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FacultyDepartment of Science and Technology on Food Safety / Graduate School of Biology-Oriented Science and Technology
PositionAssociate Professor
Commentator Guide
Last Updated :2020/09/02

Education and Career

Academic & Professional Experience

  •   2011 ,  - 現在, Faculty of Biology-Oriented Science and Technology, Kindai University

Research Activities

Research Areas

  • Life sciences, Animal production science
  • Life sciences, Animal production science
  • Nanotechnology/Materials, Molecular biochemistry
  • Life sciences, Medical biochemistry

Research Interests

  • eco-feed, Quality of meat, mathematical biology

Published Papers

  • Loosening of Side-Chain Packing Associated with Perturbations in Peripheral Dynamics Induced by the D76N Mutation of β2-Microglobulin Revealed by Pressure-NMR and Molecular Dynamic Simulations, Kazumasa Sakurai, Ryosuke Tomiyama, Takuma Shiraki, Yasushige Yonezawa, Biomolecules, Biomolecules, Sep. 2019 , Refereed
  • Comparison of thin agar layer method and flow cytometry methods for enumeration of chlorine-injured Listeria innocua, Ayano Inoue, Takashi Ano, Nobuyuki Yamawaki, Takuma Shiraki, Hidemi Izumi, Memoirs of The Faculty of Biology-Oriented Science and Technology of Kindai University, Memoirs of The Faculty of Biology-Oriented Science and Technology of Kindai University, 41, 13 - 24, Feb. 2018 , Refereed
  • Hemopexin-dependent heme uptake via endocytosis regulates the Bach1 transcription repressor and heme oxygenase gene activation., Hada H, Shiraki T, Watanabe-Matsui M, Igarashi K, Biochimica et biophysica acta, Biochimica et biophysica acta, 1840(7), 2351 - 2360, Jul. 2014 , Refereed
  • Mechanism of Peroxisome Proliferator-Activated Receptor Gamma (PPAR gamma) Transactivation by Hesperetin Glucuronides Is Distinct from That by a Thiazolidine-2,4-dione Agent, Kanae Gamo, Takuma Shiraki, Nobuyasu Matsuura, Hiroyuki Miyachi, CHEMICAL & PHARMACEUTICAL BULLETIN, CHEMICAL & PHARMACEUTICAL BULLETIN, 62(5), 491 - 493, May 2014 , Refereed
    Summary:Hesperidin, a flavanone glycoside present abundantly in citrus fruits, is predominantly metabolized to hesperetin-7-O-beta-D-glucuronide (H7-OG) and hesperetin-3'-O-beta-D-glucuronide (H3'-OG), which exhibit partial agonistic activity towards peroxisome proliferator-activated receptor gamma (PPAR gamma). Here, in order to understand the mechanism(s) of action of PPAR gamma transactivation elicited by hesperetin glucuronides, we compared the transactivation activities of PPAR gamma (ligand-binding domain (LBD)) mutants by hesperetin glueuronides and troglitazone, a thiazolidine-2,4-dione class PPAR gamma full agonist. The assay results indicated that the mechanisms of activation of PPAR gamma by hesperetin glucuronides and by troglitazone are distinct, probably due to a difference in the binding sites of these compounds on the PPAR gamma LBD. Flavanone-class PPAR gamma partial agonists, luteolin and hesperetin glucuronides, showed similar activation profiles of the PPAR gamma LBD mutants, even though they have different side chain functionalities.
  • [Transcriptional machinery sensing cellular metabolism: nuclear receptor, PPARgamma, mediates crosstalk between metabolic networks]., Shiraki T, Waku T, Morikawa K, Seikagaku. The Journal of Japanese Biochemical Society, Seikagaku. The Journal of Japanese Biochemical Society, 85(9), 749 - 761, Sep. 2013 , Refereed
  • The nuclear receptor PPAR gamma individually responds to serotonin- and fatty acid-metabolites, Tsuyoshi Waku, Takuma Shiraki, Takuji Oyama, Kanako Maebara, Rinna Nakamori, Kosuke Morikawa, EMBO JOURNAL, EMBO JOURNAL, 29(19), 3395 - 3407, Oct. 2010 , Refereed
    Summary:The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma), recognizes various synthetic and endogenous ligands by the ligand-binding domain. Fatty-acid metabolites reportedly activate PPAR gamma through conformational changes of the Omega loop. Here, we report that serotonin metabolites act as endogenous agonists for PPAR gamma to regulate macrophage function and adipogenesis by directly binding to helix H12. A cyclooxygenase inhibitor, indomethacin, is a mimetic agonist of these metabolites. Crystallographic analyses revealed that an indole acetate functions as a common moiety for the recognition by the sub-pocket near helix H12. Intriguingly, a serotonin metabolite and a fatty-acid metabolite each bind to distinct sub-pockets, and the PPAR gamma antagonist, T0070907, blocked the fatty-acid agonism, but not that of the serotonin metabolites. Mutational analyses on receptor-mediated transcription and coactivator binding revealed that each metabolite individually uses coregulator and/or heterodimer interfaces in a ligand-type-specific manner. Furthermore, the inhibition of the serotonin metabolism reduced the expression of the endogenous PPAR gamma-target gene. Collectively, these results suggest a novel agonism, in which PPAR gamma functions as a multiple sensor in response to distinct metabolites. The EMBO Journal (2010) 29, 3395-3407. doi:10.1038/emboj.2010.197; Published online 17 August 2010
