Discovery of a novel caspase-1 substrate using a cell-free based protein array

• Project/Area Number: 25290077
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: System genome science
• Research Institution: Ehime University
• Principal Investigator: SAWASAKI TATSUYA 愛媛大学, プロテオサイエンスセンター, 教授 (50314969)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥18,460,000 (Direct Cost: ¥14,200,000、Indirect Cost: ¥4,260,000); Fiscal Year 2015: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2014: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2013: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000)
• Keywords: カスパーゼ１ / 無細胞 / プロテインアレイ / 基質探索 / プロテアーゼ / 炎症 / 蛋白質 / 基質 / カスパーゼ / 無細胞タンパク質合成 / プロテオリシス / タンパク質 / 切断 / コムギ胚芽

Outline of Final Research Achievements

We were successful to make a NC-tagged protein array consisting of about 1,000 kinds of signal transduction proteins from 300 protein kinases and 700 singe transmembrane proteins, which these proteins were fused to N-terminally FLAG tag and C-terminally biotin ligation site to detect protein cleavage by AlphaScreen technology. We screened and found a protein cleaved by capase-1 (CASP1) in the protein array. A single cleavage site in a protein was identified by alanine mutagenesis. The new protein was also cleaved in the cells that CASP1 was activated and the C-terminally cleaved fragment was located in the nucleus although the N-terminal fragment seemed to be degradated. These results suggest that this protein is a novel substrate protein for CASP1.

Research Products

• [Journal Article] Characterization of RyDEN (C19orf66) as an interferon-stimulated cellular inhibitor against dengue virus replication. (2016)
  • Author(s): Suzuki Y, Chin WX, Han Q, Ichiyama K, Lee CH, Eyo ZW, Ebina H, Takahashi H, Takahashi C, Tan BH, Hishiki T, Ohba K, Matsuyama T, Koyanagi Y, Tan YJ, Sawasaki T, Chu1 JJ, Vasudevan SG, Sano K, Yamamoto N.
  • Journal Title: PLoS Pathogen Volume: 12 Issue: 1 Pages: 275-275
  • DOI: 10.1186/s12870-015-0660-9
  • NAID: 120005763386
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay (2015)
  • Author(s): Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih PJ, Morishita R, Uchigashima M, Watanabe M, Fujimoto T, Iwasaki T, Endo Y, Sawasaki T
  • Journal Title: Sci Rep Volume: 5 Issue: 1 Pages: 11333-11333
  • DOI: 10.1038/srep11333
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Members of the Plant CRK-superfamily are Capable of trans-/auto-Phosphorylation of Tyrosine Residues. (2015)
  • Author(s): Nemoto K, Takemori N, Seki M, Shinozaki K, Sawasaki T.
  • Journal Title: The Journal of Biological Chemistry Volume: 290 Issue: 27 Pages: 16665-16677
  • DOI: 10.1074/jbc.m114.617274
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Na,K-ATPaseα3 is a death target of alzheimer patient amyloid-β assembly. (2015)
  • Author(s): Ohnishi T, ..., Matsui K, ..., Hoshi M （計35名, 記載欄不足）
  • Journal Title: Proc Natl Acad Sci U S A Volume: 112 Issue: 32 Pages: 4465-4474
  • DOI: 10.1073/pnas.1421182112
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases. (2015)
  • Author(s): Kinoshita-Kikuta E, Kinoshita E, Eguchi Y, Yanagihara S, Edahiro K, Inoue Y, Taniguchi M, Yoshida M, Yamamoto K, Takahashi H, Sawasaki T, Utsumi R, Koike T.
  • Journal Title: PLoS One Volume: 10 Issue: 7 Pages: e0132598-e0132598
  • DOI: 10.1371/journal.pone.0132598
  • Peer Reviewed / Open Access
• [Journal Article] High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system. (2015)
  • Author(s): Takemori N, Takemori A, Matsuoka K, Morishita R, Matsushita N, Aoshima M, Takeda H, Sawasaki T, Endo Y, Higashiyama S.
