核因子红细胞2相关因子2（NRF2）对与氧化应激有关的基因表达非常重要。NRF2的水平由类似Kelch的ECH相关蛋白1（KEAP1）依赖性降解控制。尽管已知氧化应激能抑制KEAP1的活性以稳定NRF2的水平，但这种调控机制尚不清楚。在本研究中，我们发现KEAP1在赖氨酸39位点（K39）被SUMO1修饰。将该赖氨酸（K39）替换为精氨酸（K39R）并不影响KEAP1的稳定性、亚细胞定位或二聚化，但促进了Cullin 3泛素连接酶的形成并增加了NRF2的泛素化。这伴随着NRF2表达的降低。基因报告器实验显示，与表达KEAP1-K39R SUMO1底物突变体的细胞相比，KEAP1-WT细胞中的抗氧化应答元件的转录增强。与此一致，染色质免疫沉淀实验揭示了表达KEAP1-WT和KEAP1-K39R突变蛋白的H1299肺癌细胞中，NRF2与抗氧化基因启动子区域的结合更高。这种KEAP1活性受其SUMO化的抑制的重要性在H1299肺癌细胞系的皮下肿瘤模型中得到了验证。该模型表明，突变KEAP1的SUMO化位点会改变活性氧物质的产生并抑制肿瘤生长。总之，我们的研究认识到NRF2依赖的氧化还原控制受到KEAP1的SUMO化调控。这些发现为抵御氧化应激提供了潜在的新治疗选择。版权所有© 2023作者。由Elsevier Inc.出版。保留所有权利。

Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) is important for the expression of genes associated with oxidative stress. The levels of NRF2 are controlled by Kelch-like ECH-associated protein 1 (KEAP1) -dependent degradation. Although oxidative stress is known to suppress KEAP1 activity to stabilize the levels of NRF2, the mechanism for this control is unclear. Here, we identify that KEAP1 is modified by SUMO1 at the lysine residue position 39 (K39). Arginine replacement of this lysine (K39R) in KEAP1 did not affect its stability, subcellular localization or dimerization but promoted the formation of the Cullin 3 ubiquitin ligase and increased NRF2 ubiquitination. This was accompanied by decreased NRF2 expression. Gene reporter assays showed that the transcription of antioxidant response elements was heightened in KEAP1-WT cells compared to cells expressing the KEAP1-K39R SUMO1 substrate mutant. Consistent with this, chromatin immunoprecipitation assays revealed higher NRF2 binding to the promoter regions of antioxidant genes in cells expressing the KEAP1-WT compared to the KEAP1-K39R mutant protein in H1299 lung cancer cell. The significance of this suppression of KEAP1 activity by its SUMOylation was tested in a subcutaneous tumor model of H1299 lung cancer cell lines that differentially expressed the WT and K39R KEAP1 constructs. This model showed that mutating the SUMOylation site on KEAP1 altered the production of reactive oxygen species and suppressed tumor growth. Taken together, our study recognizes that NRF2-dependent redox control is regulated by the SUMOylation of KEAP1. These findings identify a potential new therapeutic option to counteract oxidative stress.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.