Cancer is one of the foremost causes of death worldwide with about 14.1 million new incidences and 8.2 cancer related deaths occurring globally. NF-E2 p45-related factor 2 (Nrf2), a cap-'n'-collar basic leucine zipper (CNC-bZIP) transcription factor, prevents carcinogenesis through expression of genes that ensure the excretion, enzymatic modification, and repair of oxidative damage in cells containing the antioxidant response element (ARE) in their promoter region. Beyond providing cytoprotection against oxidative stress and xenobiotics, Nrf2 pays a role in maintaining basic physiological processes such as energy metabolism and cell cycle regulation. Whilst Nrf2 plays a pivotal role in preventing degenerative and inflammatory disease, upregulation of Nrf2 promotes tumourigenesis in cancerous cells. Therefore, understanding the mechanisms controlling Nrf2 activity is important in translational medicine. Nrf2 is regulated by proteasomal degradation by Kelch-like ECH-associated protein 1 (Keap1) an E3 ubiquitin ligase substrate adaptor protein that recruits of cullin-3 (Cul3) to Nrf2 via its Neh2 domain. Nrf2 is also negatively regulated by phosphorylation by glycogen synthase kinase-3 (GSK-3) causing β-transducin repeat-containing protein (β-TrCP) to ubiquitinate Nrf2 by Skp1-Cul1-F-box (SCF) ubiquitin ligase through the Neh6 domain of Nrf2. Several research groups have shown that induction of ARE-driven genes can be regulated by phosphoinositide 3- kinase- protein kinase B (PI3K-Akt/PKB) signalling pathway. The ability of tert-butylhydroquinone (tBHQ), 1-[2-cyano-3,12-dioxooleana-1,9(11)-diene-28-oyl]imidazole (CDDO-Im), diethyl maleate (DEM), curcumin, carnosol, ferulic acid and sulforaphane (SFN) to activate Nrf2-target genes in a Keap1-dependent or Keap1-independent manner was tested. It was discovered that all compounds, except for SFN, activate Nrf2-target genes in a Keap1-independent manner, inhibiting GSK-3 and functioning through the Neh6 domain of Nrf2. Analysis of the involvement of PI3K-Akt/PKB pathway in Nrf2 activation revealed that regulation of Nrf2 through the PI3K-Akt/PKB pathway is independent of Keap1 but dependent on GSK-3. Also, it was shown that tBHQ, DEM, CDDO-Im, curcumin, ferulic acid directly decreased phosphatase and tensin homolog (PTEN) activity, thereby preventing formation of the phosphodegron in the Neh6 domain of Nrf2. With increased Nrf2 levels reported in various cancers including lung cancer, leading to the progression of these cancers, Nrf2 can be seen as a double-edged sword. Loss-of-function somatic mutations in KEAP1 as well as somatic mutation in NFE2L2 has been reported in several human cancers playing a role in the development of such cancer. Using short hairpin RNA (shRNA) and the CRISPR/Cas9 system to generate stable Nrf2 knockdown A549 and H460 cells, the second part of this thesis investigated biochemical and physiological changes that occur, when the Nrf2 is genetically downregulated, and further on to determine what mechanism(s) is responsible for decreased cell proliferation in tumours. The findings obtained confirm that downregulation of Nrf2 from the human non-small lung adenocarcinoma epithelial cell line A549 and H460, in which Nrf2 is upregulated though somatic mutations in KEAP1, results in decreased cell proliferation. Analysis of the genes involved in NADPH generation and pentose phosphate pathway (PPP) show that decrease in Nrf2 caused a decrease in the expression of genes involved in PPP. Although knockdown of Nrf2 resulted in a decrease in cell proliferation, it was shown that this decrease was not as a result of cell death. Nrf2 is able to control cell proliferation by induction of metabolic reprogramming geared towards favoring anabolic pathways and influencing the PPP as well as provide energy source required for cell proliferation.

