Canonical and non-canonical mechanisms of Nrf2 activation

Pharmacological Research 134 (2018) 92–99

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Invited Review

Canonical and non-canonical mechanisms of Nrf2 activation Silva-Islas Carlos Alfredo, Maldonado Perla D.

⁎

T

Laboratorio de Patología Vascular Cerebral, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, Insurgentes Sur 3877, La Fama, Tlalpan, 14269, CDMX, Mexico

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in this article: Sulforophane (CID: 5350) tert-Butylhydroquinone (CID: 16043) Dimethyl fumarate (CID: 637568) Bardoxolone methyl (CID: 400769) ML334 (CID: 56840728) AN-465/14458038 (CID: 11839135) CPUY192018 (CID: 73330369) Cpd16 (CID: 1073725) Dialyl disulfide (CID: 16590) Diethyl maleate (CID: 5271566)

Nuclear Factor Erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of genes involved in the metabolism, immune response, cellular proliferation, and other processes; however, the attention has been focused on the study of its ability to induce the expression of proteins involved in the antioxidant defense. Nrf2 is mainly regulated by Kelch-like ECH-associated protein 1 (Keap1), an adapter substrate of Cullin 3 (Cul3) ubiquitin E3 ligase complex. Keap1 represses Nrf2 activity in the cytoplasm by its sequestering, ubiquitination and proteosomal degradation. Nrf2 activation, through the canonical mechanism, is carried out by electrophilic compounds and oxidative stress where some cysteine residues in Keap1 are oxidized, resulting in a decrease in Nrf2 ubiquitination and an increase in its nuclear translocation and activation. In the nucleus, Nrf2 induces a variety of genes involved in the antioxidant defense. Recently a new mechanism of Nrf2 activation has been described, called the non-canonical pathway, where proteins such as p62, p21, dipeptidyl peptidase III (DPP3), wilms tumor gene on X chromosome (WTX) and others are able to disrupt the Nrf2-Keap1 complex, by direct interaction with Keap1 decreasing Nrf2 ubiquitination and increasing its nuclear translocation and activation. In this review, the regulatory mechanisms involved in both canonical and non-canonical Nrf2 activation are discussed.

Keywords: Nrf2 Keap1 Oxidative stress Non-canonical activation Protein-protein interaction p62

1. Introduction Oxygen is required in normal cellular functions such as energy metabolism in most eukaryotic organisms; part of this oxygen is partially reduced to the superoxide anion and subsequently to other reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radical. In metabolism, the reactive nitrogen species (RNS), such as peroxynitrite and nitric oxide, are also produced. ROS and RNS, at low cellular concentration, are implicated in cellular signaling process, however an increase in these species leads to an oxidative stress state in the cells, inducing cellular damage and death [1]. Cells have developed a response called the phase 2 response, where a set of genes regulated by the Nuclear Factor Erythroid 2-related factor 2 (Nrf2), are involved in the defense against oxidative stress [2].

Nrf2 is considered the master regulator against oxidative stress. In homeostatic conditions, Nrf2 levels and its activation are controlled mainly by Kelch-like ECH-associated protein 1 (Keap1). Nevertheless, in an oxidative stress state or in the presence of electrophilic compounds, Nrf2 is activated, inducing the expression of its target genes, which are involved in cell protection. This mechanism of Nrf2 activation is known as the canonical mechanism. Recently, a new mechanism known as non-canonical has been described, where the activation of Nrf2 is carried out by Keap1-Nrf2 complex disruption by some proteins such as p62, DPP3, WTX, PALB2, p21 and BRCA1 [3]. In this article, we describe the canonical and non-canonical mechanism of Nrf2 activation and some peptides and drugs involved in the non-canonical activation as potential pharmacological targets.

Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; ATF4, activating transcription factor 4; Atg5, autophagy protein 5; Atg7, autophagy protein 7; Bach, BTB and CNC homology; CBP, CREB binding protein; CHD6, chromodomain-helicase-DNA-binding protein 6; CRIF1, CR6-interacting factor 1; Crm1, chromosomal maintenance 1; Hrd1, ERAD-asociated E3 ubiquitin-protein ligase HRD1; JDP2, c-Jun dimerization protein 2; LPS, lipopolysaccharide; Maf, musculoaponeurotic fibrosarcoma; MEF2D, myocyte enhancer factor 2D; Miro2, mitochondrial Rho GTPase 2; Neh, Nrf2-ECH-homology; NES, nuclear export signal; NLS, nuclear localization signal; PPARγ, peroxisome proliferator activated receptor γ; RAC3, receptor-associated coactivator 3; Rbx1, RING box protein 1; Sp-1, specificity protein-1; TAK1, transforming growth factor betaactivated kinase 1; UBNX7, UBX domain-containing protein 7; UFD1/NPL4, ER-associated degradation protein 1/ Nuclear protein localization protein 4 homolog; WDR23, WD repeat protein; XRE, xenobiotic response elements ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (P.D. Maldonado). https://doi.org/10.1016/j.phrs.2018.06.013 Received 14 May 2018; Received in revised form 6 June 2018; Accepted 14 June 2018 Available online 18 June 2018 1043-6618/ © 2018 Published by Elsevier Ltd.

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Fig. 1. Nrf2 protein structure from Homo sapiens (isoform 1). Nrf2 protein possesses 7 Neh domains (Neh1 to Neh7) in its structure. Neh1 domain contains bZip region (amino acid sequences between 525 and 566, on the bottom) through which it dimerizes with small Maf proteins (MafK, MafF and MafG) and other transcription factors (c-Jun, Sp-1 and JDP2). Furthermore it contains 6 Lys residues that are acetylated by CBP (K438, K443, K445, K533, K536 and K538 residues, on the bottom), important in its DNA binding; a NES sequence (amino acid sequences between 553 and 562, on the top) through which Nrf2 is exported from the nucleus by Crm1 protein; a K533 residue, important in SUMOylation process by Ubc9 protein; and S558 residue that is phosphorylated by AMPK, relevant in Nrf2 nuclear translocation. Neh2 is a degron domain. Keap1 and other ubiquitin ligases such as CRIF1 and WDR23 bind to Nrf2 trough this domain. ETGE and DLG motifs (on the top) are the sequences through which Keap1 binds to Nrf2. The DIDLID motif (on the top) is the region through WDR23 binds to Nrf2. Moreover, in this domain Nrf2 possess an α-helix with seven lysine amino acid residues (K44, K50, K52, K53, K56, K64 and K68, on the bottom), which are the ubiquitin amino acid acceptors; a NLS sequence (amino acid sequences between 42 and 53, on the bottom) important in its nuclear translocation through Karyopherin α1 and Karyopherin β1 importins; and S40 residue that is phosphorylated by PKCδ, important in Nrf2 nuclear translocation. Neh3 domain contains a second NLS sequence (amino acid sequences between 595 and 601, on the bottom); two Lys residues that are acetylated by CBP (K596 and K599, on the top), important in Nrf2 nuclear translocation; and K603, important lysine residue that is SUMOylated by Ubc9 protein. Through this domain, Nrf2 recruits CDH6 protein co-activator. Neh4 and Neh5 are transactivation domains. CBP and p300 as well as RAC3 bind to Nrf2 through this domain. Hrd1 an ubiquitin ligase, also binds to Nrf2 in this domain. A second NES sequence (amino acid sequences between 191 and 202, on the top) is localized in the Neh5 domain. Neh6 is a second degron domain. βTrCP ubiquitin ligase binds to Nrf2 through DSGIS motif (on the top), previous phosphorylation in S344 and S347 by GSK3β and DSAPGS motif (on the top). Finally, through Neh7 domain, Nrf2 interacts with RXRα protein, inducing Nrf2 repression. The figure construction was carried out following the next guideline “Guidelines for preparing color figures for everyone including the colorblind” [14].

