S-adenosylhomocysteine Research Articles

Acute ethanol-induced liver injury is prevented by betaine administration.

Binge drinking is the most common form of excessive alcohol use. Repeated episodes of binge drinking cause multiple organ injuries, including liver damage. We previously demonstrated that chronic ethanol administration causes a decline in the intrahepatic ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). This decline causes impairments in essential methylation reactions that result in alcohol-induced fatty liver (steatosis) and other features of alcohol-associated liver disease (ALD). Co-treatment with betaine during chronic ethanol feeding, normalizes hepatocellular SAM:SAH ratio and alleviates many features of liver damage including steatosis. Here, we sought to examine whether betaine treatment similarly protects against liver injury in an alcohol binge-drinking model. We hypothesized that ethanol binge with prior or simultaneous betaine administration would prevent or attenuate acute alcohol-induced liver damage. Male C57Bl/6 mice were gavaged twice, 12 h apart, with either 6 g ethanol/kg BW or with an equal volume/kg BW of 0.9% NaCl. Two separate groups of mice (n = 5/group) were gavaged with 4 g betaine/kg BW, either 2 h before or simultaneously with the ethanol or saline gavages. All mice were sacrificed 8 h after the last gavage and serum and liver parameters were quantified. Ethanol binges caused a 50% decrease in hepatic SAM:SAH ratio and a >3-fold rise in liver triglycerides (p ≤ 0.05). These latter changes were accompanied by elevated serum AST and ALT activities and blood alcohol concentrations (BAC) that were ∼three-times higher than the legal limit of intoxication in humans. Mice that were treated with betaine 2 h before or simultaneously with the ethanol binges exhibited similar BAC as in mice given ethanol-alone. Both betaine treatments significantly elevated hepatic SAM levels thereby normalizing the SAM:SAH ratio and attenuating hepatic steatosis and other injury parameters, compared with mice given ethanol alone. Simultaneous betaine co-administration with ethanol was more effective in preventing or attenuating liver injury than betaine given before ethanol gavage. Our findings confirm the potential therapeutic value of betaine administration in preventing liver injury after binge drinking in an animal model.

Cystathionine Gamma-Lyase Regulate Psilocybin Biosynthesis in Gymnopilus dilepis Mushroom via Amino Acid Metabolism Pathways.

As a potential medicine for the treatment of depression, psilocybin has gradually attracted attention. To elucidate the molecular mechanism regulating psilocybin synthesis in Gymnopilus dilepis, ultra-performance liquid chromatography (UPLC) was used to detect the changes in psilocybin content after S-adenosyl-l-homocysteine (SAH) treatment and the changes of psilocybin content in different parts (stipe and pileus), and RNA-Seq was used to explore the mechanism of psilocybin content changes. In this study, the psilocybin content in G. dilepis mycelia treated with SAH was significantly lower than that in the control group, and the content of psilocybin in the stipe was significantly higher than that in the pileus. Transcriptome analysis revealed that differential expression genes (DEGs) were associated with cysteine and methionine metabolism. In particular, the transcription levels of genes encoding Cystathionine gamma-lyase (CTH) in different treatments and different parts were positively correlated with psilocybin content. In addition, we found that the exogenous addition of CTH activity inhibitor (DL-propargylglycine, PAG) could reduce the content of psilocybin and L-serine, and the content of psilocybin and L-serine returned to normal levels after L-cysteine supplementation, suggesting that psilocybin synthesis may be positively correlated with L-cysteine or CTH, and L-cysteine regulates the synthesis of psilocybin by affecting L-serine and 4-hydroxy-L-tryptophan. In conclusion, this study revealed a new molecular mechanism that affects psilocybin biosynthesis, which can provide a theoretical basis for improving psilocybin synthesis and the possibility for the development of biomedicine.

