肿瘤细胞中凋亡抑制蛋白CFLAR乙酰化调控的分子机制

CFLAR (CASP8 and FADD-like apoptosis regulator, also called c-FLIP) is an anti-apoptotic protein which consists of two splicing variants CFLARL and CFLARS. Anti-tumor small molecules degrade CFLAR, leading to activation of apoptotic pathway,while inhibition of CFLAR degradtion reduces the efficiency of apoptosis induction.It is demonstrated that CFLAR can be acetylated in cancer cells using yeast-two-hybrid and immunoprecipitation techniques.In addition,BRD7,a Bromodomain-containing protein which can recruit CBP/p300, has physical interaction with CFLAR in cancer cells.Based on the preliminary data,we raised a hypothesis: CFLAR can be acetylated by BRD7 and CBP/p300 or PCAF,and SIRT1/2 regulates CFLAR acetylation.Moreover,the acetylation promotes the CFLAR ubiquitination and degradation, resulting in the cellular apoptosis.Accordingly,the present project is going to use the cell model of CFLAR degradation to characterize the molecular mechanism underlying CFLAR acetylation and its fuction on apoptosis induction. Hopefully,the study of CFLAR acetylation can help us understand the apoptotic signal transduction in cancer cells and develop novel anti-cancer drugs which target non-histone acetylation in cancer cells.

CFLAR/c-FLIP是凋亡抑制蛋白，有CFLARL和CFLARS两个剪切异构体。抗肿瘤药物诱导CFLAR降解，可以激活细胞凋亡通路，而抑制CFLAR的下调将显著降低诱导肿瘤细胞凋亡的效率。我们的酵母双杂交和免疫共沉淀实验表明CFLAR在肿瘤细胞中存在乙酰化修饰，而且CFLAR能与招募乙酰化酶CBP/p300的BRD7结合。基于此，提出如下假说：CFLAR能够通过BRD7和CBP/p300被乙酰化修饰；去乙酰化酶SIRT1/2调节其乙酰化程度；乙酰化修饰促进CFLAR泛素化和降解，进而促进肿瘤细胞凋亡。本课题拟利用CFLAR降解的细胞模型，探究CFLAR乙酰化的分子机制，并了解乙酰化通过对CFLAR稳定性的调控，进而影响肿瘤细胞功能（如凋亡）的分子机制。对CFLAR乙酰化的研究，可以丰富我们对细胞凋亡信号转导机制的认识，为开发基于非组蛋白乙酰化的抗肿瘤药物提供新靶标和新的肿瘤治疗策略。

CFLAR/c-FLIP 属于凋亡抑制蛋白，有CFLARL 和CFLARS 两个剪切异构体。抗肿瘤小分子药物诱导CFLAR 降解，可以激活细胞凋亡通路，而抑制CFLAR 的下调将显著降低诱导肿瘤细胞凋亡的效率。我们的实验结果表明，CFLARL在肿瘤细胞中存在乙酰化修饰，CFLARL 能够通过CBP/p300被乙酰化修饰；去乙酰化酶SIRT1/2 调节其乙酰化程度；同时，发现CFLARL存在精氨酸甲基化修饰，PRMT5与PRMT1及PAID4调控这一过程。乙酰化和甲基化修饰调控CFLARL 泛素化和降解，进而调控肿瘤细胞凋亡。本课题利用CFLARL 降解的细胞模型，借助细胞生物学和分子生物学技术，探究了CFLARL 乙酰化和甲基化修饰对CFLARL稳定性的调控，进而影响肿瘤细胞凋亡的分子机制。对CFLARL修饰机制的研究，可以丰富我们对细胞凋亡信号转导机制的认识，为开发基于蛋白翻译后修饰的抗肿瘤药物提供新的靶标和新的肿瘤治疗策略。.本项目资助下发表6篇SCI论文，培养博士生3名、硕士研究生5名。