2. Nrf2 protein

transcription factors such as AhR [20], PPARγ [21], NF-κB [22], Sp-1, p53 [23], MEF2D [24], c-Jun, c-Myc [25] and BRCA1 [26]. Epigenetic mechanisms such as methylation of the Nrf2 promoter in CpG islands, H3 histone methylation and H4 histone acetylation are also involved in Nrf2 transcriptional regulation [27]. miRNAs such as miR-27a, miR-28, miR-34a, miR-93, miR129-5p, miR142-5p, miR-144, miR-153, miR155, miR-200c, miR-340, miR-340-5p, miR-450a, miR-507 and miR634 are able to regulate Nrf2 synthesis at the posttranscriptional level [28]. At the translational level, in homeostatic conditions, Nrf2 is regulated by mTORC1 in a cap-dependent process; however, in the presence of oxidant stimuli, such as H2O2 or α-lipoic acid, the regulation of Nrf2 is carried out in a cap-independent process by an Internal Ribosomal Entry Site (IRES) mechanism [29]. Finally, at the posttranslational level, Nrf2 is regulated by proteasomal degradation mainly by Keap1 dependent ubiquitination [30]; however, βTrCP [8], CRIF1 [31], Hrd1 [32] and WDR23 [33] are also able to induce Nrf2 ubiquitination and subsequent proteosomal degradation. Keap1 is a Zn metalloprotein with 625 amino acid residues and 5 domains in its structure. The domains include the N-terminal region (NTR) [34], the Bric-a-brac, tramtrac, broad-complex/proxvirus zinc fingers (BTB/POZ) domain [6], implicated in Keap1 homodimerization and Cullin 3 (Cul3) association, the intervening region (IVR) [34], a linker between BTB and DGR domains, the Double glycine repeat/Kelch (DGR/Kelch) domain [6], important in Nrf2 repression and actin interaction, and the C-terminal region (CTR) [34]. Keap1 has some reactive cysteine residues, the C273, C288 and C297 in the BTB/POZ domain [35] and the C151 in the IVR domain [36], which are oxidized by Nrf2 inducers such as ROS, RNS, electrophilic compounds such as

Nrf2 is a member of the Cap ´n´ Collar (CNC) transcription factors with a basic leucine zipper region (bZip). It has 605 amino acid residues, however the electrophoretic mobility indicates a 96–118 KDa molecular weight, caused by the abundance of acidic residues in its structure [4] and posttranslational modifications such as phosphorylation [5]. Nrf2 has 7 Neh domains (Neh1-Neh7) important in its activity and its repression [6]. Neh1 is implied in DNA binding and heterodimerization with small Maf proteins [6,7]. Neh2 and Neh6 are degron regions (an amino acid sequence or structure in a protein involved in its degradation), targeted by Keap1 and βTrCP, through 29DLG31 and 79 ETGE82 motifs and 343DSGIS347 and 382DSAPGS387 motifs, respectively [8,9]. Neh3, Neh4 and Neh5 are transactivation domains through CHD6, CBP and RAC3 proteins association [10–12]. Finally, Neh7 domain is implicated in Nrf2 repression by RXRα protein [13] (Fig. 1). Nrf2 is ubiquitously expressed [4] and regulates the expression of around 1055 genes [15], which have in its structure the cis-acting antioxidant response element (ARE, 5′-GTGACNNNGC-3′) [16]. ARE sequence is present in the promoter region of genes involved in the antioxidant and detoxifying response, cellular proliferation, metabolism, immune response, signaling, cell survival and cellular cycle. However, Nrf2 is considered the master regulator of the redox cellular state because its deletion or decrease in aging is associated with an increase in oxidative stress and cellular death [17,18]. 3. Nrf2 regulation Nrf2 is regulated at the transcriptional level by itself [19] and other 93

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This phenomenon decreases Keap1 levels able to sequester de novo Nrf2 [38], which is translocated to the nucleus [47] (Fig. 2B). Nfr2 nuclear translocation requires the phosphorylation of S40 by PKCδ [48], S558 by AMPK [49], and other unidentified residues by other kinases, as well as the acetylation of K588 and K591 (corresponding to K596 and K599 of Human Nrf2 isoform 1) by CBP acetylase [50]. Karyopherin α1 (importin α5) and karyopherin β1 (importin β1) are responsible for Nrf2 nuclear translocation through the nuclear pore complex due to NLS sequences in the Nrf2 structure [51] (Fig. 2D). In the nucleus, Nrf2 heterodimerize with small Maf proteins (MafF, MafG and MafK) through its bZip domains and binds to the ARE sequences, inducing its transcriptional activation [7]. It has been reported that Nrf2 heterodimerization with other transcription factors such as cJun, Sp-1 [52] and JDP2 [53], also increases its transcriptional activity. Moreover, the acetylation of K438, K443, K445, K533, K536, K538, K588 and K591 by p300/CBP [54] and SUMOylation in K525 and K595 (corresponding to K533 and K603 of Human Nrf2 isoform 1) by UBC9 [55], is required for Nrf2 binding to the ARE sequence. In the same way, the co-activators p300/CBP, ATF4, RAC3 and CHD6 are required for Nrf2 activity [10–12] (Fig. 2E). In addition, the nuclear Nrf2 activity is regulated negatively by cMyc dimerization, repressing the genes regulated by Nrf2 [56]. p65 competes with Nrf2 for CBP binding, decreasing Nrf2 transcriptional activation [57], whereas MafG homodimer also decreases Nrf2 transcriptional activity by competing for ARE binding [7]. Further, Bach1 and Bach2 heterodimerize with small Maf proteins and bind to the ARE sequence, also decreasing Nrf2 ARE binding [58]. Nrf2 activity is ended by its nuclear export in a Crm1-dependent process through NES sequences in Nrf2 [59,60]. Nrf2 phosphorylation by GSK3β is also an important process in this nuclear export [61]. Moreover, nuclear levels of Keap1 are increased after Nrf2 activation by its nuclear import through karyopherin α6 (KPNA6) [62]. This increase leads to Nrf2 dissociation from ARE sequences as a consequence of Nrf2 sequestering by Keap1, which induces nuclear export in a Crm1-dependent process and NES sequences in Keap1, promoting Nrf2

sulforophane (SULF), tert-butyl hydroquinone (tBHQ), and metals such as Cd2+, As3+ and Se4+. Keap1 cysteine oxidation decreases Nrf2 ubiquitination and increases its nuclear translocation and activation [36,37].

4. Nrf2 activation through canonical mechanism In homeostatic conditions, the four-residue β-hairpin conformation of the ETGE motif in Nrf2, specifically, amino acid residues E78, E79, T80, E82 and F83, interacts with the bottom surface of the six-bladed-βpropeller structure of the Kelch domain of one protomer of a Keap1 homodimer (Y334, S363, R380, D382, R415, R483, S508, Q530, S555, S602) in a first step, called the open conformation; whereas the fourresidue β-hairpin conformation of the DLG motif in Nrf2, specifically, amino acid residues Q26, D27 and D29, interacts similarly with the bottom surface of the six-bladed-β-propeller structure of the Kelch domain of the second protomer of Keap1 homodimer (D382, R415, R483, S508, S555, S602, G603) in a second step, called the closed conformation [39,40,41,42]. The Keap1-Nrf2 interaction is also known as the hinge and latch model, where ETGE motif, a strong affinity binding region (KD = 1.9 ± 0.4 × 108 M−1) represents the hinge and DLG motif, a low affinity binding region (KD = 1.0 ± 0.0 × 106 M−1) represents the latch [43]. The closed conformation in Keap1-Nrf2 complex allows an adequate Lys rich α-helix orientation in Neh2 domain for the Keap1 dependent ubiquitination [38,44]. This process also requires the presence of Neddylated-Cul3 [45], and Rbx1 protein [30]. Moreover, p97-UBNX7UFD1/NPL4 complex extracts ubiquitinated Nrf2 from Keap1-Nrf2 complex and transfers it to 26S proteasome for its degradation [46] (Fig. 2A). On the other hand, Nrf2 inducers oxidize Cys residues in Keap1, inducing a conformational change in its structure. This structural change in Keap1 induces a Keap1-Nrf2 interaction in a closed conformation; however in this interaction Lys residues of Nrf2 are not orientated properly for its ubiquitination, decreasing Nrf2 degradation.

