研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    田所 高志(タドコロ タカシ), タドコロ タカシ

所属(マスター)

  • 薬学研究院 創薬科学研究教育センター

所属(マスター)

  • 薬学研究院 創薬科学研究教育センター

独自項目

syllabus

  • 2020, 生物物理化学, Biophysics, 学士課程, 薬学部, 反応速度論,生体分子機器分析,測定原理
  • 2020, 物理化学実習, Laboratory Exercise of Physical Chemistry, 学士課程, 薬学部, 反応速度論,熱力学,表計算
  • 2020, 物理化学Ⅰ, Physical Chemistry I, 学士課程, 薬学部, 化学結合、量子化学、分光学、電子スペクトル、振動スペクトル、回転スペクトル、核磁気共鳴(NMR)、X線結晶構造解析等、機器分析

researchmap

プロフィール情報

学位

  • 博士(工学)(大阪大学)

プロフィール情報

  • 田所, Tadokoro
  • 高志, Takashi
  • ID各種

    201501086039481178

業績リスト

研究キーワード

  • 蛋白質工学   蛋白質間相互作用   安定性   

研究分野

  • ライフサイエンス / 機能生物化学
  • ライフサイエンス / 分子生物学
  • ライフサイエンス / 生物物理学

経歴

  • 2015年05月 - 現在 北海道大学 大学院薬学研究院 特任助教
  • 2013年07月 - 2015年04月 北海道大学 大学院薬学研究院 博士研究員
  • 2008年06月 - 2013年06月 National Institutes of Health(米国) National Institute on Aging Visiting Fellow
  • 2008年04月 - 2008年06月 大阪大学大学院 工学研究科 生命先端工学専攻 物質生命工学コース 博士研究員
  • 2007年10月 - 2008年02月 大阪大学大学院 工学研究科 生命先端工学専攻 物質生命工学コース 博士研究員
  • 2007年04月 - 2007年09月 大阪大学大学院 工学研究科 生命先端工学専攻 物質生命工学コース 技術員

委員歴

  • 2021年04月 - 現在   日本結晶学会   2021年会実行委員
  • 2019年04月 - 2020年03月   生物物理学会北海道支部   会計
  • 2017年 - 2017年   日本ケミカルバイオロジー学会   第12回年会 プログラム委員

論文

  • Taishi Onodera, Shunsuke Kita, Yu Adachi, Saya Moriyama, Akihiko Sato, Takao Nomura, Shuhei Sakakibara, Takeshi Inoue, Takashi Tadokoro, Yuki Anraku, Kohei Yumoto, Cong Tian, Hideo Fukuhara, Michihito Sasaki, Yasuko Orba, Nozomi Shiwa, Naoko Iwata, Noriyo Nagata, Tateki Suzuki, Jiei Sasaki, Tsuyoshi Sekizuka, Keisuke Tonouchi, Lin Sun, Shuetsu Fukushi, Hiroyuki Satofuka, Yasuhiro Kazuki, Mitsuo Oshimura, Tomohiro Kurosaki, Makoto Kuroda, Yoshiharu Matsuura, Tadaki Suzuki, Hirofumi Sawa, Takao Hashiguchi, Katsumi Maenaka, Yoshimasa Takahashi
    Immunity 2021年08月24日 
    Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.
  • Hiroyuki Miyachi, Kayoko Kanamitsu, Mayumi Ishii, Eri Watanabe, Akira Katsuyama, Satoko Otsuguro, Fumika Yakushiji, Mizuki Watanabe, Kouhei Matsui, Yukina Sato, Satoshi Shuto, Takashi Tadokoro, Shunsuke Kita, Takanori Matsumaru, Akira Matsuda, Tomoyasu Hirose, Masato Iwatsuki, Yasuteru Shigeta, Tetsuo Nagano, Hirotatsu Kojima, Satoshi Ichikawa, Toshiaki Sunazuka, Katsumi Maenaka
    Bioorganic & medicinal chemistry letters 37 127847 - 127847 2021年04月01日 
    To develop methodology to predict the potential druggability of middle molecules, we examined the structure, solubility, and permeability relationships of a diverse library (HKDL ver.1) consisting of 510 molecules (359 natural product derivatives, 76 non-natural products, 46 natural products, and 29 non-natural product derivatives). The library included peptides, depsipeptides, macrolides, and lignans, and 476 of the 510 compounds had a molecular weight in the range of 500-2000 Da. The solubility and passive diffusion velocity of the middle molecules were assessed using the parallel artificial membrane permeability assay (PAMPA). Quantitative values of solubility of 471 molecules and passive diffusion velocity of 287 molecules were obtained, and their correlations with the structural features of the molecules were examined. Based on the results, we propose a method to predict the passive diffusion characteristics of middle molecules from their three-dimensional structural features.
  • Hiroki Kusaka, Shunsuke Kita, Takashi Tadokoro, Kouki Yoshida, Yoshiyuki Kasai, Harumi Niiyama, Yukari Fujimoto, Shinya Hanashima, Michio Murata, Shigeru Sugiyama, Toyoyuki Ose, Kimiko Kuroki, Katsumi Maenaka
    Protein expression and purification 172 105631 - 105631 2020年08月 [査読有り][通常論文]
     
    CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.
  • Yuma Nagano, Aoi Sugiyama, Madoka Kimoto, Takuya Wakahara, Yasuyo Noguchi, Xinxin Jiang, Shinya Saijo, Nobutaka Shimizu, Nana Yabuno, Min Yao, Paul R. Gooley, Gregory W. Moseley, Takashi Tadokoro, Katsumi Maenaka, Toyoyuki Ose
    Journal of Virology 2020年06月24日 [査読有り][通常論文]
     
    Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies, and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interaction between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is one order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size-exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N and C-terminal domains, from pre-formed complexes of STAT2-core/IRF-associated-domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt pre-assembled interferon-stimulated gene factor 3. IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase-signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon–mediated antiviral gene expression.
  • Takashi Tadokoro, Cassandra M Modahl, Katsumi Maenaka, Narumi Aoki-Shioi
    Toxins 12 3 2020年03月12日 [査読有り][通常論文]
     
