DAPK乙酰化修饰及其调控肝癌生长新机制的研究

Acetylation of key proteins is an important way to regulate the behavior of tumor cells. We first discovered that the DAPK, one of the ser/ thr protein kinases, is acetylated at K978 in hepatocarcinoma cells. However, the mechanisms of the DAPK’s acetylation modification, its relationship with the clinical manifestation of hepatocellular carcinoma, and its biological influence on hepatocarcinoma cells, have not been clarified. In this project, we will analyze the effects of acetylation modification on the activity and stability of DAPK, and the relationship between DAPK acetylation modification and the occurrence, development and prognosis of HCC. Acetylation and deacetylation enzymes involved in DAPK acetylation modification will be determined by acetylation and deacetylation intervention experiments combined with immunoprecipitation technique. The effects of DAPK acetylation on hepatocarcinoma cell growth will be investigated by in vitro and in vivo models. We will elucidate the role of DAPK acetylation in the Warburg effect of hepatocarcinoma cells by regulation of glucose metabolism-related enzymes in hepatocarcinoma cells. This project will clarify the effect of acetylation modification on the biological function of DAPK and its upstream acetylation regulatory molecules, and reveal the molecular mechanisms of DAPK acetylation to regulate the growth of hepatoma cells from the glucose metabolism pathway. In the scientific theory, we will find a new mechanism for DAPK to regulate the progress of hepatocellular carcinoma and explore new research field of hepatocellular carcinoma and DAPK function. In medical practice, it will evaluate the significance of DAPK function in the treatment of hepatocellular carcinoma and provide a new strategy for the study and intervention / treatment of hepatocellular carcinoma.

关键蛋白乙酰化修饰是肿瘤发生与进展的重要调控方式。我们首次发现肝癌细胞中丝/苏氨酸激酶DAPK在K978位点存在乙酰化修饰并与肝癌细胞生物学行为有关，但其乙酰化修饰机制及与肝癌细胞的生物学行为和临床表现的详细关系尚待深入探索。本课题在前期线索的基础上，拟采用分子生物学方法分析乙酰化修饰对DAPK活性和稳定性的影响；通过乙酰化/去乙酰化干预实验结合免疫共沉淀技术确定参与DAPK乙酰化修饰调节的乙酰化酶和去乙酰化酶；分别应用体外和体内模型探讨DAPK乙酰化对Warburg效应中糖代谢相关酶的影响及与肝癌发生、发展和预后的关系。本课题最终将以糖代谢途径为切入点揭示DAPK乙酰化修饰调节肝癌发生与进展的分子机制。在科学理论上，本课题将发现DAPK调控肝癌进展的新机制，开拓肝癌和DAPK功能研究的新领域；在医学实践上，评价DAPK的功能对于肝癌治疗的靶点意义，将为肝癌的研究和干预/治疗提供新的策略。

我们采用免疫共沉淀结合LC-MS/MS检测首次发现肝癌细胞表达的死亡相关蛋白激酶（death-associated protein kinase，DAPK）存在乙酰化修饰，这提示DAPK可能通过一种新的翻译后修饰方式参与其生物学功能，因此我们研究了DAPK乙酰化修饰对DAPK活性和稳定性的影响；研究DAPK乙酰化修饰的分子机制；研究DAPK乙酰化修饰对肝癌细胞生长的分子机制。实验结果确定了DAPK乙酰化修饰位点在第978位赖氨酸残基（K978），并制备特异识别DAPK乙酰化位点的抗体；细胞和动物实验证明模拟DAPK乙酰化修饰可促进肝癌PLC/PRF/5和HepG2细胞增殖；DAPK乙酰化抑制了DAPK脱磷酸化激活，抑制了DAPK酶活性；确定与DAPK1存在的相互作用蛋白，包括ATG16L1、ATG5、ATG12、LDHB、G6PD、PDK3、PDHA1、PKM2、ENO1、ACAT2、ACAA1。并系统研究DAPK1与ATG16L1以非磷酸化修饰方式发生相互结合，促进细胞自噬的机制。其中PDHA1、ACAT2、ACAA1可能是DAPK1的乙酰化酶； DAPK与去乙酰化酶HDAC1、HDAC3、Sirt3发生相互作用，减弱DAPK乙酰化水平；DAPK与糖代谢相关酶LDHB、PKM2、PDK3相互作用参与调节糖酵解进程；DAPK与ACAA1、ACAT2相互作用参与脂类代谢进程；确定DAPK乙酰化修饰促进肝癌细胞Warburg效应和增殖。收集肝癌和癌旁组织标本，用制备的特异DAPK乙酰化抗体分析临床肝癌标本DAPK乙酰化修饰，分析DAPK乙酰化修饰与肝癌分期、预后的关系，分析调节DAPK药物和miRNA的抗肿瘤效应，高良姜素、Tachyplesin可激活DAPK抑制癌细胞Warburg效应和增殖、miR340-5p靶向抑制DAPK表达；黄酮类化合物如高良姜素、槲皮素等可以降低DAPK乙酰化水平激活DAPK抑制肝癌细胞生长。研究结果确定DAPK在第978位赖氨酸残基可被乙酰化修饰，抑制DAPK活性，促进DAPK泛素化降解。DAPK通过一种新的翻译后修饰方式参与其生物学功能，研究结果将有助于我们进一步了解肝癌细胞如何调控DAPK活性，为研究DAPK在肝癌细胞发生发展中所起作用提出新观点。