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: 8240894
• 负责人: PETER K VOGT
• 金额: $ 28.43万
• 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/8240894
• 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. PUBLIC HEALTH RELEVANCE: We propose to generate a freely accessible database of changes in protein synthesis induced by the inhibition of the TOR kinase. The work will examine thousands of proteins and will create a valuable basic resource that will facilitate and accelerate research on aging and on cancer.

描述（由申请人提供）：我们建议应用脉冲SILAC（细胞培养中氨基酸的脉冲稳定同位素标记）的新技术来生成并免费提供TOR抑制对从头合成蛋白质的影响的全球数据库。脉冲SILAC测定在规定的时间间隔内新合成的蛋白质水平，与测量稳态蛋白质水平的传统SILAC有根本不同。我们将确定在雷帕霉素和atp竞争性TOR抑制剂PP242存在和不存在的情况下数千种蛋白质的从头合成。本研究中使用的细胞将是人乳腺上皮细胞系MCF10A和MCF10A H1047R， MCF10A H1047R是磷酸化肌肽3-激酶（PI3K） H1047R突变敲入的同一细胞系。在MCF10A细胞中，在缺乏生长因子的情况下，TOR不受刺激；在MCF10A H1047R细胞中，TOR活性通过PI3K信号的升高而组成性上调。这项工作将与斯克里普斯研究所约翰·耶茨教授的实验室合作进行。脉冲SILAC提供了广泛的蛋白质组覆盖。该数据库将成为宝贵的资源，并将大大加快对衰老和癌症的研究。