Fig. 2. Canonical and non-canonical pathways of Nrf2 activation. A) In homeostatic conditions, Nrf2 is sequestered by Keap1-Nedd8-Cul3-Rbx1 complex in the cytoplasm, transferring ubiquitin (Ub) proteins from E2 ligases to Neh2 domain in Nrf2. UBX7-p97-UFD1/NPL4 interacts with ubiquitinated Nrf2 and Nedd8-Cul3 complex and transfers Nrf2 to 26S proteasome for its degradation. B) Oxidative stress and Nrf2 classic inducers such as sulforophane, oxidize Keap1 cysteine residues, inducing a conformational change in its structure, preventing Nrf2 ubiquitination. Keap1 modifications increase Nrf2-Keap1 interaction, decreasing the Keap1 levels able to sequester Nrf2 synthesized de novo, since the Nrf2 orientation, is inadequate for its ubiquitiniation and subsequent degradation (Nrf2 canonical activation). C) Proteins with ETGE motif in their structure such as p62, DPP3, WTX, ProTα and PALB2 interact with Kelch domain in Keap1 preventing Nrf2 sequestering and therefore its ubiquitination. On the other hand, p21 and BRCA1 interact with Nrf2, preventing its Keap1 dependent ubiquitination (Nrf2 non-canonical activation). D) Decreasing Keap1 levels with the ability to sequester Nrf2 synthesized de novo, induce Nrf2 phosphorylation (P) by PKCδ and AMPK, and acetylation (AC) by CBP, followed by its nuclear translocation through Karyopherin α1 and Karyopherin β1. E) In the nucleus, Nrf2 is acetylated by CBP (AC), SUMOylated (SUMO) by Ubc9, dimerizated with small Maf proteins and binds to ARE sequences. Moreover, Nrf2 interacts with CBP, p300, RAC3 and CHD6 coactivators, inducing gene expression. Transcriptional activity of Nrf2 is repressed by Keap1, which is translocated to the nucleus through KPNA6, dissociates Nrf2 from ARE sequences and both, Nrf2 and Keap1, are nuclear exported by Crm1. In the cytoplasm, Nrf2 is ubiquitinated and degraded. The figure construction was carried out following the next guideline “Guidelines for preparing color figures for everyone including the colorblind” [14]. 94

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phosphorylation, promotes antineoplastic drug chemoresistance to doxorubicin, cisplatin, erastin, sorafenib, buthionine sulfoxamine and carfilzomib, as well as cancer cell proliferation; whereas mutation in KIR domain in p62, that prevents Keap1-p62 interaction, is associated with a ROS increase and the etiology of amyotrophic lateral sclerosis [64,71,76–81]. Dipeptidyl peptidase III (DPP3) is a zinc aminopeptidase able to hydrolyze peptides containing from 3 to 10 amino acid residues. It is involved in protein turnover since it participates in the hydrolysis of peptides (four to eight amino acid residues) generated by the proteasome during protein degradation [82]. DPP3 also participates in the regulation of oxidative stress through the modulation of Nrf2 pathway by direct interaction with Keap1 through its 480ETGE483 motif and Kelch domain in Keap1, decreasing Nrf2-Keap1 interaction in an independent oxidative stress process and independent of catalytic activity of DPP3. Overexpression of DDP3 decreases Nrf2 ubiquitination and increases Nrf2 nuclear translocation and activation, without increasing Nrf2 total levels in HEK293 T cells. This mechanism protects cells against oxidative stress and prevents cellular death by decreasing ROS levels; however, in breast cancer cells, an increase in DPP3, is associated to metastasis and drug chemoresistance [65,83]. WTX is a protein codified by Wilms tumor gene in chromosome X that is mutated in 30% of cases of Wilms tumor. WTX negatively regulates WTN/β-catenin pathway through degradation of β-catenin protein and is also implied in oxidative stress regulation through Nrf2 activation. WTX has a similar sequence to the ETGE motif in Nrf2, the 286 SPETGE291 sequence, through which is able to interact with the Kelch domain of Keap1 in a similar process as Nrf2 does; however, WTX is not substrate of Keap1. Phosphorylation of S286 in WTX increases the binding affinity of WTX to Keap1 and mutation in this residue decreases the interaction between WTX and Keap1, as well as mutation in T289. It has been observed that an increase in WTX decreases Nrf2 ubiquitination and activates Nrf2 transcriptional activity, in an process independent of WNT/β-catenin pathway and electrophilic compounds such as tBHQ; whereas, a decrease in WTX inhibits Nrf2 activation and sensitizes cells to the chemotherapeutic agent etoposide [66]. Prothymosin α (ProTα) is a nuclear protein involved in cellular proliferation and protection against apoptosis. ProTα have a β-turn conformation (N41, E42, E43, N44, E46) through which it interacts with the bottom surface of Kelch domain (S363, D382, R380, R415, R483, S508, N530, S555, Y572, S602) in Keap1, just like Nrf2 does. The interaction between ProTα and Keap1 increases Nrf2 activation and HO-1 expression. Further, this interaction is carried out in the nucleus of HeLa cells and increases in the presence of oxidative stress induced by diethyl maleate [67,84]. The protein partner and localizer of BRCA2 (PALB2) is a nuclear protein that interacts with BRCA1 and BRCA2, controlling its nuclear localization and stabilization. The complex BRCA1-BRCA2-PALB2 is involved in DNA repair by recombination and is a damage DNA checkpoint. On the other hand, PALB2 is able to interact with the Kelch domain of Keap1 through the 91ETGE94 sequence in its structure, similar to the ETGE motif in Nrf2. In the same way that ProTα, the interaction between Keap1 and PALB2 is carried out in the nucleus in U2OS cells, since PALB2 is a nuclear protein. The nuclear interaction between Keap1 and PALB2 prevents nuclear export and subsequent 26S proteasomal degradation of Nrf2. Furthermore, the increase in PALB2, induce Nrf2 nuclear accumulation and transcriptional activation in an oxidative stress-independent process, since PALB2 is able to compete with Nrf2 for Keap1 binding. The activation of Nrf2 through PALB2 decreases cellular ROS levels, protecting cells from oxidative stress [68]. Breast cancer type 1 susceptibility protein (BRCA1) functions as a tumor suppressor protein and regulates cell cycle progression, DNA damage signaling and repair, maintenance of genomic integrity and various transcriptional pathways. Overexpression of BRCA1 increases

Table 1 Proteins with the ability to interact with Keap1 or Nrf2, inducing an increase in Nrf2 activation. Keap1 binding proteins Protein SQSTM1/p62 DPP3 WTX Prothymosin α PALB2 Nrf2 binding proteins Protein BRCA1 p21

Motif involved in the interaction with Keap1 SKEVDPSTGELQSLQ359 (KIR domain) 480 ETGE483 286 SPETGE291 41 NEENGE46 91 ETGE94 345

Motif involved in the interaction with Nrf2 N-terminal region 154 KRR156

cytoplasmic ubiquitination and 26S proteasomal degradation [63] (Fig. 2E). 5. Non-canonical activation of Nrf2 The main attention in the study of the Nrf2 activation mechanism has been focused in the canonical pathway; moreover, the study and design of molecules with a potential therapeutic effect against oxidative stress through Nrf2 signaling, have been focused on this pathway. Recently, a new mechanism of Nrf2 activation, poorly studied, with pharmacological potential, has been described. This pathway, called non-canonical Nrf2 activation pathway, involves a set of proteins (Table 1) such as p62 [64], DPP3 [65], WTX [66], Prothymosin α [67], PALB2 [68], p21 [69] and BRCA1 [70], which have the ability to disrupt the Keap1-Nrf2 complex by direct interaction with Keap1 or Nrf2, preventing Nrf2 ubiquitination and 26S proteasomal degradation and inducing an increase in the nuclear translocation and activation of Nrf2 (Fig. 2C). Nrf2 activation by SQSTM1/p62 (hereafter referred to as p62) protein is the most studied mechanism of the non-canonical pathway. p62 is a scaffold protein involved in multiple signaling pathways, such as autophagy, where it interacts with ubiquitinated proteins and functions as autophagy receptor for protein and mitochondria degradation. The impairment in the autophagy pathway is due to a depletion in Atg7 and Atg5, increases p62 levels and Nrf2 activation, as well as Keap1 cytoplasmic accumulation in inclusion bodies, in dependent and independent oxidative stress processes [64]. p62 is able to interact with Keap1 similarly to the interaction of Keap1 with Nrf2. The KIR domain in p62 (345SKEVDPSTGELQSLQ359) interacts with the bottom surface of the six-bladed-β-propeller structure of Kelch domain in Keap1. Specifically, this interaction involves D349, P350, S351, T352, E354 and L355 residues in p62 and Y334, S363, R380, D382, R415, Q530, S555 and S602 in Keap1, with affinity binding KD = 0.54 ± 0.03 × 106 M−1, similar to the affinity binding between Keap1 and DLG motif in Nrf2 (KD = 1.0 ± 0.0 × 106 M−1) [64]. Nevertheless, phosphorylation of S351 in p62 by mTORC1 [71] or TAK1 [72], increases the affinity binding between Keap1 and p-p62 (KD = 8.5 ± 2.4 × 106 M−1) due to an additional interaction between p-S351 in p62 and R483 and S508 in Keap1, suggesting that p-p62 competes with the ETGE motif of Nrf2 for Keap1 binding [71]. The interaction of Keap1 with p62, induces a dependent autophagy degradation of Keap1 and subsequent Nrf2 stabilization and activation in MEF and HEK293 cells [71]. Interestingly, p62 expression is regulated by Nrf2, due to the presence of ARE sequences in the p62 promoter, creating a feedback positive loop [73]. The Nrf2 activation dependent on p62 by compounds such as LPS in RAW cells or the overexpression of Sens2 protein in HEK293 and MEF cells, increases the Nrf2 activation and expression of NQO1, GST and anti-apoptotic proteins such as Bcl-2 and Bcl-xL, decreasing ROS levels and protecting cell against oxidative stress [74,75]. However, sustained Nrf2 activation by impairment in autophagy and increase in p62 95

