S-adenosylmethionine Metabolism Research Articles

Comparative transcriptome analysis reveals metabolic regulation of prodigiosin in Serratia marcescens

Prodigiosin is a secondary metabolite mainly produced at 30 °C in Serratia marcescens, but it can hardly be synthetized at 37 °C or higher. In this study, we provide insight into the metabolic regulation of prodigiosin synthesis in response to temperature through transcriptome sequencing. The analysis of the function of differentially expressed genes suggested that temperature resulted in significant alteration of the metabolic pathways between 30 and 37 °C. Specifically, 30 °C favored transcriptional expression of the pig gene cluster. At the same time, the carbon flux was redistributed to pathways of pyruvate, proline, serine, especially homoserine, cystathionine, homocysteine, methionine, and s-adenosylmethionine metabolism, all involved in prodigiosin biosynthesis, and was finally increased towards the prodigiosin synthesis pathway in S. marcescens at 30 °C. Interestingly, results further confirmed increased transcriptional level of five regulators (LuxS, RpoS, Hfq, EepR, CRP), and decreased content of hexS through qPCR. Finally, successful co-overexpression of mmuM and metK, related to homocysteine, methionine, and s-adenosylmethionine metabolism, in the chromosome of JNB5-1 (JNB5-1/MK) resulted in increased prodigiosin titer up to 7.57 g/L in JNB5-1/MK at 30 °C, which was 41.2% higher than that in JNB5-1. Our transcriptome analysis provides further insight into the strain's response to temperature changes at the transcription level, which is of great significance for improving the production of prodigiosin.

Duodenal-jejunal bypass maintains hepatic S-adenosylmethionine/S-homocysteine ratio in diet-induced obese rats.

We previously reported that the duodenal-jejunal bypass (DJB) surgery altered transsulfuration and purine metabolism via flux changes in 1-carbon metabolism in the liver. In this study, we extended our study to gain further insight into mechanistic details of how the DJB-induced flux changes in 1-carbon metabolism contributes to the improvement of diet-induced nonalcoholic fatty liver disease. Rodents were subjected to surgical (sham operation and DJB) or dietary (reduced food supply to follow the weight changes in the DJB group) interventions. The microscopic features of the liver were examined by immunohistochemistry. The expressions of genes in lipid synthesis and in 1-carbon cycle in the liver were analyzed by real-time polymerase chain reaction and western blotting. Metabolic changes in the liver were determined. We observed that DJB reduces hepatic steatosis and improves insulin sensitivity in both high-fat diet-fed rats and mice. Metabolic analyses revealed that the possible underlying mechanism may involve decreased S-adenosylmethionine (SAM)-to-S-adenosylhomocysteine ratio via downregulation of SAM synthesizing enzyme and upregulation of SAM catabolizing enzyme. We also found in mice that DJB-mediated attenuation of hepatic steatosis is independent of weight loss. DJB also increased hepatic expression levels of GNMT while decreasing those of PEMT and BHMT, a change in 1-carbon metabolism that may decrease the ratio of SAM to S-adenosylhomocysteine, thereby resulting in the prevention of fat accumulation in the liver. Thus, we suggest that the change in 1-carbon metabolism, especially the SAM metabolism, may contribute to the improvement of diet-induced fatty liver disease after DJB surgery.

GEN-14 Dual regulation of histone methylation by mTOR complexes drives the progression of EGFR-mutant glioblastoma