    The CAP protein superfamily (Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) proteins) is widely distributed, but for toxinologists, snake venom CRISPs are the most familiar members. Although CRISPs are found in the majority of venoms, very few of these proteins have been functionally characterized, but those that have been exhibit diverse activities. Snake venom CRISPs (svCRISPs) inhibit ion channels and the growth of new blood vessels (angiogenesis). They also increase vascular permeability and promote inflammatory responses (leukocyte and neutrophil infiltration). Interestingly, CRISPs in lamprey buccal gland secretions also manifest some of these activities, suggesting an evolutionarily conserved function. As we strive to better understand the functions that CRISPs serve in venoms, it is worth considering the broad range of CRISP physiological activities throughout the animal kingdom. In this review, we summarize those activities, known crystal structures and sequence alignments, and we discuss predicted functional sites. CRISPs may not be lethal or major components of venoms, but given their almost ubiquitous occurrence in venoms and the accelerated evolution of svCRISP genes, these venom proteins are likely to have functions worth investigating.
  • Kengo Hirao, Sophie Andrews, Kimiko Kuroki, Hiroki Kusaka, Takashi Tadokoro, Shunsuke Kita, Toyoyuki Ose, Sarah L Rowland-Jones, Katsumi Maenaka
    iScience 23 1 100758 - 100758 2020年01月24日 [査読有り][通常論文]
     
    The human immunodeficiency virus (HIV) accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2-infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The di-leucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2-mediated endocytosis for CD3. The highly conserved C-terminal region forms a α-helix, absent from HIV-1. We further determined the structure of simian immunodeficiency virus (SIV) Nef harboring this region, demonstrating similar C-terminal α-helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide insights into the distinct pathogenesis of HIV-2 infection.
  • Hiroaki Oyama, Hiroki Koga, Takashi Tadokoro, Katsumi Maenaka, Akira Shiota, Masami Yokoyama, Masanori Noda, Tetsuo Torisu, Susumu Uchiyama
    Journal of pharmaceutical sciences 109 1 308 - 315 2020年01月 [査読有り][通常論文]
     
    Aggregation of therapeutic monoclonal antibodies has a potential risk of immunogenicity, requiring minimization of aggregate formation. We have developed a fitting formula for antibody aggregation at 40°C based on physicochemical parameters, including colloidal and conformational stabilities. An IgG1 monoclonal antibody, MAb-T, was formulated in 24 combinations of different buffer types and pH with or without sodium chloride. The fitting formula for monomer loss was successfully established by nonlinear regression analysis of the results from accelerated stability testing. Calculated monomer fraction values by the fitting formula were strongly correlated with experimental values (R2 = 0.92). The model includes secondary virial coefficient, B22, as the representative parameter of colloidal stability, and aggregation temperature, Tagg, representing conformational stability. Then, we examined charge state, conformational flexibility, and thermal unfolding profile of MAb-T to clarify the molecular basis for the different aggregation propensities in sodium acetate buffer and in sodium citrate buffer at the same pH and buffer concentration. We concluded that the accumulation of citrate anions on the surface of MAb-T is the primary source of the less colloidal and conformational stabilities, resulting in the higher aggregation propensity in sodium citrate buffer.
  • Takashi Tadokoro, Mst Lubna Jahan, Yuri Ito, Maino Tahara, Surui Chen, Atsutoshi Imai, Natsumi Sugimura, Koki Yoshida, Mizuki Saito, Toyoyuki Ose, Takao Hashiguchi, Makoto Takeda, Hideo Fukuhara, Katsumi Maenaka
    The FEBS journal 287 1 145 - 159 2020年01月 [査読有り][通常論文]
     
    The measles virus (MV) is a major cause of childhood morbidity and mortality worldwide. We previously established a mouse monoclonal antibody, 2F4, which shows high neutralizing titers against eight different genotypes of MV. However, the molecular basis for the neutralizing activity of the 2F4 antibody remains incompletely understood. Here, we have evaluated the binding characteristics of a Fab fragment of the 2F4 antibody. Using the MV infectious assay, we demonstrated that 2F4 Fab inhibits viral entry via either of two cellular receptors, SLAM and Nectin4. Surface plasmon resonance (SPR) analysis of recombinant proteins indicated that 2F4 Fab interacts with MV hemagglutinin (MV-H) with a KD value at the nm level. Furthermore, we designed a single-chain Fv fragment of 2F4 antibody as another potential biopharmaceutical to target measles. The stable 2F4 scFv was successfully prepared by the refolding method and shown to interact with MV-H at the μm level. Like 2F4 Fab, scFv inhibited receptor binding and viral entry. This indicates that 2F4 mAb uses the receptor-binding site and/or a neighboring region as an epitope with high affinity. These results provide insight into the neutralizing activity and potential therapeutic use of antibody fragments for MV infection.
  • Masanori Noda, Kentaro Ishii, Mika Yamauchi, Hiroaki Oyama, Takashi Tadokoro, Katsumi Maenaka, Tetsuo Torisu, Susumu Uchiyama
    Journal of pharmaceutical sciences 108 7 2323 - 2333 2019年07月 [査読有り][通常論文]
     
    Antibody aggregates are a potential risk for immunogenicity; therefore, rational approaches to improve associated aggregation properties need to be developed. Here, we report the amino acid region responsible for aggregation initiation. Two types of therapeutic IgG1 antibody monomer samples were prepared: IgG1 mAb40-3M stored at 40°C for 3 months, which existed in monodisperse state, and the monomer mAb65-5m, which was dissociated from small soluble aggregates by heating at 65°C for 5 min. Hydrogen deuterium exchange mass spectrometry of mAb40-3M identified 2 sites in the Fc region (site 1, F239-M256; site 2, S428-G450) with increased exchange rates. Site 1 includes a region reported as being susceptible to structural change induced by stress. Exposure of site 1 was undetected after 2 months of storage at 40°C but was subsequently detectable after 3 months. As site 2 is spatially close to site 1, the structural change of site 1 could propagate site 2. Besides these 2 regions, hydrogen deuterium exchange mass spectrometry of mAb65-5m identified an exposure of I257-W281 in Fc (site 3), within which a peptide sequence with high aggregation tendency was discovered. We thus concluded that exposure of site 3 is a trigger for the association of a partially denatured antibody.
  • Narumi Shioi, Takashi Tadokoro, Seijiro Shioi, Yuki Okabe, Haruki Matsubara, Shunsuke Kita, Toyoyuki Ose, Kimiko Kuroki, Shigeyuki Terada, Katsumi Maenaka
    The Journal of biological chemistry 294 4 1250 - 1256 2019年01月25日 [査読有り][通常論文]
     
