2021/11/20 更新

写真a

ワタナベ カズノリ
渡邉 和則
WATANABE Kazunori
所属
ヘルスシステム統合科学学域 助教
職名
助教
外部リンク

学位

  • 博士(工学) ( 愛媛大学 )

研究キーワード

  • molecular and cell biology

  • 分子細胞生物学

  • ストレス顆粒形成

  • 生化学

  • ストレス応答

  • RNA分解

  • 相分離

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

経歴

  • 岡山大学学術研究院ヘルスシステム統合科学学域   助教

    2021年4月 - 現在

      詳細を見る

  • 岡山大学大学院ヘルスシステム統合科学研究科   助教

    2019年4月 - 2021年3月

      詳細を見る

  • - 岡山大学自然科学研究科 助教

    2012年 - 2019年3月

      詳細を見る

  • - Assistant Professor,Graduate School of Natural Science and Technology,Okayama University

    2012年

      詳細を見る

所属学協会

 

論文

  • Fluorophore-PNA-Quencher/Quencher-DNA probe for miRNA detection. 査読 国際誌

    Kentaro Tabara, Kazunori Watanabe, Hajime Shigeto, Shohei Yamamura, Takamasa Kishi, Mizuki Kitamatsu, Takashi Ohtsuki

    Bioorganic & medicinal chemistry letters   51   128359 - 128359   2021年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore-PNA-quencher (fPq) and a quencher-DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA-DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis.

    DOI: 10.1016/j.bmcl.2021.128359

    PubMed

    researchmap

  • Photocontrolled apoptosis induction using precursor miR-664a and an RNA carrier-conjugated with photosensitizer 査読

    Kazunori Watanabe, Tomoko Nawachi, Ruriko Okutani, Takashi Ohtsuki

    Scientific Reports   11   14936   2021年7月

     詳細を見る

    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Science and Business Media LLC  

    <title>Abstract</title>Methods to spatially induce apoptosis are useful for cancer therapy. To control the induction of apoptosis, methods using light, such as photochemical internalization (PCI), have been developed. We hypothesized that photoinduced delivery of microRNAs (miRNAs) that regulate apoptosis could spatially induce apoptosis. In this study, we identified pre-miR-664a as a novel apoptosis-inducing miRNA via mitochondrial apoptotic pathway. Further, we demonstrated the utility of photoinduced cytosolic dispersion of RNA (PCDR), which is an intracellular RNA delivery method based on PCI. Indeed, apoptosis is spatially regulated by pre-miR-664a and PCDR. In addition, we found that apoptosis induced by pre-miR-664a delivered by PCDR was more rapid than that by lipofection. These results suggest that pre-miR-664a is a nucleic acid drug candidate for cancer therapy and PCDR and pre-miR-664a-based strategies have potential therapeutic uses for diseases affecting various cell types.

    DOI: 10.1038/s41598-021-94249-7

    researchmap

    その他リンク: http://www.nature.com/articles/s41598-021-94249-7

  • Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress 査読

    Kazunori Watanabe, Takashi Ohtsuki

    International Journal of Molecular Sciences   22 ( 9 )   4982 - 4982   2021年5月

     詳細を見る

    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.

    DOI: 10.3390/ijms22094982

    researchmap

  • Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells

    Melissa Siaw Han Lim, Yuki Nishiyama, Takashi Ohtsuki, Kazunori Watanabe, Hirotsugu Kobuchi, Kazuko Kobayashi, Eiji Matsuura

    Journal of Pharmaceutical Sciences   110 ( 4 )   1788 - 1798   2021年4月

     詳細を見る

    掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    DOI: 10.1016/j.xphs.2021.01.026

    PubMed

    researchmap

  • Minimization of apoptosis-inducing CPP-Bim peptide. 査読 国際誌

    Shengli Zhou, Kazunori Watanabe, Seiichiro Koide, Mizuki Kitamatsu, Takashi Ohtsuki

    Bioorganic & medicinal chemistry letters   36   127811 - 127811   2021年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Pro-apoptotic peptides may be promising agents for cancer therapy owing to their ability to induce apoptosis in cancer cells. TatBim, a fusion peptide of Tat cell-penetrating peptide (CPP) and the BH3 domain derived from Bim apoptosis-inducing protein, is a pro-apoptotic peptide. In this study, based on the TatBim sequence, we attempted to minimize the CPP-Bim peptide while retaining apoptosis-inducing activity. The CPP and Bim parts were systematically shortened, and the pro-apoptotic activities of the shortened peptides were examined. We obtained TatBim-N1C2 and R8Bim-N1C2 as minimized peptides with efficient apoptotic activity. These peptides may have potential applications in future biomedical studies, such as cancer therapeutics.

    DOI: 10.1016/j.bmcl.2021.127811

    PubMed

    researchmap

  • Fluorescence lifetime probes for detection of RNA degradation. 査読 国際誌

    Riku Hirata, Kazutaka Hirakawa, Naotaka Shimada, Kazunori Watanabe, Takashi Ohtsuki

    The Analyst   146 ( 1 )   277 - 282   2021年1月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    To investigate RNA degradation in live cells, detection methods that do not require RNA extraction from cells are necessary. In this study, we examined the utility of fluorescence lifetime measurements using a probe attached to the end of an RNA molecule for detecting RNA degradation. We optimized a short fluorescein-labeled RNA sequence whose fluorescence lifetime varied significantly before and after degradation. The selected HHG-fluorescein sequence (H = U, C, or A) is a promising RNA labeling unit (fluorescence lifetime probe) for live cell imaging of RNA degradation.

    DOI: 10.1039/d0an01230k

    PubMed

    researchmap

  • Photoinduced endosomal escape mechanism: a view from photochemical internalization mediated by CPP-photosensitizer conjugates 査読 国際誌

    Tet Htut Soe, Kazunori Watanabe, Takashi Ohtsuki

    Molecules   26 ( 1 )   36   2020年12月

     詳細を見る

  • Cell cycle dependence of apoptosis photo-triggered using peptide-photosensitizer conjugate. 査読 国際誌

    Hyungjin Kim, Sho Watanabe, Mizuki Kitamatsu, Kazunori Watanabe, Takashi Ohtsuki

    Scientific reports   10 ( 1 )   19087 - 19087   2020年11月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G1, S, and G2/M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G1/S transition than in the G1 and S/G2/M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions.

