Ras oncogene induced protein SUMOylation changes

背景通过RNAi合成致死筛选，我们已经确定了RAS突变癌细胞生存所需的蛋白SUMO化途径。本项目旨在解决以下问题：1.抑制SUMO化途径对RAS突变具有综合致命性的机制；2.在RAS突变细胞中，哪些细胞蛋白发生了差异的SUMO化；3.在RAS突变的背景下，这些细胞中SUMO化状态的变化如何影响其功能。有意义的材料和方法1.针对SUMO连接酶的小分子RNA。2.稳定表达6倍His标记蛋白的细胞系。3.完成2010财年蛋白质ID的质谱分析我们已经验证了针对相扑E1和E2连接酶的shRNAs。我们还建立了稳定表达His标记的SUMO1或SUMO2蛋白的细胞系。我们已经进行了蛋白质组学筛选，并鉴定了一些在RAS WT和突变细胞之间差异SUMO化的候选蛋白。

BACKGROUND Through a RNAi synthetic lethal screen we have identified the protein SUMOylation pathway to be required for the viability of Ras mutant cancer cells. PURPOSE In this project we aim to address the following questions: 1. the mechanism by which the inhibition of SUMOylation pathway is synthetically lethal with Ras mutation; 2. which cellular proteins are differentially SUMOylated in Ras mutant cells; 3. How the changes in SUMOylation status in these cells affect their function in the context of Ras mutation SIGNIFICANT MATERIALS AND METHODS 1. shRNAs that target the SUMO E1 and E2 ligases. 2. Stable cell lines that express 6x His tagged proteins. 3. mass-spectrometry protocol for protein ID FY2010 ACCOMPLISHMENT We have validated shRNAs that target the SUMO E1 and E2 ligases. We have also generated cell lines stably expressing His-tagged SUMO1 or SUMO2 proteins. We have carried out a proteomics screen and identified a number of candidate proteins that are differentially SUMOylated between Ras WT and mutant cells.