Trypanosoma brucei proliferates normally even after losing all S-adenosylhomocysteine hydrolase genes.

S-adenosylhomocysteine (SAH) hydrolase is the enzyme responsible for breaking down SAH into adenosine and homocysteine. It has long been believed that a deficiency of this enzyme leads to SAH accumulation, subsequently inhibiting methyltransferases responsible for nucleic acids and proteins, which severely affects cell proliferation. To investigate whether targeting this enzyme could be a viable strategy to combat Trypanosoma brucei, the causative agent of human African trypanosomiasis, we created a null mutant of the SAH hydrolase gene in T. brucei using the Cre/loxP system and conducted a phenotype analysis. Surprisingly, the null mutant, where all five SAH hydrolase gene loci were deleted, exhibited normal proliferation despite the observed SAH accumulation. These findings suggest that inhibiting SAH hydrolase may not be an effective approach to suppressing T. brucei proliferation, making the enzyme a less promising target for antitrypanosome drug development.

[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.

Epigenetic modulation of Drp1-mediated mitochondrial fission by inhibition of S-adenosylhomocysteine hydrolase promotes vascular senescence and atherosclerosis.

AIMS: Vascular senescence, which is closely related to epigenetic regulation, is an early pathological condition in cardiovascular diseases including atherosclerosis. Inhibition of S-adenosylhomocysteine hydrolase (SAHH) and the consequent increase of S-adenosylhomocysteine (SAH), a potent inhibitor of DNA methyltransferase, has been associated with an elevated risk of cardiovascular diseases. This study aimed to investigate whether the inhibition of SAHH accelerates vascular senescence and the development of atherosclerosis. METHODS AND RESULTS: The case-control study related to vascular aging showed that increased levels of plasma SAH were positively associated with the risk of vascular aging, with an odds ratio (OR) of 3.90 (95% CI, 1.17-13.02). Elevated pulse wave velocity, impaired endothelium-dependent relaxation response, and increased senescence-associated ß-galactosidase staining were observed in the artery of SAHH+/- mice at 32 weeks of age. Additionally, elevated expression of p16, p21, and p53, fission morphology of mitochondria, and over-upregulated expression of Drp1 were observed in vascular endothelial cells with SAHH inhibition in vitro and in vivo. Further downregulation of Drp1 using siRNA or its specific inhibitor, mdivi-1, restored the abnormal mitochondrial morphology and rescued the phenotypes of vascular senescence. Furthermore, inhibition of SAHH in APOE-/- mice promoted vascular senescence and atherosclerosis progression, which was attenuated by mdivi-1 treatment. Mechanistically, hypomethylation over the promoter region of DRP1 and downregulation of DNMT1 were demonstrated with SAHH inhibition in HUVECs. CONCLUSIONS: SAHH inhibition epigenetically upregulates Drp1 expression through repressing DNA methylation in endothelial cells, leading to vascular senescence and atherosclerosis. These results identify SAHH or SAH as a potential therapeutic target for vascular senescence and cardiovascular diseases.

NAD+ enhances the activity and thermostability of S-adenosyl-L-homocysteine hydrolase from Pyrococcus horikoshii OT3.

S-Adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) are important biochemical intermediates. SAM is the major methyl donor for diverse methylation reactions in vivo. The SAM to SAH ratio serves as a marker of methylation capacity. Stable isotope-labeled SAM and SAH are used to measure this ratio with high sensitivity. SAH hydrolase (EC 3.13.2.1; SAHH), which reversibly catalyzes the conversion of adenosine and L-homocysteine to SAH, is used to produce labeled SAH. To produce labeled SAH with high efficiency, we focused on the SAHH of Pyrococcus horikoshii OT3, a thermophilic archaeon. We prepared recombinant P. horikoshii SAHH using Escherichia coli and investigated its enzymatic properties. Unexpectedly, the optimum temperature and thermostability of P. horikoshii SAHH were much lower than its optimum growth temperature. However, addition of NAD+ to the reaction mixture shifted the optimum temperature of P. horikoshii SAHH to a higher temperature, suggesting that NAD+ stabilizes the structure of the enzyme.

Transcriptional Control of hgcAB by an ArsR-Like Regulator in Pseudodesulfovibrio mercurii ND132.

