渡部 昌 (ワタナベ マサシ)

医学研究院 生理系部門 生化学分野講師
Last Updated :2024/12/06

■研究者基本情報

学位

  • 博士(医学), 北海道大学

Researchmap個人ページ

研究分野

  • ライフサイエンス, 生理学
  • ライフサイエンス, 病態医化学
  • ライフサイエンス, 医化学

■経歴

経歴

  • 2018年 - 現在
    北海道大学大学院, 医学研究院, 講師
  • 2012年
    北海道大学大学院, 医学研究院, 助教
  • 2009年
    日本学術振興会特別研究員(DC1)

■研究活動情報

受賞

  • 2017年, 北海道医学会, 北海道医学会研究奨励賞               
    渡部昌
  • 2015年, 北海道大学医学部同窓会, フラテ研究奨励賞               
    渡部昌

論文

  • 脳炎・肥厚性硬膜炎【WS】免疫沈降法とショットガンプロテオミクスによる自己抗体測定方法の開発               
    工藤 彰彦, 矢口 裕章, 渡部 昌, 藤井 信太朗, 野村 太一, 上床 恵, 上床 尚, 白井 慎一, 岩田 育子, 松島 理明, 田中 惠子, 高橋 秀尚, 畠山 鎮次, 矢部 一郎
    神経免疫学, 29, 1, 218, 218, (一社)日本神経免疫学会, 2024年10月
    日本語
  • 鼻副鼻腔粘膜悪性黒色腫におけるTRIM27の検討               
    木村 将吾, 中丸 裕爾, 鈴木 正宣, 中薗 彬, 本間 あや, 渡邉 良亮, 加納 里志, 対馬 那由多, 鈴木 崇祥, 井戸川 寛志, 本間 明宏, 渡部 昌, 近藤 豪, 畠山 鎮次
    日本鼻科学会会誌, 63, 3, 336, 336, (一社)日本鼻科学会, 2024年09月
    日本語
  • 自己免疫性小脳失調症に対する免疫沈降法と質量分析法を用いた網羅的自己抗体測定方法の開発               
    工藤 彰彦, 矢口 裕章, 渡部 昌, 上床 尚, 白井 慎一, 岩田 育子, 松島 理明, 高橋 秀尚, 畠山 鎮次, 矢部 一郎
    神経免疫学, 28, 1, 217, 217, (一社)日本神経免疫学会, 2023年09月
    日本語
  • FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA.
    Yusuke Kasuga, Ryota Ouda, Masashi Watanabe, Xin Sun, Miki Kimura, Shigetsugu Hatakeyama, Koichi S Kobayashi
    Proceedings of the National Academy of Sciences of the United States of America, 120, 24, e2218955120, 2023年06月13日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), Major histocompatibility complex (MHC) class I and II molecules play critical roles in the activation and regulation of adaptive immunity through antigen presentation to CD8+ and CD4+ T cells, respectively. Strict regulation of MHC expression is critical for proper immune responses. CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, is a master regulator of MHC class II (MHC-II) gene transcription. Although it has been known that CIITA activity is regulated at the transcriptional and protein levels, the mechanism to determine CIITA protein level has not been elucidated. Here, we show that FBXO11 is a bona fide E3 ligase of CIITA and regulates CIITA protein level through ubiquitination-mediated degradation. A nonbiased proteomic approach for CIITA-binding protein identified FBXO11, a member of the Skp1-Cullin-1-F-box E3 ligase complex, as a binding partner of CIITA but not MHC class I transactivator, NLRC5. The cycloheximide chase assay showed that the half-life of CIITA is mainly regulated by FBXO11 via the ubiquitin-proteasome system. The expression of FBXO11 led to the reduced MHC-II at the promoter activity level, transcriptional level, and surface expression level through downregulation of CIITA. Moreover, human and mouse FBXO11-deficient cells display increased levels of MHC-II and related genes. In normal and cancer tissues, FBXO11 expression level is negatively correlated with MHC-II. Interestingly, the expression of FBXO11, along with CIITA, is associated with prognosis of cancer patients. Therefore, FBXO11 is a critical regulator to determine the level of MHC-II, and its expression may serve as a biomarker for cancer.
  • Sez6l2 autoimmunity in a large cohort study.
    Megumi Abe, Hiroaki Yaguchi, Akihiko Kudo, Azusa Nagai, Shinichi Shirai, Ikuko Takahashi-Iwata, Masaaki Matsushima, Naoko Nakamura, Kenji Isahaya, Yoshihisa Yamano, Shinji Ashida, Takashi Kasai, Keiko Tanaka, Masashi Watanabe, Takeshi Kondo, Hidehisa Takahashi, Shigetsugu Hatakeyama, Akira Takekoshi, Akio Kimura, Takayoshi Shimohata, Ichiro Yabe
    Journal of neurology, neurosurgery, and psychiatry, 94, 8, 667, 668, 2023年06月01日, [査読有り], [国際誌]
    英語
  • TRIM27 expression is associated with poor prognosis in sinonasal mucosal melanoma.
    S Kimura, M Suzuki, Y Nakamaru, S Kano, M Watanabe, A Honma, A Nakazono, N Tsushima, S Hatakeyama, A Homma
    Rhinology, 2023年03月09日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), BACKGROUND: Tripartite motif-containing 27 (TRIM27) has been implicated in the progression of various cancers. However, the role of TRIM27 in sinonasal mucosal melanoma (SNMM) remains poorly understood. MATERIALS & METHODS: We retrospectively examined 28 patients with SNMM treated with between 2003 and 2021. We undertook immunohistochemical analysis of TRIM27, Ki-67, and p-Akt1 expression in SNMM tissues. We also investigated the relationship between TRIM27 expression and clinical characteristics, prognosis, Ki-67 as a tumor growth potential marker, and p-Akt1 as one of the prognostic factors in mucosal melanoma. RESULTS: TRIM27 expression was significantly higher in T4 disease than in T3 disease and was higher in stage IV than in stage III. Patients with high-TRIM27 SNMM had a significantly poorer prognosis in terms of overall survival (OS) and disease-free survival.There was also a significantly higher rate of distant metastasis. Univariate analysis for OS revealed that TRIM27 and T classification were significant poor prognostic factors. In addition, the Ki-67 positive score and the p-Akt1 total staining score were significantly higher in the high-TRIM27 group than in the low-TRIM27 group. CONCLUSIONS: High TRIM27 expression in SNMM was associated with advanced T classification, poor prognosis and distant metastasis. We suggest that TRIM27 has potential as a novel biomarker for prognosis in SNMM.
  • TRIM22 negatively regulates MHC-II expression.
    Ayano Inoue, Masashi Watanabe, Takeshi Kondo, Satoshi Hirano, Shigetsugu Hatakeyama
    Biochimica et biophysica acta. Molecular cell research, 1869, 10, 119318, 119318, 2022年06月28日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), The development of cancer treatment has recently achieved a remarkable breakthrough, and checkpoint blockade immunotherapy has received much attention. To enhance the therapeutic efficacy of checkpoint blockade immunotherapy, recent studies have revealed the importance of activation of CD4+ T cells via an increase in major histocompatibility complex (MHC) class II molecules in cancer cells. Here, we demonstrate that tripartite motif-containing (TRIM) 22, negatively regulates MHC-II expression. Gene knockout of TRIM22 using Cas9-sgRNAs led to an increase of MHC-II proteins, while TRIM22 overexpression remarkably decreased MHC-II proteins. mRNA levels of MHC-II and class II transactivator (CIITA), which plays an essential role in the regulation of MHC-II transcription, were not affected by TRIM22. Furthermore, TRIM22 knockout did not suppress the degradation of MHC-II protein but rather promoted it. These results suggest that TRIM22 decreases MHC-II protein levels through a combination of multiple mechanisms other than transcription or degradation. We showed that inhibition of TRIM22 can increase the amount of MHC-II expression in cancer cells, suggesting a possibility of providing the biological basis for a possible therapeutic target to potentiate checkpoint blockade immunotherapy.
  • Loss of FAM83H promotes cell migration and invasion in cutaneous squamous cell carcinoma via impaired keratin distribution.
    Keiko Tokuchi, Shinya Kitamura, Takuya Maeda, Masashi Watanabe, Shigetsugu Hatakeyama, Satoshi Kano, Shinya Tanaka, Hideyuki Ujiie, Teruki Yanagi
