title: Activation of Nrf2 by H2O2: de novo synthesis versus nuclear translocation.
abstract: The most common mechanism described for the activation of the transcription factor Nrf2 is based on the inhibition of its degradation in the cytosol followed by its translocation to the nucleus. Recently, Nrf2 de novo synthesis was proposed as an additional mechanism for the rapid upregulation of Nrf2 by hydrogen peroxide (H2O2). Here, we describe a detailed protocol, including solutions, pilot experiments, and experimental setups, which allows exploring the role of H2O2, delivered either as a bolus or as a steady state, in endogenous Nrf2 translocation and synthesis. We also show experimental data, illustrating that H2O2 effects on Nrf2 activation in HeLa cells are strongly dependent both on the H2O2 concentration and on the method of H2O2 delivery. The de novo synthesis of Nrf2 is triggered within 5min of exposure to low concentrations of H2O2, preceding Nrf2 translocation to the nucleus which is slower. Evidence of de novo synthesis of Nrf2 is observed only for low H2O2 steady-state concentrations, a condition that is prevalent in vivo. This study illustrates the applicability of the steady-state delivery of H2O2 to uncover subtle regulatory effects elicited by H2O2 in narrow concentration and time ranges.
authors.affiliation: Departamento de Química e Bioquímica and Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Gonçalo
authors.initials: G
authors.lastname: Covas
authors.forename: H Susana
authors.initials: HS
authors.lastname: Marinho
authors.forename: Luísa
authors.initials: L
authors.lastname: Cyrne
authors.forename: Fernando
authors.initials: F
authors.lastname: Antunes
mesh_terms: D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D051267:NF-E2-Related Factor 2 / Q000819:agonists / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction; D015533:Transcriptional Activation
chemical_list: D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D006861:Hydrogen Peroxide
keywords: Bolus; Hydrogen peroxide; Redox regulation; Steady state; Transcription factor