  • Journal Title: Mol Biosyst. Volume: 11 Issue: 2 Pages: 361-365
  • DOI: 10.1039/c4mb00556b
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] MAPK Phosphatase 5 Expression Induced by Influenza and Other RNA Virus Infection Negatively Regulates IRF3 Activation and Type I Interferon Response. (2015)
  • Author(s): James SJ, Jiao H, Teh HY, Takahashi H, Png CW, Phoon MC, Suzuki Y, Sawasaki T, Xiao H, Chow VT, Yamamoto N, Reynolds JM, Flavell RA, Dong C, Zhang Y.
  • Journal Title: Cell Rep. Volume: 10 Issue: 10 Pages: 1722-1734
  • DOI: 10.1016/j.celrep.2015.02.030
  • Peer Reviewed / Open Access
• [Journal Article] In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: The application of the enzyme-labeled antigen method. (2014)
  • Author(s): Mizutani Y, Tsuge S, Takeda H, Hasegawa Y, Shiogama K, Onouchi T, Inada KI, Sawasaki T, Tsutsumi Y.
  • Journal Title: Mol Oral Microbiol. Volume: 29 Pages: 156-173
  • DOI: 10.1111/mom.12052
  • Peer Reviewed / Open Access
• [Journal Article] Involvement of Hepatitis C Virus NS5A Hyperphosphorylation Mediated by Casein Kinase I-α in Infectious Virus Production. (2014)
  • Author(s): Masaki T, Matsunaga S, Takahashi H, Nakashima K, Kimura Y, Ito M, Matsuda M, Murayama A, Kato T, Hirano H, Endo Y, Lemon SM, Wakita T, Sawasaki T, Suzuki T
  • Journal Title: J Virol Volume: 88(13) Issue: 13 Pages: 7541-55
  • DOI: 10.1128/jvi.03170-13
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Pctaire1/Cdk16 promotes skeletal myogenesis by inducing myoblast migration and fusion. (2014)
  • Author(s): Shimizu K, Uematsu A, Imai Y, Sawasaki T.
  • Journal Title: FEBS Lett. Volume: 588 Issue: 17 Pages: 3030-3037
  • DOI: 10.1016/j.febslet.2014.05.060
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] creenCap3: Improving prediction of caspase-3 cleavage sites using experimentally verified noncleavage sites. (2014)
  • Author(s): Fu SC, Imai K, Sawasaki T, Tomii K.
  • Journal Title: Proteomics. Volume: 14 Issue: 17-18 Pages: 2042-2046
  • DOI: 10.1002/pmic.201400002
  • Peer Reviewed / Open Access
• [Journal Article] Identification of RFPL3 protein as a novel E3 ubiquitin ligase modulating the integration activity of human immunodeficiency virus, type 1 preintegration complex using a microtiter plate-based assay. (2014)
  • Author(s): Tan BH, Suzuki Y, Takahashi H, Ying PH, Takahashi C, Han Q, Chin WX, Chao SH, Sawasaki T, Yamamoto N, Suzuki Y.
  • Journal Title: J Biol Chem. Volume: 289 Issue: 38 Pages: 26368-26382
  • DOI: 10.1074/jbc.m114.561662
  • Peer Reviewed
• [Journal Article] The apoptotic initiator caspase-8: its functional ubiquity and genetic diversity during animal evolution. (2014)
  • Author(s): Sakamaki K, Shimizu K, Iwata H, Imai K, Satou Y, Funayama N, Nozaki M, Yajima M, Nishimura O, Higuchi M, Chiba K, Yoshimoto M, Kimura H, Gracey AY, Shimizu T, Tomii K, Gotoh O, Akasaka K, Sawasaki T, Miller DJ.
  • Journal Title: Mol Biol Evol. Volume: 31 Issue: 12 Pages: 3282-3301
  • DOI: 10.1093/molbev/msu260
  • Peer Reviewed
• [Journal Article] Overexpression of the PAP1 transcription factor reveals a complex regulation of flavonoid and phenylpropanoid metabolism in Nicotiana tabacum plants attacked by Spodoptera litura. (2014)
  • Author(s): Mitsunami T, Nishihara M, Galis I, Alamgir KM, Hojo Y, Fujita K, Sasaki N, Nemoto K, Sawasaki T, Arimura G.
  • Journal Title: PLoS One. Volume: 9
  • Peer Reviewed / Open Access
• [Journal Article] The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes. (2014)
  • Author(s): Arimitsu E, Ogasawara T, Takeda H, Sawasaki T, Ikeda Y, Hiasa Y, Maeyama K.