    Journal of dermatological science, 104, 2, 112, 121, 2021年09月30日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), BACKGROUNDS: FAM83H is essential for amelogenesis, but recent reports implicate that FAM83H is involved in the tumorigenesis. We previously clarified that TRIM29 binds to FAM83H to regulate keratin distribution and squamous cell migration. However, little is known about FAM83H in normal/malignant skin keratinocytes. OBJECTIVE: To investigate the expression of FAM83H in cutaneous squamous cell carcinoma (SCC) and its physiological function. METHODS: Immunohistochemical analysis and RT-PCR of human SCC tissues were performed. Next, we examined the effect of FAM83H knockdown/overexpression in SCC cell lines using cell proliferation, migration, and invasion assay. To investigate the molecular mechanism, immunoprecipitation of FAM83H was examined. Further, Immunofluorescence staining was performed. Finally, we examined the correlation between the expressions of FAM83H and the keratin distribution. RESULTS: FAM83H expression was lower in SCC lesions than in normal epidermis and correlated with differentiation grade. The mRNA expression levels of FAM83H in SCC tumors were also lower than in normal epidermis. The knockdown of FAM83H enhanced SCC cell migration and invasion, whereas the overexpression of FAM83H led to decreases in both. Furthermore, the knockdown of FAM83H enhanced the cancer cell metastasis in vivo. FAM83H formed a complex with TRIM29 and keratins. The knockdown of FAM83H altered keratin distribution and solubility. Clinically, the loss of FAM83H correlates with an altered keratin distribution. CONCLUSION: Our findings reveal a critical function for FAM83H in regulating keratin distribution, as well as in the migration/invasion of cutaneous SCC, suggesting that FAM83H could be a crucial molecule in the tumorigenesis of cutaneous SCC.
  • Fluticasone Propionate Suppresses Poly(I:C)-Induced ACE2 in Primary Human Nasal Epithelial Cells.
    Akira Nakazono, Yuji Nakamaru, Mahnaz Ramezanpour, Takeshi Kondo, Masashi Watanabe, Shigetsugu Hatakeyama, Shogo Kimura, Aya Honma, P J Wormald, Sarah Vreugde, Masanobu Suzuki, Akihiro Homma
    Frontiers in cellular and infection microbiology, 11, 655666, 655666, 2021年, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), Background: From the first detection in 2019, SARS-CoV-2 infections have spread rapidly worldwide and have been proven to cause an urgent and important health problem. SARS-CoV-2 cell entry depends on two proteins present on the surface of host cells, angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). The nasal cavity is thought to be one of the initial sites of infection and a possible reservoir for dissemination within and between individuals. However, it is not known how the expression of these genes is regulated in the nasal mucosa. Objective: In this study, we examined whether the expression of ACE2 and TMPRSS2 is affected by innate immune signals in the nasal mucosa. We also investigated how fluticasone propionate (FP), a corticosteroid used as an intranasal steroid spray, affects the gene expression. Methods: Primary human nasal epithelial cells (HNECs) were collected from the nasal mucosa and incubated with Toll-like receptor (TLR) agonists and/or fluticasone propionate (FP), followed by quantitative PCR, immunofluorescence, and immunoblot analyses. Results: Among the TLR agonists, the TLR3 agonist Poly(I:C) significantly increased ACE2 and TMPRSS2 mRNA expression in HNECs (ACE2 36.212±11.600-fold change, p<0.0001; TMPRSS2 5.598±2.434-fold change, p=0.031). The ACE2 protein level was also increased with Poly(I:C) stimulation (2.884±0.505-fold change, p=0.003). The Poly(I:C)-induced ACE2 expression was suppressed by co-incubation with FP (0.405±0.312-fold change, p=0.044). Conclusion: The activation of innate immune signals via TLR3 promotes the expression of genes related to SARS-CoV2 cell entry in the nasal mucosa, although this expression is suppressed in the presence of FP. Further studies are required to evaluate whether FP suppresses SARS-CoV-2 viral cell entry.
  • Role of intracellular zinc in molecular and cellular function in allergic inflammatory diseases.
    Masanobu Suzuki, Takayoshi Suzuki, Masashi Watanabe, Shigetsugu Hatakeyama, Shogo Kimura, Akira Nakazono, Aya Honma, Yuji Nakamaru, Sarah Vreugde, Akihiro Homma
    Allergology international : official journal of the Japanese Society of Allergology, 70, 2, 190, 200, 2020年10月27日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), Zinc is an essential micronutrient in human body and a vital cofactor for the function of numerous proteins encoded by the human genome. Zinc has a critical role in maintaining many biochemical and physiological processes at the molecular, cellular, and multiple organ and systemic levels. The alteration of zinc homeostasis causes dysfunction of many organs and systems. In the immune system, zinc regulates the differentiation, proliferation and function of inflammatory cells, including T cells, eosinophils, and B cells, by modifying several signaling pathways such as NFκB signaling pathways and TCR signals. An adequate zinc level is essential for proper immune responses and decreased zinc levels were reported in many allergic inflammatory diseases, including atopic dermatitis, bronchial asthma, and chronic rhinosinusitis. Decreased zinc levels often enhance inflammatory activation. On the other hand, the inflammatory conditions alter the intracellular homeostasis of zinc, often decreasing zinc levels. These findings implied that there could be a vicious cycle between zinc deficiency and inflammatory conditions. In this review, we present recent evidence on the involvement of zinc in atopic dermatitis, bronchial asthma, and chronic rhinosinusitis, with insights into the involvement of zinc in the underlying molecular and cellular mechanisms related to these allergic inflammatory diseases.
  • A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets.
    Masashi Watanabe, Yasushi Saeki, Hidehisa Takahashi, Fumiaki Ohtake, Yukiko Yoshida, Yusuke Kasuga, Takeshi Kondo, Hiroaki Yaguchi, Masanobu Suzuki, Hiroki Ishida, Keiji Tanaka, Shigetsugu Hatakeyama
    Communications biology, 3, 1, 592, 592, 2020年10月20日, [査読有り], [筆頭著者, 責任著者], [国際誌]
    英語, 研究論文(学術雑誌), The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.
  • The role of Mediator and Little Elongation Complex in transcription termination.
    Hidehisa Takahashi, Amol Ranjan, Shiyuan Chen, Hidefumi Suzuki, Mio Shibata, Tomonori Hirose, Hiroko Hirose, Kazunori Sasaki, Ryota Abe, Kai Chen, Yanfeng He, Ying Zhang, Ichigaku Takigawa, Tadasuke Tsukiyama, Masashi Watanabe, Satoshi Fujii, Midori Iida, Junichi Yamamoto, Yuki Yamaguchi, Yutaka Suzuki, Masaki Matsumoto, Keiichi I Nakayama, Michael P Washburn, Anita Saraf, Laurence Florens, Shigeo Sato, Chieri Tomomori-Sato, Ronald C Conaway, Joan W Conaway, Shigetsugu Hatakeyama
    Nature communications, 11, 1, 1063, 1063, 2020年02月26日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.
