title: Molecular investigation of Keap1-dependent regulation of the Nrf2 cell defence pathway
Creators: name: Bryan, Holly
Publisher: name: University of Liverpool Repository
Year: 2015

description: Mammalian cells have evolved highly regulated defence pathways, which are activated in response to stress. The transcription factor Nrf2 is activated in response to chemical and oxidative stress and induces the expression of antioxidants and detoxification enzymes. Nrf2 activity is regulated by its cysteine-rich repressor protein Keap1, which facilitates Nrf2 degradation. In the presence of electrophilic compounds and/or oxidative stress, Keap1-mediated repression of Nrf2 is hindered, allowing the nuclear localisation of Nrf2 and the up-regulation of cell defence gene expression. The dysregulation of the Nrf2 pathway has been associated with many disease pathologies, and is a promising therapeutic target. Furthering our understanding of the chemico-biological triggers for Nrf2 activation may inform the design of novel Nrf2-inducers with increased efficacy and reduced toxicity. The modification of one or more cysteine residues in Keap1 by electrophiles is believed to be central to Nrf2 activation. Therefore, the aims of the studies in this thesis were to investigate the chemical modification of Keap1 cysteine residues by Nrf2-inducers and identify novel Keap1 binding proteins, which may play a role in the regulation of Nrf2 activity. Previous work carried out in this lab identified cysteine residues in Keap1 that are covalently modified by a panel of Nrf2-inducing compounds in recombinant Keap1 protein and in cells. Additionally, Nrf2 can be induced by glutathione (GSH) depletion in the absence of Keap1 adduct formation. We hypothesised that GSH depletion permits reactive oxygen species (ROS) to accumulate and oxidise Keap1 cysteine residues, thereby inducing Nrf2. To address this, we treated Keap1-V5 expressing HEK293T cells with Nrf2-inducing compounds. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate the ability of these compounds to form adducts with Keap1 cysteine residues, or induce reversible/irreversible redox modifications of these residues. We show that compounds which form covalent adducts with Keap1 and deplete GSH (i.e. 2,4-dinitrochlorobenzene) or do not deplete GSH (i.e. dexamethasone 2,1-mesylate) induce modifications of Keap1 that could be representative of oxidation. However compounds which do not form covalent adducts with Keap1 but cause oxidative stress (i.e. hydrogen peroxide), or GSH depletion (i.e. L-buthionine-sulfoximine), do not appear to cause oxidative-like modification of Keap1 cysteines. We therefore show that some Nrf2 inducers promote the formation of reversible and/or irreversible redox modifications of Keap1 which could be due to thiol oxidation, although this is not dependent on GSH depletion. To further explore the modification of Keap1 cysteine residues by Nrf2 inducers, we investigated the ability of triterpenoids (TPs) to modify Keap1. TPs, in particular methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-Me), are potent inducers of Nrf2 and are potential therapeutic agents. However, the mechanism of Nrf2 activation by TPs has not been fully elucidated using LC-MS/MS. We identify key cysteine residues in Keap1 which are adducted by a chemically-tuned TP (CDDO-Epoxide) in recombinant Keap1 and in cells expressing Keap1-V5. Additionally, we use an in silico modelling approach to visualise the binding orientations of CDDO-Epoxide with key Keap1 cysteine residues. Correlating the potency of a panel of TPs towards the Nrf2 pathway with their in silico propensity to bind covalently to the identified residues showed no relationship. However, we show significant positive correlation between the potency of these TPs towards Nrf2 and their in silico propensity to bind non-covalently in two cysteine-containing pockets (Cys-273, -288) in Keap1. These data reveal the specific sites of interactions between potent TP Nrf2 inducers and Keap1, and highlight the non-covalent binding of Keap1 by electrophiles as a potential mechanism of Nrf2 activation. The function of Keap1 is regulated by interactions with binding partners, such as sequestosome1 (p62) which targets it for autophagic degradation, or PGAM5 which localises Keap1 to the mitochondria. We previously used an LC-MS/MS approach to identify p62 as a novel Keap1-binding partner in cells. Therefore, we reasoned that using the same experimental approach with a more sensitive MS system, we could identify additional Keap1-binding proteins. Specifically, we identified a large number of proteins that co-purified with Keap1-V5 from HEK293T cell lysates, of which 55 were found to contain a known Keap1 binding motif, such as the one found in Nrf2, p62 and PGAM5. Network analysis highlighted the potential link between Keap1/Nrf2 and the p53 cell survival pathway. We validated the LC-MS/MS data using a yeast 2-hybrid screen, which reveals HBS1L, RIC8A and PSMD3 as novel Keap1 binding partners, although the functional relevance of the interaction of these proteins with Keap1 requires further investigation. In summary, the data presented in this thesis demonstrates that whilst the covalent modification of Keap1 cysteines is an important aspect of Nrf2 induction, the oxidation of Keap1 thiols may be an alternative mechanism. We identify key cysteine residues in Keap1 covalently modified by a potent TP Nrf2 inducer in recombinant protein and cells, but show that non-covalent modification of Keap1 may be involved in the process of Nrf2 activation by this class of compound. It will be important in future studies to determine how the modification of Keap1 cysteine residues is translated to the activation of Nrf2. Additionally, we identify putative novel Keap1 binding partners which may serve to regulate the activity of the Nrf2 pathway. Overall, these findings expand our understanding of the chemical and molecular interactions that govern the activity of Nrf2, and will therefore contribute to the ongoing efforts to target this pathway as a novel therapeutic strategy in numerous diseases.