Presentations -

14-3-3ζ Interacts with FOXO3a-DBD and Competitively Dissociates DNA through the Tethering Effects via Two Binding Motifs.

Shota Enomoto, Tomoya Kuwayama, Shoichi Nakatsuka, Mariko Yokogawa, Kosaku Kawatsu, Risa Nakamura, Tomomi Kimura, Mikio Tanabe, Toshiya Senda, Jun Saitoh, Hideyuki Saya, Masanori Osawa

[International presentation]  ICMRBS 2024, 

2024.08

, 

Poster presentation

　View Summary

FOXO3a is a transcription activator of apoptosis-related genes [1]. In cancer cells, FOXO3a is phosphorylated by a Ser/Thr kinase AKT, and then recognized by an adopter protein 14-3-3ζ [1]. The 14-3-3ζ binding dissociates FOXO3a from DNA, which suppresses the transcriptional activity [1, 2]. Consequently, apoptosis of the cancer cells is impaired, resulting in the cancer exacerbation. Although the interaction between 14-3-3ζ and FOXO3a is a potential target for anti-cancer drugs, it remains unknown how 14-3-3ζ binds to FOXO3a and dissociates it from DNA. To reveal this mechanism, we investigated the interaction FOXO3a with DNA and 14-3-3ζ. We prepared human 14-3-3ζ as a functional dimer, and human FOXO3a residues 1-284 containing a DNA binding domain (DBD) as well as two 14-3-3ζ-binding motifs, Np and Cp, each of which possess a phosphorylation site, T32 and S253, respectively. Di-phosphorylated form of FOXO3a (dpFOXO3a) was prepared by phosphorylation using AKT. Isothermal titration calorimetry (ITC) analyses showed that the dissociation constant (Kd) for the dpFOXO3a-14-3-3ζ binding was 59 nM and that for the dpFOXO3a-DNA binding was 131 nM. Although their Kd values differed only 2-fold, fluorescence-detection size exclusion chromatography (FSEC) indicated that 95% of DNA bound to dpFOXO3a was substituted by 14-3-3ζ, when 1 equivalent of 14-3-3ζ dimer was added. NMR analyses demonstrated that, while 14-3-3ζ mainly interacts with Np and Cp of dpFOXO3a, it directly interacts with the DNA-binding surface on DBD of dpFOXO3a, indicating that the replacement of DNA with 14-3-3ζ is the direct competition. However, the binding affinity of 14-3-3ζ for the isolated DBD is 700-fold weaker than that of DNA, suggesting that the tethering via Np and Cp should plays an important role in the replacement of DNA. FSEC analyses with mono-phosphorylated form of the FOXO3a mutants demonstrated that 14-3-3ζ binding to N-pFOXO3a (S253A) – DNA complex did not dissociate DNA, whereas 14-3-3ζ binding to C-pFOXO3a (T32A) – DNA complex dissociates 42% of DNA from C-pFOXO3a (T32A). These results together with the FSEC results of dpFOXO3a indicated that the 14-3-3ζ binding to Cp is more necessary to dissociate DNA, and both Np and Cp are required for the complete DNA dissociation. Detailed analysis of the NMR spectra revealed that the DBD of dpFOXO3a bound and traveled to each protomer of the 14-3-3ζ dimer. These results suggest that efficient substitution of DNA with14-3-3ζ occurs by their competition on the DBD, in which direct but weak binding of 14-3-3ζ to the DBD is significantly enhanced by increase in the accessibility and the local concentration of 14-3-3ζ around the DBD through tethering via the Np and Cp.

INT-767の作用機序に基づいた胆汁酸誘導体による新規抗HBV薬の開発

奥村彰規, 荒井潤, 池田和由, 山下ユキコ, 井口裕介, 横川真梨子, 梅澤一夫, 宇根瑞穂, 大澤匡範, 伊藤清顕

[Domestic presentation]  第110回日本消化器病学会総会, 

2024.05

, 

Symposium, workshop panel (public)

Structural insights into the inhibitory mechanism of a transcription factor FOXO3a by 14-3-3ζ

