SAM-dependent Methyltransferase Research Articles

Nutritional Control of Stem Cell Division through S-Adenosylmethionine in Drosophila Intestine

The intestine has direct contact with nutritional information. The mechanisms by which particular dietary molecules affect intestinal homeostasis are not fully understood. In this study, we identified S-adenosylmethionine (SAM), a universal methyl donor synthesized from dietary methionine, as a critical molecule that regulates stem cell division in Drosophila midgut. Depletion of either dietary methionine or SAM synthesis reduces division rate of intestinal stem cells. Genetic screening for putative SAM-dependent methyltransferases has identified protein synthesis as a regulator of the stem cells, partially through a unique diphthamide modification on eukaryotic elongation factor 2. In contrast, SAM in nutrient-absorptive enterocytes controls the interleukin-6-like protein Unpaired 3, which is required for rapid division of the stem cells after refeeding. Our study sheds light upon a link between diet and intestinal homeostasis and highlights the key metabolite SAM as a mediator of cell-type-specific starvation response.

The role of molecular modelling strategies in validating the effects of chrysin on sodium arsenite-induced chromosomal and DNA damage.

Chrysin (CHR) is a food-based bioactive ingredient whereas, sodium arsenite (SA) is one of the major contaminant in drinking water. When ingested, SA contributes to tissue damage due to bioactivation by S-adenosyl methionine (SAM)-dependent methyltransferase. Hence, the needs to nullify this effect by investigating the potentials of CHR on SA-induced genotoxicity in rats. The experiment was divided into two successive stages (ameliorative and preventive, curative studies) for 1 week. Rats were divided into four groups: distilled water, 10mg/kg SA, 10mg/kg CHR and co-administration. In stage 2, the experimental groups were given either CHR or SA for 1 week, and treated in reversed order for additional week. Lipid peroxidation, protein carbonyl and DNA fragmentation in liver, blood brain and bone marrow cells micronucleus were assayed for using standard protocols. Molecular docking of SAM-dependent methyltransferase in the presence of CHR was conducted. CHR significantly ( p < 0.05) decreased the level of lipid peroxidation, protein carbonyls and DNA fragmentation in blood, liver and brain tissues as against group treated with SA. It also significantly ( p<0.05) reduced the level of micronuclei generated in bone marrow cells. The effects of CHR were shown to be ameliorative, preventive and curative in nature. Furthermore, CHR was able to dock (with binding energy of -24.81 kcal/mol and predicted inhibition kinetic constant (Ki) of 0.959 µM) into the active site of SAM-dependent methyltransferase with strong hydrogen bond and hydrophobic interactions. The study might have unravelled the potentials of CHR against SA-induced chromosomal and DNA damage, which might be due to inhibition of SAM-dependent methyltransferase.

Crystal structure of SAM-dependent methyltransferase from Pyrococcus horikoshii.

Methyltransferases (MTs) are enzymes involved in methylation that are needed to perform cellular processes such as biosynthesis, metabolism, gene expression, protein trafficking and signal transduction. The cofactor S-adenosyl-L-methionine (SAM) is used for catalysis by SAM-dependent methyltransferases (SAM-MTs). The crystal structure of Pyrococcus horikoshii SAM-MT was determined to a resolution of 2.1 Å using X-ray diffraction. The monomeric structure consists of a Rossmann-like fold (domain I) and a substrate-binding domain (domain II). The cofactor (SAM) molecule binds at the interface between adjacent subunits, presumably near to the active site(s) of the enzyme. The observed dimeric state might be important for the catalytic function of the enzyme.

Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice

There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5′-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.

A QM/MM study of the catalytic mechanism of SAM methyltransferase RlmN from Escherichia coli.

RlmN is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the C2 methylation of adenosine 2503 (A2503) in 23S rRNA and adenosine 37 (A37) in several Escherichia coli transfer RNAs (tRNA). The catalytic reaction of RlmN is distinctly different from that of typical SAM-dependent methyltransferases that employs an SN 2 mechanism, but follows a ping-pong mechanism which involves the intermediate methylation of a conserved cysteine residue. Recently, the x-ray structure of a key intermediate in the RlmN reaction has been reported, allowing us to perform combined quantum mechanics and molecular mechanics (QM/MM) calculations to delineate the reaction details of RlmN at atomic level. Starting from the Cross-Linked RlmN C118A-tRNA complex, the possible mechanisms for both the formation and the resolution of the cross-linked species (IM2) have been illuminated. On the basis of our calculations, IM2 is formed by the attack of the C355-based methylene radical on the sp2 -hybridized C2 of the adenosine ring, corresponding to energy barrier of 14.4 kcal/mol, and the resolution of IM2 is confirmed to follow a radical fragmentation mechanism. The cleavage of C'-S' bond of mC355-A37 cross-link is in concert with the deprotonation of C2 by C118 residue, which is the rate-limiting step with an energy barrier of 17.4 kcal/mol. Moreover, the cleavage of C'-S' bond of IM2 can occur independently, that is, it does not require the loss of an electron of IM2 and the formation of disulfide bond between C355 and C118 as precondition. These findings would deepen the understanding of the catalysis of RlmN.

