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FacultyDepartment of Life Science / Graduate School of Science and Engineering Research
Commentator Guide
Last Updated :2020/09/30

Education and Career

Academic & Professional Experience

  •   2011 ,  - 現在, Faculty of Science and Engineering, Kindai University

Research Activities

Research Areas

  • Nanotechnology/Materials, Molecular biochemistry
  • Life sciences, Structural biochemistry

Published Papers

  • Tsubasa Tagawa, Teruki Hagiwara, Shigeru Shimamoto, and Yuji Hidaka, 2018, 101 - 101, Mar. 2019 , Refereed
  • STRUCTURAL CONTROL AND FUNCTIONAL ANALYSIS OF THE PRECURSOR PROTEIN OF ATRIAL NATRIURETIC PEPTIDE, Hayato Ueda, Shigeru Shimamoto, Yuji HidakaHayato Ueda, Shigeru Shimamoto, Yuji Hidaka, 2018, 94 - 94, Mar. 2019 , Refereed
  • Coupling effects of thiol and urea-type groups for promotion of oxidative protein folding, Okada S, Matsusaki M, Arai K, Hidaka Y, Inaba K, Okumura M, Muraoka T, Chem Commun (Camb), Chem Commun (Camb), 55(6), 759 - 762, Jan. 2019 , Refereed
  • Disulfide-Coupled Folding of Pro-Uroguanylin on Molecular Evolution, Kenta Mori, Kosuke Toyama, Saya Nishihara, Shigeru Shimamoto, Yuji Hidaka, 2017, 214 - 215, Mar. 2018 , Refereed
  • Molecular Evolution of L-PGDS:Substrate Recognition Mechanism of Medaka L-PGDS, Kimi Torii, Takahiro Maruno, Yuji Kobayashi, Yuji Hidaka, Shigeru Shimamoto, Peptide Science, Peptide Science, 2017, 210 - 211, Mar. 2018 , Refereed
  • Regulation of Disulfide-Coupled Folding of a De Novo Designed Protein, Saya Nishihara, Kosuke Toyama, Kenta Mori, Shigeru Shimamoto, Yuji Hidaka, Peptide Science, Peptide Science, 2017, 208 - 209, Mar. 2018 , Refereed
  • Structural Analyses of a Linker Region of the Amyloid Precursor Protein, Mizuho Imamura, Shingo Kanemura, Masaki Okumura, Hiroshi Yamaguchi, Shigeru Shimamoto, Yuji Hidaka, Peptide Science, Peptide Science, 2017, 206 - 207, Mar. 2018 , Refereed
  • Preparation of an Orexin Precorsor Protein Using an E. coli Expression System by Acid Treatment, Natsumi Mitsuoka, Shigeru Shimamoto, Yuji Hidaka, Peptide Science, Peptide Science, 2016, 221 - 222, Mar. 2017 , Refereed
  • Regulation of the Disulfide-Coupled Folding of De Novo Designed Peptides by alpha-Helix Formation, Saya Nishihara, Kosuke Toyama, Shigeru Shimamoto, Yuji Hidaka, Peptide Science, Peptide Science, 2016, 233 - 234, Mar. 2017 , Refereed
  • Activation Mechanism of Cocoonase, Nagisa Tajima, Shigeru Shimamoto, Mitsuhiro Miyazawa, Yuji Hidaka, Peptide SCience, Peptide SCience, 2016, 105 - 106, Mar. 2017 , Refereed
  • Disulfide-Coupled Folding of Pro-Uroguanylin on Molecular Evolution, Kenta Mori, Kosuke Toyama, Shigeru Shimamoto, Yuji Hidaka, Peptide Science, Peptide Science, 2016, 103 - 104, Mar. 2017 , Refereed
  • An alpha,beta-structural Shift of the Mature Region in a Precursor Protein Regulates Disulfide Selectivity under Kinetic Control, Kosuke Toyama, Shigeru Shimamoto, Yuji Hidaka, Peptide Science, Peptide Science, 2015, 333 - 334, Mar. 2016 , Refereed
  • a Mis-bridged Disulfide Isomer of Prouroguanylin, Kenta Hattori, Masaki Okumura, Shigeru Shimamoto, Yuji Hidaka, Peptide Science, Peptide Science, 2015, 327 - 328, Mar. 2016 , Refereed
  • Trapping Mechanism of Bioactive Conformation by Intra-molecular Chaperone, Yukihito Yokoyama, Masaki Okumura, Shigeru Shimamoto, Hiroshi Yamaguchi, Yuji Hidaka, Peptide Science, Peptide Science, 2014, 95 - 96, Mar. 2015 , Refereed
  • Inhibition of the Functional Interplay between Endoplasmic Reticulum (ER) Oxidoreduclin-1 alpha (Ero1 alpha) and Protein-disulfide Isomerase (PDI) by the Endocrine Disruptor Bisphenol A, Masaki Okumura, Hiroshi Kadokura, Shoko Hashimoto, Katsuhide Yutani, Shingo Kanemura, Takaaki Hikima, Yuji Hidaka, Len Ito, Kohei Shiba, Shoji Masui, Daiki Imai, Susumu Imaoka, Hiroshi Yamaguchi, Kenji Inaba, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 289(39), 27004 - 27018, Sep. 2014 , Refereed
    Summary:Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b' domain, preventing PDI from binding to unfolded proteins. The b' domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1 alpha (Ero1 alpha), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1 alpha-catalyzed PDI oxidation presumably by inhibiting the interaction between the b' domain of PDI and Ero1 alpha; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding beta-hairpin of Ero1 alpha, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1 alpha and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.
  • Crystallization and Preliminary X-ray Structural Studies of Human Prouroguanylin, Len Ito, Yuji Hidaka (corresponding author, Masaki Okumura, Hironori Konishi, Adermann Knut, Hiroshi Yamaguchi, Acta Crystallogr. Sect F Struct Biol Cryst Commun., Acta Crystallogr. Sect F Struct Biol Cryst Commun., 64(8), 771 - 772, Jun. 2008 , Refereed
  • Deimination of histone H2A and H4 at arginine 3 in HL-60 granulocytes, T Hagiwara, Y Hidaka, M Yamada, BIOCHEMISTRY, BIOCHEMISTRY, 44(15), 5827 - 5834, Apr. 2005 , Refereed
    Summary:Interplay of various covalent modifications of histone tails has an essential role in regulation of chromatin function. Peptidylarginine deiminase (PADI) 4 deiminates protein arginine to citrulline in a Ca2+-dependent manner and is present in the nucleus of granulocyte-differentiated HL-60 cells. When these cells are treated with the calcium ionophore A23187, core histone deimination occurs. To determine the deimination sites of histones, histone species were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) from the cells. Immunoblotting using antimodified citrulline antibody indicated that histones H2A, H3, and H4 but not H2B were deiminated. H2A and H4 were digested with Staphylococcus aureus V8 protease, and the digests were separated by RP-HPLC. Immuno dot-blotting and mass spectrometry showed that the deiminated residues were present in H2A (1-56) and H4 (1-52) regions but not in other regions. The H2A peptide (1-56) was digested with alpha-chymotrypsin, and the deiminated peptide was separated from the corresponding nondeiminated peptide by RP-HPLC. The deiminated residue was found to be limited to residues 1-23. Similarly, digestion of the H4 peptide (1-52) with endoproteinase Asp-N and separation of the deiminated peptide from the nondeiminated peptide indicated that the deiminated residue was limited to residues 1-23. Mass spectrometry of lysylendopeptidase digests of the H2A (1-23) and H4 (1-23) peptides showed that deimination occurred at arginine 3 of the N-terminal sequence Ac-SGRGK common to H2A and H4. These results suggest that PADI4 deiminates only a restricted site of target proteins in cells. Deimination of histones is discussed in relation to chromatin structure and function.