    DOI: 10.1038/s41598-020-76100-7

    PubMed

    researchmap

  • Endosomal Escape of Peptide-Photosensitizer Conjugates Is Affected by Amino Acid Sequences near the Photosensitizer. 査読 国際誌

    Yuichi Miyoshi, Maho Kadono, Shigetoshi Okazaki, Ayano Nishimura, Mizuki Kitamatsu, Kazunori Watanabe, Takashi Ohtsuki

    Bioconjugate chemistry   31 ( 3 )   916 - 922   2020年3月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of peptides and proteins, but CPP fusion peptides and proteins are often transported by endocytosis and trapped in endosomes. Photochemical internalization (PCI) is a method for the endosomal escape of the trapped peptide or protein and release into the cytosol using light and photosensitizers. In PCI, endosomal membranes are thought to be destabilized by singlet oxygen (1O2) photogenerated from photosensitizers localized in endosomes. We previously developed CPP-cargo-photosensitizer (PS) conjugates able to photodependently enter the cytosol via the PCI mechanism. For example, TatU1A-PS (a covalent complex of Tat [CPP], U1A RNA-binding protein [cargo], and PS) can photodependently deliver RNAs into the cytosol, and TatBim-PS (a covalent complex of Tat, Bim [cargo], and PS) can photoinduce apoptosis in mammalian cells. However, for many newly created conjugates, the induction of PCI has been insufficient. We hypothesized that the amino acid linker sequence (XX) adjacent to the photosensitizer is an important determinant of PCI efficiency. In this study, using CPP-cargo-XX-PS platforms, we examined the relationship between PCI efficiency and the linker amino acid sequence near the photosensitizer. We found that hydrophobic FF and LL linkers enhanced the PCI efficiencies of both TatBim-XX-PS and TatU1A-XX-PS. The effectiveness of the linker depended, in part, on both the cargo moiety and the photosensitizer. These results may guide the design of CPP-cargo-PS conjugates conferring broad target functions for PCI and photodynamic therapy.

    DOI: 10.1021/acs.bioconjchem.0c00046

    PubMed

    researchmap

  • Relation of Photochemical Internalization to Heat, pH and Ca2+ Ions. 査読 国際誌

    Tet Htut Soe, Tomotaka Nanjo, Kazunori Watanabe, Takashi Ohtsuki

    Photochemistry and photobiology   95 ( 6 )   1395 - 1402   2019年11月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The inefficient endosomal escape of drugs or macromolecules is a major obstacle to achieving successful delivery to therapeutic targets. An efficient approach to circumvent this barrier is photochemical internalization (PCI), which uses light and photosensitizers for endosomal escape of the delivered macromolecules. The PCI mechanism is related to photogenerated singlet oxygen, but the mechanism is still unclear. In this study, we examined the relation of PCI to heat, pH and Ca2+ ions using cell penetrating peptide (CPP)-cargo-photosensitizer (Alexa546 or Alexa633) conjugates. A cell temperature changing experiment demonstrated that heat (thermal mechanism) does not significantly contribute to the photoinduced endosomal escape. Inhibition of V-ATPase proton pump activity and endosomal pH upregulation indicated that PCI-mediated endosomal escape needs endosomal acidification prior to photoirradiation. Imaging of the CPP-cargo-photosensitizer and Ca2+ ions during photostimulation showed that intracellular calcium increase is not the cause of the endosomal escape of the complex. The increment is mainly due to Ca2+ influx. These findings show the importance of extra- and intracellular milieu conditions in the PCI mechanism and enrich our understanding of PCI-related changes in cell.

    DOI: 10.1111/php.13146

    PubMed

    researchmap

  • In-stem molecular beacon targeted to a 5'-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity. 査読 国際誌

    Yuichi Miyoshi, Takashi Ohtsuki, Hiromu Kashida, Hiroyuki Asanuma, Kazunori Watanabe

    PloS one   14 ( 1 )   e0211505   2019年

     詳細を見る

    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5'- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.

    DOI: 10.1371/journal.pone.0211505

    PubMed

    researchmap

  • Red and Near-Infrared Light-Directed Cytosolic Delivery of Two Different RNAs Using Photosensitive RNA Carriers. 査読 国際誌

    Kaori Shiraga, Tet Htut Soe, Sho Matsumoto, Kazunori Watanabe, Takashi Ohtsuki

    Bioconjugate chemistry   29 ( 9 )   3174 - 3179   2018年9月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Many cellular events are thought to be controlled by the temporal upregulation of multiple RNAs; the timing of the upregulation of these RNAs is not always the same. In this study, we first show that our light-directed intracellular RNA delivery method induced high concentrations of RNA in a short period. This effect was beneficial for the temporal control of cellular events by functional RNAs. Next, we stimulated the short-term upregulation of two different RNAs at different time points. Cytosolic delivery of a first RNA was induced by red light; thereafter, cytosolic delivery of a second RNA was induced by near-infrared light. The time difference between the introduction of the first and second RNA can be short (0.5-4 h) or long (>8 h). This strategy shows the potential for future applications of the deliberate control of time-dependent RNA concentration to guide various cellular functions by multiple RNAs.

    DOI: 10.1021/acs.bioconjchem.8b00487

    PubMed

    researchmap

  • MicroRNA-664a-5p promotes neuronal differentiation of SH-SY5Y cells. 査読 国際誌

    Kazunori Watanabe, Ryuhei Yamaji, Takashi Ohtsuki

    Genes to cells : devoted to molecular & cellular mechanisms   23 ( 3 )   225 - 233   2018年3月

     詳細を見る

    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Blackwell Publishing Ltd  

    MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH-SY5Y cells. We identified 15 up-regulated and eight down-regulated miRNAs in SH-SY5Y cells treated with all-trans retinoic acid to induce differentiation. We further showed that one of the up-regulated miRNAs, miR-664a-5p, promoted neuronal differentiation of SH-SY5Y cells. These findings enhance our understanding of the miRNAs involved in the process of neurogenesis and, in particular, highlight an important role of miR-664a-5p in SH-SY5Y cell neuronal differentiation. Further studies will be required to confirm the function of miR-664-5p in neuronal development and disease and to identify its relevant target genes.

