 alpha-glucosidase with a broad substrate specificity is the only known alpha-glucosidase that can hydrolyze alpha,alpha'-trehalose efficiently. The enzyme exhibits a very high sequence similarity to the oligo-1,6-glucosidases (O16G) of Bacillus thermoglucosidasius and Bacillus cereus which cannot act on trehalose. These three enzymes share 80% identical residues within the conserved regions (CR), which have been suggested to be located near or at the active site of the alpha-amylase family enzymes. To identify by site-specific mutagenesis the critical residues that determine the broad substrate specificity of the SAM1606 enzyme we compared the CR sequences of these three glucosidases and selected five targets to be mutagenized in SAM1606 alpha-glucosidase, Met76, Arg81, Ala116, Gly273, and Thr342. These residues have been specifically replaced by in vitro mutagenesis with Asn, Ser, Val, Pro, and Asn, respectively, as in the Bacillus O16G. The 12 mutant enzymes with single and multiple substitutions were expressed and characterized kinetically. The results showed that the 5-fold mutation virtually abolished the affinity of the enzyme for alpha, alpha'-trehalose, whereas the specificity constant for the hydrolysis of isomaltose, a good substrate for both the SAM1606 enzyme and O16G, remained essentially unchanged upon the mutation. This loss in affinity for trehalose was critically governed by a Gly273 --> Pro substitution, whose effect was specifically enhanced by the Thr342 --> Asn substitution in the 5-fold and quadruple mutants. These results provide evidence for the differential roles of the amino acid residues in the CR in determining the substrate specificity of the alpha-glucosidase.
- Published
- 1997
15. Structural studies of the sugar chains of human parotid alpha-amylase.
- Author
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Yamashita, K., primary, Tachibana, Y., additional, Nakayama, T., additional, Kitamura, M., additional, Endo, Y., additional, and Kobata, A., additional
- Published
- 1980
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- View/download PDF
16. A CARBON BALANCE ON GLYCINE AND GLUCOSE IN CAROTENE SYNTHESIS
- Author
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Chichester, C.O., primary, Nakayama, T., additional, and Mackinney, G., additional
- Published
- 1956
- Full Text
- View/download PDF
17. ON THE BIOSYNTHESIS OF CAROTENOIDS
- Author
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Mackinney, G., primary, Nakayama, T., additional, and Chichester, C.O., additional
- Published
- 1956
- Full Text
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18. INCORPORATION OF LABELED LEUCINE INTO CAROTENE BY PHYCOMYCES
- Author
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Chichester, C.O., primary, Nakayama, T., additional, Mackinney, G., additional, and Goodwin, T.W., additional
- Published
- 1955
- Full Text
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19. 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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20. Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES.
- Author
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Kado Y, Mizohata E, Nagatoishi S, Iijima M, Shinoda K, Miyafusa T, Nakayama T, Yoshizumi T, Sugiyama A, Kawamura T, Lee YH, Matsumura H, Doi H, Fujitani H, Kodama T, Shibasaki Y, Tsumoto K, and Inoue T
- Subjects
- Animals, Antibodies, Monoclonal, Humanized immunology, Calorimetry, DNA Mutational Analysis, Electron Spin Resonance Spectroscopy, Humans, Immunoglobulin G chemistry, Mice, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins chemistry, Surface Plasmon Resonance, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Antibodies, Monoclonal, Humanized chemistry, Epiregulin chemistry
- Abstract
Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro(103). A molecular dynamics simulation and mutational analyses revealed that Arg(40) in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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21. Methylation of Gata3 protein at Arg-261 regulates transactivation of the Il5 gene in T helper 2 cells.
- Author
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Hosokawa H, Kato M, Tohyama H, Tamaki Y, Endo Y, Kimura MY, Tumes DJ, Motohashi S, Matsumoto M, Nakayama KI, Tanaka T, and Nakayama T
- Subjects
- Amino Acid Sequence, Animals, Arginine metabolism, Blotting, Western, Cell Differentiation, Cell Proliferation, Cells, Cultured, Chaperonin 60 antagonists & inhibitors, Chaperonin 60 genetics, Chaperonin 60 metabolism, Chromatin Assembly and Disassembly, Chromatin Immunoprecipitation, Cytokines genetics, Cytokines metabolism, Flow Cytometry, Fluorescent Antibody Technique, GATA3 Transcription Factor metabolism, Immunoprecipitation, Interleukin-5 metabolism, Mice, Mice, Inbred C57BL, Mitochondrial Proteins antagonists & inhibitors, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Molecular Sequence Data, Promoter Regions, Genetic genetics, RNA, Messenger genetics, RNA, Small Interfering genetics, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Th1 Cells immunology, Th1 Cells metabolism, Th2 Cells immunology, Arginine genetics, DNA Methylation, GATA3 Transcription Factor genetics, Interleukin-5 genetics, Th2 Cells metabolism, Transcriptional Activation
- Abstract
Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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22. Structural insights into the low pH adaptation of a unique carboxylesterase from Ferroplasma: altering the pH optima of two carboxylesterases.
- Author
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Ohara K, Unno H, Oshima Y, Hosoya M, Fujino N, Hirooka K, Takahashi S, Yamashita S, Kusunoki M, and Nakayama T
- Subjects
- Carboxylesterase chemistry, Crystallization, Hydrogen Bonding, Hydrogen-Ion Concentration, Kinetics, Carboxylesterase metabolism, Euryarchaeota enzymology
- Abstract
To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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23. GCN5 protects vertebrate cells against UV-irradiation via controlling gene expression of DNA polymerase η.
