1.

論文

論文
喜多村, 晃一 ; Kitamura, Koichi
出版情報: 平成28(2016)年度 科学研究費補助金 基盤研究(C) 研究成果報告書 = 2016 Fiscal Year Final Research Report.  2014-04-01 - 2017-03-31  pp.4p.-,  2017-05-17. 
URL: http://hdl.handle.net/2297/00051789
概要: 金沢大学医薬保健研究域医学系<br />B型肝炎ウイルス(HBV)持続感染者は国内で130~150万人と推定されており、感染を放置すれば慢性肝炎、肝硬変、肝がんへと進行するおそれがある。HBV持続感染では、cccDNAと呼ばれる 環状ウイルスDNAが、宿主細胞の核内に維持されウイルス複製の鋳型となる。cccDNAを除去する有効な治療法は無く、B型肝炎の根治が難しい理由となっているが、現在のところcccDNAを標的とする分子機構の知見が少ない。本研究では、HBV cccDNAに対してゲノム情報を改変すると考えられる宿主因子AID/APOBEC蛋白質及び関連するDNA修復機構の作用について解析を行った。<br />Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV). Despite the crucial role of cccDNA in viral persistence, little is known about the host factors that target this viral intermediate. Recent studies have revealed that AID/APOBEC3 cytidine deaminase family members can induce C-to-U hypermutation on viral genome and restrict viral replication. Uracil residues in DNA are removed by base excision repair (BER) enzyme, uracil DNA glycosylase (UNG), when cytidine deamination is occurred in host genome. We investigated whether uracil residues were generated in HBV cccDNA by APOBEC3G and removed by UNG using in vitro. We found that IFNγ stimulation of hepatocyte cell lines induced the endogenous APOBEC3G expression and HBV cccDNA hypermutation. When UNG activity was inhibited, the IFNγ-mediated hypermutation of cccDNA was enhanced. Our result indicate that BER pathway cancels the mutations of the cccDNA generated by APOBEC proteins.<br />研究課題/領域番号:26460993, 研究期間(年度):2014-04-01 - 2017-03-31 続きを見る
2.

論文

論文
喜多村, 晃一 ; Kitamura, Koichi
出版情報: 平成25(2013)年度 科学研究費補助金 若手研究(B) 研究成果報告書 = 2013 Fiscal Year Final Research Report.  2011-2013  pp.4p.-,  2014-05-21.  金沢大学医薬保健研究域医学系
URL: http://hdl.handle.net/2297/00051790
概要: AID/APOBEC蛋白質はHBVをはじめとする様々なウイルスDNAにC-to-U変換を誘導しウイルスを不活化するというモデルが提唱されている。本研究では核内cccDNAに着目し、DHBVモデルを用いてAID、APOBEC3G及び修復因子U NGの作用を検討した。その結果、APOBEC3Gはこれまで知られていたウイルス粒子内DNAよりも高頻度なcccDNA変異導入活性を持つこと、AIDとUNGが協働してcccDNAを切断していることを示した。これらは抗ウイルス活性であると同時に宿主細胞核内の変異導入活性及びHBV DNA断片の存在を示唆しており発がん機構解明においても重要な知見であると考える。<br />Recent studies have revealed that AID/APOBEC cytidine deaminase family members can induce C-to-U hypermutation on viral genome and restrict replication of various types of virus including HBV. Uracil residues in DNA are removed by base excision repair (BER) enzyme, uracil DNA glycosylase (UNG) when cytidine deamination is occurred in host genome.Here, we investigated whether uracil residues were removed by UNG from HBV and DHBV DNAs using in vitro cell culture system. When UNG activity was inhibited by the expression of UNG inhibitory protein (UGI), the APOBEC3G-mediated hypermutation of HBV nucleocapsid DNA was enhanced. The enhanced hypermutation by APOBEC3G and UGI was also observed in DHBV cccDNA, which was more frequent than in nucleocapsid DNA. We also found that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. These results indicate that UNG excises uracils from viral genome deaminated by AID/APOBEC protein during or after cccDNA formation.<br />研究課題/領域番号:23790780, 研究期間(年度):2011-2013 続きを見る
3.

