Creation of Functional Protein Oligomers Utilizing the Structural Change of the Flexible Loop Region

• Project Area: Science on Function of Soft Molecular Systems by Cooperation of Theory and Experiment
• Project/Area Number: 16H00839
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Nara Institute of Science and Technology
• Principal Investigator: 廣田 俊 奈良先端科学技術大学院大学, 物質創成科学研究科, 教授 (90283457)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥6,760,000 (Direct Cost: ¥5,200,000、Indirect Cost: ¥1,560,000); Fiscal Year 2017: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2016: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000)
• Keywords: タンパク質 / 超分子 / 多量体 / 生体分子 / バイオナノ / ヘム / ドメインスワッピング / 蛋白質 / ナノバイオ

Outline of Annual Research Achievements

3Dドメインスワッピングはタンパク質分子間で同一構造領域を交換する現象である。本研究では、様々なヘムタンパク質を対象に、ループ部位に着目してドメインスワッピングについて調査する。本年度は、好熱菌シトクロムc552と緑膿菌シトクロムc551間でN末端領域を交換した2種類のキメラタンパク質のドメインスワッピングを利用し、Met/His配位の活性部位を2つ有するヘテロ2量体タンパク質を作製した。さらに、片方のキメラタンパク質の活性部位構造を変異させることにより、Met/His配位とHis/H2O配位の異なる活性部位を有するヘテロ2量体タンパク質を作製した。異なる活性部位を有するヘテロ2量体タンパク質のHis/H2O部位への酸素分子の結合は共鳴ラマンスペクトルにより確認した。還元型ヘテロ2量体タンパク質を酸素分子と反応させると、Fe-O2伸縮振動（νFe-O2）に由来する振動バンドが580 cm-1に観測され、18O2結合型でνFe-O2は 554 cm-1に低波数シフトした。これらの結果より、ドメインスワッピングが異なる活性部位を有する多ヘムタンパク質の構築に有用であることが示された。さらに、ドメインスワッピングしたシトクロムc 2量体をレーザートラッピング法により溶液中で局所的に集め、球状のアミロイド線維凝集体を作製することに成功した。アミロイド線維の形成はチオフラビンTによる蛍光および電子顕微鏡像により確認した。作製したアミロイド線維凝集体を直線および3次元に配列することにも成功した。また、シトクロムcの多量体形成メカニズムの解明を目指して、分子動力学シミュレーションと液体の積分方程式理論を用いて単量体と二量体の熱力学安定性の解析を行った。

Research Progress Status

29年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

29年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] China West Normal University/University of South China(中国)
• [Int'l Joint Research] National Chiao Tung University(台湾)
• [Journal Article] Theoretical analysis of the domain-swapped dimerization of cytochrome c: An MD and 3D-RISM approach (2018)
  • Author(s): Yoshida Norio、Higashi Masahiro、Motoki Hideyoshi、Hirota Shun
  • Journal Title: The Journal of Chemical Physics Volume: 148 Issue: 2 Pages: 025102-025102
  • DOI: 10.1063/1.5009785
  • Peer Reviewed / Open Access
• [Journal Article] Oxidative Modification of Methionine80 in Cytochrome c by Reaction with Peroxides (2018)
  • Author(s): Ari Dwi Nugranehi, Chunguang Ren, Yorifumi Matsumoto, Satoshi Nagao, Masaru Yamanaka, Shun Hirota
  • Journal Title: J. Inorg. Biochem. Volume: 182 Pages: 200-207
  • DOI: 10.1016/j.jinorgbio.2018.02.017
  • NAID: 120006726654
  • Peer Reviewed
• [Journal Article] Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an α-Helix-Inserted Circular Permutant of Cytochrome c 555 (2018)
  • Author(s): Oda Akiya、Nagao Satoshi、Yamanaka Masaru、Ueda Ikki、Watanabe Hiroki、Uchihashi Takayuki、Shibata Naoki、Higuchi Yoshiki、Hirota Shun
  • Journal Title: Chemistry - An Asian Journal Volume: 印刷中 Issue: 8 Pages: 964-967
