Updated on 2024/09/20

写真a

 
Sakai Ryohei
 
Affiliation
Institute of Advanced Medical Sciences, Department of Molecular Oncology, Assistant Professor
Title
Assistant Professor
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Degree

  • 博士(理学) ( 2019.3   関西学院大学 )

Research Interests

  • microautophagy

  • ユビキチン

  • メンブレントラフィック

  • リソソーム

  • RNautophagy

  • 脂質分解

  • オートファジー

Research Areas

  • Life Science / Cell biology

Education

  • 関西学院大学大学院   理工学研究科 博士課程後期課程 生命科学専攻

    2016.4 - 2019.3

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  • 関西学院大学大学院   理工学研究科 博士課程前期課程 生命科学専攻

    2014.4 - 2016.3

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  • Kwansei Gakuin University   School of Science and Technology

    2010.4 - 2014.3

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Research History

  • Nippon Medical School   Department of Molecular Oncology, Institute for Advanced Medical Sciences   Assistant Professor

    2024.9

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  • National Center of Neurology and Psychiatry   National Institute of Neuroscience, Ncnp

    2023.4 - 2024.8

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  • 日本学術振興会   特別研究員(PD)

    2020.4 - 2023.3

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  • 国立精神・神経医療研究センター   神経研究所 疾病研究第四部   流動研究員

    2019.4 - 2020.3

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Professional Memberships

Papers

  • Nucleic acid uptake occurs independent of lysosomal acidification but dependent on ATP consumption during RNautophagy/DNautophagy

    Viorica Raluca Contu, Ryohei Sakai, Yuuki Fujiwara, Chihana Kabuta, Keiji Wada, Tomohiro Kabuta

    Biochemical and Biophysical Research Communications   2023.1

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    Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.bbrc.2022.12.090

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  • The Ubiquitin Ligase RNF34 Participates in the Peripheral Quality Control of CFTR (RNF34 Role in CFTR PeriQC). International journal

    Shogo Taniguchi, Yukiko Ito, Hibiki Kiritani, Asuka Maruo, Ryohei Sakai, Yuji Ono, Ryosuke Fukuda, Tsukasa Okiyoneda

    Frontiers in molecular biosciences   9   840649 - 840649   2022

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    Language:English   Publishing type:Research paper (scientific journal)  

    The peripheral protein quality control (periQC) system eliminates the conformationally defective cystic fibrosis transmembrane conductance regulator (CFTR), including ∆F508-CFTR, from the plasma membrane (PM) and limits the efficacy of pharmacological therapy for cystic fibrosis (CF). The ubiquitin (Ub) ligase RFFL is responsible for the chaperone-independent ubiquitination and lysosomal degradation of CFTR in the periQC. Here, we report that the Ub ligase RNF34 participates in the CFTR periQC in parallel to RFFL. An in vitro study reveals that RNF34 directly recognizes the CFTR NBD1 and selectively promotes the ubiquitination of unfolded proteins. RNF34 was localized in the cytoplasm and endosomes, where RFFL was equally colocalized. RNF34 ablation increased the PM density as well as the mature form of ∆F508-CFTR rescued at low temperatures. RFFL ablation, with the exception of RNF34 ablation, increased the functional PM expression of ∆F508-CFTR upon a triple combination of CFTR modulators (Trikafta) treatment by inhibiting the K63-linked polyubiquitination. Interestingly, simultaneous ablation of RNF34 and RFFL dramatically increased the functional PM ∆F508-CFTR by inhibiting the ubiquitination in the post-Golgi compartments. The CFTR-NLuc assay demonstrates that simultaneous ablation of RNF34 and RFFL dramatically inhibits the degradation of mature ∆F508-CFTR after Trikafta treatment. Therefore, these results suggest that RNF34 plays a crucial role in the CFTR periQC, especially when there is insufficient RFFL. We propose that simultaneous inhibition of RFFL and RNF34 may improve the efficacy of CFTR modulators.