    DOI: 10.1111/gtc.12559

    Scopus

    PubMed

    researchmap

  • Ultrasound-dependent cytoplasmic internalization of a peptide-sonosensitizer conjugate. 査読 国際誌

    Yuki Inaba, Kazunori Watanabe, Mizuki Kitamatsu, Eiji Nakata, Atsushi Harada, Takashi Ohtsuki

    Bioorganic & medicinal chemistry   25 ( 15 )   4212 - 4217   2017年8月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    A method to induce cytoplasmic peptide delivery, using ultrasound, was demonstrated using a molecular conjugate of a cell-penetrating peptide (CPP), a functional peptide, and a sonosensitizer. As a model of such molecular conjugates, TatBim-RB, consisting of the Tat CPP, the Bim apoptosis inducing peptide, and the sonosensitizer rose bengal was synthesized. CPPs have been widely used for intracellular delivery of various cargos; however, CPP-fused molecules tend to become entrapped in endosomes, as was observed for TatBim-RB molecules applied to cells. To promote escape of the entrapped TatBim-RB molecules, cells were irradiated with ultrasound, which successfully induced endosomal escape and cytoplasmic dispersion of TatBim-RB, and subsequently apoptosis. Our results suggest that this peptide-sonosensitizer conjugate strategy may facilitate numerous kinds of medicinal chemistry studies, and furthermore, this specific conjugate may exhibit potential as a novel therapeutic agent for the promotion of apoptosis.

    DOI: 10.1016/j.bmc.2017.06.024

    Web of Science

    PubMed

    researchmap

  • Detection of small, highly structured RNAs using molecular beacons 査読

    J. Li, C. Xu, N. Shimada, Y. Miyoshi, K. Watanabe, W. Cong, T. Ohtsuki

    ANALYTICAL METHODS   9 ( 20 )   2971 - 2976   2017年5月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ROYAL SOC CHEMISTRY  

    The use of molecular beacons is a versatile method to detect RNAs. Typically, a single-stranded region of RNA is selected as a target sequence for molecular beacons. Therefore, the detection of highly structured short RNAs, such as tRNAs, seems to be difficult. In this study, as an example of highly structured target RNA, we used human tRNA(Lys3), which is known to have functions in protein synthesis and the reverse transcription of human immunodeficiency virus (HIV)-1. No long single-stranded region more than 8 nt is present in this tRNA, which is much shorter than the standard target length of molecular beacons (similar to 20 nt). This study showed that sensitive detection of highly structured RNAs using molecular beacons was much more difficult than that of unstructured or less structured RNAs. However, efficient detection of the tRNA(Lys3) was achieved by selecting the best target region, i.e., the region around the D arm, probably due to the ease of unfolding of this arm. Accordingly, our findings suggested that molecular beacons may have applications in the detection of highly structured RNAs related to various biological functions and diseases.

    DOI: 10.1039/c7ay00341b

    Web of Science

    researchmap

  • Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs. 査読 国際誌

    Takashi Ohtsuki, Shigeto Kanzaki, Sae Nishimura, Yoshio Kunihiro, Masahiko Sisido, Kazunori Watanabe

    Nature communications   7   12501 - 12501   2016年8月

     詳細を見る

    担当区分:最終著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at ∼405 nm at 125 mW cm(-2), DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells.

    DOI: 10.1038/ncomms12501

    Web of Science

    PubMed

    researchmap

  • Photoinduced apoptosis using a peptide carrying a photosensitizer. 査読 国際誌

    Kazunori Watanabe, Hayato Fujiwara, Mizuki Kitamatsu, Takashi Ohtsuki

    Bioorganic & medicinal chemistry letters   26 ( 13 )   3115 - 3118   2016年7月

     詳細を見る

    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines.

    DOI: 10.1016/j.bmcl.2016.04.091

    Web of Science

    PubMed

    researchmap

  • Effects of polyamines from Thermus thermophilus, an extreme-thermophilic eubacterium, on tRNA methylation by tRNA (Gm18) methyltransferase (TrmH). 査読 国際誌

    Hiroyuki Hori, Yusuke Terui, Chisato Nakamoto, Chikako Iwashita, Anna Ochi, Kazunori Watanabe, Tairo Oshima

    Journal of biochemistry   159 ( 5 )   509 - 17   2016年5月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Thermus thermophilus is an extreme-thermophilic eubacterium, which grows at a wide range of temperatures (50-83°C). This thermophile produces various polyamines including long and branched polyamines. In tRNAs from T. thermophilus, three distinct modifications, 2'-O-methylguanosine at position 18 (Gm18), 5-methyl-2-thiouridine at position 54 and N(1)-methyladenosine at position 58, are assembled at the elbow region to stabilize the L-shaped tRNA structure. However, the structures of unmodified tRNA precursors are disrupted at high temperatures. We hypothesize that polyamine(s) might have a positive effect on the modification process of unmodified tRNA transcript. We investigated the effects of eight polyamines on Gm18 formation in the yeast tRNA(Phe) transcript by tRNA (Gm18) methyltransferase (TrmH). Higher concentrations of linear polyamines inhibited TrmH activity at 55°C, while optimum concentration increased TrmH activity at 45-75°C. Exceptionally, caldohexamine, a long polyamine, did not show any positive effect on the TrmH activity at 55°C. However, temperature-dependent experiments revealed that 1 mM caldohexamine increased TrmH activity at 60-80°C. Furthermore, 0.25 mM tetrakis(3-aminopropy)ammonium, a branched polyamine, increased TrmH activity at a broad range of temperatures (40-85°C). Thus, caldohexamine and tetrakis(3-aminopropy)ammonium were found to enhance the TrmH activity at high temperatures.

    DOI: 10.1093/jb/mvv130

    Web of Science

    PubMed

    researchmap

  • Enhanced cellular uptake of lactosomes using cell-penetrating peptides. 査読 国際誌

    Akiya Akahoshi, Eiji Matsuura, Eiichi Ozeki, Hayato Matsui, Kazunori Watanabe, Takashi Ohtsuki

    Science and technology of advanced materials   17 ( 1 )   245 - 252   2016年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Polymeric micelles that are composed of synthetic polymers are generally size controllable and can be easily modified for various applications. Lactosomes (A3B-type) are biodegradable polymeric micelles composed of an amphipathic polymer, including three poly(sarcosine) blocks and a poly(l-lactic acid) block. Lactosomes accumulate in tumors in vivo through the enhanced permeability and retention (EPR) effect, even on frequently administering them. However, lactosomes cannot be efficiently internalized by cells. To improve cellular uptake of lactosomes, cell-penetrating peptide (CPP)-modified lactosomes were prepared. Seven CPPs (including EB1 and Pep1) were used, and most of them improved the cellular uptake efficiency of lactosomes. In particular, EB1- and Pep1-modified lactosomes were efficiently internalized by cells. In addition, by using CPP-modified and photosensitizer-loaded lactosomes, we demonstrated the photoinduced killing of mammalian cells, including human cancer cells. Accumulation of the EB1-modified lactosomes in NCI-N87 tumors was shown by in vivo imaging. Thus, this study demonstrated that the CPP-modified lactosome is a promising drug carrier.

