New cancer therapy by degradation of specific proteins

通过降解特定蛋白质的新癌症疗法

• 批准号: 13557019
• 负责人: NAKAYAMA Keiichi
• 金额: $ 9.02万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557019/
• 关键词: Myc; Ubiquitiylation; chimeric-protein; Mad; Nedd-4; Mad; ユビキチンリガーゼ; Nedd4; プロテアゾームインヒビター

We proposed to induce to degradation of specific protein by the modification of ubiquitin-ligase, Recently, it is reported that expression level of many proteins are regulated by not only transcription but degradation rate. Ubiquitin-porteasome system is one of the most useful system to regulate expression level of protein because of its specificity. On this unique point we try to create new ubiquitin-ligase to recognize and induce to degradate proteins which are toxic for our bodies.In this project, we used oncoprotein, Myc as model protein. Because Myc is known to bind to Max, we made several chimeric proteins, Max/Nedd4, Max/b-TrCP1, Max/CHIP. Nedd-4 is a HECT-type ubiquitin-ligase. b-TrCP1 is a F-box proteins which is a component of SCF-type ubiquitin-ligase. CHIP is a U-box type ubiquitin-ligase. It was hypothesized that Myc would be bound and ubiquitylated by these chmeric proteins.We confirmed that this artificial protein Max/CHIP bound to Myc in vitro system and also immunoprecipitation assay in vivo. The half-life of Myc reduced by overexpression of Max/CHIP protein in mammalian cells by expression vector most efficiently.Plaque assays and colony-formation assay shown that Myc-induced transformation is inhibited by Max/CHIP chimeric protein expression.Now, we observe the effect on whole bode. We implanted tumor cells which overexpressed Max/CHIP chimeric protein in Nude mouse to examine the effect of tumorigenesis.

我们提出通过修饰泛素连接酶来诱导特定蛋白质的降解，最近的研究表明，许多蛋白质的表达水平不仅受转录的调节，而且受降解速率的调节。泛素-蛋白酶体系统因其特异性而成为调节蛋白质表达水平的最常用的系统之一。在这一独特的点上，我们试图创造新的泛素连接酶来识别和诱导降解对我们身体有害的蛋白质。在这个项目中，我们使用癌蛋白Myc作为模型蛋白。由于已知Myc与MAX结合，我们制备了几种嵌合蛋白，MAX/Nedd4，Max/b-TrCP1，MAX/ChIP。NIDD-4是一种羟基型泛素连接酶。B-TrCP1是一种F-box蛋白，是SCF型泛素连接酶的组成部分。CHIP是一种U盒型泛素连接酶。我们在体外实验和体内免疫沉淀实验中证实了这种人工合成的MAX/CHIP蛋白与Myc结合。通过表达载体在哺乳动物细胞中过表达MAX/CHIP蛋白可以最有效地减少Myc的半衰期。普通实验和集落形成实验表明MAX/CHIP嵌合蛋白的表达抑制了Myc诱导的转化。我们将过表达MAX/CHIP嵌合蛋白的肿瘤细胞移植到裸鼠体内，以检测其成瘤作用。

项目成果

Kanematsu, T. et al.: "Role of the PLC-related, catalytically inactive protein p130 in GABA_A receptor function"EMBO J.. 21. 1004-1011 (2002)

Kanematsu, T. 等人：“PLC 相关的催化失活蛋白 p130 在 GABA_A 受体功能中的作用”EMBO J.. 21. 1004-1011 (2002)

Shimoda K. et. al.: "Tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-α-induced suppression of B lymphocyte formation"J. Immunol.. 169. 4707-4711 (2002)

Shimoda K. 等人：“Tyk2 是 Daxx 的诱导和核易位所必需的，它调节 IFN-α 诱导的 B 淋巴细胞形成抑制”J.Immunol.. 169. 4707-4711 (2002)

Nagahama, H.: "Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27^<Kip1> and p57^<Kip2> during mouse development"Anat. Embryol. 203. 77-87 (2001)

Nagahama, H.：“小鼠发育过程中细胞周期蛋白依赖性激酶 (CDK) 抑制剂 p27^<Kip1> 和 p57^<Kip2> 的空间和时间表达模式”Anat。

Shimoda K. et al.: "Tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-α-induced suppression of B lvmphocvte formation"J. Immunol. I. 169. 4707-4711 (2002)

Shimoda K. 等人：“Tyk2 是 Daxx 的诱导和核转位所必需的，Daxx 调节 IFN-α 诱导的 B 淋巴细胞形成抑制”J.Immunol.169.4707-4711 (2002)

Koguchi, K. et al.: "Modulation of astrocyte proliferation by cyclin-dependent kinase inhibitor p27Kip1"Glia. 37. 93-104 (2002)

Koguchi, K. 等人：“细胞周期蛋白依赖性激酶抑制剂 p27Kip1 对星形胶质细胞增殖的调节”Glia。