- Author
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Kikuchi H, Kuribayashi F, Imajoh-Ohmi S, Nishitoh H, Takami Y, and Nakayama T
- Subjects
- Acetylation radiation effects, Animals, Cell Death genetics, Cell Death radiation effects, Cell Line, Chickens, CpG Islands genetics, DNA biosynthesis, DNA genetics, DNA Repair genetics, DNA-Directed DNA Polymerase genetics, Gene Deletion, Gene Expression Regulation, Enzymologic genetics, Histones genetics, Histones metabolism, Humans, Protein Binding genetics, Protein Binding radiation effects, Transcription, Genetic genetics, Transcription, Genetic radiation effects, p300-CBP Transcription Factors genetics, DNA Fragmentation radiation effects, DNA Repair radiation effects, DNA-Directed DNA Polymerase biosynthesis, Gene Expression Regulation, Enzymologic radiation effects, Ultraviolet Rays adverse effects, p300-CBP Transcription Factors metabolism
- Abstract
By UV-irradiation, cells are subjected to DNA damage followed by mutation, cell death and/or carcinogenesis. DNA repair systems such as nucleotide excision repair (NER) and translesion DNA synthesis (TLS) protect cells against UV-irradiation. To understand the role of histone acetyltransferase GCN5 in regulation of DNA repair, we studied the sensitivity of GCN5-deficient DT40, GCN5(-/-), to various DNA-damaging agents including UV-irradiation, and effects of GCN5-deficiency on the expression of NER- and TLS-related genes. After UV-irradiation, cell death and DNA fragmentation of GCN5(-/-) were appreciably accelerated as compared with those of DT40. Interestingly, GCN5(-/-) showed a remarkable sensitivity to only UV-irradiation but not to other DNA-damaging agents tested. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically down-regulated in GCN5(-/-) (to ∼25%). In addition, ectopic expression of human POLH in GCN5(-/-) dramatically reversed the sensitivity to UV-irradiation of GCN5(-/-) to almost the same level of wild type DT40. Moreover, chromatin immunoprecipitation assay revealed that GCN5 binds to the chicken POLH gene 5'-flanking region that contains a typical CpG island and acetylates Lys-9 of histone H3, but not Lys-14 in vivo. These data suggest that GCN5 takes part in transcription regulation of POLH gene through alterations in the chromatin structure by direct interaction with its 5'-flanking region, and protects vertebrate cells against UV-induced DNA damage via controlling POLH gene expression.
- Published
- 2012
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24. The histone chaperone facilitates chromatin transcription (FACT) protein maintains normal replication fork rates.
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Abe T, Sugimura K, Hosono Y, Takami Y, Akita M, Yoshimura A, Tada S, Nakayama T, Murofushi H, Okumura K, Takeda S, Horikoshi M, Seki M, and Enomoto T
- Subjects
- Animals, Cell Cycle, Chickens, Epigenesis, Genetic, Flow Cytometry methods, Histones chemistry, Humans, Molecular Chaperones metabolism, Nucleosomes metabolism, S Phase, Chromatin chemistry, DNA Replication, DNA-Binding Proteins metabolism, High Mobility Group Proteins metabolism, Histone Chaperones chemistry, Transcription, Genetic, Transcriptional Elongation Factors metabolism
- Abstract
Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.
- Published
- 2011
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25. Histone modifiers, YY1 and p300, regulate the expression of cartilage-specific gene, chondromodulin-I, in mesenchymal stem cells.
- Author
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Aoyama T, Okamoto T, Fukiage K, Otsuka S, Furu M, Ito K, Jin Y, Ueda M, Nagayama S, Nakayama T, Nakamura T, and Toguchida J
- Subjects
- Acetylation, Cells, Cultured, DNA Methylation physiology, Histone Deacetylases metabolism, Humans, Male, Mesenchymal Stem Cells cytology, Middle Aged, Organ Specificity, Repressor Proteins metabolism, Response Elements physiology, Sp3 Transcription Factor metabolism, Cartilage, Down-Regulation physiology, Intercellular Signaling Peptides and Proteins biosynthesis, Membrane Proteins biosynthesis, Mesenchymal Stem Cells metabolism, YY1 Transcription Factor metabolism, p300-CBP Transcription Factors metabolism
- Abstract
Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.
- Published
- 2010
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26. New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.
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Unno H, Yamashita S, Ikeda Y, Sekiguchi SY, Yoshida N, Yoshimura T, Kusunoki M, Nakayama T, Nishino T, and Hemmi H
- Subjects
- Amino Acid Sequence, Carbon-Carbon Double Bond Isomerases classification, Carbon-Carbon Double Bond Isomerases genetics, Catalytic Domain, Conserved Sequence, Crystallography, X-Ray, Hemiterpenes, Hydrogen-Ion Concentration, Ligands, Models, Molecular, Molecular Sequence Data, Mutation genetics, Oxidation-Reduction, Protein Binding, Protein Structure, Quaternary, Protein Structure, Tertiary, Sequence Alignment, Substrate Specificity, Sulfolobus enzymology, Acids, Alkalies, Biocatalysis, Carbon-Carbon Double Bond Isomerases chemistry, Carbon-Carbon Double Bond Isomerases metabolism, Flavins chemistry, Flavins metabolism
- Abstract
Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.
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- 2009
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27. Gfi1-mediated stabilization of GATA3 protein is required for Th2 cell differentiation.
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Shinnakasu R, Yamashita M, Kuwahara M, Hosokawa H, Hasegawa A, Motohashi S, and Nakayama T
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- Animals, Cell Differentiation, Humans, Interleukin-5 metabolism, MAP Kinase Signaling System, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Models, Biological, Proteasome Endopeptidase Complex metabolism, Ubiquitin chemistry, DNA-Binding Proteins metabolism, GATA3 Transcription Factor metabolism, Gene Expression Regulation, Th2 Cells metabolism, Transcription Factors metabolism
- Abstract
The differentiation of naive CD4 T cells into Th2 cells requires the T cell receptor-mediated activation of the ERK MAPK cascade. Little is known, however, in regard to how the ERK MAPK cascade regulates Th2 cell differentiation. We herein identified Gfi1 (growth factor independent-1) as a downstream target of the ERK MAPK cascade for Th2 cell differentiation. In the absence of Gfi1, interleukin-5 production and the change of histone modification at the interleukin-5 gene locus were severely impaired. Furthermore, the interferon gamma gene showed a striking activation in the Gfi1(-/-) Th2 cells. An enhanced ubiquitin/proteasome-dependent degradation of GATA3 protein was observed in Gfi1(-/-) Th2 cells, and the overexpression of GATA3 eliminated the defect of Th2 cell function in Gfi1-deficient Th2 cells. These data suggest that the T cell receptor-mediated induction of Gfi1 controls Th2 cell differentiation through the regulation of GATA3 protein stability.
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- 2008
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28. Radial microtubule organization by histone H1 on nuclei of cultured tobacco BY-2 cells.