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6. Nrf2 activation by molecules with the ability to disrupt proteinprotein interaction

antioxidant enzyme expression and protects cells against oxidative stress through an increase in Nrf2 transcriptional activity. Unlike p62, DPP3, WTX, ProTα and PALB2, BRCA1 interacts with the ETGE motif of Nrf2 through its N-terminal region, preventing Keap1-dependent ubiquitination of Nrf2, increasing its activation and protecting cells against oxidative stress [70]. BRCA1 also regulates Nrf2 at the transcriptional level by dimerization with ARNT and by direct interaction with the XRE sequence in Nrf2 promotor [26]. Notably, Nrf2 regulates BRCA1 by the presence of ARE sequence in BRCA1 promotor, creating a positive feedback, where an increase in Nrf2 increases BRCA1 and vice versa [85]. p21 is a cyclin-dependent kinase inhibitor that is able to inhibit cyclin/CDK complex and is involved in the regulation of cell cycle arrest, replication, cellular proliferation and apoptosis. p21 also regulates Nrf2 activation at posttranslational level by the competition with Keap1 for Nrf2 binding through the DLG motif. p21 interacts with the DLG motif in Nrf2 through the 154KRR156 sequence in its structure. This interaction does not dissociate Keap1 from Nrf2 through the ETGE motif, but prevents Nrf2 ubiquitination, since it induces the loss of the proper orientation of Lys residues for its ubiquitination, increasing Nrf2 stabilization [69]. This Nrf2 activation mechanism is increased by Nrf2 inducers such as SULF, tBHQ [69] and dialyl disulfide (DADS) [86]. Moreover, overexpression of p21 increases Nrf2 activation and HO-1 and NQO1 expression, decreasing ROS levels and protecting JB6P + cells and mouse epidermis against oxidative stress [86]. The non-canonical Nrf2 activation through p21 prevents skin carcinogenesis and the inflammatory response [86]; however, this mechanism has also been observed in cancer cell proliferation and doxorubicin, camptothecin [87] and cisplatin chemoresistence [88]. This supports the Nrf2 dual role theory and the role of Nrf2 in carcinogenic processes, where in early times, Nrf2 is able to protect cells against the carcinogenic process through the decrease of ROS levels, whereas at longer times, Nrf2 promotes cancer cell proliferation and drug chemoresistance [89]. The inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is a serine/threonine kinase which is part of the IKK complex, and phosphorylates the inhibitor of NF-κB family proteins, activating the NF-κB signaling pathway [90]. Phosphoglycerate mutase 5 (PGAM5) is a serine/threonine phosphatase that interacts with external mitochondrial membrane [91]. PGAM5 and IKKβ have 77NXE(S/T)GE82 [92] and 34 NQETGE39 [93] sequences, respectively, and both proteins are able to interact with Keap1 through the Kelch domain. However, to date, there is no evidence that indicates that Keap1 and PGAM5 or Keap1 and IKKβ interaction increases Nrf2 transcriptional activation; in fact, the increase in PGAM5 prevents Nrf2 activation [91]. In addition to Keap1 dependent Nrf2 ubiquitination and degradation, IKKβ is also a target for Keap1. The interaction between these two proteins targets IKKβ to ubiquitination and 26S proteasomal degradation, increasing NF-κB cytoplasmic repression by IκBα and decreasing nuclear translocation and activation of NF-κB. This could be indirectly increasing Nrf2 transcriptional activity since NF-κB represses Nrf2 by CBP competition. However, to date, there is no evidence supporting this notion [90,93]. On the other hand, the interaction between Keap1 and PGAM5 is important in mitochondrial retrograde movement as opposed to Nrf2 activation [94]. PGAM5 recruits Keap1 and Nrf2 to the external mitochondrial membrane [92] forming a PGAM5-Keap1-Nrf2 complex, where one molecule of Keap1 dimer binds to one molecule of PGAM5, whereas the other molecule of Keap1 dimer, binds to one molecule of Nrf2 [91], preventing ubiquitination and proteasomal degradation of Miro2, an important protein in microtubule depolymerization and mitochondrial movement, by Keap1. This indicates that Nrf2 activity is not only limited to transcription; instead it also participates in other cellular process in a transcriptional activity independent mechanism [94].

There is a lot of evidence that indicates drug chemoresistance and cancer cell proliferation in impairment Nrf2 activation [64,71,77–81,87,88]. However, the protective effect of Nrf2 has also been proven. Nrf2 activation protects cell from cellular damage and death induced by oxidative stress. In different animal models such as cerebral ischemia, Alzheimer and Huntington diseases, hepatotoxicity, and others, the protective role of Nrf2 has been demonstrated, making Nrf2 an excellent therapeutic target [95]. Despite this, dimethyl fumarate (DMF), an electrophilic compound with the ability to activate Nrf2 through canonical pathway, is the only FDA approved drug used in humans with this mechanism, for the treatment of relapsing multiple sclerosis, despite its side effects [96]. Bardoxolone methyl, another drug with the ability to induce Nrf2 activation used in the treatment of chronic kidney disease, failed in its approval for use in humans by unspecified safety reasons, probably since if not only induces Nrf2 activation, instead is able to interact with 557 different proteins [97]. For this reason, molecules designed and studied, with the ability to activate Nrf2, must be more specific. These findings suggest that potential drugs with the ability to disrupt protein-protein interaction will be excellent candidates due to its specificity, unlike classical Nrf2 inducers, that interact and modify different proteins by oxidation or alkylation. It has been reported that some peptides derived from the ETGE motif of Nrf2 and small molecules have the ability to disrupt Keap1Nrf2 complex, making them possible pharmacological targets. Heptapeptide 77DEETGEL83 (CI50 = 5.39 ± 0.58 μmol/L) is able to disrupt the Nrf2-Keap1 complex, whereas a single mutation in E78 P (77DPETGEL83, CI50 = 0.634 ± 0.064 μmol/L) or double mutation in D77 N and E78 P (77NPETGEL83, CI50 = 0.875 ± 0.082 μmol/L), increases the ability to disrupt this complex [98]. It also has been observed that an increase in amino acid residues in these peptides increases their ability to disrupt the Nrf2-Keap1 complex. The CI50 value decreases when peptides have 9 amino acids residues (76LDEETGEFL84 CI50 = 0.389 ± 0.033 μmol/L) and decreases even more, when seven amino acids residues are added to the peptides (74FQLDEETGEFLPIQ87, CI50 = 0.257 ± 0.010 μmol/L), suggesting a higher affinity to Keap1. On the other hand, peptides with fewer than 7 amino acid residues lose the ability to disrupt Nrf2-Keap1. DLG derived peptides have low affinity for Keap1, as well as heptapeptide derived from ProTα (41NEENGEQ47). Peptides derived from p62 (349DPSTGEL355, CI50 = 34.4 ± 9.4 μmol/L) interact with Keap1 dissociating the Nrf2Keap1 complex; however, S351E mutation (349DPETGEL355) that mimic physiological S351 phosphorylation of p62, increases around 300 times the interaction with Keap1 (CI50 = 0.115 ± 0.013 μmol/L) [99]. Peptides are not ideal drugs, since they are unable to cross cytoplasmic membrane; however the addition of a penetrating peptide such as trans-activating transcriptional activator (TAT) peptide derived from HIV to 74LQLDEETGEFLPIQ87 peptide (75 μM), allows Keap1-Nrf2 complex disruption in monocytes, increasing Nrf2 activation and HO-1 expression [100]. Intracerebroventricular administration of 77 DEETGE82-CAL-TAT peptide (50 μg), with additional cleavage sequence of calpain (CAL), is able to decrease Keap1-Nrf2 binding, increasing Nrf2 activation and antioxidant enzyme expression (HO-1, NQO1 and GPx1), decreasing oxidative stress and protecting hippocampal cell from cerebral ischemia damage [101]. The addition of lipophilic groups such as stearic acid (St) to N-terminal of heptapeptides (St-77DPETGEL83, CI50 = 0.022 ± 0.003 μM and St-77NPETGEL83, CI50 = 0.272 ± 0.029 μM) also increases the Nrf2 activation and NQO1 enzyme activity in hepatic cells at 50–80 μM of St-77DPETGEL83 and St-77NPETGEL83 peptides [98]. Finally, it has been described that some small molecules such as ML334, Cpd16, AN-465/14458038 and CPUY192018, have the ability to disrupt the Nrf2-Keap1 complex. ML334 inhibits Keap1-Nrf2 complex formation (CI50 = 1.6 μM) and induces Nrf2 nuclear translocation 96