Nrf2 (NF-E2-related factor 2) is a transcription factor that regulates a battery of downstream genes that contain an antioxidant response element (ARE) in their promoters, including intracellular redox-balancing proteins, phase II detoxifying enzymes, and transporters. These Nrf2-dependent proteins work in collaboration to protect against many diseases where oxidative stress plays an essential role in disease onset and progression. Consequently, it is imperative to understand the basic molecular mechanisms of how Nrf2 is regulated so we can target this pathway for disease prevention and treatment.Nrf2 is mainly regulated at the protein level by the ubiquitin proteasome system. Under basal conditions Nrf2 is constantly ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and subsequently degraded by the 26S proteasome. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. In this dissertation, we identified two molecular mechanisms that are important in understanding how the Nrf2-Keap1 pathway is regulated: (i) USP15 negatively regulates the Nrf2-Keap1 pathway by deubiquitinating Keap1 and (ii) deubiquitinated-Keap1 binds in the Cul3-Keap1-E3 ligase complex more tightly than ubiquitinated-Keap1. Additionally, (iii) we demonstrated the importance of the Nrf2-Keap1 pathway in USP15-dependent paclitaxel-chemoresistance.Under oxidative stressed or induced conditions the ability of the E3-ligase to target Nrf2 for degradation becomes impaired. As a result, Nrf2 is stabilized and free Nrf2 translocates to the nucleus and initiates transcription of ARE-bearing genes. Activation of this pathway is advantageous for chemoprevention. In Chapters 4 and 5, we identified and characterized two activators of the Nrf2 cytoprotective pathway, oridonin and cinnamic aldehyde. These compounds inhibit Cul3-Keap1-dependent degradation of Nrf2, stabilize Nrf2 protein levels, and activate the antioxidant response. Furthermore, both compounds are able to protect against cytotoxic and genotoxic stress-induced cell death. Moreover, our study on USP15 has elucidated an additional mechanism that allows small molecules, such as oridonin, to activate Nrf2 by causing a switch in ubiquitination from Nrf2 to Keap1. Taken together, these findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.

This dissertation comprises identifying the structural determinants of binding selectivity as demonstrated in three systems. The first system involves the structure determination of Keap1-small molecule fragment complexes to locate binding surfaces. The second system involves the structural determination of a NEMO/IKKbeta complex to serve as a platform for future fragment binding validation studies. The third system involves the structural investigation of a bacterial phosphoglycosyltransferase found in Campylobacter concisus to find the active site. Keap1 binding of Nrf2 is a regulatory mechanism to inhibit the transcription factor activity of Nrf2 to upregulate Nucleoporin p62 (p62). Nucleoporin p62 is a regulator of tau protein aggregates in Alzheimer's disease. The determination of binding hot spots in the Keap1 active site could serve as a starting point for the development of inhibitors as a treatment method for Alzheimer’s disease. To achieve this, I have developed a crystal form of Keap1 that allows for fragment-based study of binding in the active site via small molecule fragment screening and X-ray crystallography. Analysis of collected data has resulted in the solution of four structures, one containing a peptide fragment and three containing small molecule fragments that occupy a region of binding within the Keap1 active site, demonstrating the utility of the crystal form and affording information on binding hot spots. Nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) is a transcription factor and has been linked to cancer, inflammation, and immune dysfunction. The enzyme complex IκB kinase (IKK) is a regulator of NF-κB and consists of three subunits: IKK-α, IKK-β, and NEMO. If NEMO activity is abrogated, IKK is unable to activate NF-κB, making it a promising therapeutic target. My research has found crystallization conditions and performed trials of phase determination on an N terminal IKKβ-binding construct of NEMO containing previously uncharacterized regions of this protein. Glycosylation is a commonly occurring post-translational modification that affects a number of processes including protein folding, trafficking, cell-cell interactions and host immune response. The phosphoglycosyl transferase PglC is an essential part of the Campylobacter glycosylation pathway and a possible antibacterial target. My research determined the crystallization conditions and has developed complexes and protein constructs for phase determination of this single-pass transmembrane protein and will in the future provide a platform for structure-based inhibition of this protein.