Trimethylation of histone H3 on lysine 27 (H3K27me3) is essential for ensuring proper gene expression and chromosomal function, and its aberration is associated with the pathogenesis of various brain tumors. However, it remains unclear how histone methylation is regulated in response to genetic mutation and intracellular metabolic status to facilitate the cancer cell survival in the most malignant IDH-wildtype glioblastoma (GBM). We herein report a novel mechanism of the specific regulation of H3K27me3 by cooperative action of two mechanistic target of rapamycin (mTOR) complexes in EGFR-mutant GBM. The level of H3K27me3 is significantly associated with the mutant EGFR signaling (EGFRvIII and EGFR amplification), and integrated analyses with histopathological, NGS and metabolome examinations revealed that both mTOR complexes (mTORC1 and mTORC2) upregulate H3K27me3 downstream of aberrant EGFR signaling. mTORC1 facilitates the protein translation of enhancer of zeste homolog 2 (EZH2), which is known as H3K27-specific methyltransferase. The other mTOR complex, mTORC2, remodels the metabolism of S-adenosylmethionine (SAM), an essential substrate for histone methylation. This synergistic mechanism causes H3K27 hypermethylation which subsequently promotes tumor cell survival both in vitro and in vivo mouse tumor model via regulation of the cell cycle-related tumor suppressor genes. The findings indicate that activated mTORC1 and mTORC2 complexes under aberrant EGFR signaling cooperatively contribute to the progression of IDH-wildtype GBM through specific epigenetic regulation, nominating them as an exploitable therapeutic target against cancer-specific epigenetics.

FOXP3 promotes colorectal carcinoma liver metastases by evaluating MMP9 expression via regulating S-adenosylmethionine metabolism.

BackgroundGrowing evidence has proved that Forkhead box protein 3 (FOXP3), which is a master regulatory gene in the development and function of regulatory T-cells, is expressed in human cancer cells. This expression indicates the crucial role FOXP3 takes up as the disease progresses. However, its role in colorectal cancer (CRC) liver metastasis is still mostly unknown. This study set out to explore the molecular characteristics of FOXP3 in driving the liver metastasis within CRC.MethodsWe downloaded the RNA-seq data from the GSE50760. Weighted gene co-expression network analysis (WGCNA)WGCNA and RNA-Seq analysis were applied to find the key gene network associated with colorectal cancer liver metastasis. Then we performed pathway enrichment analysis on liver metastasis-associated gene set. Immunohistochemistry, in vitro and in vivo studies were conducted to test expression and function of FOXP3 in CRC tissues and liver metastasis tissues. Non-targeted metabolomics analysis was performed to identify the alteration of FOXP3 expression in metabolites of colorectal cancer liver metastasis. Western blot was performed to confirm changes of matrix metalloproteinase 9MMP9 expression were downstream events of S-adenosyl-methionine (SAM).ResultsWe found that FOXP3 and MMP9 exhibited co-expression relationships and affected liver metastasis in CRC. Upregulation of FOXP3 promotes cell migration and invasion in CRC, which suggests a pro-cancer effect. Moreover, metabolomics analysis showed that knockdown of FOXP3 significantly reduced SAM levels, and changes of MMP9 expression were downstream events of SAM, which is concentration-dependent. Besides, The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Western blot analysis confirmed that overexpression of FOXP3 activates the Wnt pathway to promote colon cancer metastasis.ConclusionsOur results altogether suggested that FOXP3 expression inhibited the SAM cycle to reduce SAMe levels, resulting in altered MMP9 expression and helped CRC liver metastasis.

Transcriptomic and proteomic analysis reveals mechanisms of low pollen-pistil compatibility during water lily cross breeding

BackgroundIn water lily (Nymphaea) hybrid breeding, breeders often encounter non-viable seeds, which make it difficult to transfer desired or targeted genes of different Nymphaea germplasm. We found that pre-fertilization barriers were the main factor in the failure of the hybridization of Nymphaea. The mechanism of low compatibility between the pollen and stigma remains unclear; therefore, we studied the differences of stigma transcripts and proteomes at 0, 2, and 6 h after pollination (HAP). Moreover, some regulatory genes and functional proteins that may cause low pollen-pistil compatibility in Nymphaea were identified.ResultsRNA-seq was performed for three comparisons (2 vs 0 HAP, 6 vs 2 HAP, 6 vs 0 HAP), and the number of differentially expressed genes (DEGs) was 8789 (4680 were up-regulated), 6401 (3020 were up-regulated), and 11,284 (6148 were up-regulated), respectively. Using label-free analysis, 75 (2 vs 0 HAP) proteins (43 increased and 32 decreased), nine (6 vs 2 HAP) proteins (three increased and six decreased), and 90 (6 vs 0 HAP) proteins (52 increased and 38 decreased) were defined as differentially expressed proteins (DEPs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs and DEPs were mainly involved in cell wall organization or biogenesis, S-adenosylmethionine (SAM) metabolism, hydrogen peroxide decomposition and metabolism, reactive oxygen species (ROS) metabolism, secondary metabolism, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis.ConclusionsOur transcriptomic and proteomic analysis highlighted specific genes, incuding those in ROS metabolism, biosynthesis of flavonoids, SAM metabolism, cell wall organization or biogenesis and phenylpropanoid biosynthesis that warrant further study in investigations of the pollen-stigma interaction of water lily. This study strengthens our understanding of the mechanism of low pollen-pistil compatibility in Nymphaea at the molecular level, and provides a theoretical basis for overcoming the pre-fertilization barriers in Nymphaea in the future.