  • DOI: 10.1002/asia.201800252
  • NAID: 120006726655
  • Peer Reviewed
• [Journal Article] Theoretical Study on Oligomerization of Cytochrome <i>c</i> (2018)
  • Author(s): 根木 秀佳、吉田 紀生、廣田 俊、東 雅大
  • Journal Title: Journal of Computer Chemistry, Japan Volume: 17 Issue: 1 Pages: 8-13
  • DOI: 10.2477/jccj.2018-0006
  • NAID: 130006528632
  • ISSN: 1347-1767, 1347-3824
  • Peer Reviewed / Open Access
• [Journal Article] A Single Spherical Assembly of Protein Amyloid Fibrils Formed by Laser Trapping (2017)
  • Author(s): K. Yuyama, M. Ueda, S. Nagao, S. Hirota, T. Sugiyama, and H. Masuhara
  • Journal Title: Angewandte Chemie Interanational Edition Volume: 56 Issue: 24 Pages: 6739-6743
  • DOI: 10.1002/anie.201702352
  • NAID: 120006473600
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Effect of Methionine80 Heme Coordination on Domain Swapping of Cytochrome c (2017)
  • Author(s): S. Hirota, N. Yamashiro, Z. Wang, and S. Nagao
  • Journal Title: Journal of Biological Inorganic Chemistry Volume: 22 Issue: 5 Pages: 705-712
  • DOI: 10.1007/s00775-017-1446-3
  • NAID: 120006334946
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Rational Design of Domain-Swapping-Based c-Type Cytochrome Heterodimers by Using Chimeric Proteins (2017)
  • Author(s): Mohan Zhang, Tukasa Nakanishi, Masaru Yamanaka, Satoshi Nagao, Sachiko Yanagisawa, Yasuhito Shomura, Naoki Shibata, Takashi Ogura, Yoshiki Higuchi, Shun Hirota
  • Journal Title: ChemBioChem Volume: 18 Issue: 17 Pages: 1712-1715
  • DOI: 10.1002/cbic.201700219
  • NAID: 120006726652
  • Peer Reviewed
• [Journal Article] Design of Artificial Metalloproteins/Metalloenzymes by Tuning Noncovalent Interactions (2017)
  • Author(s): Shun Hirota, Ying-Wu Lin
  • Journal Title: J. Biol. Inorg. Chem. Volume: 23 Issue: 1 Pages: 7-25
  • DOI: 10.1007/s00775-017-1506-8
  • NAID: 120006763860
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] タンパク質ドメインスワッピングの構造・機構解析 (2017)
  • Author(s): 廣田俊、長尾聡、山中優
  • Journal Title: ぶんせき Volume: 2017 Pages: 464-467
  • Peer Reviewed
• [Journal Article] Formation and Carbon Monoxide-Dependent Dissociation of Allochromatium vinosum Cytochrome c' Oligomers Using Domain-Swapped Dimers (2017)
  • Author(s): M. Yamanaka, M. Hoshizumi, S. Nagao, R. Nakayama, N. Shibata, Y. Higuchi, and S. Hirota
  • Journal Title: Protein Science Volume: 26 Issue: 3 Pages: 464-474
  • DOI: 10.1002/pro.3090
  • NAID: 120006334944
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Improved Stopped-Flow Time-Resolved Resonance Raman Spectroscopy Device for Studying Enzymatic Reactions (2017)
  • Author(s): S. Yanagisawa, M. S. Deshpande, S. Hirota, T. Nakagawa, and T. Ogura
  • Journal Title: Journal of Raman Spectroscopy Volume: 48 Issue: 5 Pages: 680-685
  • DOI: 10.1002/jrs.5100