Identification of the Early Steps in Herbicidin Biosynthesis Reveals an Atypical Mechanism of C-Glycosylation

Herbicidins are adenosine-derived nucleoside antibiotics with an unusual tricyclic core structure. Deletion of the genes responsible for formation of the tricyclic skeleton in Streptomyces sp. L-9-10 reveals the in vivo importance of Her4, Her5, and Her6 in the early stages of herbicidin biosynthesis. In vitro characterization of Her4 and Her5 demonstrates their involvement in an initial, two-stage C-C coupling reaction that results in net C5'-glycosylation of ADP/ATP by UDP/TDP-glucuronic acid. Biochemical analyses and intermediate trapping experiments imply a noncanonical mechanism of C-glycosylation reminiscent of NAD-dependent S-adenosylhomocysteine (SAH)-hydrolase catalysis. Structural characterization of the isolated metabolites suggests possible reactions catalyzed by Her6 and Her7. An overall herbicidin biosynthetic pathway is proposed based on these observations.

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

BackgroundAutologous 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.MethodsRats 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.ResultsCompared 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.ConclusionsDiabetes 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.Graphical abstractDiabetes 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.

Higher S-adenosylhomocysteine and lower ratio of S-adenosylmethionine to S-adenosylhomocysteine were more closely associated with increased risk of subclinical atherosclerosis than homocysteine.

AimTo examine the relationship of C1 metabolites of the methionine cycle with the risk of subclinical atherosclerosis (SA) in the Chinese population.MethodsA total of 2,991 participants aged 45–75 years old were included for data analyses based on the baseline data of the Guangzhou Nutrition and Health Cohort. Three core serum methionine metabolites including serum S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and homocysteine (Hcy) were measured by UPLC-MS/MS. SA was determined by B-mode ultrasound measured carotid intima-media thickness (CIMT) at the common artery and bifurcation segments. Multivariable logistic and linear regression models were performed to estimate the associations of C1 metabolites of the methionine cycle with SA risk or CIMT.ResultsAfter controlling for potential cofounders and other C1 metabolites, in comparison with the lowest quartile, participants in the highest quartile had lower risk of SA by 27.6% (OR = 0.724; 95% CI:0.563–0.93, Ptrend = 0.007) for SAM and 32.2% (OR = 0.678; 95% CI:0.538–0.855, Ptrend < 0.001) for SAM/SAH, while increased SA risk by 27.9% (OR = 1.279; 95% CI: 1.065–1.535, Ptrend < 0.001) for SAH. No significant association was observed for Hcy with SA after further adjustment of SAH and SAM. The results of multivariable linear regression showed similar findings. The highest two standardized coefficients were observed for SAH (β = 0.104 for CCA and 0.121 for BIF, P< 0.001) and SAM/SAH (β = −0.071 for CCA and −0.084 for BIF, P< 0.001). Subgroup analyses suggested more evident associations of SAH with SA were observed in participants of higher cardiovascular risk profiles.ConclusionOur cross-sectional data showed higher serum SAH, but lower SAM/SAH were independently associated with increased risk of SA among the Chinese middle-aged and elderly population.

Association of serum s-adenosylmethionine, s-adenosylhomocysteine, and their ratio with the risk of dementia and death in a community

We examined the association of serum s-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH) (methionine metabolites), and their ratio on the risk of dementia and death in a community-dwelling population of older Japanese individuals. 1371 residents of Hisayama, Japan, aged 65 years or older and without dementia, were followed for a median of 10.2 years (2007–2017). We divided serum SAM, SAH, and SAM/SAH ratio into quartiles. Cox proportional hazards models were used to estimate the hazard ratios (HRs) and their 95% confidence intervals (CIs) of serum SAM, SAH, and SAM/SAH ratio levels on the risk of a composite outcome of all-cause dementia or death, and each outcome. During the follow-up, 635 participants developed all-cause dementia and/or died, of which 379 participants developed dementia and 394 deaths occurred. The multivariable-adjusted HRs of the composite outcome decreased significantly with increasing serum SAM levels (P for trend = 0.01), while they increased significantly with higher serum SAH levels (P for trend = 0.03). Higher serum SAM/SAH ratio levels were significantly associated with a lower risk of the composite outcome (P for trend = 0.002), as well as with lower risk of each outcome. Our findings suggest that the balance of methionine metabolites may closely associate with the risk of dementia and death.

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.

A High-Throughput Continuous Spectroscopic Assay to Measure the Activity of Natural Product Methyltransferases.