PURPOSE: The Nrf2/Keap1/ARE pathway is a major regulator of cytoprotective responses to oxidants. Gluatredoxin-1 (Glrx-1), a small thiol transferase removes glutathione (GSH) adducts from proteins and participates in redox signaling. Glrx-/- mice exhibit increased protein GSH adducts (PSSG) and non-alcoholic fatty liver disease (NAFLD). Unexpectedly, our Glrx-/- mice showed increased hepatic glutathione (GSH) levels. The Nrf2/Keap1/ARE pathway, as an important regulator of glutathione synthesis, could be regulated by Glrx-1 activity. METHODS: To determine the role of Nrf2 in vivo, we treated Glrx-/- mice with high fat high sucrose (HFHS) diet to induce metabolic and oxidative stress. Livers were harvested at 10 months of age after 8 months on HFHS diet. Gene expression of Nrf2 and its down-signaling targets were determined using RT-qPCR and protein expression was accessed via WB. To determine the role of Nrf2 in Glrx-deficiency in vitro, Glrx siRNA was transfected in HEK293A and HepG2 cells and exposed to high palmitate high glucose (HPHG) to mimic metabolic stress and hydrogen peroxide to mimic oxidative stress. RESULTS: Glrx-/- deficiency increased Nrf2 activity and gene expression, and decreased Keap1 activity and gene expression. Glrx silencing in liver promoted Nrf2 activity and translocation to the nucleus, and downstream targets of Nrf2 were upregulated. CONCLUSION: Our findings indicate that the Nrf2/Keap1/ARE pathway is regulated by Glrx in vitro and in vivo.

Yes / R-loops are by-products of transcription that must be tightly regulated to maintain genomic stability and gene expression. Here, we describe a mechanism for the regulation of the Rloop- specific helicase, senataxin (SETX), and identify the ubiquitin specific peptidase 11 (USP11) as an R-loop regulator. USP11 de-ubiquitinates SETX and its depletion increases SETX K48-ubiquitination and protein turnover. Loss of USP11 decreases SETX steady-state levels and reduces R-loop dissolution. Ageing of USP11 knockout cells restores SETX levels via compensatory transcriptional downregulation of the E3 ubiquitin ligase, KEAP1. Loss of USP11 reduces SETX enrichment at KEAP1 promoter, leading to R-loop accumulation, enrichment of the endonuclease XPF and formation of double-strand breaks. Overexpression of KEAP1 increases SETX K48-ubiquitination, promotes its degradation and R-loop accumulation. These data define a ubiquitination-dependent mechanism for SETX regulation, which is controlled by the opposing activities of USP11 and KEAP1 with broad applications for cancer and neurological disease. / Wellcome Trust Investigator Award, Lister Institute of Preventative Medicine Fellowship

The Keap1-Nrf2 pathway is a major upstream regulator of xenobiotic detoxification. In Drosophila, directed activation of the protein complex of Keap1 and CncC (the homolog of human Nrf2) in principal and stellate cells of the Malpighian (renal) tubules confers resistance to lethal doses of the pesticide malathion, which is metabolized into organic anions. Dietary exposure to organic anions such as salicylate (10 mM) causes increases in fluid secretion rate and salicylate flux across Malpighian (renal) tubules. Here we used salicylate-selective microelectrodes and Ramsay assays to determine the role of Keap1/CncC in regulating these responses. Fluid secretion rate and salicylate flux across tubules isolated from adults with directed activation of Keap1/CncC in the principal cells are comparable to the values from salicylate-fed controls. Fluid secretion rate, concentration of salicylate in the secreted fluid and salicylate flux did not differ significantly between tubules isolated from adults with directed activation of Keap1/CncC in the principal cells reared on a diet containing salicylate and those reared on control media, indicating that the detoxification pathway was activated regardless of the presence of dietary salicylate. This is in contrast to the significant increase in fluid secretion rate and salicylate flux between tubules isolated from salicylate-fed adults and adults reared on a control diet with directed activation of Keap1/CncC in the stellate cells, supporting previous studies that demonstrated the inability of stellate cells to transport organic anions. Taken together, these results suggest a role for Keap1/CncC in upregulating fluid secretion in response to the presence of dietary organic anions. / Thesis / Master of Science (MSc) / The Keap1-Nrf2 pathway is a major upstream regulator of xenobiotic detoxification. In Drosophila, directed activation of the protein complex of Keap1 and CncC (the Nrf2 homolog) in principal and stellate cells of the Malpighian (renal) tubules confers resistance to lethal doses of the pesticide malathion, which is metabolized into organic anions. Dietary exposure to organic anions such as salicylate (10 mM) causes increases in fluid secretion rate and salicylate flux across Malpighian (renal) tubules that are comparable to tubules isolated from adults with activated Keap1/CncC reared on a salicylate-free diet. This suggests a role for Keap1/CncC in upregulating fluid secretion in response to the presence of dietary organic anions.

