Methionine restriction (MR) extends the lifespan and healthspan of numerous eukaryotic organisms, but the molecular mechanisms at play are unclear. Here we find that the ability of MR to extend the budding yeast chronological and replicative lifespans is the consequence of reduced methionine conversion to the methyl donor S-adenosylmethionine (SAM). Mechanistically, the key antiaging event downregulated by MR is the methylation of protein phosphatase 2A (PP2A). In chronological aging cells under MR, unmethylated PP2A no longer dephosphorylates Npr2, a component of the SEACIT complex, resulting in activation of non-nitrogen-starvation (NNS)-induced autophagy. Deletion of genes encoding components of SEACIT or ATG1 (encoding a central player in the initiation of autophagy) blocked the ability of MR to extend lifespan, showing the critical role of the NNS-induced autophagy pathway in lifespan extension by MR. We identify the relevant Npr2 site dephosphorylated by PP2A as serine 362 and show that Npr2 phosphomimetic mutants are sufficient to extend chronological and replicative lifespan. Finally, we discover that MR only during the early stages of chronological aging is sufficient to prolong autophagy and extend lifespan. In addition to elucidating the molecular mechanism of MR-mediated lifespan extension, this study highlights potential therapeutic targets to achieve lifespan and healthspan extension in humans without the challenging long-term dietary changes required to achieve MR.

The dopaminergic system constitutes a principal element of the reward neurocircuitry and is responsive to influences encountered early in life. Methylation potential, shaped by nutrients involved in one-carbon metabolism, intersects with immune signaling and dopaminergic receptor, creating a broader metabolic network through which early life nutrition may alter neurobehavioral outcomes. We have shown that methyl nutrient imbalance between folic acid and choline during pregnancy produces offspring with higher body weight and perturbed methylation potential. However, whether these effects reflect disruptions in the dopaminergic receptor expression and systemic inflammation, and the capacity of betaine (a direct methyl donor) to modify responses across different nutritional contexts remain unknown. To address this, primiparous Wistar rats were randomly assigned to either a recommended vitamin or high folic acid/low choline diet, with or without betaine supplementation during pregnancy, then switched to a control diet during lactation. One female and one male offspring from each dam were followed for 12 weeks post-weaning under an obesogenic environment. Prenatal micronutrient composition produced distinct patterns of activity in offspring across the light-dark cycle, revealing behavioral signatures of both imbalance and betaine supplementation. Betaine alone reduced body weight and food intake, and provided partial protection against the metabolic consequences of the imbalanced micronutrient diet. Dopaminergic receptor gene expression was responsive to prenatal micronutrient exposure, with treatment × $$ \times $$ sex interactions restricted to the ventral tegmental area (VTA) and betaine modifying receptor patterns across both the VTA and nucleus accumbens. Catechol-O-methyltransferase expression was altered consistent with changes in S-adenosylmethionine and the S-adenosylmethionine: S-adenosylhomocysteine ratio across groups. Higher C-reactive protein concentrations occurred under micronutrient imbalance, but remained unchanged with betaine supplementation. In conclusion, gestational intake of high folic acid and low choline disturbs dopaminergic, metabolic and immune outcomes in offspring, whereas betaine may serve as a potential modulator capable of mitigating these effects.

Methionine (Met) is an essential amino acid that limits the nutritional value of many crop plants, yet its steady-state level in plant tissues is remarkably low. At the same time, Met is a central metabolic hub supporting protein synthesis and, through S-adenosylmethionine (SAM), drives the production of hormones, vitamins, polyamines, and epigenetic marks. The primary objective of this review is to understand why and how plants maintain low steady-state Met levels, and what occurs when Met content is elevated, a question of growing importance for Met biofortification. We first summarize evidence that flux through the Met/SAM pathway is high while Met pools remain small, because Met is rapidly diverted to SAM, S-methylmethionine (SMM), and other metabolites. We then describe how increasing Met at the genetic level alters development, primary metabolism, and stress responses, enhancing amino acids and sugars in leaves and proteins and starch in seeds, but often causing growth defects and stress hypersensitivity. However, moderate exogenous Met can improve growth and stress tolerance. Finally, we highlight how changes in Met/SAM levels affect DNA and histone methylation, as well as transposable-element silencing. Together, these findings suggest that plants limit Met to control metabolic, redox, and epigenetic modification, constraining Met biofortification strategies and seed nutritional improvement efforts.