Perturbation of Methionine/S-adenosylmethionine Metabolism as a Novel Vulnerability in MLL Rearranged Leukemia.

Leukemias bearing mixed lineage leukemia (MLL) rearrangement (MLL-R) resulting in expression of oncogenic MLL fusion proteins (MLL-FPs) represent an especially aggressive disease subtype with the worst overall prognoses and chemotherapeutic response. MLL-R leukemias are uniquely dependent on the epigenetic function of the H3K79 methyltransferase DOT1L, which is misdirected by MLL-FPs activating gene expression, driving transformation and leukemogenesis. Given the functional necessity of these leukemias to maintain adequate methylation potential allowing aberrant activating histone methylation to proceed, driving leukemic gene expression, we investigated perturbation of methionine (Met)/S-adenosylmethionine (SAM) metabolism as a novel therapeutic paradigm for MLL-R leukemia. Disruption of Met/SAM metabolism, by either methionine deprivation or pharmacologic inhibition of downstream metabolism, reduced overall cellular methylation potential, reduced relative cell numbers, and induced apoptosis selectively in established MLL-AF4 cell lines or MLL-AF6-expressing patient blasts but not in BCR-ABL-driven K562 cells. Global histone methylation dynamics were altered, with a profound loss of requisite H3K79 methylation, indicating inhibition of DOT1L function. Relative occupancy of the repressive H3K27me3 modification was increased at the DOT1L promoter in MLL-R cells, and DOT1L mRNA and protein expression was reduced. Finally, pharmacologic inhibition of Met/SAM metabolism significantly prolonged survival in an advanced, clinically relevant patient–derived MLL-R leukemia xenograft model, in combination with cytotoxic induction chemotherapy. Our findings provide support for further investigation into the development of highly specific allosteric inhibitors of enzymatic mediators of Met/SAM metabolism or dietary manipulation of methionine levels. Such inhibitors may lead to enhanced treatment outcomes for MLL-R leukemia, along with cytotoxic chemotherapy or DOT1L inhibitors.

Early-life exposure to the endocrine disruptor 17-α-ethinylestradiol induces delayed effects in adult brain, liver and ovotestis proteomes of a self-fertilizing fish

Early-life represents a critically sensitive window to endocrine disrupting chemicals, potentially leading to long-term repercussions on the phenotype later in life. The mechanisms underlying this phenomenon, referred to as the Developmental Origins of Health and Disease (DOHaD), are still poorly understood. To gain molecular understanding of these effects, we exposed mangrove rivulus (Kryptolebias marmoratus) for 28 days post hatching (dph) to 4 and 120 ng/L 17-α-ethinylestradiol, a model xenoestrogen. After 28 days, fish were raised for 140 days in clean water and we performed quantitative label-free proteomics on brain, liver and ovotestis of 168 dph adults. A total of 820, 888 and 420 proteins were robustly identified in the brain, liver and ovotestis, respectively. Effects of 17-α-ethinylestradiol were tissue and dose-dependent: a total of 31, 51 and 18 proteins were differentially abundant at 4 ng/L in the brain, liver and ovotestis, respectively, compared to 20, 25 and 39 proteins at 120 ng/L. Our results suggest that estrogen-responsive pathways, such as lipid metabolism, inflammation, and the innate immune system were affected months after the exposure. In addition, the potential perturbation of S-adenosylmethionine metabolism encourages future studies to investigate the role of DNA methylation in mediating the long-term effects of early-life exposures. SignificanceThe Developmental Origins of Health and Disease (DOHaD) states that early life stages of humans and animals are sensitive to environmental stressors and can develop health issues later in life, even if the stress has ceased. Molecular mechanisms supporting DOHaD are still unclear. The mangrove rivulus is a new fish model species naturally reproducing by self-fertilization, making it possible to use isogenic lineages in which all individuals are highly homozygous. This species therefore permits to strongly reduce the confounding factor of genetic variability in order to investigate the effects of environmental stress on the phenotype. After characterizing the molecular phenotype of brain, liver and ovotestis, we obtained true proteomic reaction norms of these three organs in adults after early life stages have been exposed to the common endocrine disruptor 17-α-ethinylestradiol (EE2). Our study demonstrates long-term effects of early-life endocrine disruption at the proteomic level in diverse estrogen-responsive pathways 5 months after the exposure. The lowest tested and environmentally relevant concentration of 4 ng/L had the highest impact on the proteome in brain and liver, highlighting the potency of endocrine disruptors at low concentrations.

