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SHIMAMOTO Shigeru

    Department of Life Science　Associate Professor
Last Updated :2024/05/14

Researcher Information

URL

J-Global ID

Research Interests

  • Biophysical chemistry   protein technology   thermodynamics   structural biology   ITC   NMR   interaction analysis   protein folding   lipocalin   prostaglandin   Lipocalin-type prostaglandin D synthase   造血器型プロスタグランジンD合成酵素   bioactive peptide   Heat-stable enterotoxin   proprotein   Prouroguanylin   

Research Areas

  • Life sciences / Biophysics
  • Life sciences / Structural biochemistry
  • Nanotechnology/Materials / Molecular biochemistry
  • Life sciences / Pharmaceuticals - analytical and physicochemistry

Academic & Professional Experience

  • 2015/04 - Today  Kindai UniversityFaculty of Science and Engineering, Department of Life Science講師
  • 2011/04 - 2014/03  Kindai UniversityFaculty of Science and Engineering, Department of Life Science助教
  • 2010/04 - 2011/03  大阪大学大学院薬学研究科日本学術振興会特別研究員(PD)

Education

  • 2007/04 - 2010/03  大阪大学大学院  薬学研究科（博士後期課程）  分子薬科学専攻
  • 2005/04 - 2007/03  大阪大学大学院  薬学研究科（博士前期課程）  分子薬科学専攻
  • 2001/04 - 2005/03  Osaka University  School of Pharmaceutical Sciences  Department of Pharmaceutical Sciences

Association Memberships

  • The Japan Society of Calorimetry and Thermal Analysis   Biophysical Society   The Japanese Peptide Society   Protein Science Society of Japan   

Published Papers

  • Kyona Hiroshima; Nana Sakata; Tadafumi Konogami; Shigeru Shimamoto; Yuji Hidaka
    Molecules MDPI AG 28 (23) 7754 - 7754 2023/11 
    Proopiomelanocortin (POMC) is a precursor protein of several peptide hormones, such as ACTH and β-endorphin. Almost all of the peptide hormones in POMC have been drastically investigated in terms of their biological activities. However, the biological activity of the joining peptide region (JP) in POMC is unknown. Therefore, to explore the biological activity of JP, sequence analyses of mammalian POMC were performed. We found an -Arg-Gly-Asp- (RGD) motif in several mammalian species, such as porcine, suggesting that JP has cell adhesion activity. To validate this hypothesis, the cell adhesion activities of the synthetic porcine JP peptides were examined using 293T cells. Cell adhesions were observed in a concentration-dependent manner of the JP peptides. In addition, the JP peptide competitively inhibited cell adhesion to the POMC-coated plates. Moreover, the cell adhesion activity of the joining peptide was inhibited by the addition of EDTA, indicating that the JP peptide mediates the cell adhesion activity via a receptor protein, integrin. Interestingly, a human JP peptide, which possesses an -Arg-Ser-Asp- (RSD) sequence in place of the RGD sequence, exhibited a higher ability in the cell adhesion activity than that of the porcine JP peptide, suggesting that the cell adhesion activity of the joining peptide is developed during the molecular evolution of POMC. In conclusion, our results reveal that the joining peptide in POMC plays an important role during cell adhesion and provide useful information related to signal transduction of nerve peptide hormones derived from POMC.
  • Nana Sakata; Yuri Murakami; Mitsuhiro Miyazawa; Shigeru Shimamoto; Yuji Hidaka
    Molecules 28 (8) 3494 - 3504 2023/04 [Refereed]
  • Masaya Goto; Shinya Yoshino; Kyona Hiroshima; Toru Kawakami; Kaeko Murota; Shigeru Shimamoto; Yuji Hidaka
    Molecules 28 (1) 1128 - 1142 2023/01 [Refereed][Invited]
  • Nana Sakata; Ayumi Ogata; Mai Takegawa; Yuri Murakami; Misaki Nishimura; Mitsuhiro Miyazawa; Teruki Hagiwara; Shigeru Shimamoto; Yuji Hidaka
    Molecules 27 (2) 1 - 13 2022/11 [Refereed]
  • Nana Sakata; Ayumi Ogata; Mai Takegawa; Nagisa Tajima; Misaki Nishimura; Teruki Hagiwara; Mitsuhiro Miyazawa; Shigeru Shimamoto; Yuji Hidaka
    Biochemical and Biophysical Research Communications Elsevier BV 624 35 - 39 0006-291X 2022/10 [Refereed]
  • Shigeru Shimamoto; Yuta Nakahata; Yuji Hidaka; Takuya Yoshida; Tadayasu Ohkubo
    Biomolecular NMR Assignments Springer Science and Business Media LLC 1874-2718 2022/04 [Refereed]
  • Shigeru Shimamoto; Yusuke Nakagawa; Yuji Hidaka; Takahiro Maruno; Yuji Kobayashi; Kazuki Kawahara; Takuya Yoshida; Tadayasu Ohkubo; Kosuke Aritake; Mahesh K Kaushik; Yoshihiro Urade
    Biochemical and biophysical research communications 569 66 - 71 2021/07 [Refereed]
     