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and activation in U2OS and HepG2 cells with EC50 values between 13–18 μM, without inducing toxicity in HepG2 and HEK293 cells up to 26 μM concentrations [102]. Cpd16, belongs to benzene-disulfonamide class, interacts with Keap1 in the Nrf2 binding site, breaking down the Nrf2-Keap1 complex (CI50 = 2.7 μM). Keap1-Cpd16 interaction increases Nrf2 transcriptional activity at 30–100 μM concentration of Cpd16 in DLD1 cells. AN-465/14458038 is able to interact with Keap1 in the Nrf2 binding site, inducing Nrf2 dissociation from Keap1 (EC50 = 9.80 μM) [103]. In HepG2 cells, AN-465/14458038 increases Nrf2 activation, using 50 to 200 μM, without any cytotoxic effect at 200 μM. The Nrf2 activation induced is more sustained than that induced by tBHQ, suggesting that Nrf2 activation through protein-protein interaction (PPI) inhibitors is more prolonged than that produced by classical inducers [104]. CPUY192018 interacts with Keap1 (IC50 = 0.0144 μM), increasing Nrf2 nuclear translocation and activation as well as expression of HO-1, GCLM and GPx2 in NCM460 cells (0.1–20 μM), even more than tBHQ, protecting NCM460 cells from dextran sodium sulfate (DSS) induced damage, an ulcerative colitis model. In the same way, CPUY192018, in an in vivo model of ulcerative colitis induced by DSS in mice, induces protection (10–40 mg/Kg) in a Nrf2 dependent process [105].

[2] A.T. Dinkova-Kostova, W.D. Holtzclaw, T.W. Kensler, The role of Keap1 in cellular protective responses, Chem. Res. Toxicol. 18 (2005) 1779–1791. [3] J.D. Hayes, A.T. Dinkova-Kostova, The Nrf2 regulatory network provides an interface between redox and intermediary metabolism, Trends Biochem. Sci. 39 (2014) 199–218. [4] P. Moi, K. Chan, I. Asunis, A. Cao, Y.W. Kan, Isolation of NF-E2-related factor 2(Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 9926–9930. [5] J. Pi, Y. Bai, J.M. Reece, J. Williams, D. Liu, M.L. Freeman, W.E. Fahl, D. Shugar, J. Liu, W. Qu, S. Collins, M.P. Waalkes, Molecular mechanism of human Nrf2 activation and degradation: role of sequential phosphorylation by protein kinase CK2, Free Radic. Biol. Med. 42 (2007) 1797–1806. [6] K. Itoh, N. Wakabayashi, Y. Katoh, T. Ishii, K. Igarashi, J.D. Engel, M. Yamamoto, Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain, Genes Dev. 13 (1999) 76–86. [7] W. Li, S. Yu, T. Liu, J.H. Kim, V. Blank, H. Li, A.N. Kong, Heterodimerization with small Maf proteins enhances nuclear retention of Nrf2 via masking the NESzip motif, Biochim. Biophys. Acta 1783 (2008) 1847–1856. [8] S. Chowdhry, Y. Zhang, M. McMahon, C. Sutherland, A. Cuadrado, J.D. Hayes, Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity, Oncogene 32 (2013) 3765–3787. [9] M. McMahon, N. Thomas, K. Itoh, M. Yamamoto, J.D. Hayes, Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron, J. Biol. Chem. 279 (2004) 31556–31567. [10] Y. Katoh, K. Itoh, E. Yoshida, M. Miyagishi, A. Fukamizu, M. Yamamoto, Two domains of Nrf2 cooperatively bind CBP, CREB binding protein, and synergistically activate transcription, Genes Cells 6 (2001) 857–868. [11] J.H. Kim, S. Yu, J.D. Chen, A.N. Kong, The nuclear cofactor RAC3/AIB1/SRC-3 enhances Nrf2 signaling by interacting with transactivation domains, Oncogene 32 (2013) 514–527. [12] P. Nioi, T. Nguyen, P.J. Sherratt, C.B. Pickett, The carboxy-terminal Neh3 domain of Nrf2 is required for transcriptional activation, Mol. Cell. Biol. 25 (2005) 10895–10906. [13] H. Wang, K. Liu, M. Geng, P. Gao, X. Wu, Y. Hai, Y. Li, Y. Li, L. Luo, J.D. Hayes, X.J. Wang, X. Tang, RXRα inhibits the NRF2-ARE signaling pathway through a direct interaction with the Neh7 domain of Nrf2, Cancer Res. 73 (2013) 3097–3108. [14] R.Jr. Roskoski, Guidelines for preparing color figure for everyone including the colorblind, Pharmacol. Res. 119 (2017) 240–241. [15] D. Malhotra, E. Portales-Casamar, A. Singh, S. Srivastava, D. Arenillas, C. Happel, C. Shyr, N. Wakabayashi, T.W. Kensler, W.W. Wasserman, S. Biswal, Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-seq profiling and network analysis, Nucleic Acids Res. 38 (2010) 5718–5734. [16] T.H. Rushmore, M.R. Morton, C.B. Pickett, The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity, J. Biol. Chem. 266 (1991) 11632–11639. [17] J.M. Lee, M.J. Calkins, K. Chan, Y.W. Kan, J.A. Johnson, Identification of the NFE2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis, J. Biol. Chem. 278 (2003) 12029–12038. [18] J.H. Suh, S.V. Shenvi, B.M. Dixon, H. Liu, A.K. Jaiswal, R.M. Liu, T.M. Hagen, Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 3381–3386. [19] M.K. Kwak, K. Itoh, M. Yamamoto, T.W. Kensler, Enhanced expression of the transcription factor Nrf2 by cancer chemopreventive agents: role of antioxidant response element-like sequences in the Nrf2 promoter, Mol. Cell. Biol. 22 (2002) 2883–2892. [20] W. Miao, L. Hu, P.J. Scrivens, G. Batist, Transcriptional regulation of NF-E2 p45related factor (Nrf2) expression by the Aryl hydrocarbon receptor-xenobiotic response element signaling pathway, J. Biol. Chem. 280 (2005) 20340–20348. [21] H.Y. Cho, W. Gladwell, X. Wang, B. Chorley, D. Bell, S.P. Reddy, S.R. Kleeberger, Nrf2-regulated PPARγ expression is critical to protection against acute lung injury mice, Am. J. Respir. Crit. Care Med. 182 (2010) 170–182. [22] S.A. Rushworth, L. Zaitseva, M.Y. Murray, N.M. Shah, K.M. Bowles, D.J. MacEwan, The high Nrf2 expression in human acute myeloid leukemia is driven by NF-κB and underlies its chemo-resistance, Blood 120 (2012) 5188–5198. [23] M.C. Tung, P.L. Lin, Y.C. Wang, T.Y. He, M.C. Lee, S.D. Yeh, C.Y. Chen, H. Lee, Mutant p53 confers chemoresistance in non-small cell lung cancer by upregulatig Nrf2, Oncotarget 6 (2015) 41692–41705. [24] S. Nagar, S.M. Noveral, D. Trudler, K.M. Lopez, S.R. McKercher, X. Han, J.R. Yates III, J. Piña-Crespo, N. Nakanishi, T. Satoh, S.I. Okamoto, S.A. Lipton, MEF2D haploinsufficiency downregulates the Nrf2 pathway and renders photoreceptors susceptible to light-induced oxidative stress, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E4048–E4056. [25] G.M. DeNicola, F.A. Karreth, T.J. Humpton, A. Gopinathan, C. Wei, K. Frese, D. Mangal, K.H. Yu, C.J. Yeo, E.S. Calhoun, F. Scrimieri, J.M. Winter, R.H. Hruban, C. Iacobuzio-Donahue, S.E. Kern, I.A. Blair, D.A. Tuveson, Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis, Nature 475 (2011) 106–109. [26] H.J. Kang, Y.B. Hong, H.J. Kim, O.C. Rodriguez, R.G. Nath, E.M. Tilli, C. Albanese,

