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

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一个关于TOR抑制对从头蛋白质合成的影响的全球数据库。脉冲SILAC确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。我们将确定数千种蛋白质在雷帕霉素和ATP竞争性TOR抑制剂pp242存在和不存在的情况下的从头合成。这项研究中使用的细胞将是人乳腺上皮细胞系MCF10A和具有磷脂酰肌醇3-激酶(PI3K)H1047R突变敲入的同一细胞系MCF10A H1047R。在MCF10A细胞中，在没有生长因子的情况下，TOR不被刺激；在MCF10A H1047R细胞中，PI3K信号的升高使TOR活性结构性上调。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提供了对蛋白质组的广泛覆盖。拟议的数据库将成为一种宝贵的资源，并将极大地加快关于衰老和癌症的研究。

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.