USP9X通过去泛素化eIF4B调控蛋白翻译起始及其在肿瘤靶向治疗中的应用研究

Protein translation is one of most highly regulated and fundamental processes in cells. Precise regulation and control of this process are essential to all biological activities in vivo. Translation initiation is the first of three steps in protein synthesis, and it is also the rate limiting step. Dysregulation of translation has been reported in multiple sources of tumors, and overexpression of different translation initiation factors has been shown to cause tumor transformation. In addition, a number of signaling pathways which regulate translation have been connected to tumorigenesis. Although several specific translation inhibitors have potential anti-tumor activities and have entered pre-clinical trials for tumor therapy, understanding the mechanisms and regulation of translational machinery will further facilitate new drug development efforts against translation. After eIF4A was identified as the target of natural product Pateamine A, we used tandem affinity purification techniques to study the interaction network of eIF4A and eIF4B in the presence of Pateamine A. Ubiquitin specific peptidase 9 X (USP9X) was identified as an interacting protein with eIF4B. We have confirmed through immunoprecipitation that eIF4B can specifically interact with USP9X, and knockdown of USP9X caused the inhibition of general translation in an in vivo 35S methionine labeling experiment. Through the analysis of cellular polysome profile we observed increased 48s and decreased 80s and polysome,suggesting that USP9X regulates translation at the initiation step. At same time, we found that eIF4B can be monoubiquitinylated in vivo and in vitro. We will continue to characterize the function of monoubiquitinylated eIF4B in the process of translation initiation, and determine whether USP9X modulates translation initiation by deubiquitination of eIF4B. Once this is validated, the correlation of USP9X and eIF4B will be analyzed by detection of protein and mRNA expression in hepatocellular carcinoma. The ability of USP9X and eIF4B to induce tumor transformation will be evaluated through cell proliferation assays and clonal formation in different cell lines after manipulation of the expression of both proteins. Using specific small molecule inhibitors against USP9X and translation initiation, the synergistic effect of two inhibitors will be measured in different tumor cell lines, and the effect of optimized combinations of two compounds will be further evaluated in a nude mouse tumor xenograft model. Moreover, success in this project would lead to support the strategy of targeting translation initiation and USP9X for tumor therapies.

蛋白翻译起始是蛋白翻译过程的限速步骤。越来越多的研究证实蛋白翻译起始因子表达及调控的异常与肿瘤的发生发展密切相关，因此蛋白翻译起始因子及相关调控蛋白可作为肿瘤治疗的潜在靶点。我们的前期研究发现：翻译起始因子eIF4B可以在体内和体外被单泛素化；去泛素化酶USP9X特异性地与eIF4B相互作用；通过siRNA下调USP9X的表达会抑制细胞内的蛋白翻译起始。基于上述发现，本课题拟进一步研究USP9X是否可特异性地去泛素化eIF4B，以揭示USP9X调控蛋白翻译起始的机制。同时，将在肝癌临床样本中分析两者的mRNA和蛋白的表达，并研究其与肝癌发生发展的相关性。此外，将利用裸鼠模型，初步探索特异性地针对蛋白翻译起始和USP9X的小分子抑制剂协同治疗肿瘤的可行性。通过该研究有望揭示"去泛素化酶调节蛋白翻译起始"这一新机制，并为USP9X和蛋白翻译起始作为体内靶点进行肿瘤治疗以及新药研发提供科学依据。

蛋白翻译起始的调控是一种在转录后水平快速、有效的调控基因表达的方式。eIF4A1作为一种重要的ATP酶依赖的RNA解旋酶可直接影响蛋白翻译的效率，然而，泛素化修饰如何调控蛋白翻译起始复合物的研究尚不深入。本项目首次发现了去泛素化酶USP9X是蛋白翻译起始复合物的组成成分，可调控帽子结构依赖的蛋白翻译起始的效率；USP9X尤其可以显著调控5’-UTR具有复杂结构的mRNA,比如c-Myc, XIAP等的翻译。我们还首次发现了eIF4A1可以被泛素化修饰，Lys369是其主要的泛素化位点。USP9X可以去泛素化eIF4A1。不仅如此，USP9X可以促进多种细胞的增殖和生长以及细胞对化疗药物5-FU的敏感性。本项目首次报道了USP9X以及eIF4A1的泛素化可以参与蛋白翻译的过程，为USP9X在肿瘤以及神经退行性类疾病中的重要作用提供了一种新的解释。这些发现在蛋白翻译调控领域具有重要的理论意义。. 我们还对eIF4A1的ATP水解酶和RNA解旋酶之间的偶联的催化机制进行了解析，发现这与ATP水解后由linker区域参与的磷酸的释放有关。在对eIF4A1的ATP水解酶进行了高通量的药物筛选后，我们寻找到了数个候选化合物在体内和体外都具有抑制eIF4A1的活性，具有用于单独或者联合进行肿瘤治疗的潜力。