  • Loss of TRIM29 Alters Keratin Distribution to Promote Cell Invasion in Squamous Cell Carcinoma.
    Teruki Yanagi, Masashi Watanabe, Hiroo Hata, Shinya Kitamura, Keisuke Imafuku, Hiroko Yanagi, Akihiro Homma, Lei Wang, Hidehisa Takahashi, Hiroshi Shimizu, Shigetsugu Hatakeyama
    Cancer research, 78, 24, 6795, 6806, 2018年12月15日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), : TRIM29 (tripartite motif-containing protein 29) is a TRIM family protein that has been implicated in breast, colorectal, and pancreatic cancers. However, its role in stratified squamous epithelial cells and tumors has not been elucidated. Here, we investigate the expression of TRIM29 in cutaneous head and neck squamous cell carcinomas (SCC) and its functions in the tumorigenesis of such cancers. TRIM29 expression was lower in malignant SCC lesions than in adjacent normal epithelial tissue or benign tumors. Lower expression of TRIM29 was associated with higher SCC invasiveness. Primary tumors of cutaneous SCC showed aberrant hypermethylation of TRIM29. Depletion of TRIM29 increased cancer cell migration and invasion; conversely, overexpression of TRIM29 suppressed these. Comprehensive proteomics and immunoprecipitation analyses identified keratins and keratin-interacting protein FAM83H as TRIM29 interactors. Knockdown of TRIM29 led to ectopic keratin localization of keratinocytes. In primary tumors, lower TRIM29 expression correlated with the altered expression of keratins. Our findings reveal an unexpected role for TRIM29 in regulating the distribution of keratins, as well as in the migration and invasion of SCC. They also suggest that the TRIM29-keratin axis could serve as a diagnostic and prognostic marker in stratified epithelial tumors and may provide a target for SCC therapeutics. SIGNIFICANCE: These findings identify TRIM29 as a novel diagnostic and prognostic marker in stratified epithelial tissues.
  • Brain-Derived Neurotrophic Factor Improves Limited Exercise Capacity in Mice With Heart Failure.
    Junichi Matsumoto, Shingo Takada, Shintaro Kinugawa, Takaaki Furihata, Hideo Nambu, Naoya Kakutani, Masaya Tsuda, Arata Fukushima, Takashi Yokota, Shinya Tanaka, Hidehisa Takahashi, Masashi Watanabe, Shigetsugu Hatakeyama, Masaki Matsumoto, Keiichi I Nakayama, Yutaro Otsuka, Hisataka Sabe, Hiroyuki Tsutsui, Toshihisa Anzai
    Circulation, 138, 18, 2064, 2066, 2018年10月30日, [査読有り], [国際誌]
    英語
  • Anti-Sez6l2 antibody detected in a patient with immune-mediated cerebellar ataxia inhibits complex formation of GluR1 and Sez6l2.
    Hiroaki Yaguchi, Ichiro Yabe, Hidehisa Takahashi, Masashi Watanabe, Taichi Nomura, Takahiro Kano, Masahiko Watanabe, Shigetsugu Hatakeyama
    Journal of neurology, 265, 4, 962, 965, Dr. Dietrich Steinkopff Verlag GmbH and Co. KG, 2018年04月, [査読有り], [国際誌]
    英語
  • Mutations in bassoon in individuals with familial and sporadic progressive supranuclear palsy-like syndrome.
    Ichiro Yabe, Hiroaki Yaguchi, Yasutaka Kato, Yasuo Miki, Hidehisa Takahashi, Satoshi Tanikawa, Shinichi Shirai, Ikuko Takahashi, Mari Kimura, Yuka Hama, Masaaki Matsushima, Shinsuke Fujioka, Takahiro Kano, Masashi Watanabe, Shin Nakagawa, Yasuyuki Kunieda, Yoshio Ikeda, Masato Hasegawa, Hiroshi Nishihara, Toshihisa Ohtsuka, Shinya Tanaka, Yoshio Tsuboi, Shigetsugu Hatakeyama, Koichi Wakabayashi, Hidenao Sasaki
    Scientific reports, 8, 1, 819, 819, 2018年01月16日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), Clinical diagnosis of progressive supranuclear palsy (PSP) is sometimes difficult because various phenotypes have been identified. Here, we report a mutation in the bassoon (BSN) gene in a family with PSP-like syndrome. Their clinical features resembled not only those of PSP patients but also those of individuals with multiple system atrophy and Alzheimer's disease. The neuropathological findings showed a novel three + four repeat tauopathy with pallido-luysio-nigral degeneration and hippocampal sclerosis. Whole-exome analysis of this family identified a novel missense mutation in BSN. Within the pedigree, the detected BSN mutation was found only in affected individuals. Further genetic analyses were conducted in probands from four other pedigrees with PSP-like syndrome and in 41 sporadic cases. Three missense mutations in BSN that are very rarely listed in databases of healthy subjects were found in four sporadic cases. Western blot analysis of tau following the overexpression of wild-type or mutated BSN revealed the possibility that wild-type BSN reduced tau accumulation, while mutated BSN lost this function. An association between BSN and neurological diseases has not been previously reported. Our results revealed that the neurodegenerative disorder associated with the original proband's pedigree is a novel tauopathy, differing from known dementia and parkinsonism syndromes, including PSP.
  • Fine-tuning of thymocyte development by ubiquitination-mediated stability control of the ESCRT protein CHMP5
    Masashi Watanabe, Shigetsugu Hatakeyama
    CELLULAR & MOLECULAR IMMUNOLOGY, 14, 12, 957, 959, CHIN SOCIETY IMMUNOLOGY, 2017年12月, [査読有り], [筆頭著者]
    英語
  • Sez6l2 regulates phosphorylation of ADD and neuritogenesis
    Hiroaki Yaguchi, Ichiro Yabe, Hidehisa Takahashi, Masashi Watanabe, Taichi Nomura, Takahiro Kano, Masaki Matsumoto, Keiichi I. Nakayama, Masahiko Watanabe, Shigetsugu Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 494, 1-2, 234, 241, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2017年12月, [査読有り]
    英語, 研究論文(学術雑誌), Increasing evidence shows that immune-mediated mechanisms may contribute to the pathogenesis of central nervous system disorders including cerebellar ataxias, as indicated by the aberrant production of neuronal surface antibodies. We previously reported a patient with cerebellar ataxia associated with production of a new anti-neuronal antibody, anti-seizure-related 6 homolog like 2 (Sez6l2). Sez6l2 is a type 1 membrane protein that is highly expressed in the hippocampus and cerebellar cortex and mice lacking Sez6l2 protein family members develop ataxia. Here we used a proteomics-based approach to show that serum derived from this patient recognizes the extracellular domain of Sez6l2 and that Sez6l2 protein binds to both adducin (ADD) and glutamate receptor 1 (GluR1). Our results indicate that Sez6l2 is one of the auxiliary subunits of the AMPA receptor and acts as a scaffolding protein to link GluR1 to ADD. Furthermore, Sez6l2 overexpression upregulates ADD phosphorylation, whereas siRNA-mediated downregulation of Sez6l2 prevents ADD phosphorylation, suggesting that Sez6l2 modulates AMPAADD signal transduction. (C) 2017 Elsevier Inc. All rights reserved.
  • TRIM proteins and diseases
    Masashi Watanabe, Shigetsugu Hatakeyama
    JOURNAL OF BIOCHEMISTRY, 161, 2, 135, 144, OXFORD UNIV PRESS, 2017年02月, [査読有り], [筆頭著者]