Hepatic stellate cell (HSC) activation is a key driver of extracellular matrix (ECM) accumulation and liver fibrosis. Autophagy plays an essential role in regulating HSC activation, yet its metabolic regulation remains largely undefined. Curcumol, a bioactive compound derived from Curcuma longa, possesses potent antifibrotic activity, but the underlying metabolic mechanisms are unclear. In this study, we found that curcumol suppressed HSC activation and induced autophagy-dependent cell death, together with inhibition of methionine metabolism. Curcumol treatment reduced LX-2 cell viability and downregulated profibrogenic markers α-smooth muscle actin (α-SMA) and collagen type I (COL1A1) in a dose-dependent manner. Mechanistically, curcumol enhanced LC3-II accumulation, diminished p62 levels, and promoted autophagic vacuole formation, effects that were reversed by the autophagy inhibitor 3-methyladenine (3-MA). Silencing of ATG7 attenuated curcumol-induced autophagy-associated changes and cell death, supporting the involvement of ATG7 in this process. Furthermore, curcumol significantly reduced the expression of key methionine cycle enzymes MAT2A and AHCY. Supplementation with S-adenosylmethionine (SAM) partially reversed curcumol-associated changes in methionine metabolism and autophagy-related markers, and improved HSC viability, supporting a functional link between methionine metabolism and the observed phenotype. Collectively, these findings suggest that curcumol promotes autophagy-dependent death of HSCs in association with disrupted methionine metabolism, providing new insight into the metabolic basis of its antifibrotic action.

Environmental exposures can influence offspring health through epigenetic alterations in the male germline. Folate deficiency, a dietary perturbation that disrupts one-carbon metabolism and S-adenosylmethionine (SAM) production, has been linked to altered histone methylation and developmental abnormalities in offspring. However, when and how folate availability shapes the germline epigenome during spermatogenesis remains unclear. In this study, unbiased metabolomic profiling of spermatogenic cells uncovers stage-specific metabolic remodeling, including downregulation of serine- glycine-one-carbon (SGOC) metabolism in meiotic spermatocytes. Using a post-weaning folate-deficient mouse model, we investigate how folate availability influences germline epigenome establishment during spermatogenesis. Consistent with this metabolic transition, genome-wide chromatin accessibility profiling demonstrates extensive, stage-dependent remodeling under folate-deficient conditions, particularly in meiotic spermatocytes and post-meiotic spermatids. These accessibility changes display cell-type-specific genomic distributions and preferential localization to repressive chromatin compartments in post-meiotic cells. Histone modification analyses further reveal bidirectional redistribution of the active histone mark H3K4me3 in round spermatids. Although genome-wide distribution of the repressive mark H3K27me3 remains largely stable, folate deficiency alters its nuclear organization. Notably, a subset of H3K4me3 alterations established in post-meiotic cells is retained in mature sperm, providing a mechanistic link between paternal metabolic perturbation and the germline epigenome. Together, these findings demonstrate that folate availability shapes germline epigenome establishment through stage-specific metabolic and chromatin remodeling during spermatogenesis, revealing a metabolic basis for paternal environmental effects on the germline epigenome.

The radical S-adenosylmethionine (SAM) superfamily comprises more than 800,000 enzymes that use [Fe4S4] clusters to initiate radical chemistry that mediates an exceptionally broad range of chemical transformations. Within this superfamily, cobalamin (Cbl)-dependent radical SAM enzymes constitute a major subclass predominantly associated with methylation reactions. However, several notable members catalyze non-methylase reactions, for which the mechanistic role of Cbl is poorly understood. Bacteriochlorophyll biosynthesis enzyme BchE is a Cbl-dependent radical SAM enzyme that catalyzes a six-electron oxidation of Mg-protoporphyrin IX monomethylester (MPE) to protochlorophyllide (PChlide), installing a ketone and forming the fifth ring of bacteriochlorophyll under anaerobic conditions. Although prior in vivo and in vitro studies have demonstrated a requirement for Cbl, SAM, and a low-potential reductant, detailed mechanistic analysis has been impeded by the inability to obtain soluble, catalytically active enzyme. Here, we report the successful isolation and spectroscopic characterization of BchE, enabling the first in vitro reconstitution of its enzymatic activity. Using both chemical and biological reducing systems, we observe the formation of PChlide along with proposed reaction intermediates and several off-pathway products. These results provide new insight into the oxidative chemistry mediated by Cbl in non-methylase radical SAM enzymes and establish BchE as a tractable model for elucidating how cobalamin is deployed in this understudied subclass.

