Capillary Electrophoresis Mass Spectrometry-Based Metabolomics of Plasma Samples from Healthy Subjects in a Cross-Sectional Japanese Population Study.

For large-scale metabolomics, such as in cohort studies, normalization protocols using quality control (QC) samples have been established when using data from gas chromatography and liquid chromatography coupled to mass spectrometry. However, normalization protocols have not been established for capillary electrophoresis-mass spectrometry metabolomics. In this study, we performed metabolome analysis of 314 human plasma samples using capillary electrophoresis-mass spectrometry. QC samples were analyzed every 10 samples. The results of principal component analysis for the metabolome data from only the QC samples showed variations caused by capillary replacement in the first principal component score and linear variation with continuous measurement in the second principal component score. Correlation analysis between diagnostic blood tests and plasma metabolites normalized by the QC samples was performed for samples from 188 healthy subjects who participated in a Japanese population study. Five highly correlated pairs were identified, including two previously unidentified pairs in normal healthy subjects of blood urea nitrogen and guanidinosuccinic acid, and gamma-glutamyl transferase and cysteine glutathione disulfide. These results confirmed the validity of normalization protocols in capillary electrophoresis-mass spectrometry using large-scale metabolomics and comprehensive analysis.

Tryptophan Metabolism Regulates Proliferative Capacity of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) have a unique metabolic signature for maintenance of pluripotency, self-renewal, and survival. Although hPSCs could be potentially used in regenerative medicine, the prohibitive cost associated with large-scale cell culture presents a major barrier to the clinical application of hPSC. Moreover, without a fully characterized metabolic signature, hPSC culture conditions are not optimized. Here, we performed detailed amino acid profiling and found that tryptophan (TRP) plays a key role in the proliferation with maintenance of pluripotency. In addition, metabolome analyses revealed that intra- and extracellular kynurenine (KYN) is decreased under TRP-supplemented conditions, whereas N-formylkynurenine (NFK), the upstream metabolite of KYN, is increased thereby contributing to proliferation promotion. Taken together, we demonstrate that TRP is indispensable for survival and proliferation of hPSCs. A deeper understanding of TRP metabolism will enable cost-effective large-scale production of hPSCs, leading to advances in regenerative medicine.

Effects of Meal Timing on Postprandial Glucose Metabolism and Blood Metabolites in Healthy Adults.

We examined the effects of meal timing on postprandial glucose metabolism, including the incretin response and metabolites in healthy adults. Nineteen healthy young men completed two trials involving blood collection in a fasting state and at 30, 60 and 120 min after meal provision in a random order: (1) morning (~0900 h) and (2) evening (~1700 h). The blood metabolome of eight participants was analyzed using capillary electrophoresis-mass spectrometry. Postprandial glucose concentrations at 120 min (p = 0.030) and glucose-dependent insulinotropic polypeptide concentrations (p = 0.005) at 60 min in the evening trials were higher than those in the morning trials. The incremental area under the curve values of five glycolysis, tricarboxylic acid cycle and nucleotide-related metabolites and 18 amino acid-related metabolites were higher in the morning trials than those in the evening trials (p < 0.05). Partial least-squares analysis revealed that the total metabolic change was higher in the morning. Our study demonstrates that a meal in the evening exacerbates the state of postprandial hyperglycemia in healthy adults. In addition, this study provides insight into the difference of incretion and blood metabolites between breakfast and dinner, indicating that the total metabolic responses tends to be higher in the morning.

Targeting of Noncanonical Wnt5a Signaling by AP-1 Blocker Dominant-Negative Jun When It Inhibits Skin Carcinogenesis.

The transcription factor AP-1 (activator protein-1) regulates a number of genes that drive tumor promotion and progression. While basal levels of AP-1 activity are important for normal cell proliferation and cell survival, overactivated AP-1-dependent gene expression stimulates inflammation, angiogenesis, invasion, and other events that propel carcinogenesis. We seek to discover genes targeted by carcinogenesis inhibitors that do not also inhibit cell proliferation or survival. Transgenic TAM67 (dominant-negative c-Jun) inhibits mouse skin tumorigenesis and tumor progression without inhibiting cell proliferation or induced hyperproliferation. Expression profiling of wild-type and K14-TAM67 mouse epidermis has revealed a number of functionally significant genes that are induced by tumor promoters in wild-type mice but not in those expressing the AP-1 blocker. The current study now identifies Wnt5a signaling as a new target of TAM67 when it inhibits DMBA/TPA-induced carcinogenesis. Wnt5a is required to maintain the tumor phenotype in tumorigenic mouse JB6 cells and Ras-transformed human squamous carcinoma HaCaT-II4 cells, as Wnt5a knockdown suppresses anchorage-independent and tumor xenograft growth. The oncogenic Wnt5a-mediated pathway signals through activation of the protein kinase PKCα and oncogenic transcription factor STAT3 phosphorylation and not through the canonical Wnt/ß-catenin pathway. Similar to Wnt5a knockdown, inhibitors of PKCα blocked STAT3 activation in both mouse JB6 and human HaCaT-II4 tumor cells. Moreover, expression of STAT3-regulated genes FAS, MMP3, IRF1, and cyclin D1 was suppressed with Wnt5a knockdown. Treatment of mouse Wnt5a knockdown cells with a PKCα-specific activator rescued phosphorylation of STAT3. Thus, Wnt5a signaling is required for maintaining the tumor phenotype in squamous carcinoma cells, Wnt5a targeting by the AP-1 blockade contributes to inhibition of skin carcinogenesis, and the signaling pathway traverses PKCα and STAT3 activation. Coordinate overactivation of Wnt5a expression and STAT3 signaling is observed in human skin and colon cancers as well as glioblastoma.