    DOI: 10.1080/14686996.2016.1178056

    Web of Science

    PubMed

    researchmap

  • The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates. 査読 国際誌

    Takashi Ohtsuki, Shunya Miki, Shouhei Kobayashi, Tokuko Haraguchi, Eiji Nakata, Kazutaka Hirakawa, Kensuke Sumita, Kazunori Watanabe, Shigetoshi Okazaki

    Scientific reports   5   18577 - 18577   2015年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated (1)O2 molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of (1)O2 was confirmed using (1)O2 quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than (1)O2 lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule.

    DOI: 10.1038/srep18577

    Web of Science

    PubMed

    researchmap

  • Photo-dependent protein biosynthesis using a caged aminoacyl-tRNA. 査読 国際誌

    Akiya Akahoshi, Yoshio Doi, Masahiko Sisido, Kazunori Watanabe, Takashi Ohtsuki

    Bioorganic & medicinal chemistry letters   24 ( 23 )   5369 - 72   2014年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Translation systems with four-base codons provide a powerful strategy for protein engineering and protein studies because they enable site-specific incorporation of non-natural amino acids into proteins. In this study, a caged aminoacyl-tRNA with a four-base anticodon was synthesized. The caged aminoacyl-tRNA contains a photocleavable nitroveratryloxycarbonyl (NVOC) group. This study showed that the caged aminoacyl-tRNA was not deacylated, did not bind to EF-Tu, and was activated by light. Photo-dependent translation of an mRNA containing the four-base codon was demonstrated using the caged aminoacyl-tRNA.

    DOI: 10.1016/j.bmcl.2014.10.053

    Web of Science

    PubMed

    researchmap

  • mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress. 査読 国際誌

    Kazunori Watanabe, Kenichi Ijiri, Takashi Ohtsuki

    FEBS letters   588 ( 18 )   3454 - 60   2014年9月

     詳細を見る

    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNA(Met) (iMet) through 5'-3' exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells.

    DOI: 10.1016/j.febslet.2014.08.003

    Web of Science

    PubMed

    researchmap

  • Near-infrared light-directed RNAi using a photosensitive carrier molecule. 査読 国際誌

    Yuka Matsushita-Ishiodori, Mika Morinaga, Kazunori Watanabe, Takashi Ohtsuki

    Bioconjugate chemistry   24 ( 10 )   1669 - 73   2013年10月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Controlled activation of small RNAs, such as small interfering RNA, in cells is very useful for various biological applications. Light is an effective inducer of controlled activation; in particular, near-infrared light is favorable because it can penetrate deeper into tissues than UV or visible light. In this study, near-infrared light control of RNA interference (RNAi) was demonstrated in mammalian cells using a photosensitive RNA carrier molecule, consisting of an RNA carrier protein and a fluorochrome. The photosensitive carrier molecule was identified from six candidates, each with a different fluorochrome. Using this carrier molecule, cytosolic RNA delivery and RNAi can be triggered by near-infrared light. Cytotoxicity was not observed after photoinduction of RNAi.

    DOI: 10.1021/bc4001195

    Scopus

    PubMed

    researchmap

  • The catalytic domain of topological knot tRNA methyltransferase (TrmH) discriminates between substrate tRNA and nonsubstrate tRNA via an induced-fit process. 査読 国際誌

    Anna Ochi, Koki Makabe, Ryota Yamagami, Akira Hirata, Reiko Sakaguchi, Ya-Ming Hou, Kazunori Watanabe, Osamu Nureki, Kunihiro Kuwajima, Hiroyuki Hori

    The Journal of biological chemistry   288 ( 35 )   25562 - 25574   2013年8月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2'-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.

    DOI: 10.1074/jbc.M113.485128

    Web of Science

    PubMed

    researchmap

  • Degradation of initiator tRNAMet by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cells. 査読 国際誌

    Kazunori Watanabe, Ryu Miyagawa, Chie Tomikawa, Rie Mizuno, Akihisa Takahashi, Hiroyuki Hori, Kenichi Ijiri

    Nucleic acids research   41 ( 8 )   4671 - 85   2013年4月

     詳細を見る

    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Stress response mechanisms that modulate the dynamics of tRNA degradation and accumulation from the cytoplasm to the nucleus have been studied in yeast, the rat hepatoma and human cells. In the current study, we investigated tRNA degradation and accumulation in HeLa cells under various forms of stress. We found that initiator tRNA(Met) (tRNA(iMet)) was specifically degraded under heat stress. Two exonucleases, Xrn1 and Xrn2, are involved in the degradation of tRNA(iMet) in the cytoplasm and the nucleus, respectively. In addition to degradation, we observed accumulation of tRNA(iMet) in the nucleus. We also found that the mammalian target of rapamycin (mTOR), which regulates tRNA trafficking in yeast, is partially phosphorylated at Ser2448 in the presence of rapamycin and/or during heat stress. Our results suggest phosphorylation of mTOR may correlate with accumulation of tRNA(iMet) in heat-treated HeLa cells.

    DOI: 10.1093/nar/gkt153

    Web of Science

    PubMed

    researchmap

  • Formation of tRNA granules in the nucleus of heat-induced human cells. 査読 国際誌

    Ryu Miyagawa, Rie Mizuno, Kazunori Watanabe, Kenichi Ijiri

    Biochemical and biophysical research communications   418 ( 1 )   149 - 55   2012年2月

     詳細を見る

    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA(Met) (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA(Met) was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

    DOI: 10.1016/j.bbrc.2011.12.150

    Web of Science

    PubMed

    researchmap

  • Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme. 査読 国際誌

    Yukimatsu Toh, Tomoyuki Numata, Kazunori Watanabe, Daijiro Takeshita, Osamu Nureki, Kozo Tomita

    The EMBO journal   27 ( 14 )   1944 - 52   2008年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    CCA-adding enzyme builds the 3'-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D(73)N(74), mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75); D(73) is a discriminator nucleotide and N is either A, G, or U). The mini-D(73)N(74) complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N(74) to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D(73)C(74)C(75) complex, the mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75) complexes adopt inactive open forms. Only the mini-D(73)C(74)U(75) accepts AMP to a similar extent as mini-D(73)C(74)C(75), and ATP shifts the enzyme to a closed, active form and allows U(75) to flip for AMP incorporation. These findings suggest that the 3'-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis.

