Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Biochemistry

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Biochemistry

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Profile and Settings

Degree

• M.D., Ph.D.

Profile and Settings

• Name (Japanese)

  HATAKEYAMA

• Name (Kana)

  Shigetsugu

• Name

  200901010757908976

Alternate Names

https://hokudai-ikagaku.jp

Achievement

Research Interests

• TRIN protein   タンパク質分解   プロテアソーム   ユビキチンリガーゼ   ユビキチン   

Research Areas

• Life sciences / Cell biology
• Life sciences / Medical biochemistry
• Life sciences / Pathobiochemistry
• Life sciences / Molecular biology

Research Experience

• 2021/04 - Today Hokkaido University Faculty of Medicine and Graduate School of Medicine Dean
• 2017/04 - Today Hokkaido University Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine Professor
• 2017/04 - 2021/03 Hokkaido University Faculty of Medicine and Graduate School of Medicine Vice Dean
• 2011/04 - 2021/03 Hokkaido University Central Institute of Radioisotope Director
• 2014/04 - 2017/03 Hokkaido University
• 2007/04 - 2017/03 Hokkaido University Graduate School of Medicine, Division of Medicine
• 2013/04 - 2014/03 Hokkaido University
• 2011/09 - 2013/08 Hokkaido University Institute for Animal Experimentation Graduate School of Medicine
• 2004/07 - 2007/03 Hokkaido University Graduate School of Medicine, Division of Physiological Science
• 2000/11 - 2004/06 Kyushu University Medical Institute of Bioregulation
• 1997/03 - 2000/10 Kyushu University Medical Institute of Bioregulation
• 1995/06 - 1997/02 NIH NCI
• 1994/04 - 1995/05 ワシントン大学医学部 博士研究員

Education

• 1990/04 - 1994/03  Hokkaido University  Graduate School of Medicine
• 1984/04 - 1990/03  Hokkaido University  School of Medicine

Awards

• 2013/06 日本リウマチ財団 三浦記念リウマチ学術研究賞
   リウマチ性疾患における免疫細胞活性化を制御するユビキチン化システムの解明 

  受賞者: 畠山 鎮次
• 2012/11 公益信託・日本白血病研究基金研究助成事業 一般研究賞
   ユビキチン依存性分化制御による白血病治療への展開基盤 

  受賞者: 畠山 鎮次
• 2007 公益信託・日本白血病研究基金研究助成事業 一般研究賞
   多発性骨髄腫細胞の細胞内品質管理におけるユビキチン化酵素Ro52の役割 

  受賞者: 畠山 鎮次
• 2005 日本リウマチ財団 三浦記念リウマチ学術研究賞
   シェーグレン症候群関連自己抗原Ro52/SSAのユビキチンリガーゼとしての機能解析 

  受賞者: 畠山 鎮次

Published Papers

• Neuroimmune modulation by tryptophan derivatives in neurological and inflammatory disorders.

  Takeshi Kondo, Yuka Okada, Saika Shizuya, Naoko Yamaguchi, Shigetsugu Hatakeyama, Kenta Maruyama

  European journal of cell biology 103 (2) 151418 - 151418 2024/05/08 [Refereed]
   

  The nervous and immune systems are highly developed, and each performs specialized physiological functions. However, they work together, and their dysfunction is associated with various diseases. Specialized molecules, such as neurotransmitters, cytokines, and more general metabolites, are essential for the appropriate regulation of both systems. Tryptophan, an essential amino acid, is converted into functional molecules such as serotonin and kynurenine, both of which play important roles in the nervous and immune systems. The role of kynurenine metabolites in neurodegenerative and psychiatric diseases has recently received particular attention. Recently, we found that hyperactivity of the kynurenine pathway is a critical risk factor for septic shock. In this review, we first outline neuroimmune interactions and tryptophan derivatives and then summarized the changes in tryptophan metabolism in neurological disorders. Finally, we discuss the potential of tryptophan derivatives as therapeutic targets for neuroimmune disorders.
• 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 2023/08 [Refereed]
  
• 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  2023/06/13 [Refereed]
   

  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.
• 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 2023/03/09 [Refereed]
   

  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.
• Curcumin partly prevents ISG15 activation via ubiquitin-activating enzyme E1-like protein and decreases ISGylation.

  Nodoka Oki, Shino Yamada, Tamaki Tanaka, Hiromi Fukui, Shigetsugu Hatakeyama, Fumihiko Okumura

  Biochemical and biophysical research communications 625 94 - 101 2022/08/05 [Refereed]
   

  The expression of the ubiquitin-like molecule interferon-stimulated gene 15 kDa (ISG15) and post-translational protein modification by ISG15 (ISGylation) are strongly activated by interferons or pathogen infection, suggesting that ISG15 and ISGylation play an important role in innate immune responses. More than 400 proteins have been found to be ISGylated. ISG15 is removed from substrates by interferon-induced ubiquitin-specific peptidase 18 or severe acute respiratory syndrome coronavirus 2‒derived papain-like protease. Therefore, maintaining strong ISGylation may help prevent the spread of coronavirus disease 2019 (COVID-19). However, it is unknown whether nutrients or chemicals affect ISGylation level. Curcumin is the major constituent of turmeric and functions as an immunomodulator. Here, we investigated the effect of curcumin on ISGylation. MCF10A and A549 cells were treated with interferon α and curcumin after which the expression levels of various proteins were determined. The effect of curcumin on ubiquitylation was also determined. Curcumin treatment was found to reduce ISGylation in a dose-dependent manner. The findings suggested that curcumin partly prevents disulfide bond-mediated ISG15 dimerization directly or indirectly, thereby increasing monomer ISG15 levels. Reduced ISGylation may also occur via the prevention of ISG15 activation by ubiquitin-activating enzyme E1-like protein. In conclusion, curcumin treatment was found to reduce ISGylation, suggesting that it may contribute to severe COVID-19. This is the first study to report a relationship between ISGylation and a food component.
• 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 2022/06/28 [Refereed]
   

  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 2021/09/30 [Refereed]
   

  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.
• Post-translational Wnt receptor regulation: Is the fog slowly clearing?: The molecular mechanism of RNF43/ZNRF3 ubiquitin ligases is not yet fully elucidated and still controversial

  Tadasuke Tsukiyama, Bon-Kyoung Koo, Shigetsugu Hatakeyama

  BioEssays 43 (4) 1521-1878 2021/04/01 

  Wnt signaling plays pivotal roles during our entire lives, from conception to death, through the regulation of morphogenesis in developing embryos and the maintenance of tissue homeostasis in adults. The regulation of Wnt signaling occurs on several levels: at the receptor level on the plasma membrane, at the β-catenin protein level in the cytoplasm, and through transcriptional regulation in the nucleus. Several recent studies have focused on the mechanisms of Wnt receptor regulation, following the discovery that the Wnt receptor frizzled (Fzd) is a target of the ubiquitin ligases, RNF43 and ZNRF3. RNF43 and ZNRF3 are homologous genes that are mutated in several cancers. The details underlying their mechanism of action continue to unfold, while at the same time raising many new questions. In this review, we discuss advances and controversies in our understanding of Wnt receptor regulation.
• 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 2021/04 [Refereed]
   

  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.
• 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 [Refereed]
   

  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.
• A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets.

  Watanabe M, Saeki Y, Takahashi H, Ohtake F, Yoshida Y, Kasuga Y, Kondo T, Yaguchi H, Suzuki M, Ishida H, Tanaka K, Hatakeyama S

  Communications biology 3 (1) 2020/10 [Refereed][Not invited]
   

  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.
• RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis.

  Erika Sugisawa, Yasunori Takayama, Naoki Takemura, Takeshi Kondo, Shigetsugu Hatakeyama, Yutaro Kumagai, Masataka Sunagawa, Makoto Tominaga, Kenta Maruyama

  Cell 182 (3) 609 - 624 0092-8674 2020/06/29 [Refereed][Not invited]
   

  Gastrointestinal enterochromaffin cells regulate bone and gut homeostasis via serotonin (5-hydroxytryptamine [5-HT]) production. A recent report suggested that gut microbes regulate 5-HT levels; however, the precise underlying molecular mechanisms are unexplored. Here, we reveal that the cation channel Piezo1 in the gut acts as a sensor of single-stranded RNA (ssRNA) governing 5-HT production. Intestinal epithelium-specific deletion of mouse Piezo1 profoundly disturbed gut peristalsis, impeded experimental colitis, and suppressed serum 5-HT levels. Because of systemic 5-HT deficiency, conditional knockout of Piezo1 increased bone formation. Notably, fecal ssRNA was identified as a natural Piezo1 ligand, and ssRNA-stimulated 5-HT synthesis from the gut was evoked in a MyD88/TRIF-independent manner. Colonic infusion of RNase A suppressed gut motility and increased bone mass. These findings suggest gut ssRNA as a master determinant of systemic 5-HT levels, indicating the ssRNA-Peizo1 axis as a potential prophylactic target for treatment of bone and gut disorders.
• 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 2020/02/26 [Refereed][Not invited]
   

  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.
• NONO Is a Negative Regulator of SOX2 Promoter

  SHANSHAN LIANG, HIDEHISA TAKAHASHI, TETSURO HIROSE, YASUHIRO KURAMITSU, SHIGETSUGU HATAKEYAMA, HIRONORI YOSHIYAMA, RUOYU WANG, JUN-ICHI HAMADA, HISASHI IIZASA

  Cancer Genomics - Proteomics 17 (4) 359 - 367 1109-6535 2020 [Refereed][Not invited]
  
• Autophagy promotes citrullination of VIM (vimentin) and its interaction with major histocompatibility complex class II in synovial fibroblasts.

  Sugawara E, Kato M, Kudo Y, Lee W, Hisada R, Fujieda Y, Oku K, Bohgaki T, Amengual O, Yasuda S, Onodera T, Hatakeyama S, Atsumi T

  Autophagy 16 (5) 1 - 10 1554-8627 2019/09 [Refereed][Not invited]
   

  We aimed to investigate the involvement of macroautophagy/autophagy in autoimmunity in rheumatoid arthritis (RA) through citrullination of VIM (vimentin) and its interaction with MHC class II in synovial fibroblasts (SFs). The cell surface expression of MHC class II and B7 costimulatory molecules on SFs was analyzed by flow cytometry after treatment with IFNG/IFN-γ (interferon gamma). Intracellular citrullinated autoantigens in SFs were analyzed by immunoblotting using serum from anti-citrullinated peptide antibodies (ACPA)-positive patient as a primary antibody. SFs were incubated in serum-free medium or treated with proteasome inhibitor MG132 to induce autophagy. An autophagy inhibitor 3-methyladenin (3-MA) was used. Intracellular citrullinated VIM (cVIM) was evaluated by immunoblotting and immunocytochemistry. The interaction between MHC class II and cVIM was evaluated with co-immunoprecipitation and proximity ligation assay (PLA). We demonstrated that MHC class II, CD274/B7-H1 and PDCD1LG2/B7-DC were expressed on SFs following treatment with IFNG whereas CD276/B7-H3 was detected on SFs regardless of the presence of IFNG. ACPA-positive sera recognized a 54 kDa protein in SFs. By immunoprecipitation, the 54 kDa protein recognized by RA sera was revealed to be cVIM. Following induction of autophagy, intracellular cVIM was increased in SFs but the effect was canceled by 3-MA. The interaction between MHC class II and cVIM was demonstrated by co-immunoprecipitation. Furthermore, PLA revealed the significant increase of MHC class II-cVIM interaction following induction of autophagy. Our findings suggest that SFs may contribute to the autoimmunity in RA through citrullination of VIM and its interaction with MHC class II promoted by autophagy.Abbreviations: 3-MA: 3-methyladenine; ACPA: anti-citrullinated peptide antibodies; anti-CCP: anti-cyclic citrullinated peptide antibody; cVIM: citrullinated VIM; BECN1: beclin1; DAPI: 4',6-diamidino-2-phenylindole; FBS: fetal bovine serum; HLA: human leukocyte antigen; IFNG/IFN-γ: interferon gamma; IL6: interleukin 6; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence index; MHC: major histocompatibility complex; OA: osteoarthritis; PADI: peptidyl arginine deiminase; PepA: pepstatin A; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RA: rheumatoid arthritis; SFs: synovial fibroblasts; siRNA: small interfering RNA; VIM: vimentin.
• Loss of TRIM29 alters keratin distribution to promote cell invasion in squamous cell carcinoma.

  Yanagi T, Watanabe M, Hata H, Kitamura S, Imafuku K, Yanagi H, Homma A, Wang L, Takahashi H, Shimizu H, Hatakeyama S

  Cancer research 78 (24) 6795 - 6806 2018/11 [Refereed][Not invited]
   

  : 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 0009-7322 2018/10/30 [Refereed][Not invited]
  
• TRIM59 promotes gliomagenesis by inhibiting tc45 dephosphorylation of STAT3

  Youzhou Sang, Yanxin Li, Lina Song, Angel A. Alvarez, Weiwei Zhang, Deguan Lv, Jianming Tang, Feng Liu, Zhijie Chang, Shigetsugu Hatakeyama, Bo Hu, Shi-Yuan Cheng, Haizhong Feng

  Cancer Research 78 (7) 1792 - 1804 1538-7445 2018/04/01 [Refereed][Not invited]
   

  Aberrant EGFR signaling is a common driver of glioblastoma (GBM) pathogenesis however, the downstream effectors that sustain this oncogenic pathway remain unclarified. Here we demonstrate that tripartite motif-containing protein 59 (TRIM59) acts as a new downstream effector of EGFR signaling by regulating STAT3 activation in GBM. EGFR signaling led to TRIM59 upregulation through SOX9 and enhanced the interaction between TRIM59 and nuclear STAT3, which prevents STAT3 dephosphorylation by the nuclear form of T-cell protein tyrosine phosphatase (TC45), thereby maintaining transcriptional activation and promoting tumorigenesis. Silencing TRIM59 suppresses cell proliferation, migration, and orthotopic xenograft brain tumor formation of GBM cells and glioma stem cells. Evaluation of GBM patient samples revealed an association between EGFR activation, TRIM59 expression, STAT3 phosphorylation, and poor prognoses. Our study identifies TRIM59 as a new regulator of oncogenic EGFR/STAT3 signaling and as a potential therapeutic target for GBM patients with EGFR activation. Significance: These findings identify a novel component of the EGFR/STAT3 signaling axis in the regulation of glioma tumorigenesis.
• 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 1432-1459 2018/04/01 [Refereed][Not invited]
  
• 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 2018/01/16 [Refereed][Not invited]
   

  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.
• 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 0006-291X 2017/12 [Refereed][Not invited]
   

  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.
• 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 1672-7681 2017/12 [Refereed][Not invited]
  
• TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis

  Shigetsugu Hatakeyama

  TRENDS IN BIOCHEMICAL SCIENCES 42 (4) 297 - 311 0968-0004 2017/04 [Refereed][Invited]
   

  Tripartite motif (TRIM) family proteins, most of which have E3 ubiquitin ligase activities, have various functions in cellular processes including intracellular signaling, development, apoptosis, protein quality control, innate immunity, autophagy, and carcinogenesis. The ubiquitin system is one of the systems for post-translational modifications, which play crucial roles not only as markers for degradation of target proteins by the proteasome but also as regulators of protein-protein interactions and of the activation of enzymes. Accumulating evidence has shown that TRIM family proteins have unique, important roles and that their dysregulation causes several diseases classified as cancer, immunological disease, or developmental disorders. In this review we focus on recent emerging topics on TRIM proteins in the regulation of autophagy, innate immunity, and carcinogenesis.
• Regulation of intestinal homeostasis by the ulcerative colitis-associated gene RNF186

  Kosuke Fujimoto, Makoto Kinoshita, Hiroo Tanaka, Daisuke Okuzaki, Yosuke Shimada, Hisako Kayama, Ryu Okumura, Yoki Furuta, Masashi Narazaki, Atsushi Tamura, Shigetsugu Hatakeyama, Masahito Ikawa, Kiichiro Tsuchiya, Mamoru Watanabe, Atsushi Kumanogoh, Sachiko Tsukita, Kiyoshi Takeda

  MUCOSAL IMMUNOLOGY 10 (2) 446 - 459 1933-0219 2017/03 [Refereed][Not invited]
   

  Genome-wide association studies and subsequent deep sequencing analysis have identified susceptible loci for inflammatory bowel diseases (IBDs) including ulcerative colitis (UC). A gene encoding RING finger protein 186 (RNF186) is located within UC-susceptible loci. However, it is unclear whether RNF186 is involved in IBD pathogenesis. Here, we show that RNF186 controls protein homeostasis in colonic epithelia and regulates intestinal inflammation. RNF186, which was highly expressed in colonic epithelia, acted as an E3 ligase mediating polyubiquitination of its substrates. Permeability of small organic molecules was augmented in the intestine of Rnf186 (-/-) mice. Increased expression of several RNF186 substrates, such as occludin, was found in Rnf186(-/-) colonic epithelia. The disturbed protein homeostasis in Rnf186(-/-) mice correlated with enhanced endoplasmic reticulum (ER) stress in colonic epithelia and increased sensitivity to intestinal inflammation after dextran sulfate sodium (DSS) treatment. Introduction of an UC-associated Rnf186 mutation led to impaired E3 ligase activity and increased sensitivity to DSS-induced intestinal inflammation in mice. Thus, RNF186 maintains gut homeostasis by controlling ER stress in colonic epithelia.
• TRIM proteins and diseases

  Masashi Watanabe, Shigetsugu Hatakeyama

  JOURNAL OF BIOCHEMISTRY 161 (2) 135 - 144 0021-924X 2017/02 [Refereed][Invited]
   

  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.
• Leukemogenic kinase FIP1L1-PDGFRA and a small ubiquitin-like modifier E3 ligase, PIAS1, form a positive cross-talk through their enzymatic activities

  Makoto Ibata, Junko Iwasaki, Yoichiro Fujioka, Koji Nakagawa, Stephanie Darmanin, Masahiro Onozawa, Daigo Hashimoto, Yusuke Ohba, Shigetsugu Hatakeyama, Takanori Teshima, Takeshi Kondo

  CANCER SCIENCE 108 (2) 200 - 207 1347-9032 2017/02 [Refereed][Not invited]
   

  Fusion tyrosine kinases play a crucial role in the development of hematological malignancies. FIP1L1-PDGFRA is a leukemogenic fusion kinase that causes chronic eosinophilic leukemia. As a constitutively active kinase, FIP1L1-PDGFRA stimulates downstream signaling molecules, leading to cellular proliferation and the generation of an anti-apoptotic state. Contribution of the N-terminal FIP1L1 portion is necessary for FIP1L1-PDGFRA to exert its full transforming activity, but the underlying mechanisms have not been fully characterized. We identified PIAS1 as a FIP1L1-PDGFRA association molecule by yeast two-hybrid screening. Our analyses indicate that the FIP1L1 portion of FIP1L1-PDGFRA is required for efficient association with PIAS1. As a consequence of the association, FIP1L1-PDGFRA phosphorylates PIAS1. Moreover, the kinase activity of FIP1L1-PDGFRA stabilizes PIAS1. Therefore, PIAS1 is one of the downstream targets of FIP1L1-PDGFRA. Moreover, we found that PIAS1, as a SUMO E3 ligase, sumoylates and stabilizes FIP1L1-PDGFRA. In addition, suppression of PIAS1 activity by a knockdown experiment resulted in destabilization of FIP1L1-PDGFRA. Therefore, FIP1L1-PDGFRA and PIAS1 form a positive cross-talk through their enzymatic activities. Suppression of sumoylation by ginkgolic acid, a small molecule compound inhibiting a SUMO E1-activating enzyme, also destabilizes FIP1L1-PDGFRA, and while the tyrosine kinase inhibitor imatinib suppresses FIP1L1-PDGFRA-dependent cell growth, ginkgolic acid or siRNA of PIAS1 has a synergistic effect with imatinib. In conclusion, our results suggest that sumoylation by PIAS1 is a potential target in the treatment of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia.
• Ubiquitin-conjugating enzymes (E2S)

  Masashi Watanabe, Shigetsugu Hatakeyama

  Advances in Medicine and Biology 120 49 - 71 2017/01/01 

  Ubiquitination is one of the posttranslational modification pathways used by eukaryotic cells and is involved in a broad range of intracellular processes such as cell proliferation, cell death, damaged DNA repair, protein quality control, and signal transduction. Alterations in the ubiquitin (Ub) transfer pathway are also associated with various disease conditions including cancer, cardiovascular diseases, infectious diseases, and neuropsychiatric disorders. Ubiquitination is catalyzed by ubiquitinactivating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E1 activates Ub in an ATP-dependent manner and transfers the activated Ub to the conserved catalytic cysteine residue of E2. E3 is a substrate recognition module and catalyzes the transfer of activated ubiquitin from E2 to the lysine residue of a substrate via binding to both E2 and the substrate. Ub itself can act as a substrate, and seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) and an N-terminus (Met1) of Ub served as donors to form a polyubiquitin chain. In humans, about 35 E2 genes have been identified and have been classified into 4 classes based on the presence or absence of an additional N- or C-terminal stretch of polypeptides to the catalytic core. E2s are thought to be 'ubiquitin couriers' with supporting functions. However, recent studies have revealed that E2s have active functions in determination of the type, length and topology of ubiquitin chains and in the progression of chain assembly reactions, whereby they determine the intracellular fate of the modified substrates. Here, we show the current understanding of the functional diversity of E2 enzymes.
• 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 0022-2828 2016/11 [Refereed][Not invited]
   

  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.
• Role of apolipoprotein B100 and oxidized low-density lipoprotein in the monocyte tissue factor induction mediated by anti-2 glycoprotein I antibodies

  K. Otomo, O. Amengual, Y. Fujieda, H. Nakagawa, M. Kato, K. Oku, T. Horita, S. Yasuda, M. Matsumoto, K. I. Nakayama, S. Hatakeyama, T. Koike, T. Atsumi

  LUPUS 25 (12) 1288 - 1298 0961-2033 2016/10 [Refereed][Not invited]
   

  Objective The objective of this paper is to elucidate the not yet known plasma molecule candidates involved in the induction of tissue factor (TF) expression mediated by 2GPI-dependent anticardiolipin antibody (aCL/2GPI) on monocytes. Methods Human serum incubated with FLAG-2GPI was applied for affinity chromatography with anti- FLAG antibody. Immunopurified proteins were analyzed by a liquid chromatography coupled with mass spectrometry (LC-MS). TF mRNA induced by the identified molecules on monocytes was also analyzed. Results Apolipoprotein B100 (APOB) was the only identified serum molecule in the MS search. Oxidized LDL, containing APOB as well as ox-Lig1 (a known ligand of 2GPI), was revealed as a 2GPI-binding molecule in the immunoprecipitation assay. TF mRNA was markedly induced by oxidized LDL/2GPI complexes with either WBCAL-1 (monoclonal aCL/2GPI) or purified IgG from APS patients. The activities of lipoprotein-associated phospholipase A2, one of the component molecules of oxidized LDL, were significantly higher in serum from APS patients than in those from controls. Conclusion APOB (or oxidized LDL) was detected as a major 2GPI binding serum molecule by LC-MS search. Oxidized LDL/aCL/2GPI complexes significantly induced TF expressions on monocytes. These data suggest that complexes of oxidized LDL and aCL/2GPI may have a crucial role in the pathophysiology of APS.
• 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 1874-9399 2016/08 [Refereed][Not invited]
   

  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.
• Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes

  Yuichiro Fujieda, Olga Amengual, Masaki Matsumoto, Kimiko Kuroki, Hidehisa Takahashi, Michihito Kono, Takashi Kurita, Kotaro Otomo, Masaru Kato, Kenji Oku, Toshiyuki Bohgaki, Tetsuya Horita, Shinsuke Yasuda, Katsumi Maenaka, Shigetsugu Hatakeyama, Keiichi I. Nakayama, Tatsuya Atsumi

  RHEUMATOLOGY 55 (6) 1117 - 1126 1462-0324 2016/06 [Refereed][Not invited]
   

  Objective. Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphati dylserine-prothrombin complex, which is associated with APS. We have previously reported that aPS-PT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression. Methods. RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography-tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR). Results. RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT. Conclusion. We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS.
• TRIM39 negatively regulates the NF kappa B-mediated signaling pathway through stabilization of Cactin

  Masanobu Suzuki, Masashi Watanabe, Yuji Nakamaru, Dai Takagi, Hidehisa Takahashi, Satoshi Fukuda, Shigetsugu Hatakeyama

  CELLULAR AND MOLECULAR LIFE SCIENCES 73 (5) 1085 - 1101 1420-682X 2016/03 [Refereed][Not invited]
   

  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 inflammatory cytokine IL-1 is involved in bladder remodeling after bladder outlet obstruction in mice

  Yukiko Kanno, Takahiko Mitsui, Takeya Kitta, Kimihiko Moriya, Tadasuke Tsukiyama, Shigetsugu Hatakeyama, Katsuya Nonomura

  NEUROUROLOGY AND URODYNAMICS 35 (3) 377 - 381 0733-2467 2016/03 [Refereed][Not invited]
   

  AimsWe investigated the relationship between IL-1 and morphological and functional changes following partial bladder outlet obstruction (pBOO). MethodsFemale wild-type C57/BL6 mice (WT) and IL-1-/- mice (KO) were used. Animals were sacrificed either 1 or 3 weeks after pBOO or sham surgery, and their bladders were harvested to determine bladder weight, for RT-PCR to measure interleukin-1 (IL-1), insulin growth factor-1 (IGF-1), and transforming growth factor- (TGF-) levels, and for histological analysis with Hematoxylin-Eosin (HE) staining. Cystometry was performed on conscious animals 3 weeks after surgery to evaluate urodynamic parameters. IGF-1 was also administered intraperitoneally to KO with pBOO, and bladder weight was then investigated. ResultsIL-1-mRNA levels were significantly higher in WT-pBOO than in WT-sham. IGF-1-mRNA and TGF--mRNA levels were also significantly higher in WT-pBOO than in WT-sham; however, these increases were smaller in KO-pBOO than in WT-pBOO. Bladder weight was significantly higher in WT-pBOO than in WT-sham, while increases in bladder weight were significantly suppressed in KO-pBOO. HE staining revealed the thickened bladder wall in WT-pBOO, and this phenomenon was less in KO-pBOO than in WT-pBOO. Regarding the urodynamic parameters examined, micturition pressure and bladder capacity were significantly higher in WT-pBOO than in WT-sham, but remained unchanged in KO-pBOO. The administration of IGF-1 to KO-pBOO led to similar increases in bladder weight and the thickened bladder wall as those observed in WT-pBOO. ConclusionIL-1 has the potential to induce bladder remodeling and deteriorate urodynamic parameters in pBOO. Neurourol. Urodynam. 35:377-381, 2016. (c) 2015 Wiley Periodicals, Inc.
• Early evidence for the role of TRIM29 in multiple cancer models

  Shigetsugu Hatakeyama

  EXPERT OPINION ON THERAPEUTIC TARGETS 20 (7) 767 - 770 1472-8222 2016 [Refereed][Not invited]
  
• TRIM29 regulates the p63-mediated pathway in cervical cancer cells

  Yasushi Masuda, Hidehisa Takahashi, Shigetsugu Hatakeyama

  BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1853 (10) 2296 - 2305 0167-4889 2015/10 [Refereed][Not invited]
   

  Cell invasion and adhesion play an important role in cancer metastasis and are orchestrated by a complicated network of transcription factors including p63. Here, we show that a member of the tripartite motif protein family, TRIM29, is required for regulation of the p63-mediated pathway in cervical cancer cells. TRIM29 knockdown alters the adhesion and invasion activities of cervical cancer cells. TRIM29 knockdown and overexpression cause a significant decrease and increase of TAp63 alpha expression, respectively. TRIM29 knockdown alters the expression pattern of integrins and increases ZEB1 expression. TRIM29 is required for suppression of an increase in the adhesion activity of cells by TAp63 alpha. These findings suggest that TRIM29 regulates the p63-mediated pathway and the behavior of cervical cancer cells. (C) 2015 Elsevier B.V. All rights reserved.
• 耳鼻咽喉科領域疾患に関与するユビキチン化タンパク質修飾

  鈴木 正宣, 渡部 昌, 中丸 裕爾, 高木 大, 加納 里志, 本間 あや, 畠山 鎮次, 福田 諭

  耳鼻咽喉科免疫アレルギー 日本耳鼻咽喉科免疫アレルギー学会 33 (3) 185 - 192 0913-0691 2015/09 [Not refereed][Not invited]
   

  ユビキチン化修飾は真核生物に広く存在するタンパク質翻訳後修飾の一つであり,多岐にわたる細胞機能を制御する。ユビキチン化修飾系の異常を原因とする疾患も数多く知られており,耳鼻咽喉科領域においても内耳や鼻粘膜の環境恒常性や,自己免疫・炎症性疾患や悪性疾患の病態に関連していることが報告されている。標的タンパク質へのユビキチンの共有結合は,ユビキチン活性化酵素(E1),ユビキチン連結酵素(E2),ユビキチンリガーゼ(E3)の3つの酵素群が担っている。この中でもE3は基質の特異性を決定する酵素であり,その機能と標的である基質を明らかにすることは,さまざまな疾患の病態解明において重要な課題となっている。耳鼻科領域の疾患において,ユビキチン修飾系についての研究は始まったばかりであり,今後,病態の解明,検査法の確立や新規治療法の開発につながる可能性を秘めている。(著者抄録)
• TRIM29 regulates the assembly of DNA repair proteins into damaged chromatin

  Yasushi Masuda, Hidehisa Takahashi, Shigeo Sato, Chieri Tomomori-Sato, Anita Saraf, Michael P. Washburn, Laurence Florens, Ronald C. Conaway, Joan W. Conaway, Shigetsugu Hatakeyama

  NATURE COMMUNICATIONS 6 7299  2041-1723 2015/06 [Refereed][Not invited]
   

  Although DNA double-strand break (DSB) repair is mediated by numerous proteins accumulated at DSB sites, how DNA repair proteins are assembled into damaged chromatin has not been fully elucidated. Here we show that a member of the tripartite motif protein family, TRIM29, is a histone-binding protein responsible for DNA damage response (DDR). We found that TRIM29 interacts with BRCA1-associated surveillance complex, cohesion, DNA-PKcs and components of TIP60 complex. The dynamics of the TRIM29-containing complex on H2AX nucleosomes is coordinated by a cross-talk between histone modifications. TRIM29 binds to modified histone H3 and H4 tails in the context of nucleosomes. Furthermore, chromatin binding of TRIM29 is required for the phosphorylation of H2AX and cell viability in response to ionizing radiation. Our results suggest that TRIM29 functions as a scaffold protein to assemble DNA repair proteins into chromatin followed by efficient activation of DDR.
• Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

  Tadasuke Tsukiyama, Akimasa Fukui, Sayuri Terai, Yoichiro Fujioka, Keisuke Shinada, Hidehisa Takahashi, Terry P. Yamaguchi, Yusuke Ohba, Shigetsugu Hatakeyama

  MOLECULAR AND CELLULAR BIOLOGY 35 (11) 2007 - 2023 0270-7306 2015/06 [Refereed][Not invited]
   

  Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/beta-catenin pathway. Here, we show that RNF43 suppresses both Wnt/beta-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/beta-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/beta-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/beta-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/beta-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.
• TRIM29 regulates the p63-mediated pathway in cervical cancer cells.