Guanidine N-methylation by BlsL Is Dependent on Acylation of Beta-amine Arginine in the Biosynthesis of Blasticidin S.

The peptidyl nucleoside blasticidin S (BS) produced by Streptomyces griseochromogenes was the first non-mercurial fungicide used to prevent rice blast and increasingly used as a selection reagent in transgenic study. Acylation by addition of a leucine residue at the beta amine group of arginine side chain of demethylblasticidin S (DBS) has been proposed as a novel self-resistance to the cytotoxic biosynthetic intermediate. But the resultant product leucyldemethylblasticidin S (LDBS) has not been isolated as a metabolite, and LDBS synthetase activity remained to be demonstrated in S. griseochromogenes. In this study, we isolated LDBS in a BS heterologous producer S. lividans WJ2 upon the deletion of blsL, which encodes a S-Adenosyl methionine-dependent methyltransferase. Purified BlsL efficiently methylated LDBS at the delta N of beta-arginine to generate the ultimate intermediate LBS, but nearly didn’t methylate DBS to final product BS. Above experiments demonstrated that LDBS is indeed an intermediate in BS biosynthetic pathway, and acylation of beta-amino group of arginine side chain is prerequisite for efficient guanidine N-methylation in addition to being a self-resistance mechanism.

Molecular cloning, heterologous expression and functional characterization of gamma tocopherol methyl transferase (γ-TMT) from Glycine max

γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is the last enzyme in the tocopherol biosynthetic pathway and it catalyzes the conversion of γ-tocopherol into α-tocopherol, the nutritionally significant and most bioactive form of vitamin E. Although the γ-TMT gene has been successfully overexpressed in many crops to enhance their α-tocopherol content but still only few attempts have been made to uncover its structural, functional and regulation aspects at protein level. In this study, we have cloned the complete 909bp coding sequence of Glycine max γ-TMT (Gm γ-TMT) gene that encodes the corresponding protein comprising of 302 amino acid residues. The deduced Gm γ-TMT protein showed 74–87% sequence identity with other characterized plant γ-TMTs. Gm γ-TMT belongs to Class I Methyl Transferases that have a Rossmann-like fold which consists of a seven-stranded β sheet joined by α helices. Heterologous expression of Gm γ-TMT in pET29a expression vector under the control of bacteriophage T7 promoter produced a 37.9 kDa recombinant Gm γ-TMT protein with histidine hexamer tag at its C-terminus. The expression of recombinant Gm γ-TMT protein was confirmed by western blotting using anti-His antibody. The recombinant protein was purified by Ni2+-NTA column chromatography. The purified protein showed SAM dependent methyltransferase activity. The α-tocopherol produced in the in-vitro reaction catalyzed by the purified enzyme was detected using reverse phase HPLC. This study has laid the foundation to unveil the biochemical understanding of Gm γ-TMT enzyme which can be further explored by studying its kinetic behaviour, substrate specificity and its interaction with other biomolecules.

Proteomic studies on anti-tumor agent ansamitocin P-3 producer Actinosynnema pretiosum in response to ammonium and isobutanol.

Our previous work showed that the biosynthesis of ansamitocin P-3 (AP-3), an anti-tumor agent, by Actinosynnema pretiosum was depressed by ammonium but enhanced by isobutanol in the medium. Here we show proteomics analyses on A. pretiosum in different fermentation conditions with and without ammonium or isobutanol using two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization, and linear ion trap quadrupole mass spectrometry. Pairwise comparison of repetitive 2-DE maps was performed to find differentially expressed spots, and eight proteins were identified as functionally annotated ones. Among these proteins, D-3-phosphoglycerate dehydrogenase (PGDH) and glyceraldehyde 3-phosphate dehydrogenase showed statistically significant up-regulation in ammonium vs. basic or isobutanol medium, while fatty acid synthetase, histidine-tRNA ligase, transposase, molecular chaperone GroEL, SAM-dependent methyltransferase, and Crp/Fnr family transcriptional regulator were overexpressed in ammonium vs. basic medium. Based on the 2-DE data, exogenous L-serine which could inhibit the PGDH activity was added to the cultures with isobutanol, and a lower AP-3 production was confirmed under 2.5mM serine addition (24 or 48h).