  • NAID: 120006334945
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Observation of the Cooperative Collapse in the Spontaneous Folding Process of Cytochrome c by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy (2018)
  • Author(s): Miyuki Sakaguchi, Masaru Yamanaka, Shun Hirota, Kunihiko Ishii, Tahei Tahara
  • Organizer: 62nd Annual Meeting of Biophysical Society
  • Int'l Joint Research
• [Presentation] Effect of Rare Codon Positions on Hydrogenobacter thermophiles cyt c552 Oligomerization in E. coli Cells (2018)
  • Author(s): Hongxu Yang, Masaru Yamanaka, Satoshi Nagao, Shun Hirota
  • Organizer: 98th Chemical Society of Japan Annual Meeting
• [Presentation] Construction of a Supramolecule with an Artificial Protein Based on Cytochrome c555 (2018)
  • Author(s): Akiya Oda, Satoshi Nagao, Masaru Yamanaka, Ikki Ueda, Naoki Shibata, Yoshiki Higuchi, Shun Hirota
  • Organizer: 98th Chemical Society of Japan Annual Meeting
• [Presentation] Design and Properties of Domain-Swapped Myoglobin Dimer (2018)
  • Author(s): Satoshi Nagao, Suda Ayaka, Hisashi Kobayashi, Naoki Shibata, Yoshiki Higuchi, Shun Hirota
  • Organizer: 98th Chemical Society of Japan Annual Meeting
• [Presentation] 耐熱性シトクロムcへのCO応答性四次構造変化の付与と多量化 (2018)
  • Author(s): 山中優、中山諒子、若井暁、藤井創太郎、越澤大典、三本木至宏、廣田俊
  • Organizer: 日本化学会第98春季年会
• [Presentation] 脂質二分子膜への結合によるシトクロムc部分変性に関する溶液NMR解析 (2018)
  • Author(s): 小林紀、長尾聡、廣田俊
  • Organizer: 日本化学会第98春季年会
• [Presentation] ドメインスワッピングを実現するミニマルなデザイン (2018)
  • Author(s): 志賀翔多、山中優、廣田俊、真壁幸樹
  • Organizer: 日本化学会第98春季年会
• [Presentation] Design of Domain-Swapped Myoglobin Dimer (2017)
  • Author(s): Satoshi Nagao, Ayaka Suda, Hisashi Kobayashi, Naoki Shibata, Yoshiki Higuchi, and Shun Hirota
  • Organizer: 97th Chemical Society of Japan Annual Meeting
  • Place of Presentation: 慶応義塾大学(神奈川県横浜市)
  • Year and Date: 2017-03-16
• [Presentation] Rhodopseudomonas palustris由来シトクロムc′の多量化 (2017)
  • Author(s): 久保田真史, 宮本昂明、廣田俊
  • Organizer: 日本化学会第97春季年会
  • Place of Presentation: 慶応義塾大学(神奈川県横浜市)
  • Year and Date: 2017-03-16
• [Presentation] Laser Trapping Dynamics of Amyloid Fibril Formation of Cytochrome c Monomer and Dimers (2017)
  • Author(s): Chieh-Ju Chang, Yugo Hayashi, Shun Hirota, Ken-ichi Yuyama, Teruki Sugiyama, and Hirotashi Masuhara
  • Organizer: 97th Chemical Society of Japan Annual Meeting
  • Place of Presentation: 慶応義塾大学(神奈川県横浜市)
  • Year and Date: 2017-03-16
• [Presentation] Effect of Rare Codons on Hydrogenobacter thermophiles cyt c552 (2017)
  • Author(s): Hongxu Yang, Masaru Yamanaka, Satoshi Nagao, and Shun Hirota
  • Organizer: 97th Chemical Society of Japan Annual Meeting
  • Place of Presentation: 慶応義塾大学(神奈川県横浜市)
  • Year and Date: 2017-03-16
• [Presentation] Formation of Cytochrome c Oligomers Using Domain-Swapped Dimers and Carbon Monoxide-Dependent Control of Oligomer Association/Dissociation (2017)
