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dc.contributor.advisorJacobson, Myron Ken_US
dc.contributor.advisorLiebler, Daniel Cen_US
dc.contributor.authorHong, Fei
dc.creatorHong, Feien_US
dc.date.accessioned2011-12-06T14:21:34Z
dc.date.available2011-12-06T14:21:34Z
dc.date.issued2005en_US
dc.identifier.urihttp://hdl.handle.net/10150/196091
dc.description.abstractActivation of the transcription factor Nrf2 regulates expression of phase II enzymes and other adaptive responses to electrophile and oxidant stress. Nrf2 concentrations are regulated by the thiol-rich sensor protein Keap1, which is an adaptor protein for Cul3-dependent ubiquitination and degradation of Nrf2. However, the links between site-specificity of Keap1 modification by electrophiles and mechanisms of Nrf2 activation are poorly understood. We studied the actions of the prototypical Nrf2 inducer tert-butylhydroquinone (tBHQ) and two biotin-tagged, thiol-reactive electrophiles N-iodoacetyl-N-biotinylhexylenediamine (IAB) and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane (BMCC). Both IAB and tBHQ induce expression of ARE-directed GFP expression in ARE/TK-GFP HepG2 cells and both initiatednuclear Nrf2 accumulation and induction of heme oxygenase 1 in HEK293 cells. In contrast, BMCC produced none of these effects. Liquid chromatography tandem mass spectrometry (LC-MS-MS) analysis of human Keap1 modified by IAB or BMCC in vitro indicated that IAB adduction occurred primarily in the central linker domain, whereas BMCC modified other Keap1 domains. Treatment of FLAG-Keap1 transfected HEK293 with the Nrf2-activating compounds IAB and tBHQ generated high molecular weight Keap1 forms, which were identified as K-48-linked polyubiquitin-conjugates by immunoblotting and LC-MS-MS. Keap1 polyubiquitination coincided with Nrf2 stabilization and nuclear accumulation. In contrast, BMCC did not induce Keap1 polyubiquitination. Our results suggest that Nrf2 activation is regulated through the polyubiquitination of Keap1, which in turn is triggered by specific patterns of electrophile modification of the Keap1 central linker domain. These results suggest that Keap1 adduction triggers a switching of Cul3-dependent ubiquitination from Nrf2 to Keap1, leading to Nrf2 activation.The chemopreventive agent sulforaphane is an isothiocyanate, which was isolated from broccoli. Sulforaphane was demonstrated to induce ARE-regulated genes by stimulating the Keap1-Nrf2 system. This agent is a powerful electrophile that can react with thiols to form thionoacyl adducts. A specific sulforaphane modification pattern on Keap1 may trigger the activation of Nrf2. However, thionoacyl adducts are labile to hydrolysis and transacylation reactions, which prevent the identification of the sulforaphane modification patten on Keap1. In this study, we have developed a LC-MS-MS method to map sulforaphane modification sites formed on Keap1 in vitro. Our studies indicate that sulforaphane displays a different pattern of Keap1 modification than ARE/ERE inducers that modify Keap1 by alkylation. Moreover, the modification of Keap1 in vivo by sulforaphane does not trigger the ubiquitination of Keap1, which suggests a novel mechanism for Nrf2 stabilization by sulforaphane thionoacyl adduct formation.
dc.language.isoENen_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.subjectPharmaceutical Sciencesen_US
dc.titleANALYSIS OF ELECTROPHILE-INDUCED NRF2 GENE ACTIVATIONen_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.contributor.chairJacobson, Myron Ken_US
dc.contributor.chairLiebler, Daniel Cen_US
dc.identifier.oclc137354782en_US
thesis.degree.grantorUniversity of Arizonaen_US
thesis.degree.leveldoctoralen_US
dc.contributor.committeememberFutscher, Bernard W.en_US
dc.contributor.committeememberYang, Danzhouen_US
dc.contributor.committeememberCress, Ann E.en_US
dc.identifier.proquest1280en_US
thesis.degree.disciplinePharmaceutical Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.namePhDen_US
refterms.dateFOA2018-08-19T22:38:06Z
html.description.abstractActivation of the transcription factor Nrf2 regulates expression of phase II enzymes and other adaptive responses to electrophile and oxidant stress. Nrf2 concentrations are regulated by the thiol-rich sensor protein Keap1, which is an adaptor protein for Cul3-dependent ubiquitination and degradation of Nrf2. However, the links between site-specificity of Keap1 modification by electrophiles and mechanisms of Nrf2 activation are poorly understood. We studied the actions of the prototypical Nrf2 inducer tert-butylhydroquinone (tBHQ) and two biotin-tagged, thiol-reactive electrophiles N-iodoacetyl-N-biotinylhexylenediamine (IAB) and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane (BMCC). Both IAB and tBHQ induce expression of ARE-directed GFP expression in ARE/TK-GFP HepG2 cells and both initiatednuclear Nrf2 accumulation and induction of heme oxygenase 1 in HEK293 cells. In contrast, BMCC produced none of these effects. Liquid chromatography tandem mass spectrometry (LC-MS-MS) analysis of human Keap1 modified by IAB or BMCC in vitro indicated that IAB adduction occurred primarily in the central linker domain, whereas BMCC modified other Keap1 domains. Treatment of FLAG-Keap1 transfected HEK293 with the Nrf2-activating compounds IAB and tBHQ generated high molecular weight Keap1 forms, which were identified as K-48-linked polyubiquitin-conjugates by immunoblotting and LC-MS-MS. Keap1 polyubiquitination coincided with Nrf2 stabilization and nuclear accumulation. In contrast, BMCC did not induce Keap1 polyubiquitination. Our results suggest that Nrf2 activation is regulated through the polyubiquitination of Keap1, which in turn is triggered by specific patterns of electrophile modification of the Keap1 central linker domain. These results suggest that Keap1 adduction triggers a switching of Cul3-dependent ubiquitination from Nrf2 to Keap1, leading to Nrf2 activation.The chemopreventive agent sulforaphane is an isothiocyanate, which was isolated from broccoli. Sulforaphane was demonstrated to induce ARE-regulated genes by stimulating the Keap1-Nrf2 system. This agent is a powerful electrophile that can react with thiols to form thionoacyl adducts. A specific sulforaphane modification pattern on Keap1 may trigger the activation of Nrf2. However, thionoacyl adducts are labile to hydrolysis and transacylation reactions, which prevent the identification of the sulforaphane modification patten on Keap1. In this study, we have developed a LC-MS-MS method to map sulforaphane modification sites formed on Keap1 in vitro. Our studies indicate that sulforaphane displays a different pattern of Keap1 modification than ARE/ERE inducers that modify Keap1 by alkylation. Moreover, the modification of Keap1 in vivo by sulforaphane does not trigger the ubiquitination of Keap1, which suggests a novel mechanism for Nrf2 stabilization by sulforaphane thionoacyl adduct formation.


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