    Prostaglandin D2 (PGD2), an endogenous somnogen, is a unique PG that is secreted into the cerebrospinal fluid. PGD2 is a relatively fragile molecule and should be transported to receptors localized in the basal forebrain without degradation. However, it remains unclear how PGD2 is stably carried to such remote receptors. Here, we demonstrate that the PGD2-synthesizing enzyme, Lipocalin-type prostaglandin D synthase (L-PGDS), binds not only its substrate PGH2 but also its product PGD2 at two distinct binding sites for both ligands. This behaviour implys its PGD2 carrier function. Nevertheless, since the high affinity (Kd = ∼0.6 μM) of PGD2 in the catalytic binding site is comparable to that of PGH2, it may act as a competitive inhibitor, while our binding assay exhibits only weak inhibition (Ki = 189 μM) of the catalytic reaction. To clarify this enigmatic behavior, we determined the solution structure of L-PGDS bound to one substrate analog by NMR and compared it with the two structures: one in the apo form and the other in substrate analogue complex with 1:2 stoichiometry. The structural comparisons showed clearly that open or closed forms of loops at the entrance of ligand binding cavity are regulated by substrate binding to two sites, and that the binding to a second non-catalytic binding site, which apparently substrate concentration dependent, induces opening of the cavity that releases the product. From these results, we propose that L-PGDS is a unique enzyme having a carrier function and a substrate-induced product-release mechanism.
  • Shigeru Shimamoto; Natsumi Mitsuoka; Saki Takahashi; Toru Kawakami; Yuji Hidaka
    The protein journal 39 (6) 711 - 716 2020/12 [Refereed]
     
    Numerous studies of native proteins have been reported on protein folding in this half century. Recently, post-translationally modified proteins are also focused on protein folding. However, it is still difficult to prepare such types of proteins because it requires not only the chemical but also the recombinant techniques. Native chemical ligation (NCL) is a powerful technique for producing target proteins when combined with recombinant techniques, such as expressed protein ligation (EPL). NCL basically requires an N-terminal peptide with a thioester and a C-terminal peptide which should possess a Cys residue at the N-terminus. Numerous efforts have been made to prepare N-terminal peptides carrying a thioester or a derivative thereof. However, a method for preparing C-terminal Cys-peptides with post-translational modifications has not been well developed, making it difficult to prepare such C-terminal Cys-peptides, except for chemical syntheses or enzymatic digestion. We report here on the development of a convenient technique that involves acid hydrolysis at the -Asp-Cys- sequence, to effectively obtain a C-terminal peptide fragment that can be used for any protein synthesis when combined with EPL, even under denatured conditions. Thus, this chemical digestion strategy permits the NCL strategy to be dramatically accelerated for protein syntheses in which post-translational modifications, such as glycosylation, phosphorylation, etc. are involved. In addition, this method should be useful to prepare the post-translationally modified proteins for protein folding.
  • Shigeru Shimamoto; Mayu Fukutsuji; Toi Osumi; Masaya Goto; Hiroshi Toyoda; Yuji Hidaka
    Molecules (Basel, Switzerland) 25 (20) 2020/10 [Refereed]
     
