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.

Although it is known that the regenerative function of neural stem/progenitor cells (NSPCs) declines with age, causal mechanisms underlying this phenomenon are not understood. Here, we systematically analyze subventricular zone (SVZ) NSPCs, in various groups of rats across the aging spectrum, using in vitro and in vivo histological and behavioral techniques. These studies indicate that although NSPC function continuously declines with advancing age, there is a critical time period during middle age (13-15 months) when a striking reduction in NSPC survival and regeneration (proliferation and neuronal differentiation) occurs. The studies also indicate that this specific temporal pattern of NSPC deterioration is functionally relevant at a behavioral level and correlates with the decreasing expression of the redox-sensitive transcription factor, Nrf2, in the NSPCs. When Nrf2 expression was suppressed in 'young' NSPCs, using short interfering RNAs, the survival and regeneration of the NSPCs was significantly compromised and mirrored 'old' NSPCs. Conversely, Nrf2 overexpression in 'old' NSPCs rendered them similar to 'young' NSPCs, and they showed increased survival and regeneration. Furthermore, examination of newborn Nrf2 knockout (Nrf2-/-) mice revealed a lower number of SVZ NSPCs in these animals, when compared to wild-type controls. In addition, the proliferative and neurogenic potential of the NSPCs was also compromised in the Nrf2-/- mice. These results identify a novel regulatory role for Nrf2 in NSPC function during aging and have important implications for developing NSPC-based strategies to support healthy aging and to treat age-related neurodegenerative disorders.

Submitted by Francine Silva (francine.silva@unipampa.edu.br) on 2016-09-28T21:17:01Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Potencial protetor do Extrato de Psidium guajava frente à toxicidade induzida pelo organofosforato Clorpirifós em Drosophila melanogaster.pdf: 1278673 bytes, checksum: fb86ba9285bb81b1327ca36aa998f903 (MD5) / Made available in DSpace on 2016-09-28T21:17:01Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Potencial protetor do Extrato de Psidium guajava frente à toxicidade induzida pelo organofosforato Clorpirifós em Drosophila melanogaster.pdf: 1278673 bytes, checksum: fb86ba9285bb81b1327ca36aa998f903 (MD5) Previous issue date: 2015-05-20 / Clorpirifós (CP) é um inseticida organofosforado amplamente utilizado no controle de pragas agrícolas e domésticas. O principal dano causado pelo CP é a neurotoxicidade induzida pela inibição da enzima acetilcolinesterase, o que ocasiona um aumento no neurotransmissor acetilcolina e promove uma hiperexitação no sistema nervoso central e junções musculares, levando a perturbações do funcionamento fisiológico. A exposição ocupacional é uma das principais formas de intoxicação humana por organofosforados e as terapias atuais para estes compostos não são totalmente eficientes. Nesse sentido a procura por compostos capazes de reverter esses danos tem se intensificado e alguns estudos têm focado seus esforços sobre os efeitos de proteção de plantas ou compostos naturais em várias condições neuropatológicas. Psidium guajava é uma planta amplamente utilizada na medicina popular e a sua atividade antioxidante foi descrita, no Brasil as folhas e os frutos são utilizados para a anorexia, cólera, diarréia, problemas digestivos, disenteria, insuficiência gástrica, inflamação das membranas mucosas, laringite, problemas de pele, dor de garganta, úlceras, entre outros. Neste estudo foi avaliado o potencial antioxidante e protetor do extrato hidroalcoólico de P. guajava (HEPG) contra a toxicidade induzida por CP na mosca da fruta Drosophila melanogaster. A atividade antioxidante de HEPG in vitro foi confirmada pelos ensaios de ABTS, DPPH, fenóis totais e FRAP. A exposição das moscas ao CP causou aumento da mortalidade, deficiências locomotoras e inibição da acetilcolinesterase. Moscas expostas ao CP apresentaram aumento de ROS e peroxidação lipídica, acompanhado por uma diminuição significativa na viabilidade mitocondrial. Como resposta ao aumento do estresse oxidativo, moscas expostas ao CP mostraram aumento da atividade da GST e nos níveis de