  • [Activation mechanism of PPARgamma by its endogenous ligands]., Shiraki T, Kamiya N, Jingami H, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 50(13), 1660 - 1665, Nov. 2005 , Refereed
  • Ionic basis of cold receptors acting as thermostats., Okazawa M, Takao K, Hori A, Shiraki T, Matsumura K, Kobayashi S, The Journal of neuroscience : the official journal of the Society for Neuroscience, The Journal of neuroscience : the official journal of the Society for Neuroscience, 22(10), 3994 - 4001, May 2002 , Refereed
  • Coexpression of microsomal-type prostaglandin E synthase with cyclooxygenase-2 in brain endothelial cells of rats during endotoxin-induced fever., Yamagata K, Matsumura K, Inoue W, Shiraki T, Suzuki K, Yasuda S, Sugiura H, Cao C, Watanabe Y, Kobayashi S, The Journal of neuroscience : the official journal of the Society for Neuroscience, The Journal of neuroscience : the official journal of the Society for Neuroscience, 21(8), 2669 - 2677, Apr. 2001 , Refereed
  • l-Menthol-induced [Ca2+]i increase and impulses in cultured sensory neurons., Okazawa M, Terauchi T, Shiraki T, Matsumura K, Kobayashi S, Neuroreport, Neuroreport, 11(10), 2151 - 2155, Jul. 2000 , Refereed
  • Possible nicotinic receptor-mediated modulation of synaptic transmission in nucleus of the solitary tract., Shiraki T, Toyoda A, Sugino H, Hori A, Kobayashi S, The American journal of physiology, The American journal of physiology, 272(3 Pt 2), R869 - 73, Mar. 1997 , Refereed
  • Phosphorylation of BACH1 switches its function from transcription factor to mitotic chromosome regulator and promotes its interaction with HMMR, Jie Li, Hiroki Shima, Hironari Nishizawa, Masatoshi Ikeda, Andrey Brydun, Mitsuyo Matsumoto, Hiroki Kato, Yuriko Saiki, Liang Liu, Miki Watanabe-Matsui, Kenji Iemura, Kozo Tanaka, Takuma Shiraki, Kazuhiko Igarashi, Biochemical Journal, Biochemical Journal, 475(5), 981 - 1002, Mar. 15 2018 , Refereed
    Summary:The transcription repressor BACH1 performs mutually independent dual roles in transcription regulation and chromosome alignment during mitosis by supporting polar ejection force of mitotic spindle. We now found that the mitotic spindles became oblique relative to the adhesion surface following endogenous BACH1 depletion in HeLa cells. This spindle orientation rearrangement was rescued by re-expression of BACH1 depending on its interactions with HMMR and CRM1, both of which are required for the positioning of mitotic spindle, but independently of its DNA-binding activity. A mass spectrometry analysis of BACH1 complexes in interphase and M phase revealed that BACH1 lost during mitosis interactions with proteins involved in chromatin and gene expression but retained interactions with HMMR and its known partners including CHICA. By analyzing BACH1 modification using stable isotope labeling with amino acids in cell culture, mitosis-specific phosphorylations of BACH1 were observed, and mutations of these residues abolished the activity of BACH1 to restore mitotic spindle orientation in knockdown cells and to interact with HMMR. Detailed histological analysis of Bach1-deficient mice revealed lengthening of the epithelial fold structures of the intestine. These observations suggest that BACH1 performs stabilization of mitotic spindle orientation together with HMMR and CRM1 in mitosis, and that the cell cycle-specific phosphorylation switches the transcriptional and mitotic functions of BACH1.
  • Genomewide approaches for BACH1 target genes in mouse embryonic fibroblasts showed BACH1-Pparg pathway in adipogenesis, Mitsuyo Matsumoto, Keiichi Kondo, Takuma Shiraki, Andrey Brydun, Ryo Funayama, Keiko Nakayama, Nobuo Yaegashi, Hideki Katagiri, Kazuhiko Igarashi, GENES TO CELLS, GENES TO CELLS, 21(6), 553 - 567, Jun. 2016 , Refereed
    Summary:The transcription repressor BTB and CNC homology 1 (BACH1) represses genes involved in heme metabolism and oxidative stress response. BACH1 also suppresses the p53-dependent cellar senescence in primary mouse embryonic fibroblasts (MEFs). To investigate the role of BACH1 in MEF other than its known functions, we carried out a genomewide mapping of binding site for BACH1 and its heterodimer partner MAFK in immortalized MEFs (iMEFs) using chromatin immunoprecipitation and next-generation sequencing technology (ChIP-sequence). The comparative analysis of the ChIP-sequence data and DNA microarray data from Bach1-deficient and wild-type (WT) iMEF showed 35 novel candidate target genes of BACH1. Among these genes, five genes (Pparg, Nfia, Ptplad2, Adcy1 and Ror1) were related with lipid metabolism. Bach1-deficient iMEFs showed increased expression of mRNA and protein of PPAR gamma, which is the key factor of adipogenesis. These cells also showed a concomitant increase in ligand-dependent activation of PPAR gamma target genes compared with wild-type iMEFs. Moreover, Bach1-deficient iMEFs efficiently differentiated to adipocyte compared with wild-type cells in the presence of PPAR gamma ligands. Our results suggest that BACH1 regulates expression of adipocyte-related genes including Pparg and potentiates adipocyte differentiation capacity.
  • Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin, Masae Ikura, Kanji Furuya, Shun Matsuda, Ryo Matsuda, Hiroki Shima, Jun Adachi, Tomonari Matsuda, Takuma Shiraki, Tsuyoshi Ikura, MOLECULAR AND CELLULAR BIOLOGY, MOLECULAR AND CELLULAR BIOLOGY, 35(24), 4147 - 4157, Dec. 2015 , Refereed
    Summary:The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation of NBS1 at DNA damage sites.
  • The C113D Mutation in Human Pin1 Causes Allosteric Structural Changes in the Phosphate Binding Pocket of the PPIase Domain through the Tug of War in the Dual-Histidine Motif, Ning Xu, Naoya Tochio, Jing Wang, Yu Tamari, Jun-ichi Uewaki, Naoko Utsunomiya-Tate, Kazuhiko Igarashi, Takuma Shiraki, Naohiro Kobayashi, Shin-ichi Tate, BIOCHEMISTRY, BIOCHEMISTRY, 53(34), 5568 - 5578, Sep. 2014 , Refereed
    Summary:Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin 1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.
  • Bach1 as a regulator of mitosis, beyond its transcriptional function, Jie Li, Takuma Shiraki, Kazuhiko Igarashi, Communicative and Integrative Biology, Communicative and Integrative Biology, 5(5), 477 - 479, Sep. 2012 , Refereed
    Summary:Bach1 is a transcriptional repressor which modulates several critical transcriptional responses, such as the expression of the heme oxygenase-1 (HO-1) gene in response to oxidative stress. In our recent study, we found that Bach1 possesses a novel role in mitotic chromosome alignment during metaphase. Upon BACH1 depletion in HeLa cells, mitotic chromosomes become unstable. This defect was efficiently rescued by expressing Bach1 fragments that lack the DNA binding domain, indicating that its function in mitosis involves a transcriptionindependent mechanism. The nuclear export signal (NES/CLS) of Bach1 is required for the mitotic function. Bach1 is excluded from the mitotic chromosomes depending on its NES/CLS and the nuclear exporter Crm1. Our findings suggest that Bach1 might mediate the regulation of mitotic chromosomes under conditions of cellular stress. © 2012 Landes Bioscience.
  • Transcription-independent role of Bach1 in mitosis through a nuclear exporter Crm1-dependent mechanism, Jie Li, Takuma Shiraki, Kazuhiko Igarashi, FEBS LETTERS, FEBS LETTERS, 586(4), 448 - 454, Feb. 2012 , Refereed
    Summary:The transcriptional repressor Bach1 mediates various stress responses. Despite its role in transcription, Bach1 is predominantly exported to the cytoplasm in a Crm1-dependent manner, but the functional role of its cytoplasmic retention is still unclear. We found that Bach1 was also excluded from mitotic chromatin by a C-terminal cytoplasmic localization sequence dependent and leptomycin B sensitive process. Bach1 depletion resulted in disordered mitotic chromosome alignment, which was rescued by Bach1 mutants lacking the BTB or DNA binding domains, suggesting its transcription-independent mechanism. We thus revealed a novel role of Bach1 in the regulation of mitotic chromosome dynamics. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Detecting structural similarity of ligand interactions in the lipid metabolic system including enzymes, lipid-binding proteins and nuclear receptors, Clara Shionyu-Mitsuyama, Tsuyoshi Waku, Takuma Shiraki, Takuji Oyama, Tsuyoshi Shirai, Kosuke Morikawa, PROTEIN ENGINEERING DESIGN & SELECTION, PROTEIN ENGINEERING DESIGN & SELECTION, 24(4), 397 - 403, Apr. 2011 , Refereed
    Summary:Nuclear receptors, intracellular lipid-binding proteins and metabolic enzymes are responsible for optimal metabolic homeostasis in higher organisms. Recent studies revealed the specific cooperation/competition among the subfamilies of these proteins. In this study, the nuclear receptor-lipid-binding protein-enzyme system, in which the interactions are mostly mediated by ligand molecules, was examined in terms of their ligand-binding structures to detect the similarity of interactions between functionally related subfamilies. The complex structures were dissected into single amino acid motifs for ligand fragment binding, and the presence and evolutionary origin of the motifs were compared among the protein families. As a result, functionally related nuclear receptor and enzyme pairs were found to share more motifs than expected, in agreement with the fact that the two families compete for the same ligand, and thus our study implies the possible co-evolution of the indirectly interacting protein system.
  • Proline cis/trans-Isomerase Pin1 Regulates Peroxisome Proliferator-activated Receptor gamma Activity through the Direct Binding to the Activation Function-1 Domain, Yoshito Fujimoto, Takuma Shiraki, Yuji Horiuchi, Tsuyoshi Waku, Akira Shigenaga, Akira Otaka, Tsuyoshi Ikura, Kazuhiko Igarashi, Saburo Aimoto, Shin-ichi Tate, Kosuke Morikawa, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 285(5), 3126 - 3132, Jan. 2010 , Refereed