In vitro proteomic analysis of methapyrilene toxicity in rat hepatocytes reveals effects on intermediary metabolism.

The antihistaminic drug methapyrilene was withdrawn from the market in 1979 because of hepatocarcinogenicity in rats. Since then, the drug has been used as a model hepatotoxin especially for transcriptomic analyses using material from in vivo studies. Much less transcriptomics data are available from in vitro studies, and no studies have investigated proteomic effects of methapyrilene in vitro. Thus, the present study was aimed to characterize the proteomic response of primary rat hepatocytes to methapyrilene, to broaden our knowledge on the molecular mechanisms of methapyrilene toxicity, and to compare the results of collagen sandwich-cultured hepatocytes to in vivo data. In vitro methapyrilene concentrations (0.39µM, 6.25µM, and 100µM) were chosen to cover an in vivo-relevant range. Based on published pharmacokinetic data they correspond to concentrations in portal vein blood for previously in vivo-tested doses of methapyrilene, up to a concentration showing slight cytotoxicity. Analysis of proteomic alterations by two-dimensional gel electrophoresis and mass-spectrometric protein identification demonstrated consistent and concentration-dependent effects of methapyrilene, in particular on mitochondrial proteins. Data suggest substantial deregulation of amino acid and ammonia metabolism and effects on mitochondrial energy supply pathways. The effects identified in vitro concur well with into previous in vivo observations. Several effects, for example, the influence of methapyrilene on S-adenosylmethionine metabolism, have not been described previously. The data suggest that already non-toxic concentrations of methapyrilene alter components of the intermediary metabolism, such as branched-chain amino acid metabolism, as well as urea and tricarboxylic cycle enzymes. In summary, data substantially add to our knowledge on molecular mechanisms of methapyrilene hepatotoxicity at the protein level.

<i>In vivo</i> influence of S-adenosylmethionine supplementation on ethanol teratogenicity in the rat