    DOI: 10.3389/fmolb.2022.840649

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  • Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes. International journal

    Katsunori Hase, Viorica Raluca Contu, Chihana Kabuta, Ryohei Sakai, Masayuki Takahashi, Naoyuki Kataoka, Fumihiko Hakuno, Shin-Ichiro Takahashi, Yuuki Fujiwara, Keiji Wada, Tomohiro Kabuta

    Autophagy   16 ( 11 )   1974 - 1988   2020.11

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    Language:English   Publishing type:Research paper (scientific journal)  

    RNautophagy and DNautophagy (RDA) are unconventional autophagic pathways where nucleic acids are directly transported through the lysosomal membrane, then degraded inside lysosomes. We have previously shown that bitopic protein LAMP2C and putative RNA transporter SIDT2, both lysosomal membrane proteins, mediate the direct transport of nucleic acids into lysosomes and that LAMP2C interacts with the nucleic acids and functions as a receptor during RDA. Because SIDT2-mediated RDA occurs in isolated lysosomes that lack LAMP2C, in this study, we tested the hypothesis that SIDT2 itself could also interact with the nucleic acids. Our results show that SIDT2 directly binds RNA and DNA through an arginine-rich motif (ARM) located within its main cytosolic domain, and disruption of this motif dramatically impairs SIDT2-mediated RNautophagic activity. We also found that SIDT2 interacts with exon 1 of HTT (huntingtin) transcript through the ARM in a CAG-dependent manner. Moreover, overexpression of SIDT2 promoted degradation of HTT mRNA and reduced the levels of polyglutamine-expanded HTT aggregates, hallmarks of Huntington disease. In addition, a comparative analysis of LAMP2C and SIDT2 functions at the cellular level revealed that the two proteins exert a synergistic effect on RNautophagic activity and that the ARMs which mediate the interactions of SIDT2 and LAMP2C with RNA are essential for the synergy. Together, our results point out the importance of nucleic acid-binding capacity of SIDT2 for its function in translocating nucleic acids through the lipid bilayer and suggests a potential application of RNautophagy activation to reduce the expression levels of disease-causing toxic proteins. Abbreviations: ACTB/β-actin: actin beta; ARM: arginine-rich motif; CBB: Coomassie Brilliant Blue; CD: cytosolic domain; COX4I1/COX4: cytochrome c oxidase subunit 4I1; E. coli: Escherichia coli; EGFP: enhanced green fluorescent protein; EtBr: ethidium bromide; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GST: glutathione S-transferase; HRP: horseradish peroxidase; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; HTTex1: exon 1 of the HTT gene; LAMP2: lysosomal associated membrane protein 2; LMNA: lamin A/C; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate-buffered saline; PEI: polyethyleneimine; polyQ: polyglutamine; qPCR: quantitative PCR; RAB5A: RAB5A, member RAS oncogene family; RDA: RNautophagy and DNautophagy; SCARB2/LIMP2: scavenger receptor class B member 2; SDS: sodium dodecyl sulfate; SID-1: systemic RNA interference deficient-1; SIDT2: SID1 transmembrane family member 2; WT: wild type.

    DOI: 10.1080/15548627.2020.1712109

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  • Direct uptake mechanism in lysosome required for neuromuscular homeostasis

    Yuuki Fujiwara, Viorica Raluca Contu, Chihana Kabuta, Megumu Ogawa, Hiromi Fujita, Hisae Kikuchi, Ryohei Sakai, Katsunori Hase, Mari Suzuki, Ikuko Koyama-Honda, Michio Inoue, Yasushi Oya, Yukiko U. Inoue, Takayoshi Inoue, Ryosuke Takahashi, Ichizo Nishino, Keiji Wada, Satoru Noguchi, Tomohiro Kabuta

    2020.8

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    Publisher:Cold Spring Harbor Laboratory  

    Regulated degradation of cellular components plays an essential role in homeostasis. Accumulating evidence indicates the importance of lysosomal degradation of cellular proteins1: Dysfunctions in multiple pathways to deliver cytosolic substrates into lysosomes are related to various diseases, including cancers, neurodegenerative diseases, and myopathies2. However, much of the effort at understanding such pathways has been devoted to studies on macroautophagy, which entails vast and dynamic rearrangement of membrane structure, and knowledge on other delivery systems and functions of lysosomes <italic>per se</italic> remains scant. Here, we show that cytosolic proteins are directly imported into lysosomes by a mechanism distinct from any known pathways and degraded. We find that a lysosomal membrane protein, SIDT2, which was previously reported as a putative nucleic acid transporter, is involved in the translocation of substrate proteins in this system. Gain- and loss-of-function analyses reveal that SIDT2 contributes conspicuously to the lysosomal degradation of a wide range of cytosolic proteins in cells at the constitutive level. Furthermore, a dominant-negative type of mutation in <italic>SIDT2</italic> causes familial rimmed vacuolar myopathy in humans. <italic>Sidt2</italic> knockout mice recapitulated typical features of rimmed vacuolar myopathy, including atrophy and accumulation of cytoplasmic inclusions in skeletal muscles. These results reveal a previously unknown pathway of proteolysis in lysosomes and highlight the importance of noncanonical types of autophagy in human physiology and pathophysiology.