The hgcAB gene pair encodes mercury (Hg) methylation capability in a diverse group of microorganisms, but its evolution and transcriptional regulation remain unknown. Working from the possibility that the evolutionary function of HgcAB may not be Hg methylation, we test a possible link to arsenic resistance. Using model Hg methylator Pseudodesulfovibrio mercurii ND132, we evaluated transcriptional control of hgcAB by a putative ArsR encoded upstream and cotranscribed with hgcAB. This regulator shares homology with ArsR repressors of arsenic resistance and S-adenosylhomocysteine (SAH)-responsive regulators of methionine biosynthesis but is distinct from other ArsR/SahR proteins in P. mercurii. Using quantitative PCR (qPCR) and RNA sequencing (RNA-seq) transcriptome analyses, we confirmed this ArsR regulates hgcAB transcription and is responsive to arsenic and SAH. Additionally, RNA-seq indicated a possible link between hgcAB activity and arsenic transformations, with significant upregulation of other ArsR-regulated arsenic resistance operons alongside hgcAB. Interestingly, wild-type ND132 was less sensitive to As(V) (but not As(III)) than an hgcAB knockout strain, supporting the idea that hgcAB may be linked to arsenic resistance. Arsenic significantly impacted rates of Hg methylation by ND132; however, responses varied with culture conditions. Differences in growth and metabolic activity did not account for arsenic impacts on methylation. While arsenic significantly increased hgcAB expression, hgcAB gene and transcript abundance was not a good predictor of Hg methylation rates. Taken together, these results support the idea that Hg and As cycling are linked in P. mercurii ND132. Our results may hold clues to the evolution of hgcAB and the controls on Hg methylation in nature. IMPORTANCE This work reveals a link between microbial mercury methylation and arsenic resistance and may hold clues to the evolution of mercury methylation genes (hgcAB). Microbes with hgcAB produce methylmercury, a strong neurotoxin that readily accumulates in the food web. This study addresses a critical gap in our understanding about the environmental factors that control hgcAB expression. We show that hgcAB expression is controlled by an ArsR-like regulator responsive to both arsenic and S-adenosylhomocysteine in our model organism, Pseudodesulfovibrio mercurii ND132. Exposure to arsenic also significantly impacted Pseudodesulfovibrio mercurii ND132 mercury methylation rates. However, expression of hgcAB was not always a good predictor of Hg methylation rates, highlighting the roles of Hg bioavailability and other biochemical mechanisms in methylmercury production. This study improves our understanding of the controls on hgcAB expression, which is needed to better predict environmental methylmercury production.

Selenium nanoparticles modulate histone methylation via lysine methyltransferase activity and S-adenosylhomocysteine depletion.

At physiological levels, the trace element selenium plays a key role in redox reactions through the incorporation of selenocysteine in antioxidant enzymes. Selenium has also been evaluated as a potential anti-cancer agent, where selenium nanoparticles have proven effective, and are well tolerated in vivo at doses that are toxic as soluble Se. The use of such nanoparticles, coated with either serum albumin or the naturally occurring alkaline polysaccharide chitosan, also serves to enhance biocompatibility and bioavailability. Here we demonstrate a novel role for selenium in regulating histone methylation in ovarian cancer cell models treated with inorganic selenium nanoparticles coated with serum albumin or chitosan. As well as inducing thioredoxin reductase expression, ROS activity and cancer cell cytotoxicity, coated nanoparticles caused significant increases in histone methylation. Specifically, selenium nanoparticles triggered an increase in the methylation of histone 3 at lysines K9 and K27, histone marks involved in both the activation and repression of gene expression, thus suggesting a fundamental role for selenium in these epigenetic processes. This direct function was confirmed using chemical inhibitors of the histone lysine methyltransferases EZH2 (H3K27) and G9a/EHMT2 (H3K9), both of which blocked the effect of selenium on histone methylation. This novel role for selenium supports a distinct function in histone methylation that occurs due to a decrease in S-adenosylhomocysteine, an endogenous inhibitor of lysine methyltransferases, the metabolic product of methyl-group transfer from S-adenosylmethionine in the one-carbon metabolism pathway. These observations provide important new insights into the action of selenium nanoparticles. It is now important to consider both the classic antioxidant and novel histone methylation effects of this key redox element in its development in cancer therapy and other applications.

Revisiting One-Carbon Metabolites in Human Breast Milk: Focus on S-Adenosylmethionine.

Breastfeeding is the gold standard for early nutrition. Metabolites from the one-carbon metabolism pool are crucial for infant development. The aim of this study is to compare the breast-milk one-carbon metabolic profile to other biofluids where these metabolites are present, including cord and adult blood plasma as well as cerebrospinal fluid. Breast milk (n = 142), cord blood plasma (n = 23), maternal plasma (n = 28), aging adult plasma (n = 91), cerebrospinal fluid (n = 92), and infant milk formula (n = 11) samples were analyzed by LC-MS/MS to quantify choline, betaine, methionine, S-adenosylmethionine, S-adenosylhomocysteine, total homocysteine, and cystathionine. Differences between groups were visualized by principal component analysis and analyzed by Kruskal-Wallis test. Correlation analysis was performed between one-carbon metabolites in human breast milk. Principal component analysis based on these metabolites separated breast milk samples from other biofluids. The S-adenosylmethionine (SAM) concentration was significantly higher in breast milk compared to the other biofluids and was absent in infant milk formulas. Despite many significant correlations between metabolites in one-carbon metabolism, there were no significant correlations between SAM and methionine or total homocysteine. Together, our data indicate a high concentration of SAM in breast milk, which may suggest a strong demand for this metabolite during infant early growth while its absence in infant milk formulas may indicate the inadequacy of this vital metabolic nutrient.