GSH. A expressão de mRNA de NRF2 e MPK2 (que codifica p38MAPK em D. melanogaster) também foram significativamente super regulados. HEPG foi capaz de restaurar todos os danos e alterações bioquímicas/moleculares causados pelo CP. Os nossos resultados mostram pela primeira vez o potencial efeito protetor de P. guajava contra a toxicidade causada por clorpirifós, sugerindo a Psidium guajava como um tratamento alternativo adjunto para o envenenamento por compostos organofosforados. / Chlorpyrifos (CP) is an organophosphate insecticide widely used for control agricultural and household pests. The main damage caused by the CP is the neurotoxicity induced by inhibition of the enzyme acetylcholinesterase, which causes an increase in the neurotransmitter acetylcholine and promotes hiperexitação the central nervous system and muscle junctions, leading to disruption of physiologic function. Occupational exposure is a major form of human poisoning by organophosphates and current therapies for these compounds are not fully efficient. In this sense the search for compounds that can reverse this damage has intensified and some studies have focused their efforts on plants protection purposes or natural compounds in various neuropathological conditions. Psidium guajava is a plant widely used in popular medicine and its antioxidant activity was described in Brazil leaves and fruit are used for anorexia, cholera, diarrhea, digestive problems, dysentery, gastric insufficiency, inflammation of mucous membranes, laryngitis, skin problems, neck pain, ulcers, among others. In this study we evaluated the antioxidant and protective potential of the hydroalcoholic extract of P. guajava (HEPG) against CP induced toxicity in the fruit fly Drosophila melanogaster. HEPG in vitro antioxidant activity was confirmed by ABTS, DPPH, Total Phenolics and FRAP assays. The exposure of flies to CP caused increased mortality, locomotor deficits and inhibition of acetylcholinesterase. Flies exposed to CP presented elevated ROS and lipid peroxidation which was accompanied by a significant decrease in mitochondrial viability. As a response to increased oxidative stress, CP exposed flies showed increased in GST activity and GSH levels. The mRNA expression of NRF2 and MPK2 (which encodes D. melanogaster p38MAPK) were also significantly up-regulated. HEPG was able to restore all the damage and biochemical/molecular alterations caused by CP. Our results show for the first time the potential of P. guajava protective effect against the toxicity caused by Chlorpyrifos, suggesting Psidium guajava as an adjunct alternative treatment for poisoning by organophosphorus compounds.

Orientador: Valéria Cristina Sandrim / Resumo: A pré-eclâmpsia (PE) é a principal causa de mortalidade e morbidade entre as gestantes no Brasil e em vários países. A fisiopatologia desta doença é complexa e envolve vários processos. Um destes, amplamente validado na literatura, relaciona-se a um status oxidativo, onde há prevalência de produção de radicais livres e/ou redução da atividade antioxidante. Apesar destas evidências, a suplementação clínica com antioxidantes (vitamina C e E) não se demonstrou promissora em PE. Recentemente, vem sendo explorado como terapia em várias doenças, a ativação de um fator de transcrição, o NRF2 (do inglês - nuclear factor, erythroid 2-like 2), que atua induzindo a transcrição de diversos genes que promovem a proteção celular através da codificação de proteínas com atividade desintoxicante e antioxidante. Entre elas a heme oxigenase (HO-1) é a mais estudada, pois apresenta efeitos antiapoptóticos, antioxidantes e citoprotetor. Além disso, o aumento do estresse oxidativo na PE pode potencialmente reduzir a biodisponibilidade de óxido nítrico (NO) que pode ser modulado por alguns polimorfismos localizados no gene da óxido nítrico sintase (NOS3). Notavelmente, os haplótipos formados pela combinação de polimorfismos foram associados a diferentes subgrupos de resposta à terapia anti-hipertensiva em PE. No presente estudo, comparamos as distribuições dos polimorfismos localizados nos genes NFE2L2 rs35652124 (C/T) e no gene HMOX1 rs2071746 (A/T) em gestantes com PE que respondem à metildopa ou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Preeclampsia (PE) is the