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Nakayama T, Ishii T, Hotta T, and Mizuno K
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Cell Nucleus metabolism, Cells, Cultured, Centrosome metabolism, Cloning, Molecular, DNA metabolism, Electrophoresis, Gel, Two-Dimensional, Models, Biological, Rabbits, Recombinant Proteins metabolism, Gene Expression Regulation, Plant, Histones metabolism, Microtubules metabolism, Nicotiana metabolism
- Abstract
In acentriolar higher plant cells, the surface of the nucleus acts as a microtubule-organizing center, substituting for the centrosome. However, the protein factors responsible for this microtubule organization are unknown. The nuclear surfaces of cultured tobacco BY-2 cells possess particles that generate microtubules. We attempted to isolate the proteins in these particles to determine their role in microtubule organization. When incubated with plant or mammalian tubulin, some, but not all, of the isolated nuclei generated abundant microtubules radially from their surfaces. The substance to induce the formation of radial microtubules was confirmed by SDS-PAGE to be a protein with apparent molecular mass of 38 kDa. Partial analysis of the amino acid sequences of the peptide fragments suggested it was a histone H1-related protein. Cloning and cDNA sequence analysis confirmed this and revealed that when the recombinant protein was incubated with tubulin, it could organize microtubules as well as the 38-kDa protein. Histone H1 and tubulin formed complexes immediately, even on ice, and then clusters of these structures were formed. These clusters generated radial microtubules. This microtubule-organizing property was confined to histone H1; all other core histones failed to act as organizers. On immunoblot analysis, rabbit antibodies raised against the 38-kDa protein cross-reacted with histone H1 proteins from tobacco BY-2 cells. These antibodies virtually abolished the ability of the nucleus to organize radial microtubules. Indirect immunofluorescence showed that the antigen was distributed at the nuclear plasm and particularly at nuclear periphery independently from DNA.
- Published
- 2008
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29. Atrial glutathione content, calcium current, and contractility.
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Carnes CA, Janssen PM, Ruehr ML, Nakayama H, Nakayama T, Haase H, Bauer JA, Chung MK, Fearon IM, Gillinov AM, Hamlin RL, and Van Wagoner DR
- Subjects
- Animals, Buthionine Sulfoximine pharmacology, Cells, Cultured, Dogs, Heart Atria pathology, Humans, Kidney cytology, Muscle Cells metabolism, Myocardial Contraction, Nitrogen chemistry, Patch-Clamp Techniques, Time Factors, Calcium metabolism, Glutathione metabolism, Heart Atria metabolism, Muscle Contraction
- Abstract
Atrial fibrillation (AF) is characterized by decreased L-type calcium current (I(Ca,L)) in atrial myocytes and decreased atrial contractility. Oxidant stress and redox modulation of calcium channels are implicated in these pathologic changes. We evaluated the relationship between glutathione content (the primary cellular reducing moiety) and I(Ca,L) in atrial specimens from AF patients undergoing cardiac surgery. Left atrial glutathione content was significantly lower in patients with either paroxysmal or persistent AF relative to control patients with no history of AF. Incubation of atrial myocytes from AF patients (but not controls) with the glutathione precursor N-acetylcysteine caused a marked increase in I(Ca,L). To test the hypothesis that glutathione levels were mechanistically linked with the reduction in I(Ca,L), dogs were treated for 48 h with buthionine sulfoximine, an inhibitor of glutathione synthesis. Buthionine sulfoximine treatment resulted in a 24% reduction in canine atrial glutathione content, a reduction in atrial contractility, and an attenuation of I(Ca,L) in the canine atrial myocytes. Incubation of these myocytes with exogenous glutathione also restored I(Ca,L) to normal or greater than normal levels. To probe the mechanism linking decreased glutathione levels to down-regulation of I(Ca), the biotin switch technique was used to evaluate S-nitrosylation of calcium channels. S-Nitrosylation was apparent in left atrial tissues from AF patients; the extent of S-nitrosylation was inversely related to tissue glutathione content. S-Nitrosylation was also detectable in HEK cells expressing recombinant human cardiac calcium channel subunits following exposure to nitrosoglutathione. S-Nitrosylation may contribute to the glutathione-sensitive attenuation of I(Ca,L) observed in AF.
- Published
- 2007
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30. A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (glycine max) seedlings. Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis.
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Noguchi A, Saito A, Homma Y, Nakao M, Sasaki N, Nishino T, Takahashi S, and Nakayama T
- Subjects
- Amino Acid Sequence, Catalysis, Cloning, Molecular, Kinetics, Medicago metabolism, Models, Chemical, Molecular Sequence Data, Mutagenesis, Site-Directed, Phylogeny, Sequence Homology, Amino Acid, Vitis metabolism, Gene Expression Regulation, Plant, Glucosyltransferases chemistry, Glucosyltransferases physiology, Plant Roots metabolism, Glycine max metabolism
- Abstract
Isoflavones, a class of flavonoids, play very important roles in plant-microbe interactions in certain legumes such as soybeans (Glycine max L. Merr.). G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) is a key enzyme in the synthesis of isoflavone conjugates, which accumulate in large amounts in vacuoles and serve as an isoflavonoid pool that allows for interaction with microorganisms. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. The purified enzyme is a monomeric protein of 46 kDa, catalyzing regiospecific glucosyl transfer from UDP-glucose to isoflavones to produce isoflavone 7-O-beta-D-glucosides (k(cat) = 0.74 s(-1), K(m) for genistein = 3.6 microM, and K(m) for UDP-glucose = 190 microM). The GmIF7GT cDNA was isolated based on the amino acid sequence of the purified enzyme. Phylogenetic analysis showed that GmIF7GT is a novel member of glycosyltransferase family 1 and is distantly related to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. The purified enzyme was unexpectedly devoid of the N-terminal 49-residue segment and thus lacks the histidine residue corresponding to the proposed catalytic residue of glycosyltransferases from Medicago truncatula (UGT71G1) and Vitis vinifera (VvGT1). The results of kinetic studies of site-directed mutants of GmIF7GT showed that both His-15 and Asp-125, which correspond to the catalytic residues of UGT71G1 and VvGT1, are not important for GmIF7GT activity. The results also suggest that an acidic residue at position 392 is very important for primary catalysis of GmIF7GT. These results led to the proposal that GmIF7GT utilizes a strategy of catalysis that is distinct from those proposed for UGT71G1 and VvGT1.
