KEAP1 signaling pathway, autophagy, and apoptosis

Free Radical Biology & Medicine 50 (2011) 1186–1195

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Hypothesis Paper

Molecular cross-talk between the NRF2/KEAP1 signaling pathway, autophagy, and apoptosis Tomasz M. Stępkowski a,⁎, Marcin K. Kruszewski a,b a b

Institute of Nuclear Chemistry and Technology, Center for Radiobiology and Biological Dosimetry, 03–195 Warsaw, Poland Institute of Agricultural Medicine, 20–090 Lublin, Poland

a r t i c l e

i n f o

Article history: Received 29 November 2010 Revised 20 January 2011 Accepted 25 January 2011 Available online 2 February 2011 Keywords: NRF2 KEAP1 Oxidative stress PGAM5 FAC1(BPTF) Prothymosin α NF-κB p62 Apoptosis Autophagy Free radicals

a b s t r a c t Oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. This, in turn, may cause the induction of autophagy or apoptosis. The NRF2/KEAP1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels. The relation between NRF2/KEAP1 signaling and regulation of apoptosis and autophagy is not well understood. In this hypothesis article we discuss how KEAP1 protein and its direct interactants (such as PGAM5, prothymosin α, FAC1 (BPTF), and p62) provide a molecular foundation for a possible cross-talk between NRF2/KEAP1, apoptosis, and autophagy pathways. We present a hypothesis for how NRF2/KEAP1 may interfere with the cellular apoptosis-regulatory machinery through activation of the ASK1 kinase by a KEAP1 binding partner—PGAM5. Based on very recent experimental evidence, new hypotheses for a cross-talk between NF-κB and the NRF2/KEAP1 pathway in the context of autophagy-related “molecular hub” protein p62 are also presented. The roles of KEAP1 molecular binding partners in apoptosis regulation during carcinogenesis and in neurodegenerative diseases are also discussed. © 2011 Elsevier Inc. All rights reserved.

The NRF2 (nuclear factor erythroid 2-related factor 2)/KEAP1 (Kelch-like ECH-associated protein 1) pathway enables cell adaptation to oxidative stress caused by various stimuli, among which chemical oxidative and electrophilic agents, as well as physical agents such as UV radiation, are the most common. On the other hand, this pathway is also strongly activated in response to natural cancer-chemopreventive agents. These are mostly plant-derived compounds belonging to the isothiocyanates, organosulfurs, and polyphenols, among which the most extensively studied, in the context of NRF2/KEAP1 signaling, are phenetyl isothiocyanate, sulforaphane, quercetin, resveratrol, and curcumin [1,2]. Apart from its positive role in normal cells, the NRF2/KEAP1 pathway also has its dark side—it is responsible for drug resistance and the survival of certain cancer cells. Somatic mutations in genes coding the NRF2 and KEAP1 proteins, leading to permanent activation of this stressrelated pathway, are found in many types of cancer [3]. Paradoxically, the NRF2/KEAP1 pathway can be responsible for cancer cell survival and, on the other hand, it is activated by chemopreventive antioxidants, for example anticarcinogenic polyphenols, which were found to cause cancer cell apoptosis under certain conditions [4,5]. It seems that this

⁎ Corresponding author. Fax: + 48 22 504 13 41. E-mail address: [email protected] (T.M. Stępkowski). 0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2011.01.033

dualism may result from the fact that activation of the apoptotic machinery or its inhibition depends on the strength and duration of the oxidative stress. Extensively studied cancer-chemopreventive compounds are widely recognized as effective antioxidants; however, some of them, such as quercetin, are in fact weak pro-oxidants with the capability of inducing long-term “hormetic” NRF2 response stimulation [6]. The induction of apoptosis by polyphenolic compounds such as quercetin is proposed to be related to their potential to inhibit NF-κB and activate the ASK1/p38 pathway [5,7]. The relation between these pathways and NRF2/KEAP1 is not well understood. In this review an attempt is made to shed some new light on this interesting topic. Polyphenolic compound concentrations that are able to induce apoptosis vary significantly in different cancer cell lines and the reason for these discrepancies is still unclear—no correlation has been found so far between the basal level of NRF2 stimulation and polyphenol-induced apoptosis. It seems logical and is supported by recent studies that active early NRF2 cellular response has an antiapoptotic effect [8,9]. Nevertheless, the exact mechanism explaining this relation remains unknown. The molecular mechanisms explaining how the NRF2/KEAP1 pathway is modulated during autophagy and apoptosis also remain largely unknown. Hence, the focus of this hypothesis review is on possible direct crosstalk between the NRF2/KEAP1 pathway and the pathways responsible for regulating the process of apoptosis. The aim is to summarize the current knowledge about proteins that link the NRF2/KEAP1 pathway