leading cause of mortality and morbidity among pregnant women in Brazil and several countries. Its pathophysiology is complex and involves several processes, including the oxidative stress (increase of free radicals and/or decrease of antioxidant defense). Although evidences, clinical supplementation with vitamins (C and E) was not promising in preeclampsia. Currently, therapeutically the activation of transcription factor, NRF2 (nuclear factor, erythroid 2-like 2), has been explored in several diseases. This factor promote cytoprotection by activates the transcription of several antioxidant and detoxification proteins. Hemeoxigenase-1 (HO-1) is the most studied, because has antiapoptotic, anti-inflammatory and cytoprotection activities. In addition, the increased oxidative stress in PE can potentially reduce the bioavailability of nitric oxide (NO) which may impaired by some SNPs on endothelial nitric oxide synthase (NOS3) gene. Notably, haplotypes formed by the combination of polymorphisms of were associated with different subgroups of response to antihypertensive therapy in PE. Therefore, in the present study we compared the distributions of rs35652124 (C/T) NFE2L2 and rs2071746 (A/T) HMOX1 polymorphisms in PE patients who respond to methyldopa or antihypertensive therapy with those found in PE patients who do not respond to methyldopa or total antihypertensive therapy. We also examined whether NFE2L2 and HMOX1 polymorphisms affect plasma HO-1 leve... (Complete abstract click electronic access below) / Mestre

A hallmark of type 2 diabetes (T2D) is β-cell dysfunction and the eventual loss of functional β-cell mass. Therefore, mechanisms that improve or preserve β-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on β-cell function. INS-1 832/13 derived β-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract, oligomeric, or polymeric procyanidin-rich fractions demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in β-cell fuel utilization. Mitochondrial complex III, IV and V components were upregulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with Nrf2 nuclear localization and expression of Nrf2 target genes, including NRF-1 and GABPA, essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and upregulation of genes critical for mitochondrial respiration, and, ultimately, enhanced glucose-stimulated insulin secretion and β-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients.

Hexavalent Chromium (Cr(VI) induces malignant cell transformation in normal bronchial epithelial (BEAS-2B) cells. Cr(VI)-transformed cells exhibit increased level of antioxidants, are resistant to apoptosis, and are tumorigenic. RNAseq analysis in Cr(VI)-transformed cells showed that expression of transcripts associated with mitochondrial oxidative phosphorylation is reduced, and the expression of transcripts associated with pentose phosphate pathway, glycolysis, and glutaminolysis are increased. Sirtuin-3 (SIRT3) regulates mitochondrial adaptive response to stress, such as metabolic reprogramming and antioxidant defense mechanisms. SIRT3 was upregulated and it positively regulated mitochondrial oxidative phosphorylation in Cr(VI)-transformed cells. Our results suggests that SIRT3 plays an important role in mitophagy deficiency of Cr(VI)-transformed cells. Furthermore, SIRT3 knockdown suppressed cell proliferation and tumorigenesis of Cr(VI)-transformed cells. Nrf2 is a transcription factor that regulates oxidative stress response. This study investigated the role of Nrf2 in regulating metabolic reprogramming in Cr(VI)-transformed cells. We observed that in Cr(VI)-transformed cells p-AMPKthr172 was increased, when compared to normal BEAS-2B cells. Additionally, Nrf2 knockdown reduced p-AMPKthr172. Our results suggest that Nrf2 regulated glycolytic shift via AMPK regulation of PFK1/PFK2 pathway. Furthermore, our results showed that Nrf2 constitutive activation in Cr(VI-transformed cells increased cell proliferation and tumorigenesis. Overall this dissertation demonstrated that Cr(VI)-transformed cells undergo metabolic reprogramming. We demonstrated that Nrf2 constitutive activation plays decisive role on metabolic reprogramming induction, and SIRT3 activation contributing to increased cancer cell proliferation and tumorigenesis.