  • Methylation of the Guanidino Group of Arginine Residues Prevents Citrullination by, Teruki Hagiwara, Yuji Hidaka, Michiyuki Yamada, FEBS Letters, FEBS Letters, 579, 4088 - 4092, 2005 , Refereed
  • Side Chain Contributions to the Interconversion of the Topological Isomers of Guanylin-like Peptides, Axel Schulz, Ute Marx, Naomi Tidten, Thomas Lauber, Yuji Hidaka, Knut Adermann, Journal of Peptide Science, Journal of Peptide Science, 11, 319 - 330, Nov. 2004 , Refereed
  • Folding of a Peptide Hormone Assisted by a Propeptide: Folding of Hybrid Disulfide Isomers of Uroguanylin and Heat-stable Enterotoxin, Yuji Hidaka, Len Ito, Kaori Segawa, Hiroshi Yamaguchi, Peptide Science, Peptide Science, 2003(2003), 103 - 106, 2004 , Refereed
  • The Micro Domain Responsible for Ligand-binding of Guanylyl Cyclase C, Yuji Hidaka, Yoshiko Matsumoto, Yasutsugu Shimonishi, FEBS Lett, FEBS Lett, 526, 58 - 62, Jul. 2002 , Refereed
  • In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein., Yuji Hidaka, Megumu Ohno, Bahram Hemmasi, Oliver Hill, Wolf-Georg Forssmann, Yasutsugu Shimonishi, Biochemistry, Biochemistry, 37, 8498 - 8507, May 1998 , Refereed
  • Identification of a binding region on Escherichia coli heat-stable enterotoxin to intestinal guanylyl cyclase C, Makoto Hasegawa, Yuki Kawano, Kazuya Matsumoto, Yuji Hidaka, Takashi Sato, Yasutsugu Shimonishi, Letters in Peptide Science, Letters in Peptide Science, 4, 1 - 11, Apr. 1997 , Refereed
  • Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of escherichia coli using Ni-histidine tag affinity chromatography, Yuji Hidaka, Heiyoung Park, Masayori Inouye, FEBS Lett, FEBS Lett, 400, 238 - 242, Feb. 1997 , Refereed
  • Characterization of Carboxy-terminal Region of Heat-stable Enterotoxin Receptor (STaR), Wada, A, Padilla, P.I, Takamoto, T, Hasegawa, M, Matsumoto, K, Kawano, Y, Hidaka, Y, Kurazono, H, Shimonishi, Y, Hirayama, T, Japanese Journal of Medical Science & Biology, Japanese Journal of Medical Science & Biology, 49, 254 - 255, Mar. 1996 , Refereed
  • Novel motifs in extracellular domain of heat-stable enterotoxin receptor (STaR), Wada, A, Oshita, T, Kitaura, H, Hasegawa, M, Fujisawa, J, Hidaka, Y, Shimonishi, Y, Hirayama, T, Japanese Journal of Medical Science & Biology, Japanese Journal of Medical Science & Biology, 48, 266 - 267, Mar. 1995 , Refereed
  • Significant amino-acids of Heat-stable Enterotoxin Receptor (StaR) for STa-binding, Akihiro Wada, A, Oshita, T, Kitaura, H, Jun-ichi Fujisawa, Yuji Hidaka, Yasutsugu Shimonishi, Toshiya Hirayama, Japanese Journal of Medical Science & Biology, Japanese Journal of Medical Science & Biology, 47, 315 - 316, Apr. 1994 , Refereed
  • High-Performance Liquid Chromatographic Determination of Neutral and Amino Monosaccharides by Ultraviolet and Fluorescence Detection of Sugar 9-Fluorenylmethoxycarbonyl Hydrazones and 9-Fluorenylmethoxycarbonyl Amino Sugars at Picomole and Sub-Picomole Le, Renen Zhang, Zhidan Zhang, Guoquan Liu, Yuji Hidaka, Yasutsugu Shimonishi, Journal of Chromatography,, Journal of Chromatography,, 646, 45 - 52, Mar. 1993 , Refereed
  • Identification and Cloning of the Pig Receptor for Escherichia Coli Heat-stable Enterotoxin (STa), Akihiko Wada, Toshiya Hirayama, Jun-ichi Fujisawa, Yuji Hidaka, Yasutsugu Shimonish, Japanese Journal of Medical Science & Biology, Japanese Journal of Medical Science & Biology, 45, 295 - 296, Apr. 1992 , Refereed
  • Structure-Activity Relationship of Esherichia Coli Heat-Stable Enterotoxin : Role of Ala Residue at Position 14 in Toxin-Receptor Interaction, Shinji Yamasaki, Takashi Sato, Yuji Hidaka, Hiroshi Ozaki, Hideaki Ito, Toshiya Hirayama, Yoshifumi Takeda, Takashi Sugimura, Akira Tai, Yasutsugu Shimonishi, Bull. Chem. Soc. Jpn., Bull. Chem. Soc. Jpn., 53, 2063 - 2070, Jul. 1990 , Refereed
  • A New Method for Determination of Disulfide Pairing in Peptide, Yuji Hidaka, Yasutsugu Shimonishi, Bull. Chem. Soc. Jpn.,, Bull. Chem. Soc. Jpn.,, 62, 1986 - 1994, Jun. 1989 , Refereed
  • Structure-Activity Relationship of a Heat-stable Enterotoxin Produced by Yersinia Enterocolitica, Shoko Yoshimura, Yuji Hidaka, Saburo Aimoto, Yasutsugu Shimonishi, Tae Takeda, Toshio Miwatani, Yoshifumi Takeda, Bull. Chem. Soc. Jpn., Bull. Chem. Soc. Jpn., 60, 2481 - 2489, Jul. 1987 , Refereed
  • Gene expression of the concentration-sensitive sodium channel is suppressed in lipopolysaccharide-induced acute lung injury in mice, Teruki Hagiwara, Shigeru Yoshida, Yuji Hidaka, EXPERIMENTAL LUNG RESEARCH, EXPERIMENTAL LUNG RESEARCH, 43(3), 150 - 157, 2017 , Refereed
    Summary:Purpose: The concentration-sensitive sodium channel (Na-C) is expressed in alveolar type II epithelial cells and pulmonary microvascular endothelial cells in mouse lungs. We recently reported that Na-C contributes to amiloride-insensitive sodium transport in mouse lungs (Respiratory Physiology & Neurobiology, 2016). However, details regarding its physiological role in the lung remain unknown. To examine whether Na-C is involved in alveolar fluid clearance during an acute lung injury (ALI), we analyzed the relationship between Na-C gene expression in the lung and the development of pulmonary edema in lipopolysaccharide (LPS)-induced ALI mice. Methods: LPS-induced ALI mice were prepared by the intratracheal administration of LPS. Bronchoalveolar lavage (BAL) neutrophils and lung water content (LWCs) were used as a marker of ALI and pulmonary edema, respectively. Na-C protein production in the lung was detected by immunoblotting and immunofluorescence. The gene expressions of Na-C and the epithelial sodium channel (ENaC) of LPS-induced ALI mice were examined by quantitative RT-PCR over a time course of 14 days. Results: The BAL neutrophil count increased until day 2 after LPS administration and had nearly recovered by day 6. LWCs in LPS-induced mice gradually increased until day 8 and had recovered by day 14. The expression of the Na-C protein in the lungs of LPS-induced mice dramatically decreased from day 2 to day 6, but recovered by day 8. The mRNA expression of Na-C decreased in the lung, as well as those for alpha-, beta-, and gamma-ENaC during ALI. Thus, Na-C expression is suppressed during the development stage of pulmonary edema and then recovers in the convalescent phase. Conclusion: Our results suggest that suppression of the gene expression of Na-C is involved in the development of pulmonary edema in ALI.