Natural product methyltransferases (NPMTs) represent an emerging class of enzymes that can be of great use for the structural and functional diversification of bioactive compounds, such as the strategic modification of C‐, N‐, O‐ and S‐moieties. To assess the activity and the substrate scope of the ever‐expanding repertoire of NPMTs, a simple, fast, and robust assay is needed. Here, we report a continuous spectroscopic assay, in which S‐adenosyl‐L‐methionine‐dependent methylation is linked to NADH oxidation through the coupled activities of S‐adenosyl‐L‐homocysteine (SAH) deaminase and glutamate dehydrogenase. The assay is highly suitable for a high‐throughput evaluation of small molecule methylation and for determining the catalytic parameters of NPMTs under conditions that remove the potent inhibition by SAH. Through the modular design, the assay can be extended to match the needs of different aspects of methyltransferase cascade reactions and respective applications.

O-302 Lower expression of AHCY is associated with increased miscarriage risk

Abstract Study question Are alterations in the transcript levels of genes from the methionine cycle associated with maternal associated miscarriage risk? Summary answer Lower expression of AHCY is associated with a greater number of prior pregnancy losses What is known already Around 15% of pregnancies end in miscarriage, and the risk of recurrence increases with each pregnancy loss. Aberrant differentiation (decidualization) of endometrial stromal cells into specialised decidual cells to accommodate implantation is a key maternal factor for miscarriage risk. Our previous work identified secretory changes in cysteine and methionine metabolites upon decidualization. The methionine cycle contributes to vital cellular functions, including producing methionine for proliferation, regulating cell differentiation, and S-adenosylmethionine (SAM) production. SAM is required for protein, RNA and DNA methylation, thereby influencing pathways at the metabolic, epigenetic, and proteomic levels. AHCY clears S-adenosylhomocysteine (SAH), reducing its inhibition of methylation. Study design, size, duration Endometrial biopsies (n = 250) were collected during the luteal phase (LH + 6-11). Patients were grouped based on their miscarriage history. Accordingly, expression of genes from the methionine cycle were quantified using RT-qPCR. Participants/materials, setting, methods Endometrial biopsies were obtained, with written informed consent, from women attending the Implantation Clinic at University Hospitals Coventry and Warwickshire NHS Trust, following transvaginal ultrasounds to exclude uterine pathology. Isolated RNA was converted into cDNA. Expression of AHCY, AMD1, BHMT2, CBS, MAT2A, MAT2B, and MTR were normalised to L19. Statistical analysis was performed in Graphpad Prism; with significance accepted at p &amp;lt; 0.05. AHCY was silenced in an endometrial cell line to determine its effect on decidualization. Main results and the role of chance This study reports a distinct reduction in expression of methionine cycle genes (MAT2A, AHCY, AMD1, MTR, BHMT2) in the late-luteal phase of the cycle consistent with a reduction in proliferation. By plotting percentile graphs based on the statistical distribution in gene expression for each day of the luteal phase, comparisons have been made between groups. AHCY expression is significantly reduced in patients with increasing number of prior miscarriages, particularly between 0-2 and 5+ previous miscarriages (p = 0.0334). Neither patient age nor BMI are a factor in this reduced expression. In contrast, there is a stromal specific increase in AHCY upon decidualization in vitro, suggesting it is required in the decidua. Silencing AHCY in an endometrial cell line significantly reduces PRL expression upon decidualization. Reduction in AHCY may lead to a decreased “methylation potential” as SAH cannot be cleared. SAH accumulation inhibits methylation, and limits SAM production, thus compounding its effect. Decreased methylation potential could prevent differentiation of the stromal compartment, resulting in lower levels of PRL, and altering decidual timing. Therefore, an embryo may implant into a tissue primed for disintegration, resulting a miscarriage. In summary, AHCY may contribute to aberrant decidualization augmenting the risk of miscarriage. Limitations, reasons for caution Results are based on endometrial biopsies from an implantation clinic, therefore studies into biopsies from women with normal reproductive histories should also be analysed. Further functional studies are needed to ascertain the mechanism of action of AHCY in miscarriage. Wider implications of the findings This study identified that a decrease in AHCY in whole endometrial tissue is associated with increased risk of miscarriage. Further, silencing AHCY perturbed decidual marker expression. Thus, AHCY may act as a biomarker for atypical decidualization, and the clearance of SAH may be a potential treatment. Trial registration number N/A