    英語, Ubiquitination is one of the posttranslational modifications that regulates a number of intracellular events including signal transduction, protein quality control, transcription, cell cycle, apoptosis and development. The ubiquitin system functions as a garbage machine to degrade target proteins and as a regulator for several signalling pathways. Biochemical reaction of ubiquitination requires several enzymes including E1, E2 and E3, and E3 ubiquitin ligases play roles as receptors for recognizing target proteins. Most of the tripartite motif (TRIM) proteins are E3 ubiquitin ligases. Recent studies have shown that some TRIM proteins function as important regulators for a variety of diseases including cancer, inflammatory diseases, infectious diseases, neuropsychiatric disorders, chromosomal abnormalities and developmental diseases. In this review, we summarize the involvement of TRIM proteins in the aetiology of various diseases.
  • The novel heart-specific RING finger protein 207 is involved in energy metabolism in cardiomyocytes
    Wataru Mizushima, Hidehisa Takahashi, Masashi Watanabe, Shintaro Kinugawa, Shouji Matsushima, Shingo Takada, Takashi Yokota, Takaaki Furihata, Junichi Matsumoto, Masaya Tsuda, Ikuru Chiba, Shun Nagashima, Shigeru Yanagi, Masaki Matsumoto, Keiichi I. Nakayama, Hiroyuki Tsutsui, Shigetsugu Hatakeyama
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 100, 43, 53, ELSEVIER SCI LTD, 2016年11月, [査読有り]
    英語, 研究論文(学術雑誌), A failing heart shows severe energy insufficiency, and it is presumed that this energy shortage plays a critical role in the development of cardiac dysfunction. However, little is known about the mechanisms that cause energy metabolic alterations in the failing heart. Here, we show that the novel RING-finger protein 207 (RNF207), which is specifically expressed in the heart, plays a role in cardiac energy metabolism. Depletion of RNF207 in neonatal rat cardiomyocytes (NRCs) leads to a reduced cellular concentration of adenosine triphosphate (ATP) and mitochondrial dysfunction. Consistent with this result, we observed here that the expression of RNF207 was significantly reduced in mice with common cardiac diseases including heart failure. Intriguingly, proteomic approaches revealed that RNF207 interacts with the voltage-dependent anion channel (VDAC), which is considered to be a key regulator of mitochondria function, as an RNF207-interacting protein. Our findings indicate that RNF207 is involved in ATP production by cardiomyocytes, suggesting that RNF207 plays an important role in the development of heart failure. (C) 2016 Elsevier Ltd. All rights reserved.
  • p53 represses the transcription of snRNA genes by preventing the formation of little elongation complex
    Delnur Anwar, Hidehisa Takahashi, Masashi Watanabe, Masanobu Suzuki, Satoshi Fukuda, Shigetsugu Hatakeyama
    BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, 1859, 8, 975, 982, ELSEVIER SCIENCE BV, 2016年08月, [査読有り]
    英語, 研究論文(学術雑誌), The regulation of transcription by RNA polymerase II (Pol II) is important for a variety of cellular functions. ELL/EAF-containing little elongation complex (LEC) was found to be required for transcription of Pol II-dependent small nuclear RNA (snRNA) genes. It was shown that the tumor suppressor p53 interacts with ELL and inhibits transcription elongation activity of ELL Here, we show that p53 inhibits interaction between ELL/EAF and ICE1 in LEC and thereby p53 represses transcription of Pol II-dependent snRNA genes through inhibiting LEC function. Furthermore, induction of p53 expression by ultraviolet (UV) irradiation decreases the occupancy of ICE1 at Pol II-dependent snRNA genes. Consistent with the results, knockdown of p53 increased both the expression of snRNA genes and the occupancy of Pol II and components of LEC at snRNA genes. Our results indicate that p53 interferes with the interaction between ELL/EAF and ICE1 and represses transcription of snRNA genes by Pol II. (C) 2016 Elsevier B.V. All rights reserved.
  • TRIM39 negatively regulates the NFκB-mediated signaling pathway through stabilization of Cactin.
    Suzuki M, Watanabe M, Nakamaru Y, Takagi D, Takahashi H, Fukuda S, Hatakeyama S
    Cellular and molecular life sciences : CMLS, 73, 5, 1085, 1101, SPRINGER BASEL AG, 2016年03月, [査読有り]
    英語, 研究論文(学術雑誌), NF kappa B is one of the central regulators of cell survival, immunity, inflammation, carcinogenesis and organogenesis. The activation of NF kappa B is strictly regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. Several types of ubiquitination play important roles in multi-step regulations of the NF kappa B pathway. Some of the tripartite motif-containing (TRIM) proteins functioning as E3 ubiquitin ligases are known to regulate various biological processes such as inflammatory signaling pathways. One of the TRIM family proteins, TRIM39, for which the gene has single nucleotide polymorphisms, has been identified as one of the genetic factors in Behcet's disease. However, the role of TRIM39 in inflammatory signaling had not been fully elucidated. In this study, to elucidate the function of TRIM39 in inflammatory signaling, we performed yeast two-hybrid screening using TRIM39 as a bait and identified Cactin, which has been reported to inhibit NF kappa B- and TLR-mediated transcriptions. We show that TRIM39 stabilizes Cactin protein and that Cactin is upregulated after TNF alpha stimulation. TRIM39 knockdown also causes activation of the NF kappa B signal. These findings suggest that TRIM39 negatively regulates the NF kappa B signal in collaboration with Cactin induced by inflammatory stimulants such as TNF alpha.
  • The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ.
    Watanabe M, Takahashi H, Saeki Y, Ozaki T, Itoh S, Suzuki M, Mizushima W, Tanaka K, Hatakeyama S
    eLife, 4, ELIFE SCIENCES PUBLICATIONS LTD, 2015年04月, [査読有り], [筆頭著者]
    英語, 研究論文(学術雑誌), Adipocyte differentiation is a strictly controlled process regulated by a series of transcriptional activators. Adipogenic signals activate early adipogenic activators and facilitate the transient formation of early enhanceosomes at target genes. These enhancer regions are subsequently inherited by late enhanceosomes. PPAR gamma is one of the late adipogenic activators and is known as a master regulator of adipogenesis. However, the factors that regulate PPAR gamma expression remain to be elucidated. Here, we show that a novel ubiquitin E3 ligase, tripartite motif protein 23 (TRIM23), stabilizes PPAR gamma protein and mediates atypical polyubiquitin conjugation. TRIM23 knockdown caused a marked decrease in PPAR gamma protein abundance during preadipocyte differentiation, resulting in a severe defect in late adipogenic differentiation, whereas it did not affect the formation of early enhanceosomes. Our results suggest that TRIM23 plays a critical role in the switching from early to late adipogenic enhanceosomes by stabilizing PPAR gamma protein possibly via atypical polyubiquitin conjugation.
  • MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex
    Hidehisa Takahashi, Ichigaku Takigawa, Masashi Watanabe, Delnur Anwar, Mio Shibata, Chieri Tomomori-Sato, Shigeo Sato, Amol Ranjan, Chris W. Seidel, Tadasuke Tsukiyama, Wataru Mizushima, Masayasu Hayashi, Yasuyuki Ohkawa, Joan W. Conaway, Ronald C. Conaway, Shigetsugu Hatakeyama
    NATURE COMMUNICATIONS, 6, 5941, NATURE PUBLISHING GROUP, 2015年01月, [査読有り]