    Summary:The important roles of a nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) are widely accepted in various biological processes as well as metabolic diseases. Despite the worldwide quest for pharmaceutical manipulation of PPAR gamma activity through the ligand-binding domain, very little information about the activation mechanism of the N-terminal activation function-1 (AF-1) domain. Here, we demonstrate the molecular and structural basis of the phosphorylation-dependent regulation of PPAR gamma activity by a peptidyl-prolyl isomerase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain, thereby inhibiting the polyubiquitination of PPAR gamma. The interaction and inhibition are dependent upon the WW domain of Pin1 but are independent of peptidyl-prolyl cis/trans-isomerase activity. Gene knockdown experiments revealed that Pin1 inhibits the PPAR gamma-dependent gene expression in THP-1 macrophage-like cells. Thus, our results suggest that Pin1 regulates macrophage function through the direct binding to the phosphorylated AF-1 domain of PPAR gamma.
  • Structural Insight into PPAR gamma Activation Through Covalent Modification with Endogenous Fatty Acids, Tsuyoshi Waku, Takuma Shiraki, Takuji Oyama, Yoshito Fujimoto, Kanako Maebara, Narutoshi Kamiya, Hisato Jingami, Kosuke Morikawa, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 385(1), 188 - 199, Jan. 2009 , Refereed
    Summary:Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor that regulates lipid homeostasis, and several fatty acid metabolites have been identified as PPAR gamma ligands. Here, we present four crystal structures of the PPAR gamma ligand binding domain (LBD) covalently bound to endogenous fatty acids via a unique cysteine, which is reportedly critical for receptor activation. The structure analyses of the LBD complexed with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) revealed that the covalent binding of 15d-PGJ2 induced conformational changes in the loop region following helix H2', and rearrangements of the side-chain network around the created covalent bond in the LBD. Point mutations of these repositioned residues on the loop and helix H3 almost completely abolished PPAR gamma activation by 15d-PGJ2, indicating that the observed structural alteration may be crucial for PPAR gamma activation by the endogenous fatty acid. To address the issue of partial agonism of endogenous PPAR gamma ligands, we took advantage of a series of oxidized eicosatetraenoic acids (oxoETEs) as covalently bound ligands to PPAR gamma. Despite similar structural and chemical properties, these fatty acids exhibited distinct degrees of transcriptional activity. Crystallographic studies, using two of the oxoETE/PPAR gamma LBD complexes, revealed that transcriptional strength of each oxoETE is associated with the difference in the loop conformation, rather than the interaction between each ligand and helix H12. These results suggest that the loop conformation may be responsible for the modulation of PPAR gamma activity. Based on these results, we identified novel agonists covalently bound to PPAR gamma by in silico screening and a cell-based assay. Our crystallographic study of LBD complexed with nitro-233 demonstrated that the expected covalent bond is indeed formed between this newly identified agonist and the cysteine. This study presents the structural basis for the activation and modulation mechanism of PPAR gamma through covalent modification with endogenous fatty acids. (C) 2008 Elsevier Ltd. All rights reserved.
  • Atomic structure of mutant PPAR gamma LBD complexed with 15d-PGJ(2): Novel modulation mechanism of PPAR gamma/RXR alpha function by covalently bound ligands, Tsuyoshi Waku, Takuma Shiraki, Takuji Oyama, Kosuke Morikawa, FEBS LETTERS, FEBS LETTERS, 583(2), 320 - 324, Jan. 2009 , Refereed
    Summary:15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) activates a nuclear receptor heterodimer, peroxisome proliferators-activated receptor gamma (PPAR gamma)/retinoid X receptor (RXR alpha) through covalent binding to Cys285 in PPAR gamma ligand-binding domain (LBD). Here, we present the 1.9 angstrom crystal structure of C285S mutant LBD complexed with 15d-PGJ(2), corresponding to the non-covalently bound state. The ligand lies adjacent to a hydrogen-bond network around the helix H2 and the nearby beta-sheet. Comparisons with previous structures clarified the relationships between PPAR gamma function and conformational alterations of LBD during the process of covalently binding ligands, such as 15d-PGJ(2), and thus suggested a mechanism, by which these ligands modulate PPAR gamma/RXR alpha function through conformational changes of the loop following helix H2' and the beta-sheet. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Activation mechanism of PPAR gamma by its endogenous ligands, Takuma Shiraki, SEIKAGAKU, SEIKAGAKU, 79(10), 960 - 964, Oct. 2007 , Refereed
  • Spectroscopic analyses of the binding kinetics of 15d-PGJ(2) to the PPAR gamma ligand-binding domain by multi-wavelength global fitting, T Shiraki, TS Kodama, S Shiki, T Nakagawa, H Jingami, BIOCHEMICAL JOURNAL, BIOCHEMICAL JOURNAL, 393(Pt 3), 749 - 755, Feb. 2006 , Refereed