  • Overview of the regulation of disulfide bond formation in peptide and protein folding, Yuji Hidaka, Current Protocols in Protein Science, Current Protocols in Protein Science, 76(76), 28.6.1 - 6, 2014 , Refereed
    Summary:Disulfide bonds play a critical role in the maintenance of the native conformation of proteins under thermodynamic control. In general, disulfide bond formation is associated with protein folding, and this restricts the formation of folding intermediates such as misbridged disulfide isomers or kinetically trapped conformations, which provide important information related to how proteins fold into their native conformation. Therefore, numerous studies have focused on the structural analysis of folding intermediates in vitro. However, isolating or trapping folding intermediates, as well as the entire proteins, including mutant proteins, is not an easy task. Several chemical methods have recently been developed for examining peptide and protein folding and for producing, e.g., intact, post-translationally modified, or kinetically trapped proteins, or proteins with misbridged disulfide bonds. This overview introduces chemical methods for regulating the formation of disulfide bonds of peptides and proteins in the context of the thermodynamic and kinetic control of peptide and protein folding. © 2014 by John Wiley & Sons, Inc.
  • Chemical methods for producing disulfide bonds in peptides and proteins to study folding regulation, Masaki Okumura, Shigeru Shimamoto, Yuji Hidaka, Current Protocols in Protein Science, Current Protocols in Protein Science, 76(76), 28.7.1 - 13, 2014 , Refereed
    Summary: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. © 2014 by John Wiley & Sons, Inc.
  • Chemical methods and approaches to the regioselective formation of multiple disulfide bonds, Shigeru Shimamoto, Hidekazu Katayama, Masaki Okumura, Yuji Hidaka, Current Protocols in Protein Science, Current Protocols in Protein Science, 76(76), 28.8.1 - 28, 2014 , Refereed
    Summary: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. © 2014 by John Wiley & Sons, Inc.
  • Folding of peptides and proteins: Role of disulfide bonds, recent developments, Yuji Hidaka, Shigeru Shimamoto, Biomolecular Concepts, Biomolecular Concepts, 4(6), 597 - 604, Dec. 01 2013 , Refereed
    Summary: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.
  • Effects of positively charged redox molecules on disulfide-coupled protein folding, Masaki Okumura, Shigeru Shimamoto, Takeyoshi Nakanishi, Yu-ichiro Yoshida, Tadafumi Konogami, Shogo Maeda, Yuji Hidaka, FEBS LETTERS, FEBS LETTERS, 586(21), 3926 - 3930, Nov. 2012 , Refereed
    Summary: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. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • In Vitro Regulatory Effect of Epididymal Serpin CRES on Protease Activity of Proprotein Convertase PC4/PCSK4, P. Mishra, Q. Qiu, A. Gruslin, Y. Hidaka, M. Mbikay, A. Basak, CURRENT MOLECULAR MEDICINE, CURRENT MOLECULAR MEDICINE, 12(8), 1050 - 1067, Sep. 2012 , Refereed
    Summary:PC4 or PCSK4 belongs to the 9-member superfamily of mammalian subtilases collectively called Proprotein Convertases or Proprotein Convertase Subtilisin/Kexins that convert inactive precursor proteins into their active mature forms by endoproteolytic cleavage. PC4-activity plays a crucial role in mammalian fertilization via activation of sperm surface proteins. PC4 knockout mice exhibit severely impaired male fertility due to premature sperm acrosome reaction. Regulation of sperm-PC4 activity during its storage and transport through epididymis is an important determinant for ultimate egg-binding and fertilizing capacities of sperms. Herein we show that epididymal serpin CRES (cystatin related epididymal spermatogenic) recombinant protein inhibits PC4 activity in vitro in a differential manner when measured against the fluorogenic substrate Boc-RVRR-MCA depending on its oligomeric state. Thus while CRES-dimer exhibits K-i similar to 8 mu M, the corresponding monomer showed Ki > 100 mu M. Both forms also blocked PC4-mediated processing of human proIGF-2 in human placenta tropoblast cell line with dimer being more efficient. Using specific inhibitors and substrates, we also demonstrated the presence of PC4-like activity and CRES protein in varying levels in the fluids of various epididymal compartments. Our observations suggest a potential function of CRES as a regulator of PC4 in sperm-egg interaction and fertilization.
  • Disulfide-coupled protein folding: looking back, looking forward, Yuji Hidaka, FEBS JOURNAL, FEBS JOURNAL, 279(13), 2261 - 2261, Jul. 2012 , Refereed
  • A chemical method for investigating disulfide-coupled peptide and protein folding, Masaki Okumura, Shigeru Shimamoto, Yuji Hidaka, FEBS JOURNAL, FEBS JOURNAL, 279(13), 2283 - 2295, Jul. 2012 , Refereed
    Summary: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.