Structures and mechanisms of tRNA methylation by METTL1-WDR4.

Specific, regulated modification of RNAs is important for proper gene expression1,2. tRNAs are rich with various chemical modifications that affect their stability and function3,4. 7-Methylguanosine (m7G) at tRNA position 46 is a conserved modification that modulates steady-state tRNA levels to affect cell growth5,6. The METTL1-WDR4 complex generates m7G46 in humans, and dysregulation of METTL1-WDR4 has been linked to brain malformation and multiple cancers7-22. Here we show how METTL1 and WDR4 cooperate to recognize RNA substrates and catalyse methylation. A crystal structure of METTL1-WDR4 and cryo-electron microscopy structures of METTL1-WDR4-tRNA show that the composite protein surface recognizes the tRNA elbow through shape complementarity. The cryo-electron microscopy structures of METTL1-WDR4-tRNA with S-adenosylmethionine or S-adenosylhomocysteine along with METTL1 crystal structures provide additional insights into the catalytic mechanism by revealing the active site in multiple states. The METTL1 N terminus couples cofactor binding with conformational changes in the tRNA, the catalytic loop and the WDR4 C terminus, acting as the switch to activate m7G methylation. Thus, our structural models explain how post-translational modifications of the METTL1 N terminus can regulate methylation. Together, our work elucidates the core and regulatory mechanisms underlying m7G modification by METTL1, providing the framework to understand its contribution to biology and disease.

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.

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.

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.

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.

Epigenetic Upregulation of H19 and AMPK Inhibition Concurrently Contribute to S-Adenosylhomocysteine Hydrolase Deficiency-Promoted Atherosclerotic Calcification.

BACKGROUND: S-adenosylhomocysteine (SAH) is a risk factor of cardiovascular disease; inhibition of SAH hydrolase (SAHH) results in SAH accumulation and induces endothelial dysfunction and atherosclerosis. However, the effect and mechanism of SAHH in atherosclerotic calcification is still unclear. We aimed to explore the role and mechanism of SAHH in atherosclerotic calcification. METHODS: The relationship between SAHH and atherosclerotic calcification was investigated in patients with coronary atherosclerotic calcification. Different in vivo genetic models were used to examine the effect of SAHH deficiency on atherosclerotic calcification. Human aortic and murine vascular smooth muscle cells (VSMCs) were cultured to explore the underlying mechanism of SAHH on osteoblastic differentiation of VSMCs. RESULTS: The expression and activity of SAHH were decreased in calcified human coronary arteries and inversely associated with coronary atherosclerotic calcification severity, whereas plasma SAH and total homocysteine levels were positively associated with coronary atherosclerotic calcification severity. Heterozygote knockout of SAHH promoted atherosclerotic calcification. Specifically, VSMC-deficient but not endothelial cell-deficient or macrophage-deficient SAHH promoted atherosclerotic calcification. Mechanistically, SAHH deficiency accumulated SAH levels and induced H19-mediated Runx2 (runt-related transcription factor 2)-dependent osteoblastic differentiation of VSMCs by inhibiting DNMT3b (DNA methyltransferase 3b) and leading to hypomethylation of the H19 promoter. On the contrary, SAHH deficiency resulted in lower intracellular levels of adenosine and reduced AMPK (AMP-activated protein kinase) activation. Adenosine supplementation activated AMPK and abolished SAHH deficiency-induced expression of H19 and Runx2 and osteoblastic differentiation of VSMCs. Finally, AMPK activation by adenosine inhibited H19 expression by inducing Sirt1 (sirtuin-1)-mediated histone H3 hypoacetylation and DNMT3b-mediated hypermethylation of the H19 promoter in SAHH deficiency VSMCs. CONCLUSIONS: We have confirmed a novel correlation between SAHH deficiency and atherosclerotic calcification and clarified a new mechanism that epigenetic upregulation of H19 and AMPK inhibition concurrently contribute to SAHH deficiency-promoted Runx2-dependent atherosclerotic calcification.

S-adenosyl-L-homocysteine extends lifespan through methionine restriction effects.