The neonatal heart undergoes a rapid metabolic transition from fetal glycolysis to oxidative phosphorylation, requiring coordinated metabolic remodeling. Mechanisms driving this transition remain unclear. Here, we demonstrate that sufficient mitochondrial S-adenosylmethionine (mitoSAM), imported via the solute carrier Slc25a26 , is essential for this shift by sustaining the lipoylation of 2-oxoacid dehydrogenases, critical for TCA cycle activation. Proteomic and metabolomic profiling revealed that reduced mitoSAM availability impaired lipoylation, blocking TCA cycle function and restricting nucleotide synthesis, while mitochondrial gene expression and respiratory capacity remained largely intact. In vivo EdU labeling showed persistent cardiomyocyte proliferation imposing further strain on nucleotide pools. Supplementation with medium-chain triglycerides during the suckling-to-weaning transition restored metabolic function and normalized cardiac growth and morphology. Our data reveal a critical developmental window in which mitoSAM-dependent lipoylation ensures heart maturation.

Persistent detection of high-risk human papillomavirus (HPV) is required for cervical carcinogenesis, yet the metabolic phenotype associated with distinct HPV transition states remains incompletely defined. We analyzed vaginal metabolomics data from 71 HIV-negative, non-smoking, premenopausal women without other sexually transmitted infections, grouped by three-visit HPV trajectories: persistent negative (NNN, n=20), late incident positivity (NNP, n=9), conversion with persistence (NPP, n=13), clearance after prior positivity (PPN, n=16), and persistent positive (PPP, n=13). After detection-based filtering, 186 putative and 64 quantitatively estimated metabolites were retained for integrated univariate, multivariate, network, pathway, and machine learning analyses. Global class separation was weak by PERMANOVA and by five-class classification, indicating that the vaginal metabolome does not reorganize broadly across all HPV states. In contrast, trajectory-specific signals were reproducible. The strongest pairwise contrast was NNP versus PPP (best cross-validated ROC AUC 0.778; permutation p=0.039). Glycolic acid was the dominant single metabolite, particularly for NNP versus PPP (Mann-Whitney p=6.96×10^-4, FDR=0.0446, AUROC=0.902; detection 88.9% versus 15.4%; combined abundance+detection FDR=0.0010). Persistent positivity was characterized by a focused uracil-high, methyl-donor/redox-low signature, including lower glycolic acid, S-adenosylmethionine, NAD+, and betaine, together with higher uracil. Ratio mining further sharpened discrimination, with uracil/S-adenosylmethionine and uracil/creatinine among the best PPP classifiers, and glucose 1-phosphate/isovaleric acid-valeric acid strongly separating NNP from NPP. These data support a model in which HPV trajectory is encoded by targeted metabolic states rather than a diffuse HPV-positive versus HPV-negative metabolomic shift.

Polyamines, putrescine, spermidine and spermine, are essential polycationic metabolites present in all eukaryotic cells, where they regulate fundamental processes including nucleic acid stabilization, translation, and stress responses. Spermidine synthase (SPDS), a member of the aminopropyltransferase (APT) family, catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dc-SAM) to putrescine to form spermidine. Although genomic analyses predict the presence of SPDS homologs in multiple fungal species, polyamine biosynthesis has not been experimentally characterized in the multidrug-resistant fungal pathogen Candidozyma auris. Here, we report the biochemical and functional characterization of the C. auris spermidine synthase, CauSpe3. The CauSPE3 gene complemented a Saccharomyces cerevisiae spe3Δ mutant demonstrating conserved function in vivo. Recombinant CauSpe3 was expressed in Escherichia coli, purified and analyzed using the fluorescence-based DAB-APT assay, which uses 1,2-diacetylbenzene (DAB) for polyamine detection. CauSpe3 catalyzed efficient conversion of putrescine to spermidine in the presence of dc-SAM, with Khalf values of 65.5 ± 7.11 µM for putrescine and 66.9 ± 2.09 µM for dc-SAM, and Vmax values of 7.1 ± 0.57 and 7.9 ± 0.12 nmol·µg[-1]·min[-1], respectively. A catalytic-site mutant and heat-inactivated enzyme showed no detectable activity, and product formation was confirmed by means of thin-layer chromatography and mass spectrometry. These findings establish CauSpe3 as a functional spermidine synthase.