Studies have shown that maternal alcohol consumption during pregnancy negatively impacts on prenatal development. Ethanol-induced alteration in S-adenosylmethionine (SAMe) metabolism and subsequent depletion of SAMe may be a mechanism involved in fetal ethanol effects. The aim of the present study was to determine the teratogenicity of ethanol and to investigate protective effects of SAMe. Pregnant Sprague-Dawley rats, weighing 220-250 g were used. Pregnant animals were treated from day 6 through 12 of gestation with ethanol alone (6.4% ethanol) or ethanol plus SAMe (200 mg/kg). The groups for the day-20 and day-12 experiments included: Group I (ethanol liquid diet: unrestricted-fed); Group II (isocaloric maltose-dextrin liquid diet: restricted-fed); and Group III (ethanol liquid diet + SAMe: unrestricted-fed). Embryos and fetuses were recovered on gestational day-12 or day-20, respectively, and were quantitatively and qualitatively assessed for developmental anomalies. The data on continuous variables were analyzed using paired T-Test and categorical embryonic development data were processed using Chi-Square Analysis. In the present study, prenatal growth and developmental retardations such as significantly reduced crown-rump length, anomalies of the central nervous system, the heart, limbs and the skeleton were observed in embryos and fetuses of ethanol treated rats. SAMe supplementation of the ethanol treated pregnant rats significantly improved litter weight, from 20.9 + 1.6 g to 23.6 + 0.8 g, and crown-rump length from 25.8 + 0.8 mm to 27.6 + 0.4 mm, and decreased the incidence of resorptions from 1.9 + 0.6 to 0.5 + 0.8 in day-20 fetuses. It also significantly increased embryonic protein content from 173 + 36 μg to 251 + 29 μg and significantly improved the development of brain vesicles and closure of the posterior neuropore in day-12 embryos. The present study clearly showed that ethanol is teratogenic in vivo . SAMe supplementation of ethanol treated pregnant rats partially ameliorated the teratogenic effects of ethanol. This indicated that the observed ethanol related growth and developmental abnormalities may have been, at least partly, due to ethanol-induced SAMe deficiency. Further studies should consider extending the treatment period to the entire gestational period instead of limiting it to only the days covering the period of organogenesis. Keywords: ethanol teratogenicity, Sprague-Dawley rats, S-adenosylmethionine, embryos, fetuses

Stereo-Specific Transcript Regulation of the Polyamine Biosynthesis Genes by Enantiomers of Ornithine in Tobacco Cell Culture.

BackgroundOrnithine (Orn) plays an essential role in the metabolism of plant cells through incorporation in polyamines biosynthesis, the urea cycle and nitrogen metabolism. Physiological response of the plant cells to its two enantiomers have not been widely investigated yet.ObjectivesThis study aimed to evaluate effect of ornithine enantiomers on expression of certain polyamine (PAs) biosynthetic genes in tobacco cells.Materials and MethodsSuspension-cultured tobacco cells were treated with different concentrations of L- and D- Orn for 24 h. Cell viability was assayed by Evans Blue and hydrogen peroxide content. The changes of gene expression were analyzed by semi-quantitative RT-PCR.ResultsExogenous D-Orn resulted in enhancement of expression of genes involved in Orn, arginine and S-adenosyl methionine metabolism. Additionally, exogenous D-Orn treatment resulted in sustained viability of cultured tobacco cells and normal levels of hydrogen peroxide were maintained. Supplied L-Orn increased the hydrogen peroxide level and lowered viability of cells. Treatment with L-Orn had a negative effect on the transcript levels for most analyzed PA-related genes. It was also illustrated that transcription of putrescine methyl transferase, key enzyme for nicotine production, was highly upregulated by L-Orn.ConclusionsBased on the results, D-Orn was shown to have a stereo-selective function in regulation of the PAs-related genes.

Tumor-suppressive effect of S-adenosylmethionine supplementation in a murine model of inflammation-mediated hepatocarcinogenesis is dependent on treatment longevity.

Chronic inflammation precedes the majority of hepatocellular carcinoma (HCC) cases. We investigated the chemopreventive potential of S-adenosylmethionine (SAM), an essential donor for all methylation reactions in the cell, at the late precancerous stage of HCC development using the Mdr2-knockout (Mdr2-KO, Abcb4−/−) mice, a model of inflammation-mediated hepatocarcinogenesis. Previously, we revealed down-regulation of the genes regulating SAM metabolism in the liver of these mice at the precancerous stages. Now, we have supplied Mdr2-KO mice at the late precancerous stage with SAM during either a short-term (17 days) or a long-term (51 days) period and explored the effects of such supplementation on tumor development, DNA methylation and gene expression in the liver. The short-term SAM supplementation significantly decreased the number of small tumor nodules, proliferating hepatocytes and the total DNA methylation level, while it increased expression of the tumor suppressor proteins Mat1a and p21. Surprisingly, the long-term SAM supplementation did not affect tumor growth and hepatocyte proliferation, while it increased the total liver DNA methylation. Our results demonstrate that the short-term SAM supplementation in the Mdr2-KO mice inhibited liver tumor development potentially by increasing multiple tumor suppressor mechanisms resulting in cell cycle arrest. The long-term SAM supplementation resulted in a bypass of the cell cycle arrest in this HCC model by a yet unknown mechanism.