Shota Enomoto, Kuwayama Tomoya, Nakatsuka Shoichi, Yokogawa Mariko, Kawatsu Kosaku, Nakamura Risa, Kimura Tomomi, Tanabe Mikio, Senda Toshiya, Saitoh Jun, Saya Hideyuki, Osawa Masanori

[Domestic presentation]  第46回分子生物学会年会 (神戸) , 

2023.12

, 

Poster presentation, 日本分子生物学会

Structural mechanism for the acceleration of the Caf1-dependent deadenylation of mRNA by BTG2

Nao Kataoka, Mariko Yokogawa, Yuichiro Ishii, Erika Shiro, Hiroka Takashima, Ryoichi Sawazaki, Takeru Sagae, Koichi Ogami, Shin-ichi Hoshino, Masanori Osawa

[Domestic presentation]  第46回分子生物学会年会 (神戸) , 

2023.12

, 

Poster presentation, 日本分子生物学会

タンパク質-タンパク質相互作用をターゲットとした感染阻害大環状物質の探索と構造活性相関解析

米澤朋起, 清水祐吾, 池田和由, 山本雄一朗, 野口耕司, 酒井祥太, 深澤征義, 浅見仁太, 清水敏之, 大戸梅治, 横川真梨子, 大澤匡範

[Domestic presentation]  第51回構造活性相関シンポジウム (東京) , 

2023.11

, 

Oral presentation (general), 日本薬学会構造活性相関部会

Inhibitory mechanism of transcription factor FOXO3a by 14-3-3ζ

Shota Enomoto, Tomoya Kuwayama, Shoichi Nakatsuka, Mariko Yokogawa, Kosaku Kawatsu, Risa Nakamura, Tomomi Kimura, Mikio Tanabe, Toshiya Senda, Jun Saito, Hideyuki Saya, Masanori Osawa

[Domestic presentation]  第62回NMR討論会 (横須賀) , 

2023.11

, 

Oral presentation (general), 日本核磁気共鳴学会

Structural basis for a novel PPI inhibitor – Keap1 binding obtained by NMR interaction analyses

Koujin Kojima, Hanako Ishida, Ayaka Harada, Tomoki Yonezawa, Yugo Shimizu, Kazuyoshi Ikeda, Mariko Yokogawa, Masanori Osawa

[Domestic presentation]  第62回NMR討論会 (横須賀) , 

2023.11

, 

Poster presentation, 日本核磁気共鳴学会

NMR-based drug discovery of the entry inhibitor of SARS-CoV-2 that directly binds to hACE2

Mariko Yokogawa, Mahoro Horiuchi, Shunki Kaneichi, Taiga Otake, Tomoki Yonezawa, Yugo Shimizu, Kazuyoshi Ikeda, Yuichiro Yamamoto, Shota Sakai, Kohji Noguchi, Masayoshi Fukasawa, Masanori Osawa

[Domestic presentation]  第62回NMR討論会 (横須賀) , 

2023.11

, 

Poster presentation, 日本核磁気共鳴学会

NMR-based drug discovery of the entry inhibitor of SARS-CoV-2 that directly binds to spike protein of SARS-CoV-2

Shunki Kaneichi, Mariko Yokogawa, Mahoro Horiuchi, Taiga Otake, Tomoki Yonezawa, Yugo Shimizu, Kazuyoshi Ikeda, Yuichiro Yamamoto, Shota Sakai, Kohji Noguchi, Masayoshi Fukasawa, Masanori Osawa

[Domestic presentation]  第62回NMR討論会 (横須賀) , 

2023.11

, 

Poster presentation, 日本核磁気共鳴学会

Discovery of the middle-sized compounds inhibiting the SARS-CoV-2 viral entry, using in silico approach and NMR analysis

Mariko YOKOGAWA, Mahoro HORIUCHI, Shunki KANEICHI, Taiga OTAKE, Tomoki YONEZAWA, Yugo SHIMIZU, Kazuyoshi IKEDA, Yuichiro YAMAMOTO, Shota SAKAI, Kohji NOGUCHI, Masayoshi FUKASAWA, Masanori OSAWA

[Domestic presentation]  CBI学会2023年大会 (船堀 東京) , 

2023.10

, 

Poster presentation, CBI学会

Structural basis of the inhibition of the Keap1-Nrf2 interaction by novel PPI inhibitors discovered by machine learning

