研究者紹介システム

蜷川 暁
ニナガワ サトシ
バイオシグナル総合研究センター
助教
生物関係
Last Updated :2024/02/02

研究者情報

所属

  • 【主配置】

    バイオシグナル総合研究センター
  • 【配置】

    農学部 資源生命科学科, 大学院農学研究科 資源生命科学専攻

学位

  • 博士(理学), 京都大学
  • 小胞体におけるタンパク質品質管理機構の解析

授業科目

研究ニュース

研究活動

研究キーワード

  • タンパク質品質管理
  • mannose trimming
  • EDEM
  • 小胞体関連分解

研究分野

  • ライフサイエンス / 動物生命科学
  • ライフサイエンス / 応用分子細胞生物学
  • ライフサイエンス / 細胞生物学

受賞

  • 2023年04月 長瀬研究振興賞

  • 2020年 日本糖質学会, 日本糖質学会奨励賞, 高等動物におけるN 型糖鎖のマンノース切除を基軸とした小胞体関連分解機構の解析

  • 2020年 日本生化学会, 日本生化学会奨励賞, 遺伝子破壊法を用いた小胞体関連分解機構の解明

  • 2017年 日本細胞生物学会, 日本細胞生物学会論文賞, SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation

    杉本 岳大、蜷川 暁

  • 2016年06月 日本細胞生物学会, 若手優秀発表賞, 分解執行局域における新展開

  • 2008年 Kyoto University Evolution and Biodiversity from Genome to Ecosystem, gCOE poster Award, Identification and analysis of Derlins Interacting Protein

論文

  • Satoshi Ninagawa, Masaki Matsuo, Deng Ying, Shinya Aso, Kazutoshi Matsushita, Akane Fueki, Shunsuke Saito, Koshi Imami, Yasuhiko Kizuka, Tetsushi Sakuma, Takashi Yamamoto, Hirokazu Yagi, Koichi Kato, Kazutoshi Mori

    2023年10月19日

  • Tomoyo Ikeda, Tokiro Ishikawa, Satoshi Ninagawa, Tetsuya Okada, Masaya Ono, Kazutoshi Mori

    When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or β-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and β-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, β-oxidation, triacylglycerol, cholesterol, export.

    2023年07月19日, Cell structure and function, 48 (2), 123 - 133, 英語, 国内誌

    研究論文(学術雑誌)

  • Shengyu Jin, Byungseok Jin, Tokiro Ishikawa, Satoshi Ninagawa, Tetsuya Okada, Sho Koyasu, Hiroshi Harada, Kazutoshi Mori

    To survive poor nutritional conditions, tumor cells activate the unfolded protein response, which is composed of the IRE1, PERK and ATF6 arms, to maintain the homeostasis of the endoplasmic reticulum, where secretory and transmembrane proteins destined for the secretory pathway gain their correct three dimensional structure. The requirement of the IRE1 and PERK arms for tumor growth in nude mice is established. Here, we investigated the requirement for the ATF6 arm, which consists of ubiquitously expressed ATF6α and ATF6β, by constructing ATF6α-knockout, ATF6β-knockout and ATF6α/β-double knockout in HCT116 cells derived from human colorectal carcinoma. Results showed that these knockout cells grew similarly to wild-type cells in nude mice, contrary to expectations from our analysis of ATF6α-knockout, ATF6β-knockout and ATF6α/β-double knockout mice. We then found that the loss of ATF6α in HCT116 cells resulted in sustained activation of the IRE1 and PERK arms, in marked contrast to mouse embryonic fibroblasts, in which the loss of ATF6α is compensated for by ATF6β. Although IRE1-knockout in HCT116 cells unexpectedly did not affect tumor growth in nude mice, IRE1-knockout HCT116 cells with ATF6α knockdown grew significantly more slowly than wild-type or IRE1-knockout HCT116 cells. These results have unraveled the situation-dependent differential compensation strategies of ATF6α.

    American Society for Cell Biology (ASCB), 2023年01月25日, Molecular Biology of the Cell

    研究論文(学術雑誌)

  • Shunsuke Saito, Tokiro Ishikawa, Satoshi Ninagawa, Tetsuya Okada, Kazutoshi Mori

    A causal relationship between endoplasmic reticulum (ER) stress and the development of neurodegenerative diseases remains controversial. Here, we focused on Seipinopathy, a dominant motor neuron disease, based on the finding that its causal gene product, Seipin, is a protein that spans the ER membrane twice. Gain-of-function mutations of Seipin produce non-glycosylated Seipin (ngSeipin), which was previously shown to induce ER stress and apoptosis at both cell and mouse levels albeit with no clarified mechanism. We found that aggregation-prone ngSeipin dominantly inactivated SERCA2b, the major calcium pump in the ER, and decreased the calcium concentration in the ER, leading to ER stress and apoptosis in human colorectal carcinoma-derived cells (HCT116). This inactivation required oligomerization of ngSeipin and direct interaction of the C-terminus of ngSeipin with SERCA2b, and was observed in Seipin-deficient neuroblastoma (SH-SY5Y) cells expressing ngSeipin at an endogenous protein level. Our results thus provide a new direction to the controversy noted above.

