INrf2:Nrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 functions as an adaptor that interacts with Nrf2 and brings closer to ubiquitin ligase Cul3-Rbx1 for ubiquitination and degrading of Nrf2. Under basal conditions, INrf2/Cul3-Rbx1 complex is constantly degrading Nrf2. Nrf2 dissociates from the INrf2 in response to antioxidant and other stressors and translocates into the nucleus leading to activation of a myriad of genes that protect cells against stress and neoplasia. Immunoprecipitation and mass spectra analysis demonstrated interaction of INrf2 also with Phosphoglyerate mutase family member 5 (PGAM5)/BclXL/Bcl2, heat shock HSP90 and methylosome subunit pICln for unknown functions. Mass spectra analysis of immunoprecipitated INrf2 from antioxidant untreated and treated cells demonstrated phosphorylation of specific serine, threonine and tyrosine residues that might control INrf2 functions but remains unknown. Antioxidant treatment of cells led to genestein sensitive nuclear export of INrf2, Cul3 and Rbx1 presumably to allow nuclear import of Nrf2 without threat of degradation. However, the mechanism of export remains obscure. A feedback auto-regulatory loop between Nrf2 and INrf2 controls their cellular abundance. Interestingly, the Cul3 and Rbx1 gene expression is coordinately induced with INrf2 in response to antioxidant raising questions if Cul3 and Rbx1 are part of the INrf2-Nrf2 autoragulatory loop. The major goals of this proposal are to study role and regulation of INrf2. To this effect, we propose four specific aims.
Aim 1 will investigate INrf2 interaction with PGAM5/BclXL/Bcl2, HSP90, and pICln and its significance in cellular protection and cell survival/death. The domain deletion, transfection, immunoprecipitation, Western analysis, reporter and apoptosis assays will be used in these studies.
Aim 2 will elucidate the role of phosphorylation in control of homodimerization and stability of INrf2, nuclear localization, interaction with Cul3-Rbx1 and Nrf2 and degradation of Nrf2, antioxidant-induced release of Nrf2 from INrf2 and activation of gene expression, and interaction of INrf2 with PGAM5/BclXL/Bcl2, HSP90 and pICln. Sitedirected mutagenesis along with transfection and reporter assays, immunoprecipitation and Western analysis will be used to achieve the goals of this aim.
Aim 3 will continue with generation of INrf2 conditional knockout mice that do not express INrf2 protein in liver and epidermis of skin. The Cre-Lox system will be used to generate these mice. In addition, compare the sensitivity of conditional INrf2 knockout mice expressing the null genotype with that of mice expressing INrf2, following carcinogen exposure, to develop skin and liver tumors.
Aim 4 will test the hypothesis that Cul3 and Rbx1 are part of the auto-regulatory loop between INrf2 and Nrf2. Deletion mapping/transfection and band/super shift/ChIP assays will determine that ARE element and Nrf2 that regulate INrf2 also regulates antioxidant induction of Cul3 and Rbx1 genes. Co-transfection and ubiquitination assays will elucidate the role of coordinated induction of Cul3-Rbx1 in ubiquitination and degradation of Nrf2.

Public Health Relevance

INrf2 study is of considerable importance in prevention of xenobiotic, nutrient and radiation-induced oxidative stress. INrf2 serves as a sensor of xenobiotic-induced oxidative stress and initiates a signal cascade that leads to the Nr2-mediated coordinated activation of a battery of antioxidant/defensive genes. The products of these genes play critical roles in cellular protection against chemical induced cytotoxicity and neoplasia. In the competing continuation proposal, we plan to investigate the INrf2 interaction with Nrf2 and other proteins;mechanism of xenobiotic and nutrient modification of INrf2 especially phosphorylation and dephosphorylation control of INrf2 structure/function and interaction with other proteins, and its significance in Nrf2 activation of antioxidant gene expression;generate conditional knockout mice that do not express INrf2 in liver and epidermis of skin and investigate in vivo roles of INrf2 in coordinated activation of antioxidant genes and susceptibility to carcinogens;and test the hypothesis that ubiquitin ligase factors Cul3-Rbx1 are part of INrf2:Nrf2 auto-regulatory loop that controls their cellular abundance and expression and induction of antioxidant genes. These studies upon completion will provide a better understanding of the mechanisms of the cellular protection against oxidative/electrophilic stress and chemoprevention. The studies may also provide the basis for further analysis of identified targets (cis- and trans-elements) that could ultimately allow us to increase the chemoprotective capacity of cells.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
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Balshaw, David M
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University of Maryland Baltimore
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Khatri, Raju; Shah, Preeti; Guha, Rupa et al. (2015) Aromatase Inhibitor-Mediated Downregulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer. Mol Cancer Ther 14:1728-37
Niture, Suryakant K; Khatri, Raju; Jaiswal, Anil K (2014) Regulation of Nrf2-an update. Free Radic Biol Med 66:36-44
Shelton, Phillip; Jaiswal, Anil K (2013) The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J 27:414-23
Niture, Suryakant K; Gnatt, Averell; Jaiswal, Anil K (2013) Oncogene PKC? controls INrf2-Nrf2 interaction in normal and cancer cells through phosphorylation of INrf2. J Cell Sci 126:5657-69
Niture, Suryakant K; Jaiswal, Anil K (2013) Nrf2-induced antiapoptotic Bcl-xL protein enhances cell survival and drug resistance. Free Radic Biol Med 57:119-31
Kaspar, James W; Niture, Suryakant K; Jaiswal, Anil K (2012) Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression. J Cell Sci 125:1027-38
Niture, S K; Jaiswal, A K (2011) INrf2 (Keap1) targets Bcl-2 degradation and controls cellular apoptosis. Cell Death Differ 18:439-51
Niture, Suryakant K; Jaiswal, Anil K (2011) Inhibitor of Nrf2 (INrf2 or Keap1) protein degrades Bcl-xL via phosphoglycerate mutase 5 and controls cellular apoptosis. J Biol Chem 286:44542-56
Niture, Suryakant K; Jaiswal, Anil K (2010) Hsp90 interaction with INrf2(Keap1) mediates stress-induced Nrf2 activation. J Biol Chem 285:36865-75
Niture, Suryakant K; Kaspar, James W; Shen, Jun et al. (2010) Nrf2 signaling and cell survival. Toxicol Appl Pharmacol 244:37-42

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