title: Activation of the Nrf2/ARE pathway via S-alkylation of cysteine 151 in the chemopreventive agent-sensor Keap1 protein by falcarindiol, a conjugated diacetylene compound.
abstract: Under basal conditions, the interaction of the cytosolic protein Kelch-like ECH-associated protein 1 (Keap1) with the transcription factor nuclear factor-E2-related factor 2 (Nrf2) results in a low level of expression of cytoprotective genes whose promoter region contains the antioxidant response element (ARE). In response to oxidants and electrophiles, Nrf2 is stabilized and accumulates in the nucleus. The mechanism for this effect has been proposed to involve thiol-dependent modulation of Keap1, leading to loss of its ability to negatively regulate Nrf2. We previously reported that falcarindiol (heptadeca-1,9(Z)-diene-4,6-diyne-3,8-diol), which occurs in Apiaceae and the closely related Araliaceae plants, causes nuclear accumulation of Nrf2 and induces ARE-regulated enzymes. Here, we report the mechanism of Nrf2 induction by falcarindiol. NMR analysis revealed that the conjugated diacetylene carbons of falcarindiol acted as electrophilic moieties to form adducts with a cysteine (Cys) thiol. In addition, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and circular dichroism spectroscopy, it was demonstrated that falcarindiol alkylated Cys residues in Keap1 and altered the Keap1 secondary structure. Transfection studies using the purified Keap1 protein, a luciferase reporter construct, and an Nrf2-expressing plasmid indicated that the intact Keap1 protein suppressed Nrf2-mediated ARE-luciferase activity. On the other hand, the falcarindiol-alkylated Keap1 protein did not suppress such activity. Treatment of HEK293 cells overexpressing Keap1 with falcarindiol generated a high molecular weight (HMW) form of Keap1. Furthermore, the Cys151 residue in Keap1 was found to be uniquely required for not only the formation of HMW Keap1 but also an increase in ARE-luciferase activity by falcarindiol. Our results demonstrate that falcarindiol having conjugated diacetylene carbons covalently modifies the Cys151 residue in Keap1 and that the inactivation of Keap1 by falcarindiol leads to activation of the Nrf2/ARE pathway.
authors.affiliation: Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Shinji
authors.initials: S
authors.lastname: Nakayama
authors.forename: Eisaburo
authors.initials: E
authors.lastname: Anan
authors.forename: Takahito
authors.initials: T
authors.lastname: Nishiyama
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Ogura
authors.forename: Akira
authors.initials: A
authors.lastname: Hiratsuka
mesh_terms: D000478:Alkylation; D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine; D019610:Cytoprotection; D053280:Diynes / Q000494:pharmacology*; D005233:Fatty Alcohols / Q000494:pharmacology*; D017930:Genes, Reporter; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D000072019:Kelch-Like ECH-Associated Protein 1; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020218:Response Elements; D015398:Signal Transduction / Q000187:drug effects*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000975:Antioxidants; D053280:Diynes; D005233:Fatty Alcohols; D047908:Intracellular Signaling Peptides and Proteins; C486610:KEAP1 protein, rat; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495637:Nfe2l2 protein, rat; D011506:Proteins; C034379:falcarindiol; D003545:Cysteine