P731: INTERPLAY BETWEEN INFLAMMATION AND EPIGENETICS IN TUMOR REPROGRAMMING OF MESENCHYMAL STROMAL CELLS (MSCS) IN MYELODYSPLASTIC SYNDROMES (MDS)

Background: The alteration of hematopoiesis in MDS patients is deeply associated with microenvironment alterations, in particular in MSCs. Stromal cells are epigenetically reprogrammed to function in cooperation with leukemic cells and propagate the disease as a “tumor unit”. Notably, dysfunctions of MDS-MSCs persist following expansion ex vivo suggesting a hereditable epigenetic dysregulation which endures despite removal of disease-associated microenvironment factors. Aims: Here, we investigated the role of histone H2A variant MacroH2A1 (mH2A1) in promoting pro-tumorigenic inflammation and metabolic reprogramming of MSCs. Methods: MSCs were collected from BM of MDS patients (n=18) and matched healthy controls (n=8; HC). HS-5 cells were used as model of healthy MSCs. Real time, western blot, immunohistochemistry and immunofluorescence were used. Proteomic and metabolic analysis were performed. Results: MDS patients had significant higher signature of mH2A1 in bone marrow slides compared to age-matched control. Investigating mH2A1 expression in stromal cells, MDS-MSCs had increased levels of expression in respect of HC-MSCs. In accordance with the aberrant inflammation described in MDS microenvironment, MDS-MSCs showed higher levels of the sum of NO2+ and NO3+ associated to increased GSH and NADP+/NAPH. TLR4 also was upregulated and positively correlated to mH2A1 expression. To better investigate the relationship between mH2A1 and TLR4 in MDS-MSCs, we induced mH2A1 overexpression in HS-5 cells (mH2A1-OE) by mH2A1-CT-MYC plasmid. Our data showed that mH2A1-OE upregulated TLR4 and increased NFkB nuclear translocation compared to cells transfected with empty vector (CTL). Proteomic analysis confirmed upregulation of intracellular serine protease inhibitors (SerpinB2, B8, B6) strongly induced during inflammation and important for the maintenance of TLR4 activation. Moreover, proteomic approach identified upregulation of several proteins associated to hypermethylation of DNA and histones in mH2A1-OE. In particular, S-adenosylhomocysteine hydrolase which regulates the concentration of S-adenosylhomocysteine (SAH), a strong inhibitor of methyltransferase reactions and of the methyl donor S-adenosyl-methionine (SAM), resulted overexpressed. HPLC analysis showed higher SAM/SAH ratio associated to a significant reduction of SAH in mH2A1-OE, confirming the increase of the methylation index. In addition, the higher levels of CBX3, a suppressive epigenetic mark which recognizes histone H3K9me3, contributes to the maintenance of the heterochromatin. The higher levels of H3K9me3 in mh2A1-OE were confirmed by western blot analysis. Overexpression of mH2A1 also induced metabolic reprogramming with the acquisition of a more glycolytic metabolism characterized by decreased levels of NAD+/NADH, upregulation of LDHA and MCT4. As LDHA could translocate into the nucleus, we evaluated if it used a noncanonical enzymatic activity in mh2A1-OE. Data showed increased nuclear localization of LDHA producing histone H3K79 hypermethylation. Nuclear LDHA was also observed in MDS-MSC. Finally, we treated ex-vivo HC- and MDS-MSCs with azacytidine founding a significant reduction both of mH2A1 and TLR4 associated to lower levels of nuclear NFKB. Summary/Conclusion: Our data provide a key role of mH2A1in driving the crosstalk between inflammation, metabolism and epigenetic signatures in MDS-MSC. Future experiments will define the contribution of mH2A to ineffective hematopoiesis and leukemic evolution.

Mitochondrial transport and metabolism of the major methyl donor and versatile cofactor S-adenosylmethionine, and related diseases: A review†.