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with the main cellular signaling pathways related to apoptosis. The emphasis is put on proteins that bind to the central player in the regulation of NRF2 response—the NRF2 inhibitor—KEAP1. Presentation of hypotheses and interpretations of observations made with the use of various cellular models may help bench scientists to develop new strategies for gathering experimental evidence for a field of cell molecular biology that is at present not well understood. The NRF2/KEAP1 pathway NRF2 protein is a Cap'n’Collar (CNC) basic-region leucine zipper transcription factor responsible for activating transcription of more than 100 known, mainly cytoprotective, genes—the so-called “NRF2 battery.” This battery consists of antioxidant proteins, phase I and II detoxification enzymes, transport proteins, proteasome subunits, chaperones, growth factors, and their receptors, as well as some transcription factors [3,10–12]. The last two transcript groups provide evidence that this gene battery codes for not only a simple reactive oxygen species (ROS) scavenging protein machinery but also some proteins with regulatory functions. These may influence a large-scale change in cell physiology influencing global processes such as induction or inhibition of apoptosis. Under normal conditions NRF2 is constantly ubiquitinated by the CUL3/RBX1-dependent E3 ubiquitin ligase complex (named “KEAP1 complex” in further text) and degraded by proteasomes. KEAP1 is a master regulator of the NRF2/KEAP1 pathway and serves as an adaptor protein for this degradation [13–15]. The KEAP1 dimer binds NRF2, as described by the so-called “hinge-and-latch” model in which NRF2– KEAP1 contact is mediated by a strong binding of the NRF2 ETGE motif to one Kelch domain of KEAP1 (hinge) and a weak binding of the NRF2 DLG motif (latch) to the other Kelch domain [16]. During oxidative stress, cysteine residues in KEAP1 become oxidized and this modification changes the conformation of the whole complex—the latch (NRF2 DLG motif binding) is loosened. Although NRF2 still binds one Kelch domain with its ETGE motif, the disruption of the latch binding is sufficient to stop NRF2 ubiquitination: in the newly achieved complex conformation, NRF2 lysine residues are no longer available for ubiquitination and this process leads to NRF2 stabilization [16,17] (Fig. 1). Recently, another process, in addition to KEAP1 cysteine oxidation, was attributed with "loosening the latch". It was found that p21 protein can compete with KEAP1 for binding to the DLG motif of NRF2 [9]. To attain its functional destination, the chromatin, NRF2 protein has to be delivered to the nucleus. NRF2 has some nuclear localization sequences that were proposed to be sensitive to oxidative modification or phosphorylation and these posttranslational modifications may be involved in NRF2 nuclear import [18]. NRF2 is also a target for phosphorylation by MAPK but this modification only slightly contributes to NRF2 nuclear accumulation [19]. On the other hand, it has been shown in various cell culture models that an unknown protein kinase C isoform potentiates NRF2-dependent transcription. Furthermore, phosphorylation at Ser-40 by PKCδ and PKCι resulted in NRF2 dissociation from the KEAP1/CUL3/RBX1 complex but, surprisingly, was not required for NRF2 nuclear accumulation [20–23]. It is worth noting that the KEAP1/CUL3/RBX1 complex is also found in the nucleus in about 10–15% of its total cellular content in unstimulated cells, but there is a lack of evidence that the KEAP1 complex migration corresponds to the NRF2 nuclear import. As shown by the Jaiswal group, the KEAP1 complex can also degrade NRF2 inside the nucleus and its migration depends on another KEAP1 binding protein—prothymosin α (ProTα)—but not on NRF2 [23]. When NRF2 reaches chromatin, it can form a heterodimer with small MAF basic leucine zipper transcription factors, bind a sequence called the ARE (antioxidant response element), and activate the transcription of a particular NRF2-dependent gene [24,25]. Small MAF proteins, apart from their ability to bind NRF2, can form heterodimers with other CNC and basic leucine zipper transcription factors by interaction through the

Fig. 1. The "hinge-and-latch" model of NRF2 release from the KEAP1 complex, stabilization, and nuclear translocation [16]. (1) Under unstressed conditions NRF2 is constantly being ubiquitinated by the CUL3/RBX1-dependent E3 ubiquitin ligase complex and subsequently degraded by the 26S proteasome. NRF2 binds KEAP1 through a strong-binding ETGE motif and a weak-binding DLG motif. (1A) KEAP1 Broad-complex, Tramtrack, Bric-a-brac domain responsible for dimerization and CUL3 binding. (1B) KEAP1 intervening/linker region enriched with reactive cysteine residues. (1C) KEAP1 dimer KELCH protein docking domains. (2) Oxidative stress blocks NRF2 degradation. (2A) Appearance of ROS during oxidative stress modifies reactive cysteines in KEAP1 IVR domain—this results in conformational change of the KEAP1 complex and DLG motif binding—the latch is loosened. (2B) p21 may compete for DLG binding with KEAP1 and positively regulate NRF2 stabilization. (3) NRF2 release from the KEAP1 complex and translocation to the nucleus. The process of NRF2 phosphorylation by PKCι and PKCδ and possibly other kinases is attributed to NRF2 release from the KEAP1 complex. KEAP1 binding to prothymosin α or another protein competitive with the NRF2 ETGE motif may be also necessary for NRF2 release. Unknown NRF2 modification regulates its migration to the nucleus.

leucine zipper domain. Such dimerization was observed with various nuclear erythroid factor 2-related proteins, BACH (Bric-a-brac and CNC homology) proteins 1 and 2, and Fos and FosB. The above dimers were also found to bind to the ARE sequence. Apart from their ability to heterodimerize, small MAF proteins can form homodimers that, because of the lack of a transactivation domain, were shown to repress AREdependent transcription [26]. Various MAF recognition elements, which share part of their sequence consensus with AREs, were shown to be preferentially bound by MAF/MAF homodimers or MAF/NRF2 dimers [27]. Because of the existence of ARE types that have differential affinities to particular transcription factor dimers and because various such protein dimers were observed, the regulation of transcription by ARE cis-regulatory elements seems to be specific for a particular gene promoter.

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The transcription of two model genes from the ARE gene battery, HO-1 (heme oxygenase 1) and NQO1 (NADP(H):dehydrogenase quinone 1), was studied in detail and found to be regulated at the ARE level by BACH1 protein [28–30]. In such a model mechanism, under normal conditions the ARE sequence in the NQO1 promoter is occupied by small MAF/BACH1 protein dimers. During oxidative stress or antioxidant treatment BACH1 is phosphorylated by an unknown kinase, resulting in its export to the cytoplasm and subsequent degradation. The progressive nuclear export of BACH1 allows the binding of NRF2/MAF dimers to the ARE sequence in the NQO1 promoter and the activation of this cytoprotective gene's transcription [31]. This relation is graphically depicted in Fig. 2. Even though BACH1 was indicated as a dominant repressor of the transcription of two model NRF2-dependent genes, in the context of recent literature it is thought to be a specific rather than a universal inhibitor of ARE-dependent transcription. After the knockdown of BACH1 in HaCaT keratinocytes, only the HO-1 gene was found to be highly overexpressed [32,33]. Moreover, the knockdown of BACH1 did not cause the increase in cellular glutathione, neither did it potentiate the cells' resistance to xenobiotics [33]. To summarize— KEAP1 is thought to be a global repressor of NRF2-dependent transcription but other factors may be important in the negative regulation of individual genes. The superiority of the KEAP1 protein over the BACH1 transcription factor in the negative regulation of the transcription of the NRF2 gene battery can be illustrated by the observation that KEAP1 knockout is neonatally lethal to mice, whereas no such detrimental effects were observed after the knockout of BACH1 [34]. Currently, apart from the ProTα-dependent KEAP1 complex migration to the nucleus and the subsequent NRF2 degradation, there is only one molecular mechanism solved that corresponds to NRF2 nuclear export. It was found that to allow its translocation to the cytoplasm, NRF2 has to be phosphorylated by Fyn kinase, which like NRF2 is a nucleus/cytoplasm shuttle protein. The nuclear localization and activation of Fyn kinase is regulated by phosphorylation mediated by