  • Author(s): Masaru Yamanaka, Ryoko Nakayama, Satoshi Nagao, Makoto Hoshizumi, Naoki Shibata, Yoshiki Higuchi, and Shun Hirota
  • Organizer: 97th Chemical Society of Japan Annual Meeting
  • Place of Presentation: 慶応義塾大学(神奈川県横浜市)
  • Year and Date: 2017-03-16
• [Presentation] Protein Nanostructures Constructed by Fusing Two Oligomerization Domains with a 3-Helix Bundle Linker (2017)
  • Author(s): Takaaki Miyamoto and Shun Hirota
  • Organizer: 97th Chemical Society of Japan Annual Meeting
  • Place of Presentation: 慶応義塾大学(神奈川県横浜市)
  • Year and Date: 2017-03-16
• [Presentation] Construction of a Triangle-shaped Structure with a Unit Protein Based on Cytochrome c555 (2017)
  • Author(s): Akiya Oda, Ikki Ueda, Masaru Yamanaka, Satoshi Nagao, Naoki Shibata, Yoshiki Higuchi, and Shun Hirota
  • Organizer: 97th Chemical Society of Japan Annual Meeting
  • Place of Presentation: 慶応義塾大学(神奈川県横浜市)
  • Year and Date: 2017-03-16
• [Presentation] Cytochrome c: Domain Swapping, Self-Modification, and Molecular Recognition (2017)
  • Author(s): Shun Hirota, Satoshi Nagao, Masaru Yamanaka, and Yoshiki Higuchi
  • Organizer: 5th Symposium on Advanced Biological Inorganic Chemistry
  • Place of Presentation: Kolkata, India
  • Year and Date: 2017-01-07
  • Int'l Joint Research / Invited
• [Presentation] シトクロムcの多量体形成に関する理論的研究 (2017)
  • Author(s): 根木秀佳、吉田紀生、廣田俊、東雅大
  • Organizer: 研究会「凝集系の理論化学2017」
• [Presentation] 二次元蛍光寿命相関分光法による脂質膜上シトクロムcの構造ゆらぎの研究 (Study of Conformational Fluctuation of Cytochrome c on a Lipid Membrane by 2D Fluorescence Lifetime Correlation Spectroscopy) (2017)
  • Author(s): 坂口美幸、山中優、廣田俊、石井邦彦、田原太平
  • Organizer: 平成29年度日本分光学会年次講演会
• [Presentation] Heme Protein Supramolecules Constructed by Domain Swapping (2017)
  • Author(s): Shun Hirota
  • Organizer: 14th International Symposium on Applied Bioinorganic Chemistry (ISABC14)
  • Int'l Joint Research / Invited
• [Presentation] ミニマルなデザインで実現するライム病関連蛋白質OspAのドメインスワッピング (2017)
  • Author(s): 志賀翔多、山中優、廣田俊、真壁幸樹
  • Organizer: 第17回日本蛋白質科学会年会
• [Presentation] 耐熱性シトクロムc′の2量体界面残基置換によるCO応答性4次構造変化の付与 (2017)
  • Author(s): 中山諒子、山中優、藤井創太郎、越澤大典、三本木至宏、廣田俊
  • Organizer: 第17回日本蛋白質科学会年会
• [Presentation] 負電荷脂質二分子膜との相互作用によって生じる部分変性したシトクロムcの溶液NMR解析 (2017)
  • Author(s): 小林紀、長尾聡、廣田俊
  • Organizer: 第17回日本蛋白質科学会年会
• [Presentation] NMR Characterization of Cytochrome c Membrane-Binding Site Using Cardiolipin-Containing Bicelle (2017)
  • Author(s): 長尾聡、小林紀、廣田俊
  • Organizer: 第17回日本蛋白質科学会年会
• [Presentation] CO-Dependent Control of Oligomer Association/Dissociation Using Domain-Swapped Dimers of Cytochrome c′ (2017)
  • Author(s): 山中優、中山諒子、長尾聡、星住誠人、柴田直樹、樋口芳樹、廣田俊
  • Organizer: 第17回日本蛋白質科学会年会
• [Presentation] MD and 3D-RISM Study on the Domain-Swapped Cytochrome c Oligomer (2017)