S-adenosyl-L-methionine (SAM) is a coenzyme and the most commonly used methyl-group donor for the modification of metabolites, DNA, RNA and proteins. SAM biosynthesis and SAM regeneration from the methylation reaction product S-adenosyl-L-homocysteine (SAH) take place in the cytoplasm. Therefore, the intramitochondrial SAM-dependent methyltransferases require the import of SAM and export of SAH for recycling. Orthologous mitochondrial transporters belonging to the mitochondrial carrier family have been identified to catalyze this antiport transport step: Sam5p in yeast, SLC25A26 (SAMC) in humans, and SAMC1-2 in plants. In mitochondria SAM is used by a vast number of enzymes implicated in the following processes: the regulation of replication, transcription, translation, and enzymatic activities; the maturation and assembly of mitochondrial tRNAs, ribosomes and protein complexes; and the biosynthesis of cofactors, such as ubiquinone, lipoate, and molybdopterin. Mutations in SLC25A26 and mitochondrial SAM-dependent enzymes have been found to cause human diseases, which emphasizes the physiological importance of these proteins.

Abstract 3741: Folic acid combined chemotherapy has an immediate effect on C1 methionine cycle and consequential DNA methylation in liquid biopsy samples of colorectal cancer patients

Abstract The main function of the single-carbon metabolic cycle is maintaining nucleotide pool and DNA methylation which is highly influenced by the chemotherapy protocols. This study aimed to evaluate the ultrashort effect of reduced folate combined anticancer treatment on peripheral blood parameters of colorectal cancer (CRC) patients. Post-operative CRC patients were treated with chemotherapy using the combination of oxaliplatin, 5-FU, leucovorin, and capecitabine. Blood samples were taken until the beginning of the treatment and immediately after the therapy. Plasma fractions were separated and the levels of S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH) and the simultaneous quantification of nucleotides (adenine, cytosine, thymine, guanine and uracil) were detected by HPLC-MS/MS. Homocysteine (HCY) was also determined from plasma specimens. Cell-free DNA (cfDNA) was isolated from plasma and DNA methylation was analyzed by LINE-1 bisulfite pyrosequencing. Moreover, LINE-1 methylation statuses were also examined from PBMC cells that were separated from whole blood by a density gradient centrifugation. In each patient, the HCY level of plasma was decreased by an average of 17% after oxaliplatin treatment with the combination of capecitabine (XELOX) and also with 5-FU + leucovorin (FOLFOX) agents. Our study also confirmed that cancer patients have an elevated cfDNA level (avg. 20ng/plasma mL); however, its concentration decreased significantly (p=0.02) on average by 50% after treatment compared to the baseline. The same tendency was observed in the case of SAM after XELOX and FOLFOX adjuvant therapy in plasma samples (p=0.03). Moreover, mostly elevated SAM/SAH ratios were observed in parallel with reduced SAH level after the therapy. Significant (p=0.0001) mean LINE-1 hypomethylation was detected in PBMC (81.84%) compared to cfDNA of plasma (73.76%), furthermore, slightly DNA hypermethylation was noticed in CpG1 position of LINE-1 after chemotherapy in both mononuclear cells and circulating DNA. Lower level of plasma nucleotides was identified in case of adenine, cytosine and uracil after treatment, while thymine and guanine were below the detection limit. The present study demonstrates that the DNA-damaging chemotherapy results in a detectable decrease in cfDNA immediately after the treatment, that was supported by the decrease in the level of the nucleotides measured in the plasma fraction. The methionine cycle is responsible for the DNA methylation maintenance, and though the amount of members of this pathway was reduced, only a moderately increased DNA methylation could be detected even in such a short period time. Co-administration of high dose leucovorin as a folate derivative and a methyl donor with further chemotherapeutic agents may actively contribute to genome alterations. Citation Format: Zsófia Brigitta Nagy, Barbara Kinga Barták, Tamás Fodor, Gellért Balázs Karvaly, Sára Zsigrai, Krisztina Andrea Szigeti, Alexandra Kalmár, Magdolna Dank, István Takács, Béla Molnár. Folic acid combined chemotherapy has an immediate effect on C1 methionine cycle and consequential DNA methylation in liquid biopsy samples of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3741.