Microwave-based reaction screening: tandem retro-Diels-Alder/Diels-Alder cycloadditions of o-quinol dimers.

We have accomplished a parallel screen of cycloaddition partners for o-quinols utilizing a plate-based microwave system. Microwave irradiation improves the efficiency of retro-Diels-Alder/Diels-Alder cascades of o-quinol dimers which generally proceed in a diastereoselective fashion. Computational studies indicate that asynchronous transition states are favored in Diels-Alder cycloadditions of o-quinols. Subsequent biological evaluation of a collection of cycloadducts has identified an inhibitor of activator protein-1 (AP-1), an oncogenic transcription factor.

A selective small-molecule nuclear factor-kappaB inhibitor from a high-throughput cell-based assay for "activator protein-1 hits".

NSC 676914 has been identified as a selective nuclear factor-kappaB (NF-kappaB) inhibitor that does not inhibit cell proliferation. This compound was originally identified in a high-throughput cell-based assay for activator protein-1 (AP-1) inhibitors using synthetic compound libraries and the National Cancer Institute natural product repository. NSC 676914 shows activity against NF-kappaB in luciferase reporter assays at concentrations much less than the IC50 for AP-1. A serum response element reporter used as a specificity control and indicator of cell proliferation was relatively insensitive to the compound. Pretreatment with NSC 676914 is here shown to repress 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced IkappaB-alpha phosphorylation and translocation of p65/50 to the nucleus but not the processing of p52 from p100, suggesting the inhibition of NF-kappaB regulator IKKbeta rather than IKKalpha. Inhibition of NF-kappaB activation occurred as a consequence of blocking phosphorylation of IKK. Induction of IkappaB-alpha phosphorylation by TPA was diminished by pretreatment of NSC 676914 even at 1.1 mumol/L. In contrast, kinases c-Jun-NH2-kinase and extracellular signal-regulated kinases 1 and 2, important for AP-1 activation, showed no significant repression by this compound. Furthermore, a Matrigel invasion assay with breast cancer cell lines and a transformation assay in mouse JB6 cells revealed that TPA-induced invasion and transformation responses were completely repressed by this compound. These results suggest that NSC 676914 could be a novel inhibitor having potential therapeutic activity to target NF-kappaB for cancer treatment or prevention.

Quassinoid inhibition of AP-1 function does not correlate with cytotoxicity or protein synthesis inhibition.

Several quassinoids were identified in a high-throughput screening assay as inhibitors of the transcription factor AP-1. Further biological characterization revealed that while their effect was not specific to AP-1, protein synthesis inhibition and cell growth assays were inconsistent with a mechanism of simple protein synthesis inhibition. Numerous plant extracts from the plant family Simaroubaceae were also identified in the same screen; bioassay-guided fractionation of one extract (Ailanthus triphylla) yielded two known quassinoids, ailanthinone (3) and glaucarubinone (4), which were also identified in the pure compound screening procedure.

Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer.

Nrf2 regulates the cellular oxidative stress response, whereas Keap1 represses Nrf2 through its molecular interaction. To elucidate the molecular mechanism of the Keap1 and Nrf2 interaction, we resolved the six-bladed beta propeller crystal structure of the Kelch/DGR and CTR domains of mouse Keap1 and revealed that extensive inter- and intrablade hydrogen bonds maintain the structural integrity and proper association of Keap1 with Nrf2. A peptide containing the ETGE motif of Nrf2 binds the beta propeller of Keap1 at the entrance of the central cavity on the bottom side via electrostatic interactions with conserved arginine residues. We found a somatic mutation and a gene variation in human lung cancer cells that change glycine to cysteine in the DGR domain, introducing local conformational changes that reduce Keap1's affinity for Nrf2. These results provide a structural basis for the loss of Keap1 function and gain of Nrf2 function.