  • Author(s): Hideyoshi Motoki, Norio Yoshida, Shun Hirota, Masahiro Higashi
  • Organizer: The 9th Conference of the Asian Consortium on Computational Materials Science (ACCMS-9)
  • Int'l Joint Research
• [Presentation] Fluorescence Analysis on Laser Trapping-Induced Amyloid Fibril Formation of Cytochrome c (2017)
  • Author(s): Chieh-Ju Chang, Yugo Hayashi, Shun Hirota, Ken-ichi Yuyama, Hiroshi Masuhara, Teruki Sugiyama
  • Organizer: 2017年光化学討論会
• [Presentation] リン脂質バイセルを用いた膜結合シトクロムcの動的挙動に関する溶液NMR解析（Solution NMR Studies on the Dynamic Properties of Membrane-Bound Cytochrome c Using Phospholipid Bicelles） (2017)
  • Author(s): 長尾聡、小林紀、廣田俊
  • Organizer: 第11回バイオ関連化学シンポジウム
• [Presentation] シトクロムc555をベースに設計した人工タンパク質を用いたナノ構造体構築（Construction of a Nanostructure with a Designed Protein Based on Cytochrome c555） (2017)
  • Author(s): 小田祥也、上田一凱、山中優、長尾聡、柴田直樹、樋口芳樹、廣田俊
  • Organizer: 第11回バイオ関連化学シンポジウム
• [Presentation] 分子化学討論会MDシミュレーションと3D-RISM理論を用いたシトクロムcの多量体形成に関する理論的研究（Theoretical Study on Oligomer Formation of Cytochrome c with MD Simulation and 3D-RISM Theory） (2017)
  • Author(s): 根木秀佳、吉田紀生、廣田俊、東雅大
  • Organizer: 第11回分子化学討論会
• [Presentation] Site-selective Observation of Folding Dynamics of Cytochrome c by Two-dimensional Fluorescence Lifetime Correlation Spectroscopy（二次元蛍光寿命相関分光法によるシトクロムｃのフォールディング過程の部位選択的な観測） (2017)
  • Author(s): Miyuki Sakaguchi, Masaru Yamanaka, Shun Hirota, Kunihiko Ishii, Tahei Tahara
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] シトクロムc′ドメインスワップ2量体を用いたCO依存的な多量体形成・解離 (2017)
  • Author(s): 山中優、中山諒子、長尾聡、柴田直樹、樋口芳樹、廣田俊
  • Organizer: 日本農芸化学会関西・中四国・西日本支部2017年度合同大阪大会
• [Presentation] 異なるCO応答性を示す相同シトクロムc′の変異導入解析 (2017)
  • Author(s): 中山諒子、山中優、藤井創太郎、越澤大典、三本木至宏、廣田俊
  • Organizer: 日本農芸化学会関西・中四国・西日本支部2017年度合同大阪大会
• [Presentation] Construction of Heme Protein Oligomers be 3D Domain Swapping (2017)
  • Author(s): Shun Hirota, Satoshi Nagao, Masaru Yamanaka, Yoshiki Higuchi
  • Organizer: 2nd International Symposium on Biofunctional Chemistry (ISBC2017)
  • Int'l Joint Research
• [Presentation] NMR Characterization of Cytochrome c Membrane-Binding Site Using Cardiolipin-Containing Bicelles (2017)
  • Author(s): Satoshi Nagao, Hisashi Kobayashi, Shun Hirota
  • Organizer: 2nd International Symposium on Biofunctional Chemistry (ISBC2017)
  • Int'l Joint Research
• [Presentation] Construction of Heme Protein Supramolecules by Domain Swapping (2016)
  • Author(s): Shun Hirota, Satoshi Nagao, Masaru Yamanaka, and Yoshiki Higuchi
  • Organizer: 8th Asian Biological Inorganic Chemistry Conference
  • Place of Presentation: Auckland, New Zealand
  • Year and Date: 2016-12-04
  • Int'l Joint Research / Invited
• [Presentation] Formation of Myoglobin Dimers by Domain Swapping (2016)
  • Author(s): Satoshi Nagao, Ayaka Suda, Hisashi Kobayashi, Naoki Shibata, Yoshiki Higuchi, and Shun Hirota