Williams-Beuren Syndrome Related Methyltransferase WBSCR27: From Structure to Possible Function

Williams-Beuren syndrome (WBS) is a genetic disorder associated with the hemizygous deletion of several genes in chromosome 7, encoding 26 proteins. Malfunction of these proteins induce multisystemic failure in an organism. While biological functions of most proteins are more or less established, the one of methyltransferase WBSCR27 remains elusive. To find the substrate of methylation catalyzed by WBSCR27 we constructed mouse cell lines with a Wbscr27 gene knockout and studied the obtained cells using several molecular biology and mass spectrometry techniques. We attempted to pinpoint the methylation target among the RNAs and proteins, but in all cases neither a direct substrate has been identified nor the protein partners have been detected. To reveal the nature of the putative methylation substrate we determined the solution structure and studied the conformational dynamic properties of WBSCR27 in apo state and in complex with S-adenosyl-L-homocysteine (SAH). The protein core was found to form a canonical Rossman fold common for Class I methyltransferases. N-terminus of the protein and the β6–β7 loop were disordered in apo-form, but binding of SAH induced the transition of these fragments to a well-formed substrate binding site. Analyzing the structure of this binding site allows us to suggest potential substrates of WBSCR27 methylation to be probed in further research.

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

Background and aimsIt 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. MethodsHuman 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. ResultsHUVECs 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 β-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. ConclusionsSAHH inhibition induces endothelial senescence via downregulation of hTERT expression, which is associated with attenuated histone methylation over the hTERT promoter region.

A quantitative metabolomics assay targeting 14 intracellular metabolites associated with the methionine transsulfuration pathway using LC–MS/MS in breast cancer cells

The methionine transsulfuration pathway plays an important role in some fundamental biological processes, such as redox and methylation reactions. However, quantitative analysis of the majority of intracellular metabolites is rather challenging. In this study, we developed a simple, fast and reliable method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous detection of 14 methionine-related metabolites, including methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (HCY), cystathionine (Cysta), cysteine (CYS), glutathione (GSH), dimethylglycine (DMG), betaine, serine, folic acid (FA), dihydrofolic acid (DHF), tetrahydrofolic acid (THF) and 5-methyltetrahydrofolic acid (5-MTHF), in MCF-7 and MDA-MB-231 breast cancer cells. By taking advantage of a surrogate matrix, the linearity, sensitivity, precision, accuracy, stability, matrix effect, recovery, dilution integrity and carryover of the established method were evaluated and validated. This method enabled the precise measurement of methionine-related metabolites both in cells and in the medium and was successfully applied to profile these metabolites involved in the methionine transsulfuration pathway. The data showed that cystine deprivation or excessive supplementation with cystine had a marked impact on methionine metabolism, in addition to its effects on intracellular CYS and GSH levels, indicating that the methionine transsulfuration pathway was dependent on intracellular cystine levels. The established method provides a reliable way to target metabolomics for the quantitative determination of intracellular metabolites in the methionine transsulfuration pathway, which can greatly facilitate the understanding of the mechanisms involved in methylation and redox homeostasis in cellular metabolomic studies.

Mechanistic insights into the key marine dimethylsulfoniopropionate synthesis enzyme DsyB/DSYB.

Marine algae and bacteria produce approximately eight billion tonnes of the organosulfur molecule dimethylsulfoniopropionate (DMSP) in Earth's surface oceans annually. DMSP is an antistress compound and, once released into the environment, a major nutrient, signaling molecule, and source of climate-active gases. The methionine transamination pathway for DMSP synthesis is used by most known DMSP-producing algae and bacteria. The S-directed S-adenosylmethionine (SAM)-dependent 4-methylthio-2-hydroxybutyrate (MTHB) S-methyltransferase, encoded by the dsyB/DSYB gene, is the key enzyme of this pathway, generating S-adenosylhomocysteine (SAH) and 4-dimethylsulfonio-2-hydroxybutyrate (DMSHB). DsyB/DSYB, present in most haptophyte and dinoflagellate algae with the highest known intracellular DMSP concentrations, is shown to be far more abundant and transcribed in marine environments than any other known S-methyltransferase gene in DMSP synthesis pathways. Furthermore, we demonstrate in vitro activity of the bacterial DsyB enzyme from Nisaea denitrificans and provide its crystal structure in complex with SAM and SAH-MTHB, which together provide the first important mechanistic insights into a DMSP synthesis enzyme. Structural and mutational analyses imply that DsyB adopts a proximity and desolvation mechanism for the methyl transfer reaction. Sequence analysis suggests that this mechanism may be common to all bacterial DsyB enzymes and also, importantly, eukaryotic DSYB enzymes from e.g., algae that are the major DMSP producers in Earth's surface oceans.