    Heat-stable enterotoxin (STa) produced by enterotoxigenic E. coli causes acute diarrhea and also can be used as a specific probe for colorectal cancer cells. STa contains three intra-molecular disulfide bonds (C1-C4, C2-C5, and C3-C6 connectivity). The chemical synthesis of STa provided not only the native type of STa but also a topological isomer that had the native disulfide pairings. Interestingly, the activity of the topological isomer was approximately 1/10-1/2 that of the native STa. To further investigate the bioactive conformation of this molecule and the regulation of disulfide-coupled folding during its chemical syntheses, we examined the folding mechanism of STa that occurs during its chemical synthesis. The folding intermediate of STa with two disulfide bonds (C1-C4 and C3-C6) and two Cys(Acm) residues, the precursor peptide, was treated with iodine to produce a third disulfide bond under several conditions. The topological isomer was predominantly produced under all conditions tested, along with trace amounts of the native type of STa. In addition, NMR measurements indicated that the topological isomer has a left-handed spiral structure similar to that of the precursor peptide, while the native type of STa had a right-handed spiral structure. These results indicate that the order of the regioselective formation of disulfide bonds is important for the regulation of the final conformation of disulfide-rich peptides in chemical synthesis.
  • Kenji Yamamoto; Osamu Ishibashi; Keisuke Sugiura; Miki Ubatani; Masaya Sakaguchi; Masatoshi Nakatsuji; Shigeru Shimamoto; Masanori Noda; Susumu Uchiyama; Yuma Fukutomi; Shigenori Nishimura; Takashi Inui
    Scientific reports 9 (1) 1503 - 1503 2019/02 [Refereed]
     
    Several dog allergens cause allergic reactions in humans worldwide. Seven distinct dog allergens, designated Canis familiaris allergen 1 to 7 (Can f 1-Can f 7), have been identified thus far. Can f 6 shows high sequence similarity and cross-reactivity with Fel d 4 and Equ c 1, major cat and horse allergens, respectively. This study was conducted on the allergenic epitopes of Can f 6 based on its structural characterization. We demonstrated that sera from 18 out of 38 (47%) dog-sensitized patients reacted to recombinant Can f 6 protein (rCan f 6). We then determined the crystal structure of rCan f 6 by X-ray crystallography, which exhibited a conserved tertiary structural architecture found in lipocalin family proteins. Based on the tertiary structure and sequence similarities with Fel d 4 and Equ c 1, we predicted three IgE-recognizing sites that are possibly involved in cross-reactivity. Substituting three successive amino acids in these sites to triple alanine decreased IgE reactivity to the allergen. However, the degree of reduction in IgE reactivity largely depended on the site mutated and the serum used, suggesting that Can f 6 is a polyvalent allergen containing multiple epitopes and Can f 6-reactive sera contain varied amounts of IgE recognising individual Can f 6 epitopes including those predicted in this study. We also demonstrated that the predicted epitopes are partly involved in IgE cross-reactivity to Fel d 4. Interestingly, the effect of the mutation depended on whether the protein was structured or denatured, indicating that the bona fide tertiary structure of Can f 6 is essential in determining its IgE epitopes.
  • Yang Zheng; Shigeru Shimamoto; Takahiro Maruno; Yuji Kobayashi; Yoshiharu Matsuura; Kazuki Kawahara; Takuya Yoshida; Tadayasu Ohkubo
    Biochemical and biophysical research communications 509 (2) 590 - 595 0006-291X 2019/02 [Refereed]
     
    The Hepatitis C virus (HCV) core protein plays a crucial role in the development of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Its involvement in these diseases is reportedly abolished by a knockout of the proteasome activator PA28γ gene in transgenic mice, suggesting an interaction between the core protein and the PA28γ-proteasome system. This study found a direct interaction between the N-terminal 1-71 fragment of HCV core protein (Core71) and PA28γ in vitro, and that this interaction was found to enhance PA28γ-20S proteasome complex formation. While 20S proteasome activity was increased by PA28γ, it was significantly reduced by Core71 attachment in a dose-dependent manner. These results suggest that the Core-PA28γ interaction has an important role in regulating 20S proteasome activity and furthers our understanding of the pathogenesis of HCV.
  • 島本茂; 丸野孝浩
    熱測定 44 (3) 108 - 116 0386-2615 2017 [Refereed][Invited]
  • Shubin Qin; Shigeru Shimamoto; Takahiro Maruno; Yuji Kobayashi; Kazuki Kawahara; Takuya Yoshida; Tadayasu Ohkubo
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 468 (1-2) 234 - 239 0006-291X 2015/12 [Refereed]
     
    Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D-2 (PGD(2)) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP(+), and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. (C) 2015 Elsevier Inc. All rights reserved.
  • Shigeru Shimamoto; Hiroko Maruo; Takuya Yoshida; Tadayasu Ohkubo
    Biomolecular NMR assignments SPRINGER 8 (1) 129 - 32 1874-2718 2014/04 [Refereed]
     
    Lipocalin-type Prostaglandin D synthase (L-PGDS) acts as the PGD2-synthesizing enzyme in the brain of various mammalian species. It belongs to the lipocalin superfamily and is the first member of this family to be recognized as an enzyme. Although the solution and crystal structure of L-PGDS has been determined to understand the molecular mechanism of catalytic reaction, the structural analysis of L-PGDS in complex with its substrate remains to be performed. Here, we present the nearly complete assignment of the backbone and side chain resonances of L-PGDS/substrate analog (U-46619) complex. This study lays the essential basis for further understanding the substrate recognition mechanism of L-PGDS.
  • Masaki Okumura; Shigeru Shimamoto; Yuji Hidaka
    Current protocols in protein science 76 (76) 28.7.1-28.7.13 - 13 1934-3663 2014/04 [Refereed]
     
    Disulfide bonds play a critical role in the folding of secretory and membrane proteins. Oxidative folding reactions of disulfide bond-containing proteins typically require several hours or days, and numerous misbridged disulfide isomers are often observed as intermediates. The rate-determining step in refolding is thought to be the disulfide-exchange reaction from nonnative to native disulfide bonds in folding intermediates, which often precipitate during the refolding process because of their hydrophobic properties. To overcome this, chemical additives or a disulfide catalyst, protein disulfide isomerase (PDI), are generally used in refolding experiments to regulate disulfide-coupled peptide and protein folding. This unit describes such methods in the context of the thermodynamic and kinetic control of peptide and protein folding, including (1) regulation of disulfide-coupled peptides and protein folding assisted by chemical additives, (2) reductive unfolding of disulfide-containing peptides and proteins, and (3) regulation of disulfide-coupled peptide and protein folding using PDI.
  • Shigeru Shimamoto; Hidekazu Katayama; Masaki Okumura; Yuji Hidaka
    Current protocols in protein science 76 (76) 28.8.1-28.8.28 - 28.8.28 1934-3663 2014/04 [Refereed]
     
    Disulfide-bond formation plays an important role in the stabilization of the native conformation of peptides and proteins. In the case of multidisulfide-containing peptides and proteins, numerous folding intermediates are produced, including molecules that contain non-native and native disulfide bonds during in vitro folding. These intermediates can frequently be trapped covalently during folding and subsequently analyzed. The structural characterization of these kinetically trapped disulfide intermediates provides a clue to understanding the oxidative folding pathway. To investigate the folding of disulfide-containing peptides and proteins, in this unit, chemical methods are described for regulating regioselective disulfide formation (1) by using a combination of several types of thiol protecting groups, (2) by incorporating unique SeCys residues into a protein or peptide molecule, and (3) by combining with post-translational modification.
  • Yuji Hidaka; Shigeru Shimamoto
    Biomolecular concepts 4 (6) 597 - 604 1868-5021 2013/12 [Refereed]
     
    Disulfide-containing proteins are ideal models for studies of protein folding as the folding intermediates can be observed, trapped, and separated by HPLC during the folding reaction. However, regulating or analyzing the structures of folding intermediates of peptides and proteins continues to be a difficult problem. Recently, the development of several techniques in peptide chemistry and biotechnology has resulted in the availability of some powerful tools for studying protein folding in the context of the structural analysis of native, mutant proteins, and folding intermediates. In this review, recent developments in the field of disulfide-coupled peptide and protein folding are discussed, from the viewpoint of chemical and biotechnological methods, such as analytical methods for the detection of disulfide pairings, chemical methods for disulfide bond formation between the defined Cys residues, and applications of diselenide bonds for the regulation of disulfide-coupled peptide and protein folding.
  • Masaki Okumura; Shigeru Shimamoto; Takeyoshi Nakanishi; Yu-ichiro Yoshida; Tadafumi Konogami; Shogo Maeda; Yuji Hidaka
    FEBS letters ELSEVIER SCIENCE BV 586 (21) 3926 - 30 0014-5793 2012/11 [Refereed]
     