  Masuda Y, Takahashi H, Hatakeyama S

  Biochimica et biophysica acta 1853 (10 Pt A) 2296 - 2305 0006-3002 2015/06 [Refereed][Not invited]
  
• The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPAR gamma

  Masashi Watanabe, Hidehisa Takahashi, Yasushi Saeki, Takashi Ozaki, Shihori Itoh, Masanobu Suzuki, Wataru Mizushima, Keiji Tanaka, Shigetsugu Hatakeyama

  ELIFE 4 e05615  2050-084X 2015/04 [Refereed][Not invited]
   

  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.
• The TRIM-FLMN protein TRIM45 directly interacts with RACK1 and negatively regulates PKC-mediated signaling pathway

  T. Sato, H. Takahashi, S. Hatakeyama, A. Iguchi, T. Ariga

  ONCOGENE 34 (10) 1280 - 1291 0950-9232 2015/03 [Refereed][Not invited]
   

  The receptor for activated C-kinase (RACK1), a scaffolding protein that participates in the protein kinase C (PKC) signaling pathway, has an important role in shuttling active PKCs to its substrate. Indeed, recent studies have revealed that RACK1 has an important role in tumorigenesis and that enhancement of the feed-forward mechanism of the c-Jun N-terminal kinase (JNK)-Jun pathway via RACK1 is associated with constitutive activation of MEK (MAPK-ERK kinase)-ERK (extracellular signal-regulated kinase) signaling in human melanoma cells. Taken together, RACK1 additionally has a very important role in the mitogen-activated protein kinase (MAPK) signaling pathway. Here, we show that one of the tripartite motif-containing (TRIM) family ubiquitin ligases, TRIM45, is a novel RACK1-interacting protein and downregulates MAPK signal transduction. Importantly, the expression of TRIM45 is induced when growth-promoting extracellular stimuli activate the MAPK signaling pathway, resulting in attenuation of activation of the MAPK pathway. These findings suggest that TRIM45 functions as a member of the negative feedback loop of the MAPK pathway.
• Siglec-15 is a potential therapeutic target for postmenopausal osteoporosis

  Yusuke Kameda, Masahiko Takahata, Shintaro Mikuni, Tomohiro Shimizu, Hiroki Hamano, Takashi Angata, Shigetsugu Hatakeyama, Masataka Kinjo, Norimasa Iwasaki

  BONE 71 217 - 226 8756-3282 2015/02 [Refereed][Not invited]
   

  Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is an immunoreceptor that regulates osteoclast development and bone resorption in association with an immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein, DNAX-activating protein 12 kDa (DAP12). Although Siglec-15 has an important role in physiologic bone remodeling by modulating RANKL signaling, it is unclear whether it is involved in pathologic bone loss in which multiple osteoclastogenic factors participate in excessive osteoclastogenesis. Here we demonstrated that Siglec-15 is involved in estrogen deficiency-induced bone loss. WT and Siglec-15(-/-) mice were ovariectomized (Ovx) or sham-operated at 14 wk of age and their skeletal phenotype was evaluated at 18 and 22 wk of age. Siglec-15(-/-) mice showed resistance to estrogen deficiency-induced bone loss compared to WT mice. Although the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts increased after ovariectomy in both WT and Siglec-15(-/-) mice, the increase was lower in Siglec-15(-/-) mice than in WT mice. Importantly, osteoclasts in Siglec-15(-/-) mice were small and failed to spread on the bone surface, indicating impaired osteoclast differentiation. Because upregulated production of TNF-alpha as well as RANKL is mainly responsible for estrogen deficiency-induced development of osteoclasts, we examined whether Siglec-15 deficiency affects TNF-alpha-induced osteoclastogenesis in vitro. The TNF-alpha mediated induction of TRAP-positive multinucleated cells was impaired in Siglec-15(-/-) cells, suggesting that Siglec-15 is involved in TNF-alpha induced osteoclastogenesis. We also confirmed that signaling through osteoclast-associated receptor/Fc receptor common gamma chain, which is an alternative ITAM adaptor to DAP12, rescues multinucleation but not cytoskeletal organization of TNF-alpha and RANKL-induced Siglec-15(-/-) osteoclasts, indicating that the Siglec-15/DAP12 pathway is especially important for cytoskeletal organization of osteoclasts in both RANKL and TNF-alpha induced osteoclastogenesis. The present findings indicate that Siglec-15 is involved in estrogen deficiency-induced differentiation of osteoclasts and is thus a potential therapeutic target for postmenopausal osteoporosis. (C) 2014 Elsevier Inc. All rights reserved.
• Oxidative Stress Regulates IL-4 Gene Expression in Mast Cells through the Reduction of Histone Deacetylase

  Yuji Nakamaru, Dai Takagi, Akihiro Homma, Shigetsugu Hatakeyama, Satoshi Fukuda

  OTOLARYNGOLOGY-HEAD AND NECK SURGERY 152 (1) 48 - 52 0194-5998 2015/01 [Refereed][Not invited]
   

  Objective Many proinflammatory cytokines are regulated by the acetylation and deacetylation of the core histone. Since dysregulation of T helper 2 cytokine production is a key in the pathogenesis of allergic diseases, we examined the role of histone deacetylase (HDAC) on interleukin (IL)-4 gene expression in mast cells. We also examined whether oxidative stress has any impact on HDAC activity. Study Design In vitro study. Setting Academic research laboratory. Methods An IgE-sensitized mast cell line (RBL-2H3 cells) was treated with varying concentrations of the HDAC inhibitors trichostatin A (TSA) and H2O2 and stimulated with antigens. The amount of IL-4 gene expression was quantified by real-time polymerase chain reaction. Quantitative measurement of IL-4 in the cell supernatant was performed using enzyme-linked immunosorbent assay. Moreover, HDAC activity was measured with the use of a nonisotopic assay that utilized an HDAC Fluorescent Activity Assay Kit. Results IL-4 mRNA expression was induced by antigens in IgE-sensitized RBL-2H3 cells. Pretreatment with TSA and H2O2 enhanced IL-4 mRNA expression up to 5-fold in a dose-dependent manner. Furthermore, HDAC activity in RBL-2H3 cells was reduced after treatment with H2O2. Conclusion Our results suggest that oxidative stress may up-regulate IL-4 gene expression in mast cells via a decrease in HDAC activity.
• 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  2041-1723 2015/01 [Refereed][Not invited]
   

  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 0303-8467 2014/12 [Refereed][Not invited]
  
• TRIM29 Suppresses TWIST1 and Invasive Breast Cancer Behavior

  Lingbao Ai, Wan-Ju Kim, Merve Alpay, Ming Tang, Carolina E. Pardo, Shigetsugu Hatakeyama, W. Stratford May, Michael P. Kladde, Coy D. Heldermon, Erin M. Siegel, Kevin D. Brown

  CANCER RESEARCH 74 (17) 4875 - 4887 0008-5472 2014/09 [Refereed][Not invited]
   

  TRIM29 (ATDC) exhibits a contextual function in cancer, but seems to exert a tumor-suppressor role in breast cancer. Here, we show that TRIM29 is often silenced in primary breast tumors and cultured tumor cells as a result of aberrant gene hypermethylation. RNAi-mediated silencing of TRIM29 in breast tumor cells increased their motility, invasiveness, and proliferation in a manner associated with increased expression of mesenchymal markers (N-cadherin and vimentin), decreased expression of epithelial markers (E-cadherin and EpCAM), and increased expression and activity of the oncogenic transcription factor TWIST1, an important driver of the epithelial-mesenchymal transition (EMT). Functional investigations revealed an inverse relationship in the expression of TRIM29 and TWIST1, suggesting the existence of a negative regulatory feedback loop. In support of this relationship, we found that TWIST1 inhibited TRIM29 promoter activity through direct binding to a region containing a cluster of consensus E-box elements, arguing that TWIST1 transcriptionally represses TRIM29 expression. Analysis of a public breast cancer gene-expression database indicated that reduced TRIM29 expression was associated with reduced relapse-free survival, increased tumor size, grade, and metastatic characteristics. Taken together, our results suggest that TRIM29 acts as a tumor suppressor in breast cancer through its ability to inhibit TWIST1 and suppress EMT. (C) 2014 AACR.
• 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 0065-1281 2014 [Refereed][Not invited]
   

  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.
• Identification of anti-Sez612 antibody in a patient with cerebellar ataxia and retinopathy

  Hiroaki Yaguchi, Ichiro Yabe, Hidehisa Takahashi, Fumihiko Okumura, Akiko Takeuchi, Kazuhiro Horiuchi, Takahiro Kano, Atsuhiro Kanda, Wataru Saito, Masaki Matsumoto, Keiichi I. Nakayama, Shigetsugu Hatakeyama, Hidenao Sasaki

  JOURNAL OF NEUROLOGY 261 (1) 224 - 226 0340-5354 2014/01 [Refereed][Not invited]
  
• Siglec-15 Regulates Osteoclast Differentiation by Modulating RANKL-Induced Phosphatidylinositol 3-Kinase/Akt and Erk Pathways in Association With Signaling Adaptor DAP12

  Yusuke Kameda, Masahiko Takahata, Miki Komatsu, Shintaro Mikuni, Shigetsugu Hatakeyama, Tomohiro Shimizu, Takashi Angata, Masataka Kinjo, Akio Minami, Norimasa Iwasaki

  JOURNAL OF BONE AND MINERAL RESEARCH 28 (12) 2463 - 2475 0884-0431 2013/12 [Refereed][Not invited]
   

  Siglecs are a family of sialic acid-binding immunoglobulin-like lectins that regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. Here we show that Siglec-15 regulates osteoclast development and bone resorption by modulating receptor activator of nuclear factor B ligand (RANKL) signaling in association with DNAX-activating protein 12kDa (DAP12), an adaptor protein bearing an immunoreceptor tyrosine-based activation motif (ITAM). Among the known Siglecs expressed in myeloid lineage cells, only Siglec-15 was upregulated by RANKL in mouse primary bone marrow macrophages. Siglec-15-deficient mice exhibit mild osteopetrosis resulting from impaired osteoclast development. Consistently, cells lacking Siglec-15 exhibit defective osteoclast development and resorptive activity in vitro. RANKL-induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt and Erk pathways were impaired in Siglec-15-deficient cells. Retroviral transduction of Siglec-15-null osteoclast precursors with wild-type Siglec-15 or mutant Siglec-15 revealed that the association of Siglec-15 with DAP12 is involved in the downstream signal transduction of RANK. Furthermore, we found that the ability of osteoclast formation is preserved in the region adjacent to the growth plate in Siglec-15-deficient mice, indicating that there is a compensatory mechanism for Siglec-15-mediated osteoclastogenesis in the primary spongiosa. To clarify the mechanism of this compensation, we examined whether osteoclast-associated receptor (OSCAR)/Fc receptor common (FcR) signaling, an alternative ITAM-mediated signaling pathway to DAP12, rescues impaired osteoclastogenesis in Siglec-15-deficient cells. The ligands in type II collagen activate OSCAR and rescue impaired osteoclastogenesis in Siglec-15-deficient cells when cultured on bone slices, indicating that Siglec-15-mediated signaling can be compensated for by signaling activated by type II collagen and other bone matrix components in the primary spongiosa. Our findings indicate that Siglec-15 plays an important role in physiologic bone remodeling by modulating RANKL signaling, especially in the secondary spongiosa. (c) 2013 American Society for Bone and Mineral Research.
• ケージ内微生物の変動は ケージ内の湿度およびアンモニアの変動と相関する

  土佐紀子, 吉松組子, 畠山鎮次, 有川二郎

  実験動物と環境 21 (2) 87 - 98 2013/10 [Refereed][Not invited]
  
• 14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation

  Tohru Ichimura, Masato Taoka, Ikuo Shoji, Hiroki Kato, Tomonobu Sato, Shigetsugu Hatakeyama, Toshiaki Isobe, Naomi Hachiya

  JOURNAL OF CELL SCIENCE 126 (9) 2014 - 2026 0021-9533 2013/05 [Refereed][Not invited]
   

  Deregulated expression of tripartite motif-containing protein 32 (TRIM32, an E3 ubiquitin-protein ligase) contributes to various diseases. Here we report, using quantitative proteomics and biochemistry, that 14-3-3 proteins bind to phosphorylated TRIM32 and prevent TRIM32 autoubiquitylation and the formation of TRIM32-containing cytoplasmic bodies, which are potential autoregulatory mechanisms that can reduce the concentration of soluble free TRIM32. The 14-3-3-TRIM32 interaction is dependent on protein-kinase-A-catalyzed phosphorylation of TRIM32 at Ser651. We found that the inhibitory effect of 14-3-3 is, in part, a consequence of disrupting the propensity of TRIM32 to undergo higher-order self-association without affecting its dimerization. Consequently, dimerized TRIM32 bound to 14-3-3 was sequestered in a distinct cytoplasmic pool away from the microtubule network, whereas a TRIM32 mutant that cannot bind 14-3-3 underwent multimerization and was unavailable to facilitate cell growth. Our results reveal a novel connection between ubiquitylation and phosphorylation pathways, which could modulate a variety of cell events by stimulating the formation of the 14-3-3-TRIM32 signaling complex.
• Activation of Double-stranded RNA-activated Protein Kinase (PKR) by Interferon-stimulated Gene 15 (ISG15) Modification Down-regulates Protein Translation

  Fumihiko Okumura, Akiko J. Okumura, Keiji Uematsu, Shigetsugu Hatakeyama, Dong-Er Zhang, Takumi Kamura

  JOURNAL OF BIOLOGICAL CHEMISTRY 288 (4) 2839 - 2847 0021-9258 2013/01 [Refereed][Not invited]
   

  The ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly induced by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. However, how ISGylation contributes to innate immune responses is not clear. The dsRNA-dependent protein kinase (PKR) inhibits translation by phosphorylating eIF2 alpha to exert its anti-viral effect. ISG15 and PKR are induced by interferon, suggesting that a relationship exists between ISGylation and translational regulation. Here, we report that PKR is ISGylated at lysines 69 and 159. ISG15-modified PKR is active in the absence of virus infection and phosphorylates eIF2 alpha to down-regulate protein translation. The present study describes a novel pathway for the activation of PKR and the regulation of protein translation.
• Variation in the bacterial conditions inside cages is correlated with intracage humidity and ammonia levels.

  Tosa N, Yoshimatsu K, Tadasuke Tsukiyama, Hatakeyama S, Arikawa J

  Lab Animal and Environ 21 (2) 87 - 98 2013 [Refereed][Not invited]
  
• Ribophorin II Is Involved in the Tissue Factor Expression Mediated by Phosphatidylserine-Dependent Antiprothrombin Antibody On Monocytes.

  Yuichiro Fujieda, Olga Amengual, Yusaku Kanetsuka, Toshio Odani, Kotaro Otomo, Kenji Oku, Toshiyuki Bohgaki, Tetsuya Horita, Shinsuke Yasuda, Kimiko Kuroki, Katsumi Maenaka, Masaki Matsumoto, Shigetsugu Hatakeyama, Tatsuya Atsumi

  ARTHRITIS AND RHEUMATISM 64 (10) S741 - S741 0004-3591 2012/10 [Refereed][Not invited]
  
• [New findings of ubiquitin-proteasome system from yeast to higher organisms].

  Yamaguchi J, Hatakeyama S

  Seikagaku. The Journal of Japanese Biochemical Society 6 84 407 - 408 0037-1017 2012/06 [Refereed][Not invited]
  
• TRIM59 interacts with ECSIT and negatively regulates NF-kappa B and IRF-3/7-mediated signal pathways

  Takeshi Kondo, Masashi Watanabe, Shigetsugu Hatakeyama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 3 422 (3) 501 - 507 0006-291X 2012/06 [Refereed][Not invited]
   

  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.
• TRIM45 negatively regulates NF-kappa B-mediated transcription and suppresses cell proliferation

  Mio Shibata, Tomonobu Sato, Ryota Nukiwa, Tadashi Ariga, Shigetsugu Hatakeyama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1 423 (1) 104 - 109 0006-291X 2012/06 [Refereed][Not invited]
   

  The NF-kappa B signaling pathway plays an important role in cell survival, immunity, inflammation, carcinogenesis, and organogenesis. Activation of NF-kappa B is regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. The NF-kappa B signaling pathway is activated by two distinct signaling mechanisms and is strictly modulated by the ubiquitin-proteasome system. It has been reported that overexpression of TRIM45, one of the TRIM family ubiquitin ligases, suppresses transcriptional activities of Elk-1 and AP-1, which are targets of the MAPK signaling pathway. In this study, we showed that TRIM45 also negatively regulates TNF alpha-induced NF-kappa B-mediated transcription by a luciferase reporter assay and that TRIM45 lacking a RING domain also has an activity to inhibit the NF-kappa B signal. Moreover, we found that TRIM45 overexpression suppresses cell growth. These findings suggest that TRIM45 acts as a repressor for the NF-kappa B signal and regulates cell growth. (C) 2012 Elsevier Inc. All rights reserved.
• Ymer Acts as a Multifunctional Regulator in Nuclear Factor-kappa B and Fas Signaling Pathways

  Tadasuke Tsukiyama, Mayuko Matsuda-Tsukiyama, Miyuki Bohgaki, Sayuri Terai, Shinya Tanaka, Shigetsugu Hatakeyama

  MOLECULAR MEDICINE 18 (4) 587 - 597 1076-1551 2012/04 [Refereed][Not invited]
   

  The nuclear factor (NF)-kappa B family of transcription factors regulates diverse cellular functions, including inflammation, oncogenesis and apoptosis. It was reported that A20 plays a critical role in the termination of NF-kappa B signaling after activation. Previously, we showed that Ymer interacts and collaborates with A20, followed by degradation of receptor-interacting protein (RIP) and attenuation of NF-kappa B signaling. Here we show the function of Ymer in regulation of several signaling pathways including NF-kappa B on the basis of results obtained by using Ymer transgenic (Ymer Tg) mice. Ymer Tg mice exhibited impaired immune responses, including NF-kappa B and mitogen-activated protein kinase (MAPK) activation, cell proliferation and cytokine production, to tumor necrosis factor (TNF)-alpha, polyl:C or lipopolysaccharide ([PS) stimulation. Ymer Tg mice were more resistant to LPS-induced septic shock than wild-type mice. Transgene of Ymer inhibited the onset of glomerulonephritis in Ipr/Ipr mice as an autoimmune disease model. In contrast to the inflammatory immune response to LPS, Fas-mediated cell death was strongly induced in liver cells of Ymer Tg mice in which Ymer is abundantly expressed. These findings suggest that Ymer acts as a regulator downstream of several receptors and that Ymer functions as a positive or negative regulator in a signaling pathway-dependent manner. Online address: hffp://www.molmed.org doi: 10.2119/molmed.2011.00435
• TRIM67 Protein Negatively Regulates Ras Activity through Degradation of 80K-H and Induces Neuritogenesis

  Hiroaki Yaguchi, Fumihiko Okumura, Hidehisa Takahashi, Takahiro Kano, Hiroyuki Kameda, Motokazu Uchigashima, Shinya Tanaka, Masahiko Watanabe, Hidenao Sasaki, Shigetsugu Hatakeyama

  JOURNAL OF BIOLOGICAL CHEMISTRY 287 (15) 12050 - 12059 0021-9258 2012/04 [Refereed][Not invited]
   

  Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.
• TRIM6 interacts with Myc and maintains the pluripotency of mouse embryonic stem cells

  Tomonobu Sato, Fumihiko Okumura, Tadashi Ariga, Shigetsugu Hatakeyama

  JOURNAL OF CELL SCIENCE 125 (6) 1544 - 1555 0021-9533 2012/03 [Refereed][Not invited]
   

  The proto-oncogene product Myc is a master regulator of cell proliferation through its specific binding to the E-box motif in genomic DNA. It has been reported that Myc has an important role in the proliferation and maintenance of the pluripotency of embryonic stem (ES) cells and that the transcriptional activity of Myc is regulated by several post-translational modifications, including ubiquitination. In this study, we showed that tripartite motif containing 6 (TRIM6), one of the TRIM family ubiquitin ligases, was selectively expressed in ES cells and interacted with Myc followed by attenuation of the transcriptional activity of Myc. Knockdown of TRIM6 in ES cells enhanced the transcriptional activity of Myc and repressed expression of NANOG, resulting in the promotion of ES cell differentiation. These findings indicate that TRIM6 regulates the transcriptional activity of Myc during the maintenance of ES cell pluripotency, suggesting that TRIM6 functions as a novel regulator for Myc-mediated transcription in ES cells.
• TRIM32 promotes retinoic acid receptor alpha-mediated differentiation in human promyelogenous leukemic cell line HL60

  Tomonobu Sato, Fumihiko Okumura, Akihiro Iguchi, Tadashi Ariga, Shigetsugu Hatakeyama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1 417 (1) 594 - 600 0006-291X 2012/01 [Refereed][Not invited]
   

  Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor alpha (RAR alpha). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RAR alpha and enhances transcriptional activity of RAR alpha in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RAR alpha, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RAR alpha-mediated transcriptional activity even in the absence of RA and stabilizes RAR alpha in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RAR alpha-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL (C) 2011 Elsevier Inc. All rights reserved.
• TRIM proteins and cancer

  Shigetsugu Hatakeyama

  NATURE REVIEWS CANCER 11 (11) 792 - 804 1474-175X 2011/11 [Refereed][Not invited]
   

  Emerging clinical evidence shows that the deregulation of ubiquitin-mediated degradation of oncogene products or tumour suppressors is likely to be involved in the aetiology of carcinomas and leukaemias. Recent studies have indicated that some members of the tripartite motif (TRIM) proteins (one of the subfamilies of the RING type E3 ubiquitin ligases) function as important regulators for carcinogenesis. This Review focuses on TRIM proteins that are involved in tumour development and progression.
• TRIM8 regulates Nanog via Hsp90β-mediated nuclear translocation of STAT3 in embryonic stem cells

  Fumihiko Okumura, Akiko J. Okumura, Masaki Matsumoto, Keiichi I. Nakayama, Shigetsugu Hatakeyama

  Biochimica et Biophysica Acta - Molecular Cell Research 1813 (10) 1784 - 1792 0167-4889 2011/10 [Refereed][Not invited]
   

  TRIM8 is a member of a protein family defined by the presence of a common domain structure composed of a tripartite motif including a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with Hsp90β, which interacts with STAT3 and selectively downregulates transcription of Nanog in embryonic stem cells. Knock-down of TRIM8 increased phosphorylated STAT3 in the nucleus and also enhanced transcription of Nanog. These findings suggest that TRIM8 modulates translocation of phosphorylated STAT3 into the nucleus through interaction with Hsp90β and consequently regulates transcription of Nanog in embryonic stem cells. © 2011 Elsevier B.V.
• Role of Apolipoprotein B100 and Oxidized Low-Density Lipoprotein in Anti-beta2 Glycoprotein I Induced Tissue Factor Expression on Monocytes

  Kotaro Otomo, Tatsuya Atsumi, Yuichiro Fujieda, Hisako Nakagawa, Masaru Kato, Olga Amengual, Tetsuya Horita, Shinsuke Yasuda, Masaki Matsumoto, Shigetsugu Hatakeyama, Takao Koike

  ARTHRITIS AND RHEUMATISM 63 (10) S6 - S6 0004-3591 2011/10 [Refereed][Not invited]
  
• TRIM8 regulates Nanog via Hsp90β-mediated nuclear translocation of STAT3 in embryonic stem cells.

  Okumura F, Okumura AJ, Matsumoto M, Nakayama KI, Hatakeyama S

  Biochimica et biophysica acta 10 1813 1784 - 1792 0006-3002 2011/10 [Refereed][Not invited]
  
• TRIM32 promotes neural differentiation through retinoic acid receptor-mediated transcription

  Tomonobu Sato, Fumihiko Okumura, Satoshi Kano, Takeshi Kondo, Tadashi Ariga, Shigetsugu Hatakeyama

  JOURNAL OF CELL SCIENCE 124 (20) 3492 - 3502 0021-9533 2011/10 [Refereed][Not invited]
   

  Retinoic acid (RA), a metabolite of vitamin A, plays versatile roles in development, differentiation, cell cycles and regulation of apoptosis by regulating gene transcription through nuclear receptor activation. Ubiquitinylation, which is one of the post-translational modifications, appears to be involved in the transcriptional activity of intranuclear receptors including retinoic acid receptor alpha (RAR alpha). Mutations in the tripartite motif-containing protein 32 gene (TRIM32; also known as E3 ubiquitin-protein ligase) have been reported to be responsible for limb-girdle muscular dystrophy type 2H in humans, and its encoded protein has been shown to interact with several other important proteins. In this study, we found that TRIM32 interacts with RAR alpha and enhances its transcriptional activity in the presence of RA. We also found that overexpression of TRIM32 in mouse neuroblastoma cells and embryonal carcinoma cells promoted stability of RAR alpha, resulting in enhancement of neural differentiation. These findings suggest that TRIM32 functions as one of the co-activators for RAR alpha-mediated transcription, and thereby TRIM32 is a potential therapeutic target for developmental disorders and RA-dependent leukemias.
• TRIM40 promotes neddylation of IKK gamma and is downregulated in gastrointestinal cancers

  Keita Noguchi, Fumihiko Okumura, Norihiko Takahashi, Akihiko Kataoka, Toshiya Kamiyama, Satoru Todo, Shigetsugu Hatakeyama

  CARCINOGENESIS 7 32 (7) 995 - 1004 0143-3334 2011/07 [Refereed][Not invited]
   

  Gastrointestinal neoplasia seems to be a common consequence of chronic inflammation in the gastrointestinal epithelium. Nuclear factor-kappaB (NF-kappa B) is an important transcription factor for carcinogenesis in chronic inflammatory diseases and plays a key role in promoting inflammation-associated carcinoma in the gastrointestinal tract. Activation of NF-kappa B is regulated by several posttranslational modifications including phosphorylation, ubiquitination and neddylation. In this study, we showed that tripartite motif (TRIM) 40 is highly expressed in the gastrointestinal tract and that TRIM40 physically binds to Nedd8, which is conjugated to target proteins by neddylation. We also found that TRIM40 promotes the neddylation of inhibitor of nuclear factor kappaB kinase subunit gamma, which is a crucial regulator for NF-kappa B activation, and consequently causes inhibition of NF-kappa B activity, whereas a dominant-negative mutant of TRIM40 lacking the RING domain does not inhibit NF-kappa B activity. Knockdown of TRIM40 in the small intestinal epithelial cell line IEC-6 caused NF-kappa B activation followed by increased cell growth. In addition, we found that TRIM40 is highly expressed in normal gastrointestinal epithelia but that TRIM40 is downregulated in gastrointestinal carcinomas and chronic inflammatory lesions of the gastrointestinal tract. These findings suggest that TRIM40 inhibits NF-kappa B activity via neddylation of inhibitor of nuclear factor kappaB kinase subunit gamma and that TRIM40 prevents inflammation-associated carcinogenesis in the gastrointestinal tract.
• TRIM29 negatively regulates p53 via inhibition of Tip60

  Takuya Sho, Tadasuke Tsukiyama, Tomonobu Sato, Takeshi Kondo, Jun Cheng, Takashi Saku, Masahiro Asaka, Shigetsugu Hatakeyama

  BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1813 (6) 1245 - 1253 0167-4889 2011/06 [Refereed][Not invited]
   

  Ataxia-telangiectasia (AT) is an autosomal recessive genetic disease characterized by immunological deficiencies, neurological degeneration, developmental abnormalities and an increased risk of cancer. Ataxia-telangiectasia group D (ATDC) was initially described as a gene related to AT. Ataxia-telangiectasia group D. also known as TRIM29, is structurally a member of the tripartite motif (TRIM) family of proteins, some of which have been reported to be highly expressed in some human carcinomas, but the involvement of TRIM29 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM29 binds to Tip60, which has been reported as a cellular acetyltransferase protein. Overexpression of TRIM29 promoted degradation and changed localization of Tip60 and reduced acetylation of p53 at lysine 120 by Tip60, resulting in enhancement of cell growth and transforming activity. In addition, we found that TRIM29 suppresses apoptosis induced by UV irradiation in HCT116 cell lines. These findings suggest that TRIM29 functions as an oncogene that promotes tumor growth. (C) 2011 Elsevier B.V. All rights reserved.
• MDA-9/syntenin interacts with ubiquitin via a novel ubiquitin-binding motif

  Fumihiko Okumura, Koichi Yoshida, Fei Liang, Shigetsugu Hatakeyama

  MOLECULAR AND CELLULAR BIOCHEMISTRY 352 (1-2) 163 - 172 0300-8177 2011/06 [Refereed][Not invited]
   

  Ubiquitination appears to be involved in proteasome-dependent proteolysis and in the membrane trafficking system including endocytosis and exocytosis. In this study, we identified MDA-9/syntenin as a novel ubiquitin-binding protein by a yeast two-hybrid system using modified ubiquitin in which lysine 48 is substituted by arginine. It has been reported that MDA-9/syntenin is a membrane-associated protein and regulates a cellular process involving endocytosis and intracellular transport. We found that MDA-9/syntenin binds to ubiquitin by a non-covalent bond and is ubiquitinated covalently. MDA-9/syntenin has no ubiquitin-binding motifs that have so far been reported, suggesting that MDA-9/syntenin physically interacts with ubiquitin via a novel binding motif. MDA-9/syntenin is stable in the cell, suggesting that ubiquitin binding of MDA-9/syntenin or ubiquitination of MDA-9/syntenin is not related to proteolysis. Furthermore, we showed that overexpression of wild-type MDA-9/syntenin enhances formation of filopodia, whereas MDA-9/syntenin lacking the PDZ domain inhibits the formation of filopodia, suggesting that MDA-9/syntenin plays an important role via interaction with ubiquitin in the regulation of cancer metastasis and invasion.
• TRIM32 facilitates cell growth, migration and anti-apoptosis

  Satoshi Kano, Shigetsugu Hatakeyama, Satoshi Fukuda, Hiromitsu Hatakeyama

  CANCER RESEARCH 71 0008-5472 2011/04 [Refereed][Not invited]
  
• UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53

  Hong Wu, Scott L. Pomeroy, Manuel Ferreira, Natalia Teider, Juliana Mariani, Keiichi I. Nakayama, Shigetsugu Hatakeyama, Victor A. Tron, Linda F. Saltibus, Leo Spyracopoulos, Roger P. Leng

  NATURE MEDICINE 17 (3) 347 - U150 1078-8956 2011/03 [Refereed][Not invited]
   

  The TP53 gene (encoding the p53 tumor suppressor) is rarely mutated, although frequently inactivated, in medulloblastoma and ependymoma. Recent work in mouse models showed that the loss of p53 accelerated the development of medulloblastoma. The mechanism underlying p53 inactivation in human brain tumors is not completely understood. We show that ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase, physically interacts with p53 and Hdm2 (also known as Mdm2 in mice). UBE4B promotes p53 polyubiquitination and degradation and inhibits p53-dependent transactivation and apoptosis. Notably, silencing UBE4B expression impairs xenotransplanted tumor growth in a p53-dependent manner and overexpression of UBE4B correlates with decreased expression of p53 in these tumors. We also show that UBE4B overexpression is often associated with amplification of its gene in human brain tumors. Our data indicate that amplification and overexpression of UBE4B represent previously undescribed molecular mechanisms of inactivation of p53 in brain tumors.
• Plasma gelsolin facilitates interaction between beta(2) glycoprotein I and alpha(5)beta(1) integrin

  Miyuki Bohgaki, Masaki Matsumoto, Tatsuya Atsumi, Takeshi Kondo, Shinsuke Yasuda, Tetsuya Horita, Keiichi I. Nakayama, Fumihiko Okumura, Shigetsugu Hatakeyama, Takao Koike

  JOURNAL OF CELLULAR AND MOLECULAR MEDICINE 15 (1) 141 - 151 1582-1838 2011/01 [Refereed][Not invited]
   

  Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma beta(2)-glycoprotein I (beta(2)GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen-activated protein kinase (MAPK) pathway plays an important role in aPL-induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with beta(2)GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous beta(2)GPI interacts with plasma gelsolin, which binds to integrin a(5)beta(1) through fibronectin. The tethering of beta(2)GPI to monoclonal anti-beta(2)GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti-beta(2)GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti-integrin a(5)beta(1) antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin-integrin signalling pathway, was phosphorylated by anti-beta(2)GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti-beta(2)GPI antibody-induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS.
• RNF43 interacts with NEDL1 and regulates p53-mediated transcription

  Keisuke Shinada, Tadasuke Tsukiyama, Takuya Sho, Fumihiko Okumura, Masahiro Asaka, Shigetsugu Hatakeyama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 404 (1) 143 - 147 0006-291X 2011/01 [Refereed][Not invited]
   

  The ubiquitin-proteasomal system plays a crucial role in oncogenesis in colorectal tissues. Recent studies have shown that stability of beta-catenin, which functions as an oncogene for colorectal cancer, is regulated by ubiquitin-mediated degradation. It has been reported that a putative E3 ubiquitin ligase, RNF43, is highly expressed in human colorectal carcinoma and that RNF43 promotes cell growth. However, the involvement of RNF43 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that RNF43 binds to NEDD-4-like ubiquitin-protein ligase-1 (NEDL1), which enhances pro-apoptotic activity by p53. In addition, we found that RNF43 also interacts with p53 and that RNF43 suppresses transcriptional activity of p53 in H1299 cells and attenuates apoptosis induced by ultraviolet irradiation. These findings suggest that RNF43 is associated with p53-mediated apoptosis in collaboration with NEDL1 in colorectal carcinogenesis. (C) 2010 Elsevier Inc. All rights reserved.
• Molecular Basis for the Association of Human E4B U Box Ubiquitin Ligase with E2-Conjugating Enzymes UbcH5c and Ubc4

  Robert C. Benirschke, James R. Thompson, Yves Nomine, Emeric Wasielewski, Nenad Juranic, Slobodan Macura, Shigetsugu Hatakeyama, Keiichi I. Nakayama, Maria Victoria Botuyan, Georges Mer

  STRUCTURE 18 (8) 955 - 965 0969-2126 2010/08 [Refereed][Not invited]
   

  Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.
• TRIM8 modulates STAT3 activity through negative regulation of PIAS3

  Fumihiko Okumura, Yui Matsunaga, Yuta Katayama, Keiichi I. Nakayama, Shigetsugu Hatakeyama

  JOURNAL OF CELL SCIENCE 123 (13) 2238 - 2245 0021-9533 2010/07 [Refereed][Not invited]
   

  TRIM8 is a member of the protein family defined by the presence of a common domain structure composed of a tripartite motif: a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with protein inhibitor of activated STAT3 (PIAS3), which inhibits IL-6-dependent activation of STAT3. Ectopic expression of TRIM8 cancels the negative effect of PIAS3 on STAT3, either by degradation of PIAS3 through the ubiquitin-proteasome pathway or exclusion of PIAS3 from the nucleus. Furthermore, expression of TRIM8 in NIH3T3 cells enhances Src-dependent tumorigenesis. These findings indicate that TRIM8 enhances the STAT3-dependent signal pathway by inhibiting the function of PIAS3.
• Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines

  Fumihiko Okumura, Hiroyuki Kameda, Takao Ojima, Shigetsugu Hatakeyama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 395 (3) 352 - 355 0006-291X 2010/05 [Refereed][Not invited]
   

  We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. (C) 2010 Elsevier Inc. All rights reserved.
• Direct binding of TRAF2 and TRAF6 to TICAM-1/TRIF adaptor participates in activation of the Toll-like receptor 3/4 pathway

  Miwa Sasai, Megumi Tatematsu, Hiroyuki Oshiumi, Kenji Funami, Misako Matsumoto, Shigetsugu Hatakeyama, Tsukasa Seya

  MOLECULAR IMMUNOLOGY 47 (6) 1283 - 1291 0161-5890 2010/03 [Refereed][Not invited]
   

  Using yeast two-hybrid screening, we found three TRAF proteins TRAF1,2 and 6, bound the N-terminal region of the TLR3/4 adaptor TICAM-1 (TRIF). TRAF2, a newly identified TICAM-1-binding protein, bound the PxQxS motif (aa 333-338) of TICAM-1 using mutagenesis by alanine substitutions. TICAM-1 is known to induce the activation of NF-kappa B and IRF-3, which leads to activation of the interferon (IFN)-beta promoter, an activity that is conserved in the N + TIR fragment (aa 1-533). By mutation of the two distinct binding sites for TRAF2 and TRAF6 in N + TIR TICAM-1, the induction of IFN-beta was completely abrogated. Although the TRAF2 site single mutation only marginally affected TICAM-1-mediated type I IFN induction, it further impaired the function of the TRAF6 site mutant. Moreover, double point mutations of the TRAF2 and TRAF6 binding motifs in TICAM-1 N + TIR reduced the activation of IRF-3 and NF-kappa B, the critical transcription factors for IFN-beta expression. Furthermore, TRAF2/6 functioned as an E3 ligase to induce K63-mediated ubiquitination on N + TIR which was abrogated in the mutant lacking the TRAF2/6 sites in parallel with IFN-inducing activity. Confocal microscopy analysis indicated that TRAF2 and TRAF6 merged with oligomerized (i.e. activated) TICAM-1 N + TIR. However, TRAF3, which is another TRAF family member essential for TLR3-mediated type-IIFN signaling, still assembled in the mutant lacking the TRAF2/6 sites. Our data suggest that the binding of TRAF2 and TRAF6 to TICAM-1 cooperatively activates the IFN-inducing pathway through ubiquitination of TICAM-1, a modification which occurs unrelated to TRAF3 recruitment in the TICAM-1 signaling complex. TRAF2/6 may participate in TICAM-1-mediated IFN-beta induction besides TRAF3. (C) 2009 Elsevier Ltd. All rights reserved.
• TRIM24 mediates ligand-dependent activation of androgen receptor and is repressed by a bromodomain-containing protein, BRD7, in prostate cancer cells.

