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.

Parkin gene is associated with the development of autosomal recessive juvenile parkinsonism (Kitada et al., 1998) which is a common form of familial Parkinson’s disease (Klein and Schlossmacher, 2006). Since Parkin has multiple cell protective effects, increasing the expression level of Parkin in the brain might be able to rescue cells in danger, which in turn might prevent or slow down the development of Parkinson’s disease (Ulusoy and Kirik, 2008). In order to increase Parkin expression, it is important to understand the transcriptional mechanisms regulating Parkin expression (Maston et al., 2006). Since human Parkin is very big (~1.4 Mb) (Asakawa et al., 2001), in this study we use the smaller Fugu parkin gene, which is an ortholog of human Parkin (Yu et al., 2005), to search for the transcriptional factors and signaling pathways regulating Parkin expression. We have cloned vertebrate constructs that allow for the monitoring of an entire genomic Fugu parkin gene tagged with a reporter (eGFP or luciferase) in mammalian cells; and have established cellular model for studying the expression. According to the “TRANSFAC” transcription factor database, as well as “TFBIND” and “TFSEARCH” softwares (Wingender et al., 1996; Heinemeyer et al., 1998; Heinemeyer et al., 1999; Tsunoda and Takagi, 1999; Akiyama 1995), potential Nrf2 binding sites are conserved in the promoters of mammalian parkin (including human Parkin and mouse parkin) and in Fugu parkin. In this study, we could not find a link between the presence of the potential Nrf2 binding site(s) in the parkin promoter and the up-regulation of parkin; and we could not find an association between the Nrf2 pathway activation and the induction of parkin under the specific experimental conditions.

Les adjuvants vaccinaux permettent d’augmenter la réponse immunitaire dirigée contre un antigène donné. Certains de ces adjuvants miment des signaux de danger, tels que des agonistes des récepteurs de l’immunité, les récepteurs Toll-like (TLR) ou les récepteurs NOD-like (NLR), et permettent une activation des cellules dendritiques (DC). Les DC sont essentielles dans la mise en place d’une réponse adaptative contre un antigène : elles acquièrent un phénotype mature, contrôlé par les voies des MAPK et NF-κB, permettant la présentation de l’antigène aux lymphocyte T et l’initiation d’une réponse spécifique. La voie Nrf2/Keap1, impliquée principalement dans la détoxication des xénobiotiques et le contrôle du stress oxydant, peut être activée en réponse à des agonistes des TLR tels que le LPS (agoniste TLR 4). Nous avons mis en évidence qu’un traitement par le R848 (agoniste TLR7/8) ou le MDP (agoniste NOD2) induit la transcription des gènes cibles de Nrf2 dans les DC murines. Nrf2 participe également à la production de cytokines inflammatoires en réponse au LPS et au R848 et jouet un rôle dans la prolifération lymphocytaire induite par les DC pré-traitées avec le MDP. Par ailleurs, Nrf2 contrôle la réponse anticorps spécifiques de l’antigène chez la souris. L’injection d’anatoxine tétanique induit une production d’anticorps plus élevé chez la souris déficiente nrf2 par rapport aux souris sauvages. Cette augmentation de la production d’anticorps est corrélée avec une augmentation du nombre de lymphocyte B dans la moelle osseuse et la rate. / Vaccine adjuvants are able to boost immune response toward antigens when there are simultaneously injected. Some of these adjuvant mimic danger signals, such as Toll like receptors (TLR) agonists or NOD-like receptors agonists, required for dendritic cell (DC) activation. DC are essentiales for adaptative immune response against antigens : they acquire mature phenotype, controlled by MAPK and NF-kB signaling pathway, leading to antigen presentation and specific immune response. The Nrf2/keap1 signaling pathway, mainly involves in xenobiotics detoxication and oxidative stress control, can be activate by TLR agonists, such as LPS (TLR 4 agonist).We showed that R848 (TLR 7/8 agonist) and MDP (NOD2 agonist) could induce Nrf2’s target genes transcription in murines dendritic cells (BMDC). Nrf2 seems also to be part of inflammatory cytokines production in response to LPS or R848 and modulated T lymphocyte proliferation induced by MDP pre-treated BMDC. Moreover, Nrf2 appears to play a role in specific antibodies response against an antigen in mice. . In fact, Tetanus toxoid (TT) injection induces higher titer of antibodies anti-TT in nrf2-/- mice compared to nrf2+/+ mice. This increase is also correlated with more specific B lymphocytes in bone marrow and spleen after TT immunisation.

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.