    DOI: 10.1038/emboj.2008.124

    Web of Science

    PubMed

    researchmap

  • Protein-based peptide-bond formation by aminoacyl-tRNA protein transferase. 査読 国際誌

    Kazunori Watanabe, Yukimatsu Toh, Kyoko Suto, Yoshihiro Shimizu, Natsuhisa Oka, Takeshi Wada, Kozo Tomita

    Nature   449 ( 7164 )   867 - 71   2007年10月

     詳細を見る

    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Eubacterial leucyl/phenylalanyl-tRNA protein transferase (LF-transferase) catalyses peptide-bond formation by using Leu-tRNA(Leu) (or Phe-tRNA(Phe)) and an amino-terminal Arg (or Lys) of a protein, as donor and acceptor substrates, respectively. However, the catalytic mechanism of peptide-bond formation by LF-transferase remained obscure. Here we determine the structures of complexes of LF-transferase and phenylalanyl adenosine, with and without a short peptide bearing an N-terminal Arg. Combining the two separate structures into one structure as well as mutation studies reveal the mechanism for peptide-bond formation by LF-transferase. The electron relay from Asp 186 to Gln 188 helps Gln 188 to attract a proton from the alpha-amino group of the N-terminal Arg of the acceptor peptide. This generates the attacking nucleophile for the carbonyl carbon of the aminoacyl bond of the aminoacyl-tRNA, thus facilitating peptide-bond formation. The protein-based mechanism for peptide-bond formation by LF-transferase is similar to the reverse reaction of the acylation step observed in the peptide hydrolysis reaction by serine proteases.

    DOI: 10.1038/nature06167

    Web of Science

    PubMed

    researchmap

  • Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog. 査読 国際誌

    Kyoko Suto, Yoshihiro Shimizu, Kazunori Watanabe, Takuya Ueda, Shuya Fukai, Osamu Nureki, Kozo Tomita

    The EMBO journal   25 ( 24 )   5942 - 50   2006年12月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNA(Leu) or Phe-tRNA(Phe) as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNA(Leu) or Phe-tRNA(Phe), is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched beta-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys.

    DOI: 10.1038/sj.emboj.7601433

    Web of Science

    PubMed

    researchmap

  • Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltransferase) enzymes. 査読 国際誌

    Kazunori Watanabe, Osamu Nureki, Shuya Fukai, Yaeta Endo, Hiroyuki Hori

    The Journal of biological chemistry   281 ( 45 )   34630 - 9   2006年11月

     詳細を見る

    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes the methyl transfer from S-adenosyl-L-methionine (AdoMet) to the 2'-OH group of the G18 ribose in tRNA. To identify amino acid residues responsible for the tRNA recognition, we have carried out the alanine substitution mutagenesis of the basic amino acid residues that are conserved only in TrmH enzymes and not in the other SpoU proteins. We analyzed the mutant proteins by S-adenosyl-L-homocysteine affinity column chromatography, gel mobility shift assay, and kinetic assay of the methyl transfer reaction. Based on these biochemical studies and the crystal structure of TrmH, we found that the conserved residues can be categorized according to their role (i) in the catalytic center (Arg-41), (ii) in the initial site of tRNA binding (Lys-90, Arg-166, Arg-168, and Arg-176), (iii) in the tRNA binding site required for continuation the catalytic cycle (Arg-8, Arg-19, and Lys-32), (iv) in the structural element involved in release of S-adenosyl-L-homocysteine (Arg-11-His-71-Met-147 interaction), (v) in the assisted phosphate binding site (His-34), or (vi) in an unknown function (Arg-109). Furthermore, the difference between the Kd and Km values for tRNA suggests that the affinity for tRNA is enhanced in the presence of AdoMet. To confirm this idea, we carried out the kinetic studies, a gel mobility shift assay with a mutant protein disrupted in the catalytic center, and the analytical gel-filtration chromatography. Our experimental results clearly show that the enzyme has a semi-ordered sequential mechanism in which AdoMet both enhances the affinity for tRNA and induces formation of the tetramer structure.

    DOI: 10.1074/jbc.M606141200

    Scopus

    PubMed

    researchmap

  • Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family. 査読 国際誌

    Kazunori Watanabe, Osamu Nureki, Shuya Fukai, Ryohei Ishii, Hironori Okamoto, Shigeyuki Yokoyama, Yaeta Endo, Hiroyuki Hori

    The Journal of biological chemistry   280 ( 11 )   10368 - 77   2005年3月

     詳細を見る

    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Transfer RNA (Gm18) methyltransferase (TrmH (SpoU)) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 2'-OH of guanosine 18 in tRNA. This enzyme is a member of the SpoU family of RNA methyltransferases. Recent computational researches have shown that three amino acid sequence motifs are conserved among the SpoU members. Recently, we determined the crystal structures of the apoand AdoMet bound forms of TrmH (Nureki, O., Watanabe, K., Fukai, S., Ishii, R., Endo, Y., Hori, H., and Yokoyama, S. (2004) Structure 12, 593-602). Furthermore, we clarified the AdoMet binding site and proposed the catalytic mechanism. Since the functions of the conserved amino acid residues in the motifs remain unknown, here we have prepared 17 mutants of TrmH and carried out various biochemical studies, including determination of the kinetic parameters for both AdoMet and tRNA, S-adenosyl-l-homocysteine affinity chromatography, gel mobility shift assay, CD spectroscopy, and analytical gel filtration. Our results show that Asn(35), Arg(41), Glu(124), and Asn(152) are involved in binding tRNA and that the Asn(35) residue is involved in the release of S-adenosyl-l-homocysteine. Several residues of TrmH are important for stability of the enzyme. Taken together, our biochemical studies reinforce the previously proposed catalytic mechanism. We also discuss amino acid substitutions in general within the SPOUT superfamily of methyltransferases.

    DOI: 10.1074/jbc.M411209200

    Web of Science

    PubMed

    researchmap

  • Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus. 査読 国際誌

    Hironori Okamoto, Kazunori Watanabe, Yoshiho Ikeuchi, Tsutomu Suzuki, Yaeta Endo, Hiroyuki Hori

    The Journal of biological chemistry   279 ( 47 )   49151 - 9   2004年11月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E. coli tRNAThr. It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.