論文

論文
喜多村, 晃一 ; Kitamura, Koichi
出版情報: 平成22(2010)年度 科学研究費補助金 若手研究(B) 研究成果報告書 = 2010 Fiscal Year Final Research Report.  2009-2010  pp.4p.-,  2011-05-18.  金沢大学医薬保健研究域医学系
URL: http://hdl.handle.net/2297/00051791
概要: 慢性B型肝炎ではHBVを完全に除去できれば肝硬変、肝がんへの進行が止まるが、既存の治療法では血中HBV量を減少させることはできても、ウイルスの核内フォームであるcccDNAが細胞に維持されたままである。抗ウイルス因子として知られるAPOBE C3GをはじめとするAID/APOBECファミリーがHBVにおいても発現誘導されウイルスゲノムへ変異を導入することが報告されている。本研究では核内の塩基除去修復因子であるUNGがこの変異をcccDNAの段階で修復している可能性を示した。<br />Human APOBEC3 proteins are reported to be antiviral factors and catalyze C-to-U conversion on hepatitis B virus (HBV) DNA by cytosine deamination. Extensive G-to-A hypermutation on plus-strand of HBV DNA has been observed from APOBEC3G-expressing hepatoma cells. We investigated whether UNG also process the hypermutated HBV genome with base excision activity. We found that the UNG inhibition caused accumulation of hypermutation in nuclear cccDNA and decrease of cytoplasmic rcDNA level. Sequencing analysis showed that some of these cccDNA mutations resulted in nonsense codons within viral polymerase coding region. These results suggest that the BER pathway triggered from nuclear UNG repairs the viral DNA mutations introduced by the antiviral APOBECs.<br />研究課題/領域番号:21790654, 研究期間(年度):2009-2010 続きを見る
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論文

論文
Watashi, Koichi ; Liang, Guoxin ; Iwamoto, Masashi ; Marusawa, Hiroyuki ; Uchida, Nanako ; Daito, Takuji ; Kitamura, Kouichi ; Muramatsu, Masamichi ; Ohashi, Hirofumi ; Kiyohara, Tomoko ; Suzuki, Ryosuke ; Li, Jisu ; Tong, Shuping ; Tanaka, Yasuhito ; Murata, Kazumoto ; Aizaki, Hideki ; Wakita, Takaji ; 喜多村, 晃一 ; 村松, 正道
出版情報: Journal of Biological Chemistry.  288  pp.31715-31727,  2013-11-01.  American Society for Biochemistry and Molecular Biology Inc.
URL: http://hdl.handle.net/2297/00053812
概要: 金沢大学医薬保健研究域医学系<br />Virus infection is restricted by intracellular immune responses in host cells, and this is typ ically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection. 続きを見る
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論文

論文
Nakanishi, Yosuke ; Kondo, Satoru ; Wakisaka, Naohiro ; Tsuji, Akira ; Endo, Kazuhira ; Murono, Shigeyuki ; Ito, Makoto ; Kitamura, Kouichi ; Muramatsu, Masamichi ; Yoshizaki, Tomokazu ; 近藤, 悟 ; 脇坂, 尚宏 ; 辻, 亮 ; 遠藤, 一平 ; 室野, 重之 ; 喜多村, 晃一 ; 村松, 正道 ; 吉崎, 智一
出版情報: PLoS ONE.  8  pp.e62066-,  2013-04-25.  Public Library of Science
URL: http://hdl.handle.net/2297/00053815
概要: 金沢大学医薬保健研究域医学系<br />Purpose:In humans, activation-induced cytidine deaminase (AID) expression results due to infla mmation and this deaminase activity is also involved in carcinogenesis. The aim of this study is to investigate the correlation between AID expression and the clinical classification of oral cancer tissues.Experimental Design:The current study investigated the correlation between AID expression and the clinical classification of oral cancer tissues from 27 patients who underwent surgical resection using immunohistochemistry. Specific AID expression and its induction by cytokine stimulation were investigated in cultured HSC oral cancer cell lines by reverse transcriptase PCR.Results:AID expression was detected in 10 of 27 specimens (37.0%). AID expression was more frequently detected in early-stage cancer, especially in early stage T, than in late-stage cancer (T1/T2 vs. T3/4; P = 0.0493, N0 vs. N1/2/3; P = 0.0793). HSC-2, a nonmetastatic oral cancer cell line, abundantly expressed endogenous AID, whereas no such expression was observed in HSC-3, a metastatic oral cancer cell line. Moreover, AID expression was substantially induced in HSC-2 cells by stimulation of an inflammation-related cytokine, TNF-α.Conclusions:Aberrant AID expression in the oral epithelium would contribute to the initiation of oral squamous cell carcinoma. Avoiding persistent AID inducible condition such as frequent cleaning of oral cavity would play an important role for the prevention of developing oral cancer. © 2013 Nakanishi et al. 続きを見る
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論文