Embryonic stem (ES) cell maintenance and differentiation are dynamic processes controlled by various intrinsic and extrinsic factors. Identifying these factors will enhance the understanding about developmental process and improve the application of stem cells in clinic. Previous studies highlight a shift between non-oxidative and oxidative energy metabolism to play roles during differentiation. Oxidative metabolism is a major source of reactive oxygen species (ROS) which is regulated by a cytoprotective transcription factor, Nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, this study investigate relationship between metabolism, ROS, and Nrf2 during mouse ES cell differentiation. In vitro models representing early lineage differentiation were used. By measuring metabolic profiles, ROS, and Nrf2 levels from the models, Nrf2 was found related to pluripotency and ROS. However, relationship among metabolism and Nrf2 or ROS could not be detected. Gain- and loss-of-function experiments by pharmacological activator, short hairpin RNA knockdown, and CRISPR-Cas9 genome editing showed that Nrf2 could promote pluripotency and inhibit differentiation, especially during early differentiation toward neural lineage. This study suggested a new player in transcription control that governs pluripotency and differentiation.

Novel approaches are required to address Alzheimer’s disease (AD) in our ageing population, with recent interest focused on inflammation and oxidative stress (OS). Disruption of NF-E2-related factor 2 (Nrf2) signalling increases OS and promotes AD. Amyloid precursor protein (APP) is intimately linked with AD, with the Swedish mutation (<i>hAPP_swe</i>) used in numerous transgenic models. Furthermore, increasing importance has been placed on the suggested link between nutritional status and AD. To assess Nrf2 as a potential target in AD, we examined the effect of metabolic stress, by chronic high fat (HF) feeding or acute lipopolysaccharide (LPS) treatment on the brains of aged WT, <i>Nrf2^-/-</i>, hAPPswe and <i>Nrf2^-/-/hAPP_swe</i> mice. <i> </i><i>Nrf2^-/- mice displayed impaired enthorinal cortex-dependent cognition, with raised basal hippocampal inflammation. The inflammatory state was attenuated by chronic HF feeding, whilst maintaining insulin sensitivity. In contrast, <i>hAPP_swe</i> did not display an inflammatory response to HF feeding, but demonstrated impaired insulin signalling; in line with AD-associated insulin resistance. Additionally, <i>Nrf2^-/-</i> mice display increased glial cell activation and activation of mitogen activated protein kinases and mitochondrial impairment. These may be indicative of OS-induced cellular dysfunction and is supported by an aggravated response to LPS, which potentiates IL-1β production. Furthermore, despite an attenuated LPS response following the induction of tolerance, <i>Nrf2^-/-</i> mice maintain glial cell activation following treatment which may be suggestive of a primed immune environment within the brain. </i> In conclusion, these data indicate altered glucose homeostasis in both <i>Nrf2^-/-and <i>hAPP_swe</i> mice, as previously reported. Further, we advocate that Nrf2 plays a key role in mitochondrial function and health and may be important for the ameliorative effects of HF feeding. Taken together, mitochondrial dysregulation and associated OS may help explain the development of cognitive impairment in <i>Nrf2^-/-</i> mice. This may be relevant for AD given the age-dependent decline in Nrf2 expression in humans.

Oxidative stress is known to contribute to many forms of heart disease. Oxidants such as H₂O₂ can cause hypertrophy of cardiomyocytes (CMCs). Heart fibroblasts (HFs) also contribute to oxidant-induced heart disease by disordering the extracellular matrix and causing fibrosis. Since both of these cells encounter the same stressors in vivo, we examined the signaling pathways involved in responding to oxidative stress in both cell types. We have established the EGF Receptor, Src and matrix metalloproteinases (MMPs) as key regulators of oxidant-mediated phosphorylation of the MAPKs ERK1/2 and JNKs but not p38 in CMCs and HFs. We used oligonucleotide microarrays to examine the differences in global gene expression after H₂O₂ treatment in CMCs and HFs. Twenty-four hours after treatment, significant numbers of upregulated genes could be classified as being related to antioxidant or detoxification responses in both cell types. This trend lead us to examine the role of activation of promoters containing the Antioxidant Response Element (ARE) in the reaction of CMCs to H₂O₂. We have shown that H₂O₂ activates the ARE in CMCs in a manner that is dependant on the transcription factor Nf-E2 related factor 2 (Nrf2). ARE activation by H₂O₂ seems to induce cytoprotection. CMCs pretreated with H₂O₂ showed significantly less activation of caspase-3 when exposed to another oxidant, Doxorubicin. Overexpression of Nrf2 mediates this cytoprotection, possibly by protecting the cells from caspase-independent cell death. Although ARE-dependant genes were upregulated in the presence of excess Nrf2, two contractile proteins were repressed, suggesting that Nrf2 overexpression may have unknown side-effects in CMCs. We also studied the activation mechanism of Nrf2 in CMCs. Nrf2 protein levels increased after 10 min of exposure to 100 μM H₂O₂ and peaked at about 1 hr. Pharmacological and genetic inhibition of the PI3-Kinase pathway blocked AREluciferase activity in these cells. The PI3-Kinase inhibitor LY294002 also blocked Nrf2 protein accumulation, but not nuclear translocation. Here I present evidence that Nrf2 accumulation after H₂O₂ exposure is due to PI3-Kinase-mediated translational regulation. Since phosphorylation of translation initiation factors eIF4E and eIF2alpha are both inhibited by LY294002, Nrf2 translation initiation may be through non-5’ cap-mediated means.