7. Conclusion The protective role of Nrf2 has been demonstrated in a large number of animal models including diabetes, cerebral ischemia, Alzheimer and Parkinson diseases and multiple sclerosis where Keap1 oxidation by electrophilic compounds (classic inducers) is essential for Nrf2 activation and cell protection (canonical pathway). However, these classic inducers have a unspecific activity, because they can oxidize different cysteine residues in many proteins, producing side effects that limit the use of these classic inducers. Nevertheless, the Nrf2 pathway remains an attractive pharmacological target. Accordingly, more specific compounds with fewer side effects must be found and molecules that act through the non-canonical pathway are an excellent target. It has been observed that molecules with the ability to inhibit PPI are equal or more efficient than classic inducers to activate Nrf2 and apparently, with fewer side effects. For this reason, we consider that the design and study of these molecules is worthwhile and it has a promising future in the Nrf2 activation research field and in the treatment of diseases where oxidative stress plays a central role in the pathogenesis of diseases such as diabetes and neurological disorders. Funding This work was supported by CONACyT [Grant number 241655] to PDM. C.A.S.I was a recipient of CONACyT scholarship 276541. Declaration of interest None. Acknowledgements This publication is part of the Doctoral training of C.A.S.I. in the Biochemical Sciences PhD program of the National Autonomous University of Mexico. We are grateful to Dr. José Pedraza (Universidad Nacional Autónoma de México, Facultad de Química, Ciudad de México, México) for helpful with the revision of the article and to Hiram Pérez for helpful with the artwork. References [1] J. Li, W. O, W. Li, Z.G. Jiang, H.A. Ghanbari, Oxidative stress and neurodegenerative disorders, Int. J. Mol. Sci. 14 (2013) 24438–24475.

97

Pharmacological Research 134 (2018) 92–99

C.A. Silva-Islas, P.D. Maldonado

[27]

[28] [29]

[30]

[31]

[32]

[33] [34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45] [46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54] Z. Sun, Y.E. Chin, D.D. Zhang, Acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 during the antioxidant response, Mol. Cell. Biol. 29 (2009) 2658–2672. [55] X. He, Q. Lai, C. Chen, N. Li, F. Sun, W. Huang, S. Zhang, Q. Yu, P. Yang, F. Xiong, Z. Chen, Q. Gong, B. Ren, J. Weng, D.L. Eizirik, Z. Zhou, C.Y. Wang, Both conditional and overexpression of E2 SUMO-conjugating enzyme (UBC9) in mouse pancreatic beta cells result in impaired beta cell function, Diabetologia 61 (2018) 881–895. [56] S. Levy, H.J. Forman, c-Myc is a Nrf2 interacting protein that negatively regulates phase II genes through their electrophile responsive elements, IUBMB Life 62 (2010) 237–246. [57] G.H. Liu, J. Qu, X. Shen, NF-κB antagonizes Nrf2-ARE pathway by depriving CBP from Nrf2 and facilitating recruitment of HDAC3 to MafK, Bioch. Biophys. Acta 1783 (2008) 713–727. [58] J. Sun, M. Brand, Y. Zenke, S. Tashiro, M. Groudine, K. Igarashi, Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network, Proc. Natl. Acad. U. S. A. 101 (2004) 1461–1466. [59] W. Li, M.R. Jain, C. Chen, X. Yue, V. Hebbar, R. Zhou, A.N.T. Kong, Nrf2 possesses a redox-insensitive nuclear export signal overlapping with leucine zipper motif, J. Biol. Chem. 280 (2005) 28430–28438. [60] W. Li, S.W. Yu, A.N.T. Kong, Nrf2 possesses a redox-sensitive nuclear exporting signal in the Neh5 transactivation domain, J. Biol. Chem. 281 (2006) 27251–27263. [61] M. Salazar, A.I. Rojo, D. Velasco, R.M. de Sagarra, A. Cuadrado, Glycogen synthase kinase-3β inhibits xenobiotics and antioxidant cell response by direct phosphorylation and nuclear exclusion of the transcription factor Nrf2, J. Biol. Chem. 281 (2006) 14841–14851. [62] Z. Sun, T. Wu, F. Zhao, A. Lau, C.M. Birch, D.D. Zhang, KPNA6 (Importin α7)mediated nuclear import of Keap1 represses the Nrf2-dependent antioxidant response, Mol. Cell. Biol. 31 (2011) 1800–1811. [63] Z. Sun, S. Zhang, J.Y. Chan, D.D. Zhang, Keap1 controls postinduction repression of the Nrf2-mediated antioxidant response by escorting nuclear export Nrf2, Mol. Cell. Biol. 27 (2007) 6334–6349. [64] M. Komatsu, H. Kurokawa, S. Waguri, K. Taguchi, A. Kobayashi, Y. Ichimura, Y.S. Sou, I. Ueno, A. Sakamoto, K.I. Tong, M. Kim, Y. Nishito, S. Iemura, T. Natsume, T. Ueno, E. Kominami, H. Motohashi, K. Tanaka, M. Yamamoto, The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1, Nat. Cell Biol. 12 (2010) 213–223. [65] B.E. Hast, D. Goldfarb, K.M. Mulvaney, M.A. Hast, P.F. Siesser, F. Yan, D.N. Hayes, M.B. Major, Proteomic analysis of ubiquitin ligase Keap1 reveals associated proteins that inhibit Nrf2 ubiquitination, Cancer Res. 73 (2013) 2199–2210. [66] N.D. Camp, R.G. James, D.W. Dawson, F. Yan, J.M. Davison, S.A. Houck, X. Tang, N. Zheng, M.B. Major, R.T. Moon, Wilms tumor gene on X chromosome (WTX) inhibits degradation of Nrf2 protein through competitive binding to KEAP1 protein, J. Biol. Chem. 287 (2012) 6539–6550. [67] R.N. Karapetian, A.G. Evstafieva, I.S. Abaeva, N.V. Chichkova, G.S. Filonov, Y.P. Rubtsov, E.A. Sukhacheva, S.V. Melnikov, U. Schneider, E.E. Wanker, A.B. Vartapetian, Nuclear oncoprotein prothymosin α is a partner of Keap1: implications for expression of oxidative stress-protecting genes, Mol. Cell. Biol. 25 (2005) 1089–1099. [68] J. Ma, H. Cai, T. Wu, B. Sobhian, Y. Huo, A. Alcivar, M. Mehta, K.L. Cheung, S. Ganesan, A.N. Kong, D.D. Zhang, B. Xia, PALB2 interacts with Keap1 to promote Nrf2 nuclear accumulation and function, Mol. Cell. Biol. 32 (2012) 1506–1517. [69] W. Chen, Z. Sun, X.J. Wang, T. Jiang, Z. Huang, D. Fang, D.D. Zhang, Direct interaction between Nrf2 and p21 (Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response, Mol. Cell. 34 (2009) 663–673. [70] C. Gorrini, P.S. Baniasadi, I.S. Harris, J. Silvester, S. Inoue, B. Snow, P.A. Joshi, A. Wakeham, S.D. Molyneux, B. Martin, P. Bouwman, D.W. Cescon, A.J. Elia, Z. Winterton-Perks, J. Cruickshank, D. Brenner, A. Tseng, M. Musgrave, H.K. Berman, R. Khokha, J. Jonkers, T.W. Mak, M.L. Gauthier, BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival, J. Exp. Med. 210 (2013) 1529–1544. [71] Y. Ichimura, S. Waguri, Y.S. Sou, S. Kageyama, J. Hasegawa, R. Ishimura, T. Saito, Y. Yang, T. Kouno, T. Fukutomi, T. Hoshii, A. Hirao, K. Takagi, T. Mizushima, H. Motohashi, M.S. Lee, T. Yoshimori, K. Tanaka, M. Yamamoto, M. Komatsu, Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy, Mol. Cell. 51 (2013) 618–631. [72] K. Hashimoto, A.N. Simmons, R. Kajino-Sakamoto, Y. Tsuji, J. Ninomiya-Tsuji, TAK1 regulates the Nrf2 antioxidant system through modulating p62/SQSTM1, Antiox. Redox. Signal. 25 (2016) 953–964. [73] A. Jain, T. Lamark, E. Sjøttem, K.B. Larsen, J.A. Awuh, A. Øvervatn, M. McMahon, J.D. Hayes, T. Johansen, P62/SQSTM1 is a target gene for transcription factor Nrf2 and creates a positive feedback loop by inducing antioxidant response elementdriven gene transcription, J. Biol. Chem. 285 (2010) 22576–22591. [74] S. Bae, S.H. Sung, S.Y. Oh, J.M. Lim, S.K. Lee, Y.N. Park, H.E. Lee, D. Kang, S.G. Rhee, Sestrins activate Nrf2 by promoting p62-dependent autophagic degradation of Keap1 and prevent oxidative liver damage, Cell Metab. 17 (2012) 73–84. [75] S. Yin, W. Cao, Toll-like receptor signaling induces Nrf2 pathway activation through p62-triggered Keap1 degradation, Mol. Cell. Biol. 35 (2015) 2673–2683. [76] S. Goode, M. Rea, B. Sultana, M.S. Shaw, R. Searle, Layfield, ALS-FTLD associated mutations of SQSTM1 impact on Keap1-Nrf2 signalling, Mol. Cell. Neurosci. 76 (2016) 52–58. [77] A. Lau, X.J. Wang, F. Zhao, N.F. Villeneuve, T. Wu, T. Jiang, Z. Sun, E. White, D.D. Zhang, A noncanonical mechanism of Nrf2 activation by autophagy deficiency: direct interaction between Keap1 and p62, Mol. Cell. Biol. 30 (2010)