S-adenosylhomocysteine induces cellular senescence in rat aorta vascular smooth muscle cells via NF-κB-SASP pathway

Vascular aging plays an important role in the development and progression of atherosclerosis (AS) , and one-carbon metabolism dysfunction will lead to Vascular Smooth Muscle Cells (VSMCs) senescence, which contributes to vascular senescence. However, the mechanisms underlying the role of VSMCs senescence in AS remain unclear. This study aimed to evaluate S-adenosyl-homocysteine (SAH) as a one-carbon metabolite that affects VSMCs senescence. We treated Rat Aorta Smooth Muscle Cells (RASMCs) with S-adenosylhomocysteine Hydrolase (SAHH) inhibitor, adenosine-2,3-dialdehyde (ADA) and SAHH siRNA to examine the effect of elevated SAH levels on RASMCs phenotypes. SAHH inhibition induced RASMCs senescence, as demonstrated by the manifestation of senescence-associated secretory phenotype in cells and induction of senescence in pre-senescent RASMCs. Furthermore, we found that SAHH inhibition induced CpG island demethylation in the promoter of NF-κB, a molecule that drives the pro-inflammatory response of the cells manifesting the senescence-associated secretory phenotype (SASP). Overall, these findings indicate that the elevated intracellular SAH levels could be targeted to ameliorate vascular aging.

Clinical value and expression of Homer 1, homocysteine, S-adenosyl-l-homocysteine, fibroblast growth factors 23 in coronary heart disease

BackgroundThis study aimed to explore clinical value and expression of Homer 1, S-adenosyl-l-homocysteine (SAH), homocysteine (Hcy), fibroblast growth factors (FGF) 23 in coronary heart disease (CHD).MethodsFrom March 2020 to April 2021, a total of 137 patients with CHD and 138 healthy subjects who came to our hospital for physical examination and had no cardiovascular disease were retrospectively enrolled, and they were assigned to the CHD group and the control group, respectively. Patients in the CHD group were divided into stable angina pectoris (SAP) group (n = 48), unstable angina pectoris (UAP) group (n = 46), and acute myocardial infarction (AMI) group (n = 43) according to clinical characteristics for subgroup analysis. The degree of coronary artery stenosis was assessed by Gensini score, which is a reliable assessment tool for the severity of coronary artery disease. The levels of Homer 1, SAH, Hcy, and FGF 23 were tested and compared. Spearman correlation analysis was used to analyze the correlation between serum Homer1, SAH, Hcy, FGF23 levels and Gensini score, and multivariate unconditional Logistic regression was used to analyze the risk factors of coronary heart disease.ResultsDemographic characteristics of each group were comparable (P > 0.05). The body mass index (BMI), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and glucose levels of the SAP group, UAP group and AMI group were significantly higher than those of the control group, and the number of patients with smoking, alcohol consumption, hypertension, and diabetes history was significantly more than that of the control group, respectively (P < 0.05). The level of high-density lipoprotein cholesterol (HDL-C) of each subgroup was significantly lower than the control group (P < 0.05). The above indicators showed no significant difference among three subgroups (P > 0.05). Serum SAH, Hcy, Homer1 and FGF23 levels in each subgroup were significantly higher than those in control group (P < 0.05). And above indicators in SAP group and UAP group were significantly lower than those in AMI group (P < 0.05), and the levels of above indicators in SAP group were significantly lower than those in UAP group (P < 0.05). The results of Spearman correlation analysis showed that serum Homer1, FGF23, SAH, Hcy levels were positively correlated with Gensini score (r = 0.376, 0.623, 0.291, 0.372, all P < 0.01). Multivariate logistic regression analysis showed that smoking, hypertension, diabetes, alcohol consumption, obesity, HDL-C, FGF23, SAH, Hcy, Homer 1 were independent risk factors for coronary heart disease.ConclusionThe levels of FGF23, SAH, Hcy, and Homer1 tend to increase in patients with CHD compared with normal population, and the more severe the disease, the higher the levels, which has certain reference value for the clinical diagnosis of CHD and the evaluation and monitoring of the disease.

P53 m6A modulation sensitizes hepatocellular carcinoma to apatinib through apoptosis.

