保有資格:
第1種放射線取扱主任者
第1種衛生工学衛生管理者
2025/04/30 更新
研究活動
教育活動
2025/04/30 更新
保有資格:
第1種放射線取扱主任者
第1種衛生工学衛生管理者
博士(工学) ( 愛媛大学 )
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
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
岡山大学 学術研究院ヘルスシステム統合科学学域 准教授
2023年4月 - 現在
詳細を見る
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 - 2023年3月
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
日本分子生物学会
詳細を見る
日本生化学会
詳細を見る
日本RNA学会
詳細を見る
日本ハイパーサーミア学会
詳細を見る
日本ハイパーサーミア学会 編集委員会委員
2024年4月 - 現在
詳細を見る
団体区分:学協会
日本ハイパーサーミア学会 代議員
2023年9月 - 現在
詳細を見る
Abdul Basith Fithroni, Haruki Inoue, Shengli Zhou, Taufik Fatwa Nur Hakim, Takashi Tada, Minoru Suzuki, Yoshinori Sakurai, Manabu Ishimoto, Naoyuki Yamada, Rani Sauriasari, Wolfgang A. G. Sauerwein, Kazunori Watanabe, Takashi Ohtsuki, Eiji Matsuura
Cells 14 ( 1 ) 60 - 60 2025年1月
詳細を見る
掲載種別:研究論文(学術雑誌) 出版者・発行元:MDPI AG
Boron (B) neutron capture therapy (BNCT) is a novel non-invasive targeted cancer therapy based on the nuclear capture reaction 10B (n, alpha) 7Li that enables the death of cancer cells without damaging neighboring normal cells. However, the development of clinically approved boron drugs remains challenging. We have previously reported on self-forming nanoparticles for drug delivery consisting of a biodegradable polymer, namely, “AB-type” Lactosome® nanoparticles (AB-Lac particles)- highly loaded with hydrophobic B compounds, namely o-Carborane (Carb) or 1,2-dihexyl-o-Carborane (diC6-Carb), and the latter (diC6-Carb) especially showed the “molecular glue” effect. Here we present in vivo and ex vivo studies with human pancreatic cancer (AsPC-1) cells to find therapeutically optimal formulas and the appropriate treatment conditions for these particles. The biodistribution of the particles was assessed by the tumor/normal tissue ratio (T/N) in terms of tumor/muscle (T/M) and tumor/blood (T/B) ratios using near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG). The in vivo and ex vivo accumulation of B delivered by the injected AB-Lac particles in tumor lesions reached a maximum by 12 h post-injection. Irradiation studies conducted both in vitro and in vivo showed that AB-Lac particles-loaded with either 10B-Carb or 10B-diC6-Carb significantly inhibited the growth of AsPC-1 cancer cells or strongly inhibited their growth, with the latter method being significantly more effective. Surprisingly, a similar in vitro and in vivo irradiation study showed that ICG-labeled AB-Lac particles alone, i.e., without any 10B compounds, also revealed a significant inhibition. Therefore, we expect that our ICG-labeled AB-Lac particles-loaded with 10B compound(s) may be a novel and promising candidate for providing not only NIRF imaging for a practical diagnosis but also the dual therapeutic effects of induced cancer cell death, i.e., “theranostics”.
Photochemical internalization of mRNA using a photosensitizer and nucleic acid carriers 査読
Hayaki Maemoto, Ryohei Suzaki, Kazunori Watanabe, Keiji Itaka, Takashi Ohtsuki
European Journal of Medicinal Chemistry Reports 13 100242 - 100242 2024年12月
詳細を見る
Configuration of two cysteine residues in a ring within a stapled Bim peptide affects the secondary structure and apoptotic activity 査読
Shengli Zhou, Fuka Nishimura, Kazuhaya Wada, Kaho Fujii, Takeshi Kondo, Kazunori Watanabe, Yoshitane Imai, Takashi Ohtsuki, Mizuki Kitamatsu
Bioorganic & Medicinal Chemistry Letters 112 129915 - 129915 2024年11月
詳細を見る
In Vitro Study of Tumor-Homing Peptide-Modified Magnetic Nanoparticles for Magnetic Hyperthermia 査読
Shengli Zhou, Kaname Tsutsumiuchi, Ritsuko Imai, Yukiko Miki, Anna Kondo, Hiroshi Nakagawa, Kazunori Watanabe, Takashi Ohtsuki
Molecules 29 ( 11 ) 2632 - 2632 2024年6月
詳細を見る
掲載種別:研究論文(学術雑誌) 出版者・発行元:MDPI AG
Cancer cells have higher heat sensitivity compared to normal cells; therefore, hyperthermia is a promising approach for cancer therapy because of its ability to selectively kill cancer cells by heating them. However, the specific and rapid heating of tumor tissues remains challenging. This study investigated the potential of magnetic nanoparticles (MNPs) modified with tumor-homing peptides (THPs), specifically PL1 and PL3, for tumor-specific magnetic hyperthermia therapy. The synthesis of THP-modified MNPs involved the attachment of PL1 and PL3 peptides to the surface of the MNPs, which facilitated enhanced tumor cell binding and internalization. Cell specificity studies revealed an increased uptake of PL1- and PL3-MNPs by tumor cells compared to unmodified MNPs, indicating their potential for targeted delivery. In vitro hyperthermia experiments demonstrated the efficacy of PL3-MNPs in inducing tumor cell death when exposed to an alternating magnetic field (AMF). Even without exposure to an AMF, an additional ferroptotic pathway was suggested to be mediated by the nanoparticles. Thus, this study suggests that THP-modified MNPs, particularly PL3-MNPs, hold promise as a targeted approach for tumor-specific magnetic hyperthermia therapy.
