詳細

上垣　浩一 （ウエガキ　コウイチ）

  • 農学部 応用生命化学科　教授/応用生命化学科長
Last Updated :2024/04/25

コミュニケーション情報 byコメンテータガイド

  • コメント

    蛋白質の発見やその機能の解明、あるいは蛋白質の構造解明に対するコメントが可能。

  • 報道関連出演・掲載一覧

    ＜報道関連出演・掲載一覧＞ ●2023/2/25 　朝日新聞 　柿ワインの酒かすケーキについて

研究者情報

学位

  • 博士（理学）（大阪大学）

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J-Global ID

研究キーワード

  • 微生物   機能   構造   タンパク質   

現在の研究分野（キーワード）

    蛋白質の発見やその機能の解明、あるいは蛋白質の構造解明に対するコメントが可能。

研究分野

  • ライフサイエンス / 応用微生物学

研究活動情報

論文

  • Atsushi Kurata; Shimpei Takeuchi; Ryo Fujiwara; Kento Tamura; Tomoya Imai; Shino Yamasaki-Yashiki; Hiroki Onuma; Yasuhisa Fukuta; Norifumi Shirasaka; Koichi Uegaki
    Bioscience, biotechnology, and biochemistry 2023年05月 
    We characterized the membrane vesicle fraction (RD MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD MVs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected in the RD MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patches cells following the addition of the RD MV fraction. In the presence of the RD MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial Toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.
  • Atsushi Kurata; Shino Yamasaki-Yashiki; Tomoya Imai; Ayano Miyazaki; Keito Watanabe; Koichi Uegaki
    Bioscience, biotechnology, and biochemistry 87 1 119 - 128 2022年11月 
    Immunoglobulin A (IgA) is involved in the maintenance of gut homeostasis. Although the oral administration of bifidobacteria increases the amount of fecal IgA, the effects of bifidobacteria on intestinal immunity remain unclear. We found and characterized membrane vesicles (MVs) derived from Bifidobacterium longum subsp. infantis towards host immune cells. B. infantis MVs consisted of a cytoplasmic membrane and extracellular solute-binding protein (ESBP) was specifically detected. In the presence of B. infantis MVs or recombinant ESBP, RAW264 cells produced the pro-inflammatory cytokine IL-6. IgA was produced by Peyer's patches cells following the addition of B. infantis MVs. Therefore, ESBP of B. infantis MVs is involved in the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells.
  • Kohei Sasamoto; Tomoki Himiyama; Kunihiko Moriyoshi; Takashi Ohmoto; Koichi Uegaki; Tsutomu Nakamura; Yoshiaki Nishiya
    FEBS open bio 12 10 1875 - 1885 2022年10月 
    Acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866) has an N-terminal region (NTR; residues 23-135) between the signal sequence (residues 1-22) and the catalytic domain (residues 136-324), which is of unknown function. Our previous study revealed the crystal structure of the wild-type (WT) enzyme containing the NTR and the catalytic domain. Although the structure of the catalytic domain was successfully determined, that of the NTR was undetermined, as its electron density was unclear. In this study, we investigated the role of the NTR through functional and structural analyses of NTR truncation mutants. Based on sequence and secondary structure analyses, NTR was confirmed to be an intrinsically disordered region. The truncation of NTR significantly decreased the solubility of the proteins at low salt concentrations compared with that of the WT. The NTR-truncated mutant easily crystallized in a conventional buffer solution. The crystal exhibited crystallographic properties comparable with those of the WT crystals suitable for structural determination. These results suggest that NTR plays a role in maintaining the solubility and inhibiting the crystallization of the catalytic domain.
  • Atsushi Kurata; Shogo Kiyohara; Tomoya Imai; Shino Yamasaki-Yashiki; Nobuhiro Zaima; Tatsuya Moriyama; Noriaki Kishimoto; Koichi Uegaki
    Scientific Reports 12 1 13330 - 13330 2022年08月 [査読有り]
     
    Abstract We investigated the characteristics and functionalities of extracellular vesicles (EVs) from Lactiplantibacillus plantarum (previously Lactobacillus plantarum) towards host immune cells. L. plantarum produces EVs that have a cytoplasmic membrane and contain cytoplasmic metabolites, membrane and cytoplasmic proteins, and small RNAs, but not bacterial cell wall components, namely, lipoteichoic acid and peptidoglycan. In the presence of L. plantarum EVs, Raw264 cells inducibly produced the pro-inflammatory cytokines IL-1β and IL-6, the anti-inflammatory cytokine IL-10, and IF-γ and IL-12, which are involved in the differentiation of naive T-helper cells into T-helper type 1 cells. IgA was produced by PP cells following the addition of EVs. Therefore, L. plantarum EVs activated innate and acquired immune responses. L. plantarum EVs are recognized by Toll-like receptor 2 (TLR2), which activates NF-κB, but not by other TLRs or NOD-like receptors. N-acylated peptides from lipoprotein19180 (Lp19180) in L. plantarum EVs were identified as novel TLR2 ligands. Therefore, L. plantarum induces an immunostimulation though the TLR2 recognition of the N-acylated amino acid moiety of Lp19180 in EVs. Additionally, we detected a large amount of EVs in the rat gastrointestinal tract for the first time, suggesting that EVs released by probiotics function as a modulator of intestinal immunity.
  • Hironobu Takagi; Kazuki Yamamoto; Yoshifumi Matsuo; Miki Furuie; Yasuha Kasayuki; Rina Ohtani; Mizuki Shiotani; Tetsuya Hasegawa; Toru Ohnishi; Masataka Ohashi; Katsuki Johzuka; Atsushi Kurata; Koichi Uegaki
    Bioscience, biotechnology, and biochemistry 86 6 755 - 762 2022年05月 
    Isoamyl alcohol (i-AmOH) is produced from α-ketoisocaproate in the l-leucine biosynthetic pathway in yeast and controlled by the negative feedback regulation of α-isopropylmalate synthase (IPMS), which senses the accumulation of l-leucine. It is known that i-AmOH production increases when mutations in the regulatory domain reduce the susceptibility to feedback inhibition. However, the impact of mutations in this domain on the IPMS activity has not been examined. In this study, we obtained 5 IPMS mutants, encoding the LEU4 gene, N515D/S520P/S542F/A551D/A551V, that are tolerant to 5,5,5-trifluoro-dl-leucine. All mutant proteins were purified and examined for both IPMS activity and negative feedback activity by in vitro experiments. The results showed that not only the negative-feedback regulation by l-leucine was almost lost in all mutants, but also the IPMS activity was greatly decreased and the difference in IPMS activity among Leu4 mutants in the presence of l-leucine was significantly correlated with i-AmOH production.
  • Kohei Sasamoto; Tomoki Himiyama; Kunihiko Moriyoshi; Takashi Ohmoto; Koichi Uegaki; Yoshiaki Nishiya; Tsutomu Nakamura
    Acta crystallographica. Section F, Structural biology communications 77 Pt 11 399 - 406 2021年11月 
    The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1-22), an N-terminal region (NTR; residues 23-135) and a catalytic domain (residues 136-324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23-324) was successfully determined. The crystal diffracted to 1.9 Å resolution and belonged to space group I212121. The catalytic domain (residues 136-321) exhibited a (β/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23-135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.
  • Yasushi Shigeri; Makoto Nakata; Hiroshi Y Kubota; Naohiro Tomari; Yoshihiro Yamamoto; Koichi Uegaki; Yoshikazu Haramoto; Chloe Bumb; Yoshie Tanaka; Tomoya Kinumi; Hidetoshi Inagaki
    Zoological science 38 1 8 - 19 2021年02月 
    Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.
  • Tomoki Himiyama; Maki Oshima; Koichi Uegaki; Tsutomu Nakamura
    JOURNAL OF BIOCHEMISTRY 166 1 89 - 95 2019年07月 [査読有り]
     
    Peroxiredoxins from Pyrococcus horikoshii (PhPrx) and Thermococcus kodakaraensis (TkPrx) are highly homologous proteins sharing 196 of the 216 residues. We previously reported a pentagonal ring-type decameric structure of PhPrx. Here, we present the crystal structure of TkPrx. Despite their homology, unlike PhPrx, the quaternary structure of TkPrx was found to be a dodecamer comprised of six homodimers arranged in a hexagonal ring-type assembly. The possibility of the redox-dependent conversion of the molecular assembly, which had been observed in PhPrx, was excluded for TkPrx based on the crystal structure of a mutant in which all of the cysteine residues were substituted with serine. The monomer structures of the dodecameric TkPrx and decameric PhPrx coincided well, but there was a slight difference in the relative orientation of the two domains. Molecular assembly of PhPrx and TkPrx in solution evaluated by gel-filtration chromatography was consistent with the crystallographic results. For both PhPrx and TkPrx, the gel-filtration elution volume slightly increased with a decrease in the protein concentration, suggesting the existence of an equilibrium state between the decameric/dodecameric ring and lower-order assembly. This structural assembly difference between highly homologous Prxs suggests a significant influence of quaternary structure on function, worthy of further exploration.
  • Tsutomu Nakamura; Daisuke Koma; Maki Oshima; Hideto Hoshino; Takashi Ohmoto; Koichi Uegaki
    Journal of Bioscience and Bioengineering 126 2 266 - 272 2018年 [査読有り]
     
