A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes.

Enzymatic methylation at carbon five on cytosine (5mC) in DNA is a hallmark of mammalian epigenetic programming and is critical to gene regulation during early embryonic development. It has recently been shown that dynamic erasure of 5mC by three members of the ten-eleven translocation (TET) family plays a key role in cellular differentiation. TET enzymes belong to Fe (II)- and 2-ketoglutarate (2KG) dependent dioxygenases that successively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC), thus providing a chemical basis for the removal of 5mC which once was thought to be a permanent mark in mammalian genome. Since then a wide range of biochemical assays have been developed to characterize TET activity. Majority of these methods require multi-step processing to detect and quantify the TET-mediated oxidized products. In this study, we have developed a MALDI mass spectrometry based method that directly measures the TET activity with high sensitivity while eliminating the need for any intermediate processing steps. We applied this method to the measurement of enzymatic activity of TET2 and 3, Michaleis-Menten parameters (KM and kcat) of TET-2KG pairs and inhibitory concentration (IC50) of known small-molecule inhibitors of TETs. We further demonstrated the suitability of the assay to analyze chemoenzymatic labeling of 5hmC by ß-glucosyltransferase, highlighting the potential for broad application of our method in deconvoluting the functions of novel DNA demethylases.

Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5-methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.

Interaction between hepatic membrane type 1 matrix metalloproteinase and acireductone dioxygenase 1 regulates hepatitis C virus infection.

Membrane type 1 matrix metalloproteinase (MT1-MMP) binds to and regulates the function of tetraspanin-enriched microdomains. It also physically interacts with claudin-1 and acireductone dioxygenase 1 (ADI1), both associated with hepatitis C virus (HCV) cell entry. Here, we examined hepatic expression of MT1-MMP, ADI1 and claudin-1 as well as their physical interaction in relation to serum or intrahepatic HCV-RNA levels. A total of 104 liver biopsies obtained from chronic hepatitis C patients and 84 liver tissues obtained from noncancerous parts of surgically removed HCV-related hepatocellular carcinoma were analysed. Positive cytoplasmic ADI1 in liver biopsies was associated with higher serum HCV-RNA levels (P = 0.009). Positive MT1-MMP and ADI1 interaction assessed by co-immunoprecipitation was associated with lower tissue HCV-RNA levels (P = 0.009). Hepatic HCV-RNA levels were positively associated with ADI1 levels in the MT1-MMP and ADI1 co-immunoprecipitates (P = 0.030). Overexpression of MT1-MMP in Huh7.5 cells suppressed cell entry of HCV pseudoparticles as well as HCVcc infection. The suppression effect could be reversed by co-expression of ADI1 in a dose-dependent manner. In summary, clinical and cell-based experiments suggested that physical interaction between MT1-MMP and ADI1 led to suppression of HCV infection. This inhibitory effect could be reversed by ADI1 overexpression.

Properties of catechol 1, 2 dioxygenase in the cell free extract and immobilized extract of Mycobacterium fortuitum

Polycyclic aromatic hydrocarbons (PAH) are carcinogenic compounds which contaminate water and soil, and the enzymes can be used for bioremediation of these environments. This study aimed to evaluate some environmental conditions that affect the production and activity of the catechol 1,2-dioxygenase (C12O) by Mycobacterium fortuitum in the cell free and immobilized extract in sodium alginate. The bacterium was grown in mineral medium and LB broth containing 250 mg L-1 of anthracene (PAH). The optimum conditions of pH (4.0-9.0), temperature (5-70 ºC), reaction time (10-90 min) and the effect of ions in the enzyme activity were determined. The Mycobacterium cultivated in LB shown higher growth and the C12O activity was two-fold higher to that in the mineral medium. To both extracts the highest enzyme activity was at pH 8.0, however, the immobilized extract promoted the increase in the C12O activity in a pH range between 4.0 and 8.5. The immobilized extract increased the enzymatic activity time and showed the highest C12O activity at 45 ºC, 20 ºC higher than the greatest temperature in the cell free extract. The enzyme activity in both extracts was stimulated by Fe3+, Hg2+ and Mn2+ and inhibited by NH4+ and Cu2+, but the immobilization protected the enzyme against the deleterious effects of K+ and Mg2+ in tested concentrations. The catechol 1,2-dioxygenase of Mycobacterium fortuitum in the immobilized extract has greater stability to the variations of pH, temperature and reaction time, and show higher activity in presence of ions, comparing to the cell free extract

Overexpression of the HIF hydroxylase PHD3 is a favorable prognosticator for gastric cancer.