    Venomous snakes have endogenous proteins that neutralize the toxicity of their venom components. We previously identified five small serum proteins (SSP-1-SSP-5) from a highly venomous snake belonging to the family Viperidae as inhibitors of various toxins from snake venom. The endogenous inhibitors belong to the prostate secretory protein of 94 amino acids (PSP94) family. SSP-2 interacts with triflin, which is a member of the cysteine-rich secretory protein (CRISP) family that blocks smooth muscle contraction. However, the structural basis for the interaction and the biological roles of these inhibitors are largely unknown. Here, we determined the crystal structure of the SSP-2-triflin complex at 2.3 Å resolution. A concave region centrally located in the N-terminal domain of triflin is fully occupied by the terminal β-strands of SSP-2. SSP-2 does not bind tightly to the C-terminal cysteine-rich domain of triflin; this domain is thought to be responsible for its channel-blocker function. Instead, the cysteine-rich domain is tilted 7.7° upon binding to SSP-2, and the inhibitor appears to sterically hinder triflin binding to calcium channels. These results help explain how an endogenous inhibitor prevents the venomous protein from maintaining homeostasis in the host. Furthermore, this interaction also sheds light on the binding interface between the human homologues PSP94 and CRISP-3, which are up-regulated in prostate and ovarian cancers.
  • Takanori Matsumaru, Risa Ikeno, Yusuke Shuchi, Toshiki Iwamatsu, Takashi Tadokoro, Sho Yamasaki, Yukari Fujimoto, Atsushi Furukawa, Katsumi Maenaka
    Chemical communications (Cambridge, England) 55 5 711 - 714 2019年01月10日 [査読有り][通常論文]
     
    Mincle, expressed in activated phagocytes, recognizes the lipid ligand to activate the innate immune system. We have synthesized glycerol derivatives possessing simple alkyl chains or aromatic rings and elucidated their structure-activity relationships using a Mincle-mediated signaling assay. The activity depends on the length of the simple acyl chains of the glycerol derivatives.
  • Inoue M, Ando D, Kamada H, Taki S, Niiyama M, Mukai Y, Tadokoro T, Maenaka K, Nakayama T, Kado Y, Inoue T, Tsutsumi Y, Tsunoda SI
    The Journal of biological chemistry 292 16 6438 - 6451 2017年04月 [査読有り][通常論文]
     
    Tumor necrosis factor-alpha (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo. Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF.
  • Atsutoshi Imai, Takashi Tadokoro, Shunsuke Kita, Masataka Horiuchi, Hideo Fukuhara, Katsumi Maenaka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 478 2 580 - 585 2016年09月 [査読有り][通常論文]
     
    The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy. (C) 2016 Elsevier Inc. All rights reserved.
  • Atsushi Furukawa, Shunsuke Kita, Takashi Tadokoro, Hideo Fukuhara, Katsumi Maenaka
    C-Type Lectin Receptors in Immunity 179 - 190 2016年01月01日 [査読有り][通常論文]
     
    Numerous structural analyses (X-ray crystallography and NMR) of C-type lectin receptors (CLRs) have been performed, because CLRs are not only attractive as important molecules in immunity and infectious diseases but also as drug targets. In CLRs, high amino acid sequence similarity exists in the extracellular carbohydrate recognition domains (CRDs), which are responsible for ligand binding. However, recent functional analyses of CLRs implied that these molecules recognize a wide variety of ligands in addition to saccharides, including glycopeptides, glycolipids, and proteins. In this chapter, we focus on structural studies of CLRs. We first summarize the structural features conserved among the CRDs and then describe how each C-type lectin receptor elegantly achieves its distinct ligand specificity, by illustrating the structural aspects of several representative CLRs.
  • Prabhat Khadka, Joseph K. Hsu, Sebastian Veith, Takashi Tadokoro, Raghavendra A. Shamanna, Aswin Mangerich, Deborah L. Croteau, Vilhelm A. Bohr
    MOLECULAR AND CELLULAR BIOLOGY 35 23 3974 - 3989 2015年12月 [査読有り][通常論文]
     
    Poly(ADP-ribose) (PAR) polymerase I (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response.
  • Kimberly D. Jacob, Nicole Noren Hooten, Takashi Tadokoro, Althaf Lohani, Janice Barnes, Michele K. Evans
    FREE RADICAL BIOLOGY AND MEDICINE 63 115 - 125 2013年10月 [査読有り][通常論文]
     
    Brain tissues from Alzheimer's disease (AD) patients show increased levels of oxidative DNA damage and 7,8-dihydro-8-oxoguanine (8-oxoG) accumulation. In humans, the base excision repair protein 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme that recognizes and excises the mutagenic DNA base lesion 8-oxoG. Recently, two polymorphisms of OGG1, A53T and A288V, have been identified in brain tissues of AD patients, but little is known about how these polymorphisms may contribute to AD. We characterized the A53T and A288V polymorphic variants and detected a significant reduction in the catalytic activity for both proteins in vitro and in cells. Additionally, the A53T polymorphism has decreased substrate binding, whereas the A288V polymorphism has reduced AP lyase activity. Both variants have decreased binding to known OGG1 binding partners PARP-1 and XRCC1. We found that OGG1(-/-) cells expressing A53T and A288V OGG1 were significantly more sensitive to DNA damage and had significantly decreased survival. Our results provide both biochemical and cellular evidence that A53T and A288V polymorphic proteins have deficiencies in catalytic and protein-binding activities that could be related to the increase in oxidative damage to DNA found in AD brains. Published by Elsevier Inc.
  • Mahesh Ramamoorthy, Alfred May, Takashi Tadokoro, Venkateswarlu Popuri, Michael M. Seidman, Deborah L. Croteau, Vilhelm A. Bohr
    CARCINOGENESIS 34 10 2218 - 2230 2013年10月 [査読有り][通常論文]
     