Efficient co‐production of S‐adenosylmethionine and glutathione by Candida utilis: effect of dissolved oxygen on enzyme activity and energy supply

AbstractBACKGROUNDAs both S‐adenosylmethionine (SAM) and glutathione (GSH) are biosynthesized with ATP consumption in yeast cells, their efficient co‐production not only depends on high activities of key enzymes, but also on sufficient energy supply. In this study, dissolved oxygen (DO) was optimized to improve enzyme activity and energy supply for high co‐production of SAM and GSH.RESULTSBatch culture of Candida utilis CCTCC M 209298 under different DO control modes was carried out in a 5 L stirred fermentor. Based on the analyses of kinetic parameters and metabolic fluxes of key nodes involved in the metabolism of SAM and GSH, a two‐stage DO control strategy with an initial agitation rate of 350 rpm followed by a constant DO level of 30% was designed for the overproduction of SAM and GSH. A maximum co‐production of SAM and GSH of 593.9 mg L−1 was achieved using the two‐stage DO control strategy, which was much higher than that obtained from other DO control modes.CONCLUSIONPhysiological mechanism analysis revealed that the improved co‐production of SAM and GSH was closely related to the increase in key enzyme activities and sufficient energy supply under the two‐stage DO control strategy. © 2017 Society of Chemical Industry

Comparative Proteomic Analysis of Mature Pollen in Triploid and Diploid Populus deltoides.

Ploidy affects plant growth vigor and cell size, but the relative effects of pollen fertility and allergenicity between triploid and diploid have not been systematically examined. Here we performed comparative analyses of fertility, proteome, and abundances of putative allergenic proteins of pollen in triploid poplar ‘ZhongHuai1’ (‘ZH1’, triploid) and ‘ZhongHuai2’ (‘ZH2’, diploid) generated from the same parents. The mature pollen was sterile in triploid poplar ‘ZH1’. By applying two-dimensional gel electrophoresis (2-DE), a total of 72 differentially expressed protein spots (DEPs) were detected in triploid poplar pollen. Among them, 24 upregulated and 43 downregulated proteins were identified in triploid poplar pollen using matrix-assisted laser desorption/ionisation coupled with time of-flight tandem mass spectrometer analysis (MALDI-TOF/TOF MS/MS). The main functions of these DEPs were related with “S-adenosylmethionine metabolism”, “actin cytoskeleton organization”, or “translational elongation”. The infertility of triploid poplar pollen might be related to its abnormal cytoskeletal system. In addition, the abundances of previously identified 28 putative allergenic proteins were compared among three poplar varieties (‘ZH1’, ‘ZH2’, and ‘2KEN8‘). Most putative allergenic proteins were downregulated in triploid poplar pollen. This work provides an insight into understanding the protein regulation mechanism of pollen infertility and low allergenicity in triploid poplar, and gives a clue to improving poplar polyploidy breeding and decreasing the pollen allergenicity.

Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells.

BackgroundThe majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as “methionine dependence” or “methionine stress sensitivity” in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8.ResultsUsing a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential.ConclusionThis study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDA-MB-468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions.