    Escherichia coli is the most popular organism used for producing recombinant proteins. However, the expression of recombinant proteins in E. coli sometimes results in the aggregation of proteins as an inclusion body in host cells. In such cases, it is necessary to optimize the refolding conditions to obtain the recombinant protein in its native form. Several techniques, such as reducing the concentration of the induction reagent during E. coli cultivation, have been developed to prevent the formation of inclusion bodies by controlling protein expression levels. In this study, we inserted one copy of a target gene under the control of T7 promoter into the E. coli chromosome using the Red-mediated recombination system. This system enabled soluble expression of the putative D-aminoacylase from Pyrococcus abyssi, which is expressed in an insoluble form following the use of conventional plasmid-based T7 promoter/polymerase systems. The relationship between the number of inserted gene copies and amount of soluble recombinant protein produced was evaluated by multiple insertions of the eGFP gene into the E. coli chromosome. The results revealed that the total expression from the insertion of one copy was around 1/5 that of the pET plasmid system and that expression increased as the inserted gene copy number increased up to five copies.
  • Tsutomu Nakamura; Maki Oshima; Megumi Yasuda; Akiko Shimamura; Junji Morita; Koichi Uegaki
    JOURNAL OF BIOCHEMISTRY 162 6 415 - 422 2017年12月 [査読有り]
     
    Peroxiredoxin from Pyrococcus horikoshii (PhPrx) is a decameric protein formed by ring-type assembly of five dimers. To engineer the quaternary structure of PhPrx, we created a mutant PhPrx (PhPrx6m) by introducing six point mutations designed to dissociate PhPrx into dimers. Although PhPrx6m was a dimer in solution, the six dimers assembled into a dodecamer following crystallization. In the crystal structure, PhPrx6m was overoxidized, and the peroxidatic cysteine was in sulfonic acid form and two cysteines in the C-terminal region were linked by an intramolecular disulfide bond. Thus, we characterized the wild-type PhPrx overoxidized by hydrogen peroxide (PhPrxPer). Analytical ultracentrifugation showed that PhPrxPer had a higher molecular mass in solution than PhPrx. This was confirmed by analysis of the crystal structure of PhPrxPer, which was found to form a ring-type dodecamer composed of six dimers. The monomeric structures of PhPrx6m and PhPrxPer differed from that of PhPrx in the relative orientation of two domains, reflecting the number of dimers in the ring-type assembly. Unlike PhPrx, homologous peroxiredoxin from Aeropyrum pernix (ApPrx) did not undergo hexameric association. This property can be explained by the stronger connection between the two domains in ApPrx due to its C-terminal extension relative to PhPrx.
  • Marina Nawata; Hirotaka Tsutsumi; Yuta Kobayashi; Satoru Unzai; Shouhei Mine; Tsutomu Nakamura; Koichi Uegaki; Hironari Kamikubo; Mikio Kataoka; Daizo Hamada
    FEBS JOURNAL 284 18 3114 - 3127 2017年09月 [査読有り]
     
    Amyloid light-chain (AL) amyloidosis is a protein-misfolding disease characterized by accumulation of immunoglobulin light chains (LCs) into amyloid fibrils. Dimerization of a full length or variable domain (VL) of LC serves to stabilize the native state and prevent the formation of amyloid fibrils. We here analyzed the thermodynamic properties of dimerization and unfolding reactions by nonamyloidogenic V-L from REI LC or its monomeric Y96K mutant using sedimentation velocity and circular dichroism. The data indicate that the equilibrium shifts to native dimerization for wild-type REI V-L by elevating temperature due to the negative enthalpy change for dimer dissociation (-81.2 kJ.mol(-1)). The Y96K mutation did not affect the stability of the monomeric native state but increased amyloidogenicity. These results suggest that the heat-induced native homodimerization is the major factor preventing amyloid formation by wild-type REI VL. Heat-induced native oligomerization may be an efficient strategy to avoid the formation of misfolded aggregates particularly for thermostable proteins that are used at elevated temperatures under conditions where other proteins tend to misfold.
  • Masanori Ando; Takuya Kamimura; Koichi Uegaki; Vasudevanpillai Biju; Jennifer T. Damasco Ty; Yasushi Shigeri
    SENSORS AND ACTUATORS B-CHEMICAL 246 1074 - 1079 2017年07月 [査読有り]
     
    We report here a novel, sensitive detection method for gaseous amines (primary, secondary and tertiary) using thin films of CdSe-based core-shell type quantum dots (CdSe/ZnS and CdSeTe/ZnS) deposited on a glass substrate. The photoluminescence intensity of the quantum dot thin films rapidly decreased on exposure to amine gas in air, and it reversibly recovered after the atmosphere was changed back to air without amine gas. On the other hand, oxygen, nitrogen, argon, carbon dioxide, or hydrogen, all components of natural air, did not affect the photoluminescence intensity of the quantum dot thin films. Interestingly, the photoluminescence of quantum dot films showed higher sensitivity to n-alkylamines, such as hexylamine, when compared with bulky or branched molecules, such as diethylamine and triethylamine. Furthermore, thin films of green-emitting quantum dots showed higher sensitivity to amines when compared with red-emitting ones, which was attributed to the large surface-to-volume ratios of smaller quantum dots. The reversible, reproducible, and selective responses of CdSe-based core-shell type quantum dots to gaseous amines in air suggested that quantum dot thin films could be promising photoluminescence-based optical amine gas sensors. (C) 2017 Elsevier B.V. All rights reserved.
  • Koichi Uegaki; Haruko Kumanogoh; Toshiyuki Mizui; Takatsugu Hirokawa; Yasuyuki Ishikawa; Masami Kojima
    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 18 5 2017年05月 [査読有り]
     
    Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism.
  • Ronan Kelly; Leora Horn; James Chih-Hsin Yang; Dae Ho Lee; Bhardwaj Desai; Tanya Fleege; Fei Jie; Srinivasu Poondru; Anne Keating; Debbie Whitcomb; Tosei Murase; Koichi Uegaki; Kouji Aoyama; Kazuhiko Nakagawa
    JOURNAL OF THORACIC ONCOLOGY 12 1 S1083 - S1084 2017年01月
  • Tsutomu Nakamura; Maki Oshima; Koichi Uegaki
    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES 73 C1174 - C1174 2017年 [査読有り]
  • Masanori Ando; Takuya Kamimura; Koichi Uegaki; Vasudevanpillai Biju; Yasushi Shigeri
    MICROCHIMICA ACTA 183 11 3019 - 3024 2016年11月 [査読有り]
     
    Thin films of CdSe-based core-shell type quantum dots (CdSe/CdZnS, CdSe/ZnS and CdSeTe/ZnS) deposited on glass substrate were found to undergo a reversible change in photoluminescence (PL) on exposure to ozone in concentrations as low as 0.1 ppm in air. PL decreased rapidly on adding ozone and fully recovered after changing back to pure air. The PL of the CdSe/ZnS quantum dots (QDs) was not quenched by pure oxygen, nitrogen, argon, carbon dioxide, and air containing hydrogen. A comparison of various CdSe-based core-shell type QDs with different emission colors showed that green-emitting QDs with smaller size were more sensitive to ozone, while red-emitting QDs (of larger size) were more resistant to high concentrations of ozone. The response time of the sensor (for a 90 % signal change) was in the order of 10-20 min. The reversible, reproducible and selective response to ozone at room temperature under atmospheric pressure suggested that these QDs have a great potential in terms of fluorescent sensing of ozone.
  • Tsutomu Nakamura; Yasushige Yonezawa; Yuko Tsuchiya; Mayumi Niiyama; Kurumi Ida; Maki Oshima; Junji Morita; Koichi Uegaki
    JOURNAL OF STRUCTURAL BIOLOGY 195 3 286 - 293 2016年09月 [査読有り]
     