    2022年11月29日, eLife, 11, 英語, 国際誌

    研究論文(学術雑誌)

  • Ginto George, Satoshi Ninagawa, Hirokazu Yagi, Jun-Ichi Furukawa, Noritaka Hashii, Akiko Ishii-Watabe, Ying Deng, Kazutoshi Matsushita, Tokiro Ishikawa, Yugoviandi P Mamahit, Yuta Maki, Yasuhiro Kajihara, Koichi Kato, Tetsuya Okada, Kazutoshi Mori

    Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.

    2021年10月26日, eLife, 10, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Satoshi Ninagawa, Ginto George, Kazutoshi Mori

    BACKGROUND: The quality of proteins destined for the secretory pathway is ensured by two distinct mechanisms in the endoplasmic reticulum (ER): productive folding of newly synthesized proteins, which is assisted by ER-localized molecular chaperones and in most cases also by disulfide bond formation and transfer of an oligosaccharide unit; and ER-associated degradation (ERAD), in which proteins unfolded or misfolded in the ER are recognized and processed for delivery to the ER membrane complex, retrotranslocated through the complex with simultaneous ubiquitination, extracted by AAA-ATPase to the cytosol, and finally degraded by the proteasome. SCOPE OF REVIEW: We describe the mechanisms of productive folding and ERAD, with particular attention to glycoproteins versus non-glycoproteins, and to yeast versus mammalian systems. MAJOR CONCLUSION: Molecular mechanisms of the productive folding of glycoproteins and non-glycoproteins mediated by molecular chaperones and protein disulfide isomerases are well conserved from yeast to mammals. Additionally, mammals have gained an oligosaccharide structure-dependent folding cycle for glycoproteins. The molecular mechanisms of ERAD are also well conserved from yeast to mammals, but redundant expression of yeast orthologues in mammals has been encountered, particularly for components involved in recognition and processing of glycoproteins and components of the ER membrane complex involved in retrotranslocation and simultaneous ubiquitination of glycoproteins and non-glycoproteins. This may reflect an evolutionary consequence of increasing quantity or quality needs toward mammals. GENERAL SIGNIFICANCE: The introduction of innovative genome editing technology into analysis of the mechanisms of mammalian ERAD, as exemplified here, will provide new insights into the pathogenesis of various diseases.

    2020年12月11日, Biochimica et biophysica acta. General subjects, 129812 - 129812, 英語, 国際誌

    研究論文(学術雑誌)

  • Satoshi Ninagawa, Seiichiro Tada, Masaki Okumura, Kenta Inoguchi, Misaki Kinoshita, Shingo Kanemura, Koshi Imami, Hajime Umezawa, Tokiro Ishikawa, Robert B Mackin, Seiji Torii, Yasushi Ishihama, Kenji Inaba, Takayuki Anazawa, Takahiko Nagamine, Kazutoshi Mori

    Second-generation antipsychotics are widely used to medicate patients with schizophrenia, but may cause metabolic side effects such as diabetes, which has been considered to result from obesity-associated insulin resistance. Olanzapine is particularly well known for this effect. However, clinical studies have suggested that olanzapine-induced hyperglycemia in certain patients cannot be explained by such a generalized mechanism. Here, we focused on the effects of olanzapine on insulin biosynthesis and secretion by mouse insulinoma MIN6 cells. Olanzapine reduced maturation of proinsulin, and thereby inhibited secretion of insulin; and specifically shifted the primary localization of proinsulin from insulin granules to the endoplasmic reticulum. This was due to olanzapine's impairment of proper disulfide bond formation in proinsulin, although direct targets of olanzapine remain undetermined. Olanzapine-induced proinsulin misfolding and subsequent decrease also occurred at the mouse level. This mechanism of olanzapine-induced β-cell dysfunction should be considered, together with weight gain, when patients are administered olanzapine.

    2020年11月17日, eLife, 9, e60970, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Hirokazu Yagi, Maho Yagi-Utsumi, Rena Honda, Yusaku Ohta, Taiki Saito, Miho Nishio, Satoshi Ninagawa, Kousuke Suzuki, Takahiro Anzai, Yukiko Kamiya, Kazuhiro Aoki, Mahito Nakanishi, Tadashi Satoh, Koichi Kato

    MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest.

    2020年03月13日, Nature communications, 11 (1), 1368 - 1368, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Ginto George, Satoshi Ninagawa, Hirokazu Yagi, Taiki Saito, Tokiro Ishikawa, Tetsushi Sakuma, Takashi Yamamoto, Koshi Imami, Yasushi Ishihama, Koichi Kato, Tetsuya Okada, Kazutoshi Mori

    Sequential mannose trimming of N-glycan (Man9GlcNAc2 -> Man8GlcNAc2 -> Man7GlcNAc2) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no α1,2-mannosidase activity toward Man9GlcNAc2 in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man9GlcNAc2 to Man8GlcNAc2(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.