- Published
- 2007
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31. Structural and mutational studies of anthocyanin malonyltransferases establish the features of BAHD enzyme catalysis.
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Unno H, Ichimaida F, Suzuki H, Takahashi S, Tanaka Y, Saito A, Nishino T, Kusunoki M, and Nakayama T
- Subjects
- Acyltransferases genetics, Acyltransferases metabolism, Amino Acid Sequence, Anthocyanins metabolism, Binding Sites genetics, Chrysanthemum genetics, Coenzyme A chemistry, Coenzyme A metabolism, Crystallography, X-Ray, Flowers genetics, Molecular Sequence Data, Mutagenesis, Plant Proteins genetics, Protein Structure, Tertiary, Structure-Activity Relationship, Substrate Specificity genetics, Acyltransferases chemistry, Anthocyanins chemistry, Chrysanthemum enzymology, Flowers enzymology, Pigmentation genetics, Plant Proteins chemistry
- Abstract
The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6''-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3'', 6''-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.
- Published
- 2007
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32. Expression of claudin7 is tightly associated with epithelial structures in synovial sarcomas and regulated by an Ets family transcription factor, ELF3.
- Author
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Kohno Y, Okamoto T, Ishibe T, Nagayama S, Shima Y, Nishijo K, Shibata KR, Fukiage K, Otsuka S, Uejima D, Araki N, Naka N, Nakashima Y, Aoyama T, Nakayama T, Nakamura T, and Toguchida J
- Subjects
- Base Sequence, Cell Line, Tumor, Chromatin Immunoprecipitation, Claudins, DNA Primers, Electrophoretic Mobility Shift Assay, Epithelial Cells metabolism, Humans, Membrane Proteins genetics, Proto-Oncogene Proteins c-ets, Reverse Transcriptase Polymerase Chain Reaction, Sarcoma, Synovial pathology, DNA-Binding Proteins metabolism, Membrane Proteins metabolism, Proto-Oncogene Proteins metabolism, Sarcoma, Synovial metabolism, Transcription Factors metabolism
- Abstract
Synovial sarcoma, a soft tissue sarcoma that develops in adults, is pathologically subclassified into monophasic spindle synovial sarcoma and biphasic synovial sarcoma with epithelial components. The molecular mechanism building the epithelial components in biphasic synovial sarcoma is totally unknown. Here we investigated claudins, critical molecules in the tight junction, in biphasic synovial sarcoma. Expression profiles of 21 claudins in 17 synovial sarcoma tumor samples, including 9 biphasic tumors, identified claudin4, claudin7, and claudin10 as biphasic tumor-related claudins, and immunohistochemical analyses demonstrated the localization of these claudins in the epithelial component in biphasic tumors, with claudin7 the most closely associated with the epithelial component. The mRNA expression and protein localization of claudin7 coincided with those of the ELF3, an epithelia-specific member of the Ets family of transcription factors. Luciferase reporter assays demonstrated that the presence of the Ets-binding site at -150 in the promoter region of the claudin7 gene was critical for the transcriptional activity, and gel shift and chromatin immunoprecipitation assays confirmed the binding of ELF3 to the Ets site at -150. Inhibition of ELF3 expression by small interfering RNA simultaneously down-regulated the mRNA expression of the claudin7 gene, and the introduction of ELF3 expression in claudin7-negative cell lines induced mRNA expression of the claudin7 gene. Therefore, the induction of claudin7 expression by ELF3 appears critical to the formation of the epithelial structures in biphasic synovial sarcoma.
- Published
- 2006
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33. An isoflavone conjugate-hydrolyzing beta-glucosidase from the roots of soybean (Glycine max) seedlings: purification, gene cloning, phylogenetics, and cellular localization.
- Author
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Suzuki H, Takahashi S, Watanabe R, Fukushima Y, Fujita N, Noguchi A, Yokoyama R, Nishitani K, Nishino T, and Nakayama T
- Subjects
- Cells, Cultured, Cloning, Molecular, Cotyledon cytology, Cotyledon enzymology, Cotyledon genetics, Hydrolysis, Hypocotyl cytology, Hypocotyl enzymology, Hypocotyl genetics, Molecular Sequence Data, Plant Roots cytology, Plant Roots genetics, Seedlings cytology, Seedlings metabolism, Glycine max cytology, Glycine max metabolism, beta-Glucosidase isolation & purification, beta-Glucosidase metabolism, Isoflavones metabolism, Phylogeny, Plant Roots enzymology, Seedlings enzymology, Seedlings genetics, Glycine max enzymology, Glycine max genetics, beta-Glucosidase genetics
- Abstract
Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing beta-glucosidase. Here we report on the purification and cDNA cloning of this important beta-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing beta-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6 ''-O-malonyl-beta-d-glucoside) to produce free genistein (k(cat), 98 s(-1); K(m), 25 microM at 30 degrees C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous beta-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.
- Published
- 2006
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34. Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells.
- Author
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Sanematsu F, Takami Y, Barman HK, Fukagawa T, Ono T, Shibahara KI, and Nakayama T
- Subjects
- Animals, Cell Cycle Proteins genetics, Cell Death, Cell Survival, Chickens, Gene Deletion, Gene Expression Regulation, Mitosis physiology, Mutation, Protein Structure, Tertiary, Cell Cycle Proteins metabolism, Chromatin metabolism, DNA Replication physiology
- Abstract
Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.
- Published
- 2006
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35. Different roles of N-terminal and C-terminal halves of HIRA in transcription regulation of cell cycle-related genes that contribute to control of vertebrate cell growth.