    Enzymes of carbohydrate esterase (CE) family 14 catalyze hydrolysis of N-acetyl groups at the non-reducing end of the N-acetylglucosamine (GlcNAc) residue of chitooligosaccharides or related compounds. N,N'-diacetylchitobiose deacetylase (Dac) belongs to the CE-14 family and plays a role in the chitinolytic pathway in archaea by deacetylating N,N'-diacetylchitobiose (GlcNAc(2)), which is the end product of chitinase. In this study, we revealed the structural basis of reaction specificity in CE-14 deacetylases by solving a crystal structure of Dac from Pyrococcus horikoshii (Ph-Dac) in complex with a novel reaction intermediate analog. We developed 2-deoxy-2-methylphosphoramido-D-glucose (MPG) as the analog of the tetrahedral oxyanion intermediate of the monosaccharide substrate GlcNAc. The crystal structure of Ph-Dac in complex with MPG demonstrated that Arg92, Asp115, and His152 side chains interact with hydroxyl groups of the glucose moiety of the non-reducing-end GlcNAc residue. The amino acid residues responsible for recognition of the MPG glucose moiety are spatially conserved in other CE-14 deacetylases. Molecular dynamics simulation of the structure of the Ph-Dac-GlcNAc(2) complex indicated that the reducing GlcNAc residue is placed in a large intermolecular cleft and is not involved with specific interactions with the enzyme. This observation was consistent with results indicating that Ph-Dac displayed similar kinetic parameters for both GlcNAc and GlcNAc(2). This study provides the structural basis of reaction-site specificity of Dac and related CE-14 enzymes. (C) 2016 Elsevier Inc. All rights reserved.
  • Yasushi Shigeri; Takuya Kamimura; Masanori Ando; Koichi Uegaki; Hiroaki Sato; Fumito Tani; Ryuichi Arakawa; Tomoya Kinumi
    European Journal of Mass Spectrometry 22 2 83 - 90 2016年04月 [査読有り]
     
    The sensitivity, range of applications, and reaction mechanism of 2-hydrazinoquinoline as a reactive matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were examined. Using a reaction chamber (125 L) equipped with a stirring fan and a window for moving the MALDI-MS plate and volatile samples in and out, the sensitivities of 2-hydrazinoquinoline to gaseous aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and n-butyraldehyde) and ketones (acetone, methyl ethyl ketone, and methyl isobutyl ketone) were determined to be at least parts per million (ppm) levels. On the other hand, carboxylic acids (formic acid, acetic acid, propionic acid, and butyric acid) and esters (ethyl acetate, pentyl acetate, isoamyl acetate, and methyl salicylate) could not be detected by 2-hydrazinoquinoline in MALDI-MS. In addition to 2,4-dinitrophenylhydrazine, a common derivatization reagent for analyzing carbonyl compounds quantitatively in gas chromatography and liquid chromatography, the dissolution of 2-hydrazinoquinoline in an acidic solution, such as trifluoroacetic acid, was essential for its function as a reactive matrix for MALDI-MS.
  • Yoko Akazawa-Ogawa; Koichi Uegaki; Yoshihisa Hagihara
    JOURNAL OF BIOCHEMISTRY 159 1 111 - 121 2016年01月 [査読有り]
     
    Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heat-induced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability.
  • Yasushi Shigeri; Takuya Kamimura; Masanori Ando; Koichi Uegaki; Hiroaki Sato; Fumito Tani; Ryuichi Arakawa; Tomoya Kinumi
    EUROPEAN JOURNAL OF MASS SPECTROMETRY 22 2 83 - 90 2016年 [査読有り]
     
    The sensitivity, range of applications, and reaction mechanism of 2-hydrazinoquinoline as a reactive matrix for matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) were examined. Using a reaction chamber (125 L) equipped with a stirring fan and a window for moving the MALDI-MS plate and volatile samples in and out, the sensitivities of 2-hydrazinoquinoline to gaseous aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and n-butyraldehyde) and ketones (acetone, methyl ethyl ketone, and methyl isobutyl ketone) were determined to be at least parts per million (ppm) levels. On the other hand, carboxylic acids (formic acid, acetic acid, propionic acid, and butyric acid) and esters (ethyl acetate, pentyl acetate, isoamyl acetate, and methyl salicylate) could not be detected by 2-hydrazinoquinoline in MALDI-MS. In addition to 2,4-dinitrophenylhydrazine, a common derivatization reagent for analyzing carbonyl compounds quantitatively in gas chromatography and liquid chromatography, the dissolution of 2-hydrazinoquinoline in an acidic solution, such as trifluoroacetic acid, was essential for its function as a reactive matrix for MALDI-MS.
  • Nakamura, Tsutomu; Niiyama, Mayumi; Hashimoto, Wakana; Ida, Kurumi; Abe, Manabu; Morita, Junji; Uegaki, Koichi
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 71 657 - 662 WILEY-BLACKWELL 2015年06月 [査読有り]
     
    Native N,N'-diacetylchitobiose deacetylase from Pyrococcus furiosus (Pf-Dac) and its selenomethionine derivative (Se-Pf-Dac) were crystallized and analyzed in the presence and absence of cadmium ion. The four crystal structures fell into three different crystal-packing groups, with the cadmium-free Pf-Dac and Se-Pf-Dac belonging to the same space group, with homologous unit-cell parameters. The crystal structures in the presence of cadmium contained distorted octahedral cadmium complexes coordinated by three chlorides, two O atoms and an S or Se atom from the N-terminal methionine or selenomethionine, respectively. The N-terminal cadmium complex was involved in crystal contacts between symmetry-related molecules through hydrogen bonding to the N-termini. While all six N-termini of Se-Pf-Dac were involved in cadmium-complex formation, only two of the Pf-Dac N-termini participated in complex formation in the Cd-containing crystal, resulting in different crystal forms. These differences are discussed in light of the higher stability of the Cd-Se bond than the Cd-S bond. This work provides an example of the contribution of cadmium towards determining protein crystal quality and packing
  • Tsutomu Nakamura; Mayumi Niiyama; Wakana Hashimoto; Kurumi Ida; Manabu Abe; Junji Morita; Koichi Uegaki
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 71 Pt 6 657 - 662 2015年06月 [査読有り]
     
    Native N,N'-diacetylchitobiose deacetylase from Pyrococcus furiosus (Pf-Dac) and its selenomethionine derivative (Se-Pf-Dac) were crystallized and analyzed in the presence and absence of cadmium ion. The four crystal structures fell into three different crystal-packing groups, with the cadmium-free Pf-Dac and Se-Pf-Dac belonging to the same space group, with homologous unit-cell parameters. The crystal structures in the presence of cadmium contained distorted octahedral cadmium complexes coordinated by three chlorides, two O atoms and an S or Se atom from the N-terminal methionine or selenomethionine, respectively. The N-terminal cadmium complex was involved in crystal contacts between symmetry-related molecules through hydrogen bonding to the N-termini. While all six N-termini of Se-Pf-Dac were involved in cadmium-complex formation, only two of the Pf-Dac N-termini participated in complex formation in the Cd-containing crystal, resulting in different crystal forms. These differences are discussed in light of the higher stability of the Cd-Se bond than the Cd-S bond. This work provides an example of the contribution of cadmium towards determining protein crystal quality and packing depending on the use of the native protein or the selenomethionine derivative.
  • Toshiyuki Mizui; Yasuyuki Ishikawa; Haruko Kumanogoh; Maria Lume; Tomoya Matsumoto; Tomoko Hara; Shigeto Yamawaki; Masami Takahashi; Sadao Shiosaka; Chiaki Itami; Koichi Uegaki; Mart Saarma; Masami Kojima
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 112 23 E3067 - E3074 2015年06月 [査読有り]
     
    Most growth factors are initially synthesized as precursor proteins and subsequently processed into their mature form by proteolytic cleavage, resulting in simultaneous removal of a pro-peptide. However, compared with that of mature form, the biological role of the pro-peptide is poorly understood. Here, we investigated the biological role of the pro-peptide of brain-derived neurotrophic factor (BDNF) and first showed that the pro-peptide is expressed and secreted in hippocampal tissues and cultures, respectively. Interestingly, we found that the BDNF pro-peptide directly facilitates hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA receptors and the pan-neurotrophin receptor p75(NTR). The BDNF pro-peptide also enhances NMDA-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor endocytosis, a mechanism crucial for LTD expression. Thus, the BDNF pro-peptide is involved in synaptic plasticity that regulates a mechanism responsible for promoting LTD. The well-known BDNF polymorphism valine for methionine at amino acid position 66 (Val66Met) affects human memory function. Here, the BDNF pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings demonstrate functional roles for the BDNF pro-peptide and a naturally occurring human BDNF polymorphism in hippocampal synaptic depression.
  • Hitoshi Ando; Norioki Kawasaki; Naoko Yamano; Kouichi Uegaki; Atsuyoshi Nakayama
    POLYMER DEGRADATION AND STABILITY 114 65 - 71 2015年04月 [査読有り]
     
