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（细胞培养物中氨基酸的脉冲稳定同位素标记）新技术，以生成并免费提供TOR抑制对从头蛋白质合成影响的全球数据库。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常规SILAC有根本不同。我们将确定在存在和不存在雷帕霉素和ATP竞争性TOR抑制剂PP 242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MCF 10A（一种人乳腺上皮细胞系）和MCF 10A H1047 R（一种敲入磷酸肌醇3-激酶（PI 3 K）H1047 R突变的相同细胞系）。在MCF 10A细胞中，TOR在不存在生长因子的情况下不被刺激;在MCF 10A H1047 R细胞中，TOR活性通过来自PI 3 K的升高的信号传导而组成性上调。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提供蛋白质组的广泛覆盖。拟议中的数据库将成为一个宝贵的资源，并将大大加快对衰老和癌症的研究。

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.