GSK-3β (glycogen synthase kinase 3β). During activation of the NRF2 pathway, both NRF2 and GSK-3β are phosphorylated at a serine residue by PKC and this process allows NRF2 nuclear import and enables GSK3β-mediated activation of Fyn [35]. Modulation of the NRF2 response Although it seems likely that the very tight regulation of the NRF2/ KEAP1 signaling pathway both influences and is influenced by the global pro- and antiapoptotic signals, there is currently scarce experimental evidence available to explain how such mutual regulation may occur. Most NRF2 target genes, exemplified by antioxidant proteins, chaperones, and proteasome subunits, are considered model cytoprotective enzymes. Therefore, activation of the NRF2/KEAP1 pathway is thought to be parallel with or even to be favorable for antiapoptotic signaling. After cell treatment with oxidants, various oxidative stress-related transcription events follow NRF2-activated transcription, leading to apoptosis. Such cascade transcription events have been analyzed in detail in the study of fenrentinide-treated leukemia cells by Wang et al. [36]. Fenrentinide is a pro-oxidative and proapoptotic retinoid. In leukemic cells, apoptosis was a late response to transcriptome changes orchestrated by heat shock factor 1, responsible for triggering the unfolded protein response (UPR), and by transcription factor NRF2. Wang et al. [36] have presented evidence for a parallel regulation of the UPR and NRF2 response and point to the temporal relations between these two events as factors determining death versus survival choice. Another known cross-talk between the UPR and NRF2 is mediated by one of the three known sensors of endoplasmic reticulum stress—the PERK (pancreatic endoplasmic reticulum eIF2α) kinase, which was found to phosphorylate NRF2 and promote its nuclear translocation [37]. The survival versus death decision after UPR induction may be related to the level of oxidative stress that is produced during UPR by NADPH oxidases and protein disulfide isomerases and is proposed to be related to regulation and expression of CCAAT/enhancer-binding

Fig. 2. ARE-dependent transcription of the NQO1 gene is regulated by the nuclear/cytoplasm shuttling of basic leucine zipper transcription factors–inhibitor (BACH1) and activator (NRF2). Under oxidative stress, the ARE transcription inhibitor BACH1 is phosphorylated at tyrosine 486 and exported to the cytoplasm [28]. The progressive export of BACH1 allows binding of NRF2/MAF dimers to the ARE sequence inside the nucleus. Silencing of ARE-dependent transcription has been attributed to the activation of kinase GSK-3β, which phosphorylates the kinase FYN, allowing it to phosphorylate NRF2 and start its nuclear export.

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protein homologous protein (CHOP) (reviewed in Szumiel [38]). Supporting this hypothesis is the fact that NRF2 was found to be an inhibitor of CHOP and a target of PERK and, therefore, constitutes another known link in the indirect relation of NRF2 with apoptosisrelated pathways [39]. As the UPR–NRF2 relation was already extensively discussed by Cullinan and Diehl [39], the following text focuses on direct molecular protein–protein interactions in the KEAP1/NRF2 pathway. In the following paragraphs we present apoptosis-related proteins that bind KEAP1 and in that way may modulate NRF2 transcriptional activity. Also, the potential influence of these interactions in the regulation of apoptosis and autophagy is discussed. Binding of various proapoptotic or antiapoptotic proteins to KEAP1 or other proteins in the KEAP1 complex may regulate NRF2 ubiquitination and nuclear migration and in that way regulate the NRF2 response. It seems likely that the binding of different proteins to the KEAP1 complex and their posttranslation modifications constitute the main upstream event that determines the fate of NRF2/KEAP1 signaling. One important and recently presented piece of evidence for such regulation is the mechanism of competition between p21 and KEAP1 for binding to the DLG motif in the Neh2 domain of NRF2. p21 binding to this motif, which was previously described as a latch in the hinge-and-latch model of NRF2 activation, attenuates the rate of NRF2 ubiquitination and leads to NRF2 stabilization [9]. In most cases, other proteins binding the KEAP1 complex also affect NRF2/KEAP1 binding or may target the KEAP1/CUL3/RBX1 complex to different cell compartments. When discussing redox-dependent molecular signaling pathways, it is necessary to consider the fact that deleterious free radicals are both very short lived and short-range molecular species. Hence, the molecular and spatial relationships between the KEAP1 complex, antioxidant proteins, and free radicals are very important for the maintenance of physiological hydrogen peroxide signaling. For example, strong, permanent activation of NRF2 may blunt the physiological hydrogen peroxide signaling near the plasma membrane; this process was proposed as one of the causes of type II diabetes [40]. On the other hand, the KEAP1 complex was found to translocate to the nucleus and mitochondria [23,41]. Such a translocation may provide a stress sensor in these cellular compartments. The physiological consequences of oxidative stress in the nucleus and the “leaking respiratory chain” could be deleterious and the proper localization of the KEAP1 complex may allow the cell to react immediately when such a threat arises. The existence of NRF2 signaling regulation at the chromatin level should also be mentioned. NRF2 was found to compete with other transcription factors for promoter or transcription cofactor binding. An example is the competition between NRF2 and NF-κB p65 for cAMPresponse element-binding protein (CREB) transcription cofactor binding, or the NF-κB p65-dependent recruitment of histone deacetylase, which causes local chromatin hypoacetylation inhibiting the NRF2dependent transcription of heme oxygenase 1 [42].

The KEAP1 complex as a transducer unit The KEAP1 complex constitutes the central regulatory element in NRF2 signaling. In an excellent review presenting the NRF2/KEAP1 pathway in a systems biology perspective, it was named a “transducer” sensitive to oxidative as well as enzymatic and protein interaction signals [6]. Changing the status of the KEAP1 complex influences the operation of the whole NRF2/KEAP1 pathway. In addition to the CUL3/RBX1 complex and NRF2, KEAP1 was also found to bind other proteins, exemplified by phosphoglycerate mutase 5 (PGAM5), ProTα, fetal Alz-50 clone 1 (FAC1), and p62, also known as sequestosome 1 (SQSTM1). These four proteins were found to be involved in apoptosisrelated cellular events with a possible role in the cross-talk between NRF2 signaling and apoptosis.