Metabolome and proteome changes with aging in Caenorhabditis elegans

To expand the understanding of aging in the model organism Caenorhabditis elegans, global quantification of metabolite and protein levels in young and aged nematodes was performed using mass spectrometry. With age, there was a decreased abundance of proteins functioning in transcription termination, mRNA degradation, mRNA stability, protein synthesis, and proteasomal function. Furthermore, there was altered S-adenosyl methionine metabolism as well as a decreased abundance of the S-adenosyl methionine synthetase (SAMS-1) protein. Other aging-related changes included alterations in free fatty acid levels and composition, decreased levels of ribosomal proteins, decreased levels of NADP-dependent isocitrate dehydrogenase (IDH1), a shift in the cellular redox state, an increase in sorbitol content, alterations in free amino acid levels, and indications of altered muscle function and sarcoplasmic reticulum Ca2+ homeostasis. There were also decreases in pyrimidine and purine metabolite levels, most markedly nitrogenous bases. Supplementing the culture medium with cytidine (a pyrimidine nucleoside) or hypoxanthine (a purine base) increased lifespan slightly, suggesting that aging-induced alterations in ribonucleotide metabolism affect lifespan. An age-related increase in body size, lipotoxicity from ectopic yolk lipoprotein accumulation, a decline in NAD+ levels, and mitochondrial electron transport chain dysfunction may explain many of these changes. In addition, dietary restriction in aged worms resulting from sarcopenia of the pharyngeal pump likely decreases the abundance of SAMS-1, possibly leading to decreased phosphatidylcholine levels, larger lipid droplets, and ER and mitochondrial stress. The complementary use of proteomics and metabolomics yielded unique insights into the molecular processes altered with age in C. elegans.

Nonalcoholic fatty liver disease: update on pathogenesis, diagnosis, treatment and the role of S-adenosylmethionine.

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide affecting over one-third of the population in the U.S. It has been associated with obesity, type 2 diabetes, hyperlipidemia, and insulin resistance and is initiated by the accumulation of triglycerides in hepatocytes. Isolated hepatic steatosis (IHS) remains a benign process, while a subset develops superimposed inflammatory activity and progression to nonalcoholic steatohepatitis (NASH) with or without fibrosis. However, the molecular mechanisms underlying NAFLD progression are not completely understood. Liver biopsy is still required to differentiate IHS from NASH as easily accessible noninvasive biomarkers are lacking. In terms of treatments for NASH, pioglitazone, vitamin E, and obeticholic acid have shown some benefit. All of these agents have potential complications associated with long-term use. Nowadays, a complex hypothesis suggests that multiple parallel hits are involved in NASH development. However, the 'key switch' between IHS and NASH remains to be discovered. We have recently shown that knocking out enzymes involved in S-adenosylmethionine (SAMe) metabolism, the main biological methyl donor in humans that is abundant in the liver, will lead to NASH development in mice. This could be due to the fact that a normal SAMe level is required to establish the proper ratio of phosphatidylethanolamine to phosphatidylcholine that has been found to be important in NAFLD progression. New data from humans have also suggested that these enzymes play a role in the pathogenesis of NAFLD and that some of SAMe cycle metabolites may serve as noninvasive biomarkers of NASH. In this review, we discuss the evidence of the role of SAMe in animal models and humans with NAFLD and how studying this area may lead to the discovery of new noninvasive biomarkers and possibly personalized treatment for NASH.

A Comparison of the Metabolome of Male and Female Drosophila melanogaster

Metabolite profiles of ten batches of male and female Drosophila melanogaster were compared using three different chromatographic methods interfaced to an Orbitrap Exactive mass spectrometer. Several thousand features were observed and were reduced after data extraction and careful checking of around 390 metabolites excluding lipids. Chromatographic traces for these metabolites are presented as supplementary information. There were many significant differences between male and female flies. Female flies contained much higher levels of methylated lysines and methylated arginine suggesting differences in histone metabolism. In addition, there were differences in the methylation of nucleosides, and Sadenosylmethionine metabolism. Differences in the methylome may relate to the requirement of compensation for the single X chromosome present in males since methylated histones inhibit gene transcription. Nucleoside phosphate levels were elevated in female flies which may relate to increased requirement for DNA biosynthesis for egg production. A series of acylated amino acids previously observed in Drosophila was further characterised and these metabolites were present to a much greater extent in female flies and may be associated with the microbiome. Keywords: Drosophila melanogaster, male female metabolomics, methylation, acylated amino acids.

Proteomics of stress responses in wheat and barley-search for potential protein markers of stress tolerance.