  • Stem-Forming Regions That Are Essential for the Amyloidogenesis of Prion Proteins, Masatoshi Saiki, Yuji Hidaka, Masayuki Nara, Hisayuki Morii, BIOCHEMISTRY, BIOCHEMISTRY, 51(8), 1566 - 1576, Feb. 2012 , Refereed
    Summary:Prion diseases represent fatal neurodegenerative disorders caused by the aggregation of prion proteins. With regard to the formation of the amyloidogenic cross-beta-structure, the initial mechanism in the conversion to a beta-structure is critically important. To explore the core regions forming a stem of the amyloid, we designed and prepared a series of peptides comprised of two native sequences linked by a turn-inducing dipeptide moiety and examined their ability to produce amyloids. A sequence alignment of the peptides bearing the ability to form amyloid structures revealed that paired strands consisting of VNITI (residues 180-184) and VTTTT (residues 189-193) are the core regions responsible for initiating the formation of cross-beta-structures and for further ordered aggregation. In addition, most of the causative mutations responsible for inherited prion diseases were found to be located in these stem-forming regions on helix H2 and their counterpart on helix H3. Moreover, the volume effect of the nonstem domain, which contains similar to 200 residues, was deduced to be a determinant of the nature of the association such as oligomerization, because the stem-forming domain is only a small part of a prion protein. Taken together, we conclude that the mechanism underlying the initial stage of amyloidogenesis is the exposure of a newly formed intramolecular beta-sheet to a solvent through the partial transition of a native structure from an alpha-helix to a beta-structure. Our results also demonstrate that prion diseases caused by major prion proteins except the prions of some fungi such as yeast are inherent only in mammals, as evidenced by a comparison of the corresponding sequences to the stem-forming regions among different animals.
  • Acceleration of disulfide-coupled protein folding using glutathione derivatives, Masaki Okumura, Masatoshi Saiki, Hiroshi Yamaguchi, Yuji Hidaka, FEBS JOURNAL, FEBS JOURNAL, 278(7), 1137 - 1144, Apr. 2011 , Refereed
    Summary:Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives - glutamyl-cysteinyl-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (K(RCG) = 3.69 x 10-3 s-1) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.
  • Fiber Formation of a Synthetic Spider Peptide Derived From Nephila clavata, Yuji Hidaka, Ko-Ichi Kontani, Rina Taniguchi, Masatoshi Saiki, Sayoko Yokoi, Kenji Yukuhiro, Hiroshi Yamaguchi, Mitsuhiro Miyazawa, BIOPOLYMERS, BIOPOLYMERS, 96(2), 222 - 227, 2011 , Refereed
    Summary:Dragline silk is a high-performance biopolymer with exceptional mechanical properties. Artificial spider dragline silk is currently prepared by a recombinant technique or chemical synthesis. However, the recombinant process is costly and large-sized synthetic peptides are needed for fiber formation. In addition, the silk fibers that are produced are much weaker than a fiber derived from a native spider. In this study, a small peptide was chemically synthesized and examined for its ability to participate in fiber formation. A short synthetic peptide derived from Nephila clavata was prepared by a solid-phase peptide method, based on a prediction using the hydrophobic parameter of each individual amino acid residue. After purification of the spider peptide, fiber formation was examined under several conditions. Fiber formation proceeded in the acidic pH range, and larger fibers were produced when organic solvents such as trifluoroethanol and acetonitrile were used at an acidic pH. Circular dichroism measurements of the spider peptide indicate that the peptide has a 13-sheet structure and that the formation of a beta-sheet structure is required for the spider peptide to undergo fiber formation. (C) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 222-227, 2011.
  • Recognition of the N-Terminal Histone H2A and H3 Peptides by Peptidylarginine Deiminase IV, Masatoshi Saiki, Mayumi Watase, Hironori Matsubayashi, Yuji Hidaka, PROTEIN AND PEPTIDE LETTERS, PROTEIN AND PEPTIDE LETTERS, 16(9), 1012 - 1016, 2009 , Refereed
    Summary:Peptidylarginine deiminase IV (PAD4) catalyzes the conversion of an Arg residue to a citrulline residue in various proteins. In particular, citrullination of histone subunits, such as H2A and H3, by PAD4 is thought to be related to rheumatoid arthritis. However, the details of the citrullination mechanism of histone H2A and H3 are not yet well known. Moreover, the effects of N-terminal acetylation on histone subunits with respect to PAD4 recognition have not yet been studied. To further study the mechanism of PAD4 recognition of histone H2A and H3 subunits, a series of the N-terminal peptides was chemically synthesized and the citrullination sites were identified using MALDI-TOF/MS. N-terminal acetylation of histone H2A was not significant with respect to PAD4 recognition in vitro, but the acetylation of H3 peptide had a significant effect on PAD4 recognition in vitro, resulting in predominant citrullination at the Arg2 residue.
  • Structural basis for substrate recognition and dissociation by human transportin 1, Tsuyoshi Imasaki, Toshiyuki Shimizu, Hiroshi Hashimoto, Yuji Hidaka, Shingo Kose, Naoko Imamoto, Michiyuki Yamada, Mamoru Sato, MOLECULAR CELL, MOLECULAR CELL, 28(1), 57 - 67, Oct. 2007 , Refereed
    Summary:Transportin 1 (Trn1) is a transport receptor that transports substrates from the cytoplasm to the nucleus through nuclear pore complexes by recognizing nuclear localization signals (NLSs). Here we describe four crystal structures of human Trn1 in a substrate-free form as well as in the complex with three NLSs (hnRNP D, JKTBP, and TAP, respectively). Our data have revealed that (1) Trn1 has two sites for binding NLSs, one with high affinity (site A) and one with low affinity (site B), and NLS interaction at site B controls overall binding affinity for Trn1; (2) Trn1 recognizes the NLSs at site A followed by conformational change at site B to interact with the NLSs; and (3) a long flexible loop, characteristic of Trn1, interacts with site 13, thereby displacing transport substrate in the nucleus. These studies provide deep understanding of substrate recognition and dissociation by Trn1 in import pathways.
  • Structural basis for histone N-terminal recognition by human peptidylarginine deiminase 4, K Arita, T Shimizu, H Hashimoto, Y Hidaka, M Yamada, M Sato, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 103(14), 5291 - 5296, Apr. 2006 , Refereed
    Summary:Histone arginine methylation is a posttranslational modification linked to the regulation of gene transcription. Unlike other posttranslational modifications, methylation has generally been regarded as stable, and enzymes that demethylate histone arginine residues have not been identified. However, it has recently been shown that human peptidylarginine deiminase 4 (PAD4), a Ca2+-dependent enzyme previously known to convert arginine residues to citrulline in histones, can also convert monomethylated arginine residues to citrulline both in vivo and in vitro. Citrullination of histone arginine residues by the enzyme antagonizes methylation by histone arginine methyltransferases and is thus a novel posttranslational modification that regulates the level of histone arginine methylation and gene activity. Here we present the crystal structures of a Ca2+-bound PAD4 mutant in complex with three histone N-terminal peptides, each consisting of 10 amino acid residues that include one target arginine residue for the enzyme (H3/Arg-8, H3/Arg-17, and H4/Arg-3). To each histone N-terminal peptide, the enzyme induces a beta-turn-like bent conformation composed of five successive residues at the molecular surface near the active site cleft. The remaining five residues are highly disordered. The enzyme recognizes each peptide through backbone atoms of the peptide with a possible consensus recognition motif. The sequence specificity of the peptide recognized by this enzyme is thought to be fairly broad. These observations provide structural insights into target protein recognition by histone modification enzymes and illustrate how PAD4 can target multiple arginine sites in the histone N-terminal tails.