    DOI: 10.1074/jbc.M408209200

    Web of Science

    PubMed

    researchmap

  • Deep knot structure for construction of active site and cofactor binding site of tRNA modification enzyme. 査読 国際誌

    Osamu Nureki, Kazunori Watanabe, Shuya Fukai, Ryohei Ishii, Yaeta Endo, Hiroyuki Hori, Shigeyuki Yokoyama

    Structure (London, England : 1993)   12 ( 4 )   593 - 602   2004年4月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

    The tRNA(Gm18) methyltransferase (TrmH) catalyzes the 2'-O methylation of guanosine 18 (Gua18) of tRNA. We solved the crystal structure of Thermus thermophilus TrmH complexed with S-adenosyl-L-methionine at 1.85 A resolution. The catalytic domain contains a deep trefoil knot, which mutational analyses revealed to be crucial for the formation of the catalytic site and the cofactor binding pocket. The tRNA dihydrouridine(D)-arm can be docked onto the dimeric TrmH, so that the tRNA D-stem is clamped by the N- and C-terminal helices from one subunit while the Gua18 is modified by the other subunit. Arg41 from the other subunit enters the catalytic site and forms a hydrogen bond with a bound sulfate ion, an RNA main chain phosphate analog, thus activating its nucleophilic state. Based on Gua18 modeling onto the active site, we propose that once Gua18 binds, the phosphate group activates Arg41, which then deprotonates the 2'-OH group for methylation.

    DOI: 10.1016/j.str.2004.03.003

    Web of Science

    PubMed

    researchmap

  • Aquifex aeolicus tRNA (Gm18) methyltransferase has unique substrate specificity. TRNA recognition mechanism of the enzyme. 査読 国際誌

    Hiroyuki Hori, Susumu Kubota, Kazunori Watanabe, Jong-Myong Kim, Tomio Ogasawara, Tatsuya Sawasaki, Yaeta Endo

    The Journal of biological chemistry   278 ( 27 )   25081 - 90   2003年7月

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme. Experiments with chimeric tRNAs and mini- and micro-helix RNAs showed that the recognition region of this enzyme is included within the D-arm structure of tRNALeu and that a bulge is essentially required. Variants of tRNALeu, tRNASer, and tRNAPhe revealed that a combination of certain base pairs in the D-stem is strongly recognized by the enzyme, that 4 bp in the D-stem enhance methyl acceptance activity, and that the Py16Py17G18G19 sequence is important for efficient methyl transfer. The methyl acceptance activities of all the A. aeolicus tRNA genes, which can be classified into 14 categories on the basis of their D-arm structure, were tested. The results clearly showed that the substrate recognition mechanism elucidated by the variant experiments was applicable to their native substrates.

    DOI: 10.1074/jbc.M212577200

    Web of Science

    PubMed

    researchmap

▼全件表示

MISC

  • RNAデリバリー

    渡邉和則, 大槻高史

    核酸科学ハンドブック   524 - 527   2020年12月

     詳細を見る

  • 非天然アミノ酸による生体反応の制御法

    渡邉和則, 大槻高史

    CSJカレントレビュー36号   96 - 100   2020年4月

     詳細を見る

  • RNAキャリア・光応答的RNAデリバリー

    渡邉和則, 大槻高史

    バイオマテリアル学会誌   38 ( 2 )   2020年

     詳細を見る

  • Formation Mechanism and Cellular Functions of Nuclear Stress Bodies Induced by Heat Stress 招待 査読

    Yuichi Miyoshi, Kazunori Watanabe

    Thermal medicine   34 ( 3 )   23 - 34   2018年

     詳細を見る

    記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

    researchmap

  • 創薬を支える光技術-光とRNAを用いた遺伝子発現制御法 招待

    渡邉和則, 大槻高史

    光アライアンス   6   1 - 5   2018年

     詳細を見る

    記述言語:日本語   掲載種別:記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

    researchmap

  • 音増感剤を用いた超音波依存的なペプチドの細胞質内導入法

    大槻 高史, 稲葉 優樹, 北松 瑞生, 中田 栄司, 原田 敦史, 渡邉 和則

    日本DDS学会学術集会プログラム予稿集   33回   172 - 172   2017年6月

     詳細を見る

    記述言語:日本語   出版者・発行元:日本DDS学会  

    researchmap

  • Photocontrolled Intracellular RNA Delivery Using Nanoparticles or Carrier-Photosensitizer Conjugates. 招待 査読 国際誌

    Kazunori Watanabe, Takashi Ohtsuki

    Progress in molecular biology and translational science   139   101 - 19   2016年

     詳細を見る

    記述言語:英語   出版者・発行元:ELSEVIER ACADEMIC PRESS INC  

    Small interfering RNA (siRNA) and short hairpin RNA (shRNA) may potentially treat a wide variety of diseases through RNA interference-mediated silencing of specific genes. MicroRNAs (miRNAs) are endogenous regulatory RNA molecules that also modulate gene expression, and thus potential therapeutic approaches using miRNAs have attracted attention. For clinical application of these small RNAs, efficient and safe RNA delivery to target tissues and cells is necessary. Current challenges to RNA delivery are the penetration of negatively charged RNAs through the cell membrane and specific delivery of RNA into target cells. Photocontrolled intracellular RNA delivery is a promising strategy with high target specificity. This strategy includes photodependent endosomal escape of RNA or photodependent release of RNAs from carrier particles. In this chapter, photocontrolled intracellular RNA delivery methods employing gold or silver nanoparticles, upconversion nanoparticles, proteins, or polymers are discussed.

    DOI: 10.1016/bs.pmbts.2015.10.013

    Web of Science

    PubMed

    researchmap

  • 光でアポトーシスを誘導する方法の開発

    藤原隼人, 畑地祐里, 北松瑞生, 渡邉和則, 大槻高史

    日本化学会中国四国支部大会講演要旨集   2015   58   2015年11月

     詳細を見る

    記述言語:日本語  

    J-GLOBAL

    researchmap

  • Intracellular Delivery of RNA via RNA-Binding Proteins or Peptides 招待 査読

    Kazunori Watanabe, Takashi Ohtsuki

    Intracellular Delivery II   403 - 416   2014年

     詳細を見る

    記述言語:英語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

    DOI: 10.1007/978-94-017-8896-0_19

    researchmap

  • 光化学的に細胞質内に侵入するペプチド分子の設計

    大槻高史, 藤原隼人, 畑地祐里, 北松瑞生, 渡邉和則

    光化学討論会要旨集(CD-ROM)   2014   ROMBUNNO.2P052   2014年

     詳細を見る

    記述言語:日本語  

    J-GLOBAL

    researchmap

  • アミノアシルtRNAタンパク質転移酵素の分子機構の解明 招待 査読

    渡邉和則, 董雪松, 富田耕造

    生化学   81 ( 4 )   294 - 298   2009年

     詳細を見る

    記述言語:日本語   掲載種別:記事・総説・解説・論説等(その他)  

    researchmap

  • Molecular basis for peptide-bond formation by an aminoacyl-tRNA protein transferase