    Summary:PPAR gamma (peroxisome proliferator-activated receptor gamma) is a nuclear receptor that is activated by natural lipid metabolites, including 15d-PGJ(2) (15-deoxy-Delta(12,14)-prostaglandin J(2)). We previously reported that several oxidized lipid metabolites covalently bind to PPAR gamma through a Michael-addition to activate transcription. To separate the ligand-entering (dock) and covalent-binding (lock) steps in PPAR gamma activation, we investigated the binding kinetics of 15d-PGJ(2) to the PPAR gamma LBD (ligand-binding domain) by stopped-flow spectroscopy. We analysed the spectral changes of 15d-PGJ2 by multi-wavelength global fitting based on a two-step chemical reaction model, in which an intermediate state represents the 15d-PGJ(2)-PPAR gamma complex without covalent binding. The extracted spectrum of the intermediate state in wild-type PPAR gamma was quite similar to the observed spectrum of 15d-PGJ2 in the C285S mutant, which cannot be activated by 15d-PGJ2, indicating that the complex remains in the inactive, intermediate state in the mutant. Thus 'lock' rather than 'dock' is one of the critical steps in PPAR gamma activation by 15d-PGJ(2).
  • alpha,beta-unsaturated ketone is a core moiety of natural ligands for covalent binding to peroxisome proliferator-activated receptor, T Shiraki, N Kamiya, S Shiki, TS Kodama, A Kakizuka, H Jingami, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 280(14), 14145 - 14153, Apr. 2005 , Refereed
    Summary:Peroxisome proliferator- activated receptor gamma( PPAR gamma) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPAR gamma. Here, we report that some PPAR gamma ligands, including 15- deoxy- Delta(12,14)- prostaglandin J(2), covalently bind to a cysteine residue in the PPAR gamma ligand binding pocket through a Michael addition reaction by an alpha,beta-unsaturated ketone. Using rhodamine- maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPAR gamma, but not of BRL49653, a non- covalent synthetic agonist, indicating that covalent binding of the alpha, beta- unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the alpha,beta- unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPAR gamma. We propose that PPAR gamma senses oxidation of fatty acids by recognizing such an alpha,beta- unsaturated ketone as a common moiety.
  • Rational discovery of a novel interface for a coactivator in the peroxisome proliferator-activated receptor gamma: Theoretical implications of impairment in type 2 diabetes mellitus, T Shiraki, TS Kodama, K Jingami, N Kamiya, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 58(2), 418 - 425, Feb. 2005 , Refereed
    Summary:The peroxisome proliferator-activated receptor gamma (PPARgamma) is important to adipocyte differentiation and glucose homeostasis, and mutations in the gene have been observed in type 2 diabetes mellitus. The mutated residues, V290 and P467, bind to neither ligands nor a coactivator peptide in the reported crystal structures of the PPARgamma ligand binding domain. To understand the mechanism of type 2 diabetes mellitus caused by germline mutations in the PPARgamma ligand-binding domain, theoretical models of the PPARgamma-ligand-coactivator complex were built at an atomic resolution. In the models, the secondary coactivator peptide was docked next to the conventional coactivator peptide, which both contain the LXXLL motif. The secondary interface in PPARgamma for the secondary coactivator peptide has not been demonstrated by experiments. Binding energy calculations of the complex, considering the solvent effect, revealed that the secondary coactivator peptide, derived from nuclear receptor box 1 of steroid receptor coactivator 1, can be favorably bound to the secondary interface. The secondary coactivator peptide forms hydrogen bonds and a hydrophobic core with PPARgamma and the primary coactivator peptide. Next, we applied mutations to PPARgamma in silico and found that the V290M mutation, observed in type 2 diabetes mellitus, adversely affected the binding of the secondary peptide. Thus, our model provides structural insight into the impairment of PPARgamma function in type 2 diabetes mellitus. (C) 2004 Wiley-Liss, Inc.
  • The nuclear bile acid receptor FXR is activated by PGC-1 alpha in a ligand-dependent manner, E Kanaya, T Shiraki, H Jingami, BIOCHEMICAL JOURNAL, BIOCHEMICAL JOURNAL, 382(Pt 3), 913 - 921, Sep. 2004 , Refereed
    Summary:The nuclear bile acid receptor FXR (farnesoid X receptor) is one of the key factors that suppress bile acid biosynthesis in the liver. PGC-1 [PPARgamma (peroxisome-proliferator-activated receptor gamma) co-activator-1alpha] is known to control energy homoeostasis in adipose tissue, skeletal muscle and liver. We performed cell-based reporter assays using the expression system of a GAL4-FXR chimaera, the ligand-binding domain of FXR fused to the DNA-binding domain of yeast GAL4, to find the co-activators for FXR. We found that the transcriptional activation of a reporter plasmid by a GAL4-FXR chimaera was strongly enhanced by PGC-1alpha, in a ligand-dependent manner. Transcriptional activation of the SHP (small heterodimer partner) gene by the FXR-RXRalpha (retinoid X receptor alpha) heterodimer was also enhanced by PGC-1alpha in the presence of CDCA (chenodeoxycholic acid). Co-immunoprecipitation and pull-down studies using glutathione S-transferase-PGC-1alpha fusion proteins revealed that the ligand-binding domain of FXR binds PGC-1alpha in a ligand-influenced manner both in vivo and in vitro. Furthermore, our studies revealed that SHP represses its own transcription, and the addition of excess amounts of PGC-1alpha a can overcome the inhibitory effect of SHP. These observations indicate that PGC-1alpha mediates the ligand-dependent activation of FXR and transcription of SHP gene.
  • Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha - Possible link between xenobiotic response and nutritional state, T Shiraki, N Sakai, E Kanaya, H Jingami, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 278(13), 11344 - 11350, Mar. 2003 , Refereed
    Summary:In contrast to the classical nuclear receptors, the constitutive androstane receptor (CAR) is transcriptionally active in the absence of ligand. In the course of searching for the mediator of CAR activation, we found that ligand-independent activation of CAR was achieved in cooperation with the peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). PGC-1beta, a PGC-1alpha homologue, also activated CAR to less of an extent than PGC-1alpha. Coexpression of the ligand-binding domain of a heterodimerization partner, retinoid X receptor alpha, enhanced the PGC-1alpha-mediated activation of CAR, although it had a weak effect on the basal activity of CAR in the absence of PGC-1alpha. Both the N-terminal region, with the LXXLL motif, and the C-terminal region, with a serine/arginine-rich domain (RS domain), in PGC-1alpha were required for full activation of CAR. Pull-down experiments using recombinant proteins revealed that CAR directly interacted with both the LXXLL motif and the RS domain. Furthermore, we demonstrated that the RS domain of PGC-1alpha was required for CAR localization at nuclear speckles. These results indicate that PGC-1alpha mediates the ligand-independent activation of CAR by means of subnuclear targeting through the RS domain of PGC-1alpha.
  • Brain-specific endothelial induction of prostaglandin E-2 synthesis enzymes and its temporal relation to fever, W Inoue, K Matsumura, K Yamagata, T Takemiya, T Shiraki, S Kobayashi, NEUROSCIENCE RESEARCH, NEUROSCIENCE RESEARCH, 44(1), 51 - 61, Sep. 2002 , Refereed
    Summary:Brain endothelial cells are hypothesized to be the major source of prostaglandin E-2 (PGE(2)) responsible for fever because they express 2 PGE(2)-synthesizing enzymes (cyclooxygenase-2 and microsomal-type PGE synthase) in response to pyrogens. To further validate this hypothesis, we examined in rats whether endothelial expression of these enzymes occurs only in the brain, and whether the time course of enzyme expression in brain endothelial cells can explain the time courses of brain PGE(2) level and fever. Intraperitoneal injection of lipopolysaccharide induced these enzymes only in brain endothelial cells, but not in those of peripheral organs including the neck, heart, lung, liver and kidney. Induction of these enzymes in brain endothelial cells was first noticed at 1.5 It after lipopolysaccharide injection, at which time elevation of PGE(2) was also first detected. Fever started just after this time point. These results demonstrate the significance of brain endothelial cells in the PGE(2) production during fever. Unexpectedly, PGE(2) level markedly dropped at 5 h in spite of high levels of these enzymes, implicating the existence of an unknown mechanism that suppresses PGE(2) level during the recovery phase of fever. (C) 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved.
  • Cyclooxygenase in the vagal afferents: is it involved in the brain prostaglandin response evoked by lipopolysaccharide?, K Matsumura, S Kaihatsu, H Imai, A Terao, T Shiraki, S Kobayashi, AUTONOMIC NEUROSCIENCE-BASIC & CLINICAL, AUTONOMIC NEUROSCIENCE-BASIC & CLINICAL, 85(1-3), 88 - 92, Dec. 2000 , Refereed
    Summary:The vagal afferents are proposed to transmit abdominal immune signals to the brain. In this immune-brain communication, prostaglandins might play a mediator role. In fact, prostaglandin receptors are abundant in the vagal afferents. We examined here the presence of cyclooxygenase, an enzyme necessary for prostaglandin biosynthesis, in the vagal afferents of rats. We also tested whether the vagal afferents contribute to the elevation of prostaglandin E(2) in the brain after intraperitoneal injection of lipopolysaccharide. Under normal conditions, cyclooxygenase-1-like immunoreactivity was constitutively expressed in the vagal afferents at their central terminals and in their cell bodies. Cyclooxygenase-2-like immunoreactivity was absent in the vagal afferents under normal as well as lipopolysaccharide-challenged conditions. Instead, cyclooxygenase-2-like immunoreactivity was induced in brain endothelial cells by the lipopolysaccharide challenge. The elevation of prostaglandin E(2) in the cerebrospinal fluid after lipopolysaccharide challenge was not inhibited, but was rather enhanced, by the bilateral vagotomy. These results suggest that the vagal afferents potentially generate prostaglandins, which may locally modulate the vagal signal transmission, but that the vagal efferents are not essential to the elevation of prostaglandin E(2) in the brain after intraperitoneal challenge with LPS. (C) 2000 Elsevier Science B.V. All rights reserved.