NF-E2-related factor 2 (Nrf2) is a transcription factor that is responsible for maintaining cellular homeostasis by controlling the fate of cells through transcriptional upregulation of antioxidant response element-bearing genes critical for eliminating toxicants and carcinogens. Under quiescent conditions, basal levels of Nrf2 are relatively low due to tight regulation by Keap1, a substrate adaptor protein for a Cullin 3 (Cul3)-E3 ubiquitin ligase complex that facilitates the ubiquitination and degradation of Nrf2. It is thought that when cells are exposed to oxidative stress, naturally-occurring compounds, or synthetic chemicals, cysteine residues in Keap1, particularly cysteine 151 (C151), are modified causing a conformational change that compromises the ability of the Keap1-Cul3-E3 ubiquitin ligase complex to properly ubiquitinate Nrf2. It is then stabilized and allowed to translocate into the nucleus to transcriptionally activate downstream genes. Interestingly, recent emerging data has revealed the "dark side" of Nrf2. Epigentic alterations and somatic mutations in either Nrf2 or Keap1 disrupting the Nrf2-Keap1 axis and causing constitutive activation of Nrf2 have been found in many human cancer cell lines and tumors. Thus, Nrf2 provides mutated cells a protective advantage against cytotoxic chemotherapeutics, allowing for further cell survival and growth. It is well known that arsenic is a human carcinogen and can activate the Nrf2 pathway through a Keap1-C151 independent mechanism. It has also been shown that arsenic can activate autophagy, a bulk-lysosomal degradation pathway. In this dissertation, we establish the cross-talk between the Nrf2-Keap1 pathway and autophagy by elucidating a novel non-canonical mechanism of Nrf2 activation. We found that deregulation of autophagy causes accumulation of p62, a substrate adaptor protein, which sequesters Keap1 into autophagosomes and activates the Nrf2 pathway. Moreover, we also demonstrate how arsenic blocks autophagic flux and prolongs Nrf2 activation through this novel mechanism. Additionally, activation of the Nrf2 pathway has been shown to confer protection against arsenic-induced toxicity and carcinogenicity. We demonstrate that co-treatment with sulforaphane alleviates arsenic-mediated autophagy. These studies suggest that the Keap1-C151 dependent mechanism triggers the chemopreventive role of Nrf2 while activation through p62 elicits the dark side. Therefore, the use of Keap1-C151-dependent compounds to counteract environmental insults continuous to be a promising strategy for cancer prevention.