Identification and Characterization of Protein Arginine Methyltransferase 1 in Acanthamoeba castellanii.

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.

Discovery of Novel Disruptor of Silencing Telomeric 1-Like (DOT1L) Inhibitors using a Target-Specific Scoring Function for the (S)-Adenosyl-l-methionine (SAM)-Dependent Methyltransferase Family.

The disruptor of telomeric silencing 1-like (DOT1L) protein is a histone H3K79 methyltransferase that plays a key role in transcriptional elongation and cell cycle regulation and is required for the development and maintenance of MLL-rearranged mixed lineage leukemia. Much effort has been dedicated toward discovering novel scaffold DOT1L inhibitors using different strategies. Here, we report the development and application of a target-specific scoring function, the SAM score, for (S)-adenosyl-l-methionine (SAM)-dependent methyltransferases, for the discovery of novel DOT1L inhibitors. On the basis of the SAM score, we successfully identified a novel class of DOT1L inhibitors with a scaffold of [1,2,4]-triazolo-[3,4-b][1,3,4]-thiadiazole, in which compound 6 exhibits an IC50 value of 8.3 μM with selectivity versus other tested SAM-dependent methyltransferases. In cellular studies, 6 selectively targets DOT1L, blocks the proliferation of mixed lineage leukemia cell lines, and causes cell cycle arrest and apoptosis. Moreover, we analyzed the putative binding modes of 6 and its analogues obtained by molecular docking, which may assist with the future development of DOT1L inhibitors with improved potency and selectivity profiles.

The Catalytic Mechanism of the Class C Radical S-Adenosylmethionine Methyltransferase NosN.

S-Adenosylmethionine (SAM) is one of the most common co-substrates in enzyme-catalyzed methylation reactions. Most SAM-dependent reactions proceed through an SN 2 mechanism, whereas a subset of them involves radical intermediates for methylating non-nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM-dependent methyltransferases, NosN does not produce S-adenosylhomocysteine (SAH) as a co-product. Instead, NosN converts SAM into 5'-methylthioadenosine as a direct methyl donor, employing a radical-based mechanism for methylation and releasing 5'-thioadenosine as a co-product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme-catalyzed methylation reactions.

Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase

BackgroundControl of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans. MethodsC-5 methyl cytosines (m5C) were detected by immuno-blotting with m5C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry. ResultsMethylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m4C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m5C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc. ConclusionDR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m5C. General significanceAlthough, dispensable under optimal growth conditions, the presence of m5C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.

Structural and functional characterization of the TYW3/Taw3 class of SAM-dependent methyltransferases.

S-adenosylmethionine (SAM)-dependent methyltransferases regulate a wide range of biological processes through the modification of proteins, nucleic acids, polysaccharides, as well as various metabolites. TYW3/Taw3 is a SAM-dependent methyltransferase responsible for the formation of a tRNA modification known as wybutosine and its derivatives that are required for accurate decoding in protein synthesis. Here, we report the crystal structure of Taw3, a homolog of TYW3 from Sulfolobus solfataricus, which revealed a novel α/β fold. The sequence motif (S/T)xSSCxGR and invariant aspartate and histidine, conserved in TYW3/Taw3, cluster to form the catalytic center. These structural and sequence features indicate that TYW3/Taw3 proteins constitute a distinct class of SAM-dependent methyltransferases. Using site-directed mutagenesis along with in vivo complementation assays combined with mass spectrometry as well as ligand docking and cofactor binding assays, we have identified the active site of TYW3 and residues essential for cofactor binding and methyltransferase activity.

Clinical and Genomic Analysis of Liver Abscess-Causing Klebsiella pneumoniae Identifies New Liver Abscess-Associated Virulence Genes.