  Kikuchi M, Okumura F, Tsukiyama T, Watanabe M, Miyajima N, Tanaka J, Imamura M, Hatakeyama S

  Biochimica et biophysica acta 12 1793 1828 - 1836 0006-3002 2009/12 [Refereed][Not invited]
  
• The E3 Ligase TTC3 Facilitates Ubiquitination and Degradation of Phosphorylated Akt

  Futoshi Suizu, Yosuke Hiramuki, Fumihiko Okumura, Mami Matsuda, Akiko J. Okumura, Noriyuki Hirata, Masumi Narita, Takashi Kohno, Jun Yokota, Miyuki Bohgaki, Chikashi Obuse, Shigetsugu Hatakeyama, Toshiyuki Obata, Masayuki Noguchi

  DEVELOPMENTAL CELL 17 (6) 800 - 810 1534-5807 2009/12 [Refereed][Not invited]
   

  The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate AM activity remain unclear. We show here that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS.
• 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 0167-4889 2009/12 [Refereed][Not invited]
   

  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 0006-291X 2009/11 [Refereed][Not invited]
   

  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 0006-291X 2009/10 [Refereed][Not invited]
   

  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.
• Ubiquitin-Conjugating Enzyme UBE2Q2 Suppresses Cell Proliferation and Is Down-Regulated in Recurrent Head and Neck Cancer

  Hiroyuki Maeda, Naoto Miyajima, Satoshi Kano, Tadasuke Tsukiyama, Fumihiko Okumura, Satoshi Fukuda, Shigetsugu Hatakeyama

  MOLECULAR CANCER RESEARCH 7 (9) 1553 - 1562 1541-7786 2009/09 [Refereed][Not invited]
   

  The ubiquitin-proteasome system has a crucial role in maintaining and regulating cellular homeostasis including carcinogenesis. UBE2Q2, also designated Ubci, is one of the ubiquitin-conjugating enzymes (E2), and it has been reported that mRNA of UBE2Q2 is highly expressed in human head and neck squamous cell carcinoma, particularly hypopharyngeal carcinoma. However, the involvement of UBE2Q2 in carcinogenesis has not been fully elucidated. Most cases of head and neck carcinoma are treated with cis-diamminedichloroplatinum (II; CDDP) or docetaxel, which are the most effective chemotherapeutic agents against squamous cell carcinomas. Nevertheless, some head and neck cancers develop resistance to these drugs, although the causes and mechanisms remain unknown. In this study, we found high expression levels of UBE2Q2 in human head and neck carcinoma cell lines and cancer tissues by using an anti-UBE2Q2 antibody at the protein level. We also found that the expression level of UBE2Q2 is decreased in cell lines and cancer tissues that have resistance to CDDP or docetaxel and in cancer tissues treated with CDDP or docetaxel. Furthermore, we found that overexpression of UBE2Q2 affects cell proliferation and anchorage-independent cell growth. These findings suggest that UBE2Q2 is a novel oncosuppressor that inhibits tumor growth and is related to the resistance to anticarcinoma agents and that UBE2Q2 likely functions as a novel diagnostic tool and a potentially therapeutic target for head and neck squamous cell carcinoma. (Mol Cancer Res 2009;7(9):1553-62)
• Large-scale proteomic analysis of tyrosine-phosphorylation induced by T-cell receptor or B-cell receptor activation reveals new signaling pathways

  Masaki Matsumoto, Koji Oyamada, Hidehisa Takahashi, Takamichi Sato, Shigetsugu Hatakeyama, Keiichi I. Nakayama

  PROTEOMICS 9 (13) 3549 - 3563 1615-9853 2009/07 [Refereed][Not invited]
   

  Activation of the T-cell receptor (TCR) and that of the B-cell receptor (BCR) elicits tyrosine-phosphorylation of proteins that belongs to similar functional categories, but result in distinct cellular responses. Large-scale analyses providing an overview of the signaling pathways downstream of TCR or BCR have not been described, so it has been unclear what components of these pathways are shared and which are specific. We have now performed a systematic analysis and provide a comprehensive list of tyrosine-phosphorylated proteins (PY proteome) with quantitative data on their abundance in T cell, B cell, and nonlymphoid cell lines. Our results led to the identification of novel tyrosine-phosphorylated proteins and signaling pathways not previously implicated in immunoreceptor signal transduction, such as clathrin, zonula occludens 2, eukaryotic translation initiation factor 3, and RhoH, suggesting that TCR or BCR signaling may be linked to downstream processes such as endocytosis, cell adhesion, and translation. Thus comparative and quantitative studies of tyrosine-phosphorylation have the potential to expand knowledge of signaling networks and to promote understanding of signal transduction at the system level.
• Human synovial sarcoma proto-oncogene Syt is essential for early embryonic development through the regulation of cell migration

  Taichi Kimura, Mieko Sakai, Kouichi Tabu, Lei Wang, Ryosuke Tsunematsu, Masumi Tsuda, Hirofumi Sawa, Kazuo Nagashima, Hiroshi Nishihara, Shigetsugu Hatakeyama, Keiko Nakayama, Marc Ladanyi, Shinya Tanaka, Keiichi I. Nakayama

  LABORATORY INVESTIGATION 89 (6) 645 - 656 0023-6837 2009/06 [Refereed][Not invited]
   

  SYT-SSX protein, resulted from chromosomal translocation, causes synovial sarcoma, which is a malignant tumor accounting for 10% of soft tissue sarcoma. However, biological functions of SYT (synovial sarcoma translocation), also known as SS18, are largely unclear, whereas it has been proven that Syt-null mice die at early stages of embryonic development. Here, we generated Syt-deficient mice and confirmed the reported phenotypes, including growth retardation, open neural tube and haplo-insufficient lethality, and therefore, there is no doubt that Syt is essential for embryonic development. However, placental defects, described in the earlier report, were rarely seen in our mice and we frequently observed cardiac defect in Syt-deficient mice. As the mechanisms responsible for embryonic lethality seem to be complicate, we performed additional experiments. By using primary cultured embryonic fibroblasts, we showed that Syt(-/-) MEFs deregulate actin organization and suppressed cell migration. These observations suggest that Syt may contribute to the signaling pathway important for various cellular functions in vivo and in vitro, and we propose that Syt-deficient MEFs would be a powerful means to understand the biological roles of SYT in vitro. Laboratory Investigation (2009) 89, 645-656; doi:10.1038/labinvest.2009.25; published online 30 March 2009
• TRIM36 interacts with the kinetochore protein CENP-H and delays cell cycle progression

  Naoto Miyajima, Satoru Maruyama, Katsuya Nonomura, Shigetsugu Hatakeyama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 381 (3) 383 - 387 0006-291X 2009/04 [Refereed][Not invited]
   

  The tripartite motif-containing protein (TRIM) family is defined by the presence of a common domain structure composed of a RING finger, a B-box, and a coiled-coil motif. 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 by using yeast two-hybrid screening that TRIM36 has a ubiquitin ligase activity and interacts with centromere protein-H, one of the kinetochore proteins. We also found by immunofluorescence analysis that TRIM36 colocalizes with alpha-tubulin, One of the microtubule proteins. Moreover, we found that overexpression of TRIM36 decelerates the cell cycle and attenuates cell growth. These results indicate that TRIM36 is potentially associated with chromosome segregation and that an excess of TRIM36 May Cause chromosomal instability. (C) 2009 Elsevier Inc. All rights reserved.
• UBE2Q2は細胞増殖を抑制し、再発性頭頸部癌においてはその発現が抑制されている(UBE2Q2 suppresses cell proliferation and is down-regulated in recurrent head and neck cancer)

  真栄田 裕行, 加納 里志, 畠山 鎮次, 福田 諭

  日本耳鼻咽喉科学会会報 (一社)日本耳鼻咽喉科学会 112 (4) 315 - 315 0030-6622 2009/04 [Not refereed][Not invited]
  
• Riplet/RNF135, a RING Finger Protein, Ubiquitinates RIG-I to Promote Interferon-beta Induction during the Early Phase of Viral Infection

  Hiroyuki Oshiumi, Misako Matsumoto, Shigetsugu Hatakeyama, Tsukasa Seya

  JOURNAL OF BIOLOGICAL CHEMISTRY 284 (2) 807 - 817 0021-9258 2009/01 [Refereed][Not invited]
   

  RIG-I (retinoic acid-inducible gene-I), a cytoplasmic RNA helicase, interacts with IPS-1/MAVS/Cardif/VISA, a protein on the outer membrane of mitochondria, to signal the presence of virus-derived RNA and induce type I interferon production. Activation of RIG-I requires the ubiquitin ligase, TRIM25, which mediates lysine 63-linked polyubiquitination of the RIG-I N-terminal CARD-like region. However, how this modification proceeds for activation of IPS-1 by RIG-I remains unclear. Here we identify an alternative factor, Riplet/RNF135, that promotes RIG-I activation independent of TRIM25. The Riplet/RNF135 protein consists of an N- terminal RING finger domain, C-terminal SPRY and PRY motifs, and shows sequence similarity to TRIM25. Immunoprecipitation analyses demonstrated that the C-terminal helicase and repressor domains of RIG-I interact with the Riplet/RNF135 C-terminal region, whereas the CARD-like region of RIG-I is dispensable for this interaction. Riplet/RNF135 promotes lysine 63-linked polyubiquitination of the C-terminal region of RIG-I, modification of which differs from the N- terminal ubiquitination by TRIM25. Overexpression and knockdown analyses revealed that Riplet/RNF135 promotes RIGI-mediated interferon-beta promoter activation and inhibits propagation of the negative-strand RNA virus, vesicular stomatitis virus. Our data suggest that Riplet/RNF135 is a novel factor of the RIG-I pathway that is involved in the evoking of human innate immunity against RNA virus infection, and activates RIG-I through ubiquitination of its C-terminal region. We infer that a variety of RIG-I-ubiquitinating molecular complexes sustain RIG-I activation to modulate RNA virus replication in the cytoplasm.
• 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 0006-291X 2009/01 [Refereed][Not invited]
   

  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
• Tripartite motif protein 32 facilitates cell growth and migration via degradation of Abl-interactor 2

  Satoshi Kano, Naoto Miyajima, Satoshi Fukuda, Shigetsugu Hatakeyama

  CANCER RESEARCH 68 (14) 5572 - 5580 0008-5472 2008/07 [Refereed][Not invited]
   

  Tripartite motif protein 32 (TRIM32) mRNA has been reported to be highly expressed in human head and neck squamous cell carcinoma, but the involvement of TRIM32 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM32 binds to Abl-interactor 2 (Abi2), which is known as a tumor suppressor and a cell migration inhibitor, and we showed that TRIM32 mediates the ubiquitination of Abi2. Overexpression of TRIM32 promoted degradation of Abi2, resulting in enhancement of cell growth, transforming activity, and cell motility, whereas a dominant-negative mutant of TRIM32 lacking the RING domain inhibited the degradation of Abi2. In addition, we found that TRIM32 suppresses apoptosis induced by cis-diamminedichloroplatinum (II) in HEp2 cell lines. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis, and resistance to anticancer drugs.
• 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 5 1783 826 - 837 0006-3002 2008/05 [Refereed][Not invited]
  
• TRIM68 regulates ligand-dependent transcription of androgen receptor in prostate cancer cells

  Naoto Miyajima, Satoru Maruyama, Miyuki Bohgaki, Masahiko Shigemura, Nobuo Shinohara, Katsuya Nonomura, Shigetsugu Hatakeyama

  CANCER RESEARCH 68 (9) 3486 - 3494 0008-5472 2008/05 [Refereed][Not invited]
   

  The androgen receptor (AR) is a transcription factor belonging to the family of nuclear receptors that mediate the action of androgen. AR plays an important role in normal development of the prostate, as well as in the progression of prostate cancer. AR is regulated by several posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. In this study, we found that the putative E3 ubiquitin ligase TRIM68, which is preferentially expressed in prostate cancer cells, interacts with AR and enhances transcriptional activity of the AR in the presence of dihydrotestosterone. We also found that TRIM68 functionally interacts with TIP60 and p300, which act as coactivators of AR, and synergizes in the transactivation of AR. Overexpression of TRIM68 in prostate cancer cells caused an increase in secretion of prostate-specific antigen (PSA), one of the most reliable diagnostic markers for prostate cancer, whereas knockdown of TRIM68 attenuated the secretion of PSA and inhibited cell growth and colony-forming ability. Moreover, we showed that TRIM68 expression is sigmficantly up-regulated in human prostate cancers compared with the expression in adjacent normal tissues. These results indicate that TRIM68 functions as a cofactor for AR-mediated transcription and is likely to be a novel diagnostic tool and a potentially therapeutic target for prostate cancer.
• 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 0167-4889 2008/05 [Refereed][Not invited]
   

  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.
• Ro52 functionally interacts with IgG1 and regulates its quality control via the ERAD system

  Mutsumi Takahata, Miyuki Bohgaki, Tadasuke Tsukiyama, Takeshi Kondo, Masahiro Asaka, Shigetsugu Hatakeyama

  MOLECULAR IMMUNOLOGY 45 (7) 2045 - 2054 0161-5890 2008/04 [Refereed][Not invited]
   

  The 52-kDa form of SSA/Ro protein (Ro52) is one of autoantigens associated with autoimmune disorders such as systemic lupus erythematosus and Sjogren's syndrome. Anti-SSA/Ro antibodies, the biological function of which remains unknown, are frequently found in the serum of these patients. Recent functional genomic approaches have shown that Ro52/TRIM21 is one of the TRIM family proteins with a RING-finger domain which is closely associated with E3 ubiquitin ligase activity. We found by using yeast-two hybrid screening that Ro52 has an E3 activity in vitro and interacts with human IgG1 heavy chain. We also found that IgG1 heavy chain was modified with polyubiquitination by Ro52 and degraded through the ubiquitin-proteasome system in mammalian cells. Our results also showed that Ro52 interacts with the molecular chaperone p97/VCP, which is thought to function in the endoplasmic reticulum associated degradation (ERAD) system. It is likely that Ro52 plays a role in proteasomal degradation of unfolded IgG1, which is retrogradely transferred from the endoplasmic reticulum to the cytosol. Taken together, our findings suggest that Ro52 plays a significant role in quality control of IgG1 through the ERAD system. (C) 2007 Elsevier Ltd. All rights reserved.
• Ubiquitylation of epsilon-COP by PIRH2 and regulation of the secretion of PSA

  Satoru Maruyama, Naoto Miyajima, Miyuki Bohgaki, Tadasuke Tsukiyama, Masahiko Shigemura, Katsuya Nonomura, Shigetsugu Hatakeyama

  MOLECULAR AND CELLULAR BIOCHEMISTRY 307 (1-2) 73 - 82 0300-8177 2008/01 [Refereed][Not invited]
   

  Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the epsilon-subunit of coatmer complex, epsilon-COP. PIRH2 promotes the ubiquitylation of epsilon-COP in vitro and in vivo and consequently promotes the degradation of epsilon-COP. The interaction between PIRH2 and epsilon-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of epsilon-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.
• Involvement of Rabring7 in EGF receptor degradation as an E3 ligase

  Ayuko Sakane, Shigetsugu Hatakeyama, Takuya Sasaki

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 357 (4) 1058 - 1064 0006-291X 2007/06 [Refereed][Not invited]
   

  Rab7, a member of the Rab family small G proteins, is involved in the late stage of the endocytic pathway. We previously identified a Rab7 target protein, Rabring7, which contains a RING finger domain at its C termini, but the precise role of Rabring7 remains unknown. In this study, we demonstrate using an in vitro ubiquitination assay with recombinant E1 and E2 proteins that Rabring7 has E3 ligase activity and that it preferentially reacts with Ubc4 and Ubc5 as its E2 proteins. Rabring7 ubiquitinated itself but not Rab7, and a mutation at Cys-229 in the RING finger domain (Rabring7C229S) completely diminished its E3 ligase activity. In the ligand-induced degradation of EGF receptor (EGFR), Rabring7 accelerated the degradation of EGFR, whereas Rabring7C229S inhibited the degradation induced by cCb1, another E3 ligase. These results suggest that Rabring7 is involved in the endocytic trafficking of EGFR through its E3 ligase activity. (c) 2007 Elsevier Inc. All rights reserved.
• Ligand-dependent transcription of estrogen receptor alpha is mediated by the ubiquitin ligase EFP

  Ayako Nakajima, Satoru Maruyama, Miyuki Bohgaki, Naoto Miyajima, Tadasuke Tsukiyama, Noriaki Sakuragi, Shigetsugu Hatakeyama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 357 (1) 245 - 251 0006-291X 2007/05 [Refereed][Not invited]
   

  Estrogen-mediated ubiquitylation and subsequent degradation of the estrogen receptor alpha (ER alpha) appears to be involved in the transcriptional activity of ER alpha. We show that the estrogen-responsive finger protein (EFP) interacts with and ubiquitylates ER alpha. EFP promoted the ubiquitylation of ER alpha in vitro and in vivo and consequently promoted the degradation of ER alpha. The interaction between EFP and ER alpha was greatly enhanced in the presence of estrogen. The action of EFP on ER alpha in the presence of estrogen resulted in a robust interaction between ER alpha and Tip60, one of the transcriptional coactivators, leading to activation of ER alpha transcriptional activity. However, a dominant negative mutant of EFP lacking the RING domain prolonged the half-life of ER alpha and inhibited the transcription by ER alpha. Our results indicate that EFP functions as a cofactor for ER alpha-mediated transcription, thus suggesting that ER alpha-mediated transcription is closely linked to he ubiquitylation of ER alpha. (c) 2007 Elsevier Inc. All rights reserved.
• Establishment of a newly improved detection system for NF-kappa B activity

  Mayuko Matsuda, Tadasuke Tsukiyama, Miyuki Bohgaki, Katsuya Nonomura, Shigetsugu Hatakeyama

  IMMUNOLOGY LETTERS 109 (2) 175 - 181 0165-2478 2007/04 [Refereed][Not invited]
   

  The transcription factor nuclear factor-kappa B (NF-kappa B) plays roles in apoptosis, inflammation and oncogenesis. It is important for biological and medical research to understand when proteins of interest are activated in cells, leading to the establishment of a luciferase/EGFP assay to monitor the activation of transcription factors. Here, we describe an improved reporter system for NF-kappa B, the NF-kappa B-activated transgene (NAT) system that can detect NF-kappa B signalling with high sensitivity and specificity. The NAT system consists of large copy numbers of NF-kappa B consensus sequence and a minimal promoter derived from the mouse interleukin-2 (IL-2) gene. Furthermore, we generated NAT systems with stable or unstable luciferase/EGFP proteins. Stable and unstable types of luciferase/EGFP are suitable for analyzing the accumulation of and the real-time activity of NF-kappa B signal, respectively. Our findings suggest that the NAT system is effective for in vivo imaging of NF-kappa B signalling using cells or animals. (c) 2007 Elsevier B.V. All rights reserved.
• 頭頸部扁平上皮癌におけるユビキチンリガーゼTRIM32の機能解析

  加納 里志, 真栄田 裕行, 畠山 鎮次, 福田 諭

  日本耳鼻咽喉科学会会報 (一社)日本耳鼻咽喉科学会 110 (4) 326 - 326 0030-6622 2007/04 [Not refereed][Not invited]
  
• APS-mediated ubiquitination of the insulin receptor enhances its internalization, but does not induce its degradation

  Kazuhiro Kishi, Kazuaki Mawatari, Kikuko Sakai-Wakamatsu, Tomoyuki Yuasa, Miao Wang, Makoto Ogura-Sawa, Yutaka Nakaya, Shigetsugu Hatakeyama, Yousuke Ebina

  ENDOCRINE JOURNAL 54 (1) 77 - 88 0918-8959 2007/02 [Refereed][Not invited]
   

  APS, a tyrosine kinase adaptor protein with pleckstrin homology and Src homology 2 domains, is rapidly and strongly tyrosine-phosphorylated by insulin receptor kinase upon insulin stimulation. We have previously shown that APS knockout mice have increased insulin sensitivity, and that this enhancement is possibly due to increased insulin-response on adipose tissues. However, the function of APS in insulin signaling has so far been controversial. Here, we report that APS enhanced ligand-dependent multi-ubiquitination of the insulin receptor (IR) in CHO cells overexpressing the IR. APS-mediated ubiquitination of the IR induced enhancement of the IR internalization, but did not affect the IR degradation. This finding shows one of the pleiotropic functions of APS in insulin signaling.
• 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 0304-3940 2007/01 [Refereed][Not invited]
   

  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.
• Neurodegenerative diseases regulated by ubiquitin-proteasome system

  Shigetsugu Hatakeyama

  Clinical Neurology 46 (11) 890 - 892 0009-918X 2006/11 [Refereed][Not invited]
   

  Various inherited neurodegenerative diseases result from an increase in the number of glutamine codon repeats within the open reading frame of the responsible gene. Insoluble aggregates of polyglutamine-containing proteins in neurons, which are usually conjugated with ubiquitin, are a hallmark of the polyglutamine diseases. However, the molecular mechanism underlying the ubiquitylation and aggregate formation of polyglutamine-containing proteins has been largely unclear. Here we report the identification of critical factors involved in the ubiquitylation process as well as turnover of MJD1/Ataxin-3 protein, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease). E4 B/UFD2a (a ubiquitin chain assembly factor) and VCP (a AAA-family ATPase) were co-purified with the activity polyubiquitylating Ataxin-3. E4B mediated polyubiquitylation of MJD1/Ataxin-3, and VCP interacted with both E 4B and MJD1 Ataxin-3. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an Ataxin-3 mutant. These observations suggest that E4 is a rate-limiting factor in the degradation of proteins with expanded polyglutamine tracts.
• Degradation of Tob1 mediated by SCF (Skp2)-dependent ubiquitination

  Yoshihiro Hiramatsu, Kyoko Kitagawa, Toru Suzuki, Chiharu Uchida, Takayuki Hattori, Hirotoshi Kikuchi, Toshiaki Oda, Shigetsugu Hatakeyama, Keiichi I. Nakayama, Tadashi Yamamoto, Hiroyuki Konno, Masatoshi Kitagawa

  CANCER RESEARCH 66 (17) 8477 - 8483 0008-5472 2006/09 [Refereed][Not invited]
   

  Tob1, a member of the Tob/BTG family, is involved in the control of G(I)-S progression by suppressing cyclin D1 expression and acts as a tumor suppressor gene. Tob1 was reported to have a quick turnover through the ubiquitin-proteasome pathway, but proteins involved in this process are still unknown. We showed that Skp2, a substrate-targeting subunit of the SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complex, was involved in ubiquitin-dependent degradation of Tob1. Skp2 interacted with Tob1 and facilitated ubiquitination of Tob1 in intact cells as well as in vitro. Skp2 mutants without the F-box or leucine rich repeat were not able to bind to Tob1 and did not enhance ubiquitination of Tob1. Tob1 was stabilized in both Skp2(-/-) mouse fibroblasts and Skp2 knockdown HeLa cells. Moreover, cyclin D1 expression was suppressed in Skp2 knockdown HeLa cells. These data suggest that Tob1 is a novel target for degradation by the SCF-Skp2 ubiquitin ligase.
• Elmo1 inhibits ubiquitylation of Dock180

  Y Makino, M Tsuda, S Ichihara, T Watanabe, M Sakai, H Sawa, K Nagashima, S Hatakeyama, S Tanaka

  JOURNAL OF CELL SCIENCE 119 (5) 923 - 932 0021-9533 2006/03 [Refereed][Not invited]
   

  Dock180, a member of the CDM family of proteins, plays roles in biological processes such as phagocytosis and motility through its association with the signalling adaptor protein Crk. Recently, the complex formation between Dock180 and Elmo1 was reported to function as a bipartite guanine nucleotide exchange factor for Rac. In this study, we demonstrated that the amount of Dock180 increased when Elmo1 was co-expressed. Dock180 was found to be ubiquitylated and Dock180 protein levels could be augmented by treatment with proteasome inhibitor. The ubiquitylation of Dock180 was enhanced by epidermal growth factor (EGF), Crk and adhesion-dependent signals. Furthermore, Elmo1 inhibited labiquitylation of Dock180, resulting in the increase in Dock180 levels. The Elmo1 mutant Delta 531, which encompasses amino acids required for Dock180 binding, preserved the inhibitory effects on ubiquitylation of Dock180. Upon EGF stimulation, both Dock180 and ubiquitin were demonstrated to translocate to the cell periphery by immunofluorescence, and we found ubiquitylation of Dock180 and its inhibition by Elmo1 to occur in cellular membrane fractions by in vivo ubiquitylation assay. These data suggest that Dock180 is ubiquitylated on the plasma membrane, and also that Elmo1 functions as an inhibitor of ubiquitylation of Dock180. Therefore, an ubiquitin-proteasome-dependent protein degradation mechanism might contribute to the local activation of Rac on the plasma membrane.
• Ubiquitylation and degradation of serum-inducible kinase by hVPS18, a RING-H2 type ubiquitin ligase

  S Yogosawa, S Hatakeyama, KI Nakayama, H Miyoshi, S Kohsaka, C Akazawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (50) 41619 - 41627 0021-9258 2005/12 [Refereed][Not invited]
   

  Serum-inducible kinase (SNK) is a member of polo-like kinases that serve as regulators of multiple events during cell division. Rapid changes in the activity and abundance of SNK were reported after the serum stimulation and after the activation of synaptic transmission in the brain. Yet the detailed mechanisms that control the level of SNK protein have not been fully elucidated. In this report, we show that the RING-H2 domain of hVPS18 (human vacuolar protein sorting 18) has a genuine ubiquitin ligase (E3) activity. Using the yeast two-hybrid screening, we identify SNK as a candidate substrate of hVPS18. The half-life of SNK is increased in HeLa cells that down-regulated hVPS18 by lentivirus-mediated small hairpin RNA interference. Furthermore, the delayed entry into S phase is observed in HeLa cells overexpressing hVPS18. These results suggest that hVPS18 may play an important role in regulation of SNK activity through its ubiquitin ligase.
• Mammalian E4 is required for cardiac development and maintenance of the nervous system

  C Kaneko-Oshikawa, T Nakagawa, M Yamada, H Yoshikawa, M Matsumoto, M Yada, S Hatakeyama, K Nakayama, KI Nakayama

  MOLECULAR AND CELLULAR BIOLOGY 25 (24) 10953 - 10964 0270-7306 2005/12 [Refereed][Not invited]
   

  Ubiquitin conjugation typically requires three classes of enzyme: E1, E2, and E3. A fourth type of enzyme (E4), however, was recently shown to be required for the degradation of certain types of substrate in yeast. We previously identified UFD2a (also known as E4B) as an E4 in mammals. UFD2a is exclusively expressed in cardiac muscle during mouse embryonic development, but it is abundant in neurons of adult mice and is implicated in the pathogenesis of neurodegenerative disease. The precise physiological function of this enzyme has remained largely unknown, however. Here, we show that mice lacking UFD2a die in utero, manifesting marked apoptosis in the developing heart. Polyubiquitylation activity for an E4 substrate was greatly reduced in Ufd2a(-/-) mouse embryonic fibroblasts. Furthermore, Ufd2a(+/-) mice displayed axonal dystrophy in the nucleus gracilis, as well as degeneration of Purkinje cells accompanied by endoplasmic reticulum stress. These animals also developed a neurological disorder. UFD2a thus appears to be essential for the development of cardiac muscle, as well as for the protection of spinocerebellar neurons from degeneration induced by endoplasmic reticulum stress.
• Large-scale analysis of the human ubiquitin-related proteome

  M Matsumoto, S Hatakeyama, K Oyamada, Y Oda, T Nishimura, KI Nakayama

  PROTEOMICS 5 (16) 4145 - 4151 1615-9853 2005/11 [Refereed][Not invited]
   

  Protein ubiquitylation contributes to the regulation of many cellular processes including protein degradation, receptor internalization, and repair of DNA damage. We now present a comprehensive characterization of ubiquitin-conjugated and ubiquitin-associated proteins in human cells. The proteins were purified by immunoaffinity chromatography under denaturing or native conditions. They were then digested with trypsin, and the resulting peptides were analyzed by 2-D LC and MS/MS. A total of 670 distinct proteins were identified; 345 proteins (51%) were classified as Urp-D (ubiquitin-related proteome under the denaturing condition) and comprised ubiquitin-conjugated molecules, whereas 325 proteins (49%) were classified as Urp-N (ubiquitin-related proteome only under the native condition) and included molecules that associated with ubiquitylated proteins. The proportions of proteins in various functional categories differed substantially between Urp-D and Urp-N. Many ribosomal subunits were detected in the Urp-D group of proteins and several of these subunits were directly shown to be ubiquitylated by mass spectrometric analysis, suggesting that ubiquitylation might play an important role in the regulation and/or quality control of ribosomal proteins. Our results demonstrate the potential of proteomics analysis of protein ubiquitylation to provide important insight into the regulation of protein stability and other ubiquitin-related cellular functions.
• 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 0008-5472 2005/09 [Refereed][Not invited]
   

  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.
• Cell surface expression of CD147/EMMPRIN is regulated by cyclophilin 60

  T Pushkarsky, Yurchenko, V, C Vanpouille, B Brichacek, Vaisman, I, S Hatakeyama, KI Nakayama, B Sherry, MI Bukrinsky

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (30) 27866 - 27871 0021-9258 2005/07 [Refereed][Not invited]
   

  CD147, also known as extracellular matrix metalloproteinase inducer, is a regulator of matrix metalloproteinase production and also serves as a signaling receptor for extracellular cyclophilins. Previously, we demonstrated that cell surface expression of CD147 is sensitive to cyclophilin-binding drug cyclosporin A, suggesting involvement of a cyclophilin in the regulation of intracellular transport of CD147. In this report, we identify this cyclophilin as cyclophilin 60 (Cyp60), a distinct member of the cyclophilin family of proteins. CD147 co-immunoprecipitated with Cyp60, and confocal immunofluorescent microscopy revealed intracellular colocalization of Cyp60 and CD147. This interaction with Cyp60 involved proline 211 of CD147, which was shown previously to be critical for interaction between CD147 and another cyclophilin, cyclophilin A, in solution. Mutation of this proline residue abrogated co-immunoprecipitation of CD147 and Cyp60 and reduced surface expression of CD147 on the plasma membrane. Suppression of Cyp60 expression using RNA interference had an effect similar to that of cyclosporin A: reduction of cell surface expression of CD147. These results suggest that Cyp60 plays an important role in the translocation of CD147 to the cell surface. Therefore, Cyp60 may present a novel target for therapeutic interventions in diseases where CD147 functions as a pathogenic factor, such as cancer, human immunodeficiency virus infection, or rheumatoid arthritis.
• Neurodegenerative diseases regulated by ubiquitin-proteasome system

  S Hatakeyama

  SEIKAGAKU 77 (5) 389 - 399 0037-1017 2005/05 [Refereed][Not invited]
  
• Noncovalent SUMO-1 binding act ivity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein

  H Takahashi, S Hatakeyama, H Saitoh, KI Nakayama

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (7) 5611 - 5621 0021-9258 2005/02 [Refereed][Not invited]
   

  SUMO-1 is a member of a family of ubiquitin-like molecules that are post-translationally conjugated to various cellular proteins in a process that is mechanistically similar to ubiquitylation. To identify molecules that bind noncovalently to SUMO-1, we performed yeast two-hybrid screening with a SUMO-1 mutant that cannot be conjugated to target proteins as the bait. This screening resulted in the isolation of cDNAs encoding the b isoform of thymine DNA glycosylase (TDGb). A deletion mutant of TDGb (TDGb(Delta11)) that lacks a region shown to be required for noncovalent binding of SUMO-1 was also found not to be susceptible to SUMO-1 conjugation at an adjacent lysine residue, suggesting that such binding is required for covalent modification. In contrast, another mutant of TDGb (TDGb(KR)) in which the lysine residue targeted for SUMO-1 conjugation is replaced with arginine retained the ability to bind SUMO-1 noncovalently. TDGb was shown to interact with the promyelocytic leukemia protein (PML) in vitro as well as to colocalize with this protein to nuclear bodies in transfected cells. TDGb(KR) also colocalized with PML, whereas TDGb(Delta11) did not, indicating that the noncovalent SUMO-1 binding activity of TDGb is required for colocalization with PML. Furthermore, SUMO-1 modification of TDGb and PML enhanced the interaction between the two proteins. These results suggest that SUMO-1 functions to tether proteins to PML-containing nuclear bodies through post-translational modification and noncovalent protein-protein interaction.
• Mapping of ubiquitination sites on target proteins

  S Hatakeyama, M Matsumoto, KI Nakayama

  UBIQUITIN AND PROTEIN DEGRADATION, PT B 399 277 - 286 0076-6879 2005 [Refereed][Not invited]
   

  Although the identification of ubiquitin-conjugated lysine residues on target proteins is extremely significant, to date it is generally quite difficult to identify ubiquitinated sites by usual mutation analysis. More recently, the technology of mass spectrometry is answering these difficult questions. In this chapter, we introduce the method of purification of ubiquitinated target proteins using affinity chromatography with anti-polyubiquitin antibody and the identification of ubiquitinated lysine residues on target proteins using mass spectrometry. Using these techniques, we can obtain comprehensive information about ubiquitinated proteins in various cells and tissues.
• Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis

  F Okumura, S Hatakeyama, M Matsumoto, T Kamura, KI Nakayama

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (51) 53533 - 53543 0021-9258 2004/12 [Refereed][Not invited]
   

  E4B ( also known as UFD2a) is a mammalian homolog of Saccharomyces cerevisiae Ufd2, which was originally described as a ubiquitin chain assembly factor (E4). E4B is a U-box-type ubiquitin-protein isopeptide ligase (E3) and likely functions as either an E3 or an E4. With a yeast two-hybrid screen, we have now identified FEZ1 (fasciculation and elongation protein zeta 1) as a protein that interacts with E4B. FEZ1 is implicated in neuritogenesis when phosphorylated by protein kinase Czeta (PKCzeta). Interaction between E4B and FEZ1 in mammalian cells was enhanced by coexpression of constitutively active PKCzeta. E4B mediated the polyubiquitylation of FEZ1 but did not affect its intracellular stability, suggesting that such modification of FEZ1 is not a signal for its proteolysis. Polyubiquitylation of FEZ1 by E4B required Lys(27) of ubiquitin. Expression of a dominant-negative mutant of E4B in rat pheochromocytoma PC12 cells resulted in inhibition of neurite extension induced either by nerve growth factor or by coexpression of FEZ1 and constitutively active PKCzeta. These findings indicate that E4B serves as a ubiquitin ligase for FEZ1 and thereby regulates its function but not its degradation.
• Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase

  T Kamura, T Hara, M Matsumoto, N Ishida, F Okumura, S Hatakeyama, M Yoshida, K Nakayama, KI Nakayama

  NATURE CELL BIOLOGY 6 (12) 1229 - 1235 1465-7392 2004/12 [Refereed][Not invited]
   

  The cyclin-dependent kinase inhibitor p27(Kip1) is degraded at the G0-G1 transition of the cell cycle by the ubiquitin proteasome pathway(1,2). Although the nuclear ubiquitin ligase (E3) SCFSkp2 is implicated in p27(Kip1) degradation(3-6), proteolysis of p27(Kip1) at the G0 - G1 transition proceeds normally in Skp2(-/-) cells(7,8). Moreover, p27(Kip1) is exported from the nucleus to the cytoplasm at G0 - G1 ( refs 9 - 11). These data suggest the existence of a Skp2- independent pathway for the degradation of p27(Kip1) at G1 phase. We now describe a previously unidentified E3 complex: KPC ( Kip1 ubiquitination- promoting complex), consisting of KPC1 and KPC2. KPC1 contains a RING- finger domain, and KPC2 contains a ubiquitinlike domain and two ubiquitin- associated domains. KPC interacts with and ubiquitinates p27Kip1 and is localized to the cytoplasm. Overexpression of KPC promoted the degradation of p27(Kip1), whereas a dominant- negative mutant of KPC1 delayed p27(Kip1) degradation. The nuclear export of p27(Kip1) by CRM1 seems to be necessary for KPC- mediated proteolysis. Depletion of KPC1 by RNA interference also inhibited p27(Kip1) degradation. KPC thus probably controls degradation of p27(Kip1) in G1 phase after export of the latter from the nucleus.
• Small ubiquitin-like modifier 1 (SUMO-1) modification of the synergy control motif of Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) regulates synergistic transcription between Ad4BP/SF-1 and Sox9