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PGAM5 links NRF2 signaling, mitochondrial morphogenesis regulation, and apoptosis PGAM5, a recently characterized atypical member of the phosphoglycerate mutase family, is an example of a hypothetical mediator for direct cross talk between NRF2/KEAP1 signaling and regulation of apoptosis. This protein is a target for KEAP1-dependent ubiquitination. Overexpression of its two differentially spliced isoforms was found to cause a severe change in mitochondrial morphogenesis in COS1 cells. Moreover, PGAM5 is an outer mitochondrial membrane residue protein and both its isoforms were found to be direct binding partners of the BCL-xL antiapoptotic protein [41,43]. The binding with KEAP1 does not preclude PGAM5 from binding to BCL-xL. On the other hand, KEAP1 binding is not necessary for BCL-xL binding—the mutation in PGAM5 NXE(S/T)GE, a motif responsible for KEAP1 binding, did not alter PGAM5/BCL-xL binding [44]. Finally, PGAM5 is able to target the KEAP1 complex to the mitochondrial membrane and is responsible for activation of the main apoptosis-related higher order MAPK kinase— ASK1 (apoptosis signal-regulated kinase 1) [41,45]. Such molecular interactions place it as a good candidate for an examination of its role in the relationship between the NRF2/KEAP1 pathway and apoptosis. Moreover, PGAM5 is evolutionarily related to proteins regulating glucose metabolism and, therefore, constitutes additional evidence for the recently presented theory that the autophagy and apoptosis regulatory machinery has evolved from the cellular response to metabolic stress [46]. PGAM5 is expressed as two alternative splicing variants—the shorter 255-aa isoform, PGAM5-S, and the longer 289-aa PGAM5-L isoform. They differ in their C-terminal phosphoglycerate superfamily domain (Pfam PF00300). The C-terminus in both isoforms is coded by different exons; the longer last exon of the PGAM5-L isoform encodes a part of the Pfam superfamily domain, whereas the last exon of the shorter isoform has a short uncharacterized protein coding sequence and a long 3′ UTR. Takeda and co-workers proved that although the PGAM5 longer isoform does not possess the typical, for the PGAM family, catalytic activity of a phosphotransferase or phosphohydrolase of small molecules, it acts as a serine/threonine protein phosphatase and activator of ASK1 [45] (personal communication). ASK1 is the main MAPK kinase kinase (higher order MAPK) that is responsible for apoptosis induction by sustained activation of the JNK and p38 pathways [47]. Dephosphorylation of ASK1 by PGAM5 leads to its activation and may cause the subsequent apoptosis. The ASK1-dependent mechanism has been linked to apoptosis as the cause of neurodegenerative diseases, immune response, toxification, and many other stress responses [48]. The link between the ASK1 and the NRF2-mediated responses has been experimentally proven in the study of paraquat-induced cell death. It was found that KEAP1-deficient mouse embryonic fibroblasts (MEFs) were resistant to paraquat-mediated apoptosis and this effect was attributed to the inability to activate ASK1. Conversely, NRF2-deficient MEFs show paraquat hypersensitivity. The authors concluded that the NRF2/KEAP1 pathway regulates ASK1 activation both by controlling the ROS level and by regulating the expression of thioredoxin, which in a reduced form binds to and inhibits ASK1 activation [49]. The report that the KEAP1 binding partner PGAM5 is an activator of ASK1 brings another dimension to the observation that KEAP1/NRF2 regulates ASK1. Further experimental evidence is needed to find whether KEAP1/PGAM5 binding affects ASK1 dephosphorylation. On the other hand, BCL-2 family proteins, such as another binding partner of PGAM5 isoforms, BCL-xL, were found to be involved not only in the regulation of the apoptosis/survival balance but also in the regulation of mitochondrial dynamics [50]. Major mitochondrial morphogenesis alterations were observed after PGAM5 overexpression in COS1 cells [41]. The overexpression of the PGAM5-S isoform resulted in a disconnected punctate mitochondrial pattern typical of apoptosis but without disruption of the mitochondrial membrane permeability potential and cytochrome c release. PGAM5-L overexpression leads to

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the formation of big mitochondrial clusters surrounding the nucleus. The PGAM5 region containing amino acids 125–156 was proven to be responsible for the PGAM5/BCL-xL direct interaction [43]. This region is shared by both PGAM5 isoforms and is a part of the C-terminal phosphoglycerate mutase domain. Although PGAM5-S and -L differ in the C-terminal coding sequence of this domain, both isoforms are able to bind BCL-xL. So, one can speculate that the modification of the PGAM5/ BCL-xL complex structure (mediated by the C-terminal difference in PGAM5 sequence), and not the influence on PGAM5/BCL-xL binding, may be responsible for the observed differential effect of overexpression of PGAM5 isoforms on mitochondrial morphogenesis pattern. PGAM5-S is expressed at lower levels [41,44] and was not found by immunoblotting in mouse (K. Takeda, personal communication); it would be of particular interest to match the mRNA expression and protein expression level data as well as to compare the mRNA levels of two PGAM5 isoforms to find if there is a significant difference in the efficiency of PGAM5-L and -S mRNA degradation and the ubiquitination of these proteins. PGAM5-L overexpression also results in targeting the majority of KEAP1/NRF2 complexes for translocation from cytoplasm to mitochondria—it would be scientifically valuable to determine the relation between such targeting and oxidative stress. Another related question is how the oxidative stress regulates PGAM5 function and the expression of the two PGAM5 isoforms. It would be interesting to know the physiological significance of KEAP1/NRF2/PGAM5 ternary complex migration to the mitochondrial membrane. The siRNA-mediated silencing of PGAM5 results in a more pronounced NRF2 response, as shown by the use of an ARE-dependent luciferase reporter plasmid [41]. So, one can imagine that in a ternary complex with PGAM5 localized near mitochondria, NRF2 release from the complex is somehow hampered. This effect could be mediated by a NRF2 inability to bind p21 (the protein that was attributed with attenuation of NRF2 ubiquitination) or by an interaction of the complex with BCL-xL. It seems plausible that during the late stage of the NRF2 response, the ternary complex of KEAP1/NRF2 and PGAM5 reaches mitochondria and changes its sensory function—KEAP1 stops serving as a general oxidative sensor in the cytoplasm and starts to acquire oxidative signals in close proximity—directly from the mitochondrion. Such regulation would be crucial in the apoptosis/survival balance. The increased generation of ROS by the “leaking oxidative chain” could be a candidate signal to stop NRF2 response and start the apoptosis process. The hypothesis of PGAM5 as a mediator between NRF2 response and apoptosis definitely needs to be proven by solid experimental evidence, especially in the context of the roles of the two PGAM5 isoforms and the physiological significance of PGAM5/BCL-xL binding. Nevertheless, this branch of NRF2/KEAP1 signaling certainly merits further exploration. Prothymosin α: the orders from the disordered ProTα appeared recently as another essential player in the NRF2/ KEAP1 pathway apoptosis cross-talk. During NRF2 signaling this protein binds KEAP1 and mediates KEAP1 complex migration to the nucleus. Such migration was observed only in a subset of studies and probably takes place only in certain cell types. For example, it was shown that after reaching the nucleus ProTα disassociates from KEAP1 and is replaced by NRF2, which is then ubiquitinated and subsequently degraded. The physiological role of KEAP1 complex migration to the nucleus would be to allow a rapid switching off of NRF2-dependent transcription [23]. ProTα is a small, extremely acidic, and hydrophilic protein of only 12.1 kDa [51]. In addition to its role in modulation of NRF2 response, it was found to have many distinct physiological functions. There are various studies presenting evidence for its intracellular as well as extracellular signaling functions [52–54]. In the context of the direct impact of ProTα on apoptosis, these results sometimes seem to be contradictory, but such a great variety of functions may be the result of