Hepatocellular carcinoma (HCC) is insidious and prone to metastasis and recurrence. Currently, no effective treatment is available for HCC. Furthermore, HCC does not respond to various radio- and chemotherapies, and the molecular mechanism of treatment resistance is unclear. Here, we found that p53 n6-methyladenosine (m6A) played a decisive role in regulating HCC sensitivity to chemotherapy via the p53 activator RG7112 and the vascular endothelial growth factor receptor inhibitor apatinib. Our results reveal that p53 activation plays a crucial role in chemotherapy-induced apoptosis and reducing cell viability. Moreover, decreasing m6A methyltransferase (e.g., methyltransferase-like 3, METTL3) expression through chemotherapeutic drug combinations reduced p53 mRNA m6A modification. p53 mRNA m6A modification blockage induced by S-adenosyl homocysteine or siRNA-mediated METTL3 inhibition enhanced HCC sensitivity to chemotherapy. Importantly, we observed that downregulation of METTL3 and upregulation of p53 expression by oral administration of chemotherapy drugs triggered apoptosis and xenograft tumor growth inhibition in nude mice. Based on these findings, we hypothesize that a METTL3-m6A-p53 axis could be a potential target in HCC therapy.

DNA Methylation Underlies the Long‐Term Association Between Periodontitis and Atherosclerotic Cardiovascular Disease

Periodontitis, one of the most common inflammatory conditions, and the leading cause of adult tooth loss, has been identified as an independent risk factor for cardiovascular disease (CVD). Long‐term studies in edentulous patients with a history of periodontitis showed persistence of increased CVD risk for years after edentulism, suggesting that clinical elimination of disease is not sufficient in decreasing periodontitis‐induced CVD risk. This is similar to other CVD risk factors, such as smoking and diabetes. We hypothesized that periodontitis induces epigenetic changes in hematopoietic stem cells in the bone marrow (BM), and such changes persist after clinical elimination of the disease and underlie the induced‐CVD risk.To simulate clinical elimination of periodontitis and persistence of the hypothesized epigenetic reprogramming, we used a BM transplant approach. Using the low density lipoprotein receptor (LDLR) KO atherosclerosis mouse model, BM donor mice were fed a high fat diet (HFD) to induce atherosclerosis, and orally inoculated with Porphyromonas gingivalis(Pg), a keystone periodontal pathogen, to induce periodontitis; a second group was sham inoculated. (We previously showed that HFD‐fed, Pginoculated mice developed more atherosclerosis than sham‐inoculated). Naïve LDLR KO mice were irradiated and transplanted with BM from one of the 2 donor groups.Recipients of BM from Pg‐inoculated donors developed significantly more atherosclerosis, accompanied by more pro‐inflammatory plasma and macrophage cytokine profiles. Using whole genome bisulfite sequencing, 375 differentially methylated regions (DMR), and a global hypomethylation in recipients of BM from Pg‐inoculated donors, were identified. Some DMRs pointed to involvement of enzymes with major roles in methylation and demethylation. In recipients of BM from Pg‐inoculated donors, methionine adenosyl transferase (MAT), which catalyzes the conversion of methionine to S‐denosylmethionine (SAM), the universal methyl‐donor, was hypermethylated. The gene for S‐adenosylhomocysteine hydrolase, which catalyzes the reversible conversion of the potent methylation inhibitor, S‐adenosylhomocysteine (SAH), to homocysteine and adenosine, was also hypermethylated. In contrast, the de‐methylation enzyme, Ten‐Eleven Translocase (TET) 2, was hypomethylated. In validation assays, we found a significant increase in activity of TET2 and a decrease in activity of DNA methyltransferases. Plasma SAH levels were significantly higher and the SAM to SAH ratio was decreased, both of which have been associated with CVD. Homocysteine, is at the intersection of the methionine cycle and the transsulphuration pathway, where it can either be re‐methylated to methionine and go through the methionine cycle, or it can synthesize cysteine through the transsulphuration pathway. Glutathione, a critical antioxidant, is the end‐product of the transsulphuration pathway. In conditions of oxidative stress, the transsulphuration pathway is favored over the methionine cycle. Periodontitis is associated with oxidative stress. These data suggest a paradigm shifting mechanism linking epigenetic changes in gene methylation with the long term association between periodontitis and atherosclerotic CVD.