  • Endothelin-induced apoptosis of A375 human melanoma cells, M Okazawa, T Shiraki, H Ninomiya, S Kobayashi, T Masaki, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 273(20), 12584 - 12592, May 1998 , Refereed
    Summary:Endothelin-l (ET-1) inhibited serum-dependent growth of asynchronized A375 human melanoma cells, and the growth inhibitory effect was markedly enhanced when ET-1 was applied to the cells synchronized at G(1)/S boundary by double thymidine blocks. Flow cytometric analysis revealed that ET-1 did not inhibit the cell cycle progression after the release of the block but caused a significant increase of the hypodiploid cell population that is characteristic of apoptotic cell death. ET-l-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining and DNA fragmentation on gel electrophoresis. The increase in the hypodiploid cell peak was manifest within 16 h of exposure to 5 nM ET-1. Within the same time range, ET-1 caused actin reorganization and drastic morphological changes of the surviving cells from epithelioid to an elongated bipolar shape. These phenotypical changes were preceded by ET-l-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ETB via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ETB expressed by the cells in G(1)/early S phases, and overexpression of the receptor protein by cDNA microinjection conferred the responsiveness (both apoptosis and morphological changes) to ET-1 irrespective of the position of the cell in the cell cycle. These results indicated the presence of ETB-mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ET, expressed by individual A375 melanoma cells appeared to be regulated by a cell cycle-dependent mechanism, and the receptor density can be a limiting factor to control the apoptotic and cytoskeletal responses of the cells to ET-1. Although the molecular mechanisms remain to be elucidated, these findings added a new dimension to the diverse biological activities of ETs and also indicated a novel mechanism to control the responsiveness of the cell to the peptides.
  • Stimulating effect of 6R-tetrahydrobiopterin on CA(2+) channels in neurons of rat dorsal motor nucleus of the vagus, T Shiraki, K Koshimura, S Kobayashi, S Miwa, T Masaki, Y Watanabe, Y Murakami, Y Kato, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 221(1), 181 - 185, Apr. 1996 , Refereed
    Summary:We have recently found that 6R-tetrahydrobiopterin (6R-BH4), a natural cofactor for aromatic L-amino acid hydroxylases and nitric oxide synthase, enhances dopamine release. Here, using a slice patch method, we examined the effect of 6R-BH4 on Ca2+ channels in neurons of rat dorsal motor nucleus of the vagus, where dopaminergic neurons are densely located. 6R-BH4 enhanced N-type Ca2+ channel currents, whereas 6S-BH4, a diastereoisomer of 6R-BH4, had little effect. Neither sodium nitroprusside, a nitric oxide generator, nor L-DOPA, a product of tyrosine hydroxylation, mimicked the effect of 6R-BH4. These findings suggest that 6R-BH4 enhances N-type Ca2+ channel currents in stereospecifically and independently of its cofactor activities as observed in its dopamine releasing action, and raise possibility that 6R-BH4 enhances dopamine release by activating Ca2+ channels. (C) 1996 Academic Press, Inc.
    Summary:We have cloned cDNA encoding bovine endothelin converting enzyme (ECE). The predicted amino acid sequence of bovine ECE consisted of 758 amino acid residues. Northern blot analysis revealed that ECE mRNA was abundantly expressed in lung. Co-expression of the cloned cDNA of bovine ECE with human preproET-1 cDNA in CHO-K1 cells resulted in the production of mature ET-1 and this production was inhibited by phosphoramidon. (C) 1994 Academic Inc.
    Summary:We have established a novel method of molecular cloning of endothelin converting enzyme, a key enzyme in the production of a potent vasoconstrictor endothelin-1, by modification of the reverse hemolytic plaque assay. Also, we demonstrated that a cell line, CHO-K1, showed no detectable activity of endothelin converting enzyme. This cell line was transfected with a cDNA library of bovine endothelial cells. The modified reverse hemolytic plaque assay was shown to detect even a single CHO-K1 cell that was changed to produce mature ET-1 by transfection. Thus, this novel method is suggested to be useful for the molecular cloning of other secreted antigens and their processing enzyme.
    Summary:Nucleus tractus solitarius (NTS) has a high density of prostaglandin E2 (PGE2)-binding sites. Action of PGE2 (10(-9)-10(-6) M) was tested on neurons in a NTS slice with patch-clamp recording under synaptic blockade. PGE2 raised the firing rate in approximately half of the neurons in cell-attached patch mode. In whole-cell current clamp, PGE2 depolarized membrane potential accompanied by an increase in firing rate. In whole-cell voltage clamp (-58 mV), PGE2 induced the inward current with an increase in conductance. The current was linearly related to voltage from -100 mV to -10 mV and suppressed between -10 mV and 20 mV. The current-voltage curve remained similar under low external Cl- or high internal Cl- conditions and after external Na+ was replaced by Cs+. It is concluded that PGE2 excites NTS neurons by activating cation conductance.