    Interstrand cross-links (ICLs) are very severe lesions as they are absolute blocks of replication and transcription. This property of interstrand cross-linking agents has been exploited clinically for the treatment of cancers and other diseases. ICLs are repaired in human cells by specialized DNA repair pathways including components of the nucleotide excision repair pathway, double-strand break repair pathway and the Fanconi anemia pathway. In this report, we identify the role of RECQL5, a member of the RecQ family of helicases, in the repair of ICLs. Using laser-directed confocal microscopy, we demonstrate that RECQL5 is recruited to ICLs formed by trioxalen (a psoralen-derived compound) and ultraviolet irradiation A. Using single-cell gel electrophoresis and proliferation assays, we identify the role of RECQL5 in the repair of ICL lesions. The domain of RECQL5 that recruits to the site of ICL was mapped to the KIX region between amino acids 500 and 650. Inhibition of transcription and of topoisomerases did not affect recruitment, which was inhibited by DNA-intercalating agents, suggesting that the DNA structure itself may be responsible for the recruitment of RECQL5 to the sites of ICLs.
  • Peter Sykora, Jenq-Lin Yang, Leslie K. Ferrarelli, Jingyan Tian, Takashi Tadokoro, Avanti Kulkarni, Lior Weissman, Guido Keijzers, David M. Wilson, Mark P. Mattson, Vilhelm A. Bohr
    NEUROBIOLOGY OF AGING 34 7 1717 - 1727 2013年07月 [査読有り][通常論文]
     
    Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is downregulated in postmitotic muscle cells and neurons. Here, we characterize DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because they have robust BER capacity, which is heavily attenuated in postmitotic neurons. The reduction in BER activity in differentiated cells correlates with diminished protein levels of key long patch BER components, flap endonuclease-1, proliferating cell nuclear antigen, and ligase I. Thus, because of their higher BER capacity, proliferative neural progenitor cells are more efficient at repairing DNA damage compared with their neuronally differentiated progeny. Published by Elsevier Inc.
  • Leslie K. Ferrarelli, Venkateswarlu Popuri, Avik K. Ghosh, Takashi Tadokoro, Chandrika Canugovi, Joseph K. Hsu, Deborah L. Croteau, Vilhelm A. Bohr
    DNA REPAIR 12 7 518 - 528 2013年07月 [査読有り][通常論文]
     
    Telomeres are critical for cell survival and functional integrity. Oxidative DNA damage induces telomeric instability and cellular senescence that are associated with normal aging and segmental premature aging disorders such as Werner Syndrome and Rothmund-Thomson Syndrome, caused by mutations in WRN and RECQL4 helicases respectively. Characterizing the metabolic roles of RECQL4 and WRN in telomere maintenance is crucial in understanding the pathogenesis of their associated disorders. We have previously shown that WRN and RECQL4 display a preference in vitro to unwind telomeric DNA substrates containing the oxidative lesion 8-oxoguanine. Here, we show that RECQL4 helicase has a preferential activity in vitro on telomeric substrates containing thymine glycol, a critical lesion that blocks DNA metabolism, and can be modestly stimulated further on a D-loop structure by TRF2, a telomeric shelterin protein. Unlike that reported for telomeric D-loops containing 8-oxoguanine, RECQL4 does not cooperate with WRN to unwind telomeric D-loops with thymine glycol, suggesting RECQL4 helicase is selective for the type of oxidative lesion. RECQL4's function at the telomere is not yet understood, and our findings suggest a novel role for RECQL4 in the repair of thymine glycol lesions to promote efficient telomeric maintenance. Published by Elsevier B.V.
  • Tadokoro T, Rybanska-Spaeder I, Kulikowicz T, Dawut L, Oshima J, Croteau DL, Bohr VA
    DNA repair 12 6 414 - 421 2013年06月 [査読有り][通常論文]
  • Venkateswarlu Popuri, Takashi Tadokoro, Deborah L. Croteau, Vilhelm A. Bohr
    CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 48 3 289 - 299 2013年05月 [査読有り][通常論文]
     
    DNA helicases are ubiquitous enzymes that catalyze unwinding of duplex DNA and function in all metabolic processes in which access to single-stranded DNA is required, including DNA replication, repair, recombination and RNA transcription. RecQ helicases are a conserved family of DNA helicases that display highly specialized and vital roles in the maintenance of genome stability. Mutations in three of the five human RecQ helicases, BLM, WRN and RECQL4 are associated with the genetic disorders Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome that are characterized by chromosomal instability, premature aging and predisposition to cancer. The biological role of human RECQL5 is only partially understood and RECQL5 has not yet been associated with any human disease. Illegitimate recombination and replication stress are hallmarks of human cancers and common instigators for genomic instability and cell death. Recql5 knockout mice are cancer prone and show increased chromosomal instability. Recql5-deficient mouse embryonic fibroblasts are sensitive to camptothecin and display elevated levels of sister chromatid exchanges. Unlike other human RecQ helicases, RECQL5 is recruited to single-stranded DNA breaks and is also proposed to play an essential role in RNA transcription. Here, we review the established roles of RECQL5 at the cross roads of DNA replication, recombination and transcription, and propose that human RECQL5 provides important backup functions in the absence of other DNA helicases.
  • Takashi Tadokoro, Hirotaka Kazama, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    BIOCHEMISTRY 52 16 2839 - 2847 2013年04月 [査読有り][通常論文]
     