the partially unfolded structure of ProTα [55]. This protein belongs to a group of intrinsically unstructured proteins—proteins with variously sized regions of highly dynamic structure that do not form stable secondary and tertiary structures under normal physiological conditions. Such proteins have some unique properties that allow them to: (1) function as multiple binding partners of distinct proteins and be differentially folded upon binding; (2) be easily posttranslationally modified (their large, flexible protein interaction surface is easily available for interaction with other proteins; and (3) form pathological or functional amyloid fibrils under certain conditions. Taking advantage of its biophysical properties, ProTα is capable of participating in various signaling events. At first, when it was discovered in the nucleus, it was proposed that it serves, among other things, as a mediator for chromatin decondensation and transcription coactivator and, as mentioned above, a mediator in NRF2 signaling [56–59]. Later, a cytoplasmic pool of ProTα was discovered and its role in regulating apoptosome activation was proposed [53]. Recent discoveries show also the role of ProTα as an extracellular signaling molecule with a direct impact on the regulation of necrosis and apoptosis and as an inhibitor of lentiviral replication [52,60]. To make things clear, it is easier to divide the discussion about ProTα influence on programmed cell death into two parts—one focused on its extracellular immunomodulatory function and the other presenting its various complicated interactions inside the cell. It seems highly probable that these two ProTα activities are physiologically related but the exact relation is not clear yet. Prothymosin α cellular functions The most extensively studied involvement of ProTα in the direct regulation of apoptosis is its influence on apoptosome activation. It was found that ProTα is a potent inhibitor of this process—necessary for subsequent procaspase 9 cleavage and caspase 3 activation in the intrinsic, mitochondrion-dependent apoptosis pathway. Apoptosis inhibition by ProTα is mediated by its direct binding with the apoptosome-forming protein—APAF-1. This binding is proposed to hamper hydrolysis of APAF-1-bound ATP, a process necessary for apoptosome activation [53]. More recently, cytoplasmic apoptosomeinhibiting activity was attributed to a heterodimer complex of ProTα with another intrinsically unstructured and apoptosis-related protein— p8 (NUPR1, or nuclear protein 1) [61]. Notwithstanding its cytoplasmic antiapoptotic activity targeted against apoptosome activation, ProTα was found to strengthen the prosurvival p53 response by increasing p53 acetylation [62]. This process is proposed to be mediated by interaction of ProTα with histone acetyltransferases, p300, and CREB-binding protein. Although p53 is mostly known as a potent proapoptotic protein, recently, several prosurvival functions have been found for this protein and some of them were attributed to the increased expression of the p53 target gene p21. This protein mediates cell cycle arrest at the G1/S phase boundary, allowing prosurvival processes such as DNA repair to happen. Beyond the well-documented role of p21 in the cell cycle and apoptosis regulation, it was found to compete with KEAP1 for NRF2 DLG motif binding. p21 binding to NRF2 in the KEAP1 complex leads to attenuation of ubiquitination and stabilization of this transcription factor [9]. It is now clear that both NRF2 response and the p53/p21 axis prosurvival pathway are influenced directly by ProTα. Once p53 response is triggered, ProTα directs it to prosurvival function by increasing p53-mediated p21 transcription. Then, cytoplasmic p21 potentiates the NRF2 response by attenuating its ubiquitination. On the other hand, the cytoplasmic ProTα pool was shown to have an opposite function—to be able to target the KEAP1 complex to the nucleus for rapid degradation of NRF2 [23] and to hamper apoptosome activation [53]. KEAP1/ProTα interaction was found to be strongly potentiated by zinc ions, which interact with the unfolded

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structure of ProTα to make it partially folded [55,63]. This may constitute a mechanism for changing ProTα cellular function—high zinc ion concentration may switch off NRF2 response and facilitate apoptosis. In conclusion, it is possible that the cytoplasm/nucleus shuttle of ProTα and zinc concentration belong to the key factors responsible for the outcome of NRF2 signaling and apoptosis.

ProTα escapes stressed cells to serve its extracellular immunomodulatory functions Apart from its quite well known multitargeted activities inside the cell, ProTα is also an extracellular immunomodulatory signaling molecule. It was found to enhance the expression of MHC second class molecules in monocytes, to increase T cell proliferation in response to antigen [64,65], to potentiate integrin expression and perforin production in NK cytotoxic cells [66], to specifically inhibit HIV replication in macrophages [60], and to hamper necrosis and induce apoptosis in a rat transient focal ischemia model and neural cells cultured under serumfree conditions [52]. The mechanisms of ProTα extracellular activity are currently poorly understood. Although it has been shown that ProTα is secreted into a serum-free cell culture medium as a soluble survival factor and it is found in the serum of cancer patients, the mechanism controlling its secretion remains unknown. ProTα lacks a signal peptide sequence required for vesicular release. On the other hand, no plasma membrane receptor has been attributed as a mediator of the extracellular functions of ProTα. In neuronal cells cultured in serum-free medium, ProTα inhibits necrosis by enhancing glucose transporter GLUT1/4 plasma membrane internalization, which involves a PKCβ-dependent mechanism. Hence, the putative Gi/o (G protein coupled) receptor is one of the candidates for a ProTα receptor [52]. Another model indicates one of the Toll-like receptors as a putative ProTα receptor. This hypothesis is based on the fact that the most immunoactive C-terminal ProTα peptides, TKKQKTDEDD, are cleaved from the rest of the protein molecule at the early stage of caspase activation. Subsequently, they form polymers of β-pleated structure that are resistant to proteolysis. Such molecules are proposed to reach the extracellular space and serve as ligands of TLR receptors [66]. Another hypothesis that seems plausible is that ProTα may be secreted as a heterodimer with another protein or, taking advantage of its amyloid-forming potency, removed from the dying cell with the help of autophagy processes. ProTα, similar to some other unstructured proteins, is capable of forming amyloid fibrils. In the case of ProTα, amyloidogenesis occurs at low pH [67]. The C-terminal β-pleated globular aggregates observed by Skopeliti et al. [66] may serve as a seed for amyloidogenesis of the full-length ProTα. It is possible that ProTα resides in autophagosomes or other low-pH vesicles as proteolysisresistant amyloids or small β-pleated structures; when deleterious stress leads to necrosis, ProTα reaches the extracellular space and disaggregates to carry out its immunomodulatory function. Such a hypothesis is consistent with the results obtained by Ueda et al. [52], who showed that not only the C-terminal peptide, but the whole ProTα molecule, is secreted outside the cell. This process occurs only under serum-free conditions. Such an environment causes necrosis in a lowdensity neuronal culture, whereas in a high-density culture, apoptosis is induced. ProTα has been proven to be the secreted soluble factor that is responsible for the shift from necrosis to apoptosis in high-density neuronal cell cultures. As there are, so far, no publications concerning the relation between ProTα extracellular activity and NRF2 response activation, it would be interesting to test how the ProTα C-terminal peptide, complete ProTα molecule, and ProTα amyloid affect the NRF2-dependent transcription under metabolic stress and normal conditions (as studied in neuronal cells by Ueda et al. [52]) and what the impact is of this potential influence on apoptosis-related pathways.

