莪术醇是一种抗肿瘤效果明确的天然化合物，鉴定莪术醇在肿瘤细胞中的相互作用靶点蛋白对阐明其抗肿瘤作用机制以及探索新的肿瘤治疗靶点具有重要意义。我们已建立了莪术醇配基亲和层析方法，并且利用此方法从293T细胞中鉴定出了潜在的药物相互作用蛋白。此项目拟利用莪术醇配基亲和层析方法结合质谱技术从鼻咽癌细胞亚细胞组分提取物中鉴定莪术醇潜在相互作用蛋白；然后利用半高通量筛查方法、药物Pull down、药物Western dot blot及药物诱导靶标蛋白稳定性检测等方法确定莪术醇相互作用蛋白。在此基础上，检测莪术醇对其靶标蛋白的调控作用，包括莪术醇靶标蛋白表达水平、翻译后修饰水平、细胞内定位、酶活力、及其与染色体或下游蛋白分子的结合能力等指标；并利用慢病毒系统构建靶标蛋白获得功能与缺失功能的细胞模型，利用此模型研究莪术醇通过其相互作用蛋白发挥抗肿瘤功能及其机制。

Curcumol has been reported to be an anti-tumor natural compound, while the underlying mechanism is still unknown. It is of importance to identify direct curcumol-interaction proteins for uncovering its anti-tumor mechanisms and probing the new cancer therapy targets. Our laboratory has successfully developed a method of drug affinity chromatograph by conjugating curcumol to agarose beads via chemical modification of curcumol without interference of its antitumor function. In this project, drug affinity chromatograph and mass spectrometry methods are employed to identify potential curcumol-interaction proteins from subcellular protein extraction of human nasopharyngeal carcinoma CNE-2 cells. The candidate target proteins will be validated by the methods of half-high throughput screening, drug pull-down，drug western dot blot and drug affinity responsive target protein stability. Furthermore, the protein level, protein post translational modification level, cellular localization, enzyme activity and downstream functions of the target protein will be detected to determine whether curcumol could regulate its target function by some of these pathways. We also propose to construct cell models, gain or loss of curcumol target protein functions, using lentivirus expression systems. These cell models are used to determine whether antagonist or agonist function is exerted by curcumol to its target protein, and to explore the downstream anti-tumor mechanisms mediated by curcumol-interacting protein.