  T Komatsu, H Mizusaki, T Mukai, H Ogawa, D Baba, M Shirakawa, S Hatakeyama, KI Nakayama, H Yamamoto, A Kikuchi, KI Morohashi

  MOLECULAR ENDOCRINOLOGY 18 (10) 2451 - 2462 0888-8809 2004/10 [Refereed][Not invited]
   

  An orphan nuclear receptor, Ad4 binding protein/ steroidogenic factor 1 (Ad4BP/SF- 1), is essential for the development and function of steroidogenic tissues. To examine the transcriptional regulation of Ad4BP/SF- 1, two-hybrid screening was performed, and the sumoylation [ conjugation of a small ubiqutin-like modifier (SUMO-1)] components Ubc9, protein inhibitor of activated STAT 1 (PIAS1), and protein inhibitor of activated STAT 3 (PIAS3) were isolated. Cultured cell and in vitro studies revealed that Ad4BP/SF- 1 is sumoylated at K119 and K194. Because K194 lies within the synergy control (SC) motif defined to repress synergistic transcription from promoters containing multiple binding sites, correlation between the functions of the SC motif and sumoylation was investigated. The K194R mutant of Ad4BP/SF- 1, which cannot be sumoylated, showed enhanced synergistic transcription from a promoter containing multiple Ad4/SF-1 sites, suggesting that sumoylation is necessary for repression of transcriptional synergy through the SC motif. It has been established that the Mullerian inhibiting substance gene is transcribed predominantly under the control of Ad4BP/ SF-1 and, moreover, its transcription is regulated synergistically with Sox9, Gata4, and Wt1. Interestingly, it was found that all of these factors are sumoylated, and these sumoylation sites occur within SC motifs. Based on the observation that SC motif mutants of Ad4BP/ SF-1 and Sox9 resulted in the enhancement of their synergistic transcription, it was concluded that the SC motif regulates synergistic transcription even between distinct types of transcription factors. Considering that both mutants cannot be sumoylated, it is likely that sumoylation is implicated in this regulation. Because it was revealed with an in vitro sumoylated Ad4BP/ SF-1 that DNA binding activity and interaction with Sox9 were unaffected, sumoylation may regulate transcription through affecting selective and cooperative interaction among factors constituting transcriptional complexes.
• U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy

  S Hatakeyama, M Matsumoto, T Kamura, M Murayama, DH Chui, E Planel, R Takahashi, KI Nakayama, A Takashima

  JOURNAL OF NEUROCHEMISTRY 91 (2) 299 - 307 0022-3042 2004/10 [Refereed][Not invited]
   

  Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.
• Subcellular expression of autoimmune regulator is organized in a spatiotemporal manner

  H Akiyoshi, S Hatakeyama, J Pitkanen, Y Mouri, Doucas, V, J Kudoh, K Tsurugaya, D Uchida, A Matsushima, K Oshikawa, KI Nakayama, N Shimizu, P Peterson, M Matsumoto

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (32) 33984 - 33991 0021-9258 2004/08 [Refereed][Not invited]
   

  Autoimmune regulator ( AIRE) is responsible for the development of organ-specific autoimmune disease in a monogenic fashion. Rare and low levels of tissue expression together with the lack of AIRE-expressing cell lines have hampered a detailed analysis of the molecular dynamics of AIRE. Here we have established cell lines stably transfected with AIRE and studied the regulatory mechanisms for its subcellular expression. We found that nuclear body ( NB) formation by AIRE was dependent on the cell cycle. Biochemical fractionation revealed that a significant proportion of AIRE is associated with the nuclear matrix, which directs the functional domains of chromatin to provide sites for gene regulation. Upon proteasome inhibition, AIRE NBs were increased with concomitant reduced expression in the cytoplasm, suggesting that subcellular targeting of AIRE is regulated by a ubiquitin-proteasome pathway. We also found that AIRE NBs compete for cAMP-response element-binding protein-binding protein/p300, a common coactivator of transcription, with the promyelocytic leukemia gene product. These results suggest that the transcriptional regulating activities of AIRE within a cell are controlled and organized in a spatiotemporal manner.
• CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70

  M Urushitani, J Kurisu, M Tateno, S Hatakeyama, KI Nakayama, S Kato, R Takahashi

  JOURNAL OF NEUROCHEMISTRY 90 (1) 231 - 244 0022-3042 2004/07 [Refereed][Not invited]
   

  Over 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heat-shock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SOD1-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and polyubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1(G93A) transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome.
• Interaction of U-box-type ubiquitin-protein ligases (E3s) with molecular chaperones

  S Hatakeyama, M Matsumoto, M Yada, KI Nakayama

  GENES TO CELLS 9 (6) 533 - 548 1356-9597 2004/06 [Refereed][Not invited]
   

  Members of the U-box family of proteins constitute a class of ubiquitin-protein ligases (E3s) distinct from the HECT-type and RING finger-containing E3 families. Two representative mammalian U-box proteins, UFD2a and CHIP, interact with the molecular chaperones VCP and either Hsp90 or Hsc70, respectively, and are implicated in the degradation of damaged proteins. We have now investigated the roles of mammalian U-box proteins by performing a comprehensive screen for molecules that interact with these proteins in the yeast two-hybrid system. All mammalian U-box proteins tested were found to interact with molecular chaperones or cochaperones, including Hsp90, Hsp70, DnaJc7, EKN1, CRN, and VCP. These observations suggest that the function of U box-type E3s is to mediate the degradation of unfolded or misfolded proteins in conjunction with molecular chaperones as receptors that recognize such abnormal proteins.
• Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7

  M Yada, S Hatakeyama, T Kamura, M Nishiyama, R Tsunematsu, H Imaki, N Ishida, F Okumura, K Nakayama, KI Nakayama

  EMBO JOURNAL 23 (10) 2116 - 2125 0261-4189 2004/05 [Refereed][Not invited]
   

  The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c-Myc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of c-Myc was also apparent in mouse Fbw7(-/-) embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively.
• Skp2-mediated degradation of p27 regulates progression into mitosis

  K Nakayama, H Nagahama, YA Minamishima, S Miyake, N Ishida, S Hatakeyama, M Kitagawa, S Iemura, T Natsume, KI Nakayama

  DEVELOPMENTAL CELL 6 (5) 661 - 672 1534-5807 2004/05 [Refereed][Not invited]
   

  Although Skp2 has; been thought to mediate the degradation of p27 at the G(1)-S transition, Skp2(-/-) cells exhibit accumulation of p27 in S-G(2) phase with over-replication. We demonstrate that Skp2(-/-)p27(-/-) mice do not exhibit the overreplication phenotype, suggesting that p27 accumulation is required for its development. Hepatocytes of Skp2(-/-) mice entered the endoduplication cycle after mitogenic stimulation, whereas this phenotype was not apparent in Skp2(-/-)p27(-/-) mice. Cdc2-associated kinase activity was lower in Skp2(-/-) cells than in wild-type cells, and a reduction in Cdc2 activity was sufficient to induce overreplication. The lack of p27 degradation in G(2) phase in Skp2(-/-) cells may thus result in suppression of Cdc2 activity and consequent inhibition of entry into M phase. These data suggest that p27 proteolysis is necessary for the activation of not only Cdk2 but also Cdc2, and that Skp2 contributes to regulation of G(2)-M progression by mediating the degradation of p27.
• Mouse Fbw7/Sel-10/Cdc4 is required for Notch degradation during vascular development

  R Tsunematsu, K Nakayama, Y Oike, M Nishiyama, N Ishida, S Hatakeyama, Y Bessho, R Kageyama, T Suda, KI Nakayama

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (10) 9417 - 9423 0021-9258 2004/03 [Refereed][Not invited]
   

  Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. We generated mice deficient in Fbw7 and found that the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7(-/-) embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulated in Fbw7(-/-) embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7(-/-) embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development.
• Molecular clearance of ataxin-3 is regulated by a mammalian E4

  M Matsumoto, M Yada, S Hatakeyama, H Ishimoto, T Tanimura, S Tsuji, A Kakizuka, M Kitagawa, KI Nakayama

  EMBO JOURNAL 23 (3) 659 - 669 0261-4189 2004/02 [Refereed][Not invited]
   

  Insoluble aggregates of polyglutamine-containing proteins are usually conjugated with ubiquitin in neurons of individuals with polyglutamine diseases. We now show that ataxin-3, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3), undergoes ubiquitylation and degradation by the proteasome. Mammalian E4B (UFD2a), a ubiquitin chain assembly factor (E4), copurified with the polyubiquitylation activity for ataxin-3. E4B interacted with, and thereby mediated polyubiquitylation of, ataxin-3. Expression of E4B promoted degradation of a pathological form of ataxin-3. In contrast, a dominant-negative mutant of E4B inhibited degradation of this form of ataxin-3, resulting in the formation of intracellular aggregates. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an ataxin-3 mutant. These observations suggest that E4 is a rate-limiting factor in the degradation of pathological forms of ataxin-3, and that targeted expression of E4B is a potential gene therapy for SCA3.
• AIRE functions as an E3 ubiquitin ligase

  D Uchida, S Hatakeyama, A Matsushima, HW Han, S Ishido, H Hotta, J Kudoh, N Shimizu, Doucas, V, KI Nakayama, N Kuroda, M Matsumoto

  JOURNAL OF EXPERIMENTAL MEDICINE 199 (2) 167 - 172 0022-1007 2004/01 [Refereed][Not invited]
   

  Autoimmune regulator (AIRE) gene mutation is responsible for the development of autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy, an organ-specific autoimmune disease with monogenic autosomal recessive inheritance. AIRE is predominantly expressed in medullary epithelial cells of the thymus and is considered to play important roles in the establishment of self-tolerance. AIRE contains two plant homeodomain (PHD) domains, and the novel role of PHD as in E3 ubiquitin (Ub) ligase has just emerged. Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity. The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD 1 (C311Y and P326Q) abolished its E3 ligase activity. These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved.
• Degradation of p57(Kip2) mediated by SCFSkp2 - dependent ubiquitylation

  T Kamura, T Hara, S Kotoshiba, M Yada, N Ishida, H Imaki, S Hatakeyama, K Nakayama, KI Nakayama

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (18) 10231 - 10236 0027-8424 2003/09 [Refereed][Not invited]
   

  The abundance of the cyclin-dependent kinase (CDK) inhibitor p57(KiP2), an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57(KiP2) by targeting it for ubiquitylation and proteolysis. The elimination of p57(Kip2) was impaired in Skp2(-/-) cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57(KiP2) in vivo. Overexpression of WT Skp2 promoted degradation of p57(Kip2), whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57(KiP2). Mutation of the threonine residue (Thr-310) of human p57(KiP2) that is conserved between the COOH-terminal QT domains of p57(KiP2) and p27(KiP1) prevented the effect of Skp2 on the stability of p57(KiP2), suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57(Kip2) ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57(Kip2) and that of the related CDK inhibitor p27(KiP1).
• Impaired degradation of inhibitory subunit of NF-kappa B (I kappa B) and beta-catenin as a result of targeted disruption of the beta-TrCP1 gene

  K Nakayama, S Hatakeyama, S Maruyama, A Kikuchi, K Onoe, RA Good, KI Nakayama

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (15) 8752 - 8757 0027-8424 2003/07 [Refereed][Not invited]
   

  beta-TrCP1 (also known as Fbw1a or FWD1) is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Although biochemical studies have suggested that beta-TrCP1 targets inhibitory subunit of NF-kappaB (IkappaB) proteins and beta-catenin for ubiquitylation, the physiological role of beta-TrCP1 in mammals has remained unclear. We have now generated mice deficient in beta-TrCP1 and shown that the degradation of IkappaBalpha and IkappaBbeta is reproducibly, but not completely, impaired in the cells of these animals. The nuclear translocation and DNA-binding activity of NF-kappaB as well as the ability of this transcription factor to activate a luciferase reporter gene were also inhibited in beta-TrCP1(-1-) cells compared with those apparent in wild-type cells. The subcellular localization of beta-catenin was altered markedly in beta-TrCP1(-/-) cells. Furthermore, the rate of proliferation was reduced and both cell size and the percentage of polyploid cells were increased in embryonic fibroblasts derived from beta-TrCP1(-1-) mice pared with the corresponding wild-type cells. These results suggest that beta-TrCPI contributes to, but is not absolutely required for, the degradation Of IkappaB and beta-catenin and the consequent regulation of the NF-kappaB and Wnt signaling pathways, respectively. In addition, they implicate beta-TrCP1 in the maintenance of ploidy during cell-cycle progression.
• Ubiquitylation as a quality control system for intracellular proteins

  S Hatakeyama, KI Nakayama

  JOURNAL OF BIOCHEMISTRY 134 (1) 1 - 8 0021-924X 2003/07 [Refereed][Not invited]
   

  Quality control of intracellular proteins is essential for cellular homeostasis. Molecular chaperones recognize and contribute to the refolding of misfolded or unfolded proteins, whereas the ubiquitin-proteasome system mediates the degradation of such abnormal proteins. Ubiquitin-protein ligases (E3s) determine the substrate specificity for ubiquitylation and have been classified into HECT and RING-finger families. More recently, however, U-box proteins, which contain a domain (the U box) of about 70 amino acids that is conserved from yeast to humans, have been identified as a new type of E3. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. Yeast Ufd2 is functionally implicated in cell survival under stressful conditions. This review addresses recent progress in characterization of the role of E3 enzymes, especially that of U-box proteins, in quality control of intracellular proteins.
• Preferential interaction of TIP120A with Cul1 that is not modified by NEDD8 and not associated with Skp1

  K Oshikawa, M Matsumoto, M Yada, T Kamura, S Hatakeyama, KI Nakayama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 303 (4) 1209 - 1216 0006-291X 2003/04 [Refereed][Not invited]
   

  The SCF complex, which consists of the invariable components Skp1, Cul1, and Rbx1 as well as a variable F-box protein, functions as an E3 ubiquitin ligase. The mechanism by which the activity of this complex is regulated, however, has been unclear. The application of tandem affinity purification has now resulted in the identification of a novel Cul1-binding protein: TATA-binding protein-interacting protein 120A (TIP120A, also called CAND1). Immunoprecipitation, immunoblot, and immunofluorescence analyses with mammalian cells revealed that TIP120A physically associates with Cull in the nucleus and that this interaction is mediated by a central region of Cull distinct from its binding sites for Skp1 and Rbx1. Furthermore, TIP120A was shown to interact selectively with Cull that is not modified by NEDD8. The Cul1-TIP120A complex does not include Skp1, raising the possibility that TIP120A competes with Skp1 for binding to Cul1. These observations thus suggest that TIP120A may function as a negative regulator of the SCF complex by binding to nonneddylated Cul1 and thereby preventing assembly of this ubiquitin ligase. (C) 2003 Elsevier Science (USA). All rights reserved.
• U-box proteins as a new family of ubiquitin ligases

  S Hatakeyama, KI Nakayama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 302 (4) 635 - 645 0006-291X 2003/03 [Refereed][Not invited]
   

  Ubiquitin-protein ligases (E3s) determine the substrate specificity of ubiquitylation and, until recently, had been classified into two families, the HECT and RING-finger families. The U-box is a domain of similar to70 amino acids that is present in proteins from yeast to humans. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. We recently showed that mammalian U-box proteins, in conjunction with an El and an E2, mediate polyubiquitylation in the absence of a HECT type or RING-finger type E3. U-box proteins have thus been defined as a third family of E3s. We here review recent progress in the characterization of U-box proteins and of their role in the quality control system that underlies the cellular stress response to the intracellular accumulation of abnormal proteins. (C) 2003 Elsevier Science (USA). All rights reserved.
• Characterization of the mouse gene for the U-box-type ubiquitin lipase UFD2a

  C Kaneko, S Hatakeyama, M Matsumoto, M Yada, K Nakayama, KI Nakayama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 300 (2) 297 - 304 0006-291X 2003/01 [Refereed][Not invited]
   

  UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor. UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4. We have isolated and characterized the mouse gene (Ube4b) for UFD2a. A full-length (similar to5700 bp) Ube4b cDNA was isolated and the corresponding gene spans > 100 kb, comprising 27 exons. Luciferase reporter gene analysis of the 5' flanking region of Ube4b revealed that nucleotides -1018 to -943 (relative to the translation initiation site) possess promoter activity. This functional sequence contains two putative Sp1 binding sites but not a TATA box. Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues. We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding. These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin-proteasome pathway. (C) 2002 Elsevier Science (USA). All rights reserved.
• Formation of Mallory body-like inclusions and cell death induced by deregulated expression of keratin 18

  Nakamichi, I, S Hatakeyama, KI Nakayama

  MOLECULAR BIOLOGY OF THE CELL 13 (10) 3441 - 3451 1059-1524 2002/10 [Refereed][Not invited]
   

  Mallory bodies (MBs) are cytoplasmic inclusions that contain keratin 8 (K8) and K18 and are present in hepatocytes of individuals with alcoholic liver disease, nonalcoholic steatohepatitis, or benign or malignant hepatocellular neoplasia. Mice fed long term with griseofulvin are an animal model of MB formation. However, the lack of a cellular model has impeded understanding of the molecular mechanism of this process. Culture of HepG2 cells with griseofulvin has now been shown to induce both the formation of intracellular aggregates containing K18 as well as an increase in the abundance of K18 mRNA. Overexpression of K18 in HepG2, HeLa, or COS-7 cells also induced the formation of intracellular aggregates that stained with antibodies to ubiquitin and with rhodamine B (characteristics of MBs formed in vivo), eventually leading to cell death. The MB-like aggregates were deposited around centrosomes and disrupted the microtubular array. Coexpression of K8 with K18 restored the normal fibrous pattern of keratin distribution and reduced the toxicity of K18. In contrast, an NH2-terminal deletion mutant of K8 promoted the formation of intracellular aggregates even in the absence of K18 overexpression. Deregulated expression of K18, or an imbalance between K8 and K18, may thus be an important determinant of MB formation, which compromises the function of centrosomes and the microtubule network and leads to cell death.
• CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity

  Y Imai, M Soda, S Hatakeyama, T Akagi, T Hashikawa, K Nakayama, R Takahashi

  MOLECULAR CELL 10 (1) 55 - 67 1097-2765 2002/07 [Refereed][Not invited]
   

  Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity.
• Increased proliferation of B cells and auto-immunity in mice lacking protein kinase C delta

  A Miyamoto, K Nakayama, H Imaki, S Hirose, Y Jiang, M Abe, T Tsukiyama, H Nagahama, S Ohno, S Hatakeyama, KI Nakayama

  NATURE 416 (6883) 865 - 869 0028-0836 2002/04 [Refereed][Not invited]
   

  Protein kinase C (PKC), which comprises 11 closely related isoforms, has been implicated in a wide variety of cellular processes, such as growth, differentiation, secretion, apoptosis and tumour development(1-4). Among the PKC isotypes, PKC-delta is unique in that its overexpression results in inhibition of cell growth(5-11). Here we show that mice that lack PKC-delta exhibit expansion of the B-lymphocyte population with the formation of numerous germinal centres in the absence of stimulation. The rate of proliferation in response to stimulation was greater for B cells from PKC-delta-deficient mice than for those from wild-type mice. Adoptive transfer experiments suggested that the hyperproliferation phenotype is B-cell autonomous. Production of interleukin-6 was markedly increased in B cells of PKC-delta-null mice as a result of an increase in the DNA-binding activity of NF-IL6. Furthermore, the PKC-delta-deficient mice contain circulating autoreactive antibodies and display immune-complex-type glomerulonephritis, as well as lymphocyte infiltration in many organs. These results suggest that PKC-delta has an indispensable function in negative regulation of B-cell proliferation, and is particularly important for the establishment of B-cell tolerance.
• Identification of developmentally expressed proteins that functionally interact with Nedd4 ubiquitin ligase

  R Murillas, KS Simms, S Hatakeyama, AM Weissman, MR Kuehn

  JOURNAL OF BIOLOGICAL CHEMISTRY 277 (4) 2897 - 2907 0021-9258 2002/01 [Refereed][Not invited]
   

  Nedd4 is a HECT domain-containing ubiquitin ligase that mediates ubiquitylation and proteasome degradation of target proteins. The molecular basis for the interaction of Nedd4 with substrates lies in its WW domains, which can bind proline-rich (PY) domains in target proteins. Nedd4 is a developmentally expressed protein and may have a fundamental role to play in embryonic processes. However, whether Nedd4 has such a function is currently unknown, in part because few developmentally regulated ubiquitylation substrates have been identified or characterized. We have carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation embryo that are able to interact with Nedd4. Characterization of their functional interaction with Nedd4 in vitro and in vivo demonstrated that three of the four are bona fide Nedd4 binding partners, and two have the capacity to be ubiquitylation substrates. One of these is the first identified nonviral substrate for Nedd4-mediated monoubiquitylation. Interestingly, neither of these two ubiquitylated proteins interacts with Nedd4 through PY-mediated mechanisms. For one of the three Nedd4 binding partners, there was no discernable evidence of ubiquitylation. However, this protein clearly associates with Nedd4 through its PY domains and can alter the location of Nedd4 in cells, suggesting a role other than as a ubiquitylation substrate.
• Degradation of p27(Kip1) at the G(0)-G(1) transition mediated by a Skp2-independent ubiquitination pathway

  T Hara, T Kamura, K Nakayama, K Oshikawa, S Hatakeyama, KI Nakayama

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (52) 48937 - 48943 0021-9258 2001/12 [Not refereed][Not invited]
   

  Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the Go-G, transition of the cell cycle has now been shown to proceed normally in Shp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G,-G(1), transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Shp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of P27(Kip1) accumulation during S-G, phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of P27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Shp2(-/-) cells, even with a threonine 187 --> alanine mutant of P27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from Go to G,.
• Essential role of the prosurvival bcl-2 homologue A1 in mast cell survival after allergic activation

  Z Xiang, AA Ahmed, C Moller, K Nakayama, S Hatakeyama, G Nilsson

  JOURNAL OF EXPERIMENTAL MEDICINE 194 (11) 1561 - 1569 0022-1007 2001/12 [Not refereed][Not invited]
   

  Mast cells reside in tissues, where upon activation through the high-affinity-IgE-receptor (Fc epsilon RI) they degranulate and orchestrate the allergic reaction. Mast cells survive this activation and can thus be reactivated. In this study we demonstrate that this process depends on the pro-survival gene Al. Activation of mast cells through Fc epsilon RI resulted in degranulation, strong induction of Al mRNA and protein, and cell survival. In contrast, Al-deficient mast cells released granule mediators similar to the wild-type control, but the cells did not survive an allergic activation. Furthermore, A1(-/-) mice that had been sensitized and provocated with allergen exhibited a lower number of mast cell compared with littermate controls. The induction of Al was dependent on calcium, as EDTA prevented Al expression. The calcium ionophore, ionomycin, induced Al expression and mast cell survival, whereas compound 48/80, a well-known mast cell secretagogue, did not. This study uncovers the importance of Al for mast cell survival in allergic reactions, and it proposes Al as a potential target for the treatment of allergic diseases.
• Characterization of a mouse gene (Fbxw6) that encodes a homologue of Caenorhabditis elegans SEL-10

  S Maruyama, S Hatakeyama, K Nakayama, N Ishida, K Kawakami, K Nakayama

  GENOMICS 78 (3) 214 - 222 0888-7543 2001/12 [Refereed][Not invited]
   

  The SCF complex is a type of ubiquitin ligase that consists of the invariable components SKP1, CUL1, and RBX1 as well as a variable component, known as an F-box protein, that is the main determinant of substrate specificity. The Caenorhabditis elegans F-box- and WD40-repeat-containing protein SEL-10 functionally and physically associates with LIN-12 and SEL-12, orthologues of mammalian Notch and presenilin, respectively. We have now identified a gene (which we call Fbxw6) that encodes a mouse homologue (F-box-WD40 repeat protein 6, or FBW6) of SEL-10 and is expressed mainly in brain, heart, and testis. Co-immunoprecipitation analysis showed that FBW6 interacts with SKP1 and CUL1, indicating that these three proteins form an SCF complex. Comparison of the genomic organization of Fbxw6, which is located on mouse chromosome 3.3E3, with that of mouse Fbxw1, Fbxw2, and Fbxw4 showed only a low level of similarity, indicating that these genes diverged relatively early and thereafter evolved independently.
• U box proteins as a new family of ubiquitin-protein ligases

  S Hatakeyama, M Yada, M Matsumoto, N Ishida, KI Nakayama

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (35) 33111 - 33120 0021-9258 2001/08 [Refereed][Not invited]
   

  The U box is a domain of similar to 70 amino acids that is present in proteins from yeast to humans. The prototype U box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor that cooperates with a ubiquitin-activating enzyme (EI), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3) to catalyze ubiquitin chain formation on artificial substrates. E3 enzymes are thought to determine the substrate specificity of ubiquitination and have been classified into two families, the HECT and RING finger families. Six mammalian U box proteins have now been shown to mediate polyubiquitination in the presence of El and E2 and in the absence of E3. These U box proteins exhibited different specificities for E2 enzymes in this reaction. Deletion of the U box or mutation of conserved amino acids within it abolished ubiquitination activity. Some U box proteins catalyzed polyubiquitination by targeting lysine residues of ubiquitin other than lysine 48, which is utilized by HECT and RING finger E3 enzymes for polyubiquitination that serves as a signal for proteolysis by the 26 S proteasome. These data suggest that U box proteins constitute a third family of E3 enzymes and that E4 activity may reflect a specialized type of E3 activity.
• Regulation of the level of vesl-1S/homer-1a proteins by ubiquitin-proteasome proteolytic systems

  H Ageta, A Kato, S Hatakeyama, K Nakayama, Y Isojima, H Sugiyama

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (19) 15893 - 15897 0021-9258 2001/05 [Not refereed][Not invited]
   

  The vesl-1S/homer-1a gene is up-regulated during seizure and long term potentiation. Other members of the Vesl family, Vesl-1L, -2, and -3, are constitutively expressed in the brain. We examined the regulatory mechanisms governing the expression level of Vesl-1S protein, either an exogenously introduced one in COS7 or human embryonic kidney 293T cells or an endogenous one in rat brain neurons in cultures, In both cases, application of proteasome inhibitors increased the amount of Vesl-1S protein but not that of Vesl-1L, -2, or -3 protein. Deletion analyses revealed that the C-terminal 11-amino acid region was responsible for the proteolysis of Vesl-1S by proteasomes. Application of proteasome inhibitors promoted ubiquitination of Vesl-1S protein but not that of the Vesl-1S deletion mutant, which evaded proteasome-mediated degradation. These results indicate that ubiquitin-proteasome systems are involved in the regulation of the expression level of Vesl-1S protein.
• The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2

  S Kamizono, T Hanada, H Yasukawa, S Minoguchi, R Kato, M Minoguchi, K Hattori, S Hatakeyama, M Yada, S Morita, T Kitamura, H Kato, K Nakayama, A Yoshimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (16) 12530 - 12538 0021-9258 2001/04 [Not refereed][Not invited]
   

  Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia. TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells, This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-8 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitinligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases.
• Regulation of the cell cycle at the G(1)-S transition by proteolysis of cyclin E and p27(Kip1)

  K Nakayama, S Hatakeyama, K Nakayama

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 282 (4) 853 - 860 0006-291X 2001/04 [Not refereed][Not invited]
   

  The transition from G(1), phase to S phase of the mammalian cell cycle is controlled by many positive and negative regulators, among which cyclin E and p27(Kip1) respectively, undergo the most marked changes in concentration at this transition. The abundance of both cyclin E and p27(Kip1) is regulated predominantly by posttranslational mechanisms, in particular by proteolysis mediated by the ubiquitin-proteasome pathway. Cyclin E and p27(Kip1) each bind to and undergo polyubiquitination by the same ubiquitin ligase, known as SCFSkp2. The degradation of cyclin E and p27(Kip1) is greatly impaired in Skp2-deficient mice, resulting in intracellular accumulation of these proteins. In this article, recent progress in characterization of the molecular mechanisms that control the proteolysis of cyclin E and p27(Kip1) is reviewed. (C) 2001 Academic Press.
• Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development

  H. Nagahama, S. Hatakeyama, K. Nakayama, M. Nagata, K. Tomita, K. I. Nakayama

  Anatomy and Embryology 203 (2) 77 - 87 0340-2061 2001 [Not refereed][Not invited]
   

  The cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 are thought to regulate progression of the cell cycle. We have previously shown that the phenotypes of p27-/- mice are substantially different from those of p57-/- mice, suggesting that spatial and temporal expression patterns of p27Kip1 and p57Kip2 might be distinct. In this study, the roles of p27Kip1 and p57Kip2 in development were examined by characterizing their expression patterns during mouse embryogenesis by immunohistochemical analysis. Whereas certain organs and tissues (brain, lens, ganglion, lung, heart, liver, skin and kidney) expressed both proteins, others expressed only p27Kip1 (thymus, spleen, retina, testis and ovary) or only p57Kip2 (gut, palate, pancreas, cartilage and skeletal muscle). In addition, some organs expressed both p27Kip1 and p57Kip2 but showed mutually exclusive patterns of distribution among tissues. Thus, in the adrenal gland, p57Kip2 was expressed in the cortex but not in the medulla, whereas p27Kip1 was expressed in the medulla but not in the cortex. Whereas the expression of p57Kip2 in most tissues was restricted to embryogenesis, expression of p27Kip1 in many tissues was maintained in adult animals. Double-label immunofluorescence staining with either anti-p27Kip1 or anti-p57Kip2 and anti-BrdU revealed that the expression of p27Kip1 and p57Kip2 was inversely correlated with cell proliferation, suggesting that p27Kip1 and p57Kip2 are expressed exclusively in postmitotic cells. These complex spatial and temporal patterns of expression are consistent with the phenotypes of mice deficient in p27Kip1 or p57Kip2, and they suggest that these proteins might play important roles in tissue development.
• Down-regulation of p27(Kip1) expression is required for development and function of T cells

  T Tsukiyama, N Ishida, M Shirane, YA Minamishima, S Hatakeyama, M Kitagawa, K Nakayama, K Nakayama

  JOURNAL OF IMMUNOLOGY 166 (1) 304 - 312 0022-1767 2001/01 [Refereed][Not invited]
   

  The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation, The cyclin-dependent kinase inhibitor p27(Kip1) is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4(-)CD8(-) thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27(Kip1) expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27(Kip1) expression. The development of thymocytes in the transgenic strain in which p27(Kip1) is most abundant p27-Tg(high) mice) appeared to be blocked at the CD4(-)CD8(-)CD25(+)CD44(low) stage. Peripheral T cells from p27-Tg(high) mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells, Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tg(high) mice. These results suggest that down-regulation of p27(Kip1) expression is required for the development, proliferation, and immunoresponsiveness of T cells.
• Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability

  N Ishida, M Kitagawa, S Hatakeyama, K Nakayama

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (33) 25146 - 25154 0021-9258 2000/08 [Refereed][Not invited]
   

  The association of the p27(Kip1) protein with cyclin and cyclin-dependent kinase complexes inhibits their kinase activities and contributes to the control of cell proliferation. The p27(Kip1) protein has now been shown to be phosphorylated in vivo, and this phosphorylation reduces the electrophoretic mobility of the protein. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) phosphorylation and prevented the shift in electrophoretic mobility. Phosphopeptide mapping and phosphoamino acid analysis revealed that phosphorylation at Ser(10) accounted for similar to 70% of the total phosphorylation of p27(Kip1), and the extent of phosphorylation at this site was similar to 25- and 75-fold greater than that at Ser(178) and Thr(187), respectively. The phosphorylation of p27(Kip1) was markedly reduced when the positions of Ser(10) and Pro(11) were reversed, suggesting that a proline-directed kinase is responsible for the phosphorylation of Ser(10) The extent of Ser(10) phosphorylation was markedly increased in cells in the G(0)-G(1) phase of the cell cycle compared with that apparent for cells in S or M phase. The p27(Kip1) protein phosphorylated at Ser(10) was significantly more stable than the unphosphorylated form. Furthermore, a mutant p27(Kip1) in which Ser(10) was replaced with glutamic acid in order to mimic the effect of Ser(10) phosphorylation exhibited a marked increase in stability both in vivo and in vitro compared with the wild-type or S10A mutant proteins. These results suggest that Ser(10) is the major site of phosphorylation of p27(Kip1) and that phosphorylation at this site, like that at Thr(187), contributes to regulation of p27(Kip1) stability.
• Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication

  K Nakayama, H Nagahama, YA Minamishima, M Matsumoto, Nakamichi, I, K Kitagawa, M Shirane, R Tsunematsu, T Tsukiyama, N Ishida, M Kitagawa, K Nakayama, S Hatakeyama

  EMBO JOURNAL 19 (9) 2069 - 2081 0261-4189 2000/05 [Refereed][Not invited]
   

  The ubiquitin-proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skp1-Cullin-F-bos protein (SCF) ubiquitin ligase, were generated. Although Skp2(-/-) animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis, Skp2(-/-) cells also exhibit increased accumulation of both cyclin E and p27(Kip1). The elimination of cyclin E during S and G(2) phases is impaired in Skp2(-/-) cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27(Kip1) is mediated by the SCFSkp2 ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators.
• Delayed clearance of zymosan-induced granuloma and depressed phagocytosis of macrophages with concomitant up-regulated kinase activities of Src-family in a human monocyte chemoattractant protein-1 transgenic mouse

  M Ato, K Iwabuchi, N Matsuki, N Mukaida, C Iwabuchi, A Takahashi, T Takayanagi, EA Dondog, S Hatakeyama, H Ishikura, M Kato, Negishi, I, H Nishihori, K Watano, K Ogasawara, K Matsushima, K Onoe

  IMMUNOBIOLOGY 201 (3-4) 432 - 449 0171-2985 2000/01 [Not refereed][Not invited]
   

  A human monocyte chemoattractant protein-1 (hMCP-1) transgenic mouse (Tgm) line which constitutively produces a large amount of hMCP-1 (7-13 ng/ml in the serum) was established. Although expression of the transgene was detected in various tissues, an accumulation of macrophages (M phi) was seen in only lymphoid organs which might be attributed to the high concentration of hMCP-1 in these organs. A reduced phagocytosis by peritoneal M phi in vivo and a delayed clearance of granulomas in the liver following zymosan administration were observed in these Tgm. However, peritoneal exudate cells (PEC) from Tgm exhibited normal in vitro phagocytic activity and nitric oxide (NO) production upon stimulation with IFN-gamma as compared with those from non-Tgm. In addition, high activities of src-family protein tyrosine kinases (PTK), Fgr and Hck, were also noted in the peritoneal resident cells from Tgm, whereas the level of mitogen-activated protein kinase (MAPK) activity was almost the same as chat of non-Tgm. It was suggested that the low functional activities of Tgm M phi seen in vivo were attributed to down-regulation of the unique transducing system of hMCP-1 signals under the influence of a high concentration of the hMCP-1. It seemed that the depressed functions were recovered when the peritoneal cells were released ex vivo from such a high hMCP-1 environment.
• Cell cycle-dependent expression of mammalian E2-C regulated by the anaphase-promoting complex/cyclosome