    In vitro folding of disulfide-containing proteins is generally regulated by redox molecules, such as glutathione. However, the role of the cross-disulfide-linked species formed between the redox molecule and the protein as a folding intermediate in the folding mechanism is poorly understood. In the present study, we investigated the effect of the charge on a redox molecule on disulfide-coupled protein folding. Several types of aliphatic thiol compounds including glutathione were examined for the folding of disulfide-containing-proteins, such as lysozyme and prouroguanylin. The results indicate that the positive charge and its dispersion play a critical role in accelerating disulfide-coupled protein folding.
  • Masaki Okumura; Shigeru Shimamoto; Yuji Hidaka
    The FEBS journal WILEY-BLACKWELL 279 (13) 2283 - 95 1742-464X 2012/07 [Refereed]
     
    Investigations of protein folding have largely involved studies using disulfide-containing proteins, as disulfide-coupled folding of proteins permits the folding intermediates to be trapped and their conformations determined. Over the last decade, a combination of new biotechnical and chemical methodology has resulted in a remarkable acceleration in our understanding of the mechanism of disulfide-coupled protein folding. In particular, expressed protein ligation, a combination of native chemical ligation and an intein-based approach, permits specifically labeled proteins to be easily produced for studies of protein folding using biophysical methods, such as NMR spectroscopy and X-ray crystallography. A method for regio-selective formation of disulfide bonds using chemical procedures has also been established. This strategy is particularly relevant for the study of disulfide-coupled protein folding, and provides us not only with the native conformation, but also the kinetically trapped topological isomer with native disulfide bonds. Here we review recent developments and applications of biotechnical and chemical methods to investigations of disulfide-coupled peptide and protein folding. Chemical additives designed to accelerate correct protein folding and to avoid non-specific aggregation are also discussed.
  • Ayano Fukuhara; Hidemitsu Nakajima; Yuya Miyamoto; Katsuaki Inoue; Satoshi Kume; Young-Ho Lee; Masanori Noda; Susumu Uchiyama; Shigeru Shimamoto; Shigenori Nishimura; Tadayasu Ohkubo; Yuji Goto; Tadayoshi Takeuchi; Takashi Inui
    Journal of controlled release : official journal of the Controlled Release Society ELSEVIER SCIENCE BV 159 (1) 143 - 50 0168-3659 2012/04 [Refereed]
     
    Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily and a secretory lipid-transporter protein, which binds a wide variety of hydrophobic small molecules. Here we show the feasibility of a novel drug delivery system (DDS), utilizing L-PGDS, for poorly water-soluble compounds such as diazepam (DZP), a major benzodiazepine anxiolytic drug, and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and anticonvulsant. Calorimetric experiments revealed for both compounds that each L-PGDS held three molecules with high binding affinities. By mass spectrometry, the 1:3 complex of L-PGDS and NBQX was observed. L-PGDS of 500μM increased the solubility of DZP and NBQX 7- and 2-fold, respectively, compared to PBS alone. To validate the potential of L-PGDS as a drug delivery vehicle in vivo, we have proved the prospective effects of these compounds via two separate delivery strategies. First, the oral administration of a DZP/L-PGDS complex in mice revealed an increased duration of pentobarbital-induced loss of righting reflex. Second, the intravenous treatment of ischemic gerbils with NBQX/L-PGDS complex showed a protective effect on delayed neuronal cell death at the hippocampal CA1 region. We propose that our novel DDS could facilitate pharmaceutical development and clinical usage of various water-insoluble compounds.
  • Shigeru Shimamoto; Takuya Yoshi; Tadayasu Ohkubo
    Yakugaku Zasshi The Pharmaceutical Society of Japan 131 (11) 1575 - 1581 0031-6903 2011 [Refereed][Invited]
     
    Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a multi functional protein acting as a PGD2 synthesizing enzyme, a transporter or scavenger of various lipophilic ligands, and an amyloid b chaperon in the brain. L-PGDS is a member of the lipocalin superfamily and has the ability to bind various lipophilic molecules such as prostanoid, retinoid, bile pigment, and amyloid β peptide. However, the molecular mechanism for a wide variety of ligand binding has not been well understood. In this study, we determined by NMR the structure of recombinant ouse L-PGDS and LPGDS/ PGH2 analog complex. L-PGDS has the typical lipocalin fold, consisting of an eight-stranded b-barrel and a long α-helix. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins and the cavity contained two pockets. Kinetic studies and molecular docking studies based on the result of NMR titration experiments provide the direct evidence for two binding sites for PGH2 and retinoic acid in the large cavity of L-PGDS. Structural comparison of L-PGDS/U-46619 complex with apo-LPGDS showed that the H2-helix, CD-loop, and EF-loop located at the upper end of the β-barrel change the conformation to cover the entry of the cavity upon U-46619 binding. These results indicated that the two binding sites in the large cavity and induced fit mechanism were responsible for the broad ligand specificity of L-PGDS. © 2011 The Pharmaceutical Society of Japan.
  • Kayoko Hayashihara; Susumu Uchiyama; Shigeru Shimamoto; Shouhei Kobayashi; Miroslav Tomschik; Hidekazu Wakamatsu; Daisuke No; Hiroki Sugahara; Naoto Hori; Masanori Noda; Tadayasu Ohkubo; Jordanka Zlatanova; Sachihiro Matsunaga; Kiichi Fukui
    The Journal of biological chemistry AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 285 (9) 6498 - 507 0021-9258 2010/02 [Refereed]
     
    In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys(97)-Lys(274)), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by approximately 25 bp. The solution structure determined by NMR revealed that the globular domain (Met(153)-Thr(237)) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.
  • Yuya Miyamoto; Shigenori Nishimura; Katsuaki Inoue; Shigeru Shimamoto; Takuya Yoshida; Ayano Fukuhara; Mao Yamada; Yoshihiro Urade; Naoto Yagi; Tadayasu Ohkubo; Takashi Inui
    Journal of structural biology ACADEMIC PRESS INC ELSEVIER SCIENCE 169 (2) 209 - 18 1047-8477 2010/02 [Refereed]
     
    Lipocalin-type prostaglandin D synthase (L-PGDS) acts as both a PGD(2) synthase and an extracellular transporter for small lipophilic molecules. From a series of biochemical studies, it has been found that L-PGDS has an ability to bind a variety of lipophilic ligands such as biliverdin, bilirubin and retinoids in vitro. Therefore, we considered that it is necessary to clarify the molecular structure of L-PGDS upon binding ligand in order to understand the physiological relevance of L-PGDS as a transporter protein. We investigated a molecular structure of L-PGDS/biliverdin complex by small-angle X-ray scattering (SAXS) and multi-dimensional NMR measurements, and characterized the binding mechanism in detail. SAXS measurements revealed that L-PGDS has a globular shape and becomes compact by 1.3A in radius of gyration on binding biliverdin. NMR experiments revealed that L-PGDS possessed an eight-stranded antiparallel beta-barrel forming a central cavity. Upon the titration with biliverdin, some cross-peaks for residues surrounding the cavity and EF-loop and H2-helix above the beta-barrel shifted, and the intensity of other cross-peaks decreased with signal broadenings in (1)H-(15)N heteronuclear single quantum coherence spectra. These results demonstrate that L-PGDS holds biliverdin within the beta-barrel, and the conformation of the loop regions above the beta-barrel changes upon binding biliverdin. Through such a conformational change, the whole molecule of L-PGDS becomes compact.
  • Daisuke Irikura; Kosuke Aritake; Nanae Nagata; Toshihiko Maruyama; Shigeru Shimamoto; Yoshihiro Urade
    The Journal of biological chemistry AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 284 (12) 7623 - 30 0021-9258 2009/03 [Refereed]
     
    We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3-250 microm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH(2) (K(m) = 14 microm) with a K(i) value of 75 microm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD(2) by L-PGDS-expressing human TE-671 cells after stimulation with Ca(2+) ionophore (5 microm A23187) with an IC(50) value of about 3 microm without affecting their production of PGE(2) and PGF(2alpha) but had no effect on the PGD(2) production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD(2) production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE(2) and PGF(2alpha) and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.
  • Shigeru Shimamoto; Takuya Yoshida; Takashi Inui; Keigo Gohda; Yuji Kobayashi; Ko Fujimori; Toshiharu Tsurumura; Kosuke Aritake; Yoshihiro Urade; Tadayasu Ohkubo
    The Journal of biological chemistry AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 282 (43) 31373 - 9 0021-9258 2007/10 [Refereed]
     
    Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2), a common precursor of various prostanoids, to produce PGD(2), an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD(2)-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel beta-barrel and a long alpha-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH(2) analog, revealed that PGH(2) almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH(2) binding mode. These results indicated that the two binding sites for PGH(2) and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.