    Kazunori Watanabe, Yukimatsu Toh, Kozo Tomita

    Seikagaku   81 ( 4 )   294 - 298   2009年

     詳細を見る

    記述言語:日本語   掲載種別:書評論文,書評,文献紹介等  

    Scopus

    PubMed

    researchmap

  • tRNAを使うリボソームによらないアミノ酸付加反応:Nエンドルールへの目印

    渡邉和則, 富田耕造

    蛋白質核酸酵素   53 ( 9 )   1152 - 1157   2008年

     詳細を見る

    記述言語:日本語   掲載種別:記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

    researchmap

  • 高度好熱菌由来ポリアミンは高温環境下でRNA修飾系を安定化する

    堀弘幸, 照井祐介, 中本知里, 守谷誠人, 渡辺和則, 鴨直樹, 鈴木勉, 渡辺公綱, 遠藤弥重太, 大島泰郎

    日本分子生物学会年会講演要旨集   28th   108   2005年11月

     詳細を見る

    記述言語:日本語  

    J-GLOBAL

    researchmap

  • Identification of Essential Amino Acid Residues of tRNA (Gm18) Methyltransferase for Methyl-Transfer Activity 査読

    K. Watanabe, H. Hori, Y. Endo

    Nucleic Acids Res. Supplement   1   33 - 34   2001年

     詳細を見る

▼全件表示

講演・口頭発表等

  • 温熱依存的なSAFB顆粒形成はmTORC1が関与している

    的野 恭平, 井上 歩実, 岡田 真実, 山本 理紗子, 大槻 高史, 渡邉 和則

    日本分子生物学会  2021年12月 

     詳細を見る

    会議種別:ポスター発表  

    researchmap

  • miRNA検出のための新規PNA/DNAプローブの設計開発

    田原健太朗, 渡邉和則, 重藤元, 山村昌平, 岸高稚, 北松瑞生, 大槻高史

    日本分子生物学会  2021年12月 

     詳細を見る

    会議種別:ポスター発表  

    researchmap

  • pre-miR-664aとPCDR法を組み合わせた光依存的なアポトーシス誘導法

    渡邉和則, 縄稚朋子, 奥谷瑠璃子, 大槻高史

    日本分子生物学会  2021年12月 

     詳細を見る

    会議種別:ポスター発表  

    researchmap

  • RNA分解を検出する蛍光寿命プロープ

    平田陸, 平川和貴, 島田尚鷹, 渡邉和則, 大槻高史

    日本生化学会  2021年11月 

     詳細を見る

    会議種別:ポスター発表  

    researchmap

  • 細胞内還元環境下でCPPが離脱するCPP融合タンパク質の開発

    小林達哉, 中山真伍, 渡邉和則, 大槻高史

    日本化学会中四国支部会  2021年11月 

     詳細を見る

    会議種別:ポスター発表  

    researchmap

  • miRNA検出のための新規PNA/DNAプローブの設計開発

    田原健太朗, 大槻高史, 渡邊和則, 北松瑞生, 重藤元, 山村昌平, 岸高稚

    日本化学会中四国支部会  2021年11月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • 細胞内への物質輸送のためPCI分子素子の開発

    角菜々子, 渡邉和則, 阿部由佳, 北松瑞生, 大槻高史

    日本生化学会  2021年11月 

     詳細を見る

    会議種別:ポスター発表  

    researchmap

  • 相分離を介した核内ストレス顆粒の形成は温熱抵抗性に関与している 招待

    渡邉和則, 的野恭平, 大槻高史

    日本ハイパーサーミア学会  2021年9月 

     詳細を見る

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    researchmap

  • Fluorophore-PNA-Quencher/Quencher-DNA probe for RNA detection

    Kentaro Tabara, Kazunori Watanabe, Hajime Shigeto, Shohei Yamamura, Takamasa Kishi, Mizuki Kitamatsu, Takashi Ohtsuki

    日本RNA学会  2021年7月 

     詳細を見る

    会議種別:ポスター発表  

    researchmap

  • Development of light-controlled apoptosis induction method 招待

    Kazunori Watanabe

    The 12th International symposium for future technology creating better human health and society  2021年3月 

     詳細を見る

    会議種別:シンポジウム・ワークショップ パネル(指名)  

    researchmap

  • 核内ストレス顆粒の形成は温熱抵抗性に関与している

    渡邉和則, 大槻高史

    日本ハイパーサーミア学会  2020年9月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • mTOR複合体を介した核内ストレス顆粒構成因子SAFB, Satellite Ⅲ RNA顆粒形成機構の解明

    的野 恭平, 井上 歩実, 岡田 真実, 山本 理紗子, 大槻 高史, 渡邉 和則

    日本ハイパーサーミア学会  2020年9月 

     詳細を見る

    記述言語:日本語   会議種別:口頭発表(一般)  

    researchmap

  • mTOR複合体制御による核内ストレス顆粒形成機構の解明

    渡邉和則, 井上歩実, 岡田真実, 山本理紗子, 大槻高史

    日本分子生物学会  2019年12月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • mTOR複合体を介した核内ストレス顆粒形成機構の解明

    渡邉和則, 井上歩実, 岡田真実, 山本理紗子, 大槻高史

    日本ハイパーサーミア学会  2019年9月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • 温熱による開始tRNA分解を介した翻訳抑制と細胞周期との関係性

    渡邉和則, 梅本裕介, 長房すずか, 高橋昭久, 井尻憲一, 大槻高史

    日本ハイパーサーミア学会  2018年8月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • mTORを介した温熱による核内ストレス構造体形成機構

    渡邉和則, 岡田真実, 井上歩実, 山本理紗子, 大槻高史

    日本ハイパーサーミア学会  2017年9月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • 熱ストレスによる開始tRNAMetの細胞内動態

    渡邉和則, 宮川隆, 冨川千恵, 水野利恵, 高橋昭久, 大槻高史, 堀弘幸, 井尻憲一

    日本RNA学会  2013年7月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • 温熱によるtRNA分解促進に対する耐性獲得機構の解明

    渡邉和則, 宮川隆, 水野利恵, 高橋昭久, 井尻憲一

    日本ハイパーサーミア学会  2011年9月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • ストレス誘導によるtRNA分解耐性機構の探索

