[Interaction of DNA Methyltransferase Dnmt3a with Phosphorus Analogs of S-Adenosylmethionine and S-Adenosylhomocysteine].

Enzymatic methyltransferase reactions are of crucial importance for cell metabolism. S-Adenosyl-L-methionine (AdoMet) is a main donor of the methyl group. DNA, RNA, proteins, and low-molecular-weight compounds are substrates of methyltransferases. In mammals, DNA methyltransferase Dnmt3a de novo methylates the C5 position of cytosine residues in CpG sequences in DNA. The methylation pattern is one of the factors that determine the epigenetic regulation of gene expression. Here, interactions with the catalytic domain of Dnmt3a was for the first time studied for phosphonous and phosphonic analogs of AdoMet and S-adenosyl-L-homocysteine (AdoHcy), in which the carboxyl group was substituted for respective phosphorus-containing group. These AdoMet analogs were shown to be substrates of Dnmt3a, and the methylation efficiency was only halved as compared with that of natural AdoMet. Both phosphorus-containing analogs of AdoHcy, which is a natural methyltransferase inhibitor, showed similar inhibitory activities toward Dnmt3a and were approximately four times less active than AdoHcy. The finding that the phosphonous and phosphonic analogs are similar in activity was quite unexpected because the geometry and charge of their phosphorus-containing groups differ substantially. The phosphorus-containing analogs of AdoMet and AdoHcy are discussed as promising tools for investigation of methyltransferases.

Insufficient S-adenosylhomocysteine hydrolase compromises the beneficial effect of diabetic BMSCs on diabetic cardiomyopathy.

BACKGROUND: Autologous stem cell therapy is a promising strategy for cardiovascular diseases including diabetic cardiomyopathy (DCM), but conclusions from clinical trials were compromised. We assumed that diabetes might induce the dysfunction of stem cells and thus limit its therapeutic effect. This study aimed to compare the effect of diabetes and nondiabetes-derived bone marrow mesenchymal stem cells (BMSCs) transplantation on DCM and explored the potential mechanism. METHODS: Rats with diabetes were induced using high-fat diets and streptozotocin (STZ) injection. BMSCs harvested from diabetic and nondiabetic rats were infused into DCM rats, and the effects on the heart were identified by echocardiography and histopathology. The inhibition or overexpression of SAHH in nondiabetic and diabetic BMSCs was used to confirm its key role in stem cell activity and cardiac therapy. RESULTS: Compared with normal BMSCs, the therapeutic effects of diabetic rat-derived stem cells on improving cardiac function and adverse remodeling were significantly attenuated. In vitro, diabetic BMSCs had lower cell viability and paracrine function than nondiabetic BMSCs. It was further found that diabetic BMSCs had obvious mitochondrial oxidative stress damage and S-adenosylhomocysteine (SAH) accumulation due to S-adenosylhomocysteine hydrolase (SAHH) deficiency. SAHH inhibition by adenosine dialdehyde (ADA) or shSAHH plasmid in normal BMSCs significantly reduced the favorable effects on endothelial cell proliferation and tube-forming capacity. In contrast, SAHH overexpression in diabetic BMSCs significantly improved cellular activity and paracrine function. Transplantation of BMSCs with SAHH overexpression improved cardiac adverse remodeling and angiogenesis. Activation of the Nrf2 signaling pathway may be one of the key mechanisms of SAHH-mediated improvement of stem cell viability and cardiac repair. CONCLUSIONS: Diabetes leads to compromised bioactivity and repair capacity of BMSCs. Our study suggests that SAHH activation may improve the cardioprotective effect of autologous transplantation of diabetes-derived BMSCs on patients with DCM. Diabetes induced the inhibition of S-adenosylhomocysteine (SAH) expression and aging phenotype in BMSCs and thus decreased the cell viability and paracrine function. Compared with normal BMSCs, the therapeutic effects of diabetic rat-derived BMSCs on improving cardiac function and adverse remodeling were significantly attenuated. SAHH overexpression in diabetic BMSCs significantly rescued cellular function partly via activating Nrf2/HO-1 signal. Transplantation of diabetic BMSCs with SAHH overexpression improved angiogenesis and cardiac adverse remodeling in rats.

