mTORC1 stimulates cell growth through SAM synthesis and m6A mRNