转录因子 NRF2 在调节保护细胞免受氧化损伤的基因方面发挥着重要作用。 O-GlcNAc 修饰是一种翻译后修饰，对于细胞对应激的反应至关重要。尽管NRF2和O-GlcNAc均已被证明参与维持细胞氧化还原平衡和促进癌症恶性，但潜在的机制仍然难以捉摸。采用免疫印迹、荧光素酶报告基因、ROS测定、免疫共沉淀和免疫荧光来检测效果NRF2 上的全局细胞 O-GlcNAc 酰化。利用质谱法绘制了 NRF2 上的 O-GlcNAc 酰化位点图谱，并通过位点特异性诱变和 O-GlcNAc 酶标记进行了验证。使用人类肺癌样本来验证 O-GlcNAc 和 NRF2 之间的关联。随后，在体外和体内评估了NRF2 O-GlcNAcNAc化在肺癌恶性肿瘤和顺铂耐药中的影响。NRF2在Ser103残基处被O-GlcNAcNAc化，这阻碍了其与KEAP1的结合，从而增强了其稳定性、核定位和转录活动。氧化应激和顺铂可以通过 AMPK 激酶的激活提高 OGT Thr444 的磷酸化，从而增强 OGT 与 NRF2 的结合，并随后提高 NRF2 O-GlcNAcNA 酰化。在细胞和异种移植小鼠模型中，NRF2 Ser103 处的 O-GlcNAc 酰化可促进肺癌的恶性化。在人类肺癌组织样本中，与配对的邻近正常组织相比，整体 O-GlcNAc 基化显着增加，并且 NRF2 及其 O-GlcNAc 基化水平升高。化疗促进 NRF2 O-GlcNA 酰化，从而降低细胞 ROS 水平并促进肺癌细胞存活。我们的研究结果表明，OGT O-GlcNA 酰化 NRF2 Ser103，这种修饰在细胞抗氧化、肺癌恶性肿瘤和顺铂耐药中发挥作用.© 2024 作者。约翰·威利出版的《临床与转化医学》

The transcription factor NRF2 plays a significant role in regulating genes that protect cells from oxidative damage. O-GlcNAc modification, a type of posttranslational modification, is crucial for cellular response to stress. Although the involvement of both NRF2 and O-GlcNAc in maintaining cellular redox balance and promoting cancer malignancy has been demonstrated, the potential mechanisms remain elusive.The immunoblotting, luciferase reporter, ROS assay, co-immunoprecipitation, and immunofluorescence was used to detect the effects of global cellular O-GlcNAcylation on NRF2. Mass spectrometry was utilised to map the O-GlcNAcylation sites on NRF2, which was validated by site-specific mutagenesis and O-GlcNAc enzymatic labelling. Human lung cancer samples were employed to verify the association between O-GlcNAc and NRF2. Subsequently, the impact of NRF2 O-GlcNAcylation in lung cancer malignancy and cisplatin resistance were evaluated in vitro and in vivo.NRF2 is O-GlcNAcylated at Ser103 residue, which hinders its binding to KEAP1 and thus enhances its stability, nuclear localisation, and transcription activity. Oxidative stress and cisplatin can elevate the phosphorylation of OGT at Thr444 through the activation of AMPK kinase, leading to enhanced binding of OGT to NRF2 and subsequent elevation of NRF2 O-GlcNAcylation. Both in cellular and xenograft mouse models, O-GlcNAcylation of NRF2 at Ser103 promotes the malignancy of lung cancer. In human lung cancer tissue samples, there was a significant increase in global O-GlcNAcylation, and elevated levels of NRF2 and its O-GlcNAcylation compared to paired adjacent normal tissues. Chemotherapy promotes NRF2 O-GlcNAcylation, which in turn decreases cellular ROS levels and drives lung cancer cell survival.Our findings indicate that OGT O-GlcNAcylates NRF2 at Ser103, and this modification plays a role in cellular antioxidant, lung cancer malignancy, and cisplatin resistance.© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.