論文
Seishima, Noriko ; Kondo, Satoru ; Wakaea, Kousho ; Wakisaka, Naohiro ; Kobayashi, Eiji ; Kano, Makoto ; Moriyama-Kita, Makiko ; Nakanishi, Yosuke ; Endo, Kazuhira ; Imoto, Tomoko ; Ishikawa, Kazuya ; Sugimoto, Hisashi ; Hatano, Miyako ; Ueno, Takayoshi ; Koura, Miki ; Kitamura, Kouichi ; Muramatsu, Masamichi ; Yoshizaki, Tomokazu ; 近藤, 悟 ; 脇坂, 尚宏 ; 喜多, 万紀子 ; 遠藤, 一平 ; 波多野, 都 ; 小浦, 美樹 ; 喜多村, 晃一 ; 村松, 正道 ; 吉崎, 智一
出版情報: Scientific Reports = 8.  1  pp.18732-,  2018-02.  Springer Nature
URL: http://hdl.handle.net/2297/00053819
概要: 金沢大学医薬保健研究域医学系<br />Activation-induced cytidine deaminase (AID) and apolipoprotein B mRNA-editing catalytic polype ptide 3 (A3) family are cytidine deaminases that play critical roles in B-cell maturation, antiviral immunity and carcinogenesis. Adenoids and palatine tonsils are secondary lymphoid immune organs, in which AID and A3s are thought to have several physiological or pathological roles. However, the expression of AID or A3s in these organs has not been investigated. Therefore, we investigated the expression profiles of AID and A3s, using 67 samples of adenoids and palatine tonsils from patients, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical analyses. AID and A3s expression levels in the adenoids and the palatine tonsils of the same individual significantly correlated with each other. Of note, AID expression level in the adenoids negatively correlated with the age (r = −0.373, P = 0.003). The younger group with adenoid vegetation and tonsillar hypertrophy showed more abundant AID expression than the older group with recurrent tonsillitis and peritonsillar abscesses (P = 0.026). Moreover, immunohistochemical analysis revealed the distribution of AID and A3s in the epithelial cells as well as germinal centres. The localisation of AID expression and its relation to age may contribute to adenoid vegetation and inflammation.<br />Ministry of Education, Culture, Sports, Science and Technology B23390396,A24689064 続きを見る
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論文

論文
Wakaea, Kousho ; Nishiyama, Tomoaki ; Kondo, Satoru ; Izuka, Takashi ; Que, Lusheng ; Chen, Cong ; Kase, Kina ; Kitamura, Kouichi ; Md , Monjurul ; Wang, Zhe ; Ahasan, Md Monjurul ; Nakamura, Mitsuhiro ; Fujiwara, Hiroshi ; Yoshizaki, Tomokazu ; Hosomochi, Kazuyoshi ; Tajima, Atsushi ; Nakahara, Tomomi ; Kiyono, Tohru ; Muramatsu, Masamichi ; 西山, 智明 ; 近藤, 悟 ; 加瀬, 希奈 ; 喜多村, 晃一 ; 中村, 充宏 ; 藤原, 浩 ; 吉崎, 智一 ; 村松, 正道
出版情報: Scientific Reports = 8.  1  pp.9745-,  2018-12-01.  Springer Nature
URL: http://hdl.handle.net/2297/00053820
概要: 金沢大学医薬保健研究域医学系<br />Mitochondrial DNA (mtDNA) mutations are found in many types of cancers and suspected to be inv olved in carcinogenesis, although the mechanism has not been elucidated. In this study, we report that consecutive C-to-T mutations (hypermutations), a unique feature of mutations induced by APOBECs, are found in mtDNA from cervical dysplasia and oropharyngeal cancers. In vitro, we found that APOBEC3A (A3A) and 3B (A3B) expression, as well as mtDNA hypermutation, were induced in a cervical dysplastic cell line W12 when cultured in a differentiating condition. The ectopic expression of A3A or A3B was sufficient to hypermutate mtDNA. Fractionation of W12 cell lysates and immunocytochemical analysis revealed that A3A and A3B could be contained in mitochondrion. These results suggest that mtDNA hypermutation is induced upon keratinocyte differentiation, and shed light on its molecular mechanism, which involves A3s. The possible involvement of mtDNA hypermutations in carcinogenesis is also discussed. © 2018 The Author(s). 続きを見る
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論文