Hypervirulent variants of Klebsiella pneumoniae (hvKp) that cause invasive community-acquired pyogenic liver abscess (PLA) have emerged globally. Little is known about the virulence determinants associated with hvKp, except for the virulence genes rmpA/A2 and siderophores (iroBCD/iucABCD) carried by the pK2044-like large virulence plasmid. Here, we collected most recent clinical isolates of hvKp from PLA samples in China, and performed clinical, molecular, and genomic sequencing analyses. We found that 90.9% (40/44) of the pathogens causing PLA were K. pneumoniae. Among the 40 LA-Kp, K1 (62.5%), and K2 (17.5%) were the dominant serotypes, and ST23 (47.5%) was the major sequence type. S1-PFGE analyses demonstrated that although 77.5% (31/40) of the LA-Kp isolates harbored a single large virulence plasmid varied in size, 5 (12.5%) isolates had no plasmid and 4 (10%) had two or three plasmids. Whole genome sequencing and comparative analysis of 3 LA-Kp and 3 non-LA-Kp identified 133 genes present only in LA-Kp. Further, large scale screening of the 133 genes in 45 LA-Kp and 103 non-LA-Kp genome sequences from public databases identified 30 genes that were highly associated with LA-Kp, including iroBCD, iucABCD and rmpA/A2 and 21 new genes. Then, these 21 new genes were analyzed in 40 LA-Kp and 86 non-LA-Kp clinical isolates collected in this study by PCR, showing that new genes were present 80–100% among LA-Kp isolates while 2–11% in K. pneumoniae isolates from sputum and urine. Several of the 21 genes have been proposed as virulence factors in other bacteria, such as the gene encoding SAM-dependent methyltransferase and pagO which protects bacteria from phagocytosis. Taken together, these genes are likely new virulence factors contributing to the hypervirulence phenotype of hvKp, and may deepen our understanding of virulence mechanism of hvKp.

Crystal structure and enantioselectivity of terpene cyclization in SAM-dependent methyltransferase TleD.

TleD is a SAM (S-adenosyl-l-methionine)-dependent methyltransferase and acts as one of the key enzymes in the teleocidin B biosynthesis pathway. Besides methyl transferring, TleD also rearranges the geranyl and indole moieties of the precursor to form a six-membered ring. Moreover, it does not show homologies with any known terpenoid cyclases. In order to elucidate how such a remarkable reaction could be achieved, we determined the complex crystal structures of TleD and the cofactor analogue S-adenosyl-l-homocysteine with or without the substrate teleocidin A1. A domain-swapped pattern via an additional N-terminal α-helix is observed in TleD hexamers. Structural comparison and alignment shows that this additional N-terminal α-helix is the common feature of SAM methyltransferase-like cyclases TleD and SpnF. The residue Tyr21 anchors the additional N-terminal α-helix to a 'core SAM-MT fold' and is a key residue for catalytic activity. Molecular dynamics simulation results suggest that the dihedral angle C23-C24-C25-C26 of teleocidin A1 is preferred to 60-90° in the TleD and substrate complex structure, which tend to adopt a Re-face stereocenter at C25 position after reaction and is according to in vitro enzyme reaction experiments. Our results also demonstrate that methyl transfer can be a new chemical strategy for carbocation formation in the terpene cyclization, which is the key initial step.

A metabolomics-driven approach reveals metabolic responses and mechanisms in the rat heart following myocardial infarction

BackgroundMyocardial infarction (MI) is caused by myocardial necrosis resulting from prolonged ischemia. However, the biological mechanisms underlying MI remain unclear. MethodsWe evaluated metabolic and lipidomic changes in rat heart tissue from sham and MI at 1h, 1day and 10day after coronary ligation, using global profiling based on metabolomics. ResultsA time-dependent increase or decrease in polar and lipid metabolite levels was measured. The S-adenosylmethionine (SAM) concentration and the SAM/S-adenosylhomocysteine (SAH) ratio gradually decreased in a time-dependent manner and were significantly downregulated 10days after MI. Transcriptome analysis revealed that the levels of coenzyme Q (Coq)-3 and Coq5, both of which are SAM-dependent methyltransferases, were decreased in the MI groups. These results suggested that dysregulation of SAM may be related to down regulated COQ biosynthetic pathway. In addition, short-chain (C3) and medium-chain (C4–C12) acylcarnitine levels gradually decreased, whereas long-chain acylcarnitine (C14–18) levels increased, owing to a defect in β-oxidation during ischemia. These changes are related to energy-dependent metabolic pathways, and a subsequent decrease in adenosine triphosphate concentration was observed. ConclusionsThe comprehensive integration of various omics data provides a novel means of understanding the underlying pathophysiological mechanisms of MI.