  • Side chain contributions to the interconversion of the topological isomers of guanylin-like peptides, A Schulz, UC Marx, N Tidten, T Lauber, Y Hidaka, K Adermann, JOURNAL OF PEPTIDE SCIENCE, JOURNAL OF PEPTIDE SCIENCE, 11(6), 319 - 330, Jun. 2005 , Refereed
    Summary:The peptide hormones guanylin and uroguanylin are ligands of the intestinal guanylyl cyclase-C (GC-C) that is involved in the regulation of epithelial water and electrolyte transport. The small peptides contain 15 and 16 amino acids, respectively, and two disulfide bonds with a 1-3/2-4 connectivity. This structural feature causes the unique existence of two topological isoforms for each peptide in an approximate 3: 2 ratio, with only one of the isoforms exhibiting GC-C-activating potential. The two uroguanylin isomers can be separated by HPLC and are of sufficient stability to be studied separately at ambient temperatures while the two guanylin isomers are rapidly interconverting even at low temperatures. Both isomers show clearly distinguishable (1)H chemical shifts. To investigate the influence of certain amino acid side chains on this isomerism and interconversion kinetics, derivatives of guanylin and uroguanylin (L-alanine scan and chimeric peptides) were designed and synthesized by Fmoc solid-phase chemistry and compared by HPLC and 2D (1)H NMR spectroscopy. Amino acid residues with the most significant effects on the interconversion kinetics were predominantly identified in the COOH-terminal part of both peptides, whereas amino acids in the central part of the peptides only moderately affected the interconversion. Thus, the conformational conversion among the isomers of both peptides is under the control of a COOH-terminal sterical hindrance, providing a detailed model for this dynamic isomerism. Our results demonstrate that kinetic control of the interconversion process can be achieved by the introduction of side chains with a defined sterical profile at suitable sequence positions. This is of potential impact for the future development of GC-C peptide agonists and antagonists. Copyright (c) 2004 European Peptide Society and John Wiley & Sons, Ltd.
  • Role of disulfide bonds in the folding of prouroguanylin and heat-stable enterotoxin, Yuji Hidaka, Len Ito, Hiroshi Yamaguchi, Peptide Revolution: Genomics, Proteomics & Therapeutics, Peptide Revolution: Genomics, Proteomics & Therapeutics, 2003, 431 - 432, 2004 , Refereed
  • Dual function of the propeptide of prouroguanylin in the folding of the mature peptide - Disulfide-coupled folding and dimerization, Y Hidaka, C Shimono, M Ohno, N Okumura, K Adermann, WG Forssmann, Y Shimonishi, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 275(33), 25155 - 25162, Aug. 2000 , Refereed
    Summary:Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II), We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B,, Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498-8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells respectively, Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.
  • Determination of the binding site on the extracellular domain of guanylyl cyclase C to heat-stable enterotoxin, M Hasegawa, Y Hidaka, Y Matsumoto, T Sanni, Y Shimonishi, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 274(44), 31713 - 31718, Oct. 1999 , Refereed
    Summary:Guanylyl cyclase C, one of the family of membrane-bound guanylyl cyclases, consists of an extracellular domain and an intracellular domain, which are connected by a single transmembrane polypeptide. The extracellular domain binds unique small polypeptides with high specificity, which include the endogenous peptide hormones, guanylin and uroguanylin, as well as an exogenous enterotoxigenic peptide, heat-stable enterotoxin, secreted by pathogenic Escherichia coli. Information on this specific binding is propagated into the intracellular domain, followed by the synthesis of cGMP, a second messenger that regulates a variety of intracellular physiological processes. This study reports the design of a photoaffinity labeled analog of heat-stable enterotoxin (biotinyl-(AC(5))(2)-[Gly(4),Pap(11)]STp(4-17)), which incorporates a Pap residue (p-azidophenylalanine) at position 11 and a biotin moiety at the N terminus, and the use of this analog to determine the ligand-binding region of the extracellular domain of guanylyl cyclase C, The endoproteinase Lys-C digestion of the extracellular domain, which was covalently labeled by this ligand, and mass spectrometric analyses of the digest revealed that the ligand specifically binds to the region (residue 387 to residue 393) of guanylyl cyclase C. This region is localized close to the transmembrane portion of guanylyl cyclase C on the external cellular surface. This result was further confirmed by characterization of site-directed mutants of guanylyl cyclase C in which each amino acid residue was substituted by an Ala residue instead of residues normally located in the region. This experiment provides the first direct demonstration of the ligand-binding site of guanylyl cyclase C and will contribute toward an understanding of the receptor recognition of a ligand and the modeling of the interaction of the receptor and its ligand at the molecular level.
  • The relevance of N-linked glycosylation to the binding of a ligand to guanylate cyclase C, M Hasegawa, Y Hidaka, A Wada, T Hirayama, Y Shimonishi, EUROPEAN JOURNAL OF BIOCHEMISTRY, EUROPEAN JOURNAL OF BIOCHEMISTRY, 263(2), 338 - 345, Jul. 1999 , Refereed
    Summary:The role of carbohydrate moieties at the N-linked glycosylation sites of guanylate cyclase C (GC-C), a receptor protein for guanylin, uroguanylin and heat-stable enterotoxin, in ligand binding and structural stability was examined using site-directed mutagenesis of the putative N-linked glycosylation sites in the extracellular domain (ECD) of porcine GC-C. For this purpose, eight mutant proteins of ECD (N9A, N20A, N56A, N172A, N261A, N284A, N334A and N379A): and six mutant proteins of the complete GC-C (N9A, S11A, N172A, T174A, N379A and T381A) were prepared, in which Ala replaced Asn, Ser and Thr at the N-linked glycosylation consensus sites. All the mutant proteins showed a ligand-binding affinity (K-d) similar to those of the wild-type proteins, although the deletion of a carbohydrate moiety at each of the N-linked glycosylation sites affected the ligand-binding ability of ECD or GC-C to some degree. However, the mutant proteins of ECD (N379A) and GC-C (N379A and T381A) showed considerably decreased binding ability in the context of maximum capacity (B-max to a ligand, despite the fact that the expression levels of these mutant proteins were nearly the same as the wild-type proteins. Moreover, the mutant protein of ECD (N379A) was considerably less stable to a denaturant. These results clearly indicate a crucial role for the carbohydrate moiety at N379, which is located near the transmembrane region, in structural stability, the ability to bind to a ligand and the cyclase catalytic activity of GC-C, and provide a route for the elucidation of the mechanism of the interaction between GC-C and a ligand.