Discovery of Inhibitors of DNA Methyltransferase 2, an Epitranscriptomic Modulator and Potential Target for Cancer Treatment.

Selective manipulation of the epitranscriptome could be beneficial for the treatment of cancer and also broaden the understanding of epigenetic inheritance. Inhibitors of the tRNA methyltransferase DNMT2, the enzyme catalyzing the S-adenosylmethionine-dependent methylation of cytidine 38 to 5-methylcytidine, were designed, synthesized, and analyzed for their enzyme-binding and -inhibiting properties. For rapid screening of potential DNMT2 binders, a microscale thermophoresis assay was established. Besides the natural inhibitors S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG), we identified new synthetic inhibitors based on the structure of N-adenosyl-2,4-diaminobutyric acid (Dab). Structure-activity relationship studies revealed the amino acid side chain and a Y-shaped substitution pattern at the 4-position of Dab as crucial for DNMT2 inhibition. The most potent inhibitors are alkyne-substituted derivatives, exhibiting similar binding and inhibitory potencies as the natural compounds SAH and SFG. CaCo-2 assays revealed that poor membrane permeabilities of the acids and rapid hydrolysis of an ethylester prodrug might be the reasons for the insufficient activity in cellulo.

Inhibition of S-adenosylhomocysteine hydrolase induces endothelial senescence via hTERT downregulation.

BACKGROUND AND AIMS: It has been established that endothelial senescence plays a critical role in the development of atherosclerosis. Elevated S-adenosylhomocysteine (SAH) level induced by inhibition of S-adenosylhomocysteine hydrolase (SAHH) is one of the risk factors of atherosclerosis; however, the interplay between endothelial senescence and inhibition of SAHH is largely unknown. METHODS: Human umbilical vein endothelial cells (HUVECs) after serial passage were used. SAHH-specific inhibitor adenosine dialdehyde (ADA) and SAHH siRNA treated HUVECs and SAHH+/-mice were used to investigate the effect of SAHH inhibition on endothelial senescence. RESULTS: HUVECs exhibited distinct senescence morphology as HUVECs were passaged, together with a decrease in intracellular SAHH expression and an increase in intracellular SAH levels. SAHH inhibition by ADA or SAHH siRNA elevated SA ß-gal activity, arrested proliferation, and increased the expression of p16, p21 and p53 in HUVECs and the aortas of mice. In addition, decreased expression of hTERT and reduced occupancy of H3K4me3 over the hTERT promoter region were observed following SAHH inhibition treatment. To further verify the role of hTERT in the endothelial senescence induced by SAHH inhibition, hTERT was overexpressed with a plasmid vector under CMV promoter. hTERT overexpression rescued the senescence phenotypes in endothelial cells induced by SAHH inhibition. CONCLUSIONS: SAHH inhibition induces endothelial senescence via downregulation of hTERT expression, which is associated with attenuated histone methylation over the hTERT promoter region.

Elevated S-adenosylhomocysteine induces adipocyte dysfunction to promote alcohol-associated liver steatosis.

It has been previously shown that chronic ethanol administration-induced increase in adipose tissue lipolysis and reduction in the secretion of protective adipokines collectively contribute to alcohol-associated liver disease (ALD) pathogenesis. Further studies have revealed that increased adipose S-adenosylhomocysteine (SAH) levels generate methylation defects that promote lipolysis. Here, we hypothesized that increased intracellular SAH alone causes additional related pathological changes in adipose tissue as seen with alcohol administration. To test this, we used 3-deazaadenosine (DZA), which selectively elevates intracellular SAH levels by blocking its hydrolysis. Fully differentiated 3T3-L1 adipocytes were treated in vitro for 48 h with DZA and analysed for lipolysis, adipokine release and differentiation status. DZA treatment enhanced adipocyte lipolysis, as judged by lower levels of intracellular triglycerides, reduced lipid droplet sizes and higher levels of glycerol and free fatty acids released into the culture medium. These findings coincided with activation of both adipose triglyceride lipase and hormone sensitive lipase. DZA treatment also significantly reduced adipocyte differentiation factors, impaired adiponectin and leptin secretion but increased release of pro-inflammatory cytokines, IL-6, TNF and MCP-1. Together, our results demonstrate that elevation of intracellular SAH alone by DZA treatment of 3T3-L1 adipocytes induces lipolysis and dysregulates adipokine secretion. Selective elevation of intracellular SAH by DZA treatment mimics ethanol's effects and induces adipose dysfunction. We conclude that alcohol-induced elevations in adipose SAH levels contribute to the pathogenesis and progression of ALD.