Abstract 9: Metabolic profiling of bladder cancer cell lines reveals molecular alterations involved in methylation and novel epigenetic phenotype

Abstract Background: Bladder cancer (BCa) is primarily “carcinogen driven cancer”. Epidemiological studies indicate environmental factors play a major causative role compared to genetic factors. Xenobiotic metabolism is highly perturbed and precise mechanisms involved are poorly understood during BCa progression. We identified metabolic signature that can distinguish bladder cancer from controls and reveals major alterations in phaseI/II enzymes involved in xenobiotic metabolism and suggest a key role for epigenetic modifications. Material and Methods: In this study, we used mass spectrometry based metabolomics profiling coupled with enrichment-based bioprocess mapping to obtain insight into biochemical alterations in bladder cancer cell lines. We further validated related gene expression using real time quantitative PCR (qPCR) and proteins using western blotting. Results: In this study, we used high-throughput mass spectrometry to measure over 350 compounds in seven bladder cell lines, identifying 91 metabolites which exhibited significant changes in bladder Cancer. Most importantly, methylated, hydroxylated and acetylated metabolites are altered. Interestingly, S-Adenosyl methionine (SAM) is the most prominent pathway upregulated corroborated with our previous findings obtained using patient derived metabolomic data from two independent cohorts. Second, we observe many of phaseI/phaseII metabolic enzymes including aldehyde oxidase (AOX1), cytochrome P450 1A1 (CYP1A1), CYP1B1, Glutathione S-transferase T1 (GSTT1), Glutathione S-transferase M2 (GSTM2), N-acetyl transferase I NAT1 and NAT2 are transcriptionally repressed in BCa cell line compared to benign indicating the pivotal role of methylation in gene silencing. Interestingly, we observe differential expression of polycomb group of proteins (Pcg) associated with PRC2 and PRC1 complex. Specifically, histone-lysine N-methyl transferase (EZH2) protein, which is SAM dependent histone methyl transferase and concomitant 3meHK27 trimethylated histone K27, is highly expressed in metastatic UMUC3 BCa cell line further indicating prominence of epigenetic modifications. Conclusion: We present an integrative pathway analysis of a metabolic gene signature which has not been previously described in the context of bladder cancer cell lines. Further mechanistic analyses reveals prominent role for methylation status and associated epigenetic modifications being played in the transcriptional repression of key xenobiotic enzymes. Collectively, our novel findings provide an opportunity for development of efficient biomarker implications and epigenetic therapy targeting BCa progression. Citation Format: Rashmi Krishnapuram, Franklin Gu, Salil Kumar Bhowmik, Suman Maity, Mohan Manikkam, Friedrich-Carl von Rundstedt, Vasanta Putluri, Yair Lotan, Jonathan M. Levitt, Seth P. Lerner, Cristian Coarfa, Arun Sreekumar, Nagireddy Putlurip. Metabolic profiling of bladder cancer cell lines reveals molecular alterations involved in methylation and novel epigenetic phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 9.

Abstract 1267: Identification and characterization of oncogenic methyltransferase ESOC1 as a diagnostic and therapeutic target for esophageal cancer

Abstract Esophageal cancer is one of the most lethal malignancies of the digestive tract, and at the time of diagnosis most of the patients are at advanced stages. In spite of the use of current surgical techniques combined with various treatment modalities, such as radiotherapy and chemotherapy, the prognosis of esophageal cancer still remains poor. Therefore, further development of new anti-cancer drugs for this disease with minimum risk of adverse event is urgently awaited. To screen oncoantigens which could be used for the development of new diagnostic biomarkers as well as molecular targeted drugs and/or immunotherapy, we have established a screening system as follows; i) To identify overexpressed genes in esophageal cancers by genome-wide screening using the microarray representing 27,648 genes and pure populations of tumor cells taken from cancer tissues by laser microdissection, ii) To verify the candidate genes for their very low or absent expression in normal tissues by northern-blotting, iii) To validate the clinicopathological significance of their expression with tissue microarray covering hundreds of archived esophageal cancers, iv) To verify whether the target genes are essential for the growth or survival of cancer cells by RNAi and cell growth assays. During this process, we selected dozens of druggable oncoproteins with various enzymatic activities, and identified overexpression of metyltransferase ESOC1 (esophageal cancer-associated oncoprotein 1) in the majority of esophageal cancers and its scarce expression in normal tissues except testis. Immunohistochemical staining using tumor tissue microarrays consisting of 251 esophageal cancers demonstrated that ESOC1 expression was associated with poor prognosis (P = 0.0029 by log-rank test), and multivariate analysis confirmed its independent prognostic value (P = 0.0082). Suppression of ESOC1 expression with siRNA effectively suppressed the growth of cancer cells. ESOC1 has a SAM dependent methyltransferase domain, and we confirmed its methyltransferase activity for some small non-coding RNAs that were overexpressed in esophageal cancers. In addition, we confirmed that the exogenous expression of ESOC1 increased the growth activity of HEK293T cells, but the mutant ESOC1 did not have methyltransferase activity, and did not confer the growth promoting activity. Since ESOC1 plays a significant role in the cell growth and malignant nature of tumors, we suggest that ESOC1 is likely to be a prognostic biomarker and a potential therapeutic target for developing new anti-cancer drugs and cancer vaccines. Citation Format: Yataro Daigo, Atsushi Takano, Yusuke Nakamura. Identification and characterization of oncogenic methyltransferase ESOC1 as a diagnostic and therapeutic target for esophageal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1267.

