氧化应激诱导Neddylation修饰酶UBE2F泛素化降解的生物学效应和机制研究

Mutiple antitumor therapies are used to induce high levels of reactive oxygen species (ROS), resulting in cell death. However, the mechanisms remain unclear. Our previous study found that ROS can significantly promote the degradation of neddylation modification enzyme UBE2F. Moreover, low expression of UBE2F can inactivate Cullin5 and promote the accumulation of NOXA, leading to apoptosis. These results indicate that excess ROS promote the ubiquitin-proteasome degradation of UBE2F under oxidative stress, thereby inactivating Cullin5 and leading to the accumulation of NOXA, which inducing apoptosis. To test this hypothesis, we plan to carry out intensive studies in this project to elucidate the mechanism of cell apoptosis regulated by ROS-induced UBE2F degradation. The success of this project will reveal mechanisms of ROS-dependent antitumor therapies and provide a solid scientific basis for the discovery of UBE2F-targeting drugs.

多种抗肿瘤治疗策略均可增强细胞内氧自由基ROS的产生，进而促进肿瘤细胞发生凋亡，但是ROS诱导的肿瘤杀伤机制尚未阐明。我们发现ROS的产生显著促进Neddylation修饰酶UBE2F的泛素化蛋白酶体降解。研究表明，UBE2F表达下调显著抑制Cullin5的Neddylation修饰和活化，促进Cullin5下游底物NOXA积聚，导致细胞凋亡。基于上述发现，我们提出以下假说：ROS含量过高导致细胞处于氧化应激状态，Neddylation修饰酶UBE2F发生泛素化蛋白酶体降解，进而抑制Cullin5的Neddylation修饰水平和催化活性，促进NOXA蛋白积聚，诱导细胞凋亡。本项目旨在验证该假说，阐明氧化应激促进UBE2F泛素化蛋白酶体降解诱导细胞凋亡的分子机制，揭示UBE2F降解对ROS依赖的抗肺癌治疗的作用效果，为研发UBE2F分子靶向药物提供依据。

研究背景：放疗抵抗是非小细胞肺癌治疗的主要障碍，导致患者生存率低下。因此，迫切需要鉴定新型的放射增敏靶点来提高放射治疗的效果。.研究方法：WesternBlot检测NEDD8结合酶E2 UBE2F和NOXA的表达。用台盼蓝染色法检测放疗和H2O2处理后肺癌细胞的存活率。构建裸鼠皮下瘤模型评估靶向灭活UBE2F的肺癌放疗增敏的体内效果。.研究结果：我们发现放疗产生的活性氧（ROS）促进NEDD8结合酶E2 UBE2F的蛋白表达，而ROS清除剂N-乙酰-L-半胱氨酸（NAC）则可阻断ROS诱导的UBE2F表达上调。UBE2F的增加促进凋亡蛋白NOXA的降解，从而诱导肺癌细胞对放疗的凋亡抵抗。反之，UBE2F敲除导致NOXA蛋白降解受阻而积聚，进而促进细胞凋亡，显著提高肺癌细胞对放疗的敏感性。此外，TCGA数据库分析显示UBE2F在人肺腺癌中过表达。.研究结论：我们的结果揭示了ROS依赖的放射治疗对UBE2F蛋白表达的调控作用。下调UBE2F表达在体内外均显示出良好的放疗增敏效果，提示UBE2F作为肺癌细胞放疗增敏靶点具有良好的应用前景。