Self-assembly of Peptide Amphiphiles with Alkyl Groups for siRNA Delivery 査読
Taufik F.N. Hakim, Kazunori Watanabe, Shomu Fujimoto, Mizuki Kitamatsu, Takashi Ohtsuki
Chemistry Letters 2023年9月
詳細を見る
Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo. 査読 国際誌
Yuka Ikawa, Takuya Wakai, Hiroaki Funahashi, Tet Htut Soe, Kazunori Watanabe, Takashi Ohtsuki
Scientific reports 13 ( 1 ) 13050 - 13050 2023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)
Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.
FRET probe for detecting two mutations in one EGFR mRNA. 査読 国際誌
Myat Thu, Kouta Yanai, Hajime Shigeto, Shohei Yamamura, Kazunori Watanabe, Takashi Ohtsuki
The Analyst 148 ( 11 ) 2626 - 2632 2023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)
Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.
DOI: 10.1039/d3an00554b
Photochemical internalizationに基づく光依存的細胞質内RNA導入法の副作用低減 査読
坂東晃成, 渡邉和則, 大槻高史
日本レーザー医学会誌 44 ( 1 ) 62 - 68 2023年4月
詳細を見る
Ultrasound-dependent RNAi using TatU1A-rose bengal conjugate. 査読 国際誌
Nanako Sumi, Shota Nagahiro, Eiji Nakata, Kazunori Watanabe, Takashi Ohtsuki
Bioorganic & medicinal chemistry letters 68 128767 - 128767 2022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)
Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics.
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.
Kazunori Watanabe, Tomoko Nawachi, Ruriko Okutani, Takashi Ohtsuki
Scientific Reports 11 ( 1 ) 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.
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
Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells 査読
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
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 BV
The A(3)B type Lactosome comprised of poly(sarcosine)(3)-block-poly(t-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A(3)B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photo-induced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT. (C) 2021 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
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.
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
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.
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.
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
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.
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.
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
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.
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
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
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.
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
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.
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
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.
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.
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
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.
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
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.
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.
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
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.
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.
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.
Structural change of tRNA (Gm18) methyltransferase by binding of methyl donor analogues. 査読
Watanabe K, Nureki O, Fukai S, Endo Y, Hori H
Nucleic acids symposium series (2004) ( 49 ) 301 - 302 2005年
詳細を見る
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.
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.
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.
渡邉和則, 大槻高史
Drug Delivery System 37 ( 3 ) 229 - 236 2022年7月
詳細を見る
Tet Htut Soe, Kazunori Watanabe, Takashi Ohtsuki
Molecules 26 ( 1 ) 36 - 36 2020年12月
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) 出版者・発行元:MDPI AG
Endosomal escape in cell-penetrating peptide (CPP)-based drug/macromolecule delivery systems is frequently insufficient. The CPP-fused molecules tend to remain trapped inside endosomes and end up being degraded rather than delivered into the cytosol. One of the methods for endosomal escape of CPP-fused molecules is photochemical internalization (PCI), which is based on the use of light and a photosensitizer and relies on photoinduced endosomal membrane destabilization to release the cargo molecule. Currently, it remains unclear how this delivery strategy behaves after photostimulation. Recent findings, including our studies using CPP-cargo-photosensitizer conjugates, have shed light on the photoinduced endosomal escape mechanism. In this review, we discuss the structural design of CPP-photosensitizer and CPP-cargo-photosensitizer conjugates, and the PCI mechanism underlying their application.