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.

Differential responses of the Nrf2-Keap1 system to laminar and oscillatory shear stresses in endothelial cells.

The Nrf2-Keap1 system coordinately regulates cytoprotective gene expression via the antioxidant responsive element (ARE). The expression of several ARE-regulated genes was found to be up-regulated in endothelial cells by laminar shear stress, suggesting that Nrf2 contributes to the anti-atherosclerosis response via the ARE. To gain further insight into the roles that Nrf2 plays in the development of atherosclerosis, we examined how Nrf2 regulates gene expression in response to anti-atherogenic laminar flow (L-flow) or pro-atherogenic oscillatory flow (O-flow). Exposure of human aortic endothelial cells (HAECs) to L-flow, but not to O-flow, induced the expression of cytoprotective genes, such as NAD(P)H quinone oxidoreductase 1 (NQO1) by 5-fold and heme oxygenase-1 by 8-fold. The critical contribution of Nrf2 to the expression induced by L-flow was ascertained in siRNA-mediated knock-down experiments. Two cyclooxygenase-2 (COX-2) specific inhibitors attenuated Nrf2 nuclear accumulation in the acute phase of L-flow exposure. A downstream product of COX-2, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), activated the Nrf2 regulatory pathway in HAECs through binding to the cysteines of Keap1. These results demonstrate that 15d-PGJ2 is essential for L-flow to activate Nrf2 and induce anti-atherosclerotic gene expression. Whereas both L-flow and O-flow induced the nuclear accumulation of Nrf2 to comparable levels, chromatin immunoprecipitation analysis revealed that Nrf2 binding to the NQO1 ARE was significantly diminished in the case of O-flow compared with that of L-flow. These results suggest that O-flow inhibits Nrf2 activity at the DNA binding step, thereby suppressing athero-protective gene expression and hence predisposing the blood vessels to the formation of atherosclerosis.

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.

Evolutionary conserved N-terminal domain of Nrf2 is essential for the Keap1-mediated degradation of the protein by proteasome.

Under homeostatic conditions, Nrf2 activity is constitutively repressed. This process is dependent on Keap1, to which Nrf2 binds through the Neh2 domain. Since the N-terminal subdomain of Neh2 (Neh2-NT) contains evolutionarily conserved motifs, we examined the roles they play in the degradation of Nrf2. In Neh2-NT, we defined a novel motif that is distinct from the previously characterized DIDLID motif and designated it DLG motif. Deletion of Neh2-NT or mutation of the DLG motif largely abolished the Keap1-mediated degradation of Nrf2. These mutations were found to enfeeble the binding affinity of Nrf2 to Keap1. The Neh2-NT subdomain directed DLG-dependent, Keap1-independent, degradation of a reporter protein in the nucleus. By contrast, mutation of DLG did not affect the half-life of native Nrf2 protein in the nucleus under oxidative stress conditions. These results thus demonstrate that DLG motif plays essential roles in the Keap1-mediated proteasomal degradation of Nrf2 in the cytoplasm.

Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2.

Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.

Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.

Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The critical role of C273 and C288 was established by (i) their high reactivity when purified recombinant Keap1 was treated with dexamethasone mesylate and the dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and (ii) transfection of keap1 and nrf2 gene-deficient mouse embryonic fibroblasts with constructs expressing cysteine to alanine mutants of Keap1, and measurement of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter. Reaction of Keap1 with inducers results in formation of intermolecular disulfide bridges, probably between C273 of one Keap1 molecule and C288 of a second. Evidence for formation of such dimers was obtained by 2D PAGE of extracts of cells treated with inducers, and by the demonstration that whereas C273A and C288A mutants of Keap1 alone could not repress Nrf2 activation of the ARE-luciferase reporter, an equal mixture of these mutant constructs restored repressor activity.

Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes.

Transcription factor Nrf2 regulates basal and inducible expression of phase 2 proteins that protect animal cells against the toxic effects of electrophiles and oxidants. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a multidomain, cysteinerich protein that is bound to the actin cytoskeleton. Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keap1-Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element. Reporter cotransfection-transactivation analyses with a series of Keap1 deletion mutants revealed that in the absence of the double glycine repeat domain Keap1 does not bind to Nrf2. In addition, deletion of either the intervening region or the C-terminal region also abolished the ability of Keap1 to sequester Nrf2, indicating that all of these domains contribute to the repressor activity of Keap1. Immunocytochemical and immunoprecipitation analyses demonstrated that Keap1 associates with actin filaments in the cytoplasm through its double glycine repeat domain. Importantly, disruption of the actin cytoskeleton promotes nuclear entry of an Nrf2 reporter protein. The actin cytoskeleton therefore provides scaffolding that is essential for the function of Keap1, which is the sensor for oxidative and electrophilic stress.