    2020年02月17日, eLife, 9, e53455, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Byungseok Jin, Tokiro Ishikawa, Mai Taniguchi, Satoshi Ninagawa, Tetsuya Okada, Shigehide Kagaya, Kazutoshi Mori

    Three types of transmembrane protein, IRE1α/IRE1β, PERK, and ATF6α/ATF6β, are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1α inhibitors. IRE1α, IRE1β, ATF6α and ATF6β are ubiquitously expressed in medaka. We found that IRE1α/ATF6α-double knockout is lethal, similarly to IRE1α/IRE1β- and ATF6α/ATF6β-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6α-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1α in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1α inhibitors in vivo.Key words: endoplasmic reticulum, inhibitor screening, mRNA splicing, phenotypic assay, unfolded protein response.

    2020年02月07日, Cell structure and function, 45 (1), 23 - 31, 英語, 国内誌

    [査読有り]

    研究論文(学術雑誌)

  • Hibiki Koba, Shengyu Jin, Nanami Imada, Tokiro Ishikawa, Satoshi Ninagawa, Tetsuya Okada, Tetsushi Sakuma, Takashi Yamamoto, Kazutoshi Mori

    ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6β, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response.

    2020年01月30日, Cell structure and function, 45 (1), 9 - 21, 英語, 国内誌

    [査読有り]

    研究論文(学術雑誌)

  • Takehiro Sugimoto, Satoshi Ninagawa, Shimpei Yamano, Tokiro Ishikawa, Tetsuya Okada, Shunichi Takeda, Kazutoshi Mori

    Accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ATF6 pathway is one of the three major pathways in vertebrates. Although ATF6, a transmembrane-type glycoprotein in the ER, functions as a UPR sensor/transducer, it is an unstable protein with a half-life of approximately 2 h and is constitutively subjected to the ER-associated degradation system with the location of the misfolded part in the ER lumen (ERAD-L). ERAD-L substrates are delivered to the cytosol through the retrotranslocon, which likely contains HRD1 (E3), gp78 (E3), SEL1L (a partner of HRD1), Derlin1/2/3 and Herp1/2. We previously showed that ATF6 represents a novel transmembrane-type ERAD-L substrate requiring both EDEM1/2/3-mediated mannose trimming and SEL1L. Here, by constructing and analyzing chicken DT40 cells deficient in various components of the retrotranslocon, we show that degradation of ATF6 requires Derlin2 or Derlin3 and that Derlin2 and Derlin3 are redundant for ERAD-L of ATF6. We further show that degradation of ATF6 requires Herp1 or Herp2 and that Herp1 and Herp2 are redundant for ERAD-L of ATF6. Furthermore, by investigating five more ERAD-L substrates, we show that SEL1L-dependent substrates require Derlin2/3 and Herp1/2 regardless of their soluble or transmembrane nature. Our results suggest that ERAD-L substrates take several routes to the cytosol. The HRD1-engaged route 1 requires SEL1L, Derlin2 or Derlin3, and Herp1 or Herp2, whereas the HRD1-engaged route 2 does not require them functionally. It remains to be determined whether the latter requires Derlin1 and whether these two routes are compositionally distinct.Key words: endoplasmic reticulum, proteasome, protein degradation, protein misfolding, ubiquitin.

    JAPAN SOC CELL BIOLOGY, 2017年07月04日, Cell structure and function, 42 (2), 81 - 94, 英語, 国内誌

    [査読有り]

    研究論文(学術雑誌)

  • Tokiro Ishikawa, Takuya Toyama, Yuki Nakamura, Kentaro Tamada, Hitomi Shimizu, Satoshi Ninagawa, Tetsuya Okada, Yasuhiro Kamei, Tomoko Ishikawa-Fujiwara, Takeshi Todo, Eriko Aoyama, Masaharu Takigawa, Akihiro Harada, Kazutoshi Mori

    The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.

    ROCKEFELLER UNIV PRESS, 2017年06月05日, The Journal of cell biology, 216 (6), 1761 - 1774, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Direct Mapping of Additional Modifications on Phosphorylated O-glycans of α-Dystroglycan by Mass Spectrometry Analysis in Conjunction with Knocking Out of Causative Genes for Dystroglycanopathy.