    英語, 研究論文(学術雑誌), Regulation of transcription elongation by RNA polymerase II (Pol II) is a key regulatory step in gene transcription. Recently, the little elongation complex (LEC)-which contains the transcription elongation factor ELL/EAF-was found to be required for the transcription of Pol II-dependent small nuclear RNA (snRNA) genes. Here we show that the human Mediator subunit MED26 plays a role in the recruitment of LEC to a subset of snRNA genes through direct interaction of EAF and the N-terminal domain (NTD) of MED26. Loss of MED26 in cells decreases the occupancy of LEC at a subset of snRNA genes and results in a reduction in their transcription. Our results suggest that the MED26-NTD functions as a molecular switch in the exchange of TBP-associated factor 7 (TAF7) for LEC to facilitate the transition from initiation to elongation during transcription of a subset of snRNA genes.
  • Pathology of frontotemporal dementia with limb girdle muscular dystrophy caused by a DNAJB6 mutation
    Ichiro Yabe, Mishie Tanino, Hiroaki Yaguchi, Akihiro Takiyama, Huaying Cai, Hiromi Kanno, Ikuko Takahashi, Yukiko K. Hayashi, Masashi Watanabe, Hidehisa Takahashi, Shigetsugu Hatakeyama, Shinya Tanak, Hidenao Sasaki
    CLINICAL NEUROLOGY AND NEUROSURGERY, 127, 10, 12, ELSEVIER SCIENCE BV, 2014年12月, [査読有り]
    英語, 研究論文(学術雑誌)
  • TRIM29 as a novel prostate basal cell marker for diagnosis of prostate cancer
    Yukiko Kanno, Masashi Watanabe, Taichi Kimura, Katsuya Nonomura, Shinya Tanaka, Shigetsugu Hatakeyama
    ACTA HISTOCHEMICA, 116, 5, 708, 712, ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2014年, [査読有り]
    英語, 研究論文(学術雑誌), Tripartite motif protein 29 (TRIM29) is one of the TRIM family proteins, some of which function as E3 ubiquitin ligases. In this study, we investigated the usefulness of TRIM29 for diagnosis of prostate cancer Prostate tissues including carcinoma and non-carcinoma tissues obtained by needle biopsy and radical prostatectomy were used. Immunohistochemistry was performed according to standard procedures using an antibody against TRIM29. Immunohistochemical staining with an antibody against 34 beta E12, which recognizes cytokeratins 1, 5, 10 and 14, was performed as a control. Basal cells of normal prostatic glands were stained with anti-TRIM29 antibody in all cases, whereas prostate cancer tissues had no or little staining with anti-TRIM29 antibody. TRIM29 is selectively expressed in basal cells of the normal prostate gland, and immunohistochemical staining with anti-TRIM29 antibody showed the same expression pattern as that with 34 beta E12 in prostate cancer and its benign mimics. Our data indicate that TRIM29 may be useful for distinguishing prostate cancers from benign tissues. (C) 2013 Elsevier GmbH. All rights reserved.
  • TRIM59 interacts with ECSIT and negatively regulates NF-κB and IRF-3/7-mediated signal pathways.
    Kondo T, Watanabe M, Hatakeyama S
    Biochemical and biophysical research communications, 422, 3, 501, 507, 3, 2012年06月, [査読有り]
    英語, 研究論文(学術雑誌), Innate immune responses are triggered by pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) and then activate intracellular signaling pathways including NF-kappa B and interferon regulatory factors. Recently, it has been reported that tripartite motif (TRIM) proteins function as crucial regulators via ubiquitin-mediated modifications for these signaling pathways. In this study, we showed that one of the TRIM family ubiquitin ligases, TRIM59, interacts with ECSIT as an adaptor protein required for the TLR-mediated transduction pathway. Luciferase reporter assays using reporter plasmids including NF-kappa B responsive element, interferon beta (IFN-beta) promoter and interferon-sensitive response element (ISRE) showed that overexpression of TRIM59 repressed their transcriptional activities, whereas knockdown of TRIM59 enhanced their transcriptional activities. Furthermore, TRIM59 inhibited phosphorylation and dimerization of IRF3 and IRF7, suggesting that TRIM59 negatively regulates upstream kinases for IRFs. These findings indicate that TRIM59 may serve as a multifunctional regulator for innate immune signaling pathways. (c) 2012 Elsevier Inc. All rights reserved.
  • TRIM24 mediates ligand-dependent activation of androgen receptor and is repressed by a bromodomain-containing protein, BRD7, in prostate cancer cells
    Misato Kikuchi, Fumihiko Okumura, Tadasuke Tsukiyama, Masashi Watanabe, Naoto Miyajima, Junji Tanaka, Masahiro Imamura, Shigetsugu Hatakeyama
    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1793, 12, 1828, 1836, ELSEVIER SCIENCE BV, 2009年12月, [査読有り]
    英語, 研究論文(学術雑誌), The androgen receptor (AR) is a ligand-dependent transcription factor that belongs to the family of nuclear receptors, and its activity is regulated by numerous AR coregulators. AR plays an important role in prostate development and cancer. In this study, we found that TRIM24/transcriptional intermediary factor lot (T1F1 alpha), which is known as a ligand-dependent nuclear receptor co-regulator, interacts with AR and enhances transcriptional activity of AR by dihydrotestosterone in prostate cancer cells. We showed that TRIM24 functionally interacts with TIP60, which acts as a coactivator of AR and synergizes with TIP60 in the transactivation of AR. We also showed that TRIM24 binds to bromodomain containing 7 (BRD7), which can negatively regulate cell proliferation and growth. A luciferase assay indicated that BRD7 represses the AR transactivation activity upregulated by TRIM24. These findings indicate that TRIM24 regulates AR-mediated transcription in collaboration with TIP60 and BRD7. (C) 2009 Elsevier B.V. All rights reserved.
  • ZNRF1 interacts with tubulin and regulates cell morphogenesis
    Koichi Yoshida, Masashi Watanabe, Shigetsugu Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 389, 3, 506, 511, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009年11月, [査読有り]
    英語, 研究論文(学術雑誌), The ubiquitin-proteasome system has been implicated in neuronal degeneration and regeneration. We demonstrated that overexpression of ZNRF1, which has been identified as a crucial molecule in nerve regeneration, causes morphological changes such as neurite-like elongation. Molecular dissections showed that both the RING finger domain and zinc finger domain are required for morphological changes. Furthermore, we identified beta-tubulin type 2 (Tubb2) as a ZNRF1-binding protein by yeast two-hybrid screening. In vivo binding assay showed that ZNRF1 interacts with Tubb2 and immunofluorescent staining Suggests that ZNRF1 is colocalized with Tubb2. These results suggest that ZNRF1 mediates regulation of neuritogenesis via interaction with tubulin. (C) 2009 Elsevier Inc. All rights reserved.
  • TRIM31 interacts with p52(Shc) and inhibits Src-induced anchorage-independent growth
    Masashi Watanabe, Tadasuke Tsukiyama, Shigetsugu Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 388, 2, 422, 427, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009年10月, [査読有り], [筆頭著者]