- Author
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Ahmad A, Kikuchi H, Takami Y, and Nakayama T
- Subjects
- Amino Acid Motifs, Animals, Apoptosis, Base Sequence, Blotting, Southern, Blotting, Western, Cell Cycle, Cell Cycle Proteins metabolism, Cell Line, Cell Proliferation, Chickens, Chromatin Immunoprecipitation, Cloning, Molecular, DNA, Complementary metabolism, Down-Regulation, Exons, HeLa Cells, Histone Chaperones, Histones chemistry, Homozygote, Humans, Luciferases metabolism, Models, Biological, Models, Genetic, Molecular Sequence Data, Mutation, Nuclear Proteins metabolism, Promoter Regions, Genetic, Protein Structure, Tertiary, Repressor Proteins chemistry, Reverse Transcriptase Polymerase Chain Reaction, Saccharomyces cerevisiae metabolism, Transcription Factors metabolism, Transcription, Genetic, Transfection, Up-Regulation, Cell Cycle Proteins chemistry, Repressor Proteins physiology, Transcription Factors chemistry
- Abstract
We reported previously that chicken HIRA, a homolog of Saccharomyces cerevisiae transcriptional co-repressors Hir1p and Hir2p, possesses seven WD dipeptide motifs and an LXXLL motif in its N-terminal and C-terminal halves, respectively, required for transcription regulations. Here, by using the gene targeting technique, we generated the homozygous HIRA-deficient DT40 mutant DeltaHIRA. The HIRA deficiency caused slightly delayed cell growth and affected the opposite transcriptions of cell cycle-related genes, i.e. repressions for P18, CDC25B, and BCL-2, activations for P19 and cyclin A, and histones H2A, H2B, H3, and H4. These altered expressions were completely revived by the artificial stable expression of hemagglutinin-tagged HIRA in DeltaHIRA. The ability to rescue the delayed growth rate was preferentially aided by the N-terminal half instead of the C-terminal half. We cloned the chicken P18 genomic DNA, and we established that its promoter was located surrounding the sequence GCGGGCGC at positions -1157 to -1150. Chromatin immunoprecipitation assay revealed that the N-terminal half interacted directly or indirectly with the putative promoter region of the p18 gene, resulting in up-regulation of the gene. These results indicated that the N-terminal half of HIRA should contribute positively to the growth rate via up-regulation of a set of cell cycle-related genes, whereas the C-terminal half down-regulated another set of them without exhibiting any effect on the cell growth.
- Published
- 2005
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36. Ras-ERK MAPK cascade regulates GATA3 stability and Th2 differentiation through ubiquitin-proteasome pathway.
- Author
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Yamashita M, Shinnakasu R, Asou H, Kimura M, Hasegawa A, Hashimoto K, Hatano N, Ogata M, and Nakayama T
- Subjects
- Acetylation, Animals, Cell Differentiation, Cells, Cultured, GATA3 Transcription Factor, Histones metabolism, Mice, Mice, Inbred C57BL, Nuclear Proteins physiology, Proto-Oncogene Proteins physiology, Proto-Oncogene Proteins c-mdm2, Zinc Fingers, DNA-Binding Proteins metabolism, Extracellular Signal-Regulated MAP Kinases physiology, MAP Kinase Signaling System physiology, Proteasome Endopeptidase Complex physiology, Th2 Cells cytology, Trans-Activators metabolism, Ubiquitin metabolism, ras Proteins physiology
- Abstract
Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.
- Published
- 2005
- Full Text
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37. Cluster formation of inositol 1,4,5-trisphosphate receptor requires its transition to open state.
- Author
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Tateishi Y, Hattori M, Nakayama T, Iwai M, Bannai H, Nakamura T, Michikawa T, Inoue T, and Mikoshiba K
- Subjects
- Animals, COS Cells, Calcium analysis, Calcium Channels chemistry, Calcium Channels genetics, Chlorocebus aethiops, Cytosol chemistry, Green Fluorescent Proteins, Inositol 1,4,5-Trisphosphate analysis, Inositol 1,4,5-Trisphosphate Receptors, Mice, Microscopy, Fluorescence, Protein Conformation, Receptors, Cytoplasmic and Nuclear chemistry, Receptors, Cytoplasmic and Nuclear genetics, Transfection, Calcium Channels physiology, Receptor Aggregation, Receptors, Cytoplasmic and Nuclear physiology
- Abstract
The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) Ca(2+) channel plays pivotal roles in many aspects of physiological and pathological events. It was previously reported that IP(3)R forms clusters on the endoplasmic reticulum when cytosolic Ca(2+) concentration ([Ca(2+)](C)) is elevated. However, the molecular mechanism of IP(3)R clustering remains largely unknown, and thus its physiological significance is far from clear. In this study we found that the time course of clustering of green fluorescent protein-tagged IP(3)R type 1 (GFP-IP(3)R1), evoked by IP(3)-generating agonists, did not correlate with [Ca(2+)](C) but seemed compatible with cytoplasmic IP(3) concentration. IP(3) production alone induced GFP-IP(3)R1 clustering in the absence of a significant increase in [Ca(2+)](C) but elevated [Ca(2+)](C) without IP(3) production did not. Moreover IP(3)R1 mutants that do not undergo an IP(3)-induced conformational change failed to form clusters. Thus, IP(3)R clustering is induced by its IP(3)-induced conformational change to the open state. We also found that GFP-IP(3)R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligand-induced clustering of a ligand-gated channel protein.
- Published
- 2005
- Full Text
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38. UDP-glucuronic acid:anthocyanin glucuronosyltransferase from red daisy (Bellis perennis) flowers. Enzymology and phylogenetics of a novel glucuronosyltransferase involved in flower pigment biosynthesis.
- Author
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Sawada S, Suzuki H, Ichimaida F, Yamaguchi MA, Iwashita T, Fukui Y, Hemmi H, Nishino T, and Nakayama T
- Subjects
- Asteraceae genetics, Catalysis drug effects, Cloning, Molecular, DNA, Complementary genetics, Enzyme Inhibitors pharmacology, Flowers genetics, Gene Expression, Glucuronosyltransferase isolation & purification, Ions pharmacology, Kinetics, Metals pharmacology, Molecular Sequence Data, Phylogeny, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Solubility, Substrate Specificity, Anthocyanins metabolism, Asteraceae enzymology, Flowers enzymology, Glucuronosyltransferase genetics, Glucuronosyltransferase metabolism, Uridine Diphosphate Glucuronic Acid metabolism
- Abstract
In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2''-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6''-O-malonylglucoside with a kcat value of 34 s(-1) at pH 7.0 and 30 degrees C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6''-O-malonylglucoside, 19 microM) and UDP-glucuronate (Km, 476 microM). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28-40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.