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FAC1 ilitation of apoptosis in neurodegeneration NRF2/KEAP1 pathway is well known for its neuroprotective role in various cellular and animal models of neurodegenerative diseases [68]. The molecular mechanisms that are responsible for the massive necrotic, as well as apoptotic, neuronal cell death in these diseases are still not completely understood. Nevertheless, amyloid β and oxidative stress are without doubt the most important pathological features of neurodegenerative diseases such as Alzheimer's and Parkinson's. It has recently been found that NRF2/KEAP1 pathway activation not only protects the cells from apoptosis during neurodegeneration, but also hampers the formation of amyloid β [69–71]. In the case of a direct cross talk between NRF2/KEAP1 signaling and cell death during neurodegeneration, proteins coded in the bromodomain PHD (plant homeodomain) finger transcription factor (BPTF)/FAC1 gene locus seem to have a crucial role. FAC1 was first cloned and analyzed as an 810-aa protein by Bowser et al. during their study of Alzheimer's disease. It was named fetal Alz-50 clone 1 because it cross-reacted with Alz-50 antibody [72]. Later, it became clear that FAC1 shares its sequence with a larger protein called BPTF that constitutes a part of the NURF (nucleosome remodeling factor) [73,74]. There are various isoforms of protein-coding transcripts in the BPTF/FAC1 locus but all of them share the sequence coding the Cterminal bromodomain and PHD zinc finger motifs. The majority of early studies of FAC1 conducted by Bowser et al. were done using a FAC1 antibody that could have recognized various protein isoforms coded in the BPTF locus. Therefore, it is better to attribute the results gathered in the early FAC1 studies to the proteins encoded by the whole BPTF gene locus. The FAC1/BPTF locus codes various transcription factor isoforms, which are especially interesting in the context of NRF2 signaling during neurodegeneration and in apoptosis regulation, because at least one of the isoforms: (1) is a direct binding partner of KEAP1 [75]; (2) exhibits altered expression and cellular distribution patterns in response to amyloid β and during brain development [76,77]; (3) when overexpressed in PT67 fibroblasts leads to apoptosis, further augmented by treatment with the NRF2 response inducer diethylmaleate [78]; (4) in late response to amyloid β is redistributed in punctate structures surrounding the nucleus—this may be a clue to its localization in or near mitochondria, which often during apoptosis fuse around the nucleus forming a similar pattern [78]; and (5) is a part of NURF [74]. Although FAC1 was found to bind KEAP1, there are currently no data showing how this interaction affects the NRF2 response. On the other hand, apoptosis caused by FAC1 overexpression was augmented by NRF2 release from KEAP1, but not that caused by KEAP1 overexpression [75]. These effects were observed in cells that do not express endogenous FAC1 and, therefore, this may not be a general effect of FAC1 overexpression. It is also not certain what the effect is of larger BPTF isoform overexpression. Until it is determined that the 810-aa form of the BPTF protein overexpressed in the cellular model studied by Strachan et al. [78] is naturally expressed in a physiologically significant amount, the relevance of the observation of the FAC1-mediated apoptosis is not clear. What speaks for these data as being relevant to the physiological situation is the fact that one of the protein-coding isoforms annotated in the ENSEMBL project in BPTF locus differs in only two amino acids from the protein studied by Strachan et al. [78]. So, it may be assumed that the apoptosis mediated by the overexpression of 810-aa FAC1 has a physiological meaning. The above-mentioned alternative splicing may determine the role of BPTF gene transcription in apoptosis, NRF2 signaling, and chromatin remodeling. It is highly plausible that FAC1 induces apoptosis by its activity as a transcriptional regulator, as it was shown that the FAC1 Nterminal sequence, containing a DNA-binding domain, was the one responsible for apoptosis induction [78]. At present, the physiological significance of FAC1/KEAP1 binding remains unsolved but the analysis of FAC1 proteasomal degradation

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and its influence on NRF2 response as well as the examination of its expression patterns under oxidative stress conditions may result in important information that will shed new light on the relation between NRF2 response, apoptosis, and neurodegeneration. p62 in a KEAP1-refereed interplay between NF-κB, autophagy, and apoptosis In the preceding section we presented a possible mechanism by which the NRF2/KEAP1 pathway may interfere with the cellular apoptosis regulatory machinery, namely, activation of the ASK1 kinase by a KEAP1 binding partner—PGAM5. This molecular interaction axis may have crucial importance in apoptosis regulation. So far, there are no experimental data concerning the relation between PGAM5/KEAP1 interaction and apoptosis. Therefore, one can only speculate how this interaction affects PGAM5's ability to dephosphorylate ASK1 and, thus, hamper or attenuate the proapoptotic signaling. It seems logical that PGAM5-dependent KEAP1 complex trafficking to the outer mitochondrion membrane may have the function of sensing the level of free radicals arising from the leaking oxidative chain. When the NRF2/KEAP1 pathway is activated, the cell status (most of the NRF2 present in the nucleus and a significant amount of “free” KEAP1 pool) is generally considered to be favorable for antiapoptotic signaling. Nevertheless, the molecular links between the NRF2/KEAP1 and the antiapoptotic pathways are not fully identified. Apart from the above-mentioned “PGAM5 hypothesis,” another way for NRF2/KEAP1 to interfere with apoptosis may be by means of a direct KEAP1/p62 interaction and NF-κB pathway regulation. p62 (SQSTM1) is a multidomain adapter protein able to bind various signaling proteins, oligomerize, and form cytosolic speckles. It serves as a molecular hub mediating various protein interactions [79,80]. During autophagy, p62 binds LC3 and ubiquitin and participates in the trafficking of proteins that are substrates for autophagosome degradation. In the process of macroautophagy and mitophagy, p62 not only acts as an adaptor protein in selective autophagy of ubiquitinated proteins or mitochondria, but also becomes a substrate for degradation [81,82]. On the other hand, when autophagy is inhibited, p62 oligomers and ubiquitin-containing proteins form inclusion bodies, which are a pathological feature of several neurological diseases as well as diabetes and malignancy [83,84]. Inability to degrade p62 is an oncogenic feature that causes NF-κB activation and lowers the level of proapoptotic intracellular ROS [85]. It was recently found that knockout of p62 under conditions of autophagy deficiency, or its degradation by autophagy under normal conditions, has a tumorigenesis-suppressive effect and may possibly hamper antiapoptotic signaling [86,87]. Explanation of this observation is still elusive. It is proposed that p62 regulates apoptosis differentially because this protein has a multidomain structure and is capable of binding various proteins (Fig. 3). On the one hand, it may