F.L. Chung, S.H. Kwon, I. Bae, Detoxification: a novel function of BRCA1 in tumor suppression? Toxicol. Sci. 122 (2011) 26–37. S. Yu, T.O. Khor, K.L. Cheung, W. Li, T.Y. Wu, Y. Huang, B.A. Foster, Y.W. Kan, A.N. Kong, Nrf2 expression in regulated by epigenetic mechanisms in prostate cancer of TRAMP mice, PLoS One 5 (2010) e8579. S. Kurinna, S. Werner, Nrf2 and microRNAs: new but awaited relations, Biochem. Soc. Trans. 43 (2015) 595–601. W. Li, N. Thakor, E.Y. Xu, Y. Huang, C. Chen, R. Yu, M. Holcik, A.N. Kong, An internal ribosomal entry site mediates redox-sensitive translation of Nrf2, Nucleic Acids Res. 38 (2010) 778–788. D.D. Zhang, S.C. Lo, J.V. Cross, D.J. Templeton, M. Hannink, Keap1 is a redoxregulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex, Mol. Biol. Cell. 24 (2004) 10941–10953. H.J. Kang, Y.B. Hong, H.J. Kim, I. Bae, CR6-interacting factor 1(CRIF1) regulates NF-E2-related factor 2 (Nrf2) protein and stability by proteasome-mediated degradation, J. Biol. Chem. 285 (2010) 21258–21268. T. Wu, F. Zhao, B. Gao, C. Tan, N. Yagishita, T. Nakajima, P.K. Wong, E. Chapman, D. Fang, D.D. Zhang, Hrd1 suppresses Nrf2-mediated cellular protection during liver cirrhosis, Genes Dev. 28 (2014) 708–722. J.Y. Lo, B.N. Spatola, S.P. Curran, WDR23 regulates Nrf2 independently of Keap1, PLoS Genet. 13 (2017) e1006762. M.I. Kang, A. Kobayashi, N. Wakabayashi, S.G. Kim, M. Yamamoto, Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes, Proc. Natl. Acad. U. S. A. 101 (2004) 2046–2051. A.T. Dinkova-Kostova, W.D. Holtzclaw, R.N. Cole, K. Itoh, N. Wakabayashi, Y. Katoh, M. Yamamoto, P. Talalay, Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants, Proc. Natl. Acad. U. S. A. 99 (2002) 11908–11913. D.D. Zhang, M. Hannink, Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress, Mol. Cell. Biol. 23 (2003) 8137–8151. M. McMahon, D.J. Lamont, K.A. Beattie, J.D. Hayes, Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc and alkenals, Proc. Natl. Acad. U. S. A. 107 (2010) 18838–18843. L. Baird, D. Llères, S. Swift, A.T. Dinkova-Kostova, Regulatory flexibility in the Nrf2-mediated stress response in conferred by conformational cycling of the Keap1-Nrf2 protein complex, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 15259–15264. S.C. Lo, X. Li, M.T. Henzl, L.J. Beamer, M. Hannink, Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling, EMBO J. 25 (2006) 3605–3617. B. Padamanabhan, K.I. Tong, T. Ohta, Y. Nakamura, M. Scharlock, M. Ohtsuji, M.I. Kang, A. Kobayashi, S. Yokoyama, M. Yamamoto, Structural basis for defects of Keap1 activity provoked by its point mutation in lung cancer, Mol. Cell 21 (2006) 689–700. B. Padmanabhan, K.I. Tong, A. Kobayashi, M. Yamamoto, S. Yokoyama, Structural insights into the similar modes of Nrf2 transcription factor recognition by the cytoplasmic repressor Keap1, J. Synchotron. Rad. 15 (2007) 273–276. K.I. Tong, B. Padmanabhan, A. Kobayashi, C. Shang, Y. Hirotsu, S. Yokoyama, M. Yamamoto, Different electrostatic potential define ETGE and DLG motif as hinge and latch in oxidative stress response, Mol. Cell. Biol. 27 (2007) 7511–7521. K.I. Tong, Y. Katoh, H. Kusunoki, K. Itoh, T. Tanaka, M. Yamamoto, Keap1 recruits Neh2 through to ETGE and DLG motif: characterization of the two-site molecular recognition model, Mol. Cell. Biol. 26 (2006) 2887–2900. F. Hong, M.L. Freeman, D.C. Liebler, Identification of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane, Chem. Res. Toxicol. 18 (2005) 1917–1926. S.C. Lo, M. Hannink, CAND1-mediated substrate adaptor recycling is required for efficient repression of Nrf2 by Keap1, Mol. Cell. Biol. 26 (2006) 1235–1244. S. Tao, P. Liu, Luo G, M. Rojo de la Vega, H. Chen, T. Wu, J. Tilloston, E. Chapman, D.D. Zhang, p97 negatively regulates Nrf2 by extracting ubiquitylated Nrf2 from Keap1-Cul3 E3 complex, Mol. Cell. Biol. 37 (2017) e00660–16. K. Itoh, N. Wakabayashi, Y. Katoh, T. Ishii, T. O’Connor, M. Yamamoto, Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles, Genes Cells. 8 (2003) 379–391. H.C. Huang, T. Nguyen, C.B. Pickett, Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription, J. Biol. Chem. 277 (2002) 42769–42774. M.S. Joo, W.D. Kim, K.Y. Lee, J.H. Kim, J.H. Koo, S.G. Kim, AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at serine 550, Mol. Cell. Biol. 36 (2016) 1931–1942. Y. Kawai, L. Garduño, M. Theodore, J. Yang, I.J. Arinze, Acetylation-deacetylation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) regulates its transcriptional activity and nucleocytoplasmic localization, J. Biol. Chem. 286 (2011) 7629–7640. M. Theodore, Y. Kawai, J. Yang, Y. Kleshchenko, S.P. Reddy, F. Villalta, I.J. Arinze, Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2, J. Biol. Chem. 283 (2008) 8984–8994. P.L. Chi, C.C. Lin, Y.W. Chen, L.D. Hsiao, C.M. Yang, CO induces Nrf2-dependent heme oxygenase-1 transcription by cooperating with Sp1 and c-Jun in rat brain astrocytes, Mol. Neurobiol. 52 (2015) 277–292. S. Tanigawa, C.H. Lee, C.S. Lin, C.C. Ku, H. Hasegawa, S. Qin, A. Kawahara, Y. Korenori, K. Miyamori, M. Noguchi, L.H. Lee, Y.C. Lin, C.L. Steve Lin, Y. Nakamura, C. Jin, N. Yamaguchi, R. Eckner, D.X. Hou, K.K. Yokoyama, Jun dimerization protein 2 is a critical component of the Nrf2/MafK complex regulating the response to ROS homeostasis, Cell Death Dis. 4 (2013) e921.