- Published
- 2005
- Full Text
- View/download PDF
39. (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase from the thermoacidophilic archaeon Sulfolobus solfataricus. Molecular cloning and characterization of a membrane-intrinsic prenyltransferase involved in the biosynthesis of archaeal ether-linked membrane lipids.
- Author
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Hemmi H, Shibuya K, Takahashi Y, Nakayama T, and Nishino T
- Subjects
- Alkyl and Aryl Transferases isolation & purification, Alkyl and Aryl Transferases metabolism, Amino Acid Sequence, Autoradiography, Chromatography, High Pressure Liquid, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Phylogeny, Sequence Alignment, Alkyl and Aryl Transferases genetics, Sulfolobus solfataricus enzymology
- Abstract
The core structure of membrane lipids of archaea have some unique properties that permit archaea to be distinguished from the others, i.e. bacteria and eukaryotes. (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase, which catalyzes the transfer of a geranylgeranyl group from geranylgeranyl diphosphate to (S)-3-O-geranylgeranylglyceryl phosphate, is involved in the biosynthesis of archaeal membrane lipids. Enzymes of the UbiA prenyltransferase family are known to catalyze the transfer of a prenyl group to various acceptors with hydrophobic ring structures in the biosynthesis of respiratory quinones, hemes, chlorophylls, vitamin E, and shikonin. The thermoacidophilic archaeon Sulfolobus solfataricus was found to encode three homologues of UbiA prenyltransferase in its genome. One of the homologues encoded by SSO0583 was expressed in Escherichia coli, purified, and characterized. Radio-assay and mass spectrometry analysis data indicated that the enzyme specifically catalyzes the biosynthesis of (S)-2,3-di-O-geranylgeranylglyceryl phosphate. The fact that the orthologues of the enzyme are encoded in almost all archaeal genomes clearly indicates the importance of their functions. A phylogenetic tree constructed using the amino acid sequences of some typical members of the UbiA prenyltransferase family and their homologues from S. solfataricus suggests that the two other S. solfataricus homologues, excluding the (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase, are involved in the production of respiratory quinone and heme, respectively. We propose here that archaeal prenyltransferases involved in membrane lipid biosynthesis might be prototypes of the protein family and that archaea might have played an important role in the molecular evolution of prenyltransferases.
- Published
- 2004
- Full Text
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40. Interleukin (IL)-4-independent maintenance of histone modification of the IL-4 gene loci in memory Th2 cells.
- Author
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Yamashita M, Shinnakasu R, Nigo Y, Kimura M, Hasegawa A, Taniguchi M, and Nakayama T
- Subjects
- Acetylation, Adoptive Transfer, Animals, Cytokines metabolism, DNA Polymerase II metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Enhancer Elements, Genetic physiology, Epitopes genetics, GATA3 Transcription Factor, Interleukin-13 genetics, Methylation, Mice, Mice, Inbred BALB C, Mice, Nude, Mice, Transgenic, Promoter Regions, Genetic physiology, RNA, Messenger analysis, Trans-Activators genetics, Trans-Activators metabolism, Histones metabolism, Immunologic Memory physiology, Interleukin-4 genetics, Th2 Cells physiology
- Abstract
Interleukin (IL)-4-induced STAT6 activation and the subsequent up-regulation of GATA3 are crucial for the induction of chromatin remodeling of the Th2 cytokine gene loci as Th2 cells undergo development. This study probes the role of these molecules in the maintenance of memory Th2 cells. IL-4 was not required to maintain the capability for Th2 cytokine production in in vivo generated antigen-specific memory Th2 cells. Histone H3-K9/14 hyperacetylation and intergenic transcripts associated with the IL-4 gene locus were preserved in the absence of IL-4, but those associated with the IL-13 gene were partially IL-4-dependent. Histone H3-K4 methylation of the IL-13 and IL-4 gene loci was fully preserved in memory Th2 cells and accompanied by memory cell-specific accumulation of Pol II complex to highly restricted sites. Thus, memory Th2 cells maintain a unique Th2-specific remodeled chromatin in the IL-4 and IL-13 gene loci by active molecular events that are IL-4-independent.
- Published
- 2004
- Full Text
- View/download PDF
41. Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3.
- Author
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Aoyama T, Okamoto T, Nagayama S, Nishijo K, Ishibe T, Yasura K, Nakayama T, Nakamura T, and Toguchida J
- Subjects
- 5-Methylcytosine chemistry, Azacitidine pharmacology, Binding Sites, Blotting, Western, Bone and Bones metabolism, Cartilage metabolism, Cell Line, Tumor, Chondrocytes metabolism, Chromatin metabolism, CpG Islands, Cytosine metabolism, Decitabine, Genes, Reporter, Humans, Luciferases metabolism, Precipitin Tests, Promoter Regions, Genetic, Protein Binding, RNA Interference, RNA, Messenger metabolism, RNA, Small Interfering metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sp1 Transcription Factor metabolism, Sp3 Transcription Factor, Sulfites pharmacology, Thymine chemistry, Transcription, Genetic, Azacitidine analogs & derivatives, DNA Methylation, DNA-Binding Proteins metabolism, Intercellular Signaling Peptides and Proteins genetics, Membrane Proteins genetics, Transcription Factors metabolism
- Abstract
Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.
- Published
- 2004
- Full Text
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42. Chemokines generally exhibit scavenger receptor activity through their receptor-binding domain.
- Author
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Shimaoka T, Nakayama T, Hieshima K, Kume N, Fukumoto N, Minami M, Hayashida K, Kita T, Yoshie O, and Yonehara S
- Subjects
- Amino Acid Substitution, Animals, B-Lymphocytes cytology, B-Lymphocytes metabolism, COS Cells, Cell Line, Chemokine CXCL16, Chemokine CXCL6, Chemokines antagonists & inhibitors, Chemokines chemistry, Chemokines genetics, Chemokines, CXC metabolism, Chemotaxis, Leukocyte, Chlorocebus aethiops, Escherichia coli metabolism, Humans, Jurkat Cells, Lipoproteins, LDL metabolism, Lipoproteins, LDL pharmacology, Membrane Proteins metabolism, Mice, Phylogeny, Protein Binding, Protein Structure, Tertiary, Receptors, Scavenger, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Staphylococcus aureus metabolism, Chemokines metabolism, Receptors, Immunologic metabolism
- Abstract
Chemokines are a family of cytokines that induce directed migration of various types of leukocytes through specific interactions with a group of seven transmembrane receptors. Scavenger receptors are a heterogenous family of transmembrane molecules that commonly bind and uptake oxidized low density lipoprotein and bacteria. Here, we show that not only CXC chemokine 16 (CXCL16)/SR-PSOX, a transmembrane chemokine with scavenger receptor activity, but also 12 out of 15 chemokines examined efficiently bound scavenger receptor ligands in competition with cells expressing their specific chemokine receptors. Furthermore both the chemotactic and scavenger receptor activities of SR-PSOX/CXCL16 were similarly impaired in a series of mutants altered in the chemokine domain, indicating that SR-PSOX/CXCL16 binds scavenger receptor ligands as well as CXCR6 using highly overlapping binding motifs. Taken together, chemokines generally have scavenger receptor-like activity through their receptor-binding domain, suggesting a close evolutionary relationship between chemokines and scavenger receptors.
