Antioxidant response element-mediated gene induction represents an important component of cancer chemoprevention strategies and is regulated in part by the transcription factor Nrf2, which itself is negatively regulated by Keap1. Preliminary studies have identified a new functional Cys center that exhibits differential reactivity.
The specific aims will test the hypothesis that Keap1 activity is regulated by multiple Cys residues that exhibit differential chemical reactivity, allowing integration of chemical signals.
Aim 1 will identify and measure kinetic reactivity of recombinant Keap1 Cys residues towards chemically distinct classes of Phase II inducing agents. The approach will use liquid chromatography- tandem mass spectrometry (LC-MS-MS). LC-MS-MS spectra will be analyzed using a pattern recognition algorithm SALSA.
Aim 2 will determine if key Keap1 Cys residues regulate association between the Neh2 domain of Nrf2 and Keap1 following reaction with thiol-reactive electrophiles. Recombinant Keap1 will be used in an in vitro assay that will measure binding affinity of a Neh2 GFP fusion protein. Site directed mutagenesis will be used to verify that Keap1 Cys residues control the association between Keap1 and the Neh2 domain.
Aim 3 will determine if release of Nrf2 from Keap1 is regulated by key Cys residues in intact cells. Cells will be transiently transfected with vectors expressing wild type Keap1 or Keap1 containing Cys to Ala mutations in order to verify results in vivo. Immunoprecipitation immunoblotting of FLAG tagged Keap1 and HA tagged Nrf2 will be used to measure association and release of Nrf2 from Keap1. Thiol reactivity of mutated and wild type FLAG tagged Keap1 will be assessed in vivo using 2D Difference Gel Electrophoresis.
Aim 4 will determine if key reactive Cys residues in Keap1 regulate conformational changes that are associated with release of Nrf2. Far- and near UV CD will be used to assess secondary and tertiary structure, while conformational stability will be examined using differential scanning calorimetry.