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 ( 36 ) 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
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 ( 6 ) 1 - 5 2018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
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.
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
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
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
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
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
熱耐性と開始tRNA分解を介した翻訳抑制および細胞死との関係性の解明
青木結子, 梅本裕介, 長房すずか, 高橋昭久, 井尻憲一, 大槻高史, 渡邉和則
日本分子生物学会 2023年12月
詳細を見る
熱ストレス依存的に起こる核内ストレス顆粒形成に関与するカルシウムイオンシグナル伝達機構解明
古谷優治, 的野恭平, 大槻高史, 渡邉和則
日本分子生物学会 2023年12月
詳細を見る
熱ストレスに依存した開始tRNA分解に関与するXrn1, Xrn2活性化機構の解明
渡邉和則, 岩城香菜子, 篠原里菜, 安田奈緒, 甲斐健太郎, 大槻高史
日本分子生物学会 2023年12月
詳細を見る
BNCT 治療に向けた生分解性ミセル型ホウ素製剤の開発
井上晴喜, Abdul Basith Fithroni, 渡邉和則, 松浦栄次, 大槻高史
日本バイオマテリアル学会 2023年11月
詳細を見る
腫瘍特異性の付加とアポトーシス誘導効率を向上させた光温熱剤の開発
杉原桃香, 大槻高史, 渡邉和則
日本バイオマテリアル学会 2023年11月
詳細を見る
熱ストレス依存的なSAFB顆粒形成を誘導するCa2+-PAシグナル伝達機構解明
古谷優治, 的野恭平, 高原茉莉, 大槻高史, 渡邉和則
第47回日本分子生物学会 2024年12月
詳細を見る
光化学的内在化による細胞質内mRNAデリバリー
前本隼玖, 渡邉和則, 高原茉莉, 位髙啓史, 大槻高史
第47回日本分子生物学会 2024年12月
詳細を見る
近赤外光に依存した高効率なアポトーシス誘導を可能とする光温熱剤の開発
渡邉和則, 宮本麻衣, 松浦栄次, 大槻高史
第47回日本分子生物学会 2024年12月
詳細を見る
哺乳動物初期胚への光による時空間特異的なRNA導入
大槻高史, 井川優風, 若井拓哉, 舟橋弘晃, 渡邉和則
第18回バイオ関連化学会 2024年9月
詳細を見る
ケージド相分離液滴の光応答挙動
坂東晃成, 渡邉和則, 高原茉莉, 藤原誠一, 金崎佑紀, 北松瑞生, 大槻高史
第18回バイオ関連化学会 2024年9月
詳細を見る
酵素反応を用いた部位特異的脂質化抗体の合成とその応用
高原茉莉, 渡邉和則, 大槻高史
第18回バイオ関連化学会 2024年9月
詳細を見る
近赤外光照射により高効率なアポトーシス誘導を行う光温熱剤の開発
渡邉和則, 宮本麻衣, 松浦栄次, 大槻高史
第41回日本ハイパーサーミア学会 2024年9月
詳細を見る
SAFB顆粒の形成には温熱依存的なホスファチジン酸濃度上昇が関与する
古谷優治, 大槻高史, 渡邉和則
第41回日本ハイパーサーミア学会 2024年9月
詳細を見る
Cell-penetrating peptide/photosensitizer conjugates for photo-triggered cytosolic delivery of RNAs and peptides
Takashi Ohtsuki, Mizuki Kitamatsu, Kazunori Watanabe
International Congress on Photobiology 2024年8月
詳細を見る
磁気ハイパーサーミアのための腫瘍特異的磁性ナノ粒子
周聖力, 今井律子, 三木裕紀子, 近藤杏菜, 中川大, 堤内要, 渡邉和則, 大槻高史
第40回DDS学会 2024年7月
詳細を見る
Self-Assembly Nano Micelle of Alkylated Peptide Amphiphiles as Promising siRNA Delivery to Pancreatic Cancer Cells
Taufik F.N. Hakim, Kazunori Watanabe, Shoumu Fujimoto, Mizuki Kitamatsu, Takashi Ohtsuki
第40回DDS学会 2024年7月
詳細を見る
HSF1の液-液相分離機構と翻訳後修飾との関係性について
渡邉和則, 小笠原悠人, 大槻高史
日本ハイパーサーミア学会 2023年9月
詳細を見る
温熱依存的に形成されるSAFB顆粒形成に関与するカルシウムイオンシグナル伝達機構解明
古谷優治, 大槻高史, 渡邉和則
日本ハイパーサーミア学会 2023年9月
詳細を見る
腫瘍特異的ペプチドを用いた磁気温熱療法に向けた薬剤送達法の開発
周聖力, 今井律子, 三木裕紀子, 近藤杏菜, 中川大, 河合憲康, 堤内要, 渡邉和則, 大槻高史
日本ハイパーサーミア学会 2023年9月
詳細を見る
Development of a tumor-homing peptide-mediated drug delivery method for magnetic hyperthermia