  • Role of the prosequence of guanylin, Axel Schulz, Ute C. Marx, Yuji Hidaka, Yasutsugu Shimonishi, Paul Rösch, Wolf-Georg Forssmann, Knut Adermann, Protein Science, Protein Science, 8(9), 1850 - 1859, 1999 , Refereed
    Summary:Guanylin is a guanylyl cyclase (GC)-activating peptide that is mainly secreted as the corresponding prohormone of 94 amino acid residues. In this study, we show that the originally isolated 15-residue guanylin, representing the COOH-terminal part of the prohormone, is released from the prohormone by cleavage of an Asp-Pro amide bond under conditions applied during the isolation procedures. Thus, the 15-residue guanylin is probably a non-native, chemically induced GC-activating peptide. This guanylin molecule contains two disulfide bonds that are absolutely necessary for receptor activation. We demonstrate that the folding of the reduced 15-residue guanylin results almost completely in the formation of the two inactive disulfide isomers. In contrast, the reduced form of proguanylin containing the entire prosequence folds to a product with the native cysteine connectivity. Because proguanylin lacking the 31 NH2-terminal residues of the prosequence folds only to a minor extent to guanylin with the native disulfide bonds, it is evident that this NH2-terminal region contributes significantly to the correct disulfide- coupled folding. Structural studies using CD and NMR spectroscopy show that native proguanylin contains a considerable amount of α-helical and, to a lesser extent, β-sheet structural elements. In addition, a close proximity of the NH2- and the COOH-terminal regions was found by NOESY. It appears that this interaction is important for the constitution of the correct conformation and provides an explanation of the minor guanylyl cyclase activity of proguanylin by shielding the bioactive COOH-terminal domain from the receptor.
  • Unique synthetic peptides stimulating streptolysin S production in streptococci, T Akao, S Hashimoto, K Kobashi, Y Hidaka, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 125(1), 27 - 30, Jan. 1999 , Refereed
    Summary:A peptide has been isolated from pronase digest of bovine serum albumin as the stimulatory factor of streptolysin S (SLS) production by Streptococcus pyogenes, and its primary structure has been deduced [Akao et at. (1992) Infect. Immun. 60, 4777-4780], To determine the essential structure for the stimulation, a peptide (P-l) having the deduced structure, in which three peptide fragments are linked by two disulfide bonds, and shorter analogs (P-2 to P-4) of peptide P-l were chemically synthesized. Another peptide (P-5), in which Ala is inserted between the two Cys residues in the middle peptide chain of P-1, was also synthesized, These synthetic peptides were identified by mass spectrometry and analysis of amino acid compositions, The synthetic P-1 stimulated SLS production in a dose-dependent manner. Other peptide analogs also showed remarkable stimulation of SLS production. Treatment of P-l with performic acid resulted in loss of its stimulatory activity, indicating that disulfide bridges of the peptides are necessary for their activity on SLS production, These results suggest that the unique primary structure of three peptide chains linked by two disulfide bridges is requisite for the stimulatory effect on SLS production.
  • Expression and characterization of the extracellular domain of guanylyl cyclase C from a baculovirus and Sf21 insect cells, Makoto Hasegawa, Yuki Kawano, Yoshiko Matsumoto, Yuji Hidaka, Jun-Ichi Fujii, Naoyuki Taniguchi, Akihiro Wada, Toshiya Hirayama, Yasutsugu Shimonishi, Protein Expression and Purification, Protein Expression and Purification, 15(3), 271 - 281, 1999 , Refereed
    Summary:Guanylyl cyclase (GC)-C, a single-transmembrane receptor protein for heat-stable enterotoxin, guanylin, and uroguanylin, and its N-terminal extracellular domain were prepared at a high level of expression from a system constructed of Sf21 insect cells and recombinant baculovirus. The recombinant GC-C, containing the complete sequence, retained its binding affinity to heat-stable enterotoxin with a K(D) value (6.2 x 10-10 M) and cyclase catalytic activity at a level similar to those of GC-C expressed in mammalian cell lines, such as COS-7. The N-terminal extracellular domain was prepared in a form which contained the hexahistidine tail at its C-terminus and was purified as a homogenous protein by Con A and Ni-chelating affinity chromatography from the culture medium of the insect cells. The purified N-terminal extracellular domain of GC-C exhibited the high (K(D) = 4 x 10-10 M) and low (K(D) = 7 x 10-8 M) affinity sites in binding to heat-stable enterotoxin. These results clearly indicate that the N-terminal extracellular domain of GC-C possesses the same biochemical characteristics as the complete GC-C protein even in the membrane-free form. Moreover, the extracellular domain is able to form an oligomer in a ligand-dependent manner, suggesting that the N-terminal extracellular domains interact with one another in binding to ligands.
  • Nitrite reductase from Pseudomonas aeruginosa induces inflammatory cytokines in cultured respiratory cells, K Oishi, B Sar, A Wada, Y Hidaka, S Matsumoto, H Amano, F Sonoda, S Kobayashi, T Hirayama, T Nagatake, K Matsushima, INFECTION AND IMMUNITY, INFECTION AND IMMUNITY, 65(7), 2648 - 2655, Jul. 1997 , Refereed
    Summary:Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airway of patients with chronic airway diseases. To investigate the role of. aeruginosa infection in IL-8 production in the airway of these patients, eve examined whether cell lysates of P. aeruginosa could cause IL-S production from human bronchial epithelial cells. Diluted sonicated supernatants of P. aeruginosa (SSPA) vith a mucoid or nonmucoid phenotype stimulated human bronchial epithelial (BET-IA) cells to produce IL-8. In this study, we have purified a 59-kDa heat-stable protein with IL-8-inducing activity from the SSPA by sequential ion-exchange chromatography. The N-terminal sequence of this purified protein completely matched a sequence at the N-terminal part of the mature protein of nitrite reductase from P. aeruginosa. In addition, immunoblotting with a polyclonal immunoglobulin G (IgG) against recombinant Pseudomonas nitrile reductase demonstrated a specific binding to the purified protein. Furthermore, the immunoprecipitates of the SSPA with a polyclonal IgG against recombinant nitrite reductase induced a twofold-higher IL-8 production in the BHT-IA cell culture than did the immunoprecipitates of the SSPA with a control IgG. These lines of evidence confirmed that Pseudomonas nitrite reductase was responsible for IL-8 production in the BET-1A cells. Tie purified nitrite reductase induced maximal expression of IL-8 mRNA in the BET-1A cells at 1 to 3 h after stimulation, and the IL-IS mRNA expression declined by 8 h after stimulation. New protein translation was not required far nitrite reductase-mediated IL-S mRNA expression in the BET-1A cells. Nitrite reductase stimulated the BET-1A cells, as well as human alveolar macrophages, pulmonary fibroblasts, and neutrophils, to produce IL-8. In contrast, nitrite reductase induced significant levels of tumor necrosis factor alpha and IL-1 beta protein only in human alveolar macrophages. These data support the notion that nitrite reductase from P. aeruginosa induces the production of inflammatory cytokines by respiratory cells and may contribute to the pathogenesis of chronic airway diseases and persistent P. aeruginosa infection.