    Hirokazu Yagi, Chu-Wei Kuo, Takayuki Obayashi, Satoshi Ninagawa, Kay-Hooi Khoo, Koichi Kato

    Dystroglycanopathy is a major class of congenital muscular dystrophy caused by a deficiency of functional glycans on α-dystroglycan (αDG) with laminin-binding activity. Recent advances have led to identification of several causative gene products of dystroglycanopathy and characterization of their in vitro enzymatic activities. However, the in vivo functional roles remain equivocal for enzymes such as ISPD, FKTN, FKRP, and TMEM5 that are supposed to be involved in post-phosphoryl modifications linking the GalNAc-β3-GlcNAc-β4-Man-6-phosphate core and the outer laminin-binding glycans. Herein, by direct nano-LC-MS2/MS3 analysis of tryptic glycopeptides derived from a truncated recombinant αDG expressed in the wild-type and a panel of mutated cells deficient in one of these enzymes, we sought to define the full extent of variable modifications on this phosphorylated core O-glycan at the functional Thr317/Thr319 sites. We showed that the most abundant glycoforms carried a phosphorylated core at each of the two sites, with and without a single ribitol phosphate (RboP) extending from terminal HexNAc. At much lower signal intensity, a novel substituent tentatively assigned as glycerol phosphate (GroP) was additionally detected. As expected, tandem RboP extended with a GlcA-Xyl unit was only identified in wild type, whereas knocking out of either ISPD or FKTN prevented formation of RboP. In the absence of FKRP, glycoforms with single but not tandem RboP accumulated, consistent with the suggested role of this enzyme in transferring the second RboP. Intriguingly, the single GroP modification also required functional FKTN whereas absence of TMEM5 significantly hindered only the addition of RboP. Our findings thus revealed additional levels of complexity associated with the core structures, suggesting functional interplay among these enzymes through their interactions. The simplified analytical workflow developed here should facilitate rapid mapping across a wider range of cell types to gain better insights into its physiological relevance.

    2016年11月, Molecular & cellular proteomics : MCP, 15 (11), 3424 - 3434, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Tetsuya Okada, Satoshi Ninagawa, Kazutoshi Mori

    Japanese Biochemical Society, 2016年04月, Seikagaku. The Journal of Japanese Biochemical Society, 88 (2), 257 - 60, 日本語, 国内誌

    [査読有り]

    研究論文(学術雑誌)

  • みにれびゅう 糖タンパク質の小胞体関連分解におけるマンノーストリミング機構

    岡田 徹也, 蜷川 暁, 森 和俊

    2016年, 生化学, 88 (2), 257 - 260, 日本語

    [査読有り]

  • Trypsin Sensitivity Assay to Study the Folding Status of Proteins.

    Ninagawa, S, Mori, K

    2016年, Bio-protocol, 6 (19), e1953

    [査読有り]

  • PNGase Sensitivity Assay to Study the Folding Status of Proteins.

    Ninagawa, S, Mori, K

    2016年, Bio-protocol, 6 (19), e1952

    [査読有り]

  • Satoshi Ninagawa, Tetsuya Okada, Yoshiki Sumitomo, Satoshi Horimoto, Takehiro Sugimoto, Tokiro Ishikawa, Shunichi Takeda, Takashi Yamamoto, Tadashi Suzuki, Yukiko Kamiya, Koichi Kato, Kazutoshi Mori

    Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ-ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ-ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.

    ROCKEFELLER UNIV PRESS, 2015年11月23日, The Journal of cell biology, 211 (4), 775 - 84, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • 蜷川 暁, 加藤 晃一, 森 和俊

    公益社団法人 日本農芸化学会, 2015年, 化学と生物, 53 (9), 571 - 573, 日本語

    [査読有り]

  • Satoshi Ninagawa, Tetsuya Okada, Yoshiki Sumitomo, Yukiko Kamiya, Koichi Kato, Satoshi Horimoto, Tokiro Ishikawa, Shunichi Takeda, Tetsushi Sakuma, Takashi Yamamoto, Kazutoshi Mori

    Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease-mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2.

    ROCKEFELLER UNIV PRESS, 2014年08月04日, The Journal of cell biology, 206 (3), 347 - 56, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Satoshi Horimoto, Satoshi Ninagawa, Tetsuya Okada, Hibiki Koba, Takehiro Sugimoto, Yukiko Kamiya, Koichi Kato, Shunichi Takeda, Kazutoshi Mori

    Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 and exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis showed that the luminal region of ATF6 confers SEL1L dependence on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, T-cell receptor-α, CD3-δ, and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of α1,2-mannosidase in the ER, indicating that degradation of ATF6 requires proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependence on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought.

    AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2013年11月01日, The Journal of biological chemistry, 288 (44), 31517 - 27, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • SEL1L is required for endoplasmic reticulum-associated degradation of misfolded luminal proteins but not transmembrane proteins in chicken DT40 cell line.

