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Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
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New insights into the mechanism of Keap1-Nrf2 interaction based on cancer-associated mutations

Liangkai Cheng1, Huihui Wang2, Shehan Li2

  • 1GuangDong Second Traditional Chinese Medicine Hospital, Guangzhou 510006, China.

Life Sciences
|July 6, 2021
PubMed
Abstract

Insights

Mutations in Nrf2 at Thr80 and Pro85 disrupt Keap1 binding, activating protective genes. Cancer-associated Keap1 mutations generally do not alter Nrf2 binding, yet still activate the Keap1-Nrf2 pathway.

Area of Science:

  • Biochemistry and Molecular Biology
  • Cancer Research
  • Cell Signaling

Background:

  • The Keap1-Nrf2 signaling pathway is crucial for cellular defense against oxidative stress.
  • Aberrant activation of this pathway is implicated in cancer metastasis and drug resistance.
  • While Keap1 and Nrf2 mutations are linked to cancer, their specific binding interactions remain underexplored.

Purpose of the Study:

  • To investigate the molecular mechanisms underlying Keap1-Nrf2 binding alterations in cancer-associated mutations.
  • To elucidate the impact of specific Nrf2 mutations (Thr80, Pro85) on Keap1 interaction.
  • To assess the effect of common cancer-related Keap1 mutations on Nrf2 binding affinity.

Main Methods:

  • Crystal structure determination of the Keap1-Kelch domain bound to an Nrf2 peptide.
  • Isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF), and electrophoretic mobility shift assay (EMSA) to analyze Nrf2 mutations' effects on Keap1 binding.
  • RT-PCR and Western Blot (WB) to confirm the functional impact of Nrf2 mutations in HEK293T cells.
  • ITC to evaluate the binding affinity of six high-frequency cancer-associated Keap1 mutations with Nrf2.

Main Results:

  • Nrf2 mutations at Thr80 and Pro85 significantly disrupt the interaction with Keap1.
  • These Nrf2 mutations lead to the activation of cytoprotective gene expression in HEK293T cells.
  • Most tested cancer-associated Keap1 mutations (excluding G364C) showed minimal changes in binding affinity to Nrf2.
  • Keap1 mutants can activate the Nrf2 pathway without significantly altering their binding to Nrf2.

Conclusions:

  • Nrf2 mutations at Thr80 and Pro85 are key disruptors of Keap1 binding, leading to pathway activation.
  • Cancer-associated Keap1 mutations can activate the Nrf2 pathway through mechanisms independent of altered binding affinity.
  • This study provides critical insights into the molecular basis of Keap1-Nrf2 pathway dysregulation in cancer.
Keywords:
Binding assayCancerETGE motifKeap1-Nrf2Mutation

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