  A. Yamanaka, S. Hatakeyama, K. I. Kominami, M. Kitagawa, M. Matsumoto, K. I. Nakayama

  Molecular Biology of the Cell 11 (8) 2821 - 2831 1059-1524 2000 [Not refereed][Not invited]
   

  Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. E2-C is a ubiquitin-conjugating enzyme that plays a critical role with anaphase-promoting complex/cyclosome (APC/C) in progression of and exit from M phase. Here we report that mammalian E2-C is expressed in late G2/M phase and is degraded as cells exit from M phase. The mammalian E2-C shows an autoubiquitinating activity leading to covalent conjugation to itself with several ubiquitins. The ubiquitination of E2-C is strongly enhanced by APC/C, resulting in the formation of a polyubiquitin chain. The polyubiquitination of mammalian E2-C occurs only when cells exit from M phase. Furthermore, mammalian E2-C contains two putative destruction boxes that are believed to act as recognition motifs for APC/C. The mutation of this motif reduced the polyubiquitination of mammalian E2-C, resulting in its stabilization. These results suggest that mammalian E2-C is itself a substrate of the APC/C-dependent proteolysis machinery, and that the periodic expression of mammalian E2-C may be a novel autoregulatory system for the control of the APC/C activity and its substrate specificity.
• Structure and expression of the gene encoding mouse F-box protein, Fwd2

  M Miura, S Hatakeyama, K Hattori, K Nakayama

  GENOMICS 62 (1) 50 - 58 0888-7543 1999/11 [Not refereed][Not invited]
   

  A novel class of ubiquitin ligases, termed the SCF complex, consists of invariable components, Skp1 and Cullin, and variable components called F-box proteins, which have a primary role in determining substrate specificity. We have isolated a cDNA encoding the mouse F-box protein Fwd2 (also known as MD6) as a possible constituent of an SCF-type ubiquitin ligase. Fwd2 cDNA contains 1890 bp with a 1362-bp open reading frame and encodes an approximately 51.5-kDa protein. Fwd2 is expressed predominantly in liver and, to a lesser extent, in the testis, lung, heart, and skeletal muscle. Immunofluorescence staining for Fwd2 protein shows a pattern with the cytoplasm. A coimmunoprecipitation assay has revealed the in vivo interaction between Skp1 and Fwd2 through the F-box domain. Fwd2 also interacts with Cull through Skp1, suggesting that Skp1, Cull, and the F-box protein Fwd2 form an SCF complex (SCFFwd2). We, have also isolated and determined the nucleotide sequence and genomic organization of the gene that encodes mouse Fwd2. This gene spans approximately 17 kb and consists of six exons and five introns. Our results suggest that Fwd2 is an F-box protein that constitutes an SCF ubiquitin ligase complex and that it plays a critical role in the ubiquitin-dependent degradation of proteins expressed in the liver. (C) 1999 Academic Press.
• Molecular dissection of the interactions among I kappa B alpha, FWD1, and Skp1 required for ubiquitin-mediated proteolysis of I kappa B alpha

  K Hattori, S Hatakeyama, M Shirane, M Matsumoto, K Nakayama

  JOURNAL OF BIOLOGICAL CHEMISTRY 274 (42) 29641 - 29647 0021-9258 1999/10 [Not refereed][Not invited]
   

  The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homologue of Drosophila Slimb and Xenopus beta-TrCP) functions as the ubiquitin ligase for I kappa B alpha FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DS-GXXS motif of I kappa B alpha. The structural requirements for the interactions of FWD1 with I kappa B alpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of I kappa B alpha abolished the binding of I kappa B alpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of I kappa B alpha. The I kappa B alpha mutant D31E still exhibited binding to FWD1 and underwent ubiquitination, These results suggest that, in addition to site-specific phosphorylation at Ser(32) and Ser(36), an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of I kappa B alpha. Deletion analysis of Skp1 revealed that residues 61-143 of this protein are required for binding to FWD1, On the other hand, the highly conserved residues pro(149), ILe(160), and Leu(164) in the F-box domain of FWD1 were dispensable for binding to Skp1, Together, these data delineate the structural requirements for the interactions among I kappa B alpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex.
• Common pathway for the ubiquitination of I kappa B alpha, I kappa B beta, and I kappa B epsilon mediated by the F-box protein FWD1

  M Shirane, S Hatakeyama, K Hattori, K Nakayama, K Nakayama

  JOURNAL OF BIOLOGICAL CHEMISTRY 274 (40) 28169 - 28174 0021-9258 1999/10 [Not refereed][Not invited]
   

  FWD1 (the mouse homolog of Drosophila Slimb and Xenopus beta TrCP, a member of the F-box- and WD40 repeat-containing family of proteins, and a component of the SCF ubiquitin ligase complex) was recently shown to interact with I kappa B alpha and thereby to promote its ubiquitination and degradation. This protein has now been shown also to bind to I kappa B beta and I kappa B epsilon as well as to induce their ubiquitination and proteolysis, FWD1 was shown to recognize the conserved DSG Psi XS motif (where Psi represents the hydrophobic residue) present in the NH2,terminal regions of these three I kappa B proteins only when the component serine residues are phosphorylated, However, in contrast to I kappa B alpha and I kappa B alpha, the recognition site in I kappa B epsilon for FWD1 is not restricted to the DSG Psi XS motif; FWD1 also interacts with other sites in the NH2 terminal region of I kappa B epsilon, Substitution of the critical serine residues in the NH2-terminal regions of I kappa B alpha, I kappa B beta, and IKBE with alanines also markedly reduced the extent of FWD1-mediated ubiquitination of these proteins and increased their stability. These data indicate that the three I kappa B proteins, despite their substantial structural and functional differences, all undergo ubiquitination mediated by the SCFFWD1 complex. FWD1 may thus play an important role in NF-kappa B signal transduction through regulation of the stability of multiple I kappa B proteins.
• RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination

  Kevin L. Lorick, Jane P. Jensen, Shengyun Fang, Albert M. Ong, Shigetsugu Hatakeyama, Allan M. Weissman

  Proceedings of the National Academy of Sciences of the United States of America 96 (20) 11364 - 11369 0027-8424 1999/09/28 [Refereed][Not invited]
   

  A RING finger-containing protein (AO7) that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination was identified. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several otherwise- unrelated RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Prajal, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal- coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins, with otherwise diverse structures and functions, may play previously unappreciated roles in modulating protein levels via ubiquitination.
• An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin

  M Kitagawa, S Hatakeyama, M Shirane, M Matsumoto, N Ishida, K Hattori, Nakamichi, I, A Kikuchi, K Nakayama, K Nakayama

  EMBO JOURNAL 18 (9) 2401 - 2410 0261-4189 1999/05 [Refereed][Not invited]
   

  beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3 beta (GSK-3 beta)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/beta TrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with beta-catenin, Axin, GSK-3 beta and APC, Mutations at the signal-induced phosphorylation site of beta-catenin inhibited its association with FWD1, FWD1 facilitated ubiquitination and promoted degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized beta-catenin, These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin, FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability.
• Down-regulation of p27(Kip1) by two mechanisms, ubiquitin-mediated degradation and proteolytic processing

  M Shirane, Y Harumiya, N Ishida, A Hirai, C Miyamoto, S Hatakeyama, K Nakayama, M Kitagawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 274 (20) 13886 - 13893 0021-9258 1999/05 [Refereed][Not invited]
   

  The intracellular level of p27(Kip1), a cyclin-dependent kinase (CDK) inhibitory protein, is rapidly reduced at the G(1)/S transition phase when the cell cycle pause ceases. In this study, we demonstrated that two posttranslational mechanisms were involved in p27(Kip1) breakdown: degradation via the ubiquitin (Ub)-proteasome pathway and proteolytic processing that rapidly eliminates the cyclin-binding domain. We confirmed that p27(Kip1) was ubiquitinated in vitro as well as in vivo. The p27(Kip1) -ubiquitination activity was higher at the G(1)/S boundary than during the G(0)/G(1) phase, and p27(Kip1) ubiquitination was reduced significantly when the lysine residues at positions 134, 153, and 165 were replaced by arginine, suggesting that these lysine residues are the targets for Ub conjugation. In parallel with its Uh-dependent degradation, p27(Kip1) was processed rapidly at its N terminus, reducing its molecular mass from 27 to 22 kDa, by a ubiquitination-independent but adenosine triphosphate (ATP)-dependent mechanism with higher activity during the S than the G(0)/G(1) phase. This 22-kDa intermediate had no cyclin-binding domain at its N terminus and virtually no CDK2 kinase inhibitory activity. These results suggest that p27(Kip1) is eliminated by two independent mechanisms, ubiquitin-mediated degradation and ubiquitin-independent processing, during progression from the G(1) to S phase.
• Ubiquitin-dependent degradation of I kappa B alpha is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

  S Hatakeyama, M Kitagawa, K Nakayama, M Shirane, M Matsumoto, K Hattori, H Higashi, H Nakano, K Okumura, K Onoe, RA Good, K Nakayama

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 96 (7) 3859 - 3863 0027-8424 1999/03 [Not refereed][Not invited]
   

  Activation of the transcription factor nuclear factor kappa B (NF-kappa B) is controlled by proteolysis of its inhibitory subunit (I kappa B) via the ubiquitin-proteasome pathway, Signal-induced phosphorylation of I kappa B alpha by a large multisubunit complex containing I kappa B kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/beta TrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with I kappa B alpha only when I kappa B alpha is phosphorylated, The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of I kappa B alpha in concert with I kappa B kinases, resulting in nuclear translocation of NF-kappa B. In addition, FWD1 strikingly evoked the ubiquitination of I kappa B alpha in the ira vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of I kappa B alpha. These results suggest that the substrate-specific degradation of I kappa B alpha is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated I kappa B alpha. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-kappa B through control of I kappa B protein stability.
• Erratum: Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul1/F-box protein FWD1 (Proceedings of the National Academy of Sciences of the United States America (March 30, 1999) 96:7 (3859- 3863))

  S. Hatakeyama, M. Kitagawa, K. Nakayama, M. Shirane, M. Matsumoto, K. Hattori, H. Higashi, H. Nakano, K. Okumura, K. Onoe, R. A. Good, K. I. Nakayama

  Proceedings of the National Academy of Sciences of the United States of America 96 6571  0027-8424 1999/01/01 [Not refereed][Not invited]
  
• Induction of endometriosis and adenomyosis by transvaginal pituitary transplantation in mice with and without natural killer cell activity

  T Koujyo, S Hatakeyama, H Yamada, K Iwabuchi, K Kajino, K Ogasawara, K Onoe, S Fujimoto

  AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 40 (6) 441 - 446 8755-8920 1998/12 [Refereed][Not invited]
   

  PROBLEM: The aims of this study were to establish a mouse model of endometriosis and adenomyosis and to elucidate the necessity of reduced natural killer (NK)-cell and T-cell activities in the establishment of endometriosis and adenomyosis. METHOD OF STUDY: Pituitary glands, submandibular glands, or hypothalami were transvaginally inoculated into the uteri of syngeneic female mice. Twenty weeks later, the recipient mice were sacrificed and examined. RESULTS: Cysts, adhesion of the uteri to surrounding tissues, and adenomyosis had formed in the uteri of 7 (29.2%), 14 (58.3%), and 22 (91.7%) mice, respectively, out of 24 BALB/c mice after the transplantation of pituitary glands. Similar findings were obtained by experiments with C3H/He and C57BL/6 mice. In NK-cell-deficient C57BL/6-bg(J) and T-cell-deficient BALB/c nu/nu mice, an increase in the formation of cysts, adhesion, and adenomyosis was not observed. CONCLUSIONS: These findings indicate that transvaginal pituitary transplantation specifically induces cysts, adhesion, and adenomyosis. Reduced NK-cell activities may not be necessary in the primary development of endometriosis and adenomyosis.
• Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene

  A Hamasaki, F Sendo, K Nakayama, N Ishida, Negishi, I, K Nakayama, S Hatakeyama

  JOURNAL OF EXPERIMENTAL MEDICINE 188 (11) 1985 - 1992 0022-1007 1998/12 [Refereed][Not invited]
   

  To elucidate thr role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (A1-a(-/-) mice). Spontaneous apoptosis of peripheral blood neutrophils of A1-a(-/-) mice was enhanced compared with that of tither wild-type mice or heterozygous mutants (A1-a(+/-) mice). Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a(-/-) and A1-a(+/-) animals. On the other hand, the extent of tumor necrosis factor alpha-induced acceleration of neutrophil apoptosis did not differ among A1-a(-/-), A1-a(+/-), and wild-type mice. The descending order of A1 mRNA expression was wild-type, A1-a(+/-), and A1-a(-/-). Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis.
• Multiple gene duplication and expression of mouse bcl-2-related genes, A1

  S Hatakeyama, A Hamasaki, Negishi, I, DY Loh, F Sendo, K Nakayama, K Nakayama

  INTERNATIONAL IMMUNOLOGY 10 (5) 631 - 637 0953-8178 1998/05 [Not refereed][Not invited]
   

  Here we report the genomic cloning and characterization of the murine A1 genes, which belong to the bcl-2 gene family. Southern analysis indicated the existence of at least four A1 genes in the murine genome and four different A1 genes, designated A1-a, -b, -c and -d, were cloned from the murine genomic library. The Al-a, -b and -d genes consisted of two exons, whereas the A1-c gene contained 1 bp insertion in the coding region which may result in an aberrant and truncated protein by frame-shift. With the exception of A1-c, the coding regions among A1 genes are highly conserved at >97% at the nucleotide level and at >96% at the amino acid level. A1-a, -b and -d genes appeared to be expressed specifically in organs containing many neutrophils, In neutrophils, A1-a, -b and -d transcripts were detected at a comparable level. Our data suggest that the multiple Al genes in mice were generated by gene duplication and each of them may function as anti-apoptotic molecules in neutrophils.
• Induction of endometriosis and adenomyosis by transvaginal pituitary transplantation in mice with and without natural killer cell activity

  Tsunemaro Koujyo, Shigetsugu Hatakeyama, Hideto Yamada, Kazuya Iwabuchi, Kiichi Kajino, Kazumasa Ogasawara, Kazunori Onoe, Seiichiro Fujimoto

  American Journal of Reproductive Immunology 40 (6) 441 - 446 1046-7408 1998 [Not refereed][Not invited]
   

  PROBLEM: The aims of this study were to establish a mouse model of endometriosis and adenomyosis and to elucidate the necessity of reduced natural killer (NK)-cell and T-cell activities in the establishment of endometriosis and adenomyosis. METHOD OF STUDY: Pituitary glands, submandibular glands, or hypothalami were transvaginally inoculated into the uteri of syngeneic female mice. Twenty weeks later, the recipient mice were sacrificed and examined. RESULTS: Cysts, adhesion of the uteri to surrounding tissues, and adenomyosis had formed in the uteri of 7 (29.2%), 14 (58.3%), and 22 (91.7%) mice, respectively, out of 24 BALB/c mice after the transplantation of pituitary glands. Similar findings were obtained by experiments with C3H/He and C57BL/6 mice. In NK-cell-deficient C57BL/6-bg(J) and T-cell-deficient BALB/c nu/nu mice, an increase in the formation of cysts, adhesion, and adenomyosis was not observed. CONCLUSIONS: These findings indicate that transvaginal pituitary transplantation specifically induces cysts, adhesion, and adenomyosis. Reduced NK-cell activities may not be necessary in the primary development of endometriosis and adenomyosis.
• Subcellular localization and ubiquitin-conjugating enzyme (E2) interactions of mammalian HECT family ubiquitin protein ligases

  S Hatakeyama, JP Jensen, AM Weissman

  JOURNAL OF BIOLOGICAL CHEMISTRY 272 (24) 15085 - 15092 0021-9258 1997/06 [Refereed][Not invited]
   

  In most instances, the transfer of ubiquitin to target proteins is catalyzed by the action of ubiquitin protein ligases (E3s), Full-length cDNAs encoding murine E6-associated protein (mE6-AP) as well as Nedd-4, a protein that is homologous to E6-AP in its C terminus, were cloned. Nedd-4 and mouse E6-AP are both enzymatically active E3s and function with members of the UbcH5 family of E2s, Mouse E6-AP, like its human counterpart, ubiquitinates p53 in the presence of human papilloma virus E6 protein, while Nedd-4 does not, Consistent with its role in p53 ubiquitination, mE6-AP was found both in the nucleus and cytosol, while Nedd-4 was found only in the cytosol, Binding studies implicate a 150-amino acid region that is 40% identical between mE6-AP and Nedd-4 as a binding site for the C terminal portion of an E2 enzyme (UbcH5B), Nedd-4 was determined to have a second nonoverlapping E2 binding site that recognizes the first 67 amino acids of UbcH5B but not the more C-terminal portion of this E2. These findings provide the first demonstration of physical interactions between mammalian E2s and E3s and establish that these interactions occur independently of ubiquitin and an intact E3 catalytic domain, Furthermore, the presence of two E2 binding sites within Nedd-4 suggests models for ubiquitination involving multiple E2 enzymes associated with E3s.
• Csk overexpression reduces several monokines and nitric oxide productions but enhances prostaglandin E2 production in response to lipopolysaccharide in the macrophage cell line J774A.1

  Kzuya Iwabuchi, Shigetsugu Hatakeyama, Akio Takahashi, Manabu Ato, Masato Okada, Yuri Kajino, Ki-Ichi Kajino, Kazumasa Ogasawara, Kimitaka Takami, Hachiro Nakagawa, Kazunori Onoé

  European Journal of Immunology 27 (3) 742 - 749 0014-2980 1997 [Not refereed][Not invited]
   

  The catalytic activity of src-family protein tyrosine kinases (src-PTK) is suppressed when a C-terminal tyrosine is phosphorylated by an intracellular PTK, C-terminal Src kinase (Csk). In the present report, to study the regulatory functions of the Csk in cells of monocyte/macrophage lineage, we transfected a eukaryotic expression vector containing rat csk cDNA in a macrophage cell line, J774A.1, and examined alterations of the response to lipopolysaccharide (LPS) in the transfectants which overexpressed Csk. Csk overexpression resulted primarily in a down-regulation of Fgr activity, an src-PTK expressed in J774A.1, and hyperphosphorylation of several cellular proteins of 35, 57, 66, 97 and 120-130 kDa. Furthermore, in these Csk transfectants, production of interleukin (IL)-1α, IL-6, tumor necrosis factor-α, and nitric oxide (NO) following LPS stimulation were reduced compared with those in parental J774A.1 or J774A.1 transfected with the vector alone. The extent of reduction paralleled the amounts of Csk proteins expressed in the Csk-transfected J774A.1. The reduced NO production in these cells was associated with low levels of mRNA of inducible NO synthetase. On the other hand, an enhancement of prostaglandin E2 production was observed in the Csk-transfected J774A.1 cells upon stimulation with LPS, which appeared to result from the high level of prostaglandin-H synthetase in the transfectants. The present findings indicate that overexpression of Csk has differential effects on the regulation of production of chemical mediators and monokines, probably via modulation of signal transduction downstream of LPS-mediated signals.
• Functional significance of the Fas molecule in naive lymphocytes

  S Senju, Negishi, I, N Motoyama, FP Wang, KI Nakayama, K Nakayama, PJ Lucas, S Hatakeyama, Q Zhang, S Yonehara, DY Loh

  INTERNATIONAL IMMUNOLOGY 8 (3) 423 - 431 0953-8178 1996/03 [Not refereed][Not invited]
   

  The Fas molecule mediates apoptotic signal in many cell types, Mouse mutations (lpr, lpr(cg), gld), which impair the function of Fas, cause spontaneous autoimmune disease, We generated Fas-deficient (Fas(-/-)) mice by homologous recombination, In embryonic stem cells Fas(-/-) mice developed lpr-like disease, confirming that the abnormality of Fas is causal in the Ipr phenotype, We also made Fas(-/-) chimeric mice composed of a mixture of Fas(+/+) and Fas(-/-) cells, The chimeric mice also showed the lpr phenotype. In Fas(-/-) chimeric mice, the Fas-deficient population expanded progressively among mature T and B lymphocytes. The expansion of Fas-deficient lymphocytes occurred at the naive, pre-primed, lymphocyte stage. These results suggest that the Fas molecule functions not only after antigenic stimulation, as previously hypothesized, but also at the naive lymphocyte stage.
• Fgr expression restricted to subpopulation of monocyte macrophage lineage in resting conditions is induced in various hematopoietic cells after activation or transformation

  S Hatakeyama, K Iwabuchi, M Ato, C Iwabuchi, K Kajino, K Takami, M Katoh, K Ogasawara, RA Good, K Onoe

  MICROBIOLOGY AND IMMUNOLOGY 40 (3) 223 - 231 0385-5600 1996 [Not refereed][Not invited]
   

  The c-fgr gene product (Fgr) is a member of the src-family of protein tyrosine kinases. We have established a monoclonal antibody (2H2) which recognizes the unique N-terminal domain of the murine Fgr. In the present study, using immunohistochemical analysis and immune complex kinase assay with the 2H2, we investigated expression of Fgr in various cell populations and tissues in a murine system. In resting conditions, Fgr expression was confined to subsets of a monocyte/macrophage lineage. Thus, Fgr(+) cells were detected in paracortical areas and medullas of lymph nodes, but seen only in marginal zones of the spleen and the medulla of the thymus. No Fgr(+) macrophage was detected in other tissues, Peyer's patches, brain, heart, lung, liver, pancreas, kidney and peritoneal cavity. However, immune complex kinase assay revealed that, upon stimulation, T and B cells as well as peritoneal macrophages expressed significant levels of Fgr molecules. Transformed cell lines of lymphoid origin, EL-4 and LK35.2, which are T and B lineage lymphomas, respectively, also expressed Fgr molecules. Thus, various cells of hematopoietic origin appeared to possess a potentiality to express Fgr following activation or transformation. The present findings may help elucidate the functional significance of Fgr in immunologically committed cells in either activated or non-activated conditions.
• ESSENTIAL ROLE FOR ZAP-70 IN BOTH POSITIVE AND NEGATIVE SELECTION OF THYMOCYTES

  NEGISHI, I, N MOTOYAMA, K NAKAYAMA, K NAKAYAMA, S SENJU, S HATAKEYAMA, Q ZHANG, AC CHAN, DY LOH

  NATURE 376 (6539) 435 - 438 0028-0836 1995/08 [Not refereed][Not invited]
   

  DURING thymic development, T cells that can recognize foreign antigen in association with self major histocompatibility complex (MHC) are selected for survival (positive selection) and autoreactive T cells are eliminated (negative selection). Both of these selective events are mediated by interaction between the T-cell receptor (TCR) and the peptid-MHC complex(1) But the signalling pathways that lead to cell survival or to cell death are still unclear. ZAP-70 is a protein tyrosine kinase (PTK) that is associated with the TCR signalling subunits (CD3 and zeta) and is expressed in T cells and natural killer cell(2). It has been shown that ZAP-70 plays a crucial role in T-cell activation(2-5) and development(6-8). Here we show that mice lacking ZAP-70 had neither CD4 nor CD8 single-positive T cells, but human ZAP-70 reconstituted both CD4 and CDS single-positive populations. Moreover, ZAP-70(-/-) thomocytes were not deleted by peptide antigens. Natural killer cell function was intact in the absence of ZAP-70. These data suggest that ZAP-70 is a central signalling molecule during thymic selection for CD4 and CD8 lineage.
• A POTENTIAL PEPTIDE VACCINE AGAINST 2 DIFFERENT STRAINS OF INFLUENZA-VIRUS ISOLATED AT INTERVALS OF ABOUT 10 YEARS

  H NARUSE, K OGASAWARA, R KANEDA, S HATAKEYAMA, T ITOH, H KIDA, T MIYAZAKI, RA GOOD, K ONOE

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 (20) 9588 - 9592 0027-8424 1994/09 [Not refereed][Not invited]
   

  We have developed a strategy for making synthetic peptide vaccines, in which a peptide, HA127-133, derived from the hemagglutinin (HA) of A/Aichi/2/68(H3N2) influenza virus (Aichi/68) is introduced into the Ab binding component consisting of 43-46 and 54-58 residues of a pigeon cytochrome c analogue peptide, 46F50V54A. Indeed, this hybrid peptide, 46F/HA127-133/54A, induced impressive T-cell responses and antibody production neutralizing infectivity of Aichi/68 in vitro. In a subsequent study we found that 46F/HA127-133/54A(18mer) peptide antigen, which had been prepared by substitution at the central five residues of 46F50V54A with HA127-133, generated T-cell responses and neutralizing antibody responses as well. On the basis of these prior findings, in the present study we analyzed immunopotency of 46F/HA127-133/54A(18mer) in vivo administered in several ways to I-Ab mice. We show herein that this peptide vaccine loaded in multilamellar liposomes without adjuvant protects the mice against infection with Aichi/68 within 2 weeks after final immunization. Further, this peptide vaccine was shown to be effective in preventing infection with a naturally occurring antigenic variant, A/Texas/1/77(H3N2), carrying the same sequence at 127-133 of the HA as Aichi/68 virus. Since this part of the HA is relatively conserved among H3 subtype influenza viruses, our peptide vaccine may become the basis for a new strategy to prepare effective vaccines that will overcome the ineffectiveness of classical vaccines attributable to antigenic drift of influenza viruses,
• THE MURINE C-FGR GENE-PRODUCT ASSOCIATED WITH LY6C AND P70 INTEGRAL MEMBRANE-PROTEIN IS EXPRESSED IN CELLS OF A MONOCYTE-MACROPHAGE LINEAGE

  S HATAKEYAMA, K IWABUCHI, K OGASAWARA, RA GOOD, K ONOE

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 (8) 3458 - 3462 0027-8424 1994/04 [Not refereed][Not invited]
   

  The c-fgr gene is a member of the Src family of protooncogene tyrosine kinases. A monoclonal antibody (2H2) that recognizes the specific region of the N-terminal domain of the murine c-fgr gene product (Fgr) has been established. With an immune complex kinase assay in a monocytic leukemia cell line, 2H2 monoclonal antibody was shown to precipitate a 59-kDa protein that corresponds in molecular mass to murine Fgr. Fgr was expressed highly in lymph nodes, slightly in spleen and peripheral blood leukocytes, and barely in the thymus and was not detected in bone marrow. In the presence of a mild detergent, Fgr was coimmunoprecipitated with a 70-kDa protein (p70) or with p70 plus several other molecules that were expressed on the cell-surface membrane of macrophage tumor cell lines PU5-1.8 and J774.1, respectively. By contrast, Fgr was not coimmunoprecipitated with a low-affinity receptor for the Fc portion of IgG that is associated with human Fgr. The molecule was also coimmunoprecipitated with the Ly6C molecule from a macrophage cell line (J774.1) that showed protein-tyrosine kinase activity. Peptide mapping revealed that this kinase activity was derived from Fgr. The similarity of relationship between this intramembrane p70 and/or Ly6C and cytoplasmic Fgr to relationships previously reported between T-cell antigen receptor complex, including CD4 and CD8 coreceptors, and Lck or Fyn in T cells and between surface IgM and Lyn or Blk in B cells, suggests that the Fgr and p70 or Ly6C are, indeed, associated with each other and in the murine system may be responsible for recognition of extracellular substances (either cellular or noncellular) and for signal transduction in cells of monocyte/macrophage lineage.
• Sequential Analysis of the Thymocyte Differentiation in Fully Allogeneic Bone Marrow Chimera in Mice.I. Relationship between Functions and Surface Characteristics of Thymocytes

  Kazumasa Ogasawara, Noriko Fukushi, Hisashi Arase, Chikako Iwabuchi, Shigetsugu Hatakeyama, Kazuya Iwabuchi, Robert A. Good, Kazunori Onoá

  Immunobiology 180 (2-3) 149 - 166 0171-2985 1990 [Refereed][Not invited]
   

  Differentiation of thymocytes according to surface phenotype, functional status and cell size was investigated using fully allogeneic bone marrow chimeras. Most of the donor-derived thymocytes obtained from chimeras 9 days after hematopoietic reconstitution were CD4-8and IL2R+. At day 14, CD4+8+ cells became prominent in the thymus. Eighty-six per cent of thymocytes were CD4+8+ and 9 % were CD4-8- at this stage. After day 21, the proportion of CD4+8- or CD4-8+ single positive cells transiently increased and then declined to normal level at day 42. Further, the mean size of CD4+ or CD8+ single positive cells in chimeric thymuses at day 21 after reconstitution was markedly larger than that at day 35. When proliferative responses to various stimuli (PMA + rIL2, anti-CD3 mAb (2C11) and anti-Vβ8 mAb (F23.1)) were evaluated, significant responses were generated by thymocytes for the first time at around day 28 and the responses reached their peaks at day 35. These findings demonstrated that the process of thymocyte differentiation in the fully allogeneic chimeras was similar to ontogenic development as observed in fetal mice. However, the tempo at which the differentiation of surface phenotypes and development of functions proceeded was quite different from that seen in normal mice. The relationship among surface phenotypes, cell size and functions of developing thymocytes of bone marrow chimeras is discussed. © 1990, Gustav Fischer Verlag · Stuttgart · New York. All rights reserved.
• Sequential Analysis of Distributions of Donor‐derived Thymocytes Bearing T‐cell Antigen Receptor (TCR) and Donor‐derived la+ Cells in Thymuses of Fully Allogeneic Bone Marrow Chimera in Mice

  Shigetsugu Hatakeyama, Kazumasa Ogasawara, Noriko Fukushi, Chikako Iwabuchi, Kazuya Iwabuchi, Bingyan Wang, Masaharu Kajiwara, Robert A. Good, Kazunori Onoè

  Pathology International 40 (6) 391 - 401 1440-1827 1990 [Refereed][Not invited]
   

  Lethally irradiated SJL/J mice were reconstituted with B10 bone marrow cells, and the process of thymic reconstitution by donor derived cells positive for I‐ A or Vβ8 molecules was investigated. The donor‐derived la+ cells appeared in the medulla on day 7 after reconstitution. The la+ cells became confluent up to day 14, and the cellularity in the medulla on day 17 was almost the same as that in the normal thymus. Dull Vβ8+ thymocytes were first recognized in the cortex on day 10 and were identifiable in the medulla by day 14. The Vβ8+ cells seemed to be mainly CD4+8+ double‐positive. Furthermore, most of the Vg8’cells in the medulla of chimeras given cyclosporin A for 3 weeks after reconstitution appeared to be CD4+8+. The present findings demonstrate that CD4 8+ thymocytes which bear a low concentration of TCR exist in the thymic medulla at a relatively early stage when donor‐derived la+ cells have already settled there. The coincidental appearance and coexistence of la+ cells and TCR+ thymocytes in the medulla suggest that these histological characteristics may be related to the selection of thymocytes in this area. Copyright © 1990, Wiley Blackwell. All rights reserved
• DONOR AND RECIPIENT SPECIFIC TOLERANCE IN CELLS FROM SEMI-ALLOGENEIC, H-2 SUBREGION COMPATIBLE OR FULLY ALLOGENEIC BONE-MARROW CHIMERAS ATTRIBUTABLE TO CLONAL DELETION

  K ONOE, K IWABUCHI, C IWABUCHI, H ARASE, S HATAKEYAMA, PP WAMBUA, N FUKUSHI, NEGISHI, I, RA GOOD, K OGASAWARA

  IMMUNOBIOLOGY 179 (2-3) 172 - 189 0171-2985 1989/06 [Not refereed][Not invited]
   

  Specificities of tolerance induced in allogeneic bone marrow (BM) chimeras which had been established by injecting allogeneic BM cells pretreated with anti-Thy-1 mAb alone (without complement (C)) were analyzed using Simonsen's splenomegaly assay. Lymphocytes from fully allogeneic, semi-allogeneic and H-2 subregion compatible BM chimeras were specifically unresponsive to donor and recipient antigens (Ag). However, cells from H-2 subregion compatible chimeras initiated as vigorously a GVHR in F1 recipient mice, which were disparate at H-2K and I-A regions, as did spleen cells of donor mice, which were incompatible at the entire H-2 and minor histocompatibility regions of the recipients. The donor cells from such chimeras that initiated these considerable GVHR were either CD4+ or CD8+ T cells. Furthermore, synergistic effects by the CD4+ and CD8+ T lymphocytes were also observed. We found no evidence for a suppressive mechanism(s) in maintenance of the specific tolerance in allogeneic chimeras. Further, when lymphoid cells from these chimeras were adoptively transferred to irradiated mice of the donor strain and maintained for 5 days in the absence of recipient Ag (tolerogen), the adoptively transferred cells were shown to retain their unresponsiveness to the recipient Ag. These results reveal that T lymphocytes from allogeneic BM chimeras prepared by our method had been specifically induced to a tolerant state to both donor and recipient Ag and that the major mechanism of induction and maintenance of long-lasting tolerance is attributable to clonal deletion of both CD4+ and CD8+ T cell subsets rather than to the development of a population of suppressor cells of any sort.
• A Study on Graft‐versus‐Host Reaction (GVHR) by Simonsen's Splenomegaly Assay Cells and Antigen Systems Involved in Induction of GVHR

  Kazunori Onoé, Kazuya Iwabuchi, Chikako Katsume, Toshihiko Gotohda, Akira Arase, Shigetsugu Hatakeyama, Machiko Mishima, Robert A. Good, Kazumasa Ogasawara

  Pathology International 39 (2) 101 - 110 1440-1827 1989 [Refereed][Not invited]
   

  Cells and histocompatibility antigen systems involved in graft‐versus‐host reactions (GVHR) were analyzed using Simonsen's splenomegaly assay employing various combinations of donor and F1 hybrid recipients mice. Most of the cells proliferating in spleens of mice undergoing GVHR were J11d+, and had histological features of cells of the hematopoietic lineage. The proportions of CD3+ T cells were decreased in the spleens. Disparity at minor histocompatibility determinants of AKR, I‐E and H‐2D regions between B10.A(4R) donors and (4R × AKR) F1 recipients evoked only negligible GVHR. On the contrary, disparity at H‐2K and/or I‐A regions appeared to be sufficient to permit induction of full GVHR. When surface markers of donor spleen cells were analyzed, it was shown that Thy‐1+ and/or MEL‐14+ cells caused a strong effect on GVHR. Further, either CD4+ or CD8+ T cell subset could induce significant GVHR. However, synergistic influences of these two T cell subsets on one another in GVHR were observed. The present results raise the possibility of using Simonsen's assay along with a number of reagents to identify the contribution of subsets of T lymphocytes and in analyzing precise contributions of cellular components from both donor and recipient, and also of the target antigen systems of the recipient that contribute to early events involved in GVHR. Copyright © 1989, Wiley Blackwell. All rights reserved
• A study on graft-versus-host reaction (GVHR) by Simonsen's splenomegaly assay. Cells and antigen systems involved in induction of GVHR