    Satoshi Ninagawa, Tetsuya Okada, Shunichi Takeda, Kazutoshi Mori

    Proteins misfolded in the endoplasmic reticulum (ER) are degraded in the cytosol by a ubiquitin-dependent proteasome system, a process collectively termed ER-associated degradation (ERAD). Unraveling the molecular mechanisms of mammalian ERAD progresses more slowly than that of yeast ERAD due to the laborious procedures required for gene targeting and the redundancy of components. Here, we utilized the chicken B lymphocyte-derived DT40 cell line, which exhibits an extremely high homologous recombination frequency, to analyze ERAD mechanisms in higher eukaryotes. We disrupted the SEL1L gene, which encodes the sole homologue of yeast Hrd3p in both chickens and mammals; Hrd3p is a binding partner of yeast Hrd1p, an E3 ubiquitin ligase. SEL1L-knockout cells grew only slightly more slowly than the wild-type cells. Pulse chase experiments revealed that chicken SEL1L was required for ERAD of misfolded luminal proteins such as glycosylated NHK and unglycosylated NHK-QQQ but dispensable for that of misfolded transmembrane proteins such as NHK(BACE) and CD3-δ, as in mammals. The defect of SEL1L-knockout cells in NHK degradation was restored by introduction of not only chicken SEL1L but also mouse and human SEL1L. Deletion analysis showed the importance of Sel1-like tetratricopeptide repeats but not the fibronectin II domain in the function of SEL1L. Thus, our reverse genetic approach using the chicken DT40 cell line will provide highly useful information regarding ERAD mechanisms in higher eukaryotes which express ERAD components redundantly.

    JAPAN SOC CELL BIOLOGY, 2011年, Cell structure and function, 36 (2), 187 - 95, 英語, 国内誌

    [査読有り]

    研究論文(学術雑誌)

MISC

  • 小胞体におけるN型糖鎖による糖タンパク質の品質管理機構

    蜷川 暁

    2021年, Trends in glycoscience and glycotechnology, 33 (191-193), J55 - 62, 日本語

  • 小胞体におけるN型糖鎖による糖タンパク質の品質管理機構

    蜷川暁

    2021年, Trends in Glycoscience and Glycotechnology (Web), 33 (193)

  • 高等動物の小胞体におけるタンパク質の構造形成経路と分解経路

    蜷川暁

    2021年, 生化学, 93 (4)

  • 第二世代抗精神病薬オランザピンによって,ジスルフィド結合形成異常に起因したプロインスリンの構造異常が引き起こされる

    蜷川暁, 多田誠一郎, 奥村正樹, 井ノ口健太, 木下岬, 金村進吾, 金村進吾, 今見考志, 梅澤元, 石川時郎, MACKIN Robert, 鳥居征司, 石濱泰, 稲葉謙次, 穴澤貴行, 長嶺敬彦, 森和俊

    2021年, 日本細胞生物学会大会(Web), 73rd

  • 第二世代抗精神病薬オランザピンは,膵β細胞において,小胞体でのプロインスリンの構造形成阻害と,それに伴う分解を誘発し,非典型的な糖尿病を惹起する

    蜷川暁, 多田誠一郎, 奥村正樹, 井ノ口健太, 木下岬, 金村進吾, 今見考志, 梅澤元, 石川時郎, MACKIN Robert, 鳥居征司, 石濱泰, 稲葉謙次, 穴澤貴行, 長嶺敬彦, 森和俊

    2021年, 日本薬学会年会要旨集(Web), 141st

  • OS9,XTP3Bによる小胞体関連分解の制御は分解基質の構造異常の度合いに依存する

    松下和智, 蜷川暁, 杉本岳大, 岡田徹也, 石川時郎, 佐久間哲史, 山本卓, 森和俊

    2021年, 日本糖質学会年会要旨集, 40th

  • タンパク質の異種会合による小胞体マンノシダーゼの機能発現

    蜷川暁, GEORGE Ginto, 矢木宏和, 古川潤一, 橋井則貴, 石井明子, DENG Ying, 石川時郎, 今見考志, 石濱泰, 加藤晃一, 加藤晃一, 岡田徹也, 森和俊

    2021年, 日本分子生物学会年会プログラム・要旨集(Web), 44th

  • 高等動物におけるN型糖鎖のマンノース切除を基軸とした小胞体関連分解機構の解析

    蜷川暁, GEORGE Ginto, 矢木宏和, 住友嘉樹, 真木勇太, 石川時郎, 佐久間哲史, 山本卓, 今見考志, 石濱泰, 梶原康宏, 加藤晃一, 加藤晃一, 岡田徹也, 森和俊

    2021年, 日本糖質学会年会要旨集, 40th

  • 第二世代抗精神病薬オランザピンは、ジスルフィド結合の形成異常を起因としたプロインスリンの小胞体関連分解を誘発する

    蜷川 暁, 多田 誠一郎, 井ノ口 健太, 今見 考志, 梅澤 元, 石川 時郎, 岡田 徹也, Robert Mackin, 鳥居 征司, 石濱 泰, 穴澤 貴行, 長嶺 敬彦, 森 和俊

    (公社)日本薬学会, 2020年03月, 日本薬学会年会要旨集, 140年会, 26F - pm18, 日本語

  • 遺伝子破壊法を用いた小胞体関連分解機構の解明

    蜷川暁

    2020年, 日本生化学会大会(Web), 93rd

  • 小胞体関連分解因子EDEM2は,TXNDC11とジスルフィド結合を介した複合体を形成し,マンノシダーゼ活性を発揮する

    蜷川暁, GEORGE Ginto, 矢木宏和, 住友嘉樹, 石川時郎, 佐久間哲史, 山本卓, 今見考志, 石濱泰, 加藤晃一, 加藤晃一, 岡田徹也, 森和俊