  • Journal Title: Eur J Pharmacol. Volume: 745 Pages: 117-122
  • DOI: 10.1016/j.ejphar.2014.10.011
  • Peer Reviewed
• [Journal Article] Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis. (2013)
  • Author(s): Iwasaki T, Katayama T, Kohama K, Endo Y, Sawasaki T.
  • Journal Title: Mol Biol Cell Volume: 24 Issue: 6 Pages: 748-756
  • DOI: 10.1091/mbc.e11-08-0740
  • Peer Reviewed
• [Journal Article] Anti-interleukin-5 and multiple autoantibodies are associated with human atherosclerotic diseases and serum interleukin-5 levels (2013)
  • Author(s): Ishigami T, Abe K, Aoki I, Minegishi S, Ryo A, Matsunaga S, Matsuoka K, Taleda H, Sawasaki T, Umemura S, Endoh Y.
  • Journal Title: FASEB J Volume: 27 Issue: 9 Pages: 3437-45
  • DOI: 10.1096/fj.12-222653
  • Peer Reviewed
• [Journal Article] Nek5, a novel substrate for caspase-3, promotes skeletal muscle differentiation by un-reaulatine casnase activity (2013)
  • Author(s): 清水康平、澤崎達也
  • Journal Title: FEBS Letters Volume: Vol.587 Issue: 14 Pages: 2219-2225
  • DOI: 10.1016/j.febslet.2013.05.049
  • Peer Reviewed
• [Journal Article] Suppression of DS1 phosphatidic acid phosphatase confirms resistance to Ralstonia solanacearum in Nicotiana benthamiana (2013)
  • Author(s): Masahiro Nakano, Masahiro Nishihara, Hirofumi Yoshioka, Kouhei Ohnishi, Yasufumi Hikichi, A kinori Kiba
  • Journal Title: PLoS One Volume: 8 Issue: 9 Pages: e75124-e75124
  • DOI: 10.1371/journal.pone.0075124
  • Peer Reviewed
• [Journal Article] Cell-free based protein array technology for analyses of protein kinases and ubiquitin ligases. (2013)
  • Author(s): Sawasaki T, Takeda H, Takahashi H, Nemoto K.
  • Journal Title: Seikagaku Volume: 85 Pages: 438-446
  • NAID: 10031184264
• [Presentation] コムギ無細胞系を基盤としたサイトカイン関連プロテインアレイを用いた カスパーゼ1 新規基質の探索と解析 (2015)
  • Author(s): 増原 有紀, 高橋 宏隆, 桑原 誠, 山下 政克, 澤崎 達也
  • Organizer: BMB2015（第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会）
  • Place of Presentation: 神戸ポートアイランド（兵庫県神戸市）
  • Year and Date: 2015-12-01
• [Presentation] 脱ユビキチン化酵素CYLDを分解し、がん化を促進するE3リガーゼの同定・解析 (2015)
  • Author(s): 上松 篤史, 高橋 宏隆, 竹田 浩之, 徳永 文稔, 山田 六平, 宮城 洋平, 澤崎 達也
  • Organizer: BMB2015（第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会）
  • Place of Presentation: 神戸ポートアイランド（神戸市）
  • Year and Date: 2015-12-01
• [Presentation] コムギ無細胞系を用いた287種類のヒトとマウスからなるE3ユビキチンリガーゼプロテインアレイの作製と生化学的解析への応用 (2015)
  • Author(s): 高橋 宏隆, 中島 達朗, 今村 芽依, 高橋 千佳子, 澤崎 達也
  • Organizer: BMB2015（第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会）
  • Place of Presentation: 神戸ポートアイランド（兵庫県神戸市）
  • Year and Date: 2015-12-01
• [Presentation] コムギ無細胞系を基盤とした脱ユビキチン化酵素CYLDおよびOTULINの阻害剤開発 (2015)
  • Author(s): 山中 聡士, 高橋 宏隆, 徳永 文稔, 澤崎 達也