No description available.

Els macròfags desenvolupen un paper clau en la formació de la lesió d'ateroma. Les LDL oxidades (LDLox) indueixen l'expressió de la adipocyte fatty acid-binding protein (FABP4) en els macròfags, fet que constitueix un dels desencadenants de l'evolució d'aquestes cèl·lules al fenotip de cèl·lula escumosa. Les LDLox són una font d'aldehids, que són els productes finals de l'oxidació dels àcids grassos poliinsaturats. El treball realitzat ha estat dissenyat per provar la hipòtesi de la participació dels aldehids en la inducció de l'expressió de FABP4 en macròfags. A causa del caràcter prooxidant d'aquestes espècies, s'ha avaluat si aquesta inducció podria relacionar-se amb el sistema antioxidant cel·lular dirigit pel factor de transcripció Nrf2. El treball experimental es va dur a terme amb monòcits de la línia cel·lular THP-1 que es van diferenciar a macròfags mitjançant èsters de forbol. Els aldehids apolars estudiats van ser el 2,4-decadienal (DDE) i l'hexanal. La valoració dels efectes sobre les expressions gènica i proteica de FABP4 es va realitzar per RT-PCR a temps real i western blot, respectivament. Ambdós aldehids van produir augments en l'expressió gènica i proteica de la FABP4 en els macròfags. L'estudi in silico del promotor de la FABP4 humana va revelar la presència d'un possible lloc antioxidant response element (ARE) per la unió de Nrf2 amb un elevat grau d'homologia amb la seqüència consens. L'assaig d'immunoprecipitació de la cromatina va confirmar al unió in vivo de Nrf2 a aquest lloc ARE. L'estudi de l'efecte dels aldehids sobre l'activació del factor de transcripció Nrf2 va evidenciar un comportament diferencial entre els dos compostos. El DDE augmentava la forma fosforilada i activa de Nrf2, mentre que l'hexanal no produïa cap efecte. Es va provar que en l'activació de Nrf2 produïda pel DDE estaven implicades dues de les principals vies de senyalització intracel·lular: PI-3k/Akt i ERK-MAPK. Considerant els resultats obtinguts, podem suggerir un nou paper de la via Nrf2-ARE com a desencadenant de la formació de cèl·lules escumoses a través de la inducció de FABP4 en resposta a l'oxidació. Aquests resultats proposa la via de Nrf2 com a nova diana per la intervenció terapèutica dirigida a la prevenció i el control del desenvolupament de l'aterosclerosi. / Macrophages play a crucial role in the development of atherosclerosis. It has been shown that oxidized LDL (oxLDL) induces adipocyte fatty acid-binding protein (FABP4) in human macrophages, which constitutes one of the major contributors to foam cell formation. oxLDL is a source of apolar aldehydes formed as end-products of polyunsaturated fatty acid oxidation in LDL. This thesis has been designed to study the hypothesis that apolar aldehydes participate in the induction of FABP4 expression in human macrophages. According to the prooxidant nature of these species, we have assessed whether FABP4 expression could be related to the cellular antioxidant system leadered by the transcription factor Nrf2. The experimental procedure was mainly performed by using human monocytic leukemia THP-1 cells which were differentiated to macrophages through a 72-hour phorbol ester treatment. 2,4-decadienal (DDE) and hexanal were the two apolar aldehydes studied. Reverse transcription and real time-PCR (RT-rtPCR) and Western blotting were used to assess FABP4 mRNA and protein expression, respectively. Both aldehydes produced a markedly increase in FABP4 expression at mRNA and protein levels. In silico analysis of human FABP4 promoter revealed the presence of a putative antioxidant response element (ARE) where Nrf2 could bind. This putative binding site had a high matrix similarity score with the consensus sequence. Chromatin immunoprecipitation (ChIP) assay using an Nrf2-specific antibody confirmed the in vivo binding of Nrf2 to this ARE found in human FABP4 promoter. The assessment of the effect of the two aldehydes on Nrf2 activation evidenced a differential behaviour between DDE and hexanal. Whereas DDE increased nuclear phosphorylated Nrf2 levels, hexanal showed no effect. We observed that two of the major intracellular signal transduction pathways, PI-3k/Akt and ERK-MAPK, are implicated in DDE-induced Nrf2 activation. According to our results, we propose a novel role of Nrf2-ARE pathways as a triggering step on foam cell formation mediated by FABP4 induction in response to oxidation. These results open new therapeutic targets addressed to control arteriosclerosis development.