    A biodegradable polymer titanium dioxide (TiO2) nanocomposite with antimicrobial activity arising from photooxidation of the composited TiO2, was investigated. Poly(epsilon-caprolactone-co-L-lactide) visible-light-sensitive TiO2 nanocomposite films were prepared by the solvent cast method. At a TiO2 concentration of 0.5 wt%, the nanocomposite films showed no antibacterial activity in the dark, whereas they exhibited high antibacterial activity against Escherichia coli under fluorescent light irradiation at 4000 lx. In contrast, nanocomposite films with TiO2 concentrations of 0.7 wt% and higher showed antibacterial activity even in the dark. In an enzymatic hydrolysis test, the degradation of nanocomposite films with 0.5 wff of TiO2 was suppressed 21% by fluorescent light irradiation for 4 days at 8000 lx. In addition, the degradation of films containing TiO2 was suppressed even under alternating 12-h periods of fluorescent light irradiation and non-irradiation. These results indicate that biodegradation of polymer TiO2 nanocomposite films was suppressed even when the films were not continuously irradiated with light In a soil degradation test, the degradation of films with TiO2 placed on a soil surface exposed to sunlight was obviously suppressed compared with that of films without TiO2. In contrast films with and without TiO2 placed on a soil surface in the dark or buried in soil showed no significant differences in percent degradation. A copolymer-degrading bacterium isolated from soil was resistant to the nanocomposite film with 5 wt% of TiO2 in the dark, in contrast to E. coli, suggesting that such resistant microorganisms are involved in biodegradation of the nanocomposite under dark conditions. In this study, we were able to impart an on/off biodegradation function, in which biodegradation is suppressed by light irradiation and progresses in the dark, to a biodegradable polymer by compositing the polymer with a small amount of visible-light-sensitive TiO2. (C) 2015 Elsevier Ltd. All rights reserved.
  • Shouhei Mine; Mayumi Niiyama; Wakana Hashimoto; Takahisa Ikegami; Daisuke Koma; Takashi Ohmoto; Yohta Fukuda; Tsuyoshi Inoue; Yoshito Abe; Tadashi Ueda; Junji Morita; Koichi Uegaki; Tsutomu Nakamura
    FEBS JOURNAL 281 11 2584 - 2596 2014年06月 [査読有り]
     
    In order to develop a structure-based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on N,N-diacetylchitobiose deacetylases (Dacs) from Pyrococcus horikoshii (Ph-Dac) and Pyrococcusfuriosus (Pf-Dac). Neither Ph-Dac nor Pf-Dac was expressed in the soluble fraction of Escherichiacoli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E.coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were active and thermostable up to 85 degrees C. The crystal structures of Ph-Dac and Pf-Dac were determined at resolutions of 2.0 angstrom and 1.54 angstrom, respectively. The Pyrococcus Dac forms a hexamer composed of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph-Dac, catalytic Zn situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N-acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the N epsilon atom of His264 (from the adjacent polypeptide in the Ph-Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. Mutation analysis also indicated that His264 was essential to the catalysis. These factors give the archaeal Dacs an unprecedented active site architecture a Zn-dependent deacetylases. Database Structural data are available in the Protein Data Bank database under accession numbers 3WL3, 3WL4, and 3WE7. Structured digital abstract Ph-DacandPh-Dacbindbyx-ray crystallography(View interaction) Pf-DacandPf-Dacbindbyx-ray crystallography(View interaction)
  • Yoko Akazawa-Ogawa; Mizuki Takashima; Young-Ho Lee; Takahisa Ikegami; Yuji Goto; Koichi Uegaki; Yoshihisa Hagihara
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 22 15666 - 15679 2014年05月 [査読有り]
     
    The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 degrees C and was insensitive to the number of heating (90 degrees C)-cooling (20 degrees C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.
  • Shouhei Mine; Tsutomu Nakamura; Takaaki Sato; Takahisa Ikegami; Koichi Uegaki
    JOURNAL OF BIOCHEMISTRY 155 2 115 - 122 2014年02月 [査読有り]
     
    A chitinase, from Pyrococcus furiosus, is a hyperthermophilic glycosidase that effectively hydrolyses both alpha and beta crystalline chitin. This chitinase has unique structural features; it contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBD1 and ChBD2). We have determined the structure of ChBD1, which significantly enhances the activity of the catalytic domains, by nuclear magnetic resonance spectroscopy. The overall structure of ChBD1 had a compact and globular architecture consisting of three anti-parallel beta-strands, similar to those of other proteins classified into carbohydrate-binding module (CBM) family 5. A mutagenesis experiment suggested three solvent-exposed aromatic residues (Tyr112, Trp113 and Tyr123) as the chitin-binding sites. The involvement of Tyr123 or the corresponding aromatic residues in other CBMs, has been demonstrated for the first time. This result indicates that the binding mode may be different from those of other chitin-binding domains in CBM family 5. In addition, the binding affinities of ChBD1 and ChBD2 were quite different, suggesting that the two ChBDs each play a different role in efficiently increasing the activities of AD1 and AD2.
  • Mari Satoh; Keiko Tawa; Koichi Uegaki; Tomoko Hara; Mitsuo Umetsu; Hikaru Nakazawa; Makoto Itakura; Masami Takahashi; Hiroyuki Aota; Masami Kojima
    Transactions of the Materials Research Society of Japan 39 3 361 - 364 2014年
  • Nakamura Tsutomu; Oshima Maki; Uegaki Koichi
    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES 70 C1174  2014年 [査読有り]
  • Tsutomu Nakamura; Aika Mori; Mayumi Niiyama; Hiroyoshi Matsumura; Chisa Tokuyama; Junji Morita; Koichi Uegaki; Tsuyoshi Inoue
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 69 7 719 - 722 2013年07月 [査読有り]
     
    The crystal structure of peroxiredoxin from the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii (PhPrx) was determined at a resolution of 2.25 angstrom. The overall structure was a ring-type decamer consisting of five homodimers. Citrate, which was included in the crystallization conditions, was bound to the peroxidatic cysteine of the active site, with two O atoms of the carboxyl group mimicking those of the substrate hydrogen peroxide. PhPrx lacked the C-terminal tail that forms a 32-residue extension of the protein in the homologous peroxiredoxin from Aeropyrum pernix (ApPrx).
  • Keiko Tawa; Mari Satoh; Koichi Uegaki; Tomoko Hara; Masami Kojima; Haruko Kumanogoh; Hiroyuki Aota; Yoshiki Yokota; Takahiko Nakaoki; Mitsuo Umetsu; Hikaru Nakazawa; Izumi Kumagai
    JAPANESE JOURNAL OF APPLIED PHYSICS 52 6 2013年06月 [査読有り]
     
    Plasmonic chips, which are grating replicas coated with thin metal layers and overlayers such as ZnO, were applied in immunosensors to improve their detection sensitivity. Fluorescence from labeled antibodies bound to plasmonic chips can be enhanced on the basis of a grating-coupled surface plasmon resonance (GC-SPR) field. In this study, as one of the representative candidate protein markers for brain disorders, the brain-derived neurotrophic factor (BDNF) was quantitatively measured by sandwich assay on a plasmonic chip and detected on our plasmonic chip in the concentration of 5-7 ng/mL within 40 min. Furthermore, BDNF was detected in the blood sera from three types of mice: wild-type mice and two types of mutant mice. This technique is promising as a new clinical diagnosis tool for brain disorders based on scientific evidence such as blood test results. (C) 2013 The Japan Society of Applied Physics
  • 上垣浩一; 中村努; 峯昇平; 西村重徳
    生化学 85 7 577 - 582 日本生化学会 2013年 [査読有り]
  • Tsutomu Nakamura; Yasuhiro Kashima; Shouhei Mine; Takashi Oku; Koichi Uegaki
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 114 2 150 - 154 2012年08月 [査読有り]
     
    We characterized and determined the crystal structure of a putative glucokinase/hexokinase from Therm us thermophilus that belongs to the ROK (bacterial repressors, uncharacterized open reading frames, and sugar kinases) family. The protein possessed significant enzymatic activity against glucose and mannose, with V-max values of 260 and 68 mu mol.min(-1).mg(-1) protein, respectively. Therefore, we concluded that the enzyme is a hexokinase. However, the hexokinase showed little catalytic capacity for galactose and fructose. Circular dichroism measurements indicated that the enzyme was structurally stable at 90 degrees C. The crystal structure of the enzyme was determined at a resolution of 2.02 angstrom, with R-cryst and R-free values of 18.1% and 22.6%, respectively. The polypeptide structure was divided into large and small domains. The ROK consensus sequences 1 and 2 were included in the large domain. The cysteine-rich consensus sequence 2 folded into a zinc finger, and the bound zinc was confirmed by both electron density and X-ray absorption fine structure (XAFS) spectrum. The overall structure was a homotetramer that consisted of a dimer of dimers. The accessible surface area buried by the association of the dimers into the tetrameric structures was significantly higher in the T. thermophilus enzyme than in a homologous tetrameric ROK sugar kinase. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.
  • Shouhei Mine; Takahisa Ikegami; Kazunori Kawasaki; Tsutomu Nakamura; Koichi Uegaki
    PROTEIN EXPRESSION AND PURIFICATION 84 2 265 - 269 2012年08月 [査読有り]
     