    DOI: 10.1101/2020.08.11.245688

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  • Discovery of a protein uptake pathway in lysosomes

    Yuuki Fujiwara, Viorica Raluca Contu, Chihana Kabuta, Megumu Ogawa, Hiromi Fujita, Hisae Kikuchi, Ryohei Sakai, Katsunori Hase, Mari Suzuki, Ikuko Koyama-Honda, Michio Inoue, Yasushi Oya, Yukiko U. Inoue, Takayoshi Inoue, Ryosuke Takahashi, Ichizo Nishino, Keiji Wada, Satoru Noguchi, Tomohiro Kabuta

    bioRxiv   2020.8

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    Language:English   Publisher:Cold Spring Harbor Laboratory  

    Regulated degradation of cellular components plays an essential role in homeostasis. Accumulating evidence indicates the importance of lysosomal degradation of cellular proteins1: Dysfunctions in multiple pathways to deliver cytosolic substrates into lysosomes are related to various diseases, including cancers, neurodegenerative diseases, and myopathies2. However, much of the effort at understanding such pathways has been devoted to studies on macroautophagy, which entails vast and dynamic rearrangement of membrane structure, and knowledge on other delivery systems and functions of lysosomes <italic>per se</italic> remains scant. Here, we show that cytosolic proteins are directly imported into lysosomes by a mechanism distinct from any known pathways and degraded. We find that a lysosomal membrane protein, SIDT2, which was previously reported as a putative nucleic acid transporter, is involved in the translocation of substrate proteins in this system. Gain- and loss-of-function analyses reveal that SIDT2 contributes conspicuously to the lysosomal degradation of a wide range of cytosolic proteins in cells at the constitutive level. Furthermore, a dominant-negative type of mutation in <italic>SIDT2</italic> causes familial rimmed vacuolar myopathy in humans. <italic>Sidt2</italic> knockout mice recapitulated typical features of rimmed vacuolar myopathy, including atrophy and accumulation of cytoplasmic inclusions in skeletal muscles. These results reveal a previously unknown pathway of proteolysis in lysosomes and highlight the importance of noncanonical types of autophagy in human physiology and pathophysiology.

    DOI: 10.1101/2020.08.11.245688

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  • The integral function of the endocytic recycling compartment is regulated by RFFL-mediated ubiquitylation of Rab11 effectors. International journal

    Ryohei Sakai, Ryosuke Fukuda, Shin Unida, Misaki Aki, Yuji Ono, Akinori Endo, Satoshi Kusumi, Daisuke Koga, Toshiaki Fukushima, Masayuki Komada, Tsukasa Okiyoneda

    Journal of cell science   132 ( 3 )   2019.2

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    Language:English   Publishing type:Research paper (scientific journal)  

    Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors in vitro, but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.

    DOI: 10.1242/jcs.228007

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  • Chaperone-Independent Peripheral Quality Control of CFTR by RFFL E3 Ligase. International journal

    Tsukasa Okiyoneda, Guido Veit, Ryohei Sakai, Misaki Aki, Takeshi Fujihara, Momoko Higashi, Seiko Susuki-Miyata, Masanori Miyata, Norihito Fukuda, Akihiko Yoshida, Haijin Xu, Pirjo M Apaja, Gergely L Lukacs

    Developmental cell   44 ( 6 )   694 - 708   2018.3

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    Language:English   Publishing type:Research paper (scientific journal)  

    The peripheral protein quality control (QC) system removes non-native membrane proteins, including ΔF508-CFTR, the most common CFTR mutant in cystic fibrosis (CF), from the plasma membrane (PM) for lysosomal degradation by ubiquitination. It remains unclear how unfolded membrane proteins are recognized and targeted for ubiquitination and how they are removed from the apical PM. Using comprehensive siRNA screens, we identified RFFL, an E3 ubiquitin (Ub) ligase that directly and selectively recognizes unfolded ΔF508-CFTR through its disordered regions. RFFL retrieves the unfolded CFTR from the PM for lysosomal degradation by chaperone-independent K63-linked poly-ubiquitination. RFFL ablation enhanced the functional expression of cell-surface ΔF508-CFTR in the presence of folding corrector molecules, and this effect was further improved by inhibiting the Hsc70-dependent ubiquitination machinery. We propose that multiple peripheral QC mechanisms evolved to dispose of non-native PM proteins and to preserve cellular proteostasis, even at the cost of eliminating partially functional polypeptides.