    2020年, 日本生化学会大会(Web), 93rd

  • EDEM2酵素活性発揮には,TXNDC11とのジスルフィド結合形成が必要である

    蜷川暁, GEORGE Ginto, 矢木宏和, 斎藤泰輝, 住友嘉樹, 石川時郎, 佐久間哲史, 山本卓, 今見考志, 石濱泰, 加藤晃一, 加藤晃一, 岡田徹也, 森和俊

    2020年, 日本糖質学会年会要旨集, 39th

  • メダカを用いた小胞体ストレスに起因するアポトーシス分子機構の解析

    陳 炳碩, 石川 時郎, 蜷川 暁, 岡田 徹也, 森 和俊

    (公社)日本生化学会, 2019年09月, 日本生化学会大会プログラム・講演要旨集, 92回, [1T05m - 01], 日本語

  • タンパク質の運命を制御する生体システムと疾病のフロンティア 第二世代抗精神病薬オランザピンは、副作用としてProinsulinの適切なジスルフィド結合形成を阻害し糖尿病を誘発する

    蜷川 暁, 岡田 徹也, 梅澤 元, 石川 時郎, 鳥居 征司, Mackin Robert, 今見 考志, 石濱 泰, 長嶺 敬彦, 森 和俊

    (公社)日本生化学会, 2019年09月, 日本生化学会大会プログラム・講演要旨集, 92回, [2S03m - 06], 日本語

  • 小胞体ストレスセンサーATF6αはジスルフィド結合を介して2種類の二量体を形成する(Endoplasmic reticulum stress sensor ATF6α forms two types of dimers via disulfide-bonding)

    古場 玲, 金 聖宇, 岡田 徹也, 石川 時郎, 蜷川 暁, 森 和俊

    (公社)日本生化学会, 2019年09月, 日本生化学会大会プログラム・講演要旨集, 92回, [3T03m - 01], 日本語

  • 小胞体膜タンパク質Seipinの神経変性誘導変異体に起因する小胞体ストレス発生機構の解析

    齊藤 峻介, 石川 時郎, 蜷川 暁, 岡田 徹也, 森 和俊

    (公社)日本生化学会, 2019年09月, 日本生化学会大会プログラム・講演要旨集, 92回, [3T17a - 03], 日本語

  • N型糖鎖のマンノーストリミングを基軸とした小胞体関連分解の解析

    蜷川暁

    2019年, 細胞内糖修飾の統合的ケミカルバイオロジー講演要旨集 理研シンポジウム 平成31年

  • 高等動物におけるN型糖鎖トリミングを基軸とした小胞体関連分解機構の解析

    蜷川暁, GEORGE Ginto, 矢木宏和, 住友嘉樹, 神谷由紀子, 石川時郎, 武田俊一, 佐久間哲史, 山本卓, 加藤晃一, 加藤晃一, 岡田徹也, 森和俊

    2019年, 日本糖質学会年会要旨集, 38th

  • 小胞体タンパク質品質管理機構の中心を担うN型糖鎖依存/非依存小胞体関連分解経路の分子メカニズムの解析

    蜷川暁, GINTO George, 矢木宏和, 今見考志, 石濱泰, 石川時郎, 佐久間哲史, 山本卓, 武田俊一, 加藤晃一, 加藤晃一, 岡田徹也, 森和俊

    2019年, 日本分子生物学会年会プログラム・要旨集(Web), 42nd

  • 第二世代抗精神病薬オランザピンがインスリン分泌不全を引き起こし、糖尿病を誘起する分子メカニズムの解析

    蜷川 暁, 岡田 徹也, 今見 考志, 梅澤 元, 石川 時郎, 鳥居 征司, Mackin Robert, 石濱 泰, 長嶺 敬彦, 森 和俊

    (公社)日本生化学会, 2018年09月, 日本生化学会大会プログラム・講演要旨集, 91回, [3T14a - 304)], 日本語

  • 第二世代抗精神病薬オランザピンが、副作用として糖尿病を引き起こす分子メカニズムの解析 インスリンは、適切なジスルフィド結合形成を阻害され、小胞体関連分解により処理される