    渡邉和則

    愛媛大学応用化学科セミナー  2011年7月 

     詳細を見る

    会議種別:公開講演,セミナー,チュートリアル,講習,講義等  

    researchmap

  • アミノアシルtRNAタンパク質転移酵素によるペプチド結合形成機構の解明

    渡辺和則, 董雪松, 富田耕造

    RNA研究若手の会  2007年9月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

  • アミノアシルtRNAタンパク質転移酵素によるペプチド結合反応触媒機構

    渡辺和則, 董雪松, 須藤恭子, 清水義弘, 岡夏央, 和田猛, 富田耕造

    日本RNA学会  2007年7月 

     詳細を見る

    会議種別:口頭発表(一般)  

    researchmap

▼全件表示

受賞

  • 研究助成 優秀賞

    2021年3月   日本健康開発財団  

    渡邉 和則

     詳細を見る

  • 第30回岡山工学振興会科学技術賞

    2018年7月   公益財団法人岡山工学振興会  

    渡邉 和則

     詳細を見る

  • 研究奨励賞

    2017年9月   日本ハイパーサーミア学会  

    渡邉 和則

     詳細を見る

  • 研究功績賞

    2017年3月   岡山大学工学部  

    渡邉 和則

     詳細を見る

  • 生物学研究奨励賞

    2014年10月   両備てい園記念財団  

    渡邉 和則

     詳細を見る

共同研究・競争的資金等の研究

  • 温熱抵抗性に関与するSAFB顆粒を標的にした新規温熱増感剤の開発

    2021年05月 - 2022年03月

    公益財団法人ウエスコ学術振興財団  研究助成金 

      詳細を見る

    担当区分:研究代表者 

    researchmap

  • pre-miR-664a搭載ラクトソームによる光依存的なアポトーシス誘導法の開発

    研究課題/領域番号:21K05277  2021年04月 - 2024年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    渡邉 和則

      詳細を見る

    担当区分:研究代表者 

    配分額:4160000円 ( 直接経費:3200000円 、 間接経費:960000円 )

    researchmap

  • 核内ストレス顆粒形成を標的にした効果的な温熱による細胞死誘導薬剤の探索

    2020年 - 2021年

    一般財団法人 日本健康開発財団  研究助成 

    渡邉 和則

      詳細を見る

  • miR-664a前駆体によるアポトーシス誘導機構の解明

    2019年 - 2021年

    公益財団法人 テルモ生命科学振興財団  研究助成 

    渡邉 和則

      詳細を見る

  • マイクロRNA-664aを用いたアポトーシス制御法の開発とアポトーシス誘導機構の解明

    2018年 - 2019年

    岡山工学振興会  第30回岡山工学振興会科学技術賞 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • RNA分解酵素Xrn1によるRNA認識機構の解明

    2017年 - 2020年

    JSPS  科研費若手B 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • Xrn2による開始tRNA分解を介した翻訳抑制機構の解明

    2017年 - 2019年

    内藤記念科学振興財団  内藤記念科学奨励金・研究助成 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • マイクロRNAを用いた時空間制御可能なアポトーシス誘導法の開発

    2017年 - 2018年

    ノバルティス科学振興財団  ノバルティス研究奨励金 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • 時空間制御可能な神経分化制御法の開発

    2017年 - 2018年

    公益財団法人ウエスコ学術振興財団  研究助成金 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • The degradation of initiator tRNA depended on the cell cycle under heat stress

    2016年

    倉田記念日立科学技術財団  海外渡航費補助金 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • 細胞内におけるRNA分解の観測法の開発とストレス応答研究への応用

    2015年 - 2017年

    JSPS  科研費 挑戦的萌芽 

    大槻 高史

      詳細を見る

    資金種別:競争的資金

    researchmap

  • 光誘導RNA導入法を用いた細胞周期制御法の開発

    2015年 - 2016年

    中島記念国際交流財団  日本人若手研究者研究助成金 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • 光制御による細胞分化誘導法の開発

    2015年 - 2016年

    服部報公会  工学研究奨励援助金 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • PCDR法による2種RNAの時間差導入および細胞機能の制御

    2014年 - 2017年

    JSPS  科研費基盤研究B 

    大槻 高史

      詳細を見る

    資金種別:競争的資金

    researchmap

  • 熱ストレス下での細胞周期依存性tRNA分解促進機構の解明

    2014年 - 2016年

    倉田記念日立科学技術財団  倉田奨励金 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • 熱ストレス下でのtRNA輸送機構の解明

    2014年 - 2016年

    JSPS  科研費若手B 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • マイクロRNAによる細胞分化制御法の開発

    2014年 - 2015年

    両備てい園記念財  研究奨励賞 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • mTOR regulates the degradation of initiator tRNAMet under heat stress

    2014年

    倉田記念日立科学技術財団  海外渡航費補助金 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • 熱ストレス誘導によるtRNA凝集体の形成機構の解明

    2012年 - 2014年

    内藤記念科学振興財団  内藤記念科学奨励金・研究助成 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • タンパク質分解経路における初期シグナル付加反応の分子機構の解明

    2009年 - 2010年

    JSPS  特別研究員奨励費 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • TrmHファミリーの生産物難解メカニズムの解明

    2005年 - 2006年

    理工学振興会  研究奨励賞 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

  • タンパク質構造と機能を元にしたSPOUT酵素群の機能進化についての研究

    2004年 - 2005年

    理工学振興会  研究奨励賞 

    渡邉 和則

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    researchmap

▼全件表示

 

担当授業科目

  • ヘルスシステム統合科学専門英語 (2021年度) 後期  - その他

  • ヘルスシステム統合科学特別研究 (2021年度) 通年  - その他

  • 情報処理入門2(情報機器の操作を含む) (2021年度) 第2学期  - 月1~2

  • 情報処理入門2(情報機器の操作を含む) (2021年度) 第2学期  - 木1~2

  • 生命工学実験1 (2021年度) 第4学期  - 月5,月6,月7,月8,木5,木6,木7,木8

  • 生命工学実験1 (2021年度) 第4学期  - 月5,月6,月7,月8,木5,木6,木7,木8

  • RNA工学 (2021年度) 前期  - 火5~6

  • ヘルスシステム統合科学専門英語 (2020年度) 後期  - その他

  • ヘルスシステム統合科学特別研究 (2020年度) 通年  - その他

  • ヘルスシステム統合科学総合演習 (2020年度) 後期  - その他

  • 生命工学実験1 (2020年度) 第4学期  - 月4,月5,月6,月7,木4,木5,木6,木7

  • 生命工学実験1 (2020年度) 第4学期  - 月4,月5,月6,月7,木4,木5,木6,木7

▼全件表示