論文
Kitamura, Kouichi ; Que, Lusheng ; Shimadu, Miyuki ; Koura, Miki ; Ishihara, Yuuki ; Wakaea, Kousho ; Nakamura, Takashi ; Watashi, Koichi ; Wakita, Takaji ; Muramatsu, Masamichi ; 喜多村, 晃一 ; 島津, 美幸 ; 小浦, 美樹 ; 村松, 正道
出版情報: PLoS Pathogens = 14.  6  pp.e1007124-,  2018-06-21.  Public Library of Science
URL: http://hdl.handle.net/2297/00053821
概要: 金沢大学医薬保健研究域医学系<br />Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepa tocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An in vitro FEN activity assay demonstrated cleavage of 5′-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated in vitro when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the in vitro cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA in vitro. 続きを見る
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論文

論文
村松, 正道 ; 喜多村, 晃一 ; リャン, グオシン
出版情報: 日本臨床免疫学会会誌 = Japanese journal of clinical immunology.  31  pp.259-,  2008-08-31.  日本臨床免疫学会 = The Japan Society for clinical immunology
URL: http://hdl.handle.net/2297/29514
概要: 我々はこれまで、活性化B細胞に特異的に発現する遺伝子Activation-induced cytidine deaminase (AID)を単離し、次にAIDが抗体遺伝子の2つの遺伝子改編(クラススイッチ組換えとsomatic hyperm utation)のトリガーとなる事を示してきた。すなわち、生体が抗原刺激を受けた際、B細胞が活性化されAIDを新規に発現誘導し、AIDが抗体遺伝子にDNA切断を誘導する。S領域のDNA切断はクラススイッチ組換えにつながり、AIDが可変領域に作用するとsomatic hypermutationが起こる。実際、変異によりAIDの機能不全が起こると常染色体劣性遺伝の液性免疫不全症である高IgM症候群となる事がわかっている。その後の解析より、AIDによる抗体遺伝子座改編システムは、B細胞による獲得免疫の根幹を形成するメカニズムであるのみならず、そのシステムの些細なほころびが発がんのリスクとなる事もわかってきた。ここではAIDと発がんの関連に関する研究の一端を紹介する。 もう一つのトピックとしてAIDにまつわる新たな展開を紹介したい。AIDは、DNAやRNA上の塩基を修飾し塩基の遺伝情報を改変する活性を持つDNA/RNA deaminaseの1員である。この一群のdeaminaseは遺伝情報を再編集し生体の様々な局面に関わっている事が少しずつ明らかになってきた。AIDに最も近いdeaminaseメンバーであるAPOBEC3は、ここ数年HIVウイルスにhypermutationを導入することでHIVを不活性化する自然免疫のeffecter分子として注目されている。HIV以外のウイルスでも、このdeaminaseの働きが相次いで報告されている。AIDも含むdeaminaseの自然免疫における役割を考察する。 続きを見る
10.

論文

論文
村松, 正道 ; 喜多村, 晃一 ; 若江, 亨祥
出版情報: 生化学 = The journal of Japanese Biochemical Society.  88  pp.557-562,  2016-10-25.  日本生化学会 = Japanese Biochemical Society
URL: http://hdl.handle.net/2297/46611
概要: APOBEC3がHIV-1の逆転写のいずれかのプロセスに作用し抗ウイルス活性を示すことが報告されて以来,APOBECタンパク質が新しいタイプの抗ウイルス因子として脚光を浴びることとなった.逆転写を必要とするウイルスで忘れがちなウイルスとして B型肝炎ウイルス(HBV)がある. APOBEC3のHIV-1への抗ウイルス活性が報告されてまもなく,HBVもやはりAPOBECタンパク質の抗ウイルス活性の標的になることが報告された. HIV-1への抗ウイルス作用と類似するところも多々あるが,HBVは肝細胞特異的感染症であり,感染細胞の核にウイルスエピソームDNAを形成するなど,HBVにユニークな点もある.本稿ではAPOBECタンパク質の抗HBV活性および病態形成との関連を概説する. 続きを見る