    A goal of protein engineering technology is developing methods to increase protein stability. However, rational design of stable proteins is difficult because the stabilization mechanism of proteins is not fully understood. In this study, we examined the structural dependence of protein stabilization by introducing single amino acid substitution into ribonuclease H1 from the psychotropic bacterium Shewanella oneidensis MR-1 (So-RNase H1), which was our model protein. We performed saturation mutagenesis at various sites. Mutations that stabilized So-RNase H1 were screened using an RNase H-dependent temperature-sensitive Escherchia coli strain. Stabilizing mutations were identified by the suppressor mutagenesis method. This method yielded 39 stabilized mutants from 513 mutations at 27 positions. This suggested that more than 90% of mutations caused destabilization even in a psychotropic protein. However, 17 positions had stabilizing mutations, indicating that the stabilization factors were dispersed over many positions. Interestingly, the identified mutations were distributed mainly at exposed or nonconserved sites. These results provide a novel strategy for protein stabilization.
  • Venkateswarlu Popuri, Jing Huang, Mahesh Ramamoorthy, Takashi Tadokoro, Deborah L. Croteau, Vilhelm A. Bohr
    NUCLEIC ACIDS RESEARCH 41 2 881 - 899 2013年01月 [査読有り][通常論文]
     
    Humans have five RecQ helicases, whereas simpler organisms have only one. Little is known about whether and how these RecQ helicases co-operate and/or complement each other in response to cellular stress. Here we show that RECQL5 associates longer at laser-induced DNA double-strand breaks in the absence of Werner syndrome (WRN) protein, and that it interacts physically and functionally with WRN both in vivo and in vitro. RECQL5 co-operates with WRN on synthetic stalled replication fork-like structures and stimulates its helicase activity on DNA fork duplexes. Both RECQL5 and WRN re-localize from the nucleolus into the nucleus after replicative stress and significantly associate with each other during S-phase. Further, we show that RECQL5 is essential for cell survival in the absence of WRN. Loss of both RECQL5 and WRN severely compromises DNA replication, accumulates genomic instability and ultimately leads to cell death. Collectively, our results indicate that RECQL5 plays both co-operative and complementary roles with WRN. This is an early demonstration of a significant functional interplay and a novel synthetic lethal interaction among the human RecQ helicases.
  • Takashi Tadokoro, Mahesh Ramamoorthy, Venkateswarlu Popuri, Alfred May, Jingyan Tian, Peter Sykora, Ivana Rybanska, David M. Wilson, Deborah L. Croteau, Vilhelm A. Bohr
    MOLECULAR BIOLOGY OF THE CELL 23 21 4273 - 4285 2012年11月 [査読有り][通常論文]
     
    Human RECQL5 is a member of the RecQ helicase family, which maintains genome stability via participation in many DNA metabolic processes, including DNA repair. Human cells lacking RECQL5 display chromosomal instability. We find that cells depleted of RECQL5 are sensitive to oxidative stress, accumulate endogenous DNA damage, and increase the cellular poly(ADP-ribosyl)ate response. In contrast to the RECQ helicase family members WRN, BLM, and RECQL4, RECQL5 accumulates at laser-induced single-strand breaks in normal human cells. RECQL5 depletion affects the levels of PARP-1 and XRCC1, and our collective results suggest that RECQL5 modulates and/or directly participates in base excision repair of endogenous DNA damage, thereby promoting chromosome stability in normal human cells.
  • Venkateswarlu Popuri, Mahesh Ramamoorthy, Takashi Tadokoro, Dharmendra Kumar Singh, Parimal Karmakar, Deborah L. Croteau, Vilhelm A. Bohr
    DNA REPAIR 11 7 624 - 635 2012年07月 [査読有り][通常論文]
     
    RECQL5 is one of the five human RecQ helicases, involved in the maintenance of genomic integrity. While much insight has been gained into the function of the Werner (WRN) and Bloom syndrome proteins (BLM), little is known about RECQL5. We have analyzed the recruitment and retention dynamics of RECQL5 at laser-induced DNA double strand breaks (DSBs) relative to other human RecQ helicases. RECQL5-depleted cells accumulate persistent 53BP1 foci followed by gamma-irradiation, indicating a potential role of RECQL5 in the processing of DSBs. Real time imaging of live cells using confocal laser microscopy shows that RECQL5 is recruited early to laser-induced DSBs and remains for a shorter duration than BLM and WRN, but persist longer than RECQL4. These studies illustrate the differential involvement of RecQ helicases in the DSB repair process. Mapping of domains within RECQL5 that are necessary for recruitment to DSBs revealed that both the helicase and KIX domains are required for DNA damage recognition and stable association of RECQL5 to the DSB sites. Previous studies have shown that MRE11 is essential for the recruitment of RECQL5 to the DSB sites. Here we show that the recruitment of RECQL5 does not depend on the exonuclease activity of MRE11 or on active transcription by RNA polymerase II, one of the prominent interacting partners of RECQL5. Also, the recruitment of RECQL5 to laser-induced damage sites is independent of the presence of other DNA damage signaling and repair proteins BLM, WRN and ATM. Published by Elsevier B.V.
  • Takashi Tadokoro, Tomasz Kulikowicz, Lale Dawut, Deborah L. Croteau, Vilhelm A. Bohr
    AGING-US 4 6 418 - 430 2012年06月 [査読有り][通常論文]
     
    Werner protein (WRN), member of the RecQ helicase family, is a helicase and exonuclease, and participates in multiple DNA metabolic processes including DNA replication, recombination and DNA repair. Mutations in the WRN gene cause Werner syndrome, associated with premature aging, genome instability and cancer predisposition. The RecQ C-terminal (RQC) domain of WRN, containing alpha 2-alpha 3 loop and beta-wing motifs, is important for DNA binding and for many protein interactions. To better understand the critical functions of this domain, we generated recombinant WRN proteins (using a novel purification scheme) with mutations in Arg-993 within the alpha 2-alpha 3 loop of the RQC domain and in Phe-1037 of the ?-wing motif. We then studied the catalytic activities and DNA binding of these mutant proteins as well as some important functional protein interactions. The mutant proteins were defective in DNA binding and helicase activity, and interestingly, they had deficient exonuclease activity and strand annealing function. The RQC domain of WRN has not previously been implicated in exonuclease or annealing activities. The mutant proteins could not stimulate NEIL1 incision activity as did the wild type. Thus, the Arg-993 and Phe-1037 in the RQC domain play essential roles in catalytic activity, and in functional interactions mediated by WRN.
  • Cahyo Budiman, Takashi Tadokoro, Clement Angkawidjaja, Yuichi Koga, Shigenori Kanaya
    FEBS JOURNAL 279 6 976 - 986 2012年03月 [査読有り][通常論文]
     