Influenza viruses cause significant mortality and morbidity worldwide due to seasonal oubreaks as well as occassional, and sometimes devastating, pandemics. Estimates state that approximately 5% of the adult and 20% of the child population is infected yearly, leading to approximately a half-million deaths and three million severe infections in non-pandemic years. Aside from globally-circulating strains, zoonotic outbreaks caused by avian strains are a constant threat. In 1997, the first human cases of H5N1 infections occurred and since then strains of this subtype have killed approximately 700 people causing a severe disease with as high as 60% lethality rate. In March 2013, a strain of the H7N9 subtype started an epizootic in China causing a severe respiratory disease reminiscent of H5N1 infections and with a 20% case fatality rate. In this thesis, we have studied the host responses as well the viral replication kinetics of H5N1 and H7N9 strains and compared then to those of mild H1N1 seasonal and 2009 pandemic strains. During early infections of A549 cells, we have shown that the H5N1 virus induced a more profound and functional change to the host proteome. All viruses induced the NRF2-mediated oxidative stress responses and the H7N9 and H5N1 strains downregulated fibronectin, a host protein vital to infection for human strains. Using mathematical modeling and extensive growth kinetic analysis, we showed that the H5N1 and H7N9 strains had higher peak titers and faster growth kinetics. This was due to an higher infection rate for the H7N9 strain and an higher production rate for the H5N1 strain, compared to the human viruses. Conversely, the 2009 pandemic H1N1 strain had the poorest replication kinetics, longest eclipse phase and lowest infection rates. These results point towards the higher level of cellular disruption during infection with highly pathogenic strains of influenza, which may be indicative of the more profound changes required to support growth of viruses with faster kinetics to higher titers. Furthermore, the greater changes in the cellular proteome that we have characterized in vitro may be connected to the significantly greater virulence associated with infection by avian viruses in vivo, opening a novel and productive avenue of investigation to understand viral virulence mechanisms. / October 2015

Coronary heart disease (CHD) remains the single leading cause of natural death worldwide. Despite significant advances in the diagnosis and treatment, CHD accounts for 1 out of every 6 deaths in the United States. Myocardial infarct (MI) as a result of CHD causes irreversible damage to the heart through the loss of viable myocardial tissue. Patients surviving the initial MI are at risk of developing heart failure due to lost contractile function and adverse cardiac remodeling. Improvement in the survival rates for MI have led to an increase in the incidence of heart failure, affecting approximately 5 million people in the United States. Although treatment of heart failure has improved, the mortality rates of heart failure remain high with 1 in 5 dying within the first year of diagnosis and 50% dying within 5 years. The cost of caring for heart failure patients ranks number one in Medicare. Oxidative stress plays an important role in the etiology and pathophysiology of CHD and heart failure. The transcription factor Nrf2 is a master regulator of cellular antioxidant defense mechanisms, controlling the expression of numerous antioxidant and detoxification genes through the Antioxidant Response Element (ARE) in the promoter regions. The cytoprotective effects of Nrf2 have been demonstrated in a variety of organs and disease states; however, the role of Nrf2 in the heart and heart disease has not been defined. The work presented here defines roles of Nrf2 in limiting cardiac injury and the progression to heart failure (Chapter II), protecting cardiac myocytes from oxidative stress through the preservation of mitochondria (Chapter III), and mediating the infarct reducing effects of statins, one of the most prescribed pharmacological agent (Chapter IV). In order to investigate a role of Nrf2 in the pathology of ischemic injury in the heart, a mouse model of ischemia and myocardial infarct by occlusion of the left anterior descending coronary artery was used. Nrf2 knockout mice subjected to ischemia/reperfusion injury experienced a larger infarct size than wild-type mice. Furthermore, mice lacking Nrf2 experienced a higher mortality rate and an accelerated progression to heart failure, indicated by severely compromised contractile function and reduced cardiac output, within 10 days following an MI. Morphological examination revealed maladaptive remodeling, including myocyte hypertrophy, heart enlargement, and dilated left ventricle, in Nrf2 KO mice that was absent in WT mice. Analysis of cardiac function by echocardiogram revealed increased left ventricular mass, increased systolic volume, decreased fraction shortening, reduced ejection fraction, and decreased