  K. Onoe, K. Iwabuchi, C. Katsume, T. Gotohda, A. Arase, S. Hatakeyama, M. Mishima, R. A. Good, K. Ogasawara

  Acta Pathologica Japonica 39 101 - 110 0001-6632 1989/01/01 [Not refereed][Not invited]
  

MISC

• 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  2023/09
• FAM83Hの発現低下はケラチン分布の変化を介して皮膚有棘細胞癌の遊走と浸潤を促進する

  得地 景子, 北村 真也, 前田 拓哉, 氏家 英之, 柳 輝希, 渡部 昌, 畠山 鎮次, 加納 里志, 田中 伸哉  日本皮膚科学会雑誌  133-  (2)  265  -265  2023/02
• Proteolytic mechanism of the oncogene product PPM1D

  渡部昌, 高橋正樹, 畠山鎮次  日本分子生物学会年会プログラム・要旨集(Web)  46th-  2023
• さまざまなユビキチン結合ドメインを用いたTUBEs(Tandem Ubiquitin-Binding Entities)の作製

  渡部 昌, 畠山 鎮次  日本生化学会大会プログラム・講演要旨集  95回-  1P  -086  2022/11
• 香辛料によるISG15修飾の抑制

  大木 のどか, 山田 詩乃, 田中 環, 福井 裕美, 畠山 鎮次, 奥村 文彦  日本生化学会大会プログラム・講演要旨集  95回-  1P  -111  2022/11
• 効率的な遺伝子発現制御を可能にするCRISPRi法の開発

  大木 のどか, 近藤 豪, 畠山 鎮次  日本生化学会大会プログラム・講演要旨集  95回-  2P  -260  2022/11
• Identification of a novel protein on MHC class I transcriptional regulation

  應田涼太, 坐間のゆり, XIN Sun, 渡部昌, 畠山鎮次, 小林弘一  日本分子生物学会年会プログラム・要旨集(Web)  45th-  2022
• Macroscopic order formation in myoblast populations driven by intracellular molecular dynamics

  麓佳月, 高田真吾, 小野寺康仁, 川口喬吾, 畠山鎮次, 佐邊壽孝, 及川司  日本分子生物学会年会プログラム・要旨集(Web)  45th-  2022
• 免疫介在性小脳失調症における新規自己抗体の検討

  工藤彰彦, 矢口裕章, 阿部恵, 江口克紀, 長井梓, 脇田雅大, 白井慎一, 岩田育子, 松島理明, 水戸泰紀, 田島康敬, 渡部昌, 畠山鎮次, 米田誠, 田中惠子, 矢部一郎  日本神経学会学術大会プログラム・抄録集  63rd-  2022
• TRIM28はサイクリンA2のユビキチン化を介しS期への早期進行を抑制する

  渡部 昌, 畠山 鎮次  日本生化学会大会プログラム・講演要旨集  94回-  [P  -996]  2021/11
• COVID-19収束後の短期臨床留学再開に向けた展望と課題

  石森 直樹, 畠山 鎮次, 村上 学, Shane Peter, 高橋 誠  医学教育  52-  (Suppl.)  115  -115  2021/07
• TRIMファミリータンパク質と標的タンパク質分解

  渡部 昌, 畠山 鎮次  細胞  53-  (3)  160  -163  2021/03  

  タンパク質を選択的かつ急速に分解する薬剤の開発法として、Specific and Nongenetic Inhibitor of apoptosis proteindependent Protein Eraser(SNIPER)法やPROteolysis TArgeting Chimeras(PROTAC)法が近年注目を集めている。SNIPER/PROTAC法は、言わばユビキチン修飾系を小分子により言わば「ハイジャック」することで目的のタンパク質を分解する手法である。また、筆者らが着目しているTripartite Motif proteins(TRIM)型ユビキチンリガーゼの1つであるTRIM21に基づいたTrim-Away法もまた、新たな手法として注目されている。本稿ではまずユビキチン修飾系を概説し、TRIM型ユビキチンリガーゼのSNIPER/PROTAC法への応用の可能性およびTrim-Away法について紹介したい。(著者抄録)
• ユビキチン様修飾分子UFM1によるロイコトリエン生合成の制御

  渡部 昌, 畠山 鎮次  日本生化学会大会プログラム・講演要旨集  93回-  [P  -168]  2020/09
• 数理・情報科学を活用したシグナル伝達と疾患研究の最前線 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  日本生化学会大会プログラム・講演要旨集  92回-  [2S07m  -06]  2019/09
• ユビキチン関連タンパク質TRIM29による癌化制御の解明

  畠山鎮次  ライフサイエンス振興財団助成成果報告書  2018-  18‐20  2019/05/31  [Not refereed][Not invited]
  
• 扁平上皮癌におけるTripartite motif‐containing protein 29(TRIM29)の検討

  柳輝希, 秦洋郎, 北村真也, 清水宏, 柳紘子, 本間明宏, 渡部昌, 畠山鎮次  日本皮膚科学会雑誌  129-  (5)  1170  -1170  2019/05/15  [Not refereed][Not invited]
  
• ユビキチン化酵素の基質タンパク質同定法の確立

  畠山鎮次  日本応用酵素協会誌  (53)  51  -51  2019/03/01  [Not refereed][Not invited]
  
• 扁平上皮におけるTripartite motif‐containing protein 29(TRIM29)の機能解析

  柳輝希, 秦洋郎, 北村真也, 今福恵輔, 清水宏, 柳紘子, 本間明宏, 渡部昌, 畠山鎮次  日本皮膚科学会雑誌  129-  (1)  45  -45  2019/01/20  [Not refereed][Not invited]
  
• ユビキチンリガーゼが関与する疾患

  畠山鎮次  医学のあゆみ  267-  (13)  1090‐1095  2018/12/29  [Not refereed][Not invited]
  
• ユビキチンリガーゼTRIM29基質認識ドメインのX線結晶構造解析

  松田拳, 高木賢治, 畠山鎮次, 水島恒裕  日本結晶学会年会講演要旨集  2018-  67  2018/11/01  [Not refereed][Not invited]
  
• E3ユビキチンリガーゼRNF113Aによる自然免疫応答制御に関する研究

  石田 寛樹, 渡部 昌, 畠山 鎮次  日本生化学会大会プログラム・講演要旨集  91回-  [2P  -096]  2018/09
• 免疫介在性小脳失調症例で同定した抗Sez6l2抗体はSez6l2とGluR1の結合を阻害することにより病原性を呈する

  矢口 裕章, 高橋 秀尚, 矢部 一郎, 渡辺 雅彦, 畠山 鎮次  神経免疫学  22-  (1)  130  -130  2017/10  [Not refereed][Not invited]
  
• メディエーター複合体による転写終結制御機構

  高橋秀尚, 柴田美音, 瀧川一学, 渡部昌, 築山忠維, 山本淳一, 山口雄輝, 藤井聡, 飯田緑, RANJAN Amol, SATO Shigeo, TOMOMORI‐SATO Chieri, CONAWAY Joan, CONAWAY Ronald, 畠山鎮次  日本生化学会大会(Web)  90th-  ROMBUNNO.4P2T15‐06(3P‐0635) (WEB ONLY)  -0635)]  2017  [Not refereed][Not invited]
  
• 前立腺癌診断におけるTripartite motif‐containing protein29(TRIM29)染色の有用性の検討

  吉田一彦, 吉田一彦, 谷野美智枝, 堀井理絵, 木村太一, 津田真寿美, 近藤恒徳, 秋山太, 畠山鎮次, 田邉一成, 田中伸哉  日本泌尿器科学会東部総会プログラム・抄録集  82nd-  222  2017  [Not refereed][Not invited]
  
• 髄膜炎・臍周囲炎・呼吸障害を呈したOTULIN‐related autoinflammatory syndrome(ORAS)の1例

  植木将弘, 山田雅文, 戸澤雄介, 竹崎俊一郎, ABDRABOU ShimaaSaid MohamedAli, 小林一郎, 有賀正, 渡部昌, 畠山鎮次, 藤田宏明, 岩井一宏, 三宅紀子, 松本直通  日本免疫不全症研究会プログラム・抄録  10th-  12  2017  [Not refereed][Not invited]
  
• 癌化制御に関与するTRIM型ユビキチンリガーゼの解析

  畠山鎮次  上原記念生命科学財団研究報告集(CD-ROM)  30-  ROMBUNNO.51  -6  2016/12  [Not refereed][Not invited]
  
• ユビキチンリガーゼTRIM23は非定型ポリユビキチン化によるPPARγの安定化を介して脂肪細胞分化を制御する

  渡部昌, 高橋秀尚, 畠山鎮次  日本生化学会大会(Web)  89th-  ROMBUNNO.3T11‐02(3P‐108) (WEB ONLY)  -02(3P  2016/09  [Not refereed][Not invited]
  
• メディエーター複合体による転写制御機構

  高橋秀尚, 畠山鎮次  日本生化学会大会(Web)  89th-  ROMBUNNO.2T15‐01(2P‐273) (WEB ONLY)  -01(2P  2016/09  [Not refereed][Not invited]
  
• The Novel Heart-specific Ring-finger Protein 207 Regulates Energy Metabolism in Cardiomyocytes

  Wataru Mizushima, Hidehisa Takahashi, Masashi Watanabe, Shintarou Kinugawa, Shouji Matsushima, Shingo Takada, Takashi Yokota, Takaaki Furuhata, Junichi Matsumoto, M. Tsuda, T. Katayama, T. Nakajima, S. Hatakeyama, H. Tsutsui  CIRCULATION RESEARCH  119-  2016/07
• ユビキチンE3リガーゼTRIM23は非定型ポリユビキチン化によるPPARγの安定化を介して脂肪細胞分化を制御する

  渡部昌, 高橋秀尚, 畠山鎮次  日本細胞生物学会大会(Web)  68th-  ROMBUNNO.P1‐14 (WEB ONLY)  -70  2016/05  [Not refereed][Not invited]
  
• ユビキチンリガーゼTRIM23はPPARγの安定化を介して脂肪細胞分化を制御する

  渡部昌, 高橋秀尚, 畠山鎮次  日本内分泌学会雑誌  92-  (1)  201  2016/04/01  [Not refereed][Not invited]
  
• メディエーター複合体のサブユニットMed26による転写終結制御機構

  高橋秀尚, 畠山鎮次  日本分子生物学会年会プログラム・要旨集(Web)  39th-  ROMBUNNO.3AS10‐6(3P‐0189) (WEB ONLY)  2016  [Not refereed][Not invited]
  
• Med26はLittle elongation complexをリクルートすることでsmall nuclear RNA遺伝子の発現を制御する

  TAKAHASHI HIDEHISA, HATAKEYAMA SHIGETSUGU  日本RNA学会年会要旨集  17th-  78  2015/07/15  [Not refereed][Not invited]
  
• IDENTIFICATION OF FIP1L1-PDGFRA ASSOCIATING MOLECULE THAT LOCATES IN THE NUCLEUS AND AUGMENTS THE ACTIVITY OF FIP1L1-PDGFRA

  T. Kondo, M. Ibata, J. Iwasaki, Y. Fujioka, K. Nakagawa, S. Darmanin, M. Onozawa, D. Hashimoto, Y. Ohba, S. Hatakeyama, T. Teshima  HAEMATOLOGICA  100-  327  -327  2015/06  [Not refereed][Not invited]
  
• Bechet病疾患感受性遺伝子Tripartite motif protein 39(TRIM39)の機能解析

  鈴木 正宣, 渡部 昌, 中丸 裕爾, 高木 大, 本間 あや, 畠山 鎮次, 福田 諭  耳鼻咽喉科免疫アレルギー  33-  (2)  38  -39  2015/06  [Not refereed][Not invited]
  
• メディエーター複合体のサブユニットMED26はLittle elongation complexをリクルートすることでsnRNA遺伝子の転写を制御する

  高橋秀尚, 瀧川一学, 渡部昌, Delnur Anwar, 柴田美音, 佐藤チエリ, 佐藤滋生, Amol Ranjan, Chris W. Seidel, 築山忠維, 水島航, 林正康, 大川恭行, Joan, W. Conaway,Ronal, C. Conaway, 畠山鎮次  北海道医学雑誌  90-  (1)  76  -76  2015  [Not refereed][Not invited]
  
• Ubiquitin modification in otolaryngology

  鈴木正宣, 鈴木正宣, 渡部昌, 中丸裕爾, 高木大, 加納里志, 本間あや, 畠山鎮次, 福田諭  耳鼻咽喉科免疫アレルギー(Web)  33-  (3)  185-192 (J-STAGE)  2015  [Not refereed][Not invited]
  
• 抗神経抗体抗原であるSez6l2はAMPA受容体とADDの複合体を形成しシグナル伝達を促進する

  矢口裕章, 矢口裕章, 矢口裕章, 高橋秀尚, 矢部一郎, 渡辺雅彦, 畠山鎮次, 佐々木秀直  Neuroimmunology  20-  (1)  79  2015  [Not refereed][Not invited]
  
• TRIMファミリーユビキチンリガーゼによる生体制御機構

  畠山鎮次  日本生化学会大会(Web)  88th-  1W11-P-9 (WEB ONLY)  2015  [Not refereed][Not invited]
  
• ベーチェット病感受性遺伝子Tripartite motif protein 39(TRIM39)の機能解析

  SUZUKI MASANOBU, SUZUKI MASANOBU, WATABE MASASHI, NAKAMARU YUJI, TAKAGI DAI, HONMA AYA, HATAKEYAMA SHIGETSUGU, FUKUDA SATOSHI  日本耳鼻咽喉科免疫アレルギー学会プログラム・抄録集  33rd-  82  2015  [Not refereed][Not invited]
  
• 皮膚組織において紫外線傷害を制御するユビキチン化システムの解析

  HATAKEYAMA SHIGETSUGU  コスメトロジー研究報告  22-  143  -146  2014/09/01  [Not refereed][Not invited]
  
• 破骨細胞誘導因子Siglec‐15は閉経後骨粗鬆症の治療ターゲットとなりうるか

  KAMEDA YUSUKE, TAKAHATA MASAHIKO, MIKUNI SHINTARO, HATAKEYAMA SHIGETSUGU, SHIMIZU TOMOHIRO, HAMANO HIROKI, ANGATA TAKASHI, KINJO MASATAKA, IWASAKI NORIMASA  日本整形外科学会雑誌  88-  (8)  S1477  2014/08/29  [Not refereed][Not invited]
  
• RNA‐DNA結合タンパク質NonOは,SOX2プロモーター制御を介して乳癌幹細胞様表現型をコントロールする

  IIZASA HISASHI, LIANG SHANSHAN, TAKAHASHI HIDEHISA, KURAMITSU YASUHIRO, HIROSE TETSURO, NAKAZAWA SEITARO, HATAKEYAMA SHIGETSUGU, KAWAUCHI HIDEYUKI, YOSHIYAMA HIRONORI, HAMADA JUN'ICHI  日本RNA学会年会要旨集  16th-  139  2014/07/23  [Not refereed][Not invited]
  
• 破骨細胞誘導因子Siglec‐15は閉経後骨粗鬆症の治療ターゲットとなり得るか?

  KAMEDA YUSUKE, TAKAHATA MASAHIKO, MIKUNI SHINTARO, HATAKEYAMA SHIGETSUGU, SHIMIZU TOMOHIRO, HAMANO HIROKI, ANGATA TAKASHI, KINJO MASATAKA, IWASAKI NORIMASA  日本骨形態計測学会雑誌  24-  (2)  S116  2014/05/19  [Not refereed][Not invited]
  
• プロポロリスコーティング床敷はケージ内常在菌の増殖とマウスアレルゲンの発生を抑制する

  TOSA NORIKO, YOSHIMATSU, KUMIKO, TSUKIYAMA TADAYUI, HATAKEYAMA SHIGETSUGU, ARIKAWA JIRO  日本実験動物学会総会講演要旨集  61st-  280  2014/05/01  [Not refereed][Not invited]
  
• ベーチェット病感受性遺伝子Tripartite motif protein39(TRIM39)の機能解析

  鈴木正宣, 渡部昌, 高木大, 中丸裕爾, 畠山鎮次, 福田諭  日本耳鼻咽喉科学会会報  117-  (4)  504  -504  2014/04/20  [Not refereed][Not invited]
  
• 脂質代謝制御転写因子PPARγの活性化を制御するユビキチン化酵素と診断治療への応用

  HATAKEYAMA SHIGETSUGU  医科学応用研究財団研究報告(CD-ROM)  31-  377  -381  2014/02  [Not refereed][Not invited]
  
• Med26はLittle elongation complexをリクルートすることでsmall nuclear RNA遺伝子の発現を制御する

  高橋秀尚, 瀧川一学, 渡部昌, ANWAR Delnur, 柴田美音, 佐藤チエリ, 佐藤滋生, RANJAN Amol, SEIDEL Chris, 築山忠維, 林正康, 大川恭行, CONAWAY Joan, CONAWAY Ronald, 畠山鎮次  日本生化学会大会(Web)  87th-  WEB ONLY 2T15P-16(3P-502)  -16]  2014  [Not refereed][Not invited]
  
• メディエーター複合体による転写伸長制御

  高橋秀尚, 瀧川一学, 渡部昌, ANWAR Delnur, 柴田美音, 佐藤チエリ, 佐藤滋生, RANJAN Amol, SEIDEL Chris W, 築山忠維, 林正康, 大川恭行, CONAWAY Joan, CONAWAY Ronald C, 畠山鎮次  日本分子生物学会年会プログラム・要旨集(Web)  37th-  1W15-3(1P-0237) (WEB ONLY)  2014  [Not refereed][Not invited]
  
• AN INFLAMMATORY CYTOKINE IL-1B IS INVOLVED IN BLADDER REMODELLING AFTER PARTIAL BLADDER OUTLET OBSTRUCTION IN MICE

  Y. Kanno, T. Mitsui, T. Kitta, K. Moriya, T. Tsukiyama, S. Hatakeyama, K. Nonomura  NEUROUROLOGY AND URODYNAMICS  32-  (6)  925  -926  2013/08  [Not refereed][Not invited]
  
• TRIM6はc-Mycの機能を調節することで, マウスES細胞の多能性維持機構に関与する

  佐藤 智信, 奥村 文彦, 有賀 正, 畠山 鎮次  北海道醫學雜誌 = Acta medica Hokkaidonensia  88-  (2)  85  -85  2013/04/01
• ダイオキシン受容体内因性リガンドによる前立腺癌細胞におけるアンドロゲンレセプター発現低下と増殖抑制

  丸山覚, 築山忠維, 宮島直人, 土屋邦彦, 安部崇重, 佐澤陽, 篠原信雄, 畠山鎮次, 野々村克也  日本泌尿器科学会雑誌  104-  (2)  415  2013/03/20  [Not refereed][Not invited]
  
• Med26はLittle elongation complexをリクルートすることでsmall nuclear RNA遺伝子の発現を制御する

  TAKAHASHI Hidehisa, 瀧川一学, ANWAR Delnur, 柴田美音, TOMOMORI‐SATO Chieri, SATO Shigeo, RANJAN Amol, SEIDEL Chris, 築山忠維, 渡部昌, 林正康, 大川恭行, CONAWAY Joan, CONAWAY Ronald, 畠山鎮次  日本分子生物学会年会プログラム・要旨集(Web)  36th-  1P-0182 (WEB ONLY)  2013  [Not refereed][Not invited]
  
• ユビキチン修飾系の分子機構と疾患 TRIM型ユビキチンリガーゼと癌—TRIM蛋白質の多様性と特異性

  渡部昌, 畠山鎮次  医学のあゆみ  243-  (6)  508-514  -514  2012/11/10  [Not refereed][Not invited]
  
• 免疫と癌を制御するTRIM型ユビキチンリガーゼ

  畠山鎮次  日本臨床免疫学会会誌  35-  (4)  292  -292  2012/08/31  [Not refereed][Not invited]
   

  多くの細胞内タンパク質の分解にユビキチン-プロテアソーム系が関与している．この分解系により，細胞内シグナル伝達分子や癌関連遺伝子産物などの発現量は厳密に調節されている．ユビキチンはATP依存性にE1，E2，E3という一連の酵素カスケードによって標的タンパク質に結合され，その後ユビキチン化された多くのタンパク質はプロテアソームにより分解される．したがって，特にユビキチンリガーゼE3は標的タンパク質を認識し，最終的にユビキチンを付加するため，重要な酵素サブユニットと認識されている．ヒト遺伝子上にE3と予想されるタンパク質の遺伝子は約数百あることが推測されており，今回紹介するTRIMファミリータンパク質の遺伝子も，ヒトゲノムにおいて大きな遺伝子ファミリーを形成していることが判明している．最近になり，我々はTRIM21，TRIM25，TRIM32，TRIM68及びTRIM40が，癌化や免疫反応の制御に関与することを報告している［Nature Reviews Cancer (2011); Mol Med (2012); Carcinogenesis (2011); Cancer Res (2008); Mol Immunol (2008)]．今後，免疫系の制御や癌化に対するTRIMファミリータンパク質の関与が明らかとなることで，免疫疾患や癌の治療法の新規シーズとしての可能性が期待できる．
  
• TRIM67は80K-Hを介してRasを抑制し, 神経突起形成を誘導する

  矢口 裕章, 奥村 文彦, 高橋 秀尚, 加納 崇裕, 亀田 浩之, 内ヶ島 基政, 田中 伸哉, 渡辺 雅彦, 佐々木 秀直, 畠山 鎮次  北海道醫學雜誌 = Acta medica Hokkaidonensia  87-  (4)  175  -175  2012/08/01
• 単球系細胞における酸化LDL(APOB)および抗β2GPI抗体によって誘導される組織因子発現の検討

  大友耕太郎, 渥美達也, 藤枝雄一郎, 中川久子, 加藤将, AMENGUAL Olga, 近祐次郎, 堀田哲也, 保田晋助, 松本雅記, 畠山鎮次, 小池隆夫, 小池隆夫  日本血栓止血学会誌  23-  (2)  166  -166  2012/04/01  [Not refereed][Not invited]
  
• 亜急性小脳失調症における自己抗体の検討

  矢口裕章, 高橋秀尚, 奥村文彦, 加納崇裕, 加納崇裕, 竹内朗子, 堀内一宏, 廣谷真, 矢部一郎, 畠山鎮次, 佐々木秀直  日本神経免疫学会学術集会抄録集  24th-  90  2012  [Not refereed][Not invited]
  
• 自己免疫と炎症に関与するユビキチン化システム

  畠山鎮次  自己抗体と自己免疫  2011-  37-42,5  2011/10/26  [Not refereed][Not invited]
  
• ユビキチンの多彩な生理機能 3.複雑化するユビキチンリガーゼの機能と多様性

  畠山鎮次, 渡部昌  実験医学  29-  (12)  1921-1932  -1932  2011/08/01  [Not refereed][Not invited]
  
• 消化管疾患における新規NF‐κB制御タンパク質TRIM‐G1の発現変動解析

  野口慶太, 崎浜秀康, 本間重紀, 高橋典彦, 畠山鎮次, 藤堂省  日本外科学会雑誌  112-  (1)  442  -442  2011/05/25  [Not refereed][Not invited]
  
• 「筋萎縮性側索硬化症の病態に基づく画期的治療法の開発」神経突起様伸長及び増殖を制御するTRIM‐EN1/2の機能解析

  佐々木秀直, 矢口裕章, 矢口裕章, 奥村文彦, 加納崇裕, 加納崇裕, 渡部昌, 内ケ島基政, 渡邊雅彦, 畠山鎮次  筋萎縮性側索硬化症の病態に基づく画期的治療法の開発 平成22年度 総括研究報告書  59-61  2011  [Not refereed][Not invited]
  
• TRIMファミリー分子によるPPARγシグナルの制御

  渡部昌, 畠山鎮次  生化学  ROMBUNNO.4P-0130  2011  [Not refereed][Not invited]
  
• PKA/14‐3‐3によるユビキチンリガーゼTRIM32の制御

  市村徹, 八谷如美, 小俣結子, 畠山鎮次, 佐藤智信, 勝二郁夫  生化学  ROMBUNNO.4P-0128  2011  [Not refereed][Not invited]
  
• Ubiquitin ligase regulating intracellular traffic of prostate specific antigen

  畠山 鎮次  細胞科学研究財団助成研究報告集  21-  43  -48  2010
• TRIM68 REGULATES LIGAND-DEPENDENT TRANSCRIPTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER CELLS

  Naoto Miyajima, Satoru Maruyama, Takashige Abe, Ataru Sazawa, Nobuo Shinohara, Shigetsugu Hatakeyama, Katsuya Nonomura  JOURNAL OF UROLOGY  181-  (4)  398  -398  2009/04  [Not refereed][Not invited]
  
• プレセニリン1と2の比活性の違いを決定しているドメインの同定(Characterization of the domain responsible for the different specific activity of PS1 and PS2)

  城谷 圭朗, 橋本 康弘, 畠山 鎮次  神経化学  47-  (2-3)  242  -242  2008/08  [Not refereed][Not invited]
  
• 細胞内適応制御系としてのユビキチン‐プロテアソームシステム

  HATAKEYAMA SHIGETSUGU  適応医学  11-  (2)  13  -18  2008/01/15  [Not refereed][Not invited]
  
• 細胞内適応制御系としてのユビキチン‐プロテアソームシステム

  HATAKEYAMA SHIGETSUGU  適応医学  11-  (1)  16  2007/05/20  [Not refereed][Not invited]
  
• 新規ユビキチン結合タンパク質シンテニンの生化学的解析

  LIANG Fei, 城谷圭朗, 畠山鎮次  生化学  2007
• Gdx, an X-linked ubiquitin-like modifier, is conjugated to cyclin it and regulates cytokinesis

  Noriko Ishidal, Shigetsugu Hatakeyama, Keiko Nakayama, Keiichi Nakayama  CELL STRUCTURE AND FUNCTION  30-  100  -100  2005/06  [Not refereed][Not invited]
  
• AIRE(autoimmune regulator)はPHDドメインを介してUbiquitin E3 ligase活性を発揮する

  黒田範行, 内田大亮, 畠山鎮次, 松島明美, 石戸聡, 工藤純, 清水信義, 中山敬一, 松本満  日本分子生物学会年会プログラム・講演要旨集  26th-  520  2003/11/25  [Not refereed][Not invited]
  
• 細胞周期の開始と停止の制御機構 静止期から増殖期へ p27分解に関わる二つのユビキチンリガーゼ (生化学)

  中山敬一, 嘉村巧, 原太一, 畠山鎮次, 中山啓子  生化学  73-  (8)  636  -636  2001/08  [Not refereed][Not invited]
  
• 蛋白質分解制御による細胞分裂周期の調節 サイクリンE及びp27Kip1のユビキチン化因子のノックアウトマウスによる機能解析 (生化学)

  中山敬一, 畠山鎮次, 北川雅敏, 小南欽一郎, 中山啓子  生化学  72-  (8)  596  -596  2000/08  [Not refereed][Not invited]
  
• NAK is an I kappa B kinase-activating kinase

  Y Tojima, A Fujimoto, M Delhase, Y Chen, S Hatakeyama, K Nakayama, Y Kaneko, Y Nimura, N Motoyama, K Ikeda, M Karin, M Nakanishi  NATURE  404-  (6779)  778  -782  2000/04  [Not refereed][Not invited]
   

  Phosphorylation of I kappa B by the I kappa B kinase (IKK) complex is a critical step leading to IkB degradation and activation of transcription factor NF-kappa B-1. The IKK complex contains two catalytic subunits, IKK alpha and IKK beta, the latter being indispensable for NF-kappa B activation by pro-inflammatory cytokines(2-7). Although IKK is activated by phosphorylation of the IKK beta activation loop(8), the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure(1,9). Here we describe an IKK-related kinase, named NAK (NF-kappa B-activating kinase), that can activate IKK through direct phosphorylation. NAK induces I kappa B degradation and NF-kappa B activity through IKKb. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappa B by protein kinase C-epsilon (PKC epsilon). Thus, NAK is an IKK kinase that may mediate IKK and NF-kappa B activation in response to growth factors that stimulate PKC epsilon activity.
• 新規NF‐κB活性化キナーゼ(NAK)遺伝子の機能解析

  藤本淳司, 東島由一郎, CHEN Y, 畠山鎮次, 中山敬一, 金子葉子, 本山昇, KARIN M, 中西真  日本分子生物学会年会プログラム・講演要旨集  22nd-  671  1999/11/22  [Not refereed][Not invited]
  
• IκBαとβ‐cateninの分解に関与するユビキチン化酵素複合体SCF^FWDI

  畠山鎮次, 北川雅敏, 松本雅記, 白根道子, 服部公彦, 中野裕康, 奥村康, 菊池章, 中山敬一  日本分子生物学会年会プログラム・講演要旨集  22nd-  256  1999/11/22  [Not refereed][Not invited]
  
• 細胞周期制御因子およびシグナル伝達因子の分解の分子機構 (生化学)

  北川雅敏, 畠山鎮次, 白根道子, 中山啓子, 中山敬一  生化学  71-  (8)  1042  -1042  1999/08  [Not refereed][Not invited]
  
• NFκB活性化シグナルと生命現象 IκBのユビキチンリガーゼSCFFWD1複合体の同定 (生化学)

  中山敬一, 畠山鎮次, 北川雅敏, 白根道子, 松本雅記, 服部公彦, 中山啓子  生化学  71-  (8)  686  -686  1999/08  [Not refereed][Not invited]
  
• Ubiquitin-dependent degradation of I kappa B alpha is mediated by a ubiquitin ligase Skp1/Cul1/F-box protein FWD1 (vol 96, pg 3859, 1999)

  S Hatakeyama, M Kitagawa, K Nakayama, M Shirane, M Matsumoto, K Hattori, H Higashi, H Nakano, K Okumura, K Onoe, RA Good, K Nakayama  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA  96-  (11)  6571  -6571  1999/05  [Not refereed][Not invited]
  
• C-terminal src kinase overexpression reduces production of atherogenic cytokines and tissue factor in macrophage cell line

  K Watano, N Ishimori, K Iwabuchi, S Hatakeyama, A Takahashi, M Ato, K Ogasawara, K Takami, K Onoe, M Okada, H Nakagawa, S Fujii  CIRCULATION  98-  (17)  40  -40  1998/10  [Not refereed][Not invited]
  
• 細胞周期のブレーキp27及びp57の分化と発癌に関する役割

  中山敬一, 永濱裕康, 石田典子, 白根道子, 森正樹, 高橋勝彦, 畠山鎮次, 北川雅敏, 中山啓子  日本癌学会57回総会記事  57回-  20  -20  1998/08  [Not refereed][Not invited]
  
• Cell biological analysis and identification of binding proteins of murine ubiquitin protein ligase, E6-AP and Nedd-4.

  畠山鎮次, 豊野孝, WEISSMAN A M, 中山敬一  日本分子生物学会年会プログラム・講演要旨集  20th-  1997
• The murine c-fgr gene product associated with Ly6C and p70 inte @@S gral membrane protein is expressed in cells of a monocyte/macro @@S phage lineage.