    FKBP22 from the psychotropic bacterium Shewanella sp. SIB1 is a homodimeric protein with peptidyl prolyl cistrans isomerase (PPIase) activity. According to a tertiary model, several nonpolar residues including Trp157 and Phe197 form a substrate-binding cavity, and Asp137 and Arg142, which form a salt bridge, are located at the edge of this cavity. To analyze the role of these residues, nine single (D137A, R142A, W157A/F/Y, F197A/L/Y/W) and one double (D137A/R142A) mutant protein of SIB1 FKBP22 were constructed. The far- and near-UV CD spectra of these mutant proteins suggest that the mutations at Asp137 and Arg142 do not seriously affect the protein structure, while those at Trp157 and Phe197 cause a local conformational change around the mutation site. Each mutation decreased the PPIase activities of SIB1 FKBP22 for peptide and protein substrates similarly without seriously affecting chaperone function. This result indicates that SIB1 FKBP22 does not require PPIase activity for chaperone function. The PPIase activities of R142A, D137A and D137A/R142A decreased in this order, suggesting that Asp137 and Arg142 play a principal and auxiliary role in catalytic function, respectively, but Arg142 can function as a substitute of Asp137. Because the PPIase activity of SIB1 FKBP22 was not fully lost by the removal of all polar residues around the active site, the desolvation effect may also contribute to the enzymatic activity. However, the mutations of Trp157 to Phe or Phe197 to Leu greatly decrease the enzymatic activity, suggesting that the shape of the substrate-binding cavity is also important for enzymatic activity.
  • Mahesh Ramamoorthy, Takashi Tadokoro, Ivana Rybanska, Avik K. Ghosh, Robert Wersto, Alfred May, Tomasz Kulikowicz, Peter Sykora, Deborah L. Croteau, Vilhelm A. Bohr
    NUCLEIC ACIDS RESEARCH 40 4 1621 - 1635 2012年02月 [査読有り][通常論文]
     
    DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase II alpha with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase II alpha stimulates the decatenation activity of Topoisomerase II alpha. Consistent with these observations, RECQL5 co-localizes with Topoisomerase II alpha during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.
  • Seiko Miyashita, Takashi Tadokoro, Clement Angkawidjaja, Dong-Ju You, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    FEBS LETTERS 585 14 2313 - 2317 2011年07月 [査読有り][通常論文]
     
    Ribonuclease H3 from Bacillus stearothermophilus (Bst-RNase H3) has the N-terminal TBP-like substrate-binding domain. To identify the substrate binding site in this domain, the mutant proteins of the intact protein and isolated N-domain, in which six of the seventeen residues corresponding to those involved in DNA binding of TBP are individually mutated to Ala, were constructed. All of them exhibited decreased enzymatic activities and/or substrate-binding affinities when compared to those of the parent proteins, suggesting that the N-terminal domain of RNase H3 uses the flat surface of the beta-sheet for substrate binding as TBP to bind DNA. This domain may greatly change conformation upon substrate binding. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Aya Sato, Sonoko Yokotani, Takashi Tadokoro, Shun-ichi Tanaka, Clement Angkawidjaja, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    JOURNAL OF SYNCHROTRON RADIATION 18 1 6 - 10 2011年01月 [査読有り][通常論文]
     
    CutA1 is widely found in bacteria, plants and animals, including humans. The functions of CutA1, however, have not been well clarified. It is known that CutA1s from Pyrococcus horikoshii, Thermus thermophilus and Oryza sativa unfold at temperatures remarkably higher than the growth temperatures of the host organisms. In this work the crystal structure of CutA1 from the psychrotrophic bacterium Shewanella sp. SIB1 (SIB1-CutA1) in a trimeric form was determined at 2.7 A resolution. This is the first crystal structure of a psychrotrophic CutA1. The overall structure of SIB1-CutA1 is similar to those of CutA1 from Homo sapiens, Escherichia coli, Pyrococcus horikoshii, Thermus thermophilus, Termotoga maritima, Oryza sativa and Rattus norvergicus. A peculiarity is observed in the beta 2 strand. The beta 2 strand is divided into two short beta strands, beta 2a and beta 2b, in SIB1-CutA1. A thermal denaturation experiment revealed that SIB1-CutA1 does not unfold completely at 363 K at pH 7.0, although Shewanella sp. SIB1 cannot grow at temperatures exceeding 303 K. These results indicate that the trimeric structural motif of CutA1 is the critical factor in its unusually high stability and suggest that CutA1 needs to maintain its high stability in order to function, even in psychrotrophs.
  • Jenq-Lin Yang, Takashi Tadokoro, Guido Keijzers, Mark P. Mattson, Vilhelm A. Bohr
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 36 28191 - 28199 2010年09月 [査読有り][通常論文]
     
    Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca(2+) influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca(2+) chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H, 23H-porphine chloride tetrakis( methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca(2+)/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca(2+)- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca(2+)-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.
  • Jun Okada, Tomohiro Okamoto, Atsushi Mukaiyama, Takashi Tadokoro, Dong-Ju You, Hyongi Chon, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    BMC EVOLUTIONARY BIOLOGY 10 207  2010年07月 [査読有り][通常論文]
     
    Background: The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI). Results: To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins. Conclusions: These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.
  • Takashi Tadokoro, Shigenori Kanaya
    FEBS JOURNAL 276 6 1482 - 1493 2009年03月 [査読有り][通常論文]
     
    The prokaryotic genomes, for which complete nucleotide sequences are available, always contain at least one RNase H gene, indicating that RNase H is ubiquitous in all prokaryotic cells. Coupled with its unique substrate specificity, the enzyme has been expected to play crucial roles in the biochemical processes associated with DNA replication, gene expression and DNA repair. The physiological role of prokaryotic RNases H, especially of type 1 RNases H, has been extensively studied using Escherichia coli strains that are defective in RNase HI activity or overproduce RNase HI. However, it is not fully understood yet. By contrast, significant progress has been made in this decade in identifying novel RNases H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H. We review the results of these studies.
  • Kazufumi Takano, Ryogo Higashi, Jun Okada, Atsushi Mukaiyama, Takashi Tadokoro, Yuichi Koga, Shigenori Kanaya
    JOURNAL OF BIOCHEMISTRY 145 1 79 - 85 2009年01月 [査読有り][通常論文]
     
    Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a robust monomeric protein under kinetic control, which possesses some proline residues at the N-terminal of -helices. Proline residue at the N-terminal of an -helix is thought to stabilize a protein. In this work, the thermostability and folding kinetics of Tk-RNase HII were measured for mutant proteins in which a proline residue is introduced (Xaa to Pro) or removed (Pro to Ala) at the N-terminal of -helices. In the folding experiments, the mutant proteins examined exhibit little influence on the remarkably slow unfolding of Tk-RNase HII. In contrast, E111P and K199P exhibit some thermostabilization, whereas P46A, P70A and P174A have some thermodestabilization. E111P/K199P and P46A/P70A double mutations cause cumulative changes in stability. We conclude that the proline effect on protein thermostability is observed in a hyperthermophilic protein, but each proline residue at the N-terminal of an -helix slightly contributes to the thermostability. The present results also mean that even a natural hyperthermophilic protein can acquire improved thermostability.
  • Muhammad S. Rohman, Takashi Tadokoro, Clement Angkawidjaja, Yumi Abe, Hiroyoshi Matsumura, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    FEBS JOURNAL 276 2 603 - 613 2009年01月 [査読有り][通常論文]
     
    The Arg97 -> Gly and Asp136 -> His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7 degrees C, respectively, in T(m), and 3.5 and 6.1 kJ.mol(-1), respectively, in Delta G(H(2)O). These mutations also stabilized the So-RNase HI derivative (4x-RNase HI) with quadruple thermostabilizing mutations in an additive manner. As a result, the resultant sextuple mutant protein (6x-RNase HI) was more stable than the wild-type protein by 28.8 degrees C in T(m) and 27.0 kJ.mol(-1) in Delta G(H(2)O). To analyse the effects of the mutations on the protein structure, the crystal structure of the 6x-RNase HI protein was determined at 2.5 angstrom resolution. The main chain fold and interactions of the side-chains of the 6x-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites. These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive. The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6x-RNase HI protein. We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability.
  • Takashi Tadokoro, Kyoko Matsushita, Yumi Abe, Muhammad Saifur Rohman, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    BIOCHEMISTRY 47 31 8040 - 8047 2008年08月 [査読有り][通常論文]
     
    Ribonuclease HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is much less stable than Escherichia coli RNase HI (Ec-RNase HI) by 22.4 degrees C in T(m) and 12.5 kJ mol(-1) in Delta G(H(2)O), despite their high degrees of structural and functional similarity. To examine whether the stability of So-RNase HI increases to a level similar to. that of Ec-RNase HI via introduction of several mutations, the mutations that stabilize So-RNase HI were identified by the suppressor mutation method and combined. So-RNase HI and its variant with a C-terminal four-residue truncation (154-RNase HI) complemented the RNase H-dependent temperature-sensitive (ts) growth phenotype of E. coli strain MIC3001, while 153-RNase HI with a five-residue truncation could not. Analyses of the activity and stability of these truncated proteins suggest that 153-RNase HI is nonfunctional in vivo because of a great decrease in stability. Random mutagenesis of 153-RNase HI using error-prone PCR, followed by screening for the revertants, allowed us to identify six single suppressor mutations that make 153-RNase HI functional in vivo. Four of them markedly increased the stability of the wild-type protein by 3.6-6.7 degrees C in T. and 1.7-5.2 kJ mol(-1) in Delta G(H(2)O). The effects of these mutations were e nearly additive, and combination of these mutations increased protein stability by 18.7 degrees C in T(m) and 12.2 kJ mol(-1) in Delta G(H(2)O)These results suggest that several residues are not optimal for the stability of So-RNase HI, and their replacement with other residues strikingly increases it to a level similar to that of the mesophilic counterpart.
  • Hongju Dong, Atsushi Mukaiyama, Takashi Tadokoro, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    JOURNAL OF MOLECULAR BIOLOGY 378 1 264 - 272 2008年04月 [査読有り][通常論文]
     
    Ribonuclease HIT from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HIT) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HIT were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/ Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol(-1) as compared with the wild-type protein. The removal of each -CH2- group burial decreased the stability by 5.1 kJ mol(-1) on average in the mutant proteins of Tk-RNase HIT examined. This is comparable with the value of 5.3 kJ mol(-1) obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HIT examined exhibited faster unfolding compared with the wildtype protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HIT. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.
  • Mitsuru Haruki, Masaki Tanaka, Takehiko Motegi, Takashi Tadokoro, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    FEBS JOURNAL 274 22 5815 - 5825 2007年11月 [査読有り][通常論文]
     
    A combination of five thermostabilizing mutations, Gly23 -> Ala, His62 -> Pro, Val74 -> Leu, Lys95 -> Gly, and Asp134 -> His, has been shown to additively enhance the thermostability of Escherichia coli RNase HI [Akasako A, Haruki M, Oobatake M & Kanaya S (1995) Biochemistry 34, 8115-8122]. In this study, we determined the crystal structure of the protein with these mutations (5H-RNase HI) to analyze the effects of the mutations on the structure in detail. The structures of the mutation sites were almost identical to those of the mutant proteins to which the mutations were individually introduced, except for G23A, for which the structure of the single mutant protein is not available. Moreover, only slight changes in the backbone conformation of the protein were observed, and the interactions of the side chains were almost conserved. These results indicate that these mutations almost independently affect the protein structure, and are consistent with the fact that the thermostabiling effects of the mutations are cumulative. We also determined the protein stability curve describing the temperature dependence of the free energy of unfolding of 5H-RNase HI to elucidate the thermostabilization mechanism. The maximal stability for 5H-RNase HI was as high as that for the cysteine-free variant of Thermus thermophilus RNase HI. In contrast, the heat capacity of unfolding for 5H-RNase H was similar to that for E. coli RNase HI, which is considerably higher than that for T. thermophilus RNase HI. These results suggest that 5H-RNase HI is stabilized, in part, by the thermostabilization mechanism adopted by T. thermophilus RNase HI.
  • Takashi Tadokoro, Hyongi Chon, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    FEBS JOURNAL 274 14 3715 - 3727 2007年07月 [査読有り][通常論文]
     
