ケミカルジェネティクスを用いた可逆的な遺伝子不活法の開発

2016 Fiscal Year Research-status Report

• Project/Area Number: 16K15289
• Research Institution: Osaka University
• Principal Investigator: 黒崎 知博 大阪大学, 免疫学フロンティア研究センター, 特任教授(常勤) (50178125)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Keywords: syｋ / ケミカルジェネティクス / 時空間 / 可逆的阻害

Outline of Annual Research Achievements

特定の遺伝子の活性を、「ある時空間のみ変異させ」、将来どのような効果をもたらすかの検定の重要性は言うまでもない。例えば、B細胞の場合胚中心(GC)反応を経て、メモリー細胞が形成されるわけであるが、GCの過程で生じる変化が、メモリー細胞機能にどのような影響を与えるかは非常に重要な命題にもかかわらず、この命題へのアプローチが困難を極めている。
ある酵素の阻害剤は、in vivo投与した場合、その特異性を十分に担保さすことが非常に困難である。又、個体の中で、どの細胞の酵素を阻害して、表現系が表出しているのかを特定できない。従って、その結果の解釈に常に疑義がはさまれることになる。この特異性の点を克服するために、本研究では酵素遺伝子サイドに変異を加え、この阻害剤の特異性を飛躍的に上昇させ、この変異遺伝子と新規阻害剤の組み合わせで、可逆的in vivo阻害をおこなおうとするものである。
具体的には、SykのATP結合ポケットを伸長する変異を数種類作成し培養細胞DT40にノックインした。このノックイン変異が野生型と同様に機能することは生化学的検定、細胞応答検定より確認した。更に、この細胞を用いて、変異Syk特異的阻害剤をスクリーニングして、一番効果的にSykの酵素活性のみ阻害した阻害剤を見つけた。さらにこの阻害剤を、wash outの後、酵素活性が復活するものを選択した。現在、このSyk変異体のtransgenic mouseの作成中である。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Syk変異体の作成、この変異体を可逆的に阻害する薬剤の選別も順調に進んだ。このdataをもとに現在変異Syk transgenic mouseを作成中である。