  Hatakeyama Shigetsugu, Iwabuchi Kazuya, Ogasaware Kazumasa, Good Robert A. [et al]  Collected papers from the Institute of Immunological Science Hokkaido University  17-  285  -289  1994
• マクロファージ

  畠山鎮次, 小野江和則  アレルギーの領域  1-  (1)  63  -71  1994  [Not refereed][Not invited]
  
• SEQUENTIAL-ANALYSIS OF DISTRIBUTIONS OF DONOR-DERIVED THYMOCYTES BEARING T-CELL ANTIGEN RECEPTOR (TCR) AND DONOR-DERIVED IA+ CELLS IN THYMUSES OF FULLY ALLOGENEIC BONE-MARROW CHIMERA IN MICE

  S HATAKEYAMA, K OGASAWARA, N FUKUSHI, C IWABUCHI, K IWABUCHI, BY WANG, M KAJIWARA, RA GOOD, K ONOE  ACTA PATHOLOGICA JAPONICA  40-  (6)  391  -401  1990/06  [Not refereed][Not invited]
  
• SEQUENTIAL-ANALYSIS OF THE THYMOCYTE DIFFERENTIATION IN FULLY ALLOGENEIC BONE-MARROW CHIMERA IN MICE .2. FURTHER CHARACTERIZATION OF THE CD4+ OR CD8+ SINGLE POSITIVE THYMOCYTES

  H ARASE, N FUKUSHI, S HATAKEYAMA, K OGASAWARA, K IWABUCHI, C IWABUCHI, NEGISHI, I, RA GOOD, K ONOE  IMMUNOBIOLOGY  180-  (2-3)  167  -183  1990/02  [Not refereed][Not invited]
  
• マウス骨髄キメラにおける胸腺内MHC class II陽性細胞とＴ細胞抗原受容体発現の経時的観察

  畠山鎮次, 荒瀬 尚, 小笠原一誠, 岩渕和也, 勝目千雅子, 小野江和則  臨床免疫  20-  (8)  770  -776  1988  [Not refereed][Not invited]
  

Books etc

• 系統看護学講座 専門基礎分野 人体の構造と機能[2] 生化学

  畠山 鎮次 (Single work)
  医学書院 2019/01
• Medical Biochemistry

  Shigetsugu HATAKEYAMA (Joint translation30 Role of the Liver in Metabolism)
  丸善 2018/12
• Advances in Medicine and Biology. Volume 120

  Masashi Watanabe, Shigetsugu HATAKEYAMA (Joint workUbiquitin-Conjugating Enzymes (E2s))
  Nova Science Publishers 2017
• 次世代がん戦略研究 がん基盤生物学 −革新的シーズ育成に向けて−

  畠山 鎮次 (Contributorユビキチン関連酵素を標的としたがん治療シーズの開発)
  南山堂 2013/10
• 標準生化学

  畠山 鎮次 (Others)
  医学書院 2012/08
• PSA and Prostate Cancer

  Shigetsugu HATAKEYAMA (ContributorMolecular mechanism of PSA expression in prostate cancer cells)
  Nova Science Publishers 2010
• Handbook of Prostate Cancer Cell Research: Growth, Signalling and Survival

  Shigetsugu HATAKEYAMA (ContributorPost-translational regulation of androgen receptor in proliferation of prostate cancer cells)
  Nova Science Publishers 2009
• The Frontiers in Medical Sciences「神経変性疾患のサイエンス」

  畠山 鎮次 (ContributorII. 神経変性の分子細胞生物学、4. ユビキチン・プロテアソームシステム)
  2007/12
• Ubiquitin-proteasome system (Methods in Enzymology)

  Shigetsugu HATAKEYAMA (ContributorMapping of ubiquitination sites on target proteins)
  Academic press 2005

Presentations

• TRIM proteins and diseases  [Invited]

  Shigetsugu Hatakeyama

  ATAGO Respiratory Expert Seminar  2022/06
• TRIMファミリータンパク質による細胞機能制御  [Invited]

  畠山 鎮次

  GU Cancer Forum 2017  2017/07
• TRIMファミリータンパク質が関与する疾患  [Invited]

  畠山 鎮次

  第24回分子皮膚科学フォーラム  2017/04
• Functions of TRIM family proteins in metabolism and carcinogenesis  [Invited]

  Shigetsugu HATAKEYAMA

  International Symposium for New Aspects of the Ubiquitin Research  2016/12
• Pleomorphic modes regulation of diverse biological phenomena mediated by ubiquitin  [Not invited]

  Shigetsugu HATAKEYAMA

  第39回日本分子生物学会年会  2016/12
• TRIMファミリーユビキチンリガーゼによる生体制御機構  [Invited]

  畠山 鎮次

  日本生化学会  2015/12
• TRIMタンパク質の多彩な機能  [Invited]

  畠山 鎮次

  日本生化学会大会  2013/09
• Regulation of cellular functions by TRIM proteins  [Invited]

  Shigetsugu HATAKEYAMA

  The 35th Naito Conference  2013/07
• 免疫と癌を制御するTRIM型ユビキチンリガーゼ  [Invited]

  畠山 鎮次

  第４０回日本臨床免疫学会  2012/09
• TRIM proteins in inflammation and carcinogenesis  [Invited]

  Shigetsugu HATAKEYAMA

  2nd international symposium: Infection-related cancers  2012/05
• 癌化を制御するＴＲＩＭファミリーユビキチンリガーゼ  [Invited]

  畠山 鎮次

  第21回泌尿器科分子・細胞研究会  2012/02
• TRIM proteins and cancers  [Invited]

  Shigetsugu HATAKEYAMA

  第６９回日本癌学会学術総会  2010/09
• ユビキチンシステム関連酵素の多様性と機能  [Invited]

  畠山 鎮次

  第１０回日本蛋白質科学会年会  2010/06
• Ubiquitination of ε-COP by PIRH2 and regulation of the secretion of PSA  [Invited]

  Shigetsugu HATAKEYAMA

  International Symposium on Membrane Traffic  2007/11
• 細胞内適応制御系としてのユビキチン-プロテアソームシステム  [Not invited]

  畠山 鎮次

  第４７回日本適応医学会総会  2007/06
• 神経変性疾患に関与するユビキチンリガーゼ  [Invited]

  畠山 鎮次

  第４７回日本神経学会総会  2006/05
• ユビキチンリガーゼエンジニアリングによるがん治療  [Invited]

  畠山 鎮次

  第３回日本癌学会カンファレンス（動物モデルによる新時代のがん研究 発症機構から治療まで  2006/03
• ユビキチン化による細胞機能制御 –細胞内シグナル・転写・細胞周期・癌-  [Invited]

  畠山 鎮次

  第３３回細胞情報伝達系北海道研究会  2005/11
• ホルモン受容体の制御系としてのユビキチンリガーゼ群  [Invited]

  畠山 鎮次

  第６４回日本癌学会学術集会  2005/09
• 炎症反応に関与するユビキチン化とリン酸化  [Invited]

  畠山 鎮次

  第４２回日本臨床分子医学会学術集会  2005/07

Association Memberships

• THE JAPANESE CANCER ASSOCIATION   日本分子生物学会   日本生化学会   

Research Projects

• TRIM型ユビキチンリガーゼによるタンパク質寿命制御の解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2024/04 -2026/03 

  Author : 畠山 鎮次
• MHC発現調節による抗原提示能の人為的操作技術の開発

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  Date (from‐to) : 2022/06 -2024/03 

  Author : 畠山 鎮次
• Regulation of signal transduction by TRIM family ubiquitin ligases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2024/03 

  Author : 畠山 鎮次

   

  申請者らの研究により、免疫反応やがん化に関する細胞内シグナル伝達における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。特に細胞増殖のみならず、細胞分化過程を制御するシグナル伝達系やオートファジーの制御に、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、最近では細胞内情報伝達系の多くの分野で注目されている。本申請においては、網羅的ノックダウンスクリーニングやユビキチン化に特化したプロテオミクス的手法により、TRIMファミリーユビキチンリガーゼを基軸とした細胞内シグナルネットワークシステムを網羅的に解析する。さらに、TRIMファミリーの細胞の増殖分化および免疫系での機能を解明することで、臨床医学（自己免疫疾患、アレルギー疾患及びがん等）に貢献する知見（疾患特異的バイオマーカーや創薬シーズ等）を得る。 具体的には、全TRIMタンパク質のsiRNA等を使用することで、さまざまな細胞内シグナルを網羅的に解析し、TRIMファミリー全体のシステム構造を解明を進めた。また、各TRIMタンパク質の基質タンパク質を同定するために、TUBEを使用した質量分析解析を進めた。 実際、獲得免疫シグナルに重要となるMHCの発現に関与するTRIMタンパク質を新規同定し、機能解析を進めた。さらに、ヘッジホッグシグナルに重要な1次繊毛に関与するTRIMタンパク質を同定し、機能解析を進めた。今後は、今回の研究成果をもとに、臨床医学（自己免疫疾患、アレルギー疾患及びがん等）に貢献する知見を得ることを進める。
• Physical properties and molecular regulation of TRIM-type ubiquitin ligases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2023/03 

  Author : 畠山 鎮次

   

  「ユビキチン化」はタンパク質分解を制御する翻訳後修飾であり、多くの生命現象（タンパク質分解のみならず酵素の活性なども制御）を支える極めて重要な翻訳後修飾の一つである。TRIMタンパク質ファミリーの多様性と機能：TRIMファミリーはRING型E3ユビキチンリガーゼの一つのファミリー（ヒトでは約７０種）であり、様々な機能（がん、免疫、発生、オートファジーなどの制御）に関与することが報告されている。本研究の目的は、TRIMタンパク質の「タンパク質」としての物理化学的な性質（物性）を解析することで機能との関連性を検討し、将来的には創薬研究への知見を集積することである。TRIMタンパク質の機能を解明するためには、その基質タンパク質を同定することと、およびTRIMタンパク質の構造を決定することが重要となる。 具体的には、ユビキチン化タンパク質を精製することに優れたポリユビキチン鎖に高親和性結合を示す人工タンパク質TUBE（Tandem Ubiquitin Binding Entity）を用いた。本研究で用いるユビキチン関基質の新規同定法において、①FLAGタグ、②ユビキチン高親和性人工タンパク質（TUBE）、③TRIMタンパク質（E3酵素）の3つのドメインを融合した人工タンパク質である精製用プローブを用いた。 本年度は、シグナル伝達・選択的オートファジー・DNA修復・ERAD等に関与すると考えられているTRIMタンパク質を中心に、質量分析器（Hybrid IonTrap-Orbitrap LC-MS）を使用し、ユビキチン化基質タンパク質の同定を試み、複数のTRIMタンパク質に関して、基質タンパク質候補を同定し、各TRIMタンパク質の機能解析を進めた。
• Bassoon proteinopathyの病態解析研究

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  Date (from‐to) : 2020/04 -2023/03 

  Author : 矢部 一郎, 原 太一, 矢口 裕章, 池内 健, 高橋 秀尚, 大塚 稔久, 若林 孝一, 畠山 鎮次

   

  われわれは先行研究において、一部のタウオパチー発症に神経終末アクティブゾーンに存在するbassoon蛋白を翻訳するbassoon(BSN)遺伝子の変化を見出した。そのタウオパチーは３リピート(3R)と４リピート(4R)のタウ蛋白質が蓄積する新しい病態であることも示した。さらに、今までPSPと臨床診断していた患者を対象に遺伝子解析を実施したところ、その約10％にBSN遺伝子変化を同定した。このメカニズムとして分子生物学的手法を用いて、BSN遺伝子変異がタウタンパク質を不溶性分画に移行させる病原性があることを解明した。その結果、われわれの論文を引用し、Bassoon proteinopathyという疾患概念が提唱され、BSNタンパク質やBSN遺伝子と多発性硬化症、ハンチントン病、若年性パーキンソン病などの重要な神経疾患との関連が近年相次いで報告されている（Montenegro-Venegas C, et al. Autophagy 2020, Huang TT, et al. Acta Neuropathol Commun 2020, Hoffmann-Conaway S et al. Elife 2020）。当教室では現在このBSN遺伝子変化のPSPを始めとする神経難病への関与についてさらに検討を進めている。具体的には今回の研究では、①モデル動物と細胞株においてBSN遺伝子変異がタウオパチーを惹起する病態機序と同変異が治療標的となり得る可能性、②過去の研究報告からBSNが病態に関与している可能性がある認知症関連疾患、パーキンソン症候群、多系統萎縮症、多発性硬化症などの多様な神経疾患におけるBSN遺伝子変異関与の可能性、③血漿や髄液検体を用いてBSNタンパク質を測定し、BSNタンパク質量が診断および重症度バイオマーカーになり得る可能性、の3課題を中心に研究を遂行している。
• プロテオーム解析とCROP-seq技術を用いた新規MHC-I遺伝子誘導因子の同定

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  Date (from‐to) : 2020/07 -2022/03 

  Author : 小林 弘一, 畠山 鎮次, 應田 涼太
• TRIM型ユビキチンリガーゼの物性と動作原理の解析

  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)

  Date (from‐to) : 2019/04 -2021/03 

  Author : 畠山 鎮次

   

  ユビキチン化はタンパク質分解を制御する翻訳後修飾であり、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る。TRIMファミリーはRING型E3ユビキチンリガーゼの一つのファミリー（ヒトでは約７０種）であり、様々な機能（がん、免疫、オートファジーなど）に関与することが知られている。本研究目的は、E3ユビキチンリガーゼであるTRIMタンパク質の「タンパク質」としての物理化学的な性質（物性）を解析し、将来的には創薬研究への知見を集積することである。実際に、TRIMタンパク質の機能を解明するために、基質タンパク質とユビキチンデコーダータンパク質を同定することと、およびTRIMタンパク質の構造を 決定することが重要となる。本年度は、シグナル伝達・選択的オートファジー・DNA修復・等に関与すると考えられているTRIMタンパク質を中心に、質量分析器（Hybrid IonTrap-Orbitrap LC-MS）を使用し、ユビキチン化基質タンパク質の同定を試み、複数のTRIMタンパク質に関して、基質タンパク質候補が同定された。 また、クライオ電顕によってTRIM68の構造解析を行ったところ、コイルドコイルドメインで折れ曲がる構造をしていることが判明し、それが酵素活性において何らかの役割を果たしている可能性が示唆された。 さらに、温度変化などのストレスによってもたらされる物性変化で、どのようにユビキチンリガーゼの酵素活性が影響を受けるかを明らかにするために、ストレス関連E3ユビキチンリガーゼであるCHIPを用いて解析したところ、ヒトの体温である37度周辺に至適温度があり、許容範囲が狭いことが判明した。今後、ストレスに反応して機能すべきユビキチン化酵素が、生体において、いかに温度変化等に対応して機能しているかを明らかにすることが重要である。
• A project intended for new diagnostic marker and therapy for mesothelioma based on ubiquitin-proteasome system

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2018/04 -2021/03 

  Author : Tanino Mishie

   

  Malignant mesothelioma(MM) is an aggressive tumor which originates from the mesothelial cells of the serosal tissues. It has been reported to be associated with inhalation exposure to asbestos. MM caused by asbestos exposure, and it will become tumor after 30 to 40 years latency period. MM is treated by multidisciplinary treatment combined with surgical excision, chemotherapy and radiation, but it shows treatment resistance and poor prognosis. A novel treatment method has been desired. In this study, we focused on the role of OTUB1, one of the deubiquitinating enzyme. OTUB1 was highly expressed in MM tissue and cell lines. It regulated TGF-beta/SMAD pathway, followed by their motility and invasion. These findings suggest OTUB1 is one of the candidate protein for new therapy of MM.
• TRIMファミリータンパク質によるユビキチンネットワーク制御の解析

  文部科学省：科学研究費補助金（基盤研究(B)）

  Date (from‐to) : 2018 -2020 

  Author : 畠山 鎮次
• TRIMファミリータンパク質による抗原提示制御

  文部科学省：科学研究費補助金（新学術領域研究）

  Date (from‐to) : 2017 -2018 

  Author : 畠山 鎮次
• 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

  文部科学省：科学研究費補助金（挑戦的研究（萌芽））

  Date (from‐to) : 2017 -2018 

  Author : 畠山 鎮次
• Mechanism of selective substrate recognition by the ubiquitin ligase

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/06 -2017/03 

  Author : Kamura Takumi

   

  Ubiquitin system controls a variety of cellular processes. In this research, we try to clarify the new substrates and function of Cullin-type and TRIM-type E3s. Now we show that, in the Cullin type E3s, Ucc1, SSB4, ASB7 and Prame is related to the regulation of citric acid composition, signal transduction, spindle formation and transcription, respectively. Furthermore, we demonstrate that, in the TRIM-type E3s, TRIM45, TRIM23, TRIM29 and RNF207 controls tumor suppression, fat cell differentiation, DNA repair and glucose metabolism, respectively. Our findings shed the light on the new function of Cullin-type and TRIM-type E3s.
• Regulation of signal transduction by TRIM family proteins

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2015 -2017 

  Author : Hatakeyama Shigetsugu, TAKAHASHI Hidehisa, WATANABE Masashi

   

  Our previous studies have shown the importance of TRIM family ubiquitin ligase in intracellular signal transduction. Especially, we showed that TRIM family ubiquitin ligase regulates signal transduction involved in cell proliferation and differentiation. Therefore, in this application, we identified and analyzed TRIM family ubiquitin ligases which regulates the ubiquitination in various intracellular signalling pathways. Furthermore, we identified and analyzed candidate genes regulating metabolism and immune signal by comprehensive knockdown of TRIM family genes using their siRNA library.
• Molecular role of RNF43 in Wnt signaling and tumorigenesis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2016/03 

  Author : Tsukiyama Tadasuke, Hatakeyama Shigetsugu, Takahashi Hidehisa

   

  Wnt signaling pathways are tightly regulated by ubiquitination of their signal transducers and dysregulation of these pathways promotes the tumorigenesis. It has been reported that an ubiquitin ligase RNF43 plays important role in the Fzd-dependent regulation of Wnt signaling pathways. We recently reported that N-terminal of RNF43 is indespensable to regulate Fzd-dependent suppression of Wnt signaling pathways although C-terminal of RNF43 is able to inhibit noncanonical Wnt signaling in a Fzd-independent but Dvl-dependent fashion. In addition missense mutations found in cancer patients convert the function of RNF43 to the positive regulator of Wnt/βcatenin signaling and establish a positive feedback loop of Wnt/βcatenin signaling to accelerate tumorigenesis. These results have been published in a scientific journal [ Tsukiyama, MCB, 35:2007 (2015)].
• Establishment of gene expression system in Bombyx mori

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013 -2014 

  Author : HATAKEYAMA Shigetsugu

   

  It is important to use animal models in order to analyze the etiology of human diseases in medical researches. We tried to establish gene expression system in Bombyx more to analyze genetic diseases. We established viral expression system using special Bombyx strains, which can be infected by AcNPV. We made AcNPV virus containing a target gene (GFP gene as a model gene), infected the virus and then could express GFP protein in pupae of Bombyx.
• Comprehensive analysis of TRIM type ubiquitin ligases in cell proliferation and differentiation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012 -2014 

  Author : HATAKEYAMA Shigetsugu, TSUKIYAMA Tadasuke, TAKAHASHI Hidehisa

   

  TRIM family ubiquitin ligases are important for regulation of carcinogenesis in several cancers. It has been reported that TRIM family ubiquitin ligases regulate the activity of transcription factors in the process of cell proliferation and differentiation. By using proteomics analysis, we comprehensively analyzed TRIM family ubiquitin ligases which are related to ubiquitination of transcription factors. The analysis clarified the molecular mechanism in carcinogenesis of several cancers, maybe leading to the contribution to clinical applications for diagnosis and therapies.
• New application in treatment of head and neck cancer using genome and proteome analysis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2010 -2012 

  Author : FUKUDA Satoshi, HOMMA Akihiro, HATAKEYAMA Shigetugu

   

  In this study, we could get the genome, proteome and microRNAs profile using the head and neck cancer samples from our institute. Furthermore, salivary grand samples could be added to this study and the molecular which showed difference depending on prognosis were confirmed their expression with the immunohistochemical methods. A lot of data were presented on meetings of American head and neck society, Asian society of head and neck, and other international meetings.We got the tremendous volume of data of head and neck cancer, so we need to continue the data-mining and the examination of protein-protein reaction to apply clinically.
• Regulation of mitochondrial ROS signaling in the cardiovascular stress response

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2012 

  Author : TSUTSUI Hiroyuki, MAKITA Naomasa, KINUGAWA Shintaro, MATSUI Yutaka, ISHIMORI Naoki, HATAKEYAMA Shigetsugu

   

  The role of mitochondria in maintaining the biological function is dynamically regulated by mitochondrial DNA (mtDNA), a closed-circular double-stranded DNA molecule of～16.5 kb. MtDNA decline, mitochondrial defects, and mitochondrial oxidative stress are now well recognized in a variety of diseases such as neurodegenerative diseases, diabetes mellitus, cancer, and even aging. The present study clarified the crucial role of mitochondrial transcription factor A (TFAM) in cardiovascular stress response. With further knowledge on the mechanisms of TFAM for maintenance of mtDNA copy number and mitochondrial function, it may eventually be possible to develop novel strategies for the treatment ofcardiovascular diseases including heart failure.
• Disease models using gene expression system in Bombyx mori

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011 -2011 

  Author : HATAKEYAMA Shigetsugu

   

  We tried to establish gene expression system in Bombyx mori in order to use Bombyx as a model for analyzing human disease. We used specific Bombyx strains which can be infected by AcNPV. After AcNPV containing a target gene (GFP gene) was infected in this specific Bombyx strains, GFP was transiently expressed in Bombyx.
• Comprehensive analysis of the regulation of carcinogenesis through TRIM type ubiquitin ligases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2011 

  Author : HATAKEYAMA Shigetsugu, OKUMURA Fumihiko, TSUKIYAMA Tadasuke

   

  Ubiquitination is one of the posttranslational modifications used by eukaryotic cells, and the ubiquitin-mediated proteolytic pathway plays a crucial role in the elimination of short-lived regulatory proteins. Members of the family of tripartite motif (TRIM)-containing proteins are idefined as E3 ubiquitin ligases. There are now more than 70 known TRIM proteins in humans and mice. In this study, we analyzed the relationship between TRIM proteins and cancinogenesis.
• Analysis of diversity of ubiquitin ligase

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(特定領域研究)

  Date (from‐to) : 2006 -2010 

  Author : Kamura TAKUMI, Hatakeyama SHIGETSUGU

   

  Ubiquitylation and subsequent proteasomal degradation of regulatory proteins control a variety of cellular processes. The ubiquitin conjugation to target proteins is processed by three enzymes, E1, E2, and E3. The E3s are responsible for recognizing and recruiting target proteins for polyubiquitylation. Cullin-based E3 complexes and TRIM E3 families are thought to regulate many cellular events. In this research, we try to clarify the function of Cullin-based E3 complexes and TRIM E3 families. Now we show that Cullin-based E3 complexes are responsible for regulating the function of Mrc1, Ctf...
• New approach for diagnosis and treatment of head and neck cancer by proteomics analysis

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2007 -2009 

  Author : Satoshi FUKUDA, Akihiro HOMMA, Shigetugu HATAKEYAMA, Hiroki SHIRATO

   

  We analyzed the function of Ubiquitin in head and neck cancer. We considered TRIM32 and UBE2Q2 as a important factor to control the treatment of advanced head and neck Cancer.
• 性ホルモン受容体のユビキチン化介在性分解による癌細胞増殖制御機構

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2006 -2007 

  Author : 畠山 鎮次

   

  多くの細胞質・核質に存在するタンパク質の分解にユビキチンープロテアソーム系が関与している。この分解系の特徴は、基質特異性が高く、分解速度が速いことである。この特徴を利用して、転写因子や細胞シグナル伝達や癌遺伝子産物や癌抑制遺伝子産物は、自分自身のタンパク質としての発現量を調節している。性ホルモン受容体も最近ユビキチン化により発現量(分解量)が制御させていることが報告されている。特にエストロゲン受容体α(ERα)やアンドロゲン受容体(AR)などのホルモン受容体は、リガンドと複合体を形成して、転写因子として標的遺伝子の転写を活性化し、生殖器の発達やホルモン依存性癌(乳癌や子宮体癌など)の腫瘍形成に影響を与えることが知られている。本研究計画では、ホルモン依存性癌において重要分子であるホルモン受容体(男性ホルモンと女性ホルモン)に対して、そのユビキチン化の分子論的機序を解明することを目的とする。我々はERαを特異的にユビキチン化するユビキチンガーゼE3、TRIM25/EFPを新たに同定し、生化学的及び細胞生物学的解析を行った。TRIM25はERαと直接結合し、in vitroユビキチン化アッセイにおいてERαをユビキチン化することが確認された。また細胞内にTRIM25を過剰発現させると、ERαのユビキチン化を促進することが判明した。また、各進行度の子宮体部癌の病理検体に対して、ウエス...
• U-ボックスタンパク質による神経変性疾患関連タンパク質の分解制御

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2006 -2007 

  Author : 畠山 鎮次

   

  研究成果報告書我々はU-ボックス型ユビキチンリガーゼ(CHIPやE4Bなど)を同定し、ユビキチン化におけるタンパク質品質管理における重要性を検討した。U-ボックスタンパク質のひとつであるE4Bが、、脊髄小脳変性症3型(マシャドージョセフ病)の原因遺伝子犀物であるMJD1のユビキチン化に関与し、実験レベルで本疾患の発症を抑制させる活性があることを証明した。また、軸索変性異常マウスであるWld^sマウスにおいて発現しているE4B遺伝子とNmnat1遺伝子の融合遺伝子産物(Wld^sタンパク質)の軸索変性における機能責任部位を明らかにした。さらに今回、E4Bの結合タンパク質として我々が以前に同定した分子シャペロンp97/VCPの関連タンパク質を検索したところ、新たにRINGフィンガードメインを有するユビキチンリガーゼTRIM21/Ro52を同定した。そこで、TRIM21/Ro52の基質タンパク質候補を検索したところ、B細胞においてIgG1(IgG2、IgG4)と相互作用することが判明した。つまり、p97/VCPと協調して機能するユビキチンリガーゼとしてTRIM21/Ro52はB細胞(形質細胞)における抗体分子(IgG1_1など)の品質管理に関与することを判明した。また現在、神経組織においてTRIM21/Ro52と相互作用する分子(神経細胞死を制御する分子候補)を同定している。本研究...
• Pirh2ユビキチンリガーゼによる膜輸送制御

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2006 -2007 

  Author : 畠山 鎮次

   

  研究成果報告書多くの細胞質・核質に存在するタンパク質の分解にユビキチン・プロテアソーム系が関与している。そして、標的タンパク質のユビキチン化に必要な酵素群の中で、特にユビキチンリガーゼE3は標的タンパク質を認識し、最終的にユビキチンを付加する重要な酵素サブユニットである。最近になり、ユビキチン化タンパク質修飾が分解のためのシグナルだけではなく膜輸送(メンブラントラフィック)に重要な機能を有することが報告されている。申請者はPirh2(p53をユビキチン化するE3)が、細胞内膜輸送に関与するε-COPと相互作用することを見出しており、本申請課題においては、Pirh2による細胞内膜輸送関連タンパク質のユビキチン化の機能を解明することを目的とする。in vivo結合実験を行なったところ、PIRH2とε-COPの共沈が認められた。さらにPIRH2がε-COPをユビキチン化するかを確認するためにin vitroユビキチン化アッセイを行なったところ、PIRH2を添加時にのみ、ε-COPのポリユビキチン化が認められた。さらに細胞内でε-COPのユビキチン化が起こっているかを確認するためにin vivoユビキチン化アッセイを行なったところ、PIRH2の共発現によりε-COPのユビキチン化の増強が認められた。これらの結果により、PIRH2の過剰発現がε-COPのユビキチン化を引き起こすことが判...
• Comprehensive analysis of TRIM family ubiquitin ligases

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2006 -2007 

  Author : Shigetsugu HATAKEYAMA, 築山 忠維

   

  Ubiquitination is a versatile post-translational modification mechanism used by eukaryotic cells mainly to control protein levels through proteasome-mediated proteolysis. Ubiquitin conjugation is achieved by several enzymes that act in concert, a ubiquitin-protein ligase (E3). E3 is thought to be the component of the ubiquitin conjugation system that is most directly responsible for substrate recognition. Enzymes belonging to class E3 that have so far been identified include members of the HECT (homologous to E6-AP carboxyl terminus), RING-finger and U-box families of proteins. So far; we h...
• ハイブリッド型ユビキチン化酵素による神経変性疾患の治療

  文部科学省：科学研究費補助金(萌芽研究)

  Date (from‐to) : 2006 -2007 

  Author : 畠山 鎮次, 築山 忠維

   

  多くの細胞質・核質に存在するタンパク質の分解に関わっているのはユビキチン-プロテアソーム系である。この分解系の特徴は、基質特異性が高く、分解速度が速いことである。分解されるべきタンパク質はユビキチンが鎖状に結合され(ポリユビキチン鎖)、タンパク質分解装置であるプロテアソームがポリユビキチン鎖を認識して標的タンパク質ごと分解する。標的タンパク質のユビキチン化に必要な酵素群の中で、特にユビキチンリガーゼは標的タンパク質を認識し、最終的にユビキチンを付加する重要な因子である。本研究課題においては、各ポリグルタミン病関連タンパク質(ハンチンティン、Atrophin-1、Ataxin(ATX)-1,-2,-3,-17等)には特異的に結合するタンパク質が同定されているので、この結合タンパク質とユビキチンリガーゼであるU-ボックスタンパク質のキメラ酵素(人工ハイブリッドユビキチンリガーゼ)を作製し、ポリグルタミン含有タンパク質が人工ハイブリッドユビキチンリガーゼによってユビキチン化を受け、プロテアソームによって分解される系を構築する。さらに実際の遺伝子治療に応用できるかどうかを検討するために、ウイルスベクターを利用して細胞やマウスへの導入実験を行い、神経機能異常に対する効果を判定する。特に、ATX-3/MJD1とVCP/p97の結合に注目し、VCPにおける最小結合領域を同定しているところで...
• 分子シャペロン関連ユビキチンリガーゼによる異常構造タンパク質の分解制御

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2005 -2006 

  Author : 畠山 鎮次

   

  神経細胞内封入体が各種の神経変性疾患の組織病理学的所見として多く報告されており、封入体は異常構造をとったタンパク質であることが報告されている。病理組織学的検索によりこれらの封入体を構成するタンパク質の多くがユビキチン化というタンパク質分解のための修飾が起きていることが知られている。本研究では、封入体構成タンパク質(ポリグルタミンタンパク質、タウ、α-シヌクレインなど)の安定性を調節するメカニズムとして分子シャペロン系とユビキチン-プロテアソーム系がどのような役割を果たしているのかを検討する。特に、封入体構成タンパク質をユビキチン化させる酵素系であるU-ポックスタンパク質との関係を生化学的に明らかにすることを目的とする。我々はヒト及びマウスからU-ボックスドメインを含むタンパク質のcDNAをクローニングし、そのE3活性を証明した(U-ボックス型E3)。ほとんどのU-ボックスタンパク質は分子シャペロンとの関係が認められ、U-ボックス型E3はシャペロン依存型E3である可能性が高い。特に哺乳類のUfd2ホモログであるUFD2aが、ポリグルタミン病のひとつであるマシャド-ジョセブ病の原因遺伝子産物である、神経細胞内に蓄積されるMJD1タンパク質を認識し分解できることを見いだした。また、CHIPが、アルツハイマー病関連タンパク質であるタウや筋萎縮性側索硬化症に関連するSOD1のユビキチン...
• 性ホルモン受容体のユヒキチン化介在性分解による癌細胞増殖制御機構

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2005 -2005 

  Author : 畠山 鎮次

   

  多くの細胞質・核質に存在するタンパク質の分解にユビキチン-プロテアソーム系が関与している。この分解系の特徴は、基質特異性が高く、分解速度が速いことである。この特徴を利用して、転写因子や細胞シグナル伝達や癌遺伝子産物や癌抑制遺伝子産物は、自分自身のタンパク質としての発現量を調節している。性ホルモン受容体も最近ユビキチン化により発現量(分解量)が制御させていることが報告されている。特にエストロゲン受容体α(ERα)やアンドロゲン受容体(AR)などのホルモン受容体は、リガンドと複合体を形成して、転写因子として標的遺伝子の転写を活性化し、生殖器の発達やホルモン依存性癌(乳癌や子宮体癌など)の腫瘍形成に影響を与えることが知られている。本研究計画では、ホルモン依存性癌において重要分子であるホルモン受容体(男性ホルモンと女性ホルモン)に対して、そのユビキチン化の分論的機序を解明することを目的とする。我々はERαを特異的にユビキチン化するユビキチンガーゼE3、ERα-associated Ring-_finger protein(EARF)を新たに同定し、生化学的及び細胞生物学的解析を行った。EARFはERαと直接結合し、in vitroユビキチン化アッセイにおいてERαをユビキチン化することが確認された。また細胞内にEARFを過剰発現させると、ERαのユビキチン化を促進することが判明した。...
• U-ボックスタンパク質による神経変性疾患関連タンパク質の分解制御

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2005 -2005 

  Author : 畠山 鎮次

   

  神経細胞内封入体が各種の神経変性疾患の組織病理学的所見として多く報告されており、封入体は異常構造をとったタンパク質であることが報告されている。病理組織学的検索によりこれらの封入体を構成するタンパク質の多くがユビキチン化というタンパク質分解のための修飾が起きていることが知られている。本研究では、封入体構成タンパク質(ポリグルタミンタンパク質、タウ、α-シヌクレインなど)の安定性を調節するメカニズムとして分子シャペロン系とユビキチン-プロテアソーム系がどのような役割を果たしているのかを検討する。特に、封入体構成タンパク質をユビキチン化させる酵素系であるU-ボックスタンパク質との関係を生化学的に明らかにすることを目的とする。我々はヒト及びマウスからU-ボックスドメインを含むタンパク質のcDNAをクローニングし、そのE3活性を証明した(U-ボックス型E3)。ほとんどのU-ボックスタンパク質は分子シャペロンとの関係が認められ、U-ボックス型E3はシャペロン依存型E3である可能性が高い。異常蛋白の蓄積は対応するために、正確な蛋白フォールディングを起こすためには分子シャペロンが、一方、異常タンパク質を分解する場合はU-ボックス型E3が関与している可能性を示した。機能レベルでは、哺乳類のUfd2ホモログであるUFD2aが、ポリグルタミン病のひとつであるマシャド-ジョセフ病の原因遺伝子産物であ...
• Comprehensive analysis of molecules related for antigen receptor signaling

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2004 -2005 

  Author : Shigetsugu HATAKEYAMA, 中山 敬一, 嘉村 巧

   

  This application is to analyze comprehensively about post-translational modification regarding to phosphorylation and ubiquitylation using affinity-chromatography. The system we analyzed is signal transduction system of antigen receptor signals in immune cells, especially phosphorylation and ubiquitylation. The purpose of this project is to clarify the down-stream signal transduction molecules comprehensively from the point of protein modifications.Ubiquitylation and phosphorylation are implicated in the many intracellular phenomenons including signal transduction such as TCR,BCR,FcR signal...
• 細胞内タンパク質の特異的分解制御による癌に対する遺伝子治療法の確立

  文部科学省：科学研究費補助金(萌芽研究)

  Date (from‐to) : 2004 -2005 

  Author : 畠山 鎮次, 中山 敬一, 嘉村 巧

   

  多くの細胞質・核質に存在するタンパク質の分解に関わっているのはユビキチン-プロテアソーム系である。この分解系の特徴は、基質特異性が高く、分解速度が速いことである。分解されるべきタンパク質はユビキチンが鎖状に結合され(ポリユビキチン鎖)、タンパク質分解装置であるプロテアソームがポリユビキチン鎖を認識して標的タンパク質ごと分解する。標的タンパク質のユビキチン化に必要な酵素群の中で、特にユビキチンリガーゼは標的タンパク質を認識し、最終的にユビキチンを付加する重要な因子である。正常細胞に比べ癌細胞ではいくつかのタンパク質においてその発現の異常が報告されている。癌細胞において、癌遺伝子発現の増加(もしくは安定化)、もしくは癌抑制遺伝子産物の発現低下(もしくは不安定化)が認められることがある。例えば、Myc等の癌遺伝子においては遺伝子増幅が起こり、その発現の増加が認められる。Mycに特異的に結合する分子Maxが存在するので、このMaxとユビキチンリガーゼであるU-ボックスタンパク質のキメラ分子(Max-U)を作製し、MycがMax-Uによってユビキチン化を受け、プロテアソームによって分解される系を構築した。Max-Uにより、Mycは特異的に認識され、ユビキチン化を受けることがin vitroユビキチン化アッセイにより判明した。また、Max-Uを細胞内に発現させると、Mycの分解速度が速く...
• Analysis of degradation of cell cycle regulators Kip family

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2004 -2005 

  Author : Takumi KAMURA, 中山 敬一, 畠山 鎮次

   

  The cyclin-dependent kinase (CDK) inhibitor p27 is degraded at the G(0)-G(1) transition of the cell cycle by the ubiquitin-proteasome pathway in a Skp2-independent manner. We recently identified a novel ubiquitin ligase, KPC (Kip1 ubiquitylation-promoting complex), consisting of KPC1 and KPC2, which regulates the ubiquitin-dependent degradation of p27 at G(1) phase. We have now investigated the structural requirements for the interactions of KPC1 with KPC2 and p27. The NH(2)-terminal region of KPC1 was found to be responsible for binding to KPC2 and to p27. KPC1 mutants that lack this regio...
• 異常タンパク質蓄積による神経変性疾患発症の分子機構の解明

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2004 -2004 

  Author : 嘉村 巧, 中山 敬一, 畠山 鎮次

   

  ユビキチン・プロテアソーム系は基質特異的なタンパク質分解機構であり、基質に特異的にユビキチンを多数結合させる反応(ポリユビキチン化)は、ユビキチン結合酵素(E1)、ユビキチン活性化酵素(E2)、ユビキチンリガーゼ(E3)の複合酵素系によって行われると考えられてきた。さらに、最近になって基質の種類によっては、E1-E2-E3に加えてユビキチン鎖伸長因子(E4)を必要とするポリユビキチン化反応があることが報告された。われわれは、出芽酵母で同定された、ユビキチン鎖伸長因子(E4)であるUfd2の哺乳類ホモログE4B/UFD2aを同定した。E4BはそのC末端にU-ボックスドメインを有す。E4Bは、成体マウスでは主に大脳、小脳などの神経組織に発現が高く認められ、またポリグルタミン病の原因遺伝子産物(MJD1)のユビキチン化にE4として関わっており、神経組織におけるユビキチンシステムとの関連が示唆された。哺乳類個体におけるE4Bの分子機能を解析するためにE4Bノックアウト(-/-)マウスを作製したところ、E4B-/-マウスは胎生12〜13日で、頭部、頸部、心臓、腹部などに血管の拡張や出血を認め、致死に至った。また、心筋組織には広汎なアポトーシスが認められ、心臓の発生、成熟過程に異常が認められた。プロテオーム解析では、E4B-/-マウスにおいて脱ユビキチン化酵素であるUCHL1の分子量に変...
• 人工ハイブリット型ユビキチン連結酵素による癌治療法の開発

