Correction: Histone Demethylase JMJD2B Functions as a Co-Factor of Estrogen Receptor in Breast Cancer Proliferation and Mammary Gland Development.

[This corrects the article DOI: 10.1371/journal.pone.0017830.].

Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains.

Keap1 is a substrate adaptor of a Cullin 3-based E3 ubiquitin ligase complex that recognizes Nrf2, and also acts as a cellular sensor for xenobiotics and oxidative stresses. Nrf2 is a transcriptional factor regulating the expression of cytoprotective enzyme genes in response to such stresses. Under unstressed conditions Keap1 binds Nrf2 and results in rapid degradation of Nrf2 through the proteasome pathway. In contrast, upon exposure to oxidative and electrophilic stress, reactive cysteine residues in intervening region (IVR) and Broad complex, Tramtrack, and Bric-à-Brac domains of Keap1 are modified by electrophiles. This modification prevents Nrf2 from rapid degradation and induces Nrf2 activity by repression of Keap1. Here we report the structure of mouse Keap1 homodimer by single particle electron microscopy. Three-dimensional reconstruction at 24-A resolution revealed two large spheres attached by short linker arms to the sides of a small forked-stem structure, resembling a cherry-bob. Each sphere has a tunnel corresponding to the central hole of the beta-propeller domain, as determined by x-ray crystallography. The IVR domain appears to surround the core of the beta-propeller domain. The unexpected proximity of IVR to the beta-propeller domain suggests that any distortions generated during modification of reactive cysteine residues in the IVR domain may send a derepression signal to the beta-propeller domain and thereby stabilize Nrf2. This study thus provides a structural basis for the two-site binding and hinge-latch model of stress sensing by the Nrf2-Keap1 system.

Association of C-reactive protein with future development of diabetes: a population-based 7-year cohort study among Norwegian adults aged 30 and older in the Tromsø Study 2007-2016.

OBJECTIVES: The extent to which observed associations between high-sensitivity C-reactive protein (hs-CRP) and incident diabetes are explained by obesity and hypertension remains unclear. This study aimed to investigate the association of hs-CRP with developing diabetes in a Norwegian general population sample. DESIGN: A cohort study using two population-based surveys of the Tromsø Study: the sixth survey Tromsø6 (2007-2008) as baseline and the seventh survey Tromsø7 (2015-2016) at follow-up. SETTING: Tromsø municipality of Norway, a country with increasing proportion of older adults and a high prevalence of overweight, obesity and hypertension. PARTICIPANTS: 8067 women and men without diabetes, aged 30-87 years, at baseline Tromsø6 who subsequently also participated in Tromsø7. OUTCOME MEASURES: Diabetes defined by self-reported diabetes, diabetes medication use and/or HbA1c≥6.5% (≥48 mmol/mol) was modelled by logistic regression for the association with baseline hs-CRP, either stratified into three quantiles or as continuous variable, adjusted for demographic factors, behavioural and cardiovascular risk factors, lipid-lowering medication use, and hypertension. Interactions by sex, body mass index (BMI), hypertension or abdominal obesity were assessed by adding interaction terms in the fully adjusted model. RESULTS: There were 320 (4.0%) diabetes cases after 7 years. After multivariable adjustment including obesity and hypertension, individuals in the highest hs-CRP tertile 3 had 73% higher odds of developing diabetes (OR 1.73; p=0.004; 95% CI 1.20 to 2.49) when compared with the lowest tertile or 28% higher odds of incidence per one-log of hs-CRP increment (OR 1.28; p=0.003; 95% CI 1.09 to 1.50). There was no evidence for interaction between hs-CRP and sex, hypertension, BMI or abdominal obesity. CONCLUSIONS: Raised hs-CRP was associated with future diabetes development in a Norwegian adult population sample. The CRP-diabetes association could not be fully explained by obesity or hypertension.

Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151-independent mechanism.