  • Organizer: BMB2015（第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会）
  • Place of Presentation: 神戸ポートアイランド（兵庫県神戸市）
  • Year and Date: 2015-12-01
• [Presentation] コムギ無細胞系を用いたキナーゼ基質の探索技術 (2015)
  • Author(s): 澤崎 達也
  • Organizer: 第１５回日本蛋白質科学会年会
  • Place of Presentation: あわぎんホール（徳島県徳島市）
  • Year and Date: 2015-06-24
  • Invited
• [Presentation] 大規模生化学的解析はバイオインフォマティクス解析を凌駕するか？ (2014)
  • Author(s): 澤崎 達也
  • Organizer: 産総研・東京大学合同 創薬等支援技術基盤プラットフォーム(PDIS)ワークショップ ～実験とバイオインフォマティクスの協奏による構造生命科学～
  • Place of Presentation: 産総研臨海副都心センター別館（東京都江東区）
  • Year and Date: 2014-12-18
  • Invited
• [Presentation] コムギ無細胞技術を基盤とした化合物スクリーニング (2014)
  • Author(s): 澤崎 達也
  • Organizer: 第37回日本分子生物学会大会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2014-11-25 – 2014-11-27
  • Invited
• [Presentation] コムギ無細胞HTSを用いた創薬基盤技術 (2014)
  • Author(s): 澤崎 達也
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 京都国際会議場（京都府京都市）
  • Year and Date: 2014-10-15 – 2014-10-18
  • Invited
• [Presentation] 無細胞技術を基盤とした自己抗体探索によるアンチエイジング医学のブレークスルーをめざして (2014)
  • Author(s): 澤崎 達也
  • Organizer: 第14回 日本抗加齢医学会総会
  • Place of Presentation: 大阪国際会議場（大阪府大阪市）
  • Year and Date: 2014-06-06 – 2014-06-08
  • Invited
• [Presentation] GPCR研究のためのコムギ無細胞系プロテオリポソーム技術 (2014)
  • Author(s): 澤崎 達也
  • Organizer: 第１１回GPCR研究会
  • Place of Presentation: 日本科学未来館 みらいCANホール（東京都江東区）
  • Year and Date: 2014-05-09 – 2014-05-10
  • Invited
• [Presentation] 創薬等プラットフォームにおけるコムギ無細胞基盤膜蛋白質生産と高親和膜蛋白質抗体作成技術 (2013)
  • Author(s): 澤崎 達也、竹田 浩之
  • Organizer: 第13回日本蛋白質科学会年会
  • Place of Presentation: とりぎん文化会館（鳥取県）
  • Invited
• [Presentation] コムギ無細胞蛋白質合成系を基盤とした膜タンパク質解析 (2013)
  • Author(s): 澤崎 達也
  • Organizer: 第65回日本細胞生物学会大会
  • Place of Presentation: ウインクあいち（愛知）
  • Invited
• [Presentation] コムギ無細胞蛋白質合成系を基盤とした膜蛋白質の合成・精製と細胞への導入技術 (2013)
  • Author(s): 澤崎 達也、竹田 浩之
  • Organizer: 第65回日本細胞生物学会大会
  • Place of Presentation: ウインクあいち（愛知）
  • Invited
• [Presentation] 無細胞プロテインアレイを用いたプロテインキナーゼおよびユビキチンリガーゼの基質タンパク質探索技術 (2013)
  • Author(s): 澤崎 達也, 根本 圭一郎, 高橋 宏隆, 竹田 浩之
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: パシフィコ横浜
  • Invited
• [Presentation] コムギ無細胞蛋白質合成技術と乳がんバイオマーカー探索
  • Author(s): 澤崎 達也
  • Organizer: 日本プロテオーム学会主催プロテオサイエンスセンター JHUPOサテライトジョイントシンポジウム
  • Place of Presentation: 愛媛大学総合情報メデイアセンター・メディアホール
  • Invited
• [Presentation] 創薬等プラットフォーム事業におけるコムギ無細胞基盤蛋白質生産支援と高親和抗体構築技術開発
  • Author(s): 澤崎 達也
  • Organizer: よこはまNMR構造生物学研究会 第47回ワークショップ「創薬等プラットフォーム」
  • Place of Presentation: 理化学研究所横浜研究所
  • Invited
• [Remarks] CELL-FREE Technology
  • URL: http://www.pros.ehime-u.ac.jp/cell-free/index.html
• [Remarks] CELL-FREE Technology
  • URL: http://www.pros.ehime-u.ac.jp/cell-free/
• [Remarks] CELL-FREE Technology
  • URL: http://www.pros.ehime-u.ac.jp/cell-free/index.html