bind, oligomerize, and activate TRAF6 and in that way promote the antiapoptotic NF-κB pathway. On the other hand, it can also bind and oligomerize polyubiquitinated caspase-8 and lead to induction of apoptosis [80,88]. In a very interesting review, Moscat and Diaz-Meco [89] discuss the problem of the role of p62 in tumorigenesis. Based on the results of Mathew et al. [87] they conclude that p62 is a central player at critical decision points that control cell death and survival. The proapoptotic mechanisms that control caspase-8 polyubiquitination and aggregation with p62 in an atypical extrinsic apoptosis pathway are not well understood [80]. So, the authors focus on NF-κB regulation by p62 as the main factor that may influence tumorigenesis [85]. They present a mechanism that may be responsible for NF-κB activation by p62. RAS indirectly activates p62 transcription by induction of the ERK and PI 3kinase pathways and by potentiating the AP-1-dependent transcription of p62. Accumulation of p62 allows TRAF6 oligomerization and RASdependent TRAF6 self-polyubiquitination, which leads to activation of the main upstream activators of the canonical NF-κB pathway, the IKK (IκB kinase) catalytic subunits IKKα and IKKβ, which subsequently phosphorylate IκB (NF-κB inhibitory protein), marking it for proteasomal degradation. This cascade of events leads to the release of active NFκB dimers from the IκB-mediated cytosol sequestration and allows their migration to the nucleus for transcriptional activation of target genes, in resemblance to the so-called canonical NF-κB pathway [90]. Recently, three important papers concerning the NRF2/KEAP1 pathway have shed new light on the role of p62 in tumorigenesis. The first came from Komatsu et al. [86], who have shown that p62 is capable of binding KEAP1, similar to NRF2, and therefore, acts as a KEAP1 binding competitor, thus promoting NRF2 release from KEAP1 and strongly enhancing antioxidant gene expression. The second paper shows KEAP1 as a mediator of IKKβ ubiquitination and degradation [91]. The third paper presents evidence that p62 is a NRF2 target gene, which creates a positive feedback loop in the NRF2-mediated transcriptional response [92]. Putting these recent observations together, one can see that NF-κB regulation during autophagy and its inhibition, a process that has crucial implications in tumorigenesis, is mediated by a subtle interplay of ROS-dependent pathways. An example is the IκB degradation control by p62: it is due to the ability of p62 to bind not only to the IKKβ activators—atypical PKC and TRAF6—but also to its own inhibitor— KEAP1. Moreover, p62 acts in a molecular context that is highly dependent on ROS level, as discussed below. p62 can bind and sequester KEAP1 protein [86,93]. This process was found to enhance the NRF2-dependent transcription but—as can be reasonably assumed—also must influence KEAP1-mediated degradation of IKKβ. Therefore, p62 may mediate another previously unknown interaction between the NRF2/KEAP1 pathway and NF-κB (Fig. 4). In their review article Moscat and Diaz-Meco [89] try to find the answer to a puzzling question: why does p62 overexpression lead to NF-κB activation [94] or inhibition [87]? Deletion of p62 in RAS-induced

Fig. 3. Multidomain structural organization of p62. p62 comprises PB1 (Phox and Bem1p) protein–protein interaction domain, it mediates binding of other PB1-containing proteins such as aPKC and MEK as well as oligomerization; ZZ, zinc finger RIP1-binding domain; TB, TRAF6 binding site; LIR, LC3-interacting region; KIR, KEAP1-interacting region; and UBA, polyubiquitin-binding domain.

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Fig. 4. p62 can participate in both apoptosis induction and apoptosis inhibition. p62 may trigger apoptosis through interaction with caspase-8 or mediate its antiapoptotic effect by activating NRF2 and NF-κB-dependent transcription. p62 can bind and oligomerize KEAP1—this process potentiates the NRF2 response and may also influence KEAP1-mediated IKKβ degradation. Activation processes are represented as green arrows, inhibition is represented by red lines. The indirect protein–protein interactions are represented as dashed lines and arrows.

adenocarcinoma reduced the level of NF-κB stimulation and caused an increase in ROS, whereas it enhanced the NF-κB response and lowered ROS under conditions of metabolic stress in iBMK cells used in the study by Mathew et al. [87]. The reason for this discrepancy remained unclear. Taking advantage of the knowledge of the role of KEAP1 in inhibition of NF-κB one can assume that the observed differential activation or inhibition of NF-κB after p62 overexpression and deletion in different studies may be related to the NRF2/KEAP1 pathway and ROS level. A recent observation supports this assumption: ROS were found to significantly increase the amount of p62 that immunoprecipitated with KEAP1 [95]. The fact that p62 deletion in cell culture with plentiful nutrients and no oxidative stress lowered NF-κB stimulation and caused an increase in ROS [85] can be explained simply by an increase in the free KEAP1 pool. In the absence of oxidative stress, KEAP1 liberated from p62 sequestration will bring about more ubiquitination and degradation of NRF2 and IKKβ molecules. That will hamper the NF-κB and NRF2dependent transcription of antioxidant proteins and be the cause of a subsequent increase in ROS originating as a by-product of oxidative phosphorylation. In contrast, under conditions of metabolic and high oxidative stress, in a cell system with functional KEAP1, several mechanisms activating NRF2 and NF-κB are present. At first, as in the classical model of NRF2 activation, ROS may oxidize the reactive cysteines in KEAP1. This will hamper KEAP1-dependent ubiquitination of NRF2 as well as IKKβ, thus leading to NF-κB activation and lowering the level of ROS. NRF2 will subsequently increase the expression of p62 in a positive feedback loop; p62 will sequester more KEAP1 proteins, leading to a further increase in NF-κB and NRF2 signaling (Fig. 4). As a result, activation of NRF2dependent transcription of antioxidant genes lowers ROS levels and,