    The gene encoding a bacterial type 1 RNase H, termed RBD-RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD-RNase HI has a double-stranded RNA binding domain (RBD) at the N-terminus, which is commonly present at the N-termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD-RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD-RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD-RNase HI showed poor cleavage-site specificity for oligomeric substrates. SIB1 RBD-RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD-RNase HI. This is the first report on the biochemical characterization of RBD-RNase HI.
  • Takashi Tadokoro, Dong-Ju You, Yumi Abe, Hyongi Chon, Hiroyoshi Matsumura, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
    BIOCHEMISTRY 46 25 7460 - 7468 2007年06月 [査読有り][通常論文]
     
    Ribonuclease (RNase) HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 was overproduced in Escherichia coli, purified, and structurally and biochemically characterized. The amino acid sequence of MR-1 RNase HI is 67% identical to that of E. coli RNase HI. The crystal structure of MR-1 RNase HI determined at 2.0 A resolution was highly similar to that of E. coli RNase HI, except that the number of intramolecular ion pairs and the fraction of polar surface area of MR-1 RNase HI were reduced compared to those of E. coli RNase HI. The enzymatic properties of MR-1 RNase HI were similar to those of E. coli RNase HI. However, MR-1 RNase HI was much less stable than E. coli RNase HI. The stability of MR-1 RNase HI against heat inactivation was lower than that of E. coli RNase HI by 19 degrees C. The conformational stability of MR-1 RNase HI was thermodynamically analyzed by monitoring the CD values at 220 nm. MR-1 RNase HI was less stable than E. coli RNase HI by 22.4 degrees C in T-m and 12.5 kJ/mol in Delta G(H2O). The thermodynamic stability curve of MR-1 RNase HI was characterized by a downward shift and increased curvature, which results in an increased Delta C-p value, compared to that of E. coli RNase HI. Site-directed mutagenesis studies suggest that the difference in the number of intramolecular ion pairs partly accounts for the difference in stability between MR-1 and E. coli RNases HI.
  • H Chon, T Tadokoro, N Ohtani, Y Koga, K Takano, S Kanaya
    FEBS JOURNAL 273 10 2264 - 2275 2006年05月 [査読有り][通常論文]
     
    The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half-lives of SIB1 and E. coli RNases HII at 30 degrees C were similar to 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by similar to 0.2 M and similar to 5 degrees C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 degrees C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low- and high-activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high- and low-activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with RNase H families.

MISC

書籍等出版物

  • Silkworm Biofactory: Silk to Biology
    Takashi Tadokoro (担当:分担執筆範囲:BacMam System in Silkworm)
    CRC Press 2018年 
    edited by Katsumi Maenaka & Enoch Y. Park
  • Advanced Methods in Structural Biology
    Shunsuke Kita, Katsumi Maenaka, Takashi Tadokoro (担当:分担執筆範囲:Expression of Proteins in Insect and Mammalian Cells)
    © Springer Japan 2016年01月 25-45 
    edited by Toshiya Senda & Katsumi Maenaka
  • C-Type Lectin Receptors in Immunity
    Atsushi Furukawa, Shunsuke Kita, Takashi Tadokoro, Hideo Fukuhara, Katsumi Maenak (担当:分担執筆範囲:Structural aspects of C-type lectin receptors)
    © Springer Japan 2016年01月 179-190 
    edited by Sho Yamasaki
  • Protein Structure
    Azumi Hirata, Aya Sato, Takashi Tadokoro, Shigenori Kanaya, Kazufumi Takano (担当:分担執筆範囲:A Stable Protein - CutA1)
    InTech 2012年04月 
    edited by Eshel Faraggi

講演・口頭発表等

  • Structural, thermodynamic, and mutational analyses of a psychrotrophic RNase HI.
    Tadokoro T, You DJ, Abe Y, Chon H, Matsumura H, Koga Y, Takano K, Kanaya S
    International Symposium on Advanced Science and Biotechnology 2007 2007年03月 Suita, Osaka
  • Thermolabile RNase HI from the mesophilic bacterium Shewanella oneidensis MR-1.
    Tadokoro T, Koga Y, Takano K, Kanaya S
    9th International Conference on RIBONUCLEASE H 2006 2006年09月 Maryland
  • Studies on a Cold-adaptation Mechanism of RNase HI from a Psychrotrophic Bacterium.
    Tadokoro T, Tsunaka Y, Takano K, Kanaya S
    8th International Conference on RIBONUCLEASE H 2004 2004年09月 Strasbourg

担当経験のある科目(授業)

  • 物理化学Ⅰ北海道大学 薬学部
  • 医薬ゲノム機能学特論北海道大学 大学院薬学研究院
  • 医療薬学特論北海道大学 大学院薬学研究院
  • バイオ医薬学基礎特論北海道大学 大学院薬学研究院
  • 先端創薬学特論北海道大学 大学院薬学研究院
  • 物理化学実習北海道大学 薬学部
  • 生物物理化学北海道大学 薬学部

所属学協会

  • 日本ケミカルバイオロジー学会   日本生化学会   日本蛋白質科学会   日本結晶学会   日本生物物理学会   

共同研究・競争的資金等の研究課題

  • 高機能抗体薬物複合体創成を目指した多量体型抗体フラグメントの分子基盤
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 田所 高志
  • 免疫制御に関わるNKRP1A-LLT1複合体の立体構造解析
    文部科学省日本学術振興会:科学研究費補助金 若手研究(B)
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 田所 高志

産業財産権

社会貢献活動

  • 札幌市立日新小学校 訪問
    期間 : 2016年03月04日
    役割 : 講師
  • やましろ実験サイエンスセミナー
    期間 : 2007年11月04日
    役割 : 講師
    主催者・発行元 : きっず光科学館ふぉとん(国立研究開発法人 量子科学技術研究開発機構)


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