    A chitinase from the hyperthermophilic archaeon Pyrococcus furiosus degrades chitin to produce diacetylchitobiose [(GIcNAc)(2)] as the end product. To further investigate the degradation mechanism of (GIcNAc)(2) in Pyrococcus spp., we cloned the gene of PH0499 from Pyrococcus horikoshii, which encodes a protein homologous to the diacetylchitobiose deacetylase of Thermococcus kodakaraensis. The deacetylase (Ph-Dac) was overexpressed as inclusion bodies in Escherichia coli Rosetta (DE3) pLys. The insoluble inclusion body was solubilized and reactivated through a refolding procedure. After several purification steps, 40 mg of soluble, thermostable (up to 80 degrees C) Ph-Dac was obtained from 1 L of culture. The apparent molecular mass of the refolded Ph-Dac was 180 kDa, indicating Ph-Dac to be a homohexamer. The refolded Ph-Dac also exhibited deacetylase activity toward (GIcNAc)(2), and the deacetylation site was revealed to be specific to the nonreducing end residue of (GIcNAc)(2). These expression and purification systems are useful for further characterization of Ph-Dac. (C) 2012 Elsevier Inc. All rights reserved.
  • Hiroyoshi Matsumura; Nanoha Kusaka; Taichi Nakamura; Naoko Tanaka; Keita Sagegami; Koichi Uegaki; Tsuyoshi Inoue; Yukio Mukai
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 32 26528 - 26538 2012年08月 [査読有り]
     
    The yeast Cyc8p-Tup1p protein complex is a general transcriptional corepressor of genes involved in many different physiological processes. Herein, we present the crystal structure of the Tup1p N-terminal domain (residues 1-92), essential for Tup1p self-assembly and interaction with Cyc8p. This domain tetramerizes to form a novel antiparallel four-helix bundle. Coiled coil interactions near the helical ends hold each dimer together, whereas interdimeric association involves only two sets of two residues located toward the chain centers. A mutagenesis study confirmed that the nonpolar residues responsible for the association of the protomers as dimers are also required for transcriptional repression. An additional structural study demonstrated that the domain containing an Leu(62) -> Arg mutation that had been shown not to bind Cyc8p exhibits an altered structure, distinct from the wild type. This altered structure explains why the mutant cannot bind Cyc8p. The data presented herein highlight the importance of the architecture of the Tup1p N-terminal domain for self-association.
  • Mayumi Ishida; Hideaki Shimojo; Aki Hayashi; Rika Kawaguchi; Yasuko Ohtani; Koichi Uegaki; Yoshifumi Nishimura; Jun-ichi Nakayama
    MOLECULAR CELL 47 2 228 - 241 2012年07月 [査読有り]
     
    Centromeric heterochronnatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.
  • Yuka Watanabe; Shinichi Kitamura; Kazunori Kawasaki; Tomoki Kato; Koichi Uegaki; Kota Ogura; Kazuhiko Ishikawa
    BIOPOLYMERS 95 12 833 - 839 2011年12月 [査読有り]
     
    Plant cellulose is the most abundant organic compound on earth. Technologies for producing cellulose fiber or improving the enzymatic saccharification of cellulose hold the key to biomass applications. A technology for atomizing biomass without strong acid catalysis remains to be developed. The water jet is a well-known device used in machines (e. g., washing machines, cutters, and mills) that use high-pressure water. In this study, we examined whether a water jet system could be used to atomize crystalline cellulose, which comprises approximately 50% of plant biomass. The Star Burst System manufactured by Sugino Machine Limited (Sugino Machine; Toyama, Japan) is a unique atomization machine that uses a water jet to atomize materials and thereby places lower stress on the environment. After treatment with this system, the crystalline cellulose was converted into a gel-like form. High-angular annular dark-field scanning transmission electron microscopy showed that the cellulose fibers had been converted from a solid crystalline into a matrix of cellulose nanofibers. In addition, our results show that this system can improve the saccharification efficiency of cellulases by more than three-fold. Hence, the Star Burst System provides a new and mild pretreatment system for processing biomass materials. (C) 2011 Wiley Periodicals, Inc. Biopolymers 95: 833-839, 2011.
  • Tsutomu Nakamura; Yasuhiro Kashima; Shouhei Mine; Takashi Oku; Koichi Uegaki
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 67 1559 - 1562 2011年12月 [査読有り]
     
    Glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. The open reading frame TTHA0299 of the extreme thermophile Thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. In this study, the glucokinase/hexokinase from T. thermophilus was purified and crystallized using polyethylene glycol 8000 as a precipitant. Diffraction data were collected and processed to 2.02 angstrom resolution. The crystal belonged to space group P21, with unit-cell parameters a = 70.93, b = 138.14, c = 75.16 angstrom, beta = 95.41 degrees.
  • Kyoko Hiragami-Hamada; Kaori Shinmyozu; Daizo Hamada; Yoshiro Tatsu; Koichi Uegaki; Shinsuke Fujiwara; Jun-ichi Nakayama
    MOLECULAR AND CELLULAR BIOLOGY 31 6 1186 - 1200 2011年03月 [査読有り]
     
    The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1 alpha plays a central role in its targeting to chromatin. Recombinant HP1 alpha prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1 alpha was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1 alpha's affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1 alpha to H3K9me by mediating the interaction between HP1 alpha and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1 alpha exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1 alpha's N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.
  • Tsutomu Nakamura; Kasumi Torikai; Koichi Uegaki; Junji Morita; Kodai Machida; Atsushi Suzuki; Yasushi Kawata
    FEBS JOURNAL 278 4 598 - 609 2011年02月 [査読有り]
     
    Aeropyrum pernix K1, an aerobic hyperthermophilic archaeon, produces a cambialistic superoxide dismutase that is active in the presence of either of Mn or Fe. The crystal structures of the superoxide dismutase from A. pernix in the apo, Mn-bound and Fe-bound forms were determined at resolutions of 1.56, 1.35 and 1.48 A, respectively. The overall structure consisted of a compact homotetramer. Analytical ultracentrifugation was used to confirm the tetrameric association in solution. In the Mn-bound form, the metal was in trigonal bipyramidal coordination with five ligands: four side chain atoms and a water oxygen. One aspartate and two histidine side chains ligated to the central metal on the equatorial plane. In the Fe-bound form, an additional water molecule was observed between the two histidines on the equatorial plane and the metal was in octahedral coordination with six ligands. The additional water occupied the postulated superoxide binding site. The thermal stability of the enzyme was compared with superoxide dismutase from Thermus thermophilus, a thermophilic bacterium, which contained fewer ion pairs. In aqueous solution, the stabilities of the two enzymes were almost identical but, when the solution contained ethylene glycol or ethanol, the A. pernix enzyme had significantly higher thermal stability than the enzyme from T. thermophilus. This suggests that dominant ion pairs make A. pernix superoxide dismutase tolerant to organic media.
  • Toshiyuki Mizui; Koichi Uegaki; Yasuyuki Ishikawa; Tomoko Hara; Masami Takahashi; Sadao Shiosaka; Chiaki Itami; Haruko Kumanogoh; Masami Kojima
    NEUROSCIENCE RESEARCH 71 E138 - E138 2011年 [査読有り]
  • Haruko Kumanogoh; Keiko Tawa; Yoshiki Yokota; Tomoko Hara; Koichi Uegaki; Takahiko Nakaoki; Masami Kojima
    NEUROSCIENCE RESEARCH 71 E208 - E208 2011年 [査読有り]
  • Tsutomu Nakamura; Kasumi Torikai; Junji Morita; Yasushi Kawata; Koichi Uegaki
    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES 67 C633 - C634 2011年 [査読有り]
  • Hiroaki Tsuji; Shigenori Nishimura; Takashi Inui; Yuji Kado; Kazuhiko Ishikawa; Tsutomu Nakamura; Koichi Uegaki
    FEBS JOURNAL 277 12 2683 - 2695 2010年06月 [査読有り]
     