Wheat (Triticum aestivum; T. durum) and barley (Hordeum vulgare) agricultural production is severely limited by various abiotic and biotic stress factors. Proteins are directly involved in plant stress response so it is important to study proteome changes under various stress conditions. Generally, both abiotic and biotic stress factors induce profound alterations in protein network covering signaling, energy metabolism (glycolysis, Krebs cycle, ATP biosynthesis, photosynthesis), storage proteins, protein metabolism, several other biosynthetic pathways (e.g., S-adenosylmethionine metabolism, lignin metabolism), transport proteins, proteins involved in protein folding and chaperone activities, other protective proteins (LEA, PR proteins), ROS scavenging enzymes as well as proteins affecting regulation of plant growth and development. Proteins which have been reported to reveal significant differences in their relative abundance or posttranslational modifications between wheat, barley or related species genotypes under stress conditions are listed and their potential role in underlying the differential stress response is discussed. In conclusion, potential future roles of the results of proteomic studies in practical applications such as breeding for an enhanced stress tolerance and the possibilities to test and use protein markers in the breeding are suggested.

Comparative Analysis and Modeling of the Severity of Steatohepatitis in DDC-Treated Mouse Strains

BackgroundNon-alcoholic fatty liver disease (NAFLD) has a broad spectrum of disease states ranging from mild steatosis characterized by an abnormal retention of lipids within liver cells to steatohepatitis (NASH) showing fat accumulation, inflammation, ballooning and degradation of hepatocytes, and fibrosis. Ultimately, steatohepatitis can result in liver cirrhosis and hepatocellular carcinoma.Methodology and ResultsIn this study we have analyzed three different mouse strains, A/J, C57BL/6J, and PWD/PhJ, that show different degrees of steatohepatitis when administered a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) containing diet. RNA-Seq gene expression analysis, protein analysis and metabolic profiling were applied to identify differentially expressed genes/proteins and perturbed metabolite levels of mouse liver samples upon DDC-treatment. Pathway analysis revealed alteration of arachidonic acid (AA) and S-adenosylmethionine (SAMe) metabolism upon other pathways. To understand metabolic changes of arachidonic acid metabolism in the light of disease expression profiles a kinetic model of this pathway was developed and optimized according to metabolite levels. Subsequently, the model was used to study in silico effects of potential drug targets for steatohepatitis.ConclusionsWe identified AA/eicosanoid metabolism as highly perturbed in DDC-induced mice using a combination of an experimental and in silico approach. Our analysis of the AA/eicosanoid metabolic pathway suggests that 5-hydroxyeicosatetraenoic acid (5-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE) and prostaglandin D2 (PGD2) are perturbed in DDC mice. We further demonstrate that a dynamic model can be used for qualitative prediction of metabolic changes based on transcriptomics data in a disease-related context. Furthermore, SAMe metabolism was identified as being perturbed due to DDC treatment. Several genes as well as some metabolites of this module show differences between A/J and C57BL/6J on the one hand and PWD/PhJ on the other.

Metilentetrahidrofolat Redüktaz Aktivitesi ve Folat Metabolizması

Folat, suda cozunebilen onemli bir B vitaminidir. Bitkisel ve hayvansal kaynakli gidalarda dogal olarak bulunmaktadir. Folat, metabolizmada onemli gorevler ustlenmektedir. Bunlardan biri de plazma homosistein duzeyinin ayarlanmasidir. Homosistein metabolizmasinda gorevli olan MTHFR (Metilentetrahidrofolat reduktaz) aktivitesinde azalma, homosistein seviyesini etkilemektedir. Bu nedenle MTHFR folat metabolizmasinda onemli bir enzimdir. MTHFR geninde meydana gelen bazi mutasyonlar cesitli hastaliklar icin risk faktoru olan hiperhomosisteinemi ve homosisteinuriye sebep olabilmekte, metabolik sorunlara yol acabilmektedir. MTHFR, DNA sentezi, metilasyon reaksiyonlari ve RNA sentezi gibi onemli yolaklarda etkilidir. MTHFR geninde meydana gelen en yaygin polimorfizmler C677T ve A1298C' dir. Bu polimorfizmlerin sikligi populasyonlarda farklilik gostermektedir. MTHFR, folat dagilimini, homosistein metabolizmasini ve S-adenosilmetionin vasitasiyla metilasyonu etkiledigi icin MTHFR' de genetik kusurlar hastalik riskini negatif ve pozitif olarak ayarlama potansiyeline sahiptir.