  • Identification of ligand recognition sites in heat-stable enterotoxin receptor, membrane-associated guanylyl cyclase C by site-directed mutational analysis, A Wada, T Hirayama, H Kitaura, JI Fujisawa, M Hasegawa, Y Hidaka, Y Shimonishi, INFECTION AND IMMUNITY, INFECTION AND IMMUNITY, 64(12), 5144 - 5150, Dec. 1996 , Refereed
    Summary:Guanylyl cyclase C (STaR), a receptor protein for heat-stable enterotoxin (STa) elaborated by Escherichia coli, is associated with and spans the plasma membrane of mammalian intestinal cells. The extracellular domain functions in the binding of STa and the association of each domain to an oligomeric form. Two amino acid residues, Arg-136 and Asp-347, were identified as the residues binding to STa in the extracellular domain of pig STaR by site-directed mutagenesis and analysis of expression on 293T cells. Replacement of these residues bg other amino acid residues resulted in the toss of binding of pig STaR to STa, and as a result, STa-induced guanylyl cyclase activity was eliminated. Furthermore, mutation in a region (from Asp-347 to Val-401) which is close to the transmembrane domain caused a significant reduction in both STa-binding activity and guanylyl cyclase catalytic activity. These results suggest that the region adjacent to the transmembrane domain plays an important role in facilitating a favorable conformation of STaR for STa binding.
  • The significance of Ser1029 of the heat-stable enterotoxin receptor (STaR): Relation of STa-mediated guanylyl cyclase activation and signaling by phorbol myristate acetate, Akihiro Wada, Makoto Hasegawa, Kazuya Matsumoto, Takuro Niidome, Yuki Kawano, Yuji Hidaka, Philip Ian Padilla, Hisao Kurazono, Yasutsugu Shimonishi, Toshiya Hirayama, FEBS Letters, FEBS Letters, 384(1), 75 - 77, Apr. 08 1996 , Refereed
    Summary:To characterize Ser1029 in STaR at a consensus sequence of phosphorylation site by PKC, two mutants of mS1029A with replacement of Ser1029 to Ala1029 and CΔ1029 lacking 22 amino acids including Ser1029 were prepared. Preincubation of the wild type-STaR (wt-STaR) transfectant with 1 μM PMA caused additional STa-mediated guanylyl cyclase (GC) activation compared to control, whereas the mS1029A- and CΔ1029-transfected cells did not show a similar enhanced GC activation by PMA. After metabolic labeling with [32P]phosphate, transfected cells with wt-STaR and mutants were incubated with 1 μM PMA. Subsequent 32P-radiolabeled proteins were immunoprecipitated using anti-STaR antibody, and analyzed by autoradiography after separation on SDS-PAGE. The immunoprecipitated wt-STaR but not mS1029A and CΔ1029 had a significant radioactivity. These results suggest that the effect of PMA on wt-STaR transfectants may be caused by phosphorylation of Ser1029. The CΔ1012 mutant, with further truncation (Gln1012-Phe1050) of the carboxy terminus, did not show STa-mediated GC activation. Based on these data, these 17 amino acids (Gln1012-Ala1028), essential for signaling of GC activation by STa, have an abundance of basic amino acids which might be functionally influenced by phosphorylation of Ser1029.
    Summary:Two IgE epitope sequences comprising Ser(56)-Pro-Val-Thr-Lys-Arg-Ala-Ser-Leu-Lys-Ile-Asp-Ser-Lys-Lys(70) and Asp(104)-Val-Glu-Leu-Ser-Leu-Arg-Ser-Ser-Asp-Ile-Ala(115) were identified by deletion analysis of the cDNA encoding a new 39-kD protein of mite allergen. A synthetic dodecapeptide corresponding to the latter epitope sequence functioned as a monovalent and mite-specific hapten. Replacement of each of the 12 amino acid residues with Gly, using site-directed mutagenesis, indicated that Arg(110) may play a central role in IgE binding. However, the 8 allergic sera tested exhibited a wide variation in their amino acid residues required for reactivity to IgE in allergic sera.
    Summary:A cDNA encoding the receptor protein for a heat-stable enterotoxin (STa) produced by enterotoxigenic Eschrerichia coli was cloned from intestinal epithelial cells of a 10-week-old pig. The cDNA had an open reading frame of 3,219 base pairs and coded for a protein with 1,073 amino acid residues. The mature protein consisted of 1,050 amino acid residues with a molecular mass of ca. 121 kDa and was 87% and 82% identical with the human and rat protein, respectively. The CHO cell line overexpressing the pig recombinant STa receptor specifically bound to a photoaffinity-labeled analog of STa and showed marked elevation of the cellular content of cGMP in response to STa.
  • Expression of a truncated guanylate cyclase (GC-C), a receptor for heat-stable enterotoxin of enterotoxigenic escherichia coli, and its dimer formation in COS-7 cells, Toshiya Hirayama, Akihiro Wada, Yuji Hidaka, Jun-Ichi Fujisawa, Yoshifumi Takeda, Yasutsugu Shimonishi, Microbial Pathogenesis, Microbial Pathogenesis, 15(4), 283 - 291, 1993 , Refereed
    Summary:A fragment of guanylate cyclase C (GC-C) of about 1.7 k bp corresponding to amino acids 1-553 spanning the extracellular and transmembrane domains and a portion of the intracellular region was amplified using template cDNA prepared from rat intestinal cells by the polymerase chain reaction method. The cloned 1.7 k bp fragment was inserted into the mammalian expression vector pCGUT and the truncated GC-C expressed on the surface of COS-7 cells was demonstrated to bind heat-stable enterotoxin by photo affinity labeling with 125 I-N-5-azidonitrobenzoyl-STh[5-19]. Analysis by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis showed that the truncated GC-C formed dimers on the surface of COS-7 cells. The intracellular region of GC-C was found not to be necessary for dimer formation by the GC-C. Comparison of the molecular weights of the truncated GC-C expressed in COS-7 cells and Escherichia coli suggested that the truncated GC-C was glycosylated in the mammalian expression system. © 1993 Academic Press. All rights reserved.