Nitro-fatty acids (NO(2)-FAs) are electrophilic signaling mediators formed in vivo via nitric oxide (NO)- and nitrite (NO(2)(-))-dependent reactions. Nitro-fatty acids modulate signaling cascades via reversible covalent post-translational modification of nucleophilic amino acids in regulatory proteins and enzymes, thus altering downstream signaling events, such as Keap1-Nrf2-antioxidant response element (ARE)-regulated gene expression. In this study, we investigate the molecular mechanisms by which 9- and 10-nitro-octadec-9-enoic acid (OA-NO(2)) activate the transcription factor Nrf2, focusing on the post-translational modifications of cysteines in the Nrf2 inhibitor Keap1 by nitroalkylation and its downstream responses. Of the two regioisomers, 9-nitro-octadec-9-enoic acid was a more potent ARE inducer than 10-nitro-octadec-9-enoic acid. The most OA-NO(2)-reactive Cys residues in Keap1 were Cys(38), Cys(226), Cys(257), Cys(273), Cys(288), and Cys(489). Of these, Cys(273) and Cys(288) accounted for ∼50% of OA-NO(2) reactions in a cellular milieu. Notably, Cys(151) was among the least OA-NO(2)-reactive of the Keap1 Cys residues, with mutation of Cys(151) having no effect on net OA-NO(2) reaction with Keap1 or on ARE activation. Unlike many other Nrf2-activating electrophiles, OA-NO(2) enhanced rather than diminished the binding between Keap1 and the Cul3 subunit of the E3 ligase for Nrf2. OA-NO(2) can therefore be categorized as a Cys(151)-independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes.

The critical role of nitric oxide signaling, via protein S-guanylation and nitrated cyclic GMP, in the antioxidant adaptive response.

A nitrated guanine nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 microm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys(434) of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.

Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy.

The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase.

Combined EZH2 Inhibition and IKAROS Degradation Leads to Enhanced Antitumor Activity in Diffuse Large B-cell Lymphoma.

PURPOSE: The efficacy of EZH2 inhibition has been modest in the initial clinical exploration of diffuse large B-cell lymphoma (DLBCL), yet EZH2 inhibitors are well tolerated. Herein, we aimed to uncover genetic and pharmacologic opportunities to enhance the clinical efficacy of EZH2 inhibitors in DLBCL. EXPERIMENTAL DESIGN: We conducted a genome-wide sensitizing CRISPR/Cas9 screen with tazemetostat, a catalytic inhibitor of EZH2. The sensitizing effect of IKZF1 loss of function was then validated and leveraged for combination treatment with lenalidomide. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing analyses were performed to elucidate transcriptomic and epigenetic changes underlying synergy. RESULTS: We identified IKZF1 knockout as the top candidate for sensitizing DLBCL cells to tazemetostat. Treating cells with tazemetostat and lenalidomide, an immunomodulatory drug that selectively degrades IKAROS and AIOLOS, phenocopied the effects of the CRISPR/Cas9 screen. The combined drug treatment triggered either cell-cycle arrest or apoptosis in a broad range of DLBCL cell lines, regardless of EZH2 mutational status. Cell-line-based xenografts also showed slower tumor growth and prolonged survival in the combination treatment group. RNA-seq analysis revealed strong upregulation of interferon signaling and antiviral immune response signatures. Gene expression of key immune response factors such as IRF7 and DDX58 were induced in cells treated with lenalidomide and tazemetostat, with a concomitant increase of H3K27 acetylation at their promoters. Furthermore, transcriptome analysis demonstrated derepression of endogenous retroviruses after combination treatment. CONCLUSIONS: Our data underscore the synergistic interplay between IKAROS degradation and EZH2 inhibition on modulating epigenetic changes and ultimately enhancing antitumor effects in DLBCL.

Different electrostatic potentials define ETGE and DLG motifs as hinge and latch in oxidative stress response.

Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.

Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model.

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central alpha-helix followed by a mini antiparallel beta-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the beta-propeller structure of Keap1. In contrast, the central alpha-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two beta-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central alpha-helix in a correct position to benefit ubiquitin signaling.

Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1.