98

Pharmacological Research 134 (2018) 92–99

C.A. Silva-Islas, P.D. Maldonado

3275–3285. [78] I. Riz, T.S. Hawley, J.W. Marsal, R.G. Hawley, Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic and translational reprogramming, Oncotarget. 7 (2016) 66360–66385. [79] I.G. Ryoo, B.H. Choi, M.K. Kwak, Activation of Nrf2 by p62 and proteasome reduction in sphere-forming breast carcinoma cells, Oncotarget. 6 (2015) 8167–8184. [80] X. Sun, Z. Ou, R. Chen, X. Niu, D. Chen, R. Kang, D. Tang, Activation of the p62Keap1-Nrf2 pathway protects against ferroptosis in hepatocellular carcinoma cells, Hepatology. 63 (2016) 173–184. [81] M. Xia, H. Yu, S. Gu, Y. Xu, J. Su, H. Li, J. Kang, M. Cui, p62/SQSTM1 is involved in cisplatin resistance in human ovarian cancer cells via Keap1-Nrf2-ARE system, Int. J. Oncology. 45 (2014) 2341–2348. [82] S.C. Prajapati, S.C. Chauhan, Dipeptidyl peptidase III: a multifaceted oligopeptide N-end cutter, FEBS J. 278 (2011) 3256–3276. [83] K. Lu, A.L. Alcivar, J. Ma, T.K. Foo, S. Zywea, A. Mahdi, Y. Hou, T.W. Kensler, M.L. Gatza, B. Xia, Nrf2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-Keap1 interaction, Cancer Res. 77 (2017) 2881–2892. [84] B. Padmanabhan, Y. Nakamura, S. Yokoyama, Structural analysis of the complex of Keap1 with a prothymosin α peptide, Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 64 (2008) 233–238. [85] Q. Wang, J. Li, X. Yang, H. Sun, S. Gao, H. Zhu, J. Wu, W. Jin, Nrf2 is associated with the regulation of basal transcription activity of the BRCA1 gene, Acta Biochim. Biophys. Sin. (Shanghai). 45 (2013) 179–187. [86] Y. Shan, Z. Wei, L. Tao, S. Wang, F. Zhang, C. Shen, H. Wu, Z. Liu, P. Zhu, A. Wang, W. Chen, Y. Lu, Prophylaxis of diallyl disulfide on skin carcinogenic model via p21-dependent Nrf2 stabilization, Sci. Rep. 6 (2016) 35676. [87] S. Achuthan, T.R. Santhoshkumar, J. Prabhakar, S.A. Nair, M.R. Pillai, Drug-induced senescence generates chemoresistant stemlike cells with low reactive oxygen species, J. Biol. Chem. 286 (2011) 37813–37829. [88] N. Oshimori, D. Oristian, E. Fuchs, TGF-β promotes heterogeneity and drug resistance in squamous cell carcinoma, Cell. 160 (2015) 963–976. [89] A. Lau, N.F. Villeneuve, Z. Sun, P.K. Wong, D.D. Zhang, Dual roles of Nrf2 in cancer, Pharmacol. Res. 58 (2008) 262–270. [90] D.F. Lee, H.P. Kuo, M. Liu, C.K. Chou, W. Xia, Y. Du, J. Shen, C.T. Chen, L. Huo, M.C. Hsu, C.W. Li, Q. Ding, T.L. Liao, C.C. Lai, A.C. Lin, Y.H. Chang, S.F. Tsai, L.Y. Li, M.C. Hung, Keap1 E3 ligase-mediated downregulation of NF-κB signaling by targeting IKKβ, Mol. Cell. 36 (2009) 131–140. [91] S.C. Lo, M. Hannink, PGAM5 tethers a ternary complex containing Keap1 and Nrf2 to mitochondria, Exp. Cell Res. 314 (2008) 1789–1803. [92] S.C. Lo, M. Hannink, PGAM5, a Bcl-XL-interacting protein, is a novel substrate for the redox-regulated Keap1-dependent ubiquitin ligase complex, J. Biol. Chem. 281 (2006) 37893–37903. [93] J.E. Kim, D.J. You, C. Lee, C. Ahn, J.Y. Seong, J.I. Hwang, Suppression of NF-κB

[94]

[95]

[96]

[97] [98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

99

signaling by Keap1 regulation of IKKβ activity through autophagic degradation and inhibition of phosphorylation, Cell. Signal. 22 (2010) 1645–1654. G.B. O’Mealey, K.S. Plafker, W.L. Berry, R. Janknecht, J.Y. Chan, S.M. Plafker, A PGAM5-Keap1-Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking, J. Cell Sci. 130 (2017) 3467–3480. M. Zhang, C. An, Y. Gao, R.K. Leak, J. Chen, F. Zhang, Emerging role of Nrf2 and phase II antioxidant enzymes in neuroprotection, Prog. Neurobiol. 100 (2013) 30–47. T. Satoh, S. Lipton, Recent advances in understanding Nrf2 as a druggable target: development of pro-electrophilic and non-covalent Nrf2 activators to overcomes systemic side effects of electrophilic drugs like dimethyl fumarate, F1000Res. 6 (2017) 2138. D.D. Zhang, Bardoxolone brings Nrf2-based therapies to light, Antiox. Redox. Signal. 19 (2013) 517–518. R. Hancock, M. Schaap, H. Pfister, G. Wells, Peptide inhibitors of the Keap1-Nrf2 protein-protein interaction with improved binding and cellular activity, Org. Biomol. Chem. 11 (2013) 3553–3557. R. Hancock, H.C. Bertrand, T. Tsujita, S. Naz, A. El-Bakry, J. Laoruchupong, J.D. Hayes, G. Wells, Peptide inhibitors of the Keap1-Nrf2 protein-protein interaction, Free Radic. Biol. Med. 52 (2012) 444–451. R. Steel, J. Cowan, E. Payerne, M.A. O’Connell, M. Searcey, Anti-inflammatory effect of a cell-penetrating peptide targeting the Nrf2/Keap1 interaction, ACS Med. Chem. Lett. 3 (2012) 407–410. J. Tu, X. Zhang, Y. Zhu, Y. Dai, N. Li, F. Yang, Q. Zhang, D.W. Brann, R. Wang, Cell-permeable peptide targeting Nrf2-Keap1 interaction: a potential novel therapy for global cerebral ischemia, J. Neurosci. 35 (2015) 14727–14739. L. Wang, T. Lewis, Y.L. Zhang, C. Khodier, S. Magesh, L. Chen, D. Inayoma, Y. Chen, J. Zhen, L. Hu, L.J. Beamer, P.W. Faloon, S. Danapani, J.R. Perez, B. Munoz, M. Palmer, S. Schreiber, The identification and characterization of nonreactive inhibitor of Keap1-Nrf2 interaction through HTS using fluorescence polarization assay, Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US), 2012. D. Marcotte, W. Zeng, J.C. Hus, A. McKenzie, C. Hession, P. Jin, C. Bergeron, A. Logovskoy, I. Enyedy, H. Cuervo, D. Wang, C. Atmanene, D. Roecklin, M. Vecchi, V. Vivat, J. Kraemer, D. Winkler, V. Hong, J. Chao, M. Lukashev, L. Silvian, Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism, Bioorg. Med. Chem. 21 (2013) 4011–4019. H.P. Sun, Z.Y. Jiang, M.Y. Zhang, M.C. Lu, T.T. Yang, Y. Pan, H.Z. Huang, X.J. Zhang, Q.D. You, Novel protein-protein interaction inhibitor of Nrf2-Keap1 discovered by structure-based virtual screening, Med. Chem. Comm. 5 (2014) 93–98. M.C. Lu, J.A. Ji, Y.L. Jiang, Z.Y. Chen, Z.W. Yuan, Q.D. You, Z.Y. Jiang, An inhibitor of the Keap1-Nrf2 protein-protein interaction protects NCM460 colonic cells and alleviates experimental colitis, Sci. Rep. 6 (2015) 26585.