Books etc

  • 日本熱測定学会 (Contributorタンパク質と低分子の結合（ITC）ー生理活性物質ー)丸善 2020/08 9784621305072 xxiii, 363p
  • 島本, 茂; 他; 中村, 宜督; 榊原, 啓之; 室田, 佳恵子 (Contributor第五章 アミノ酸とタンパク質)講談社 2018/12 9784065133415 viii, 247p

MISC

Research Grants & Projects

  • Study of the Antiviral Effects of 5-ALA
    Neopharma Japan：
    Date (from‐to) : 2019/10 -2021/03 
    Author : 島本 茂; 石川 知弘
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2016/04 -2019/03 
    Author : Urade Yoshihiro
     
    Prostaglandin(PG) D2 is produced by L or H type of synthases, stimulates DP or CRTH2 receptors, and is involved in regulation of sleep and inflammation. By generating antibodies and various types of gene-manipulated mice for each enzyme and receptor, we demonstrated that PGD2 is produced as the sleep substance by L-PGD2 synthase in the arachnoid membrane, induces a release of adenosine as the second somnogen to stimulate A2A receptor-possessing neurons in the nucleus accumbens, a novel sleep center, and induces sleep; DP receptors are induced in endothelial cells in angiogenic microvessels around tumors and acts as a negative regulator of tumor growth; L-PGD2 synthase and CRTH2 receptors are induced by high fat diet in immature adipocytes and involved in obesity and insulin-resistance; and H-PGD2 synthase and CRTH2 receptors are involved in muscular necrosis of Ducchenne's muscular dystrophy. We developped potent inhibitors for human H-PGD2 synthase and started clinical trials.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2016/04 -2019/03 
    Author : SHIMAMOTO SHIGERU
     
    Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) belongs to the lipocalin superfamily which consists of transporter proteins for lipophilic ligands in the extracellular space, and is known as the PGD2-synthesizing enzyme responsible for the sleep regulation. The binding affinity and stoichiometry to each ligand were analyzed by isothermal titration calorimetry (ITC), and the binding region for each ligand on L-PGDS was estimated by NMR titration experiment. The ITC results showed that PGD2 and PGF2α bound to L-PGDS with a stoichiometry of 2 to 1 but PGE2 with a stoichiometry of 1 to 1. In addition, the NMR experiments indicated that PGD2 and PGF2α bound to both the catalytic site containing the Cys65 and non-catalytic site of L-PGDS, while PGE2 bound to only the non-catalytic site.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2012/04 -2016/03 
    Author : HIDAKA Yuji; YAMAGUCHI Hiroshi; SHIMAMOTO Shigeru
     
    To investigate how peptide hormones maintain/develop their biologically active conformation during molecular evolution, the folding of precursor proteins including prouroguanylin were estimated in vitro. Our results indicated that i)the stability of the biologically active conformation of the mature region is strongly related to the correct folding of their precursor proteins, ii)precursor protein possesses a region to accelerate molecular evolution, iii)precursor protein possesses a region to inhibit molecular evolution, iv)correct folding of prevursor protein requires mis-folded molecular species to establish the correct conformation of prouroguanylin
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2012/04 -2014/03 
    Author : SHIMAMOTO Shigeru
     
