New insights into the mechanism of Keap1-Nrf2 interaction based on cancer-associated mutations

Aims: Keap1-Nrf2 signaling pathway is one of the most important antioxidant signaling pathways, and its abnormal activation is related to cancer metastasis and drug resistance. Many studies have shown Keap1 and Nrf2 mutations are closely associated with cancer occurrence. However, few studies focus on Keap1-Nrf2 binding characteristics of cancer-associated mutations. The study investigated the molecular mechanism between Keap1/Nrf2 mutations and cancer. Main methods: We have determined the crystal structure of the Keap1-Kelch domain with Nrf2 25-mer peptide. What's more, we clarified the molecular effects of Nrf2(Thr80) and Nrf2(Pro85) on the binding of Keap1 by the method isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF) and electrophoretic mobility shift assay (EMSA). Especially, we confirmed the effect of Thr80 and Pro85 mutations on Keap1/Nrf2 signaling pathway in HEK293T cells by RT-PCR and western blot (WB). Finally, we verified the effect of six cancer-related high-frequency somatic mutations Keap1(G364C), Keap1(D422N), Keap1(R47)(0C), Keap1(G480W), Keap1(E493)(Q) and Keap1(R6)(0)(1L) on binding with Nrf2 through ITC experiments. Key findings: Nrf2(Thr80) and Nrf2(Pro85) play a vital role in the Keap1-Nrf2 interaction. Mutant or modification at position Thr80 will disrupt the interaction. Especially, Nrf2(Thr8)(0) and Nrf2(Pro85) mutations activate the expression of cytoprotective genes in HEK293T cells. As for Keapl, except G364C, the binding affinity of other cancer-related mutants to Nrf2 hardly changed, which means that Keapl mutants can activate Nrf2 without disrupting the binding to Nrf2. Significance: The study provides new insight into Keap1/Nrf2 signaling pathway and cancer.

Automated summary

This study revealed the significance of Nrf2(Thr80) and Nrf2(Pro85) in the Keap1-Nrf2 interaction, as well as the impact of Keap1 mutations on Nrf2 binding. These findings provide important insights for further understanding the role of the Keap1/Nrf2 signaling pathway in cancer development.