Books etc

  • Nanzando's medical dictionary, Takuma Shiraki, Joint author,   2015 04
  • Obesity, Takuma Shiraki, Joint author, Bardet-Biedl syndrome,   2014 05
  • Functional and Structural Biology on the Lipo-network., Takuma Shiraki, Joint author, Covalent binding: Connection between structure and function of PPARγ., Transworld Research Network,   2006


  • Transcriptional machinery sensing cellular metabolism : nuclear receptor, PPARγ, mediates crosstalk between metabolic networks, 白木 琢磨, 和久 剛, 森川 耿右, 生化学, 85, 9, 749, 761,   2013 09 ,
  • Activation mechanism of PPARγ by its endogenous ligands, SHIRAKI Takuma, 生化学, 79, 10, 960, 964,   2007 10 25 , 招待有り,
  • 2P052 Development of the semi-flexible docking tool and application to the docking between PPARgamma and coactivator peptide, Kamiya N, Shiraki T, Kodama T.S, Jingami H, Biophysics, 44, 1,   2004 11 10 ,

Research Grants & Projects

  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Regulation of nuclear receptor activity by intracellular signals and ligands, According to our previous findings of endogenous ligands for PPARg, we hypothesized that extracellular signaling molecules regulate PPARg activity through not only their intracellular metabolites acting as ligands, but also intracellular signals evoked by membrane associated receptors.To analyze the temporal and spatial regulation of PPARg activity, we developed novel reporter system, by which six independent transcriptional activities were simultaneously detected. We found that expression of phosphorylation induced stabilization of PPARg proteins by itself and blocked nuclear export of PPARg protein only in the presence of SDP-1. We also analyzed the effects of PPARg mutation identified in German obese subjects on the phoshphorylation-dependent regulation, and found that P85Q mutation blocked the effects of SDP-1. Thus, we conclude that phosphorylation signal regulates PPARg activity in the distinct mechanism from that of ligands.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(若手研究(B)), Kinetic analyses of nuclear receptor activation