    Summary:Heat-stable enterotoxin (ST(p)) produced by a porcine strain of enterotoxigenic Escherichia coli is a peptide of 18 amino acid residues. The full toxicity is generated by a core peptide of 13 amino acids from residue 5 to 17 (ST(p)(5-17)). Detailed conformational analysis of ST(p)(5-17) was performed by NMR spectroscopy and distance geometry calculations. The tetriary structure of ST(p)(5-17) was found to have a right-handed spiral fold throughout the whole molecule which was stabilized by the network of disulfide linkages and hydrogen bonds. This folding pattern was demonstrated in both organic and aqueous solutions.

Conference Activities & Talks

  • Understanding the mechanism by which PDI family catalyze oxidative folding of precursor protein,   2019 09 20
  • Acceleration of oxidative protein folding by newly-developed redox molecules,   2019 09 20
  • Cloning and Functional Analysis of Digestive Enzyme Derived from N ephila Clavata, Tsubasa Tagawa, Teruki Hagiwara, Shigeru Shimamoto, Yuji Hidaka,   2018 12 07
  • Structural Control and Functional Analysis of the Precursor Protein of Atrial Natriuretic Peptide, HayatoUeda, Shigeru Shimamoto, Yuji Hidaka,   2018 12 06
  • Structural Analyses of an N-terminal Extracellular Domain of the Amyloid Precursor Protein, Mizuho Imamura, Shingo Kanemura, Masaki Okumura, Hiroshi Yamaguchi, Shigeru Shimamoto, Yuji Hidaka, 10th International Peptide Symposium,   2018 12 05
  • MOLECULAR RECOGNITION MECHANISM OF HEMATOPOIETIC PROSTAGLANDIND SYNTHASE WITH COFACTOR AND ITS SUBSTRATE, Shigeru Shimamoto, Keisuke Asada, Yuji Hidaka, 62th Annual Meeting of Biophysycal Society,   2018 02 21
  • ACTIVATION MECHANISM OF COCOONASE, Nagisa Tajima, Mitsuhiro Miyazawa, Shigeru Shimamoto, Yuji Hidaka, 62th Annual Meeting of Biophysycal Society,   2018 02 21
  • STRUCTURAL ANALYSES OF A LINKER REGION OF THE AMYLOID PRECURSOR PROTEIN, Mizuho Imamura, Shingo Kanemura, Masaki Okumura, Shigeru Shimamoto, Yuji Hidaka, 62th Annual Meeting of Biophysycal Society,   2018 02 18
  • FOLDING ANALYSES OF A DE NOVO DESIGNED PROUROGUANYLIN., Yuji Hidaka, Saya Nishihara, Kenta Mori, Shigeru Shimamoto, 62th Annual Meeting of Biophysical Society,   2018 02 18
  • DISULFIDE-COUPLED FOLDING OF PROUROGUANYLIN ON MOLECULAR EVOLUTION, Kenta Mori, Saya Nishihara, Shigeru Shimamoto, Yuji Hidaka, 62th Annual Meeting of Biophysical Society,   2018 02 18
  • MOLECULAR EVOLUTION OF L-PGDS: SUBSTRATE RECOGNITION MECHANISM, Kimi Torii, Yuji Hidaka, Shigeru Shimamoto, 62th Annual Meeting of Biophysical Society,   2018 02 18
  • Molecular evolution of L-PGDS: substrate recognittion mechanism of medaka L-PGDS, Kimi Torii, Takahiro Maruno, Yuji Kobayashi, Yuji Hidaka, Shigeru Shimamoto,   2017 11 20
  • Regulation of disulfide-coupled folding of a de novo designed protein, Saya Nishihara, Kosuke Toyama, Kenta Mori, Shigeru Shimamoto, Yuji Hidaka,   2017 11 20
  • Structural analyses of a linker region of the amyloid precursor protein, Mizuho Imamura, Shingo Kanemura, Masaki Okumura, Shigeru Shimamoto, Yuji Hidaka,   2017 11 20
  • Lipocalin-Type Prostaglandin D Synthase Possesses Two Binding Sites for its product., Shigeru Shimamoto, Yusuke Nakagawa, Yuta Nakahata, Takahiro Maruno, Yuji Kobayashi, Kosuke Aritake, Yoshihiro Urade, Yuji Hidaka, 61st Annual Meeting of Biophysical Scoiety,   2017 02 14
  • Molecular recognition mechanism of Hematopoietic Prostaglandin D Synthase with cofactor and its substrate, Keisuke Asada, Shigeru Shimamoto, Tomohiro Oonoki, Takahiro Maruno, Yuji Kobayashi, Kosuke Aritake, Yoshihiro Urade, Yuji Hidaka, 61st Annual Meeting of Biophysical Scoiety,   2017 02 14
  • Regulation of protein folding using organic solvents and ion liquid, Yuji Hidaka, Wataru Ito, Ryosuke, Nishimura, Shigeru Shimamoto, 61st Annual Meeting of Biophysical Scoiety,   2017 02 12
  • Structural analysis of the precursor protein of atrial natriuretic peptide, Sumika Futori, Satomi Higashigawa, Shigeru Shimamoto, Yuji Hidaka, 61st Annual Meeting of Biophysical Scoiety,   2017 02 12
  • Regulation of folding of de novo designed peptides by alpha-helix formation, Saya Nishihara, Kosuke Toyama, Shigeru Shimamoto, Yuji Hidaka, 61st Annual Meeting of Biophysical Scoiety,   2017 02 12
  • Preparation of a orexin precursor protein by chemical digestion, Natsumi Mitsuoka, Shigeru Shimamoto, Yuji Hidaka, 61st Annual Meeting of Biophysical Society,   2017 02 12
  • DISULFIDE-COUPLED FOLDING OF PRO-UROGUANYLIN ON MOLECULAR EVOLUTION, Kenta Mori, Kosuke Toyama, Shigeru Shimamoto, Yuji Hidaka, 10th International Peptide Symposium,   2016 10 26
  • ACTIVATION MECHANISM OF COCOONASE, Nagisa Tajima, Shigeru Shimamoto, Mitsuhiro Miyazawa, Yuji Hidaka, 10th International Peptide Symposium,   2016 10 26
  • REGULATION OF DISULFIDE-COUPLED FOLDING OF DE NOVO DESIGNED PEPTIDES BY alpha-HELIX FORMATION, Saya Nishihara, Kosuke Toyama, Shigeru Shimamoto, Yuji Hidaka, 10th International Peptide Symposium,   2016 10 26
  • PREPARATION OF A OREXIN PRECORSOR PROTEIN USING E. COLI EXPRESSION SYSTEM BY ACID TREATMENT, Natsumi Mitsuoka, Shigeru Shimamoto, Yuji Hidaka, 10th International Peptide Symposium,   2016 10 26


  • Structure and activity of heat-stable enterotoxin, HIDAKA Yuji, 68, 8, 1448, 1452,   1996 08 25 ,