Background/Objectives: DNA methylation is a key epigenetic modification involved in regulating many cellular processes, including gene expression and the maintenance of genome stability. Ultraviolet (UV) radiation induces DNA damage in the form of pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] and cyclobutane pyrimidine dimers (CPDs), which can lead to mutations if not efficiently repaired. While cytosine methylation has been implicated in influencing UV-induced DNA damage formation, the effect of DNA methylation modulators such as S-adenosyl-L-methionine (SAM) and RG108 on UV damage formation and repair remains unclear. Methods: Here, using immunoslot blot assays, we investigated the effects of SAM and RG108 on UV-induced DNA damage formation and repair in human lymphoblastoid cells. Results: We found that SAM, but not RG108, rapidly suppresses the formation of both (6-4)PP and CPD, with detectable effects within minutes of exposure. Although SAM pretreatment was associated with modestly accelerated early (6-4)PP repair, this effect was accompanied by substantially lower initial damage levels. When cells were treated with SAM or RG108 immediately after UV irradiation to ensure equivalent initial damage burden, no significant differences in repair were observed for either lesion type, demonstrating that the accelerated early (6-4)PP repair reflects reduced lesion burden rather than increased intrinsic nucleotide excision repair (NER). Global 5-methylcytosine (5mC) levels remained stable following SAM or RG108 treatment and during UV damage repair, suggesting that these effects occur independently of global alterations in DNA methylation. Conclusions: Together, our findings reveal that SAM modulates UV damage susceptibility at the level of lesion formation without altering repair, highlighting a previously unrecognized role for DNA methylation modulators in regulating genome stability.

Betaine-homocysteine methyltransferase (BHMT) is an enzyme involved in one-carbon metabolism and plays a crucial role in maintaining liver health. In this study, we investigated the impact of liver-specific deletion of BHMT on liver dysfunction using a mouse model. We generated BHMT floxed mice and bred them with albumin Cre to generate liver-specific BHMT knockout (BHMT LKO) mice. Liver tissues harvested from six-month-old chow-fed BHMT floxed and LKO mice were characterized through histological, biochemical, and molecular analyses. BHMT LKO mice displayed a complete loss of hepatic expression of BHMT mRNA, protein and enzyme activity. Histopathological analysis revealed the development of hepatic steatosis in BHMT LKO mice compared to the floxed mice. These morphological changes were supported by biochemical analysis showing elevated levels of hepatic triglycerides in conjunction with a profound decrease in the methylation potential (i.e., reduced S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) ratio), which was mainly driven by a six- to sevenfold increase in SAH levels. BHMT LKO mice also exhibited increased lipid peroxidation and lysosomal dysfunction compared to floxed mice. Early signs of inflammation were seen in the livers of BHMT LKO mice of both sexes, as evident from significant increase in CD68-positive cells and interleukin 1β levels. Additionally, there was a moderate increase in fibrosis, as evidenced by the upregulated expression of α-smooth muscle actin and collagen II levels and the histological assessment of picrosirius red-stained liver sections of BHMT LKO mice of both sexes compared to their respective counterparts. These findings demonstrate that hepatic BHMT deficiency promotes lipid accumulation, lysosomal/proteasomal dysfunction, and early inflammatory and fibrotic changes in the liver by reducing the methylation potential. Collectively, our results underscore BHMT as a critical regulator of liver homeostasis and a potential therapeutic target in liver-related disorders.

Choline is an essential nutrient required for the synthesis of key molecules, such as phosphatidylcholine, sphingomyelin, acetylcholine, and S-adenosylmethionine. Choline metabolism encompasses two phases, namely the postprandial and postabsorptive states. The former enables the digestion, absorption, distribution, and storage of choline derivatives after a meal, while the latter allows the cellular utilization of choline and the mobilization of stored choline-containing molecules during fasting. Understanding choline metabolism is fundamental to the study of lipid disorders such as steatohepatitis or atherosclerosis, as well as neurodegenerative diseases, including Alzheimer's disease, and inflammatory signaling pathways. Members of the alkaline phosphatase (AP) superfamily are prominent contributors to extracellular choline metabolism. Within this family, several APs and ectonucleotide pyrophosphatases/phosphodiesterases (ENPP) members are required for physiological choline metabolism. While intestinal alkaline phosphatase (IAP) and alkaline sphingomyelinase/ENPP7 both participate in the digestion of choline-containing derivatives in the gut during the postprandial phase, circulating ENPP2, ENPP6, and tissue-nonspecific alkaline phosphatase (TNAP) act during the postabsorptive phase to generate choline. In this review we first provide a comprehensive overview of choline metabolism and then describe how APs and ENPPs have functionally and structurally co-evolved to catalyze sequential reactions within this metabolic pathway.