Shengli Zhou, Ritsuko Imai, Yukiko Miki, Anna Kondo, Hiroshi Nakagawa, Noriyasu Kawai, Kaname Tsutsumiuchi, Kazunori Watanabe, Takashi Ohtsuk
Asian chemical biology conference 2023年8月
詳細を見る
Development and Characterization of Novel Peptide Amphiphiles for siRNA Delivery
Taufik F.N. Hakim, Kazunori Watanabe, Mizuki Kitamatsu, Takashi Ohtsuki
第七回日本核酸医薬学会 2023年7月
詳細を見る
光増感剤と核酸キャリアの併用による光依存的mRNAデリバリー法
前本隼玖, 渡邉和則, 位髙啓史, 大槻高史
第45回日本光医学・光生物学会 2023年6月
詳細を見る
温熱による開始tRNA分解と細胞周期依存的な翻訳抑制の関係性について
竹本理恵, 梅本裕介, 長房すずか, 高橋昭久, 井尻憲一, 大槻高史, 渡邉和則
日本分子生物学会 2022年12月
詳細を見る
HSF1、SASFB顆粒形成の抑制は温熱によるアポトーシスを増強する
渡邉和則, 大槻高史
日本分子生物学会 2022年12月
詳細を見る
PCI法の副作用を減らす試み
坂東晃成, 渡邉和則, 大槻高史
日本分子生物学会 2022年12月
詳細を見る
哺乳動物における初期胚発生の光制御法の開発
井川優風, 若井拓哉, 舟橋弘晃, 渡邉和則, 大槻高史
日本バイオマテリアル学会 2022年11月
詳細を見る
効率的なsiRNAの光依存的細胞内送達能を持つ生分解性ナノキャリアの開発
田中七星, 渡邉和則, 小渕浩嗣, 久保貴紀, 松浦栄次, 大槻高史
日本バイオマテリアル学会 2022年11月
詳細を見る
pre-miR-664a搭載ラクトソームを用いた光依存的なアポトーシス誘導法の開発
宮本麻衣, 大槻高史, 松浦栄次, 渡邉和則
バイオ関連化学シンポジウム 2022年9月
詳細を見る
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
生物学研究奨励賞
2023年11月 両備てい園記念財団
渡邉和則
詳細を見る
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
ケージド相分離ペプチドからなる相分離液滴の光制御と応用
研究課題/領域番号:25K01897 2025年04月 - 2028年03月
日本学術振興会 科学研究費助成事業 基盤研究(B)
大槻 高史、渡邉和則、北松瑞生、高原茉莉
詳細を見る
第一および第二近赤外光に依存したRNA輸送による高効率アポトーシス誘導剤の開発
2024年06月 - 2025年03月
公益財団法人岡山工学振興会 第36回 一般研究助成
詳細を見る
第一、第二近赤外光を用いた高効率アポトーシス誘導法の開発
2024年06月 - 2025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
SAFB顆粒形成阻害を標的とした新規作用機序を介した温熱増感剤の開発
2024年04月 - 2025年03月
公益財団法人 天野工業技術研究所 2024年度(前期募集)研究助成金
詳細を見る
高効率なアポトーシス誘導を可能とする新規光温熱剤の開発
2023年11月 - 2025年03月
公益財団法人 テルモ生命科学振興財団 研究助成
詳細を見る
がん細胞選択的光温熱剤の開発
2023年11月 - 2024年08月
公益財団法人両備檉園記念財団 研究助成
詳細を見る
pre-miR-664aと腫瘍特異性を持ったペプチドを組み合わせることで高効率な細胞死誘導を可能とする光温熱剤の開発
2023年05月 - 2024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
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
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 )
本研究は、【アポトーシスを誘導するpre-miR-664aの標的mRNAの解明】、【pre-miR-664aとラクトソームを組み合わせた光依存的なアポトーシスの誘導法の開発】を目的に研究を進めている。2021年度は下記の課題を実施しました。
(1) pre-miR-664aにより発現変動するmRNAの同定の試み
マイクロアレイを用いてpre-miR-664aにより発現減少するmRNAをいくつか同定することができた。
(2) 光依存的なアポトーシス誘導
ラクトソームに細胞膜透過性ペプチド (CPP) を介してpre-miR-664aを搭載することに成功した。そこで、粒径測定やゼータ電位の測定を行なった。次に、光依存的にアポトーシスを誘導することができるか検証した。その結果、pre-miR-664aを搭載したラクトソームを用いることで光照射をしなければアポトーシスが誘導されず、光照射依存的にアポトーシスを誘導することができた。しかし、標的のないpre-miR-controlでも光依存的にアポトーシスが誘導されてしまった。そこで、ラクトソームに搭載している光増感剤を変更したpre-miR-664a搭載ラクトソームの調製に成功した。しかしながら、pre-miR-664a及びpre-miR-controlともに光依存的にアポトーシスが誘導されていた。このことから、光増感剤の力によりアポトーシスが誘導されていることが明らかになった。
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
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