Kojima Koujin, Ishida Hanako, Harada Ayaka, Yonezawa Tomoki, Shimizu Yugo, Ikeda Kazuyoshi, Yokogawa Mariko, Osawa Masanori

[Domestic presentation]  CBI学会2023年大会 (船堀 東京) , 

2023.10

, 

Poster presentation, CBI学会

ALSの原因となるTDP-43の沈殿形成を抑制するRNAの探索

西田優理華, 西村錬, 横川真梨子, 坂上史佳, 横田隆徳, 大澤 匡範

[Domestic presentation]  第６７回日本薬学会関東支部大会 (東京) , 

2023.09

, 

Poster presentation, 日本薬学会

HBV PreS1/NTCP相互作用を標的とした阻害剤簡易スクリーニング系の構築

堀渕慶太, 入間田早瑛, 山本雄一朗, 横川真梨子, 清水祐吾, 米澤朋起, 池田和由, 浅見仁太, 清水敏之, 大戸梅治, 深澤征義, 大澤匡範, 野口耕司

[Domestic presentation]  第６７回日本薬学会関東支部大会 (東京) , 

2023.09

, 

Poster presentation, 日本薬学会

ALSの原因となるTDP-43沈殿を抑制するRNAの創製

西村 錬, 西田 優理華, 横川 真梨子, 坂上 史佳, 横田 隆徳, 大澤 匡範

[Domestic presentation]  第20回次世代を担う若手のためのフィジカル・ファーマフォーラム (PPF2023) (京都) , 

2023.08

-

2023.09

, 

Oral presentation (general), 日本薬学会 物理系薬学部会

リン酸化および14-3-3ζの結合による転写因子FOXO3aの機能抑制機構の解明

榎本 翔太, 桑山知也, 横川真梨子, 中塚将一, 河津光作, 中村吏佐, 木村友美, 齋藤潤, 佐谷秀行, 大澤匡範

[Domestic presentation]  第20回次世代を担う若手のためのフィジカル・ファーマフォーラム (PPF2023) (京都) , 

2023.08

-

2023.09

, 

Oral presentation (general), 日本薬学会 物理系薬学部会

NMRを活用したSARS-CoV-2スパイクタンパク質とhACE2の相互作用を標的とするSARS-CoV-2侵入阻害化合物の分子設計

横川真梨子, 堀内まほろ, 金一駿希, 大竹帝河, 米澤朋起, 清水祐吾, 池田和由, 山本雄一朗, 酒井祥太, 野口耕司, 深澤征義, 大澤匡範

[Domestic presentation]  第23回日本蛋白質科学会年会 (京都) , 

2023.07

, 

Poster presentation, 日本蛋白質科学会

Oolonghomobisflavan CによるB型肝炎ウイルス阻害機構の解明

石場智彬, 横川真梨子, 横田旭美, 大澤匡範

[Domestic presentation]  日本薬学会第143年会 (札幌市) , 

2023.03

, 

Poster presentation, 日本薬学会

医薬品特許化合物データベースの構築とその構造発生への応用

清水祐吾, 幸瞳, 大田雅照, 本間光貴, 大澤匡範, 池田和由

[Domestic presentation]  日本薬学会 第143年会 (札幌市) , 

2023.03

, 

Poster presentation, 日本薬学会

NMRを活用したSARS-CoV-2スパイクタンパク質とhACE2の相互作用を標的とするSARS-CoV-2侵入阻害化合物の分子設計

横川真梨子, 堀内まほろ, 金一駿希, 大竹帝河, 米澤朋起, 清水祐吾, 池田和由, 山本雄一朗, 酒井祥太, 野口耕司, 深澤征義, 大澤匡範

[Domestic presentation]  日本薬学会 第143年会 (札幌市) , 

2023.03

, 

Poster presentation, 日本薬学会

COVID-19の原因となるタンパク質-タンパク質相互作用を標的とする合成中分子阻害剤のインシリコスクリーニング

米澤朋起, 清水祐吾, 池田和由, 山本雄一朗, 野口耕司, 酒井祥太, 深澤征義, 横川真梨子, 大澤匡範

日本薬学会 第143年会 (札幌市) , 

2023.03

, 

Poster presentation, 日本薬学会