A Mass Spectrometric Assay of METTL3/METTL14 Methyltransferase Activity.

A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (m6A) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, m6A can modulate mRNA splicing, export, localization, degradation, and translation. Although potent and selective inhibitors exist for several members of the Type I S-adenosylmethionine (SAM)-dependent methyltransferase family, no inhibitors have been reported for METTL3/METTL14 to date. To facilitate drug discovery efforts, a sensitive and robust mass spectrometry-based assay for METTL3/METTL14 using self-assembled monolayer desorption/ionization (SAMDI) technology has been developed. The assay uses an 11-nucleotide single-stranded RNA compared to a previously reported 27-nucleotide substrate. IC50 values of mechanism-based inhibitors S-adenosylhomocysteine (SAH) and sinefungin (SFG) are comparable between the SAMDI and radiometric assays that use the same substrate. This work demonstrates that SAMDI technology is amenable to RNA substrates and can be used for high-throughput screening and compound characterization for RNA-modifying enzymes.

Procaine and S-Adenosyl-l-Homocysteine Affect the Expression of Genes Related to the Epigenetic Machinery and Change the DNA Methylation Status of In Vitro Cultured Bovine Skin Fibroblasts.

Cloning using somatic cell nuclear transfer (SCNT) has many potential applications such as in transgenic and genomic-edited animal production. Abnormal epigenetic reprogramming of somatic cell nuclei is probably the major cause of the low efficiency associated with SCNT. Strategies to alter DNA reprogramming in donor cell nuclei may help improve the cloning efficiency. In the present study, we aimed to characterize the effects of procaine and S-adenosyl-l-homocysteine (SAH) as demethylating agents during the cell culture of bovine skin fibroblasts. We characterized the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A), DNA methyltransferase 3 beta (DNMT3B), TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts. We found that DNA methylation levels of satellite I were reduced by SAH (p = 0.0495) and by the combination of SAH and procaine (p = 0.0479) compared with that in the control group. Global DNA methylation levels were lower in cells that were cultivated with both compounds than in control cells (procaine [p = 0.0116], SAH [p = 0.0408], and both [p = 0.0163]). Regarding gene expression, there was a decrease in the DNMT1 transcript levels in cells cultivated with SAH (p = 0.0151) and SAH/procaine (0.0001); a decrease in the DNMT3A transcript levels in cells cultivated with SAH/procaine (p = 0.016); and finally, a decrease in the DNMT3B transcript levels in cells cultivated with procaine (p = 0.0007), SAH (p = 0.0060), and SAH/procaine (p = 0.0021) was found. Higher levels of TET3 transcripts in cells cultivated with procaine (p = 0.0291), SAH (p = 0.0373), and procaine/SAH (p = 0.0013) compared with the control were also found. Regarding the OCT4 gene, no differences were found. Our results showed that the use of procaine and SAH during bovine cell culture was able to alter the epigenetic profile of the cells. This approach may be a useful alternative strategy to improve the efficiency of reprogramming the somatic nuclei after fusion, which in turn will improve the SCNT efficiency.

Characterization of Inhibitor Binding Through Multiple Inhibitor Analysis: A Novel Local Fitting Method.

Understanding inhibitor binding modes is a key aspect of drug development. Early in a drug discovery effort these considerations often impact hit finding strategies and hit prioritization. Multiple inhibitor experiments, where enzyme inhibition is measured in the presence of two simultaneously varied inhibitors, can provide valuable information about inhibitor binding. These experiments utilize the inhibitor concentration dependence of the observed combined inhibition to determine the relationship between two compounds. In this way, it can be determined whether two inhibitors bind exclusively, independently, synergistically, or antagonistically. Novel inhibitors can be tested against each other or reference compounds to assist hit classification and characterization of inhibitor binding. In this chapter, we discuss the utility and design of multiple inhibitor experiments and present a new local curve fitting method for analyzing these data utilizing IC50 replots. The IC50 replot method is analogous to that used for determining mechanisms of inhibition with respect to substrate, as originally proposed by Cheng and Prusoff (Cheng and Prusoff Biochem Pharmacol 22: 3099-3108, 1973). The IC50 replot generated by this method reveals distinct patterns that are diagnostic of the nature of the interaction between two inhibitors. Multiple inhibition of the histone methyltransferase EZH2 by EPZ-5687 and the reaction product S-adenosylhomocysteine is presented as an example of the method.