Hypoxia-induced factors (HIFs) play a central role in the adaptive mechanisms of cancer cells to survive under conditions of hypoxia. HIFs are regulated by prolyl hydroxylases (PHDs) among which PHD3 is implicated as a tumor suppressor. We aimed to correlate PHD3 expression with clinicopathologic parameters and to evaluate its prognostic significance in gastric cancer. The 101 tissue samples were collected from 83 resected stages I­IV gastric cancer patients, which were grouped as non-cancerous mucosa (n=18) and primary carcinoma (n=83). PHD3 expression was evaluated by immunohistochemistry. We adopted Pearson chi-square test, univariate analysis, multivariate analysis and Kaplan­Meier method. The positive frequency of PHD3 in cancer cells was 42.2%, whereas non-cancerous mucosa had no detectable PHD3. The expression of PHD3 increased significantly from non-cancerous mucosa to cancer. A significant difference was observed between PHD3 expression and tumor differentiation (P=0.007). The overexpression of PHD3 was associated with well differentiation. In univariate analyses, American Joint Committee on Cancer (AJCC) stage (P<0.0001), pT classification (P<0.0001), pN classification (P<0.0001), differentiation (P=0.0121), peritoneal metastasis (P=0.0006) and gross features (P=0.0104) were significantly associated with survival except PHD3 (P=0.2228) (Table 3). In multivariate analysis, AJCC stage was prognostically independent [hazard ratio (HR), 3.078; 95% confidence interval (CI), 2.228­4.252; P<0.0001]. Overexpression of PHD3 is a favorable prognosticator for gastric cancer. AJCC stage is an independent prognostic factor of gastric cancer.

Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution.

The early functional response of a bacterial community from the sediments of a chronically oil-polluted retention basin located at the Etang de Berre (France) was investigated just after petroleum addition. After removing hydrocarbon compounds by natural abiotic and biotic processes, the sediments were maintained in microcosms and Vic Bilh petroleum was added. The diversity and the expression of genes encoding ring-hydroxylating dioxygenases (RHD) were examined just after the petroleum addition until 14 days focussing on the first hours following the contamination. RHD gene copy numbers and diversity were maintained throughout all the incubation period; however, transcripts were detected only during the first 2 days. One dominant RHD gene, immediately and specifically expressed in response to petroleum contamination, was related to RHD gene carried by a plasmid found in Pseudomonas spp. The expression of the RHD genes was correlated with high biodegradation levels observed for low molecular weight PAHs at 7 days of incubation. The study shows that the bacterial metabolism induced just after the oil input is a key stage that could determine the bacterial community structure changes. Monitoring the expression of RHD genes, key genes involved in hydrocarbon degradation, may provide useful information for managing bioremediation processes.

Overexpression of the HIF hydroxylases PHD1, PHD2, PHD3 and FIH are individually and collectively unfavorable prognosticators for NSCLC survival.