    蜷川 暁, 岡田 徹也, 梅澤 元, 石川 時郎, 鳥居 征司, Mackin Robert, 長嶺 敬彦, 森 和俊

    生命科学系学会合同年次大会運営事務局, 2017年12月, 生命科学系学会合同年次大会, 2017年度, [1P - 1141], 日本語

  • α-ジストログリカンのラミニン結合性糖鎖形成するポストリン酸修飾の構造解析

    矢木 宏和, Kuo Chu-Wei, 尾林 卓幸, 蜷川 暁, Khoo Kay-Hooi, 加藤 晃一

    (公社)日本薬学会, 2017年03月, 日本薬学会年会要旨集, 137年会 (3), 53 - 53, 日本語

  • オルガネラ局域の間取り図 分解執行局域における新展開

    蜷川 暁, 岡田 徹也, 住友 嘉樹, 堀本 賢, 鈴木 匡, 武田 俊一, 佐久間 哲史, 山本 卓, 神谷 由紀子, 加藤 晃一, 森 和俊

    (一社)日本細胞生物学会, 2016年05月, 日本細胞生物学会大会講演要旨集, 68回, 30 - 30, 日本語

  • 糖鎖非依存小胞体関連分解経路によるシビアな構造異常糖タンパク質の強制分解

    蜷川 暁, 岡田 徹也, 住友 嘉樹, 堀本 賢, 杉本 岳大, 石川 時郎, 武田 俊一, 山本 卓, 神谷 由紀子, 加藤 晃一, 森 和俊

    (公社)日本生化学会, 2015年12月, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [2P0296] - [2P0296], 日本語

  • Derlin2/3およびHerp1/2はSEL1L依存的な構造異常タンパク質分解経路に必要である

    杉本 岳大, 蜷川 暁, 山野 晋平, 石川 時郎, 岡田 徹也, 武田 俊一, 森 和俊

    (公社)日本生化学会, 2015年12月, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [4T17 - 09(3P0384)], 日本語

  • EDEM1/2/3 are alpha 1,2-mannosidases essential for endoplasmic reticulum-associated degradation of glycoproteins

    Satoshi Ninagawa, Tetsuya Okada, Yoshiki Sumitomo, Yukiko Kamiya, Satoshi Horimoto, Tokiro Ishikawa, Shunichi Takeda, Tetsushi Sakuma, Takashi Yamamoto, Koichi Kato, Kazutoshi Mori

    OXFORD UNIV PRESS INC, 2014年11月, GLYCOBIOLOGY, 24 (11), 1097 - 1097, 英語

    研究発表ペーパー・要旨(国際会議)

  • ERにおける蛋白質品質管理システムに関する逆遺伝学的解析(Reverse Genetic Analysis of Protein Quality Control System in the ER)

    蜷川 暁, 住友 嘉樹, 岡田 徹也, 武田 俊一, 森 和俊

    (公社)日本生化学会, 2011年09月, 日本生化学会大会プログラム・講演要旨集, 84回, 3S11p - 4, 英語

  • トリDT40細胞のERADの解析 Sel1Lノックアウトの影響(Analysis of ERAD in chicken DT40 cells: effects of Sel1L knockout)

    蜷川 暁, 岡田 徹也, 武田 俊一, 森 和俊

    (公社)日本生化学会, 2010年12月, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 83回・33回, 4T10 - 5, 英語

  • 小胞体関連分解構成因子Derlinsに結合するタンパク質の同定と解析

    蜷川暁, 岡田徹也, 小田裕香子, 大川克也, 森和俊

    2007年, 生化学, 4P-0199, 日本語

所属学協会

  • 日本糖質学会

    2014年04月 - 現在
  • 日本細胞生物学会

    2010年04月 - 現在
  • 日本分子生物学会

    2010年04月 - 現在
  • 日本生化学会

    2008年04月 - 現在

共同研究・競争的資金等の研究課題

  • 哺乳動物の小胞体関連分解における小胞体内イベントの全容解明

    森 和俊, 蜷川 暁

    日本学術振興会, 科学研究費助成事業, 基盤研究(A), 京都大学, 2022年04月01日 - 2025年03月31日

  • 公益財団法人 武田科学振興財団, ライフサイエンス研究助成, 2020年10月 - 2023年05月

  • 公益財団法人 小林財団, 第9回研究助成, 2021年03月 - 2023年03月

  • 小胞体品質管理に関わる選別輸送ゾーンの解明

    森 和俊, 尾野 雅哉, 西頭 英起, 片桐 豊雅, 名黒 功, 蜷川 暁

    日本学術振興会, 科学研究費助成事業, 新学術領域研究(研究領域提案型), 京都大学, 2017年06月30日 - 2022年03月31日

    本研究では、3つのオルガネラ・ゾーンについて以下の成果を得た。選別輸送ゾーン:細胞内輸送を介した小胞体ストレスセンサーATF6およびIRE1の活性化過程を可視化し、制御因子を同定した。タンパク質分解ゾーン:複合体形成に着目し、Derlin1とDerlin2の機能的差異を見出した。また、中枢神経系においてDerlin1/2が神経突起伸張に必須であることを明らかにした。さらに、TXNDC11が糖タンパク分解を制御する糖鎖トリミングに必須であることを見出した。ミトコンドリア-小胞体連携ゾーン:褐色脂肪細胞分化において、PERKがミトコンドリアの機能制御に必須であることを明らかにした。

  • 小胞体における構造異常タンパク質の分解への基質運搬機構の解明

    蜷川 暁

    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), 京都大学, 2018年04月01日 - 2021年03月31日