Cloning, expression and characterization of histidine-tagged biotin synthase of Mycobacterium tuberculosis.

The emergence of Mycobacterium tuberculosis strains that are resistant to the current anti-tuberculosis (TB) drugs necessitates a need to develop a new class of drugs whose targets are different from the current ones. M. tuberculosis biotin synthase (MtbBS) is one such target that is essential for the survival of the bacteria. In this study, MtbBS was cloned, overexpressed and purified to homogeneity for biochemical characterization. It is likely to be a dimer in its native form. Its pH and temperature optima are 8.0 and 37 °C, respectively. Km for DTB and SAM was 2.81 ± 0.35 and 9.95 ± 0.98 µM, respectively. The enzyme had a maximum velocity of 0.575 ± 0.015 µM min(-1), and a turn-over of 0.0935 min(-1). 5'-deoxyadenosine (dAH), S-(5'-Adenosyl)-l-cysteine (AdoCy) and S-(5'-Adenosyl)-l-homocysteine (AdoHcy) were competitive inhibitors of MtbBS with the following inactivation parameters: Ki = 24.2 µM, IC50 = 267.4 µM; Ki = 0.84 µM, IC50 = 9.28 µM; and Ki = 0.592 µM, IC50 = 6.54 µM for dAH, AdoCy and AdoHcy respectively. dAH could inhibit the growth of M. tuberculosis H37Ra with an MIC of 392.6 µg/ml. This information should be useful for the discovery of inhibitors of MtbBS.

SAH derived potent and selective EZH2 inhibitors.

A series of novel enhancer of zeste homolog 2 (EZH2) inhibitors was designed based on the chemical structure of the histone methyltransferase (HMT) inhibitor SAH (S-adenosyl-l-homocysteine). These nucleoside-based EZH2 inhibitors blocked the methylation of nucleosomes at H3K27 in biochemical assays employing both WT PRC2 complex as well as a Y641N mutant PRC2 complex. The most potent compound, 27, displayed IC50's against both complexes of 270 nM and 70 nM, respectively. To our knowledge, compound 27 is the most potent SAH-derived inhibitor of the EZH2 PRC2 complex yet identified. This compound also displayed improved potency, lipophilic efficiency (LipE), and selectivity profile against other lysine methyltransferases compared with SAH.

Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

Synthesis and antiviral activities of 3-deaza-3-fluoroaristeromycin and its 5' analogues.

The naturally occurring adenine based carbocyclic nucleosides aristeromycin and neplanocin A and their 3-deaza analogues have found a prominent place in the search for diverse antiviral activity agent scaffolds because of their ability to inhibit S-adenosylhomocysteine (AdoHcy) hydrolase. Following the lead of these compounds, their 3-deaza-3-fluoroaristeromycin analogues have been synthesized and their effect on S-adenosylhomocysteine hydrolase and RNA and DNA viruses determined.

S-adenosylmethionine mediates inhibition of inflammatory response and changes in DNA methylation in human macrophages.