  • Organizer: 第54回日本生物物理学会年会
  • Place of Presentation: つくば国際会議場（茨城県つくば市）
  • Year and Date: 2016-11-25
• [Presentation] Structure and Function of Heme Protein Oligomers Constructed by Domain Swapping (2016)
  • Author(s): Shun Hirota
  • Organizer: Japan-Korea-Taiwan Bioinorganic Chemistry Symposium
  • Place of Presentation: 岡崎コンファレンスセンター（愛知県岡崎市）
  • Year and Date: 2016-09-29
  • Int'l Joint Research / Invited
• [Presentation] Application of Supramolecular Science for Drug Design (2016)
  • Author(s): Shun Hirota
  • Organizer: The Symposium of Annual Scientific Meeting 2016 of Indonesian Pharmacist Association
  • Place of Presentation: Jogjakarta, Indonesia
  • Year and Date: 2016-09-27
  • Int'l Joint Research / Invited
• [Presentation] 過酸との反応によるシトクロムcのMet80の酸化修飾 (2016)
  • Author(s): 松本順文、REN Chunguang、山中優、長尾聡、廣田俊
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 石川県立音楽堂・もてなしドーム地下イベント広場（石川県金沢市）
  • Year and Date: 2016-09-07
• [Presentation] ミオグロビンのループ領域アミノ酸変異によるドメインスワップ二量体のデザイン (2016)
  • Author(s): 須田綾香、小林紀、長尾聡、柴田直樹、樋口芳樹、廣田俊
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 石川県立音楽堂・もてなしドーム地下イベント広場（石川県金沢市）
  • Year and Date: 2016-09-07
• [Presentation] Domain Swapping in c-Type Cytochromes and Myoglobin (2016)
  • Author(s): Shun Hirota, Satoshi Nagao, Partha Pratim Parui, Megha Subhash Deshpande, Chunguang Ren, Yugo Hayashi, Takaaki Miyamoto, Yin-Wu Lin, and Yoshiki Higuchi
  • Organizer: 30th Anniversary Symposium of the Protein Society
  • Place of Presentation: Baltimore, USA
  • Year and Date: 2016-07-16
  • Int'l Joint Research
• [Presentation] Construction of Zn-SO4 Cluster-encapsulating Protein Nanocage by Domain Swapping (2016)
  • Author(s): Takaaki Miyamoto, Mai Kuribayashi, Satoshi Nagao, Yasuhito Shomura, Yoshiki Higuchi, and Shun Hirota
  • Organizer: 30th Anniversary Symposium of the Protein Society
  • Place of Presentation: Baltimore, USA
  • Year and Date: 2016-07-16
  • Int'l Joint Research
• [Presentation] Domain Swapping of c-Type Cytochromes and Myoglobin (2016)
  • Author(s): Shun Hirota, Satoshi Nagao, Yugo Hayashi, Takaaki Miyamoto, Chunguang Ren, Masaru Yamanaka, Ying-Wu Lin, Hirofumi Komori, Yasuhito Shomura, and Yoshiki Higuchi
  • Organizer: Ninth International Conference on Porphyrins and Phthalocyanines
  • Place of Presentation: Nanjing, China
  • Year and Date: 2016-07-03
  • Int'l Joint Research / Invited
• [Presentation] 四量体シトクロムc’の作製とCO分子結合・解離によるサイズ制御 (2016)
  • Author(s): 山中優、星住誠人、長尾聡、廣田俊
  • Organizer: 第16回日本蛋白質科学会年会
  • Place of Presentation: 福岡国際会議場（福岡県福岡市）
  • Year and Date: 2016-06-07
• [Presentation] ドメインスワップしたシトクロムcb562二量体が形成するZn-SO4クラスター内包タンパク質ナノケージの結晶構造 (2016)
  • Author(s): 宮本昂明、長尾聡、庄村康人、樋口芳樹、廣田俊
  • Organizer: 第16回日本蛋白質科学会年会
  • Place of Presentation: 福岡国際会議場（福岡県福岡市）
  • Year and Date: 2016-06-07