Strategy for Future Research Activity

必要な実験材料も樹立しているので、予定どおり、個体レベルでSykの可逆的阻害の行えるマウスを用いての研究を行うようにする。

Research Products

• [Journal Article] BACH transcription factors in innate and adaptive immunity (2017)
  • Author(s): Igarashi Kazuhiko、Kurosaki Tomohiro、Roychoudhuri Rahul
  • Journal Title: Nature Reviews Immunology Volume: 17 Pages: 437～450
  • DOI: 10.1038/nri.2017.26
  • Open Access / Int'l Joint Research
• [Journal Article] Regulation of memory B and plasma cell differentiation. (2017)
  • Author(s): Shinnakasu R, Kurosaki T.
  • Journal Title: Curr. Opin. Immunol. Volume: 45 Pages: 126-131
  • DOI: 10.1016/j.coi.2017.03.003.
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] The transcription factor Foxo1 controls germinal center B cell proliferation in response to T cell help. (2017)
  • Author(s): Inoue T, Shinnakasu R, Ise W, Kawai C, Egawa T, Kurosaki T.
  • Journal Title: J. Exp. Med. Volume: 214 Pages: 1181-1198
  • DOI: 10.1084/jem.20161263.
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis. (2016)
  • Author(s): Ohmi, Y., Ise, W., Harazono, A., Takakura, D., Fukuyama, H., Baba, Y., Narazaki, M., Shoda, H., Takahashi, N., Ohkawa, Y., Ji, S., Sugiyama, F., Fujio, K., Kumanogoh, A., Yamamoto, K., Kawasaki, N., Kurosaki, T., Takahashi, Y., Furukawa, K.
  • Journal Title: Nat. Commun. Volume: 7 Pages: 11205
  • DOI: 10.1038/ncomms11205
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Whole-Virion Influenza Vaccine Recalls an Early Burst of High-Affinity Memory B Cell Response through TLR Signaling. (2016)
  • Author(s): Onodera T, Hosono A, Odagiri T, Tashiro M, Kaminogawa S, Okuno Y, Kurosaki T, Ato M, Kobayashi K, Takahashi Y.
  • Journal Title: J. Immunol. Volume: 196 Pages: 4172-84
  • DOI: 10.4049/jimmunol.1600046.
  • Peer Reviewed / Open Access
• [Journal Article] Regulated selection of germinal center cells into the memory B cell compartment. (2016)
  • Author(s): Shinnakasu, R., Inoue, T., Kometani, K., Moriyama, S., Adachi, Y., Nakayama, M., Takahashi, Y., Fukuyama, H., Okada, T.,Kurosaki,T.
  • Journal Title: Nat. Immunol. Volume: 17 Pages: 861-9
  • DOI: 10.1038/ni.3460
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Bach2-Batf interactions control Th2-type immune response by regulating the IL-4 amplification loop. (2016)
  • Author(s): Kuwahara M, Ise W, Ochi M, Suzuki J, Kometani K, Maruyama S, Izumoto M, Matsumoto A, Takemori N, Takemori A, Shinoda K, Nakayama T, Ohara O, Yasukawa M, Sawasaki T, Kurosaki T, Yamashita M.
  • Journal Title: Nat. Commun. Volume: 7 Pages: 12596
  • DOI: 10.1038/ncomms12596.
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Protein kinase D regulates positive selection of CD4＋ thymocytes through phosphorylation of SHP-1. (2016)
  • Author(s): Ishikawa E, Kosako H, Yasuda T, Ohmuraya M, Araki K, Kurosaki T, Saito T, Yamasaki S.
  • Journal Title: Nat. Commun. Volume: 7 Pages: 12756
  • DOI: 10.1038/ncomms12756
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Conversion of T cells to B cells by inactivation of polycomb-mediated epigenetic suppression of the B-lineage program. (2016)
  • Author(s): Ikawa T, Masuda K, Endo TA, Endo M, Isono K, Koseki Y, Nakagawa R, Kometani K, Takano J, Agata Y, Katsura Y, Kurosaki T, Vidal M, Koseki H, Kawamoto H.
  • Journal Title: Genes. Dev. Volume: 30 Pages: 2475-2485
  • DOI: 10.1101/gad.290593.116
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation. (2016)
  • Author(s): Kuroda E, Ozasa K, Temizoz B, Ohata K, Koo CX, Kanuma T, Kusakabe T, Kobari S, Horie M, Morimoto Y, Nakajima S, Kabashima K, Ziegler SF, Iwakura Y, Ise W, Kurosaki T, Nagatake T, Kunisawa J, Takemura N, Uematsu S, Hayashi M, Aoshi T, Kobiyama K, Coban C, Ishii KJ.
  • Journal Title: Immunity Volume: 45 Pages: 1299-1310
  • DOI: 10.1016/j.immuni.2016.11.010.
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Selection of germinal-center B cells into memory B cell or plasma cell Compartment (2017)
  • Author(s): Kurosaki, T.
  • Organizer: The 6th NIF Winter School on Advanced Immunology
  • Place of Presentation: Singapore
  • Year and Date: 2017-01-22 – 2017-01-26
  • Invited
• [Presentation] Selection mechanisms of germinal center B cells into the memory B cell compartment (2016)
  • Author(s): Shinnakasu, R.
  • Organizer: 45th Annual Meeting of the Japanese Society for Immunology.
  • Place of Presentation: 沖縄コンベンションセンター、沖縄
  • Year and Date: 2016-12-05 – 2016-12-07
  • Invited
• [Presentation] Fate decisions of germinal center B cells into the memory B cell or plasma cell compartment (2016)
  • Author(s): Kurosaki, T.
  • Organizer: 4th Antibody Symposium
  • Place of Presentation: Singapore
  • Year and Date: 2016-11-14
  • Invited
• [Presentation] Fate decisions of germinal center B cells into the memory B cell or plasma cell compartment (2016)
  • Author(s): Kurosaki, T.
  • Organizer: The International Symposium for Advanced Immunology Commemorating the 10th anniversary of IFReC and the 77th anniversary of Prof. Tadamitsu Kishimoto's birth
  • Place of Presentation: Osaka International Convention Center, Osaka, Japan
  • Year and Date: 2016-11-01 – 2016-11-02
  • Int'l Joint Research / Invited
• [Presentation] Overview of germinal center immunity (2016)
  • Author(s): Kurosaki, T.
  • Organizer: The 13th International Workshop on Autoantibodies and Autoimmunity (IWAA2016)
  • Place of Presentation: The Westin Miyako Kyoto, Kyoto, Japan
  • Year and Date: 2016-10-11 – 2016-10-13
  • Int'l Joint Research / Invited
• [Presentation] Memory B cell generation mechanisms (2016)
  • Author(s): Kurosaki, T.
  • Organizer: Annual program of monthly lectures
  • Place of Presentation: Madrid, Spain
  • Year and Date: 2016-09-08
  • Invited
• [Presentation] Transcriptional regulation in memory B cell development (2016)
  • Author(s): Kurosaki, T.
  • Organizer: EMBO conference “Lymphocyte Antigen Receptor Signaling”
  • Place of Presentation: Siena, Italy
  • Year and Date: 2016-09-03 – 2016-09-07
  • Int'l Joint Research / Invited
• [Presentation] Instructive selection of germinal center B cells into the memory compartment (2016)
  • Author(s): Kurosaki, T.
  • Organizer: International congress of Immunology 2016
  • Place of Presentation: Melbourne, Australia
  • Year and Date: 2016-08-21 – 2016-08-26
  • Int'l Joint Research / Invited
• [Presentation] Instructive selection of germinal center B cells into the memory compartment (2016)
  • Author(s): Kurosaki, T.
  • Organizer: Antibody and Fc Receptor Biology: Bench to Bedside A Special Symposium to Honor Jeffrey V. Ravetch on his 65th Birthday
  • Place of Presentation: New York, USA
  • Year and Date: 2016-05-09
  • Int'l Joint Research / Invited
• [Presentation] B細胞を介する自己免疫抑制 (2016)
  • Author(s): 黒崎知博
  • Organizer: 第60回日本リウマチ学会総会・学術集会
  • Place of Presentation: パシフィコ横浜、横浜
  • Year and Date: 2016-04-21 – 2016-04-23
  • Invited
• [Remarks] Laboratory of Lymphocyte Differentiation
  • URL: http://lymph.ifrec.osaka-u.ac.jp/index_j.html