Methionine restriction (MetR) can extend lifespan and delay the onset of aging-associated pathologies in most model organisms. Previously, we showed that supplementation with the metabolite S-adenosyl-L-homocysteine (SAH) extends lifespan and activates the energy sensor AMP-activated protein kinase (AMPK) in the budding yeast Saccharomyces cerevisiae. However, the mechanism involved and whether SAH can extend metazoan lifespan have remained unknown. Here, we show that SAH supplementation reduces Met levels and recapitulates many physiological and molecular effects of MetR. In yeast, SAH supplementation leads to inhibition of the target of rapamycin complex 1 (TORC1) and activation of autophagy. Furthermore, in Caenorhabditis elegans SAH treatment extends lifespan by activating AMPK and providing benefits of MetR. Therefore, we propose that SAH can be used as an intervention to lower intracellular Met and confer benefits of MetR.

Comparative Analysis of Sulfonium-π, Ammonium-π, and Sulfur-π Interactions and Relevance to SAM-Dependent Methyltransferases.

We report the measurement and analysis of sulfonium-π, thioether-π, and ammonium-π interactions in a ß-hairpin peptide model system, coupled with computational investigation and PDB analysis. These studies indicated that the sulfonium-π interaction is the strongest and that polarizability contributes to the stronger interaction with sulfonium relative to ammonium. Computational studies demonstrate that differences in solvation of the trimethylsulfonium versus the trimethylammonium group also contribute to the stronger sulfonium-π interaction. In comparing sulfonium-π versus sulfur-π interactions in proteins, analysis of SAM- and SAH-bound enzymes in the PDB suggests that aromatic residues are enriched in close proximity to the sulfur of both SAM and SAH, but the populations of aromatic interactions of the two cofactors are not significantly different, with the exception of the Me-π interactions in SAM, which are the most prevalent interaction in SAM but are not possible for SAH. This suggests that the weaker interaction energies due to loss of the cation-π interaction in going from SAM to SAH may contribute to turnover of the cofactor.

Pathogenic SLC25A26 variants impair SAH transport activity causing mitochondrial disease.

The SLC25A26 gene encodes a mitochondrial inner membrane carrier that transports S-adenosylmethionine (SAM) into the mitochondrial matrix in exchange for S-adenosylhomocysteine (SAH). SAM is the predominant methyl-group donor for most cellular methylation processes, of which SAH is produced as a by-product. Pathogenic, biallelic SLC25A26 variants are a recognized cause of mitochondrial disease in children, with a severe neonatal onset caused by decreased SAM transport activity. Here, we describe two, unrelated adult cases, one of whom presented with recurrent episodes of severe abdominal pain and metabolic decompensation with lactic acidosis. Both patients had exercise intolerance and mitochondrial myopathy associated with biallelic variants in SLC25A26, which led to marked respiratory chain deficiencies and mitochondrial histopathological abnormalities in skeletal muscle that are comparable to those previously described in early-onset cases. We demonstrate using both mouse and fruit fly models that impairment of SAH, rather than SAM, transport across the mitochondrial membrane is likely the cause of this milder, late-onset phenotype. Our findings associate a novel pathomechanism with a known disease-causing protein and highlight the quests of precision medicine in optimizing diagnosis, therapeutic intervention and prognosis.

AHCYL1 senses SAH to inhibit autophagy through interaction with PIK3C3 in an MTORC1-independent manner.

S-adenosyl-l-homocysteine (SAH), an amino acid derivative, is a key intermediate metabolite in methionine metabolism, which is normally considered as a harmful by-product and hydrolyzed quickly once formed. AHCY (adenosylhomocysteinase) converts SAH into homocysteine and adenosine. There are two other members in the AHCY family, AHCYL1 (adenosylhomocysteinase like 1) and AHCYL2 (adenosylhomocysteinase like 2). Here we define AHCYL1 function as a SAH sensor to inhibit macroautophagy/autophagy through PIK3C3. The C terminus of AHCYL1 interacts with SAH specifically and the interaction with SAH promotes the binding of the N terminus to the catalytic domain of PIK3C3, resulting in inhibition of PIK3C3. More importantly, this observation was further validated in vivo, indicating that SAH functions as a signaling molecule. Our study uncovers a new axis of SAH-AHCYL1-PIK3C3, which senses the intracellular level of SAH to inhibit autophagy in an MTORC1-independent manner.Abbreviations: ADOX: adenosine dialdehyde; AHCY: adenosylhomocysteinase; AHCYL1: adenosylhomocysteinase like 1; cLEU: cycloleucine; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; SAH: S-adenosyl-l-homocysteine; SAM: S-adenosyl-l-methionine.

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.