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2004 -2004 

  Author : 嘉村 巧, 畠山 鎮次

   

  癌遺伝子産物の発現量を、そのタンパク質分解を促進することによって低下させるシステムを構築し、臨床応用の可能性を検討することが本研究の目的である。最近、われわれはU-box型ユビキチンリガーゼを同定し報告した。このU-box型ユビキチンリガーゼはSCF型ユビキチンリガーゼとは異なり、単体よりなりその単一分子中に基質と結合する領域および基質にユビキチンを付加する領域(U-box)の両方を持っているのが特徴である。よってハイブリットU-boxタンパク質は、より効率よく基質をユビキチン化し分解に導く可能性がある。われわれは、U-box型ユビキチンリガーゼのU-boxタンパク質と癌遺伝子産物(Myc)と結合することが知られているタンパク質(Max)とのハイブリッドU-boxタンパク質(U-box/Maxハイブリッドタンパク質)を作製し、培養細胞を用いてMycに対する影響を検討した。その結果としてこの人工ハイブリッド型ユビキチンリガーゼによってMycのユビキチン化が促進され、その半減期が短縮されることを確認した。また培養細胞にこの人工ハイブリッド型ユビキチンリガーゼとMycを共同発現させることによりMyc誘導性のコロニー形成能が阻害された。さらにはこの人工ハイブリッド型ユビキチンリガーゼとMycを共同発現する細胞をヌードマウスに移植したところ明らかに造腫瘍能の低下を認められた。この方法が...
• プロテアソームへのユビキチン化基質のターゲッテング機構の解析

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2003 -2004 

  Author : 嘉村 巧, 中山 啓子, 中山 敬一, 畠山 鎮次

   

  ユビキチン・プロテアソーム系を介したタンパク質分解は細胞周期・シグナル伝達・脳変性疾患等に中心的な役割を果たしている。タンパク質へのユビキチン化反応には、E1,E2,E3の酵素群が関与し、この分野に関する研究は急速に進んでいるが、その後のステップであるポリユビキチン化タンパク質のプロテアソームへのターゲッテング機構に関してはほとんど明らかになっていない。最近、我々は生化学的手法を用いて、KPC1(C末側にRINGフィンガー配列を持つ)とKPC2(N末側にUBL配列、C末側に2つのUBA配列を持つ)の2量体からなる複合体を、CDKインヒビターp27Kip1に対する新たなE3として細胞抽出液より分離・精製した。近年UBLおよびUBA配列をもつタンパク質の機能解析が盛んに行われているが、我々が同定したKPC2に関する報告はいまだなされていない。そこで本研究ではKPC2のユビキチン・プロテアソーム系に対する影響を生化学的、細胞生物学的方法を用いて解析することを目的とする。試験管内および細胞内でKPC2がプロテアソームおよびユビキチンと結合することを確認している。これらの結合にはそれぞれKPC2のUBL及びUBAドメインが必要であった。また試験管内でのp27のユビキチン化反応に対してKPC2のSTI1ドメインが必要であることを明らかにした。さらにRNA干渉法をもちいて細胞内でp27の分...
• Analysis of the PKC-δ Signaling Pathway by Proteomics with Embryonic and Genetic Engeneering

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2003 -2004 

  Author : Keiichi NAKAYAMA, 谷内 一郎, 嘉村 巧, 畠山 鎮次, 中山 啓子

   

  The accumulation of genome sequence data has facilitated the establishment of new approaches to systematic characterization of gene expression profiles at the mRNA level (transcriptome). Such strategies do not, however, necessarily provide direct information about the profile of protein expression (proteome), given that the abundance of a given mRNA does not necessarily correlate with that of the encoded protein. Furthermore, numerous characteristics of proteins, including their subcellular localization, interactions with other molecules, stability, and posttranslational modification, are a...
• Study of molecular mechanisms of CD4 silencing and role s of Runx proteins during lymphocyte development

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2003 -2004 

  Author : Ichiro TANIUCHI, 畠山 鎮次, 嘉村 巧, 中山 敬一, 中山 啓子

   

  The aim of this research is to understand the molecular mechanisms of CD4 silencing and function of Runx transcriptional factors during lymphocyte development. Because a main experimental approach was in vivo analyses of gene manipulated mice, we have generated several mutant mouse strains including gene targeted mice and transgenic mice. By using gene targeting in Es cells and Cre-loxP system, we have generated flox mutant allele for Runx1,Runx3 or CBFβ gene for conditional inactivation by induction of Cre recombinase by tissue and stage specific manner. We have also generated Runx3 transg...
• シグナル伝達を調節するSCFリガーゼ

  文部科学省：科学研究費補助金(特定領域研究(B), 特定領域研究)

  Date (from‐to) : 2000 -2004 

  Author : 小南 欽一郎, 畠山 鎮次, 畠山 鎮次

   

  SCF複合体の既知の構成分子Skp1、Cul1、Rbx1、F-boxタンパク質等にTAPタグをつけ、TAPシステムによって会合因子を単離する実験系を確立し、マイクロシークエンサー及び質量分析器でそのタンパク質配列を明らかにした。そのなかには転写調節に重要であるTIP120AがSCF複合体構成成分と結合していることが判明した。またSCF^の機能解析を目的に、Fbw7ノックアウトマウスを作製したところ、心臓奇形等で胎生致死となり、Fbw7が発生学上Notch系のシグナルに関与することを明らかにした。また、Fbw7がリン酸化依存性に癌遺伝子であるMycのユビキチン化及び分解の制御に関与することを報告した。さまざまなタンパク質合成および修飾ミスにより細胞質で不溶化するタンパク質が存在するが、分子シャペロン等が異常たんぱく質の修復に関与することが報告されている。申請者はもうひとつの異常タンパク質処理メカニズムとしてユビキチン-プロテアソーム系に注目した。つまり、物性が正常にとれないタンパク質をユビキチン化し分解するユビキチンリガーゼの存在を明らかにすることを目的として研究を遂行した。申請者は実際に、タンパク質の品質管理に関する酵素として新たなユビキチンリガーゼ(U-ボックスタンパク質群)を同定し、疾患との関係を解析している。その基質タンパク質や調節タンパク質を同定するために...
• Investigation into the molecular mechanisms underlying proteolysis of CDK inhibitor p27

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(特定領域研究(C), 特定領域研究)

  Date (from‐to) : 2000 -2004 

  Author : Keiichi NAKAYAMA, 畠山 鎮次, 北川 雅敏

   

  We have studied the mechamism underlying the regulation of the abundance of the CDK inhibitor p27. p27 is degraded at the GO-G1 transition, and its abundance remains low during cell proliferation. The SCF/Skp2 ubiquitin ligase complex has been thought to play a critical role in p27 degradation. However, analysis of Skp2-deficient mice revealed that degradation of p27 at the GO-G1 transition proceeds normally in Skp2-/-cells, whereas p27 proteolysis during S-G2 phases is impaired in these Skp2-deficient cells. These results suggested the presence of Skp2-independent pathway to control ubiqui...
• 異常タンパク質蓄積による神経変性疾患発症の分子機構の解明

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2003 -2003 

  Author : 嘉村 巧, 谷内 一郎, 畠山 鎮次

   

  ポリグルタミン病と呼ばれる神経変性疾患群は、原因遺伝子上でトリプレット・リピートが異常に伸長することによってタンパク質内に異常ポリグルタミン領域を生じ、この異常タンパク質が神経細胞内封入体に蓄積し、最終的に変性、脱落することによって発症することが知られている。これら封入体成分は高度にユビキチン化されていることより、ユビキチン・プロテアソーム系によるタンパク質分解が、ポリグルタミン病の病態へ関与していることが示唆されている。我々はポリグルタミン病の病因解明の手がかりとしてMachado-Joseph病の原因遺伝子産物MJD1に対するE3ユビキチンリガーゼを生化学的手法で精製しているが、その過程でMJD1と結合するタンパク質VCPと、VCPに結合してMJD1のユビキチン化鎖の伸長を誘導する酵素UFD2を発見した。そこで本研究では、VCP-UFD2複合体の神経細胞における異常MJD1の分解についての検討を行うと同時に、異常MJD1を発現させたモデル動物に対してVCPとUFD2を過剰発現させ、病理学的・神経学的な変化を解析する。すでにUFD2を過剰発現することによりMJDの分解を促進できることを確認している。そして現在MJDトランスジェニックマウスおよびUFD2ノックアウトマウスおよびタランスジェニックマウスの作製をすすめているところである。個体レベルにおいてもUFD2を神経細胞内に...
• 組織特異的Runx不活性化による発がんモデルマウスの作製と発がんの分子機構の解明

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2003 -2003 

  Author : 谷内 一郎, 畠山 鎮次, 嘉村 巧

   

  Runxファミリーの発がんにおける役割をマウス個体内で検討する目的に、Runx遺伝子の組織特異的不活性化マウスの作製を行っている。本年度は既に作製したRunx1コンヂィショナルノックアウトマウスを血液幹細胞でCreを発現するFes-Creマウスと交配した。その結果、末梢血球系細胞では一部の細胞でのみRunx1の不活性化が見られ、全ての幹細胞でRunx1の不活性化が起きていない事が考えられた。また、数カ月の観察では、著名ながん化は見られず、長期観察が必要と考えられた。次に、その変異が固形がん、特に胃がんの発生に関与すると考えられているRunx3遺伝子に関して、Runx3を組織特異的に不活性化する事の出来るRunx3コンヂィショナルノックアウトマウスを作製した。Runx3の場合、ES細胞に変異を導入した際に使用したneo遺伝子を除去したESクローンからは生殖系列を介し変異が伝わらなかったので、生殖系列でCreを発現するトランスジェニックマウスを用いneo遺伝子カセットマウス交配により除去する方法で、Runx3コンヂィショナルノックアウトマウスを作製した。次に、Runxファミリーの共通のβユニットであるCbfβをコードするCbfb遺伝子に関してもコンヂィショナルノックアウトマウスの作製を行った。現在、ES細胞のクローニングは終了し、ES細胞のインジェクションを行っている所である。ま...
• 人工ハイブリット型ユビキチン連結酵素による癌治療法の開発

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2003 -2003 

  Author : 嘉村 巧, 畠山 鎮次

   

  癌遺伝子産物の発現量を蛋白分解を促進することによって低下させるシステムを構築し、その臨床応用の可能性を検討することが本研究の目的である。そこで本研究においてはSCF型ユビキチン連結酵素の基質認識コンポーネントであるF-box蛋白と、癌遺伝子産物(ここでは代表例として活性化Ras)と結合することが知られている蛋白(Sos)とのハイブリッドF-box蛋白(F-box/Sosハイブリッド蛋白)を作成し、この人工ハイブリッド型ユビキチン連結酵素によってRasの分解に対する影響を検討している。現在までにこのF-box/Sosハイブリッドタンパク質が試験管内および細胞内で実際にSkp1やRasと結合することを確認している。また昆虫細胞で発現・精製したF-box/Sosハイブリッドタンパク質、Skp1、Cul1およびRbx1からなるE3ユビキチンリガーゼ複合体がE1、E2、およびユビキチンの存在下でRasのユビキチン化反応を再構成できることも確認している。そして活性化Rasの過剰発現によるトランスフォームに対する影響を検討しようとしているところである。転写因子などの調節蛋白質の多くはユビキチンプロテアゾーム系による蛋白分解によってその発現量が調節されているが、このユビキチン化の特徴は厳密な基質特異性が認められることである。そこで、この特異性の高いシステムを癌遺伝子産物に適応すれば、過剰蛋白...
• マウスGemininの発生工学的手法を用いた機能解析

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2002 -2003 

  Author : 中山 啓子, 嘉村 巧, 畠山 鎮次

   

  Gemininは、mitosisの間にだけに分解される分子を網羅的にスクリーングすることによつて発見された分子である。G1期には全く存在しないが、S期からG2期、M期の開始にかけて蓄積し、有糸分裂の中期(metaphase)から後期(anaphase)への移行期に急速に消失する。このタンパク量の急激な変化はAPC/Cによるユビキチン化によって分解を受けるためである。また、Gemininを過剰発現するとDNA複製が抑制されることから、DNA複製のライセンシングに関与する分子であると考えられてきた。このように細胞周期の進行に強く関わっていると考えられる分子Gemininのノックアウトマウスを作製することによって、そのin vivoでの生物学的役割を検討することが本研究の目的である。Geminin遺伝子をクローニングし遺伝子構造を明らかにした後、Cdt1結合領域をコードするエクソンをネマイシン耐性遺伝子に置換するターゲティングベクターを作製し、定法に則りノックアウトマウスを作製したところ、胎生早期に致死であることが確認された。現在までの解析では、ノックアウト胚は胎生7.5日には観察されていない。このような胎生初期の死亡からも細胞の正常な分裂に必須の分子であることが予想される。一方、2004年には、Gemininは、Polycomb遺伝子との相互作用によって、Hox遺伝子を制御するこ...
• 人工ハイブリット型ユビキチン連結酵素による癌治療法の開発

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2002 -2002 

  Author : 嘉村 巧, 畠山 鎮次

   

  癌遺伝子産物の発現量を蛋白分解を促進することによって低下させるシステムを構築し、その臨床応用の可能性を検討することが本研究の目的である。そこで本研究においてはSCF型ユビキチン連結酵素の基質認識コンポーネントであるF-box蛋白と、癌遺伝子産物(ここでは代表例として活性化Ras)と結合することが知られている蛋白(Sos)とのハイブリッドF-box蛋白(F-box/Sosハイブリッド蛋白)を作成し、この人工ハイブリッド型ユビキチン連結酵素によってRasの分解に対する影響を検討している。現在までにこのF-box/Sosハイブリッドタンパク質が試験管内および細胞内で実際にSkp1やRasと結合することを確認している。また昆虫細胞で発現・精製したF-box/Sosハイブリッドタンパク質、Skp1、Cu11およびRbx1からなるE3ユビキチンリガーゼ複合体がE1、E2、およびユビキチンの存在下でRasのユビキチン化反応を再構成できることも確認している。そして活性化Rasの過剰発現によるトランスフォームに対する影響を検討しているところである。転写因子などの調節蛋白質の多くはユビキチンプロテアゾーム系による蛋白分解によってその発現量が調節されているが、このユビキチン化の特徴は厳密な基質特異性が認められることである。そこで、この特異性の高いシステムを癌遺伝子産物に適応すれば、過剰蛋白のみを除...
• プロテアソームへのユビキチン化基質のターゲッテング機構の解析

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2002 -2002 

  Author : 嘉村 巧, 中山 敬一, 中山 啓子, 畠山 鎮次

   

  ユビキチン・プロテアソーム系を介したタンパク質分解は細胞周期・シグナル伝達・脳変性疾患等に中心的な役割を果たしている。タンパク質へのユビキチン化反応には、E1,E2,E3の酵素群が関与し、この分野に関する研究は急速に進んでいるが、その後のステップであるポリユビキチン化タンパク質のプロテアソームへのターゲッテング機構に関してはほとんど明らかになっていない。最近、我々は生化学的手法を用いて、p140 (C末側にRINGフィンガー配列を持つ)とp50 (N末側にUBL配列、C末側に2つのUBA配列を持つ)の2量体からなる複合体を、CDKインヒビターp27Kip1に対する新たなE3として細胞抽出液より分離・精製した。近年UBLおよびUBA配列をもつタンパク質の機能解析が盛んに行われているが、我々が同定したp50に関する報告はいまだなされていない。そこで本研究ではp50のユビキチン・プロテアソーム系に対する影響を生化学的、細胞生物学的方法を用いて解析することを目的とする。現在までに、試験管内および細胞内でp50がプロテアソームおよびユビキチンと結合することを確認している。また試験管内でのp27のユビキチン化反応に対してp50が抑制的に作用することも確認している。そしてp27の分解に対する影響をp50の過剰発現あるいはp50に対するRNA干渉法をもちいて検討中である。さらにはp50のノッ...
• Establishment of therapeutic method for neurodegenerative diseases by specific protein degradation system for abnormal proteinsEstablishment of therapeutic method for neurodegenerative diseases by specific protein degradation system for abnormal proteins

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2001 -2002 

  Author : Shigetsugu HATAKEYAMA, 山下 順範, 中山 啓子, 中山 敬一, 山下 順範囲

   

  Intracellular protein degradation is involved in the regulation of biologically important phenomenon including cell cycle, transcriptional regulation and signal transduction. Ubiquitylation is mediated by a multienzyme cascade and involves the formation by ubiquitin of thiol esters with at least two, and, in most instances, three, distinct types of enzyme: an E1, an E2, and an E3.Then Polyubiquitylated proteins are recognized and degraded by proteasomes. In this cascade, E3 enzymes are thought to determine the substrate specificity of ubiquitylation and have been classified into two familie...
• New clearance system of abnormal protein in Polyglutammine disease

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2001 -2002 

  Author : Keiichi NAKAYAMA, 中山 啓子, 畠山 鎮次

   

  Machado-Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3) is neurodegenerative disease which is caused by polyglutamine expansion in a responsible gene product, MJD1/Ataxin3. MJD1 has now been shown to undergo ubiquitylation and degradation by proteasome-dependent pathway. MJD1 with expanded polygiutamine tract was more resistant to degradation than normal MJD1. We established an in vitro system of ubiquitylation of MJD1, thereby biochemically purified activity to mediate polyubiquitylation of MJD1 from rabbit reticulocyte lysate. An AAA-family ATPase VCP was isolated from the activ...
• New cancer therapy by degradation of specific proteins

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2001 -2002 

  Author : Keiichi NAKAYAMA, 山下 順範, 中山 啓子, 畠山 鎮次

   

  We proposed to induce to degradation of specific protein by the modification of ubiquitin-ligase, Recently, it is reported that expression level of many proteins are regulated by not only transcription but degradation rate. Ubiquitin-porteasome system is one of the most useful system to regulate expression level of protein because of its specificity. On this unique point we try to create new ubiquitin-ligase to recognize and induce to degradate proteins which are toxic for our bodies.In this project, we used oncoprotein, Myc as model protein. Because Myc is known to bind to Max, we made sev...
• 神経変性疾患における細胞内封入体のユビキチン介在性分解系の解析と臨床応用

  文部科学省：科学研究費補助金(特定領域研究(C))

  Date (from‐to) : 2001 -2001 

  Author : 畠山 鎮次
• ユビキチン化酵素複合体の同定及び細胞生物学的解析

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 2000 -2001 

  Author : 畠山 鎮次

   

  現在まで蛋白分解機構が報告されているが、特に、酵母におけるユビキチン依存性プロテアソーム介在性蛋白分解に関する研究により、Skp1、Cu1-1、F-box蛋白複合体(SCF複合体)と言われるユビキチン化を実行する酵素複合体は、細胞周期をはじめ、さまざまな細胞機能に関与する分子の発現調節(分解調節)を行っていることが明らかになったSCFとはSkpl、Cul1、F-ボックス蛋白(基質認識受容体)からなるユビキチンリガーゼ(E3)複合体である。我々は、酵母におけるアナロジーを利用して、マウスにおけるSkp1、Cul-1及び複数のFボックス蛋白を同定した。特に、我々がFWD1/β-TrCP(1つのF boxと7つのWD40リピートを有する)として同定したF-box蛋白は、免疫学上重要であるNF-KκBの抑制分子であるIκBαと、細胞接着もしくは癌の分野で注目されるβ-cateninのユピキチン化、及び分解に関与していることを明らかにした。またSkp2というFボックス蛋白を同定し、Skp2の標的分子として、サイタリンEとp27を生化学的及び遺伝学的手法により見い出した。さらに、実際にサイクリンEとp27のユピキチンリガーゼであるかを確証するために、Skp2ノックアウトマウスを作製し、Skp2ノックアウトマウスは野生型マウスと比較して、体重が少なく、全体的に成長が遅い以外、肉眼レベルでは...
• Functional analysis of ubiquitin ligase by knock-out mise

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2000 -2001 

  Author : Keiko NAKAYAMA, 小南 欽一郎, 畠山 鎮次, 中山 敬一, 北川 雅俊

   

  1) We have reported that F-box protein, FWD1 works as a ubiquitin ligase on the degradation of lκB or β-catenin. FWD1 constitutes SCF complex with Cul-1, Skp1, and Rbx1. In this study, we generated knock-out mice of Cul-1 and Skp1, which are components of SCF complex, and another F-box protein, Skp2. Analysis of these mice shows the biological role of protein degradation by SCF complex system, one category of proteolysis by ubiquitin-proteasome system.2) Skp2 is a ubiquitin ligase of cyclin E and p27^. In Skp2 knock-out mice, cyclin E and p27^ was accumulated abnormally. Cells i...
• Identification of the factors mediating ubiquitination of inclusion body in neurodegenerative diseases

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1999 -2000 

  Author : Kei-ichi NAKAYAMA, 畠山 鎮次, 北川 雅敏, 中山 啓子, 小南 欽一郎

   

  It has been reported that most of inclusion bodies which are found in patients' neurons of neurodegenerative diseases are ubiquitinated. The aim of this research project is to identify the factors to mediate ubiquitination of the proteins constituting the inclusion body. As a model system, we chose Machado-Joseph disease (MJD), in which polyglutamine stretch of MJD1 protein is abnormally expanded. We found that MJD1 protein undergoes ubiquitination both in vivo and in vitro. Using the in vitro ubiquitination system as an assay for detection of ubiquitinating activity, we purified the factor...
• 細胞周期調節分子の分解制御に関する研究-マウスSCF複合体の遺伝子クローニングと解析-

  文部科学省：科学研究費補助金(特定領域研究(A))

  Date (from‐to) : 1999 -1999 

  Author : 畠山 鎮次, 中山 啓子, 北川 雅敏, 中山 敬一

   

  細胞内に存在する分子の分解制御は、多方面にわたる医学生物学領域の蛋白分子の機能に重要な働きを果たしている。今まで、いくつかの蛋白分解機構が報告されているが、特に、酵母におけるユビキチン依存性プロテアソーム介在性蛋白分解に関する研究により、Skp1、Cul-1、F-box蛋白複合体(SCF複合体)と言われるユビキチン化を実行する酵素複合体は、細胞周期をはじめ、さまざまな細胞機能に関与する分子の発現調節(分解調節)を行っていることが明らかになった。この構成成分のなかで、F-box蛋白が基質分子をユビキチン化させるための基質特異性を担っていることが推定されている。我々は、酵母におけるアナロジーを利用して、マウスにおけるSkp1、Cul-1及び複数のF-box蛋白を同定した。特に、我々がFWD1/β-TrCP(1つのF boxと7つのWD40リピートを有する)として同定したF-box蛋白は、免疫学上重要であるNF-κBの抑制分子であるIκBαと、細胞接着もしくは癌の分野で注目されるβ-cateninのユビキチン化、及び分解に関与していることが判明した。IκBαとβ-cateninの発現量にかかわるアミノ酸配列に存在するセリン/スレオニンをリン酸化する上流のシグナル及びキナーゼは異なるが、そのモチーフ自体は非常に似ており、リン酸化が起こった場合、FWD1/β-TrCPは、IκBαとβ-...
• 細胞周期制御因子の分解機構の解明とノックアウトマウスを用いた発がん研究

  文部科学省：科学研究費補助金(特定領域研究(A))

  Date (from‐to) : 1999 -1999 

  Author : 中山 啓子, 小南 欽一郎, 畠山 鎮次, 北川 雅敏

   

  細胞周期進行に不可欠であると考えられているサイクリンEの分解機構の解明をめざし、サイクリンEのユビキチンリガーゼの同定を試みた。酵母ですでに知られている、ユビキチンリガーゼのマウス相同遺伝子を単離し、サイクリンEへの結合及び蛋白の安定性への効果を調べ、サイクリンEのユビキチンリガーゼである複合体を同定することができた。その複合体を構成するSkp2、Skp1、Cul1のノックアウトマウスを作製し、その表現型を解析することによって、生物学的機能を知るとともに、サイクリンEの分解が発がんに果たす役割を検討した。1.サイクリンEはSCF複合体をE3とするユビキチン/プロテアゾーム系によって分解された。2.SCF複合体を構成するF-boxタンパクはSkp2であった。3.Skp2ノックアウトマウスは発生段階では体重が少ないこと以外に肉眼的な以上は認めないが、肝細胞や気管上皮細胞などで細胞および核の腫大が認められ、肝細胞では染色体倍数性が異常となり、4倍体、8倍体の核が多数観察された。4.Skp2ノックアウトマウスの胎仔線維芽細胞は増殖が遅く、正常に比しアポトーシスが多く見られた。5.Skp2ノックアウトマウスでは生後1年までで明らかな発がん傾向を認めていない。6.Skp1及びCul1ノックアウトマウスはいずれも胎生6.5〜7.5日目に死亡する早期の胎生致死であった。7.Skp1及びCul...
• Yeast three-hybrid法によるp53分解に関与する分子の研究

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1998 -1999 

  Author : 畠山 鎮次

   

  p53はE6とユビキチンリガーゼE6-APにより3量体を形成し、E6-APによってエビキチン化され、その後、プロテアソームにより分解を受ける。これは、HPV感染化での病理学的状況下であるが、生理学的にもp53はユビキチン化を介して、比較的短い半減期(t1/2=20分)で分解を受ける。よって、E6に相当する内因性分子が存在し、p53分解を調節している可能性がある。本研究は、E6に相当する内因性分子が存在すると仮定し、p53とE6-APと3分子複合体を形成する新たなる遺伝子をクローニングすることを目的とし、3分子が複合体を形成するときに、目的とする第3の遺伝子をクローニングする方法(Yeast three-hybrid法)を開発を行った。Yeast three-hybrid法は、p53の生理的分解機序のみならず、細胞生物学上において重要となる分子間結合を検索する画期的な手段となることが期待される。また、p53を始め、細胞周期を調節する分子(サイクリン、CDKインヒビターなど)の発現が合成のみならず、分解(特にユビキチン化)により制御されていることが報告され始めている。特に、酵母における先駆的研究により、Skp1/Cul-1/F-box蛋白複合体(SCF複合体)は、細胞周期をはじめ、さまざまな細胞機能に関与する分子の発現調節を行っていることが報告されている。我々は、酵母におけるアナ...
• Identification and functional analysis of S phase promoting complex in mammalian cell growth

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1998 -1999 

  Author : Masatoshi KITAGAWA, 中山 敬一, 畠山 鎮次, 中山 啓子

   

  We study the mechanisms of growth promotion via ubiquitin-dependent proteolysis. β-catenin plays an essential role for growth regulation and colon cancer formation. Cytoplasmic β-catenin is regulated by ubiquitin-proteasome system via Wnt signaling pathway. In this study, we identified FWD1, an ubiquitin ligase for β-catenin. FWD1 formed a complex with β-catenin, axin, APC, GSK3βvia its WD40 repeats in the phosphorylation-dependent manner. Furthermore, FED1 also bound with Skp1 and Cull as an ubiquitination machinery named SCF through its F-box. The SCFィイD1FWD1ィエD1 complex ubiquitinated the...
• Regulation of degradation of p27Kip1, one of CDK inhibitory protein, for cancer therapy

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1998 -1999 

  Author : Masatoshi KITAGAWA, 平井 愛山, 中山 敬一, 畠山 鎮次, 中山 啓子

   

  It has been reported that the cyclin dependent kinase inhibitory proteins (CDK inhibitor) , such as p27ィイD1Kip1ィエD1 negatively regulates the G1-CDK activity in normal cells. In most cancer cells, up-regulation of G1-cyclin dependent kinase (G1-CDK) activity contributes in their malignant growth. First of all, we study the expression of p27ィイD1Kip1ィエD1 in human lung cancer using immunohistoichemistry. We found that p27ィイD1Kip1ィエD1 labeling index was decreased and p27ィイD1Kip1ィエD1 degradation activity was up-regulated in cancerious lung tissues, compared with nonneoplastic lung tissues. Theref...
• 細胞周期調節分子の分解制御に関する研究 - Yeast three-hybrid法の確立と応用 -

  文部科学省：科学研究費補助金(特定領域研究(A))

  Date (from‐to) : 1998 -1998 

  Author : 畠山 鎮次, 中山 啓子, 北川 雅敏, 中山 敬一

   

  細胞周期を調節するサイクリンとサイクリン依存性キナーゼ(CDK)に直接結合し、機能抑制するCDK阻害分子(CDK inhibitors;CKI)であるP27はユビキチン化を受けることにより、その発現レベルが調節されている。本研究は、p27のユビキチン化に関与するE3が存在すると仮定し、p27とUBC3と3分子複合体を形成する新たなる遺伝子を同定することを目的とした。さらに、本研究では、3分子が複合体を形成するときに、目的とする第3の遺伝子をクローニングする方法であるYeast three-hybrid法を開発した。また、p53、p27を始め、細胞周期を調節する分子(サイクリン、CKIなど)の発現が合成のみならず、分解(特にユビキチン化)により制御されていることが報告され始めている。特に、酵母における先駆的研究により、Skp1/CUl-1/E-box蛋白複合体(SCF複合体)は、CKIを含む細胞周期調節分子をはじめ、さまざまな細胞機能に関与する分子の発現調節を行っていることが報告されている。我々は、酵母におけるアナロジーを利用して、マウスにおけるSkp1、Cul-1及び複数のF-box蛋白を同定した。我々がFWD1として同定したF-box蛋白は、免疫学上重要であるNF-κBの抑制分子であるIκBαと、大腸癌に関与するβ-cateninのユビキチン化、及び分解に関与していることが...
• サイクリン依存性キナーゼ阻害分子の分解機構の解明

  文部科学省：科学研究費補助金(特定領域研究(A))

  Date (from‐to) : 1998 -1998 

  Author : 中山 啓子, 畠山 鎮次, 北川 雅敏, 中山 敬一

   

  p27などCDKインヒビターと呼ばれる分子群は、CDK/Cyclin複合体に結合し酵素活性を阻害することから細胞周期調節に直接関わる重要な因子と考えられる。p27の蛋白量は蛋白分解に規定されていることが既に報告されていたがその分子機構はは不明であった。そこでその分解のメカニズムの解明を試み以下の点が明らかとなった。1. 培養細胞(NIH3T3)を用い接触阻害によって細胞周期を同調させ、細胞周期によるp27の変動を観察した。p27は細胞周期依存性にG1-S移行期にもっとも強くユビキチン化された。2. ユビキチン付加部位を特定するためp27の変異体を作製しユビキチン化を調べたところ,アミノ酸残基134番、153番、165番目のリジンをアルギニンに置換したところユビキチン化が抑制された。3. p27をウェスタンブロットで観察すると、p27のユビキチン化と同時に22kDaの蛋白も同時に認識された。これはp27がユビキチン化以外にプロセシングを受けていることを示唆する。この反応はATP依存的であり、lactacystin、chymostatin、PMSFで抑制されたが、antipain、pepstatin、leupeptin、E64では影響を受けない。このプロセシング反応はプロテアゾームに関与しているchymotrypsin様プロテアーゼによるものと考えられた。4. プロセシングされた...
• Analysis of sinnalling pathway of apoptosis degradation using PKCδ gene-targeting mice

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1997 -1998 

  Author : Kei-ichi NAKAYAMA, 真貝 洋一, 大野 茂男, 畠山 鎮次, 中山 啓子, 北川 雅敏

   

  It has been reported that protein kinase Cδ (PKCδ) a subtype of PKC family members, is cleaved by caspase resulting in conversion to activated PKCδ upon apoptosis. The aim of this research project is to elucidate the physiological function of PKCδ by generating mice deficient in PKCδ gene.We constructed the targeting vector and transfect it into embryonic stem (ES) cells to obtain the homologous recombinant of ES cells for PKCδ locus. According to the standard method to generate gene-ablated mice, PKCδ-deficient mice were produced with the mutant ES cells. The PKCδ-deficient mice were viabl...

Industrial Property Rights

• 特願PCT/JP2013/083915:前立腺基底細胞の検出方法  2013年/12/18

  畠山 鎮次, 田中 伸哉
• 特開2001-224380:サイクリンEをユビキチン化分解するユビキチンリガーゼSCF複合体のF-ボックスタンパク  2001/08/21

  中山 敬一, 中山 啓子, 畠山 鎮次  科学技術振興事業団  200903021511213439
• 特開2000-166542:IκBまたはβカテニンのユビキチン化を促進するユビキチンリガーゼSCF複合体のF-ボックスタンパク  2000/06/20

  中山 敬一, 中山 啓子, 北川 雅敏, 畠山 鎮次  科学技術振興事業団  200903015715513148
• 特開平7-286000:c-fgr遺伝子産物に特異的に反応するモノクローナル抗体  1995/10/31

  畠山 鎮次, 岩淵 和也, 小笠原 一誠, 小野江 和則  住友電気工業株式会社  200903003243646027

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• 高校生メディカル講座（地域医療を支える人づくりプロジェクト事業)「医学生・医師としての勉強と進路、そして北海道の医療問題」

  Date (from-to) : 2019/07/15

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道教育委員会
• 高校生メディカル講座（地域医療を支える人づくりプロジェクト事業)「からだの構成成分とがんの関係」

  Date (from-to) : 2019/07/15

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道教育委員会
• 北大セミナーin旭川「がんの原因はどんなことなの？」

  Date (from-to) : 2016/09/11-2016/09/11

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道大学
• 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」

  Date (from-to) : 2015/11/18

  Role : Lecturer

  Sponser, Organizer, Publisher  : 立命館慶祥中学校
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  Date (from-to) : 2014/09/20

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道留萌高等学校
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  Date (from-to) : 2014/07/21

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道大学
• 「がん」はどうしてできるの -遺伝子とタンパク質の研究から-

  Date (from-to) : 2014/05/27

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海学園札幌高等学校「進路ガイダンス」
• 人が病気にかかると体の中で何が起きるのだろうか（札幌市立中央中学校）

  Date (from-to) : 2013/06/25

  Role : Lecturer

  Sponser, Organizer, Publisher  : JST「サイエンス・パートナーシップ・プログラム」事業
• 高校生メディカル講座（地域医療を支える人づくりプロジェクト事業）『生化学・分子生物学の基礎』

  Date (from-to) : 2010/07/11

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道教育委員会
• 高校生メディカル講座（地域医療を支える人づくりプロジェクト事業）『医学部に入学後、何をして何になるのか？』

  Date (from-to) : 2010/07/11

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道教育委員会
• 職業セミナー「医系希望の後輩たちへ」

  Date (from-to) : 2009/10/15

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道旭川東高等学校
• 職業セミナー「医系希望の後輩たちへ」

  Date (from-to) : 2008/10/16

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道旭川東高等学校
• 北大セミナーin 旭川「北大医学部の教育システムと医学研究の進歩」

  Date (from-to) : 2006/07/17

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道大学