    英語, 研究論文(学術雑誌), Tripartite motif-containing protein (TRIM) family proteins are involved in a broad range of biological processes and, consistently, their alterations result in diverse pathological conditions such as genetic diseases, viral infection and cancer development. In this study, we found that one of the TRIM family proteins, TRIM31, is highly expressed in the gastrointestinal tract and interacts with p52(Shc), one of the signal transducers. We also found by a binding assay that almost the whole region other than the RING domain is required for the binding to p52(Shc) but found by pulse-chase analysis that overexpression of TRIM31 does not affect the stability of p52(Shc). Moreover, we found that overexpression of TRIM31 suppresses anchorage-independent cell growth induced by the active form of c-Src. These results suggest that TRIM31 attenuates c-Src signaling via p52(Shc) under anchorage-independent growth conditions and is potentially associated with growth activity of cells in the gastrointestinal tract. (C) 2009 Elsevier Inc. All rights reserved.
  • Inhibition of NF-kappa B signaling via tyrosine phosphorylation of Ymer
    Hiroyuki Kameda, Masashi Watanabe, Miyuki Bohgaki, Tadasuke Tsukiyama, Shigetsugu Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 378, 4, 744, 749, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009年01月, [査読有り]
    英語, 研究論文(学術雑誌), Cytoplasmic zinc finger protein A20 functionally dampens inflammatory signals and apoptosis via inhibition of NF-kappa B activation. We have reported that Ymer interacts with A20 and lysine (K)-63-linked polyubiquitin chain and that Ymer inhibits NF-kappa B signaling in collaboration with A20. It has also been reported that Ymer is phosphorylated by EGF stimulation. We found that Ymer was considerably phosphorylated on tyrosine residues also via Src family kinases such as Lck. A luciferase reporter assay showed that mutation of tyrosines on Ymer (YmerY217/279/304F) results in loss of the inhibitory activity for NF-kappa B signaling. Furthermore, a soft agar colony formation assay showed that the combination of SFCY527F and YmerY217/279/304F has no ability for anchorage-independent growth, suggesting that tyrosine phosphorylation of Ymer is important for inhibition of the NF-kappa B-mediated apoptotic pathway. These findings demonstrate that Ymer is likely to be a negative regulator for the NF-kappa B signaling pathway. (C) 2008 Elsevier Inc. All rights reserved
  • Involvement of Ymer in suppression of NF-kappaB activation by regulated interaction with lysine-63-linked polyubiquitin chain.
    Bohgaki M, Tsukiyama T, Nakajima A, Maruyama S, Watanabe M, Koike T, Hatakeyama S
    Biochimica et biophysica acta, 1783, 826, 837, 5, 2008年05月, [査読有り]
  • Involvement of Ymer in suppression of NF-kappa B activation by regulated interaction with lysine-63-linked polyubiquitin chain
    Miyuki Bohgaki, Tadasuke Tsukiyama, Ayako Nakajima, Satoru Maruyama, Masashi Watanabe, Takao Koike, Shigetsugu Hatakeyama
    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1783, 5, 826, 837, ELSEVIER SCIENCE BV, 2008年05月, [査読有り]
    英語, 研究論文(学術雑誌), It is known that the cytoplasmic zinc finger protein A20 functionally dampens inflammatory signals and apoptosis via inhibition of NF-kappa B activation and biochemically acts as a unique ubiquitin-modifying protein with dcubiquitmating activity and ubiquitin ligase activity. However, the molecular mechanisms of A20-modulated signal transduction that influence normal immune responses or tumor immunity have not been fully elucidated. Using a yeast two-hybrid system to search for proteins interacting with A20, we identified one novel binding protein, Ymer. Ymer, which has been reported to be highly phosphorylated on tyrosine residues via EGF stimulation, bound to lysine (K)-63-linked polyubiquitin chain on receptor-interacting serine/threonine-protein kinase 1 (RIP 1), which is essential for NF-kappa B signaling in collaboration with A20. A luciferase assay showed that NF-kappa B signaling was down-regulated by overexpression of Ymer, whereas knock-down of Ymer up-egulated NF-kappa B signaling even without stimulation. These findings demonstrate that Ymer is likely to be a negative regulator for the NF-kappa B signaling pathway. (C)007 Elsevier B.V All rights reserved.
  • Protection of vincristine-induced neuropathy by Wld(S) expression and the independence of the activity of Nmnat1
    Masashi Watanabe, Tadasuke Tsukiyama, Shigetsugu Hatakeyama
    NEUROSCIENCE LETTERS, 411, 3, 228, 232, ELSEVIER IRELAND LTD, 2007年01月, [査読有り], [筆頭著者]
    英語, 研究論文(学術雑誌), The slow Wallerian degeneration protein (Wld(S)), a fusion protein containing amino-terminal E4B and full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by physical damages, toxins and genetic mutations which result in patients being diagnosed with neurodegenerative diseases. It is still controversial whether the suppression of axonal degeneration by Wld(S) is due to Nmnat1 or other portion. We generated Wld(S) or Nmnat1-overexpressing Neuro2A cell lines, in which neuronal differentiation including neurite elongation can be induced by retinoic acid. The overexpression of Wld(S) delayed the neurite degeneration by vincristine, whereas that of Nmnat1 did not delay it much. Taken together, Nmnat1 is considerably weaker than Wld(S) for protection from toxic injury in vitro, suggesting that amino-terminal region of Wld(S) is likely to be more significant for protection from axonal degeneration. (c) 2006 Elsevier Ireland Ltd. All rights reserved.
  • Targeted destruction of c-Myc by an engineered ubiquitin ligase suppresses cell transformation and tumor formation
    S Hatakeyama, M Watanabe, Y Fujii, KL Nakayama
    CANCER RESEARCH, 65, 17, 7874, 7879, AMER ASSOC CANCER RESEARCH, 2005年09月, [査読有り]
    英語, 研究論文(学術雑誌), Given that expression of c-Myc is up-regulated in many human malignancies, targeted inactivation of this oncoprotein is a potentially effective strategy for cancer treatment The ubiquitin-proteasome pathway of protein degradation is highly specific and can be engineered to achieve the elimination of undesirable proteins such as oncogene products. We have now generated a ftision protein (designated Max-U) that is composed both of May, which forms a heterodimer with c-Myc, and of CRW, which is a U box-type ubiquitin ligase (E3). Max-U physically interacted with c-Myc in transfected cells and promoted the ubiquitylation of c-Myc in vitro. It also reduced the stability of c-Myc in vivo, resulting in suppression of transcriptional activity dependent on c-Myc. Expression of Max-U reduced both the abundance of endogenous c-Myc in and the proliferation rate of a Burkitt lymphoma cell line. Furthermore, expression of Max-U but not that of a catalytically inactive mutant thereof markedly inhibited both the anchorage-independent growth in vitro of NIH 3T3 cells that overexpress c-Myc as well as tumor formation by these cells in nude mice. These findings indicate that the targeted destruction of c-Myc by an artificial E3 may represent an effective therapeutic strategy for certain human malignancies.