Maize OXIDATIVE STRESS2 Homologs Enhance Cadmium Tolerance in Arabidopsis through Activation of a Putative SAM-Dependent Methyltransferase Gene.

Previously the Arabidopsis (Arabidopsis thaliana) zinc finger protein OXIDATIVE STRESS2 (AtOXS2) and four OXS2-like (AtO2L) family members were described to play a role in stress tolerance and stress escape. For stress escape, SOC1 was a target of AtOXS2. However, for stress tolerance, the downstream targets were not identified. We cloned two OXS2 homolog genes from sweet corn, ZmOXS2b and ZmO2L1 Both genes are transiently inducible by Cd treatment. When expressed in Arabidopsis, each enhances tolerance against cadmium. Further analysis showed that ZmOXS2b and ZmO2L1 proteins enhance Cd tolerance in Arabidopsis by activating at least one target gene, that encoding a putative S-adenosyl-l-Met-dependent methyltransferase superfamily protein (AT5G37990), which we named CIMT1 This activation involves the in vivo interaction with a segment of the CIMT1 promoter that contains a BOXS2 motif previously identified as the binding element for AtOXS2. More importantly, CIMT1 is induced by Cd treatment, and overexpression of this gene alone was sufficient to enhance Cd tolerance in Arabidopsis. The connection of ZmOXS2b and ZmO2L1 to Arabidopsis CIMT1 suggests a similar network may exist in maize (Zea mays) and may provide a clue to possibly using a CIMT1 maize homolog to engineer stress tolerance in a major crop.

Identification of candidate genes for drought tolerance in coffee by high-throughput sequencing in the shoot apex of different Coffea arabica cultivars.

BackgroundDrought is a widespread limiting factor in coffee plants. It affects plant development, fruit production, bean development and consequently beverage quality. Genetic diversity for drought tolerance exists within the coffee genus. However, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. In this study, we compared the molecular responses to drought in two commercial cultivars (IAPAR59, drought-tolerant and Rubi, drought-susceptible) of Coffea arabica grown in the field under control (irrigation) and drought conditions using the pyrosequencing of RNA extracted from shoot apices and analysing the expression of 38 candidate genes.ResultsPyrosequencing from shoot apices generated a total of 34.7 Mbp and 535,544 reads enabling the identification of 43,087 clusters (41,512 contigs and 1,575 singletons). These data included 17,719 clusters (16,238 contigs and 1,575 singletons) exclusively from 454 sequencing reads, along with 25,368 hybrid clusters assembled with 454 sequences. The comparison of DNA libraries identified new candidate genes (n = 20) presenting differential expression between IAPAR59 and Rubi and/or drought conditions. Their expression was monitored in plagiotropic buds, together with those of other (n = 18) candidates genes. Under drought conditions, up-regulated expression was observed in IAPAR59 but not in Rubi for CaSTK1 (protein kinase), CaSAMT1 (SAM-dependent methyltransferase), CaSLP1 (plant development) and CaMAS1 (ABA biosynthesis). Interestingly, the expression of lipid-transfer protein (nsLTP) genes was also highly up-regulated under drought conditions in IAPAR59. This may have been related to the thicker cuticle observed on the abaxial leaf surface in IAPAR59 compared to Rubi.ConclusionsThe full transcriptome assembly of C. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. Using these data, candidate genes were selected and their differential expression profiles were confirmed by qPCR experiments in plagiotropic buds of IAPAR59 and Rubi under drought conditions. As regards the genes up-regulated under drought conditions, specifically in the drought-tolerant IAPAR59, several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as ABA and lipid metabolism, for example. The identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0777-5) contains supplementary material, which is available to authorized users.