    The hyperthermostable chitinase from the hyperthermophilic archaeon Pyrococcus furiosus has a unique multidomain structure containing two chitin-binding domains and two catalytic domains, and exhibits strong crystalline chitin hydrolyzing activity at high temperature. In order to investigate the structure-function relationship of this chitinase, we analyzed one of the catalytic domains (AD2) using mutational and kinetic approaches, and determined the crystal structure of AD2 complexed with chito-oligosaccharide substrate. Kinetic studies showed that, among the acidic residues in the signature sequence of family 18 chitinases (DXDXE motif), the second Asp (D-2) and Glu (E) residues play critical roles in the catalysis of archaeal chitinase. Crystallographic analyses showed that the side-chain of the catalytic proton-donating E residue is restrained into the favorable conformer for proton donation by a hydrogen bond interaction with the adjacent D-2 residue. The comparison of active site conformations of family 18 chitinases provides a new criterion for the subclassification of family 18 chitinase based on the conformational change of the D-2 residue.
  • Toshiyuki Mizui; Koichi Uegaki; Yasuyuki Ishikawa; Tomoko Hara; Sadao Shiosaka; Masami Takahashi; Haruko Kumanogoh; Masami Kojima
    NEUROSCIENCE RESEARCH 68 E141 - E141 2010年 [査読有り]
  • Akihiro Ito; Taka-aki Okamura; Koichi Uegaki; Han-Woo Kim; Kazuhiko Ishikawa; Tsutomu Nakamura; Hitoshi Yamamoto; Norikazu Ueyama
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 2 428 - 430 2009年02月 [査読有り]
     
    Analysis of products digested by glycosyl hydrolases helps understanding of the hydrolysis mechanism and the substrate recognition in the enzymes. We developed a new universal technique, which consists of ruthenium (II) complex labeling and mass spectrometry analysis, to identify the reducing sugars released from oligosaccharides by enzymatic digestion. This method was applied to enzymatic digestion by chitinase and cellulase of the hyperthermophilic archaea Pyrococcus fusiosus and Pyrococcus horikoshii respectively.
  • Hisatsugu Koshimizu; Kazuyuki Kiyosue; Tomoko Hara; Shunsuke Hazama; Shingo Suzuki; Koichi Uegaki; Guhan Nagappan; Eugene Zaitsev; Takatsugu Hirokawa; Yoshiro Tatsu; Akihiko Ogura; Bai Lu; Masami Kojima
    MOLECULAR BRAIN 2 2009年 [査読有り]
     
    Background: Proneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75(NTR)) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood.Results: Based on two rare single nucleotide polymorphisms (SNPs) of the human brain-derived neurotrophic factor (BDNF) gene, we generated R125M-, R127L- and R125M/R127L-BDNF, which have amino acid substitution(s) near the cleavage site between the pro-and mature-domain of BDNF. Western blot analyses demonstrated that these BDNF variants are poorly cleaved and result in the predominant secretion of proBDNF. Using these cleavage-resistant proBDNF (CR-proBDNF) variants, the molecular and cellular roles of proBDNF on the CNS neurons were examined. First, CR-proBDNF showed normal intracellular distribution and secretion in cultured hippocampal neurons, suggesting that inhibition of proBDNF cleavage does not affect intracellular transportation and secretion of BDNF. Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS neurons. Treatment with CR-proBDNF elicited apoptosis of cultured cerebellar granule neurons (CGNs), while treatment with mature BDNF (matBDNF) promoted cell survival. Third, we examined the effects of CR-proBDNF on neuronal morphology using more than 2-week cultures of basal forebrain cholinergic neurons (BFCNs) and hippocampal neurons. Interestingly, in marked contrast to the action of matBDNF, which increased the number of cholinergic fibers and hippocampal dendritic spines, CR-proBDNF dramatically reduced the number of cholinergic fibers and hippocampal dendritic spines, without affecting the survival of these neurons.Conclusion: These results suggest that proBDNF has distinct functions in different populations of CNS neurons and might be responsible for specific physiological cellular processes in the brain.
  • Tsutomu Nakamura; Shouhei Mine; Yoshihisa Hagihara; Kazuhiko Ishikawa; Takahisa Ikegami; Koichi Uegaki
    JOURNAL OF MOLECULAR BIOLOGY 381 3 670 - 680 2008年09月 [査読有り]
     
    A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both alpha and beta crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded beta-sheets, was composed of a typical beta-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass. (C) 2008 Elsevier Ltd. All rights reserved.
  • Koichi Uegaki; Tsutomu Nakamura; Kazuhiko Ishikawa; Shouhei Mine
    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES 64 C267 - C268 2008年
  • Yoshihisa Hagihara; Shouhei Mine; Koichi Uegaki
    JOURNAL OF BIOLOGICAL CHEMISTRY 282 50 36489 - 36495 2007年12月 [査読有り]
     
    We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala(49) and Ile(70) are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 degrees C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala(49) and Ile(70) are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.
  • Hee-Jin Kang; Koichi Uegaki; Harumi Fukada; Kazuhiko Ishikawa
    EXTREMOPHILES 11 2 251 - 256 2007年03月 [査読有り]
     
    A hyperthermophilic beta-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).
  • Tsutomu Nakamura; Shouhei Mine; Yoshihisa Hagihara; Kazuhiko Ishikawa; Koichi Uegaki
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 63 7 - 11 2007年01月 [査読有り]
     
    The crystal structure of the catalytic domain of a chitinase from the hyperthermophilic archaeon Pyrococcus furiosus (AD2(PF-ChiA)) has been determined at 1.5 angstrom resolution. This is the first structure of the catalytic domain of an archaeal chitinase. The overall structure of AD2(PF-ChiA) is a TIM-barrel fold with a tunnel-like active site that is a common feature of family 18 chitinases. Although the catalytic residues (Asp522, Asp524 and Glu526) are conserved, comparison of the conserved residues and structures with those of other homologous chitinases indicates that the catalytic mechanism of PF-ChiA is different from that of family 18 chitinases.
  • Susumu Iiizumi; Yuji Nomura; Sairei So; Koichi Uegaki; Kayoko Aoki; Kei-ichi Shibahara; Noritaka Adachi; Hideki Koyama
    BIOTECHNIQUES 41 3 311 - + 2006年09月 [査読有り]
     
    Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway (R) technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human preB cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest call be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian-particularly human-genes.
  • Shouhei Mine; Tsutomu Nakamura; Yoshihisa Hagihara; Kazuhiko Ishikawa; Takahisa Ikegami; Koichi Uegaki
    JOURNAL OF BIOMOLECULAR NMR 36 70 - 70 2006年 [査読有り]
  • Tsutomu Nakamura; Takahiko Yamamoto; Tsuyoshi Inoue; Hiroyoshi Matsumura; Atsuko Kobayashi; Yoshihisa Hagihara; Koichi Uegaki; Mitsuo Ataka; Yasushi Kai; Kazuhiko Ishikawa
    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES 61 C262 - C262 2005年
  • K Uegaki; A Taga; Y Akada; S Suzuki; S Honda
    ANALYTICAL BIOCHEMISTRY 309 2 269 - 278 2002年10月 [査読有り]
     
    The efficacy Of Our capillary electrophoresis method for simultaneous estimation of the association constants of glycoprotein Glycoforms to a common target protein was demonstrated using ribonuclease and ovalburnin glycoforms as glycoforni models and Lens culinaris agglutinin (LCA) as a protein model. The ribonuclease glycoforms were fairly well separated in the absence of LCA at pH 5.8, but the peaks were retarded without any change of separation profile in the presence of LCA, the retardation becorning greater as LCA concentration increased. The estimated values of apparent association constant (&) were at the 106 M-1 level for all the ribonuclease glycoforms, and there was no significant difference among glycoforms. The high-mannose-type N-glycans released from a mixture of ribonuclease glycoforms gave lower values of K-a at the 10(4) 10(5) M-1 level to the same protein, and the glycans having a larger number of the mannose residue gave larger & values. These results imply that the glycan moiety in this glycoprotein might contribute to its binding to the protein, but the polypeptide core played the major role. In contrast, ovalburnin glycoforms gave poorly resolved peaks in the absence of LCA, but they were separated into several peaks in the presence of LCA, which were tentatively assigned based on the knowledge of affinity to this lectin, and K-a values were estimated simultaneously. The estimated & values were smaller than those of the ribonuclease glycoforms, suggesting the major role of the N-glycan moiety. Thus, capillary electrophoresis allowed simultaneous estimation of K-a values under common conditions using small amounts of glycoform mixtures and proteins without prior isolation and purification. Comparison of the obtained values will provide useful information on the glycan structure-affinity correlation. (C) 2002 Elsevier Science (USA). All rights reserved.
  • T Otomo; H Sakahira; K Uegaki; S Nagata; T Yamazaki
    NATURE STRUCTURAL BIOLOGY 7 8 658 - 662 2000年08月 [査読有り]
     