The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

Crystallographic evidence for water-assisted photo-induced peptide cleavage in the stony coral fluorescent protein Kaede.

A coral fluorescent protein from Trachyphyllia geoffroyi, Kaede, possesses a tripeptide of His62-Tyr63-Gly64, which forms a chromophore with green fluorescence. This chromophore's fluorescence turns red following UV light irradiation. We have previously shown that such photoconversion is achieved by a formal beta-elimination reaction, which results in a cleavage of the peptide bond found between the amide nitrogen and the alpha-carbon at His62. However, the stereochemical arrangement of the chromophore and the precise structural basis for this reaction mechanism previously remained unknown. Here, we report the crystal structures of the green and red form of Kaede at 1.4 A and 1.6 A resolutions, respectively. Our structures depict the cleaved peptide bond in the red form. The chromophore conformations both in the green and red forms are similar, except a well-defined water molecule in the proximity of the His62 imidazole ring in the green form. We propose a molecular mechanism for green-to-red photoconversion, which is assisted by the water molecule.

The H3K27 demethylase, Utx, regulates adipogenesis in a differentiation stage-dependent manner.

Understanding the molecular mechanisms that drive adipogenesis is important in developing new treatments for obesity and diabetes. Epigenetic regulations determine the capacity of adipogenesis. In this study, we examined the role of a histone H3 lysine 27 demethylase, the ubiquitously transcribed tetratricopeptide repeat protein on the X chromosome (Utx), in the differentiation of mouse embryonic stem cells (mESCs) to adipocytes. Using gene trapping, we examined Utx-deficient male mESCs to determine whether loss of Utx would enhance or inhibit the differentiation of mESCs to adipocytes. Utx-deficient mESCs showed diminished potential to differentiate to adipocytes compared to that of controls. In contrast, Utx-deficient preadipocytes showed enhanced differentiation to adipocytes. Microarray analyses indicated that the ß-catenin/c-Myc signaling pathway was differentially regulated in Utx-deficient cells during adipocyte differentiation. Therefore, our data suggest that Utx governs adipogenesis by regulating c-Myc in a differentiation stage-specific manner and that targeting the Utx signaling pathway could be beneficial for the treatment of obesity, diabetes, and congenital utx-deficiency disorders.

Attenuated DNA damage repair delays therapy-related myeloid neoplasms in a mouse model.

Therapy-related cancers are potentially fatal late life complications for patients who received radio- or chemotherapy. So far, the mouse model showing reduction or delay of these diseases has not been described. We found that the disruption of Aplf in mice moderately attenuated DNA damage repair and, unexpectedly, impeded myeloid neoplasms after exposure to ionizing radiation (IR). Irradiated mutant mice showed higher rates of p53-dependent cell death, fewer chromosomal translocations, and a delay in malignancy-induced mortality. Simultaneous deficiency of p53 abrogated IR-induced apoptosis and the benefit of impaired DNA repair on mortality in irradiated Aplf­/­ mice. Depletion of APLF in non-tumorigenic human cells also markedly reduced the risk of radiation-induced chromosomal aberrations. We therefore conclude that proficient DNA damage repair may promote chromosomal aberrations in normal tissues after irradiation and induce malignant evolution, thus illustrating the potential benefit in sensitizing p53 function by manipulating DNA repair efficiency in cancer patients undergoing genotoxic therapies.

Purification, crystallization and preliminary X-ray diffraction analysis of the Kelch-like motif region of mouse Keap1.

Keap1 (Kelch-like ECH-associating protein 1) is a negative regulator of the Nrf2 transcription factor in the cytoplasm. The Kelch/DGR (double-glycine repeat) domain of Keap1 associates with Nrf2 as well as with actin filaments. A recombinant protein containing both the Kelch/DGR domain and the C-terminal region of mouse Keap1 was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The crystal belongs to space group P6(1) or P6(5), with unit-cell parameters a = b = 102.95, c = 55.21 A, and contains one molecule in the asymmetric unit. A complete diffraction data was collected to 2.25 A resolution using an R-AXIS IV++ imaging plate mounted on an RA-Micro7 Cu Kalpha rotating-anode X-ray generator.

Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles.

Electrophile responsive element (EpRE)-mediated gene induction is a pivotal mechanism of cellular defense against the toxicity of electrophiles and reactive oxygen species (ROS). Nrf2, which belongs to the cap'-n'-collar family of basic region-leucine zipper transcription factors, has emerged as an essential component of an EpRE-binding transcriptional complex. Detailed analysis of the regulatory mechanism governing Nrf2 activity led to the identification of Keap1, which represses Nrf2 activity by directly binding to the N-terminal Neh2 domain. Keap1 interaction with Neh2 leads to the sequestration of Nrf2 in the cytoplasm and to the enhancement of Nrf2 degradation by proteasomes conferring tight regulation on the response. Electrophiles act to counteract sequestration of Nrf2 by Keap1 and provoke Nrf2 activation. Constitutive activation of Nrf2-regulated transcription in Keap1 knockout mice clearly demonstrated that the disruption of Keap1 repression is sufficient for the activation of Nrf2. These observations indicated that the mechanism that modulates Nrf2-Keap1 interaction is pivotal for the cellular sensing mechanism for electrophiles. Recent analyses argue that the redox mechanism that modifies cysteine residues of Keap1 governs the Keap1-Nrf2 interaction and therefore is critical for sensing of electrophiles.

Selective isotope labeling of recombinant proteins in Escherichia coli.

Selective stable-isotope labeling is a useful technique to study structures of proteins, especially intrinsically disordered proteins, by nuclear magnetic resonance spectroscopy. Here, we describe a simple method for amino acid selective isotope labeling of recombinant proteins in E. coli. This method only requires addition of an excess of unlabeled amino acids and, if necessary, enzyme inhibitors to the culture medium. Its efficiency has been demonstrated even in labeling with glutamine or glutamate that is easily converted to other amino acid types by the metabolic pathways of E. coli.

Histone demethylase JMJD2B functions as a co-factor of estrogen receptor in breast cancer proliferation and mammary gland development.

Estrogen is a key regulator of normal function of female reproductive system and plays a pivotal role in the development and progression of breast cancer. Here, we demonstrate that JMJD2B (also known as KDM4B) constitutes a key component of the estrogen signaling pathway. JMJD2B is expressed in a high proportion of human breast tumors, and that expression levels significantly correlate with estrogen receptor (ER) positivity. In addition, 17-beta-estradiol (E2) induces JMJD2B expression in an ERα dependent manner. JMJD2B interacts with ERα and components of the SWI/SNF-B chromatin remodeling complex. JMJD2B is recruited to ERα target sites, demethylates H3K9me3 and facilitates transcription of ER responsive genes including MYB, MYC and CCND1. As a consequence, knockdown of JMJD2B severely impairs estrogen-induced cell proliferation and the tumor formation capacity of breast cancer cells. Furthermore, Jmjd2b-deletion in mammary epithelial cells exhibits delayed mammary gland development in female mice. Taken together, these findings suggest an essential role for JMJD2B in the estrogen signaling, and identify JMJD2B as a potential therapeutic target in breast cancer.

The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.

Impaired selective turnover of p62 by autophagy causes severe liver injury accompanied by the formation of p62-positive inclusions and upregulation of detoxifying enzymes. These phenotypes correspond closely to the pathological conditions seen in human liver diseases, including alcoholic hepatitis and hepatocellular carcinoma. However, the molecular mechanisms and pathophysiological processes in these events are still unknown. Here we report the identification of a novel regulatory mechanism by p62 of the transcription factor Nrf2, whose target genes include antioxidant proteins and detoxification enzymes. p62 interacts with the Nrf2-binding site on Keap1, a component of Cullin-3-type ubiquitin ligase for Nrf2. Thus, an overproduction of p62 or a deficiency in autophagy competes with the interaction between Nrf2 and Keap1, resulting in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Our findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.