cardiac output in Nrf2 KO mice. Nrf2 KO mice also demonstrated expression of biomarkers of heart failure, such as expression of fetal gene program, with elevated levels of β-MHC, ANF, and BNP mRNA in the myocardium. Interestingly, a lack of immune cell infiltrate and myofibroblasts as well as a deficiency in collagen deposition were observed in the infarcted region of hearts from Nrf2 KO mice. These data indicate that Nrf2 plays an important role in protecting the myocardium from ischemic injury and the progression to heart failure. Lack of Nrf2 response results in deficiency of wound healing and instead initiation of maladaptive remodeling, leading to heart failure. Mitochondria are key sources of reactive oxygen species (ROS) generation, as well as important targets for ROS-induced cell injury. Cardiac myocytes have the highest content of mitochondria among all cell types and can be particularly susceptible to mitochondrial dysfunction due to the high metabolic demand associated with the contractile function of the heart. With cardiomyocytes (CMCs) isolated from neonatal rats and kept under tissue culture conditions, mitochondria exist in elaborated networks. Such networks were replaced by predominately individual punctate mitochondria 24 hours after exposure to a sublethal dose of H₂O₂. Mitochondrial morphology was altered with membrane swelling and disorganization of inner cristae with areas of condensation. Disrupted mitochondrial morphology was associated with a loss of membrane potential and decreased expression of mitochondrial proteins involved in the electron transport chain, such as cytochrome b and cytochrome c. Nrf2 overexpression prevented H₂O₂ from inducing morphological changes in mitochondria and the reduction of cytochrome b and cytochrome c expresssion. Although Nrf2 is known as a transcription factor regulating antioxidant and detoxification genes, Nrf2 overexpression did not significantly reduce the level of protein oxidation as measured by carbonyl formation. Instead, we found that Nrf2 localizes to the outer mitochondrial membrane, suggesting a direct role of Nrf2 in mitochondrial protection. As further evidence of a direct role in mitochondrial protection, a cell-free system of mitochondria isolated from the myocardium of Nrf2 knockout mice were more sensitive to permeability transition, an indicator of mitochondrial dysfunction. Combined, these data suggest that Nrf2 protects mitochondria from oxidant injury likely through direct interaction with mitochondria. In the clinic, statins are now commonly administered for patients experiencing MI or CHD. Statins have become mainstays in the treatment of hypercholesterolemia and atherosclerosis as inhibitors of the rate limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. In addition, statins have been shown to elicit pleiotropic effects, including plaque stabilization, maintenance of endothelial function, anti-inflammatory actions, and antioxidant capabilities, independent of effects on cholesterol synthesis. Recently, these pleiotropic effects have been implicated in providing acute protection against ischemia and reperfusion injury, which has led to the use of high dose statins clinically before revascularization of an ischemic event. I have found that administration of atorvastatin in mice induced Nrf2 protein levels in the heart, brain, lung, and liver. While atorvastatin reduced infarct size following an MI in wild-type mice, this protective effect was lost in mice lacking Nrf2. Failure of atorvastatin to protect against MI in Nrf2 knockout mice indicates that Nrf2 plays a critical role in mediating the protective effects of acute statin treatment. Nrf2 induction by statins is a novel discovery. In order to understand the mechanism of such statin effect, I used an in vitro cell system, in which a variety of statins, atorvastatin, simvastatin, lovastatin, and pravastatin, were found to elevate Nrf2 protein levels. Elevation of Nrf2 by statins was independent of increased protein stability or transcriptional regulation. Instead, statins increased Nrf2 mRNA association with ribosomal complexes and induced Nrf2 protein through a translational mechanism. Recruitment of Nrf2 mRNA to ribosomes and induction of Nrf2 protein was dependent on activation of PI3 kinase. These studies provide evidence that Nrf2 plays a critical role in protecting cardiac myocytes and the heart from oxidative stress and MI. In the absence of Nrf2, mice experienced worse cardiac injury following MI and quickly advanced to heart failure. Mechanistically, this work has identified a novel role of Nrf2 in preserving mitochondrial morphology and integrity during oxidative stress through a direct interaction with the outer mitochondrial membrane. Finally, a newly defined role of Nrf2 induction by statins in mediating protection against MI by acute statin therapy indicates that modulation of Nrf2 may represent a viable pharmacological target for cardiac protection in humans.

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.