    小胞体内において、構造異常タンパク質は小胞体関連分解によって処理される。小胞体関連分解は、大きく3つのステップに分かれており、1、分解基質の逆行輸送チャネルへの運搬 2、逆行輸送 3、細胞質プロテアソームによる分解 である。特に1に関して、どのように基質が分解へ選別され、逆行輸送チャネルへ運搬されて行くのか?は最重要問題の1つとして挙げられている。また当研究室の結果より、小胞体は均質な空間ではなく、構造形成ゾーン、逆行輸送ゾーン(分解ゾーン)といった機能的な区画に分かれていることが示唆されている。本研究では、糖鎖刈り込みの進んだ糖鎖を認識し、分解に導くとも考えられている2つのホモロジーレクチン分子OS-9、XTP3Bが、分解基質を構造形成ゾーンから逆行輸送ゾーンに運搬し、分解へ導く運命決定因子かどうか明らかにすることを目的とした。 まずOS-9/XTP3B Double KO (DKO)細胞を作製すると、刈り込みの進んだ糖鎖が分解されず、低分子量にバンドが検出された。これは、OS-9とXTP3Bの両者の機能が重複し、刈り込みの進んだ糖鎖を持つ基質の分解を促進することを明確に示す結果である。これをサポートする結果として、OS-9 Single KO、XTP3B single KO、OS-9/XTP3B DKO細胞全体の糖鎖を糖鎖分析にかけると糖鎖のトリミングの進んだ糖鎖が分解されず蓄積していることが分かった。また現在は小胞体がさらに細分化したゾーンに分かれていることを示すことが出来始めている。これらの2つを合わせて現在、OS-9、XTP3Bが基質を構造形成ゾーンから分解ゾーンへと運搬しているかどうかを検証しようとしている。

  • 群馬大学, 生体調節研究所「内分泌・代謝学共同研究拠点」共同研究, 2018年04月 - 2019年03月, 研究代表者

  • 蜷川 暁

    日本学術振興会, 科学研究費助成事業 若手研究(B), 若手研究(B), 京都大学, 2016年04月01日 - 2018年03月31日

    小胞体関連分解機構において、糖タンパク質の構造異常の度合いが低いと糖鎖依存分解経路のみで分解され、構造異常の度合いが高いと糖鎖依存、非依存分解経路の両者で分解される現象を発表した。今回その分子メカニズムの解析を行った。 過去の報告より糖鎖依存分解経路から糖鎖非依存分解経路へtargetingする候補分子に着目し、遺伝子破壊株の作製を試みた。すると、細胞にとって非常に重要であるこの分子は、欠失すると細胞は死に至るため、この分子の変異体細胞を作出した。その変異体細胞では、少なくとも糖鎖非依存分解経路の分解が抑制されていた。現在も論文化に向けさらなる解析を続けている。

  • 蜷川 暁

    日本学術振興会, 科学研究費助成事業 若手研究(B), 若手研究(B), 2014年04月01日 - 2016年03月31日

    小胞体のN型糖鎖からのマンノースの2ステップトリミングが糖鎖依存的分解経路に必須である。我々は最初のマンノーストリミングがEDEM2によって遂行され、次のマンノーストリミングがEDEM3/1によって遂行されるということを示した(Ninagawa et al., JCB 2014)。 またEDEM1/2/3をすべてKOしたEDEM triple KO細胞を用いて、シビアな構造異常糖タンパク質は糖鎖依存分解経路に加えて糖鎖非依存分解経路でも処分される」ことを立証した(Ninagawa et al., JCB 2015)。

  • 小胞体関連分解構成因子Derlinsに結合するタンパク質の同定と解析

    蜷川 暁

    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, 京都大学, 2008年 - 2009年

    小胞体関連分解とは小胞体において正しく立体構造をとることができないタンパク質を小胞体から細胞質に逆行輸送した後、細胞質のプロテアソームで分解する機構である。 研究計画に沿って研究を進めていたが、RNAi法を用いた遺伝子抑制実験では、きちんとした実験結果が出にくいということが判明したため、遺伝子破壊法であるknock out実験によって研究を進めた。具体的には、組み換えが非常におこりやすく遺伝子破壊しやすい、ニワトリBリンパ球細胞を用いて解析をした。現在までに小胞体関連分解の中心因子であるSellLや他3種のKnock out細胞の作成に成功した。 SellL-/-細胞は、小胞体関連分解の基質であるNHK(NNN)の分解遅延が見られた。また予想された通り糖鎖を持たないNHK(QQQ)の分解遅延も確認されている。これらのことによりDT40を用いた遺伝子破壊は期待された効果を得ており、今後、簡単に遺伝子操作できることから様々な遺伝子のknock outを得、解析することができ、現在までにきちんと調べられていなかった遺伝子もどのように4小胞体関連分解に関わっているかきちんと調べることができる。コントロールはとれてきたので、今後機能未知の遺伝子に関しても詳細に調べて行きたい。