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
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
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
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
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
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
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
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
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
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
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
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
PCDR技術による細胞機能の光制御
研究課題/領域番号:25282232 2013年04月 - 2016年03月
日本学術振興会 科学研究費助成事業 基盤研究(B)
大槻 高史, 渡邉 和則
詳細を見る
配分額:18590000円 ( 直接経費:14300000円 、 間接経費:4290000円 )
本研究では、筆者らが開発した「光依存的な細胞質内RNA導入技術」(Photoinduced Cytosolic Dispersion of RNA; PCDR技術)をベースに「可視光および近赤外光による細胞機能の制御系」を開発した。近赤外光による細胞質内RNA導入法の開発、複数波長の光による複数のRNA導入のタイミング制御、3次元的な細胞集団への適用、光による細胞内翻訳や分化の制御などに取り組んだ。
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
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
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
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
ヘルスシステム統合科学アドバンストインターンシップ (2024年度) 通年 - その他
ヘルスシステム統合科学専門英語 (2024年度) 後期 - その他
ヘルスシステム統合科学特別研究 (2024年度) 通年 - その他
ヘルスシステム統合科学特別研究 (2024年度) 通年 - その他
分子輸送学 (2024年度) 前期 - その他
化学・生命系実験2 (2024年度) 第4学期 - 月5~8
合成化学実験1 (2024年度) 第4学期 - 月5~8
情報処理入門2(情報機器の操作を含む) (2024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) (2024年度) 第2学期 - 木1~2
材料プロセス実験1 (2024年度) 第4学期 - 月5~8
生体分子輸送学 (2024年度) 後期 - 金1~2
生命工学実験1 (2024年度) 第4学期 - 月5~8
遺伝子工学 (2024年度) 第2学期 - 月1~2
遺伝子工学 (2024年度) 第2学期 - 月1~2
ヘルスシステム統合科学アドバンストインターンシップ (2023年度) 通年 - その他
ヘルスシステム統合科学専門英語 (2023年度) 後期 - その他
ヘルスシステム統合科学特別研究 (2023年度) 通年 - その他
ヘルスシステム統合科学特別研究 (2023年度) 通年 - その他
分子輸送学 (2023年度) 前期 - その他
化学・生命系実験2 (2023年度) 第4学期 - 月5~8
合成化学実験1 (2023年度) 第4学期 - 月5~8
情報処理入門2(情報機器の操作を含む) (2023年度) 第2学期 - 木1~2
情報処理入門2(情報機器の操作を含む) (2023年度) 第2学期 - 月1~2
材料プロセス実験1 (2023年度) 第4学期 - 月5~8
生命工学実験1 (2023年度) 第4学期 - 月5~8
遺伝子工学 (2023年度) 第2学期 - 月1~2
遺伝子工学 (2023年度) 第2学期 - 月1~2
ヘルスシステム統合科学専門英語 (2022年度) 後期 - その他
ヘルスシステム統合科学特別研究 (2022年度) 通年 - その他
化学・生命系実験2 (2022年度) 第4学期 - 月5~8
合成化学実験1 (2022年度) 第4学期 - 月5~8
情報処理入門2(情報機器の操作を含む) (2022年度) 第2学期 - 木1~2
情報処理入門2(情報機器の操作を含む) (2022年度) 第2学期 - 月1~2
材料プロセス実験1 (2022年度) 第4学期 - 月5~8
生命工学実験1 (2022年度) 第4学期 - 月5~8
RNA工学 (2022年度) 前期 - 不開講
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 - 木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 - 月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
Copyright Okayama University