INTRODUCTION: Hypoxia induced factors (HIFs) are at the heart of the adaptive mechanisms cancer cells must implement for survival. HIFs are regulated by four hydroxylases; Prolyl hydroxylase (PHD)-1,-2,-3 and factor inhibiting HIF (FIH). We aimed to investigate the prognostic impact of these oxygen sensors in NSCLC. METHODS: Tumor tissue samples from 335 resected stages I to IIIA NSCLC patients was obtained and tissue microarrays (TMAs) were constructed. Hydroxylase expression was evaluated by immunohistochemistry. PRINCIPAL FINDINGS: There was scorable expression for all HIF hydroxylases in tumor cells, but not in stroma. In univariate analyses, high tumor cell expression of all the HIF hydroxylases were unfavorable prognosticators for disease-specific survival (DSS); PHD1 (P = 0.023), PHD2 (P = 0.013), PHD3 (P = 0.018) and FIH (P = 0.033). In the multivariate analyses we found high tumor cell expression of PHD2 (HR = 2.03, CI 95% 1.20-3.42, P = 0.008) and PHD1 (HR = 1.45, CI 95% 1.01-2.10, P = 0.047) to be significant independent prognosticators for DSS. Besides, there was an additive prognostic effect by the increasing number of highly expressed HIF hydroxylases. Provided none high expression HIF hydroxylases, the 5-year survival was 80% vs. 23% if all four were highly expressed (HR = 6.48, CI 95% 2.23-18.8, P = 0.001). CONCLUSIONS: HIF hydroxylases are, in general, poor prognosticators for NSCLC survival. PHD1 and PHD2 are independent negative prognostic factors in NSCLC. Moreover, there is an additive poor prognostic impact by an increasing number of highly expressed HIF hydroxylases.

Regulação cruzada entre peroxidases e indolamina 2.3 dioxigenase no controle da metabolização do triptofano / Peroxidases and indoleamine 2.3 dioxygenase crosstalk modulating tryptophan metabolization

Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no...

Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by...

BRCA1 tumours correlate with a HIF-1alpha phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression.

BACKGROUND: There are limited data regarding the hypoxia pathway in familial breast cancers. We therefore performed a study of hypoxic factors in BRCA1, BRCA2 and BRCAX breast cancers. METHODS: Immunoperoxidase staining for HIF-1alpha, PHD1, PHD2, PHD3, VEGF and FIH was carried out in 125 (38 BRCA1, 33 BRCA2 and 54 BRCAX) breast carcinomas. These were correlated with clinicopathological parameters and the intrinsic breast cancer phenotypes. RESULTS: BRCA1 tumours correlated with positivity for HIF-1alpha (P=0.008) and negativity for PHD3 (P=0.037). HIF-1alpha positivity (P=0.001), PHD3 negativity (P=0.037) and nuclear FIH negativity (P=0.011) was associated with basal phenotype. HIF-1alpha expression correlated with high tumour grade (P=0.009), negative oestrogen receptor (ER) status (P=0.001) and the absence of lymph node metastasis (P=0.028). Nuclear FIH expression and PHD3 correlated with positive ER expression (P=0.024 and P=0.035, respectively). BRCA1 cancers with positive HIF-1alpha or cytoplasmic FIH had a significantly shorter relapse-free survival (P=0.007 and P=0.049, respectively). CONCLUSIONS: The aggressive nature of BRCA1 and basal-type tumours may be partly explained by an enhanced hypoxic drive and hypoxia driven ER degradation because of suppressed PHD and aberrantly located FIH expression. This may have important implications, as these tumours may respond to compounds directed against HIF-1alpha or its downstream targets.

Isolation and characterization of a novel strain of Stenotrophomonas maltophilia possessing various dioxygenases for monocyclic hydrocarbon degradation / Isolamento e caracterização de uma nova cepa de Stenotrophomonas maltophilia com várias dioxigenases para degradação de hidrocarbonetos monocíclicos

A Gram-negative bacterium, designated as strain KB2, was isolated from activated sludge and was found to utilize different aromatic substrates as sole carbon and energy source. On the basis of morphological and physiochemical characteristics and 16S rRNA gene sequence analysis, the isolated strain KB2 was identified as Stenotrophomonas maltophilia. Strain KB2 is from among different Stenotrophomonas maltophilia strains the first one described as exhibiting the activities of three types of dioxygenases depending on the structure of the inducer. The cells grown on benzoate and catechol showed mainly catechol 1,2- dioxygenase activity. The activity of 2,3-dioxygenase was detected after phenol induction. Protocatechuate 3,4-dioxygenase was found in crude cell extracts of this strain after incubation with 4-hydroxybenzoic acid, protocatechuic acid and vanillic acid. Because of broad spectrum of dioxygenases' types that Stenotrophomonas maltophilia KB2 can exhibit, this strain appears to be very powerful and useful tool in the biotreatment of wastewaters and in soil decontamination.