S-adenosylmethionine (SAM), the unique methyl donor in DNA methylation, has been shown to lower lipopolysaccharide (LPS)-induced expression of the proinflammatory cytokine TNF-α and increase the expression of the anti-inflammatory cytokine IL-10 in macrophages. The aim of this study was to assess whether epigenetic mechanisms mediate the anti-inflammatory effects of SAM. Human monocytic THP1 cells were differentiated into macrophages and treated with 0, 500, or 1,000 µmol/l SAM for 24 h, followed by stimulation with LPS. TNFα and IL-10 expression levels were measured by real-time PCR, cellular concentrations of SAM and S-adenosylhomocysteine (SAH), a metabolite of SAM, were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and DNA methylation was measured with LC-MS/MS and microarrays. Relative to control (0 µmol/l SAM), treatment with 500 µmol/l SAM caused a significant decrease in TNF-α expression (-45%, P < 0.05) and increase in IL-10 expression (+77%, P < 0.05). Treatment with 1,000 µmol/l SAM yielded no significant additional benefits. Relative to control, 500 µmol/l SAM increased cellular SAM concentrations twofold without changes in SAH, and 1,000 µmol/l SAM increased cellular SAM sixfold and SAH fourfold. Global DNA methylation increased 7% with 500 µmol/l SAM compared with control. Following treatment with 500 µmol/l SAM, DNA methylation microarray analysis identified 765 differentially methylated regions associated with 918 genes. Pathway analysis of these genes identified a biological network associated with cardiovascular disease, including a subset of genes that were differentially hypomethylated and whose expression levels were altered by SAM. Our data indicate that SAM modulates the expression of inflammatory genes in association with changes in specific gene promoter DNA methylation.

Elevated S-adenosylhomocysteine alters adipocyte functionality with corresponding changes in gene expression and associated epigenetic marks.

Maternal deficiencies in micronutrients affecting one-carbon metabolism before and during pregnancy can influence metabolic status and the degree of insulin resistance and obesity of the progeny in adulthood. Notably, maternal and progeny plasma S-adenosylhomocysteine (SAH) levels are both elevated after vitamin deficiency in pregnancy. Therefore, we investigated whether this key one-carbon cycle intermediate directly affects adipocyte differentiation and function. We found that expansion and differentiation of murine 3T3-L1 preadipocytes in the presence of SAH impaired both basal and induced glucose uptake as well as lipolysis compared with untreated controls. SAH did not alter preadipocyte factor 1 (Dlk1) or peroxisome proliferator-activated receptor-γ 2 (Pparγ2) but significantly reduced expression of CAAT enhancer-binding protein-α (Cebpα), Cebpß, and retinoid x receptor-α (Rxrα) compared with untreated adipocytes. SAH increased Rxrα methylation on a CpG unit (chr2:27,521,057+, chr2:27,521,049+) and CpG residue (chr2:27,521,080+), but not Cebpß methylation, relative to untreated adipocytes. Trimethylated histone H3-Lys27 occupancy was significantly increased on Cebpα and Rxrα promoters in SAH-treated adipocytes, consistent with the reduction in gene expression. In conclusion, SAH did not affect adipogenesis per se but altered adipocyte functionality through epigenetic mechanisms, such that they exhibited altered glucose disposal and lipolysis. Our findings implicate micronutrient imbalance in subsequent modulation of adipocyte function.

Combination of S-adenosylhomocysteine and scriptaid, a non-toxic epigenetic modifying reagent, modulates the reprogramming of bovine somatic-cell nuclear transfer embryos.

The goal of this study was to improve the development of bovine somatic-cell nuclear transfer (SCNT) embryos by optimizing the combination of DNA methyltransferases inhibitor S-adenosylhomocysteine (SAH) and histone deacetylase inhibitor Scriptaid (SPD). A. 4 × 4-factor design of different drug combinations (0, 0.75, 1.0, and 1.5 mM SAH and 0, 5, 250, and 500 nM SPD) was used to identify an optimal combination of 0.75 mM SAH and 250 nM SPD that improved the developmental competence of bovine SCNT embryos. Further experiments using this combination revealed that methylation levels of CpG islands near exon 1 of the pluripotent gene SOX2; the epigenetic-related gene HDAC3 and DNMT3a; imprinted genes XIST and PEG3; as well as apoptosis-related genes BCL2 and BAX were returned to levels similar to those of in vitro fertilized (IVF) embryo after treatment, which also normalized transcript levels for these genes. This combination also returned global DNA methylation to a normal level, correcting H4K12ac levels while enhancing H3K9ac levels. Thus, the combined application of 0.75 mM SAH and 250 nM SPD can significantly improve the reprogramming of bovine SCNT embryos by stabilizing how embryos utilize their genomes.

S-adenosyl-homocysteine is a weakly bound inhibitor for a flaviviral methyltransferase.

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.