therefore, abrogates ROS-dependent NF-κB activation. If the ROS level attains a critical threshold at which antioxidant proteins cannot further deal with the increasing amount of free radicals, apoptosis may be initiated and NRF2 response downregulated by the p53-dependent apoptotic pathway [8,96]. The puzzling question is why p62 overexpression correlates with reduced NF-κB activation in defective autophagy [87]. It may be explained by the following hypothesis. Under conditions of suppressed autophagy, when p62 constantly accumulates in cytoplasmic speckles, what matters is the proportions of p62-binding protein (NF-κB activators and inhibitors) levels: caspase-8, aPKC, TRAF6, and KEAP1. It is probable that p62 has differential binding affinity to its various partners; thus, binding of KEAP1, aPKC, and TRAF6, which are considered antiapoptotic, favors NF-κB signaling, whereas it impairs p62/caspase-8 binding. When p62 is overexpressed, this may favor signaling from the proapoptotic ubiquitinated caspase-8 binding partner, which, under “normal” conditions, would not have a chance to be oligomerized by p62 and would be degraded. Although the binding mechanisms and domains are known, there are currently no studies describing the molecular interplay between p62 and its binding partners under defined conditions. We can only speculate that the mechanism of competitive binding may exist and be modulated by oxidative stress. In the context of recent observations, it seems to be of special interest to experimentally test how the NRF2/KEAP1 pathway affects p62dependent NF-κB activation and caspase-8-mediated apoptosis under conditions of mild or high oxidative stress and elucidate its influence on tumorigenesis. KEAP1 mutations and constitutive NRF2 activation are found in various tumors, most often in lung cancers [97]. When KEAP1 is dysfunctional, it cannot mediate IKKβ ubiquitination and this may be one of the mechanisms that keep antiapoptotic NF-κB signaling

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constantly active in certain cancer cells. Moreover, permanent activation of NRF2-dependent antioxidant gene expression keeps ROS at a level that, although typically much higher than in normal cells, is still insufficient to start apoptosis. An alternative explanation of this paradox appeared very recently: Niture and Jaiswal discovered that KEAP1 not only is responsible for the previously characterized degradation of NRF2 and IKKβ but also mediates ubiquitination of the main antiapoptotic protein, BCL-2 [98]. This constitutes another mechanism by which the activated KEAP1/ NRF2 pathway interacts with the apoptosis-regulatory machinery. Concluding remarks KEAP1 mutations in lung cancers [3] are an example of how one mutation may provide a considerable benefit for cancer cell survival: because of an inability to mediate KEAP1-dependent ubiquitination, lung cancer cells keep NRF2, NF-κB, and BCL-2 antiapoptotic pathways constantly active. As mentioned in the introduction, Zhang et al. presented a systems biology view of KEAP1 being a “transducer” protein module in NRF2/KEAP1 signaling [6]. The data reviewed above significantly broaden this concept. By its various protein interactions, KEAP1 is a transducer, not only in controlling the transcription of the NRF2 gene battery and in counteracting ROS, but also in a much broader context involving regulation of apoptosis and autophagy. Acknowledgments The authors acknowledge the assistance of Monica Borrin-Flint in the preparation of the manuscript and thank Professor Irena Szumiel for editorial help and for fruitful discussion and Dariusz Stępkowski for valuable comments. Support from an INCT statutory grant is gratefully acknowledged. References [1] Kaspar, J. W.; Niture, S. K.; Jaiswal, A. K. Nrf2:INrf2 (Keap1) signaling in oxidative stress. Free Radic. Biol. Med. 47:1304–1309; 2009. [2] Kensler, T. W.; Wakabayashi, N. Nrf2: friend or foe for chemoprevention? Carcinogenesis 31:90–99; 2010. [3] Hayes, J. D.; McMahon, M. NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem. Sci. 34:176–188; 2009. [4] Kwak, M. K.; Kensler, T. W. Targeting NRF2 signaling for cancer chemoprevention. Toxicol. Appl. Pharmacol. 244:66–76; 2010. [5] Lee, Y. K.; Hwang, J. T.; Kwon, D. Y.; Surh, Y. J.; Park, O. J. Induction of apoptosis by quercetin is mediated through AMPKalpha1/ASK1/p38 pathway. Cancer Lett. 292: 228–236; 2010. [6] Zhang, Q.; Pi, J.; Woods, C. G.; Andersen, M. E. A systems biology perspective on Nrf2-mediated antioxidant response. Toxicol. Appl. Pharmacol. 244:84–97; 2010. [7] Sun, Z. J.; Chen, G.; Hu, X.; Zhang, W.; Liu, Y.; Zhu, L. X., et al. Activation of PI3K/ Akt/IKK-alpha/NF-kappaB signaling pathway is required for the apoptosisevasion in human salivary adenoid cystic carcinoma: its inhibition by quercetin. Apoptosis 15:850–863; 2010. [8] Toledano, M. B. The guardian recruits cops: the p53–p21 axis delegates prosurvival duties to the Keap1–Nrf2 stress pathway. Mol. Cell 34:637–639; 2009. [9] Chen, W.; Sun, Z.; Wang, X. J.; Jiang, T.; Huang, Z.; Fang, D., et al. Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response. Mol. Cell 34:663–673; 2009. [10] Kensler, T. W.; Wakabayashi, N.; Biswal, S. Cell survival responses to environmental stresses via the Keap1–Nrf2–ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47:89–116; 2007. [11] Kobayashi, M.; Yamamoto, M. Molecular mechanisms activating the Nrf2–Keap1 pathway of antioxidant gene regulation. Antioxid. Redox Signaling 7:385–394; 2005. [12] Zhang, D. D. Mechanistic studies of the Nrf2–Keap1 signaling pathway. Drug Metab. Rev. 38:769–789; 2006. [13] Itoh, K.; Wakabayashi, N.; Katoh, Y.; Ishii, T.; Igarashi, K.; Engel, J. D., et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13:76–86; 1999. [14] Kobayashi, A.; Kang, M. I.; Okawa, H.; Ohtsuji, M.; Zenke, Y.; Chiba, T., et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol. Cell. Biol. 24:7130–7139; 2004. [15] Dhakshinamoorthy, S.; Jaiswal, A. K. Functional characterization and role of INrf2 in antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase 1 gene. Oncogene 20:3906–3917; 2001.

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