    We present here the structure of the complex between the CAD domain of caspase activated deoxyribonuclease (CAD) and the CAD domain of its inhibitor (ICAD), determined by nuclear magnetic resonance spectroscopy. The two domains adopt a very similar fold, which consists of an alpha-helix and a beta-sheet, and are aligned side by side in the complex. Notably the positive charges on the strand beta 2 at one end of the beta-sheet of CAD and negative charges around the opposite end of the beta-sheet of ICAD are paired in the complex. Point mutations of the charged amino acids at this interface, on either CAD or ICAD, prevented formation of the functional CAD-ICAD complex. This implies that the interaction between the CAD domains of CAD and ICAD is an essential step in the correct folding of CAD in the complex.
  • EH Morita; T Murakami; K Uegaki; T Yamazaki; N Sato; Y Kyogoku; H Hayashi
    JOURNAL OF BIOMOLECULAR NMR 17 4 351 - 352 2000年08月 [査読有り]
  • K Uegaki; T Otomo; H Sakahira; M Shimizu; N Yumoto; Y Kyogoku; S Nagata; T Yamazaki
    JOURNAL OF MOLECULAR BIOLOGY 297 5 1121 - 1128 2000年04月 [査読有り]
     
    Caspase-activated DNase (CAD), which causes a genome fragmentation at the final stage of apoptosis, is a protein of about 40 kDa and exists as a complex form with the inhibitor ICAD in Living cells. There is sequence homology of about 80 amino acid residues at the N termini of CAD and ICAD (called the CAD domain). Here, we report the three-dimensional structure of the CAD domain of CAD determined by multi-dimensional NMR spectroscopy and the property of CAD domains investigated by a surface plasmon resonance experiment. The CAD domain of CAD is an independently folded domain composed of one alpha-helix and five beta-strands forming a single sheet. The overall structure is categorized in the ubiquitin superfold. This domain can bind strongly to the isolated CAD domain of ICAD (dissociation constant: 5.48(+/-0.003)x 10(-8) M). It suggests the function of the CAD domains in the CAD-ICAD system, that the protein-protein interaction through the CAD domains plays an important role in the inhibition of CAD DNase activity and in the correct folding of CAD. On the basis of structural comparison with other protein complexes containing the ubiquitin superfold, the interaction mode of the CAD domains is proposed. (C) 2000 Academic Press.
  • M Petukhov; K Uegaki; N Yumoto; S Yoshikawa; L Serrano
    PROTEIN SCIENCE 8 10 2144 - 2150 1999年10月 [査読有り]
     
    The assumption that the intrinsic cu-helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and lie). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gin) at different positions of a model polyalanine-based peptide. We found that Thr is more favorable (by similar to 0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gin the differences are smaller (+/-0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (+/-0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.
  • Otomo T; Teruya K; Uegaki K; Yamazaki T; Kyogoku Y
    J Biomol NMR 14 2 105 - 114 1999年06月 [査読有り]
  • T Yamazaki; T Otomo; N Oda; Y Kyogoku; K Uegaki; N Ito; Y Ishino; H Nakamura
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 120 22 5591 - 5592 1998年06月
  • J Furui; K Uegaki; T Yamazaki; M Shirakawa; MB Swindells; H Harada; T Taniguchi; Y Kyogoku
    STRUCTURE WITH FOLDING & DESIGN 6 4 491 - 500 1998年04月 [査読有り]
     
    Background: The transcription of interferon (IFN) and IFN-inducible genes is mainly regulated by the interferon regulatory factor (IRF) family of proteins, which recognize a unique AAGTGA hexamer repeat motif in the regulatory region of IFN genes, A DNA-binding domain of approximately 100 amino acids has been commonly found in the IRF family of proteins, but it has no sequence homology to known DNA-binding motifs. Elucidation of the structures of members of the IRF family is therefore useful to the understanding of the regulation and evolution of the immune system at the structural level, Results: The solution structure of the DNA-binding domain of interferon regulatory factor-e (IRF-2) has been determined by NMR spectroscopy. It is composed of a four-stranded antiparallel beta sheet and three a helices, and its global fold is similar to those of the winged helix-turn-helix (wHTH) family of proteins. A long loop (Pro37-Asp51) is found immediately before the HTH motif, which is not found in other wHTH proteins. The NMR signals of residues in this long loop, as well as the second helix of the HTH motif, are strongly affected upon the addition of the hexamer repeat DNA, suggesting that these structural elements participate in DNA recognition and binding, Conclusions: The structural similarity of the DNA-binding domain of IRF-2 with those of proteins in the wHTH family shows that the IRF proteins belong to the wHTH family, even though there is no apparent sequence homology among proteins of the two families. The sequential structure alignment program (SSAP) shows that IRF-2 has a slightly different structure from typical wHTH proteins, mainly in the orientation of helix 2. The IRF family of proteins should therefore be categorized into a subfamily of the wHTH family. The evidence here implies that the evolutional pathway of the IRF family is distinct from that of the other wHTH proteins, in other words, the immune system diverged from an evolutional stem at an early stage.
  • Ishida, A; Shigeri, Y; Tatsu, Y; Uegaki, K; Kameshita, I; Okuno, S; Kitani, T; Yumoto, N; Fujisawa, H
    FEBS LETTERS 427 1 115 - 118 1998年
  • K Uegaki; S Murase; N Nemoto; Y Kobayashi; S Yoshikawa; N Yumoto
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 241 3 737 - 743 1997年12月 [査読有り]
     
    To determine whether or not the dimeric structure of neuropeptide Y (NPY) that is found in solution is necessary for its function, we investigated the effects of covalent dimerization on the structure and function of NPY using the carboxy-terminal fragment, NPY(12-36), in which residues 12 and 31 (located at both ends of alpha-helical region) were replaced by Cys residues. Among the three species (the parallel dimer, the anti-parallel dimer, and the intramolecularly cross-linked monomer) obtained by oxidation of the fragment, the anti-parallel dimer was pre-dominant. NMR analysis showed that both parallel and anti-parallel dimers had alpha-helices similar to that of intact NPY, suggesting that covalent dimerization might have little effect on the helical structure. A binding assay with Y2 receptors on porcine hippocampal membranes revealed that the IC50 value of the anti-parallel dimer was almost the same as that of NPY(13-36), which is known as a Y2-specific ligand. By contrast, the binding by the parallel dimer was weaker by more than one order of magnitude. Our results suggest that the formation of dimers of NPY is not essential for binding to the receptor. (C) 1997 Academic Press.
  • K Uegaki; N Nemoto; M Shimizu; T Wada; Y Kyogoku; Y Kobayashi
    FEBS LETTERS 379 1 47 - 50 1996年01月 [査読有り]
     
    For structure analysis of peptides by multinuclear MMR, stable isotope-labeled samples are required. A direct overexpression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of N-15- labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear MMR analysis.
  • K UEGAKI; M SHIRAKAWA; H HARADA; T TANIGUCHI; Y KYOGOKU
    FEBS LETTERS 359 2-3 184 - 188 1995年02月 [査読有り]
     
    The secondary structure elements of the DNA-binding domain of mouse interferon regulatory factor 2 [IRF-2(113)] were determined by heteronuclear multidimensional NMR spectroscopy. The sequential NOE connectivities, amide proton exchange rates, and (3)J(NH alpha) coupling constants indicated the presence of three alpha-helical regions and four short beta-strands connected through relatively long loops, The long range NOEs indicated the four strands form an antiparallel beta-sheet and the three cu-helices form a bundle on the sheet, The arrangement of the secondary structure elements and the overall folding topology resemble those of the DNA binding domains of bacterial activator CAP, heat shock transcription factors, and fork-head proteins, although there is no sequence homology among them.
  • K UEGAKI; M SHIRAKAWA; H HARADA; T TANIGUCHI; Y KYOGOKU
    PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES 70 10 200 - 204 1994年12月 [査読有り]
     
    The secondary structure elements and the folding topology of the DNA-binding domain of mouse interferon regulatory factor 2 [IRF-2(113)] were determined by heteronuclear multidimensional NMR spectroscopy. The sequential NOE (nuclear Overhauser effect) connectivities indicated the presence of three alpha-helical regions and four short beta-strands connected by relatively long loops. The long range NOEs indicated the four strands form an antiparallel beta-sheet and the three alpha-helices form a bundle on the sheet. The arrangement of the secondary structure elements and the overall folding topology resemble those of the DNA binding domains of bacterial activator CAP, heat shock transcription factors and fork-head family transcription factors, although there is no sequence homology among them.
  • K UEGAKI; M SHIRAKAWA; T FUJITA; T TANIGUCHI; Y KYOGOKU
    PROTEIN ENGINEERING 6 2 195 - 200 1993年02月 [査読有り]
     
    The DNA binding domain of the interferon regulatory factor-2 protein (IRF-2) has been produced and characterized. alpha-chymotrypsin digestion of the purified IRF-2 protein bound to a synthetic binding site yields a peptide fragment of 14 K in molecular weight. N-terminal analysis of this peptide fragment showed that its sequence is the same as that of the intact IRF-2. A peptide fragment of approximately 14 K, IRF-2(113), which corresponds to the N-terminal 113 amino acids of the intact IRF-2 protein, has been expressed in a functional form in Escherichia coli. The first methionine was processed during the expression and the purified IRF-2(113) thus contains 112 amino acids. DNase I footprinting and gel retardation assaying showed that IRF-2(113) binds to a synthetic DNA having the consensus binding site and to the upstream regulatory sequence of the IFN-beta gene as intact IRF-2 does. These results showed that this peptide fragment, IRF-2(113), may be a good material for investigation of the DNA binding domain of IRF-2 and of the DNA-protein interaction.