Bromo-deaza-SAH: a potent and selective DOT1L inhibitor.

Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia.

Involvement of Src and the actin cytoskeleton in the antitumorigenic action of adenosine dialdehyde.

Transmethylation is an important reaction that transfers a methyl group in S-adenosylmethionine (SAM) to substrates such as DNA, RNA, and proteins. It is known that transmethylation plays critical roles in various cellular responses. In this study, we examined the effects of transmethylation on tumorigenic responses and its regulatory mechanism using an upregulation strategy of adenosylhomocysteine (SAH) acting as a negative feedback inhibitor. Treatment with adenosine dialdehyde (AdOx), an inhibitor of transmethylation-suppressive adenosylhomocysteine (SAH) hydrolase (SAHH), enhanced the level of SAH and effectively blocked the proliferation, migration, and invasion of cancer cells; the treatment also induced the differentiation of C6 glioma cells and suppressed the neovascular genesis of eggs in a dose-dependent manner. Through immunoblotting analysis, it was found that AdOx was capable of indirectly diminishing the phosphorylation of oncogenic Src and its kinase activity. Interestingly, AdOx disrupted actin cytoskeleton structures, leading to morphological changes, and suppressed the formation of a signaling complex composed of Src and p85/PI3K, which is linked to various tumorigenic responses. In agreement with these data, the exogenous treatment of SAH or inhibition of SAHH by specific siRNA or another type of inhibitor, 3-deazaadenosine (DAZA), similarly resulted in antitumorigenic responses, suppressive activity on Src, the alteration of actin cytoskeleton, and a change of the colocalization pattern between actin and Src. Taken together, these results suggest that SAH/SAHH-mediated transmethylation could be linked to the tumorigenic processes through cross-regulation between the actin cytoskeleton and Src kinase activity.

Increased plasma S-adenosyl-homocysteine levels induce the proliferation and migration of VSMCs through an oxidative stress-ERK1/2 pathway in apoE(-/-) mice.

AIMS: Although S-adenosyl-homocysteine (SAH) is considered to be a more sensitive predictor of cardiovascular disease than homocysteine, the underlying mechanisms of its effects remain unknown. We investigated the in vivo and in vitro effects of SAH on vascular smooth muscle cells (VSMCs) proliferation and migration related to the development of atherogenesis in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: A total of 72 apoE(-/-) mice were randomly divided into six groups (n= 12 for each group). The control group was fed a conventional diet, the M group was fed a 1% methionine-supplemented diet, the A group was fed a diet that was supplemented with the SAH hydrolase (SAHH) inhibitor adenosine-2, 3-dialdehyde (ADA), the M+A group was fed a diet that was supplemented with methionine plus ADA, and two of the groups were intravenously injected with retrovirus that expressed either SAHH shRNA (SAHH(+/-)) or scrambled shRNA semi-weekly for 8 weeks. Compared with the controls, the mice in the A, M+A, and SAHH(+/-) groups had higher plasma SAH levels, larger atheromatous plaques, elevated VSMC proliferation, and higher aortic reactive oxygen species and malondialdehyde levels. In cultured VSMCs, 5 µM ADA or SAHH shRNA caused SAH accumulation, which resulted in increased cell proliferation, migration, oxidative stress, and extracellular-regulated kinase 1/2 (ERK1/2) activation. These effects were significantly attenuated by preincubation with superoxide dismutase (300 U/mL). CONCLUSION: Our results suggest that elevated SAH induces VSMC proliferation and migration through an oxidative stress-dependent activation of the ERK1/2 pathway to promote atherogenesis.

Structure-guided design of a methyl donor cofactor that controls a viral histone H3 lysine 27 methyltransferase activity.

vSET (a viral SET domain protein) is an attractive polycomb repressive complex 2 (PRC2) surrogate to study the effect of histone H3 lysine 27 (H3K27) methylation on gene transcription, as both catalyze histone H3K27 trimethylation. To control the enzymatic activity of vSET in vivo with an engineered S-adenosyl-l-methionine (SAM) analogue as methyl donor cofactor, we have carried out structure-guided design, synthesis, and characterization of orthogonal vSET methyltransferase mutant/SAM analogue pairs using a "bump-and-hole" strategy.