Uma bactéria Gram-negativa, denominada KB2, foi isolada de lodo ativado, verificando-se ser capaz de utilizar substratos aromáticos com única fonte de carbono e energia. Com base nas características morfológicas e físico-químicas, e na análise da sequencia do gene 16SrRNA, esta bactéria foi identificada como Stenotrophomonas maltophilia. Entre as diversas cepas de S. maltophilia já descritas, essa cepa é a primeira com atividade de três tipos de dioxigenases, dependendo da estrutura do indutor. As células cultivadas em benzoato e catecol apresentaram atividade de catecol 1,2-dioxigenase principalmente. A atividade de 2,3-dioxigenase foi detectada após indução com fenol. Após incubação com ácidos 4-hidrobenzoico, ácido protocatecuico evanílico, encontrou-se protocatecuato 3,4-dioxigenase no extrato celular. Devido ao amplo espectro de atividade das diferentes dioxigenases de S. maltophilia KB2, esta cepa parece ser uma ferramenta poderosa e útil para o biotratamento de efluentes e descontaminação do solo.

JARID1B is a histone H3 lysine 4 demethylase up-regulated in prostate cancer.

Histone methylation is a dynamic process that participates in a diverse array of cellular processes and has been found to associate with cancer. Recently, several histone demethylases have been identified that catalyze the removal of methylation from histone H3 lysine residues. Through bioinformatic and biochemical analysis, we identified JARID1B as a H3K4 demethylase. Overexpression of JARID1B resulted in loss of tri-, di-, and monomethyl H3K4 but did not affect other histone lysine methylations. In vitro biochemical experiments demonstrated that JARID1B directly catalyzes the demethylation. The enzymatic activity requires the JmjC domain and uses Fe(II) and alpha-ketoglutarate as cofactors. Furthermore, we found that JARID1B is up-regulated in prostate cancer tissues, compared with benign prostate samples. We also demonstrated that JARID1B associates with androgen receptor and regulates its transcriptional activity. Thus, we identified JARID1B as a demethylase capable of removing three methyl groups from histone H3 lysine 4 and up-regulated in prostate cancer.

Regulated nucleo-cytoplasmic shuttling of human aci-reductone dioxygenase (hADI1) and its potential role in mRNA processing.

Bacterial aci-reductone dioxygenase (ARD), a member of the cupin superfamily, has evolutionarily primitive protein folding and functions in the methionine recycling pathway. Recently, a human ARD orthologue (human ADI1, hADI1) has been identified and exhibits functions other than ARD activity. The hADI1 localizes mainly to the cytoplasm, but a substantial fraction is nuclear, suggesting functions in both cellular compartments. In this study, we report that nucleo-cytoplasmic transport of hADI1 is regulated by a non-canonical nuclear export signal (NES) located in the N-terminal region of hADI1. The NES is composed of multiple basic amino-acid residues instead of the canonical leucine-rich sequence. Nuclear export of hADI1 was not mediated by CRM1, a major transporter that binds to leucine-rich NES. Substitution of the basic residues with alanines abolished NES activity. Mutant hADI1 accumulated in the nucleus and formed speckles frequently observed with splicing factors and some transcription factors. Indeed, hADI1 specifically co-localized with the splicing factor U1-70K to the nucleus but not with another splicing factor, SC35. U1-70K over-expression induced nuclear accumulation of hADI1. Nuclear hADI1 expression significantly altered the splicing pattern of the adenovirus E1A mini-gene, which generates multiple alternatively spliced transcripts. Thus, hADI1 may have acquired a novel role in nuclear mRNA processing possibly by modulating U1-70K-related functions, an activity negatively regulated by a non-classical NES sequence.