Oxidative stress is a feature of many chronic neurodegenerative diseases as well as a contributing factor in acute disorders including stroke. Fork head class of transcription factors (Foxos) play a key role in promoting oxidative stress-induced apoptosis in neurons through the upregulation of a number of pro-apoptotic genes. Here I demonstrate that synaptic NMDA receptor activity not only promotes Foxos nuclear exclusion but also suppresses the expression of Foxo1 in a PI3K-dependent fashion. I also found that Foxo1 is in fact, a Foxo target gene and that it is subject to a feed-forward inhibition by synaptic activity, which is thought to result in longerterm suppression of Foxo downstream gene expression than previously thought. The nuclear factor (erythroid 2-related) factor 2 (Nrf2) is another transcription factor involved in oxidative stress and the key regulator of many genes, whose products form important intrinsic antioxidant systems. In the CNS, artificial activation of Nrf2 in astrocytes has been shown to protect nearby neurons from oxidative insults. However, the extent to which Nrf2 in astrocytes could respond to endogenous signals such as mild oxidative stress is less clear. The data presented herein, demonstrate for the first time that endogenous Nrf2 could be activated by mild oxidative stress and that this activation is restricted to astrocytes. Contrary to the established dogma, I found that mild oxidative stress induces the astrocytic Nrf2 pathway in a manner distinct from the classical Keap1 antagonism employed by prototypical Nrf2 inducers. The mechanism was found to involve direct regulation of Nrf2's transactivation properties. Overall these results advance our knowledge of the molecular mechanism(s) associated with the control of endogenous antioxidant defences by physiological signals.

To maintain intracellular redox homeostasis, genes encoding many endogenous antioxidants and phase II detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis- antioxidant responsive elements (AREs) in their promoter regions. Nrf2 has emerged as the pivatol transcription factor responsible for ARE-dependent transcription, and has been shown to play critical roles in hepatotoxicity, chemical carcinogenesis, pulmonary inflammatory diseases, neurodegenerative diseases and aging. Therefore, understanding the molecular mechanism of the Nrf2-dependent cytoprotective system is important for development of drugs for therapeutic intervention.Nrf2 is targeted by Keap1 for ubiquitin-mediated degradation under basal conditions. Upon oxidative stress, distinct cysteine residues of Keap1 are alkylated, leading to inhibition of Keap1 and activation of Nrf2. However, it was not clear how Nrf2 is re-entered into the repression status when redox homeostasis is re-achieved. In this dissertation, we establish that the post-induction repression of Nrf2 is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination/ degradation machinery. We show that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2 signaling; ubiquitination of Nrf2 is carried out in the cytosol; Keap1 nuclear translocation is independent of Nrf2; and the Nrf2-Keap1 complex does not bind the ARE. Collectively, these results suggest that Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE and mediates nuclear export of Nrf2 followed by ubiquitination and degradation of Nrf2 in the cytoplasm.In addition to Keap1-mediated negative regulation, we identified a novel positive regulatory mechanism of Nrf2 mediated by transcription co-activator p300/CBP. We show that p300/CBP directly binds and acetylates Nrf2 in response to oxidative stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner both in vitro and in vivo. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.

Le stress électrophile et oxydant est un souci grandissant pour la santé avec l'évolution de nos modes de vie. L'exposition aux ultraviolets, à la pollution, aux substances carcinogènes, à la fumée de cigarette et la pratique intensive d'activités sportives sont autant de causes de stress oxydant pour l'organisme. Ces dommages sont associés à plusieurs maladies et conditions pathologiques telles que cancers, diabètes, infections pulmonaires et maladies neurodégénératives. L'élément de réponse antioxydant (ARE) est un des composants principaux des défenses de la cellule contre ce phénomène. Ce promoteur agit sous le contrôle de Nrf2 (Nuclear factor erythroid 2-related factor 2). Une stratégie populaire pour l'activation de ce mécanisme est l'utilisation d'inducteurs covalents. Ces molécules agissent par la formation de liens covalents avec les nombreux résidus cystéine de Keap1 (Kelch-like ECH-associated protein 1), une protéine chaperonne qui contrôle l'activité de Nrf2. Cette thèse présente la synthèse, les propriétés biologiques et l'étude des relations structure-activité d'une librairie d'électrophiles capables d'induire la transcription des gènes cibles de la voie de signalisation Keap1/Nrf2/ARE. Le premier volet fait état de la comparaison d'une variété d'électrophiles simples pour étudier les préférences de la cible. Le deuxième volet montre que la présence d'une seconde fonction capable de piéger un résidu cystéine fournit des analogues très puissants.