その他活動・業績

書籍等出版物

  • Advances in Medicine and Biology. Volume 120               
    Masashi Watanabe, Shigetsugu Hatakeyama, Chapter 3. Ubiquitin-Conjugating Enzymes (E2s)
    Nova Science Publishers, 2017年, 9781536118070

講演・口頭発表等

  • 自然免疫シグナルに関わるユビキチンリガーゼ群の網羅的な基質同定と解析               
    渡部 昌, 畠山鎮次
    第92回日本生化学会大会, 2019年09月19日
    [招待講演]
  • The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARgamma               
    渡部昌
    第39回日本分子生物学会年会, 2016年12月01日, 英語, シンポジウム・ワークショップパネル(指名)
    [招待講演], [国内会議]

担当経験のある科目_授業

  • 遺伝学               
    北海道大学
  • 生化学実習               
    北海道大学
  • 生化学               
    北海道大学
  • 細胞生物学               
    北海道大学

所属学協会

  • 日本生理学会               
  • 日本分子生物学会               
  • 日本生化学会               

共同研究・競争的資金等の研究課題

  • 新規に開発した基質同定法を用いたがんドライバーユビキチンリガーゼの機能解析
    科学研究費助成事業
    2021年04月01日 - 2024年03月31日
    渡部 昌, 近藤 豪
    (1)がん関連ユビキチンリガーゼ基質同定プローブの作製と安定発現細胞株の樹立:解析対象の18種類のユビキチンリガーゼ遺伝子を入手し、基質を捕獲するためのユビキチンリガーゼプローブをレトロウイルス発現ベクター上に組み込んだ。今年度は変異していない野生型のユビキチンリガーゼ遺伝子について作製を行った。作製したプローブベクターを用いてレトロウイルスを作製し、プローブを安定に発現する細胞株作製を試みたところ、13種類のプローブについて作製に成功した。残り5種については細胞毒性のために作製できなかった。
    (2) イオントラップ・オービトラップ型質量分析器による基質・結合分子同定:樹立した13種類の細胞からユビキチン化タンパク質を精製し、高感度質量分析計にて網羅的な同定を行った。具体的には、それぞれの細胞を可溶化し、抗FLAG抗体で免疫沈降を行い、第一段階の精製を行った。トリクロロ酢酸を用いて沈殿後、アセトンによる洗浄、乾燥、ジチオスレイトールにて還元、ヨードアセタミドにてアルキル化を行った後にトリプシンにてペプチドへと分解後、脱塩処理を行った。さらに抗ユビキチンレムナント抗体で再度免疫沈降を行い、第二段階の精製を行い、再度脱塩処理を行った。得られたサンプルについて質量分析を行った。実験は全て3回繰り返した。得られた結果は、過去に我々が行い蓄積している同様の基質同定結果と比較してスコアを算出することで、個々のユビキチンリガーゼ特異的な基質候補を抽出した。抗ユビキチンレムナント抗体について、これまではアガロースビーズに結合したものを使用していたが、今年度はマグネットビーズに結合した抗体についても検討を行い、アガロースビーズと同等またはそれ以上の結果が得られることを確認した。
    日本学術振興会, 基盤研究(B), 北海道大学, 21H02690
  • 「ユビキチンコード」解明へ向けたポリユビキチン化基質の網羅的同定
    科学研究費助成事業 挑戦的研究(萌芽)
    2019年06月28日 - 2022年03月31日
    渡部 昌
    ユビキチンによるタンパク質の可逆的修飾は、様々な生命現象を支える重要な翻訳後修飾の一つであり、特に近年、自身の分子内に7個存在するリジン残基とアミノ末端のメチオニンを介した多彩なポリユビキチン鎖と、その機能との関係性の解明に焦点があたっている。本研究では、ポリユビキチン鎖を効率よく抽出するプローブを複数開発し、そのポリユビキチン鎖特異性の検証を行った。その結果、バリエーションに富んだ特異性を保持する複数のプローブを得た。
    日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 19K22408
  • 信頼性の高いユビキチン化基質タンパク質の新規同定法の確立
    科学研究費助成事業
    2017年06月30日 - 2019年03月31日
    畠山 鎮次, 高橋 秀尚, 渡部 昌
    「ユビキチン化」はタンパク質分解を制御する多くの生命現象を支える極めて重要な翻訳後修飾の一つであり、ユビキチンリガーゼ(E3)が選択的に基質タンパク質を認識する。したがって個々のE3に特異的な基質を同定し、基質タンパク質のユビキチン化部位を決定することは、それらが関連する生命現象を理解する上で重要である。しかし、さまざまな問題により、ユビキチン化を受けた基質同定法の標準化には未だほど遠い。本研究では、TUBEとユビキチンレムナント抗体を用いた新規のテクノロジーにより、さまざまなタイプのユビキチンリガーゼ(E3)へ網羅的に適用し、E3-基質関係を俯瞰的に解析した。
    日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 17K19506
  • ゲノムに局在するユビキチン様修飾分子Ufm1による生活習慣病防御機構の解明               
