RIP1磷酸化调控细胞死亡信号转导复合体Ripoptosome形成机制的研究

Ripoptosome is a newly discovered mammalian intracellular signalling complex involved in cell death pathways. Its formation and subsequent caspase activation depends on RIP1 kinase activity. As reported previously, RIP1 kinase activity is regulated by its phosphorylation. However, the machinery among RIP1 phosphorylation, kinase activity and Ripoptosome formation is still unknown. It therefore becomes the main component in this proposal. In this proposal, relevant molecular and cellular methods including gel filtration, immunoprecipitation, point mutagenesis, RNAi, western blot for phophorylated protein are outlined to further investigate the effect of RIP1 phosphorylation on its kinase activity, Ripoptosome formation, cell fate as well as RIP1 self stability. Role of the molecular chaperon, Hsp90 in protecting phosphorylated RIP1 from degradation and relationship between RIP1 phosphorylation and FADD phosphorylation will also be studied. Innovative discovery is hopefully to be obtained in the mechanism of Ripoptosome formation regulated by RIP1, RIP1 and FADD recognition to initiate Ripoptosome and Ripoptosome half-life control. This research will eventually further our understanding in cell death signalling pathway, tumour pathological knowledge and benefit the innovation of anti-cancer drugs.

Ripoptosome是在哺乳动物肿瘤细胞中新发现的一个细胞死亡信号复合体。它的形成和下游caspase激活依赖于核心组分RIP1的磷酸激酶活性。据报道，RIP1磷酸激酶活性受RIP1磷酸化调控。但RIP1磷酸化、RIP1磷酸激酶活性与Ripoptosome形成之间的关系并不明确，这也是本课题的核心内容。本课题拟使用分子筛、免疫共沉淀、定点突变、RNAi、磷酸化免疫印迹等手段，研究RIP1磷酸化对RIP1磷酸激酶活性、Ripoptosome形成和细胞死亡的作用，分析RIP1磷酸化对自身稳定性的影响，探究分子伴侣Hsp90保护磷酸化RIP1的机制以及RIP1磷酸化与FADD磷酸化的关系。本项申请有望在RIP1调控Ripoptosome形成、RIP1与FADD相互识别、Ripoptosome稳定性调控机制等方面取得创新性成果，为更好地阐述细胞死亡通路奠定基础，为肿瘤病理和药物研发提供依据。

本项课题根据申请书所列内容开展，采用免疫共沉淀、分子筛、定点突变、TALENs介导的基因编辑等技术开展研究，研究内容包括RIP1磷酸化对Ripoptosome形成和细胞死亡的作用、RIP1磷酸化影响其自身稳定性的机制(包括RIP1磷酸化对自身结构稳定性的影响和Hsp90对RIP1的保护作用)和RIP1磷酸化与FADD磷酸化的关系等三个方面的内容。在本课题执行期间，以上三部分研究内容均按计划进行，完成或部分完成了计划内容，取得了相应的实验数据和成果。研究进展包括：阐明RIP1Ser161、Ser166和Ser303位点磷酸化对Ripoptosome形成和细胞死亡的决定作用；阐明细胞内RIP1为FADD和caspase-8提供docking位点；证明Hsp90不参与RIP1 docking和Ripoptosome组装；探索了RIP1的磷酸激酶活性底物，发现RIP1在Ripoptosome组装过程中可能参与FADD磷酸化。本项研究在RIP1调控Ripoptosome形成、RIP1与FADD相互识别、Ripoptosome稳定性控制方面取得部分创新性成果，最终为更好的阐述细胞死亡通路奠定基础，为肿瘤病理和治疗研究提供理论依据。