Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2.
The risks of cancer and other degenerative diseases caused by reactive oxygen species and electrophiles can be reduced by the up-regulation of detoxifying enzymes. A major mechanism whereby these protective enzymes are induced occurs through activation of the antioxidant response element (ARE) by the oxidative-stress sensor protein Kelch-like ECH-associated protein 1 (Keap1) and the transcription factor NF-E2-related factor 2 (Nrf2). Under basal conditions, Keap1 sequesters Nrf2 in the cytoplasm by binding to its Neh2 domain. Chemical inducers such as sulforaphane are known to react with Keap1 cysteine residues, thereby promoting Nrf2 nuclear accumulation and hence ARE activation. A widely accepted model for Nrf2 nuclear accumulation is that modification of Keap1 cysteines leads directly to dissociation of the Keap1-Nrf2 complex. This model is based on studies with mouse proteins and has served as the experimental basis and hypothesis for numerous investigations. Through a combination of chemical, mass spectrometry, and isothermal titration calorimetry methods, we have tested the direct-dissociation model using a series of ARE inducers: sulforaphane, isoliquiritigenin, 15-deoxy-Delta12,14-prostaglandin-J2, menadione, 1-Cl-2,4-dinitrobenzene, and biotinylated iodoacetamide. Surprisingly, these data suggest that the direct disruption model for Keap1-Nrf2 is incorrect. The relative reactivity of human Keap1 cysteines was determined. In addition to the same five cysteines identified for mouse Keap1, two highly reactive and previously unobserved cysteines were identified. Based on these results, a model is proposed that should aid in the understanding of Keap1-Nrf2 signaling mechanisms.
Eggler
Aimee L
AL
Center for Pharmaceutical Biotechnology, University of Illinois, Chicago, IL 60607, USA.
Liu
Guowen
G
Pezzuto
John M
JM
van Breemen
Richard B
RB
Mesecar
Andrew D
AD
D000818:Animals; D002151:Calorimetry; D002599:Chalcone / Q000031:analogs & derivatives / Q000378:metabolism; D047188:Chalcones; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004136:Dinitrobenzenes / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction / Q000502:physiology*; D006801:Humans; D007155:Immunologic Factors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007460:Iodoacetamide / Q000378:metabolism; D017879:Isothiocyanates; D000072019:Kelch-Like ECH-Associated Protein 1; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism*; D051267:NF-E2-Related Factor 2; D015230:Prostaglandin D2 / Q000031:analogs & derivatives / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D013454:Sulfoxides; D013861:Thiocyanates / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D024483:Vitamin K 3 / Q000378:metabolism
C097240:15-deoxy-delta(12,14)-prostaglandin J2; D047188:Chalcones; D004268:DNA-Binding Proteins; D004136:Dinitrobenzenes; D007155:Immunologic Factors; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D046912:Multiprotein Complexes; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D011506:Proteins; D013454:Sulfoxides; D013861:Thiocyanates; D015534:Trans-Activators; D002599:Chalcone; D024483:Vitamin K 3; C040920:isoliquiritigenin; C016766:sulforaphane; D003545:Cysteine; D015230:Prostaglandin D2; D007460:Iodoacetamide