    科学研究費補助金(若手研究(A))
    2016年 - 2019年
    渡部昌
    文部科学省, 研究代表者, 競争的資金
  • TRIMファミリータンパク質によるシグナル伝達制御
    科学研究費助成事業
    2015年04月01日 - 2018年03月31日
    畠山 鎮次, 高橋 秀尚, 渡部 昌
    申請者らの研究により、細胞内シグナル伝達制御におけるTRIMファミリーユビキチンリガーゼ群の重要性が明らかとなった。特に、細胞増殖や細胞分化の過程を制御するシグナル伝達系に、TRIMファミリーユビキチンリガーゼが関与していることが示された。そこで本研究申請においては、網羅的ノックダウンスクリーニングやプロテオミクス的手法により、さまざまな細胞内シグナル系におけるユビキチン化を制御するTRIM型ユビキチンリガーゼを同定・解析した。さらには、siRNAライブラリーを用いたTRIMファミリー遺伝子の網羅的ノックダウンにより、代謝や免疫シグナルに関する候補遺伝子を同定し、その機能解析を行った。
    日本学術振興会, 基盤研究(B), 北海道大学, 15H04690
  • 自然免疫シグナルにおけるユビキチン化基質の網羅的同定と解析               
    科学研究費補助金(新学術領域研究)
    2017年 - 2018年
    渡部昌
    文部科学省, 研究代表者, 競争的資金
  • 特異性の高い新規ユビキチンリガーゼ基質同定法の開発
    科学研究費補助金(挑戦的萌芽研究)
    2015年 - 2016年
    渡部 昌
    ユビキチン化修飾の基質特異性はユビキチンリガーゼ(E3)が決定しているため、個々のE3に特異的な基質を同定しそのユビキチン化部位を決定することは様々な生命現象を理解する上で重要となる。近年、ユビキチン結合ドメイン(UBA)とE3の融合プローブによるLigase trap法、ユビキチン高親和性ドメイン(TUBE)とユビキチン化を受けたペプチドを認識するK-εGG抗体を組み合わせるTR-TUBE法が提唱された。私たちはこの手法にいくつかの改良を加えてさらに進展させた基質同定法の開発を試み、感度の向上に成功している。この手法を用いることで、E3の機能解析が加速することが期待できると思われる。
    文部科学省, 挑戦的萌芽研究, 北海道大学, 研究代表者, 競争的資金, 15K15058
  • 直鎖状ユビキチン化による脂質代謝制御転写因子PPARガンマー活性化制御機構の解明
    科学研究費補助金(若手研究(B))
    2013年 - 2014年
    渡部 昌
    脂肪細胞分化は転写因子群によって厳密に制御されている。分化誘導刺激を行うと、まず早期転写因子群が誘導され、標的遺伝子上で早期エンハンシオソームが形成される。これは後に後期転写因子群に引き継がれ、脂肪細胞の成熟に必須な遺伝子の発現が誘導されることで分化が進行する。PPARガンマーは脂肪細胞分化のマスター遺伝子であり、後期転写因子の1つである。本研究では、新規ユビキチンリガーゼTRIM23がPPARガンマータンパク質の非定型ユビキチン化を促進し、安定化させることで脂肪細胞分化を円滑に進行させる機能を持っていることを見出した。
    文部科学省, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 25860201
  • ユビキチン化による脂質代謝制御転写因子PPARガンマーの活性化制御機構の解明
    科学研究費補助金(研究活動スタート支援)
    2012年 - 2012年
    渡部 昌
    ①PTRIMは直接PPARγをユビキチン化することを解明
    申請者は、少なくとも細胞内ではPPARγと結合しユビキチン化を促進するユビキチンリガーゼPTRIMを同定していたが、直接結合することでユビキチン化を促進しているかは明らかではなかった。リコンビナントPTRIM、PPARγを作製し、In vitroユビキチン化アッセイを行ったところ、PTRIMによりPPARγを直接ユビキチン化することを明らかにした。
    ②免疫沈降法と質量分析器を用いた新たなTRIM23相互作用タンパク質の探索
    FLAG-TRIM23タンパク質を細胞内で発現させ、抗FLAG抗体にてプルダウンし、質量分析器にて相互作用分子を探索し、複数の結合タンパク質を得た。このうち、メディエーター複合体サブユニットのMed24が含まれていた。PTRIMがMed24のリクルートを介してPPARγの活性化を調節している可能性が示唆された。
    文部科学省, 研究活動スタート支援, 北海道大学, 研究代表者, 競争的資金, 24890002
  • 疾患関連TRIMファミリーユビキチンリガーゼ群の網羅的解析               
    科学研究費助成事業(特別研究員奨励費)
    2009年 - 2011年
    渡部昌
    文部科学省, 研究代表者, 競争的資金

産業財産権

  • 標的タンパク質分解誘導剤を介してユビキチン化されるタンパク質の同定方法               
    特許権, 渡部昌, 畠山鎮次, 佐伯泰
    特願2021-170461, 2021年10月18日

社会貢献活動

  • さっぽろサイエンスフェスティバル2015 in 北大               
    2015年12月19日
    講師, 実演
    フェスティバル
  • 立命館慶祥中学校 中学3年生キャリア教育 北海道大学キャンパスツアー「ヒトの遺伝子の異常によって起きる病気」               
    2015年11月18日
    講師
    セミナー・ワークショップ
    立命館慶祥中学校

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