MISC

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 清水 真人; 上垣 浩一; 野崎 剛徳; 三浦 治郎
     
    糖は生体にとってエネルギー源として重要であるが、活性の高いアルデヒド基を有しており、無酵素、無加熱の生理学的条件下でタンパク質中アミノ基と反応し、シッフ塩基、アマドリ転位、酸化反応を経て不可逆的に糖化最終産物（Advanced Glycation End products: AGEｓ）を形成してしまう。AGEsは結合したタンパク質とともに代謝されない限り、生体に蓄積する。 加齢に伴う動脈硬化、生体内の異所性石灰化、β‐アミロイド中への蓄積、骨代謝においてはコラーゲンが変質することによる骨の脆弱化。また後縦靭帯骨化症などの難病疾患にも関与していると考えられている。またAGEsはRAGE（Receptor for AGEs)との相互作用を介して様々な生体シグナルを発していると考えられている。 これまで免疫染色法、免疫電子顕微鏡やウェスタンブロット、蛍光寿命測定でAGEsの分布状態を解析してきた。またタンパク質を酸でアミノ酸レベルにまで完全分解してAGEsを逆相カラムで分離しAGEsの存在をHPLC解析や質量分析など物理化学手的な解析進めてきた。これらの多面的な解析により高齢者、糖尿病の患者の象牙質中にAGEｓが蓄積していること、う蝕表面がAGEs化されるとう蝕の進行が抑制傾向にあることなどを見出してきた。また糖尿病モデルラットの臼歯のAGEs蓄積を質量イメージングで捉えることができた。また糖尿病モデルラットを用いることでラットの臼歯部にヒトの糖尿病患者に多く見出される歯髄内結石が増加することも捉えた。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 茂里 康; 上垣 浩一; 絹見 朋也; 稲垣 英利
     
    本研究課題では、モリアオガエルが産卵時に形成する泡巣の特異的な性質・謎に注目した。そこで質量分析法・次世代シーケンサー等の解析手法を駆使したところ、泡巣中に少なくとも22種類の新規タンパク質が存在し、それらのアミノ酸・遺伝子配列の解読に成功した。相同性検索の結果、22種類のタンパク質は、「受精・抗ストレス・抗炎症関連」、「抗菌・抗ウイルス関連」、「保湿・潤滑・細胞保護・構造体維持関連」に類似・分類できる事が判明した。また還元剤を用いた実験から、泡巣の形成にはジスルフィド結合が重要な働きを担っている事を明らかにした。
  • 柿の酒粕を用いた特産品開発推進事業
    奈良県：受託研究
    研究期間 : 2020年04月 -2021年03月
  • D-アミノ酸デアセチラーゼの基質認識機構解明に向けた構造学的アプローチ
    日本応用酵素協会：寄付研究費
    研究期間 : 2019年04月 -2020年09月 
    代表者 : 上垣 浩一
  • 県産ワイン商品開発推進事業
    奈良県：受託研究費
    研究期間 : 2019年04月 -2020年03月 
    代表者 : 上垣 浩一
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2015年04月 -2019年03月 
    代表者 : 中村 努; 上垣 浩一; 中山 敦好; 松村 浩由
     
    CE-14ファミリーに属するデアセチラーゼPhDacと基質アナログとの複合体の立体構造を解明した。それにより、PhDacの基質認識と反応機構を明らかにした。PhDacの基質認識に関わるアミノ酸残基がCE-14ファミリー一般に空間的に保存されていたことから、この基質認識メカニズムがCE-14ファミリー一般に共有されることを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2014年04月 -2019年03月 
    代表者 : 早川 典子; 川野邊 渉; 木川 りか; 本多 貴之; 山中 勇人; 佐藤 嘉則; 酒井 清文; 楠 京子; 長田 武; 上垣 浩一
     
    本研究は、三つの調査研究から構成された。 一つは材料化学的な調査で、除去すべき汚れの化学構造を把握することで酵素探索の方向性を明確にし、酵素による文化財材料への影響を評価した。探索した酵素が、文化財の構成材料および修理材料に与える影響も分析した。二つ目は微生物酵素学的調査であり、材料調査の結果を基に汚れの主成分を分解する酵素を探索した。使用した酵素が文化財上で機能を十分発現する条件についての検討も行った。以上を踏まえ、三つ目の調査研究である文化財修復現場での適用を検討した。安全性の確認や、より効果的な適用方法について、十分に協議検討し適切な酵素によるクリーニング方法を開発した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 星野 英人; 上垣 浩一; 丹羽 一樹
     
    従来の蛍光顕微鏡システムを駆使して、研究者の独自技術である自己励起蛍光蛋白質・BAF融合体を導入した安定細胞株を対象に、化学発光による細胞観察を試み、生細胞に対する、ルシフェリン-ルシフェラーゼ反応に基づく化学発光イメージングの可能性を提示すると共に、幾つかの課題を新たに見出した。最大の問題は、培地成分に依存し、閉鎖型の灌流細胞培養チャンバーシステムでの培地輸液過程に伴う気泡発生であり、シンプルな原理に基づく、気泡除去装置の開発を試みたが、研究期間中には完成に至らなかった。また、更なる高輝度化のためにBAF分子内で起こる共鳴エネルギー転移の効率を更に改善する手法を見出し、国内特許出願を行った。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2010年 -2012年 
    代表者 : 上垣 浩一; 中村 努; 池上 貴久
     
    好熱菌由来セルラーゼ触媒ドメインにセルロース結合能を付加すべくリンカーを介して他の好熱菌由来のキチン吸着ドメインを C 末端に融合させた耐熱性人工セルラーゼを創製する際に考慮する必要のあるパラメーター(リンカー長の最適化、吸着ドメインの多重化による活性向上効果等)を調べ、リンカー長には最適な長さがあること、吸着ドメインの多重化の効果は結晶性基質かアモルファス基質かで異なる事、N 末に融合した場合の最適なリンカー長は C 末の場合と異なることを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2010年 -2012年 
    代表者 : 小島 正己; 上垣 浩一; 石川 保幸
     
    成長因子をはじめとする生体タンパク質は、活性化型になるまでにプロセッシングと呼ばれる酵素反応を受けるものが存在する。本研究においては神経栄養因子のプロセッシングに反応proBDNF→mature BDNF+BDNF pro-peptide に注目した。その結果、BDNF pro-peptide には BDNF伝達を抑制する活性があること、その活性発現に必要な受容体等を決定した。プロセッシングは多くの成長因子の活性化に共通するメカニズムであるが、本研究の成果は成長因子研究分野に新たなメカニズムの存在の可能性
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2006年 -2007年 
    代表者 : 小島 正己; 上垣 浩一
     
    神経栄養因子類は神経細胞の生存・分化・シナプス機能の亢進を行う神経系の成長因子であり、その生理作用とレセプターを介したシグナル伝達機構の研究が世界的に重要となってきている。しかし、神経栄養因子自身の分泌と細胞内輸送のメカニズムについては研究が遅れている。その理由のひとつは、神経栄養因子自身の発現量が少ないため解析が困難なことにある。我々は、神経栄養因子BDNF(Brain-derived neurotrophic factor)を研究しているが、この分子についても分泌と輸送のメカニズムはほとんど解明されていない。神経栄養因子が複雑な神経回路の中でどのような時空間制御をもって分泌・輸送されるのかを解明することは、レセプターを介したシグナル伝達の理解と同様に重要である。本研究では、この問題を解明できる分子基盤としてBDNFとの相互作用分子候補を生化学的手法と質量分析法を用いて100以上同定した。相互作用分子は細胞機能の観点から6群に分類されていたが直接的相互作用を行う分子と間接的に相互作用する分子が含まれると考えられた。いくつかの分子についてはビアコアを用いて結合定数の決定までを行った。また、コレステロールをはじめとする脂質分子の相互作用性も解析した。結合定数を決定した分子種については、抗体を用いた相互作用の阻害実験、結合環境の決定などを含めて行った。これらの結果に基づいたBDNFの新たな分子メカニズムが解明されるものと期待されると同時に、蛋白質同士の相互作用研究への貢献が期待される。

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