Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.
Level 5 - mechanism / opinion, no new human data
Mechanistic bench and cell culture study with no human data.
PubMed 14764894 · doi:10.1073/pnas.0307301101
What was done
The authors investigated the molecular mechanism by which electrophilic inducers disrupt the Keap1-Nrf2 complex to activate the phase 2 antioxidant response. Purified recombinant Keap1 was treated with dexamethasone mesylate and analyzed by mass spectrometry to identify reactive cysteine residues. Keap1- and Nrf2-deficient mouse embryonic fibroblasts were transfected with wild-type or cysteine-to-alanine Keap1 mutants alongside an antioxidant response element (ARE)-luciferase reporter. In addition, 2D PAGE was performed on cell extracts to assess inducer-triggered Keap1 dimerization.
What was found
The abstract reports no quantitative values or statistical metrics. Out of 25 cysteine residues in Keap1, C273 and C288 were identified as the primary sites modified by inducers. Single C273A and C288A mutant constructs lost the ability to repress Nrf2 activation of the ARE-luciferase reporter, but co-transfecting an equal mixture of both mutants restored repression. Reaction with inducers promoted intermolecular disulfide dimer formation, likely bridging C273 on one Keap1 monomer with C288 on another.
Why it matters
This study defines the specific molecular sensor mechanism (cysteines C273 and C288) by which Keap1 detects oxidants and electrophiles, providing a structural basis for pharmacologic targeting of the Nrf2 phase 2 cytoprotective pathway.
Limits
The findings are derived entirely from cell-free recombinant assays and transfected mouse embryonic fibroblasts. The abstract provides no quantitative measures or statistical uncertainty, and in vivo efficacy, safety, and human applicability were not evaluated.
Cited by
- supports Isothiocyanates bind to reactive cysteine sulfhydryl groups on Keap1, inducing a conformational change in the protein.