    DOI: 10.1016/j.devcel.2018.02.001

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Misc.

  • The lysosomal membrane protein LAMP2B regulates mammalian microlipophagy

    酒井 了平, 相澤 修, 小山 郁子[本田], 李 賢哲, 株田 千華, 株田 智弘

    日本生化学会大会プログラム・講演要旨集   95回   2T17m - 04   2022.11

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    Language:Japanese   Publisher:(公社)日本生化学会  

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  • リサイクリングエンドソーム機能制御におけるユビキチンリガーゼRFFLの機能解析

    酒井 了平, 宇仁田 伸, 福田 亮介, 沖米田 司

    日本生化学会大会プログラム・講演要旨集   91回   [1T13e - 179)]   2018.9

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    Language:Japanese   Publisher:(公社)日本生化学会  

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  • 形質膜タンパク質品質管理に関わるエンドソーム局在ユビキチンリガーゼの機能解析

    沖米田司, 酒井了平, 東桃子, 藤原健

    日本生化学会大会(Web)   90th   ROMBUNNO.1P‐0169 (WEB ONLY) - 0169]   2017.12

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    Language:Japanese   Publisher:生命科学系学会合同年次大会運営事務局  

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  • エンドソーム膜タンパク品質管理に関わるRING型ユビキチンリガーゼの役割

    酒井了平, 安藝美咲, 鈴木真治, 宮田(薄)聖子, 宮田将徳, 沖米田司

    日本生化学会大会(Web)   88th   1P0430 (WEB ONLY) - [1P0430]   2015.12

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    Language:Japanese   Publisher:(公社)日本生化学会  

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Awards

  • 最優秀ポスター発表賞

    2023.3   国立精神・神経医療研究センター 神経研究所  

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  • 仁田記念賞

    2019.2   関西学院大学  

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  • 若手優秀発表賞

    2018.9   第91回日本生化学会大会  

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  • ポスター優秀賞

    2016.11   新学術領域ユビキチンバイオロジー 第三回班会議  

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Research Projects

  • Comprehensive and Functional Analysis of Microlipophagy Regulators

    Grant number:24K18376  2024.4 - 2026.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Early-Career Scientists

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    Grant amount:\4680000 ( Direct Cost: \3600000 、 Indirect Cost:\1080000 )

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  • オートファジー系新規脂質分解システム制御因子の網羅的探索とその機能解明

    2023.7 - 2028.3

    公益財団法人 武田科学振興財団 医学系研究助成(基礎) 

    酒井 了平

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  • オートファジー系新規脂質分解システムの分子メカニズム及び生体機能の解明

    Grant number:20J00363  2020.4 - 2023.3

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    酒井 了平

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    Grant amount:\4030000 ( Direct Cost: \3100000 、 Indirect Cost:\930000 )

    リソソームによる細胞内分解システム「オートファジー」には、マクロオートファジーを含む4つの形態が存在する。これまで、マクロオートファジーに関しては精力的に研究されてきた。ところが、その他の非典型的オートファジーに関する分子機構や普遍性の多くは解明途中にあるため、リソソームそのものの機能解明が強く求められている。また、オートファジーによる分解基質の研究対象は、殆どがタンパク質であり核酸・脂質の分解機構の解明は遅れている。当研究室では、これまでに核酸を標的とした非典型的オートファジーに関する分子機構の研究が進められてきた。その研究過程において、採用者は脂肪滴内の脂質がリソソームで分解されることを発見し、その制御因子を同定し解析を進めてきた。
    2021年度は標的因子による脂質分解の分子メカニズムの解明を行った。脂質分解に関わると考えられるアミノ酸変異体を作製したところ、脂質分解が抑制され、脂質分解に必須なアミノ酸の同定に成功した。また、新規脂質分解における脂肪滴とリソソームの接触部位の解析を行うため、電子顕微鏡を用いた画像解析の取得に成功し、新規脂質分解経路の詳細なメカニズムの一端を解明した。さらに、in vivo解析においては標的因子のトランスジェニックマウスを作製し、高脂肪食飼料投与に伴う生活習慣病病態モデルマウスの解析に取り組んできた。標的因子トランスジェニックマウスは高脂肪食飼料を投与しても体重や脂肪、肝臓の重量増加が観察されなかったことから、in vivoにおいても標的因子は脂質分解に寄与していることが明らかとなった。

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