Inhibition of TOR and de novo protein synthesis: a pulsed SILAC database

TOR 和从头蛋白质合成的抑制：脉冲 SILAC 数据库

• 批准号: 8328955
• 负责人: PETER K VOGT
• 金额: $ 23.69万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8328955
• 关键词: Aging; Amino Acids; Breast; Cell Culture Techniques; Cell Line; Cells; Characteristics; Chest; Collaborations; Complex; Data Set; Databases; Epithelial Cells; Genetic Transcription; Growth Factor; Human; Investigation; Knock-in Mouse; Laboratories; Life; Longevity; MCF10A cells; Malignant Neoplasms; Mammary Gland Parenchyma; Measures; Mediating; Metabolism; Mus; Mutation; Normal Cell; Phosphatidylinositols; Phosphotransferases; Physiologic pulse; Protein Biosynthesis; Protein-Serine-Threonine Kinases; Proteins; Proteome; Proteomics; Research; Research Institute; Resources; Role; Signal Transduction; Sirolimus; Stable Isotope Labeling; Techniques; Time; Translations; Work; anticancer research; cell growth; design; gain of function mutation; inhibitor/antagonist; professor; response; time interval

DESCRIPTION (provided by applicant): We propose to apply the new technique of pulsed SILAC (pulsed stable isotope labeling by amino acids in cell culture) to generate and make freely available a global database of the effects of TOR inhibition on de novo protein synthesis. Pulsed SILAC determines the levels of proteins that are newly synthesized during a defined time interval and is fundamentally different from conventional SILAC which measures steady-state protein levels. We will determine de novo synthesis for several thousand proteins in the presence and absence of rapamycin and of the ATP-competitive TOR inhibitor PP242. The cells to be used in this study will be MCF10A, a human breast epithelial cell line, and MCF10A H1047R, the same cell line with a knock-in of the H1047R mutation of phosphoinositide 3-kinase (PI3K). In MCF10A cells, TOR is not stimulated in the absence of growth factors; in MCF10A H1047R cells, TOR activity is constitutively upregulated by elevated signaling from PI3K. The work will be carried out in collaboration with the laboratory of Professor John Yates at the Scripps Research Institute. Pulsed SILAC provides extensive coverage of the proteome. The proposed database will become a valuable resource and will greatly accelerate research on aging and on cancer.

描述（由申请人提供）：我们建议应用脉冲SILAC的新技术（氨基酸在细胞培养中通过氨基酸标记）来生成并免费获得Tor抑制对DE NOVO蛋白质的影响的全局数据库。脉冲Silac确定了在定义的时间间隔内新合成的蛋白质水平，并且与测量稳态蛋白水平的常规SILAC根本不同。在存在和不存在雷帕霉素和ATP竞争力的TOR抑制剂PP242的情况下，我们将确定几千种蛋白质的从头合成。在这项研究中使用的细胞将是MCF10A，是人类乳腺上皮细胞系和MCF10A H1047R，该细胞与磷酸肌醇3-激酶（PI3K）的H1047R突变相同的细胞系（PI3K）。在MCF10A细胞中，在没有生长因子的情况下不会刺激Tor。在MCF10A H1047R细胞中，TOR活性由PI3K的信号升高而组成地上调。这项工作将与Scripps研究所的John Yates教授的实验室合作进行。 脉冲Silac提供了蛋白质组的广泛覆盖范围。拟议的数据库将成为一种宝贵的资源，并将大大加速有关衰老和癌症的研究。

项目成果

A butterfly effect in cancer.

癌症中的蝴蝶效应。

• DOI: 10.1080/23723556.2015.1029063
• 发表时间: 2016
• 期刊: Molecular & cellular oncology
• 影响因子: 2.1
• 作者: Vogt,PeterK;Hart,JonathanR;Yates3rd,JohnR
• 通讯作者: Yates3rd,JohnR