    Lipocalin-type Prostaglandin D synthase (L-PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to produce prostaglandin D2 (PGD2), which acts as a potent endogenous somnogen in the brain. A number of studies of L-PGDS, as a drug target for sleep disorders, have been reported in attempts to understand its catalytic mechanism, and several substrate recognition models of L-PGDS have been proposed. However, details of the mechanism by which L-PDGS recognizes its substrates and products are obscure, since essential information, such as its binding affinity and stoichiometry, of the interactions between L-PGDS and its substrates and products remains unclear. Therefore, we carried out ITC and NMR experiments to characterize the binding properties. The results of the ITC and NMR measurements revealed that both the substrate and the product bind to L-PGDS in a stoichiometry of 2 to 1 and two binding sites, namely a high and a low affinity binding site, are present.
  • 日本学術振興会：科学研究費助成事業 特別研究員奨励費
    Date (from‐to) : 2009 -2010 
    Author : 島本 茂
     
    本研究は、脳内の主要蛋白質であるL-PGDSの機能を原子レベルで解明することで、生体内のL-PGDSをターゲットとした睡眠調節薬やL-PGDSを利用した疎水性有害物質除去薬(解毒剤)、または、アミロイドβ(Aβ)凝集阻害によるアルツハイマー病治療薬などの開発を目指す。 L-PGDSとPGH_2(基質)安定誘導体U-46619を結合させ、複合体が溶液中で安定な条件を模索し、NMR測定(約2週間)に十分耐えうる安定な溶液状態を決定後、NMRによりL-PGDS/U-46619複合体の溶液構造を解析した。結果として、L-PGDSは基質結合により構造変化を起こし、基質を厳密に固定することで酵素反応を可能にすることを明らかにした。得られた成果は、学会等で発表した。 また、アミロイド繊維形成過程における脳内シャペロンL-PGDSの役割解明のためL-PGDSとAβペプチドの相互作用解析を行った。具体的には、^<15>Nラベル体L-PGDSにAβペプチドを滴下し、それに伴うNMRシグナルの変化を見ることでL-PGDSにおけるAβペプチドの相互作用領域を推定した。L-PGDSがAβペプチドに結合すること、さらに、バレル構造を有するL-PGDSのバレル内部にAβペプチドが結合することが示唆された。

Teaching Experience

  • Advanced studies in Life ScienceAdvanced studies in Life Science Kindai University
  • Advanced ResearchAdvanced Research Kindai University
  • Environmental Science LaboratoryEnvironmental Science Laboratory Kindai University
  • Chemistry LaboratoryChemistry Laboratory Kindai University
  • Social WorkSocial Work Kindai University
  • Bioorganic ChemistryBioorganic Chemistry Kindai University
  • Organic ChemistryOrganic Chemistry Kindai University
  • Instrumental AnalysisInstrumental Analysis Kindai University
  • Analytical ChemistryAnalytical Chemistry Kindai University
  • BiochemistryBiochemistry Kindai University

Committee Membership

  • 2021/08 - Today   The Japan Society of Calorimetry and Thermal Analysis   editorial board member
  • 2017 - Today   Protein Science Society of Japan   editorial board member
  • 2018/08 -2020/07   The Japan Society of Calorimetry and Thermal Analysis   Secretary, Planning Affairs

Academic Contribution

  • The 55th Japan Conference Calorimetry and Thermal Analysis
    Date (from-to) :2019/10/24-2019/10/26
    Role: Planning etc
    Type: Academic society etc
    Organizer, responsible person: KAMIYAMA Tadashi
  • 第56回 熱測定ワークショップ｜熱測定のための電子工作とプログラミング 講習会 A to Z
    Date (from-to) :2019/08/30-2019/08/31
    Role: Planning etc
    Type: Academic society etc
    Organizer, responsible person: 岩間 世界
  • Summer School 2019 | The Calorimetry and Thermal Analysis
    Date (from-to) :2019/08/20-2019/08/21
    Role: Planning etc
    Type: Academic society etc
    Organizer, responsible person: SHIMAMOTO Shigeru
  • 熱測定スプリングスクール2019
    Date (from-to) :2019/03/07-2019/03/08
    Role: Planning etc
    Type: Academic society etc
    Organizer, responsible person: 鈴木 俊之
  • Physical Pharma Forum 2016
    Date (from-to) :2016/08/27-2016/08/28
    Role: Planning etc
    Type: Academic society etc
    Organizer, responsible person: 吉田 卓也
  • 第44回 若手ペプチド夏の勉強会
    Date (from-to) :2012/08/05-2012/08/07
    Role: Planning etc
    Type: Academic society etc
    Organizer, responsible person: 島本 茂；真鍋 良幸

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