- Published
- 2004
- Full Text
- View/download PDF
43. Essential role of GATA3 for the maintenance of type 2 helper T (Th2) cytokine production and chromatin remodeling at the Th2 cytokine gene loci.
- Author
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Yamashita M, Ukai-Tadenuma M, Miyamoto T, Sugaya K, Hosokawa H, Hasegawa A, Kimura M, Taniguchi M, DeGregori J, and Nakayama T
- Subjects
- Acetylation, Adenoviridae genetics, Animals, CD4-Positive T-Lymphocytes metabolism, Cell Differentiation physiology, Cytokines genetics, DNA-Binding Proteins biosynthesis, DNA-Binding Proteins genetics, GATA3 Transcription Factor, Gene Expression, Green Fluorescent Proteins, Histones metabolism, Humans, Integrases genetics, Luminescent Proteins genetics, Mice, Mice, Inbred C57BL, Mice, Transgenic, Recombination, Genetic, Retroviridae genetics, Th2 Cells immunology, Th2 Cells metabolism, Trans-Activators biosynthesis, Trans-Activators genetics, Transgenes genetics, Viral Proteins genetics, Chromatin metabolism, Cytokines biosynthesis, DNA-Binding Proteins physiology, Th2 Cells physiology, Trans-Activators physiology
- Abstract
GATA3 expression is essential for type-2 helper T (Th2) cell differentiation. GATA3-mediated chromatin remodeling at the Th2 cytokine gene loci, including Th2-specific long range histone hyperacetylation of the interleukin (IL)-13/IL-4 gene loci, occurs in developing Th2 cells. However, little is known about the role of GATA3, if any, in the maintenance of established remodeled chromatin at the Th2 cytokine gene loci. Here, we established a Cre/LoxP-based site-specific recombination system in cultured CD4 T cells using a unique adenovirus-mediated gene transfer technique. This system allowed us to investigate the effect of loss of GATA3 expression in in vitro differentiated Th2 cells. After ablation of GATA3, we detected reduced production of all Th2 cytokines, increased DNA methylation at the IL-4 gene locus, and decreased histone hyperacetylation at the IL-5 gene locus but not significantly so at the IL-13/IL-4 gene loci. Thus, GATA3 plays important roles in the maintenance of the Th2 phenotype and continuous chromatin remodeling of the specific Th2 cytokine gene locus through cell division.
- Published
- 2004
- Full Text
- View/download PDF
44. Activation of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, by phorbol 12-myristate 13-acetate (PMA) suppresses glucose transport into astroglioma cells via the glucose transporter-1 (GLUT-1).
- Author
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Nakayama T, Mikoshiba K, Yamamori T, and Akagawa K
- Subjects
- Alternative Splicing, Antigens, Surface genetics, Biological Transport drug effects, Cell Line, Tumor, Glucose Transporter Type 1, Humans, Molecular Sequence Data, Nerve Tissue Proteins genetics, Protein Isoforms genetics, Protein Isoforms metabolism, Signal Transduction drug effects, Syntaxin 1, Tetradecanoylphorbol Acetate pharmacology, Antigens, Surface metabolism, Astrocytoma metabolism, Glucose metabolism, Monosaccharide Transport Proteins metabolism, Nerve Tissue Proteins metabolism
- Abstract
Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/syntaxin 1A. We found previously that syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of syntaxin 1C is not known. In this study, we examined the relationship between syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed syntaxin 1C. Based on these results, we postulate that syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.
- Published
- 2004
- Full Text
- View/download PDF
45. CD28 costimulation controls histone hyperacetylation of the interleukin 5 gene locus in developing th2 cells.
- Author
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Inami M, Yamashita M, Tenda Y, Hasegawa A, Kimura M, Hashimoto K, Seki N, Taniguchi M, and Nakayama T
- Subjects
- Acetylation, Animals, CD28 Antigens genetics, DNA Repair, Interleukin-5 metabolism, Mice, T-Lymphocytes cytology, CD28 Antigens metabolism, Cell Differentiation physiology, Histones metabolism, Interleukin-5 genetics, T-Lymphocytes metabolism
- Abstract
Interleukin 5 (IL-5) plays a unique role in allergic inflammatory responses, and the understanding of molecular mechanisms underlying the generation of IL-5-producing cells is crucial for the regulation of allergic disorders. Differentiation of naive CD4 T cells into type-2 helper (Th2) cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13, and IL-5 genes. Histone hyperacetylation of the IL-5 gene displayed a delayed kinetics compared with that of the IL-4 and IL-13 genes, suggesting a distinct remodeling mechanism for the IL-5-gene locus. Here we studied the role of CD28 costimulation in the generation of IL-5-producing cells and the histone hyperacetylation of the IL-5 gene locus. CD28-costimulation selectively enhanced histone hyperacetylation of the IL-5 gene locus that appeared to be mediated through NF-kappaB activation and subsequent up-regulation of GATA3. The CD28 costimulation-sensitive histone hyperacetylation spanned almost the entire intergenic region between the IL-5 and RAD50 accompanied with intergenic transcript. Thus, this is the first demonstration that CD28 costimulation controls a chromatin-remodeling process during Th2 cell differentiation.
- Published
- 2004
- Full Text
- View/download PDF
46. Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity.
- Author
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Wlodawer A, Li M, Gustchina A, Tsuruoka N, Ashida M, Minakata H, Oyama H, Oda K, Nishino T, and Nakayama T
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Cloning, Molecular, Collagenases chemistry, Crystallography, X-Ray, Escherichia coli enzymology, Escherichia coli genetics, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Protein Structure, Secondary, Substrate Specificity, Thermodynamics, Aspartic Acid Endopeptidases chemistry, Aspartic Acid Endopeptidases metabolism, Carboxypeptidases chemistry, Carboxypeptidases metabolism, Collagenases metabolism
- Abstract
Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.
- Published
- 2004
- Full Text
- View/download PDF
47. Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts.
- Author
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Watanabe H, Shimizu T, Nishihira J, Abe R, Nakayama T, Taniguchi M, Sabe H, Ishibashi T, and Shimizu H
- Subjects
- Animals, Cells, Cultured, Collagenases biosynthesis, Fibroblasts enzymology, Humans, Matrix Metalloproteinase 1 genetics, Matrix Metalloproteinase 13, Mice, Mice, Inbred BALB C, Mitogen-Activated Protein Kinases metabolism, Phosphorylation, Protein Kinase C metabolism, Proto-Oncogene Proteins c-raf metabolism, RNA, Messenger analysis, Signal Transduction, Skin cytology, Skin enzymology, Tissue Inhibitor of Metalloproteinase-1 genetics, Macrophage Migration-Inhibitory Factors physiology, Matrix Metalloproteinase 1 biosynthesis, Skin radiation effects, Ultraviolet Rays
- Abstract
Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.
- Published
- 2004
- Full Text
- View/download PDF
48. N(epsilon)-(3-methylpyridinium)lysine, a major antigenic adduct generated in acrolein-modified protein.
- Author
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Furuhata A, Ishii T, Kumazawa S, Yamada T, Nakayama T, and Uchida K
- Subjects
- Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Lysine analogs & derivatives, Spectrometry, Mass, Electrospray Ionization, Acrolein metabolism, Antigens biosynthesis, Lysine biosynthesis
- Abstract
Acrolein, a representative carcinogenic aldehyde, that could be ubiquitously generated in biological systems under oxidative stress shows facile reactivity with a nucleophile such as a protein. In this study, to gain a better understanding of the molecular basis of acrolein modification of protein, we characterized the acrolein modification of a model peptide (the oxidized B chain of insulin) by electrospray ionization-liquid chromatography/mass spectrometry method and established a novel acrolein-lysine condensation reaction. In addition, we found that this condensation adduct represented the major antigenic adduct generated in acrolein-modified protein. To identify the modification site and structures of adducts generated in the acrolein-modified insulin B chain, both the acrolein-pretreated and untreated peptides were digested with V8 protease and the resulting peptides were subjected to electrospray ionization-liquid chromatography/mass spectrometry. This technique identified nine peptides, which contained the acrolein adducts at Lys-29 and the N terminus, and revealed that the reaction of the insulin B chain with acrolein gave multiple adducts, including an unknown adduct containing two molecules of acrolein per lysine. To identify this adduct, we incubated N(alpha)-acetyllysine with acrolein and isolated a product having the same molecular mass as the unknown acrolein-lysine adduct. On the basis of the chemical and spectroscopic evidence, the adduct was determined to be a novel pyridinium-type lysine adduct, N(epsilon)-(3-methylpyridinium)lysine (MP-lysine). The formation of MP-lysine was confirmed by amino acid analysis of proteins treated with acrolein. More notably, this condensation adduct appeared to be an intrinsic epitope of a monoclonal antibody 5F6 that had been raised against acrolein-modified protein.
- Published
- 2003
- Full Text
- View/download PDF
49. Nep98p is a component of the yeast spindle pole body and essential for nuclear division and fusion.
- Author
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Nishikawa S, Terazawa Y, Nakayama T, Hirata A, Makio T, and Endo T
- Subjects
- Amino Acid Sequence, G2 Phase, Genes, Fungal, HSP70 Heat-Shock Proteins metabolism, Membrane Proteins physiology, Microscopy, Electron, Microscopy, Fluorescence, Mitosis, Molecular Chaperones, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Nuclear Proteins, Plasmids metabolism, Protein Binding, Protein Structure, Tertiary, Saccharomyces cerevisiae Proteins physiology, Temperature, Two-Hybrid System Techniques, Cell Nucleus metabolism, Membrane Proteins chemistry, Membrane Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Spindle Apparatus
- Abstract
During the mating of yeast Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. This process requires the functions of BiP/Kar2p, a member of the Hsp70 family in the endoplasmic reticulum, and its partner protein, Jem1p. To investigate further the role of BiP and Jem1p in nuclear fusion, we screened for partner proteins for Jem1p by the yeast two-hybrid system and identified Nep98p. Nep98p is an essential integral membrane protein of the nuclear envelope and is enriched in the spindle pole body (SPB), the sole microtubule-organizing center in yeast. Temperature-sensitive nep98 mutant cells contain abnormal SPBs lacking the half-bridge, suggesting the essential role of Nep98p in the organization of the normal SPB. Additionally, nep98 mutant cells show defects in mitotic nuclear division and nuclear fusion during mating. Because Jem1p is not required for nuclear division, Nep98p probably has dual functions in Jem1p-dependent karyogamy and in Jem1p-independent nuclear division.
- Published
- 2003
- Full Text
- View/download PDF
50. Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells.
- Author
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Zhang S, Mizutani A, Hisatsune C, Higo T, Bannai H, Nakayama T, Hattori M, and Mikoshiba K
- Subjects
- Animals, Base Sequence, Basement Membrane metabolism, Blotting, Western, Brain metabolism, Cell Line, Cytoplasm metabolism, DNA Primers, Dogs, Inositol 1,4,5-Trisphosphate Receptors, Membrane Proteins chemistry, Membrane Proteins metabolism, Mice, Precipitin Tests, Protein Binding, Protein Transport, Calcium Channels metabolism, Cytoskeletal Proteins, Membrane Proteins physiology, Neuropeptides, Receptors, Cytoplasmic and Nuclear metabolism
- Abstract
Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP(3)R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP(3)R1 translocation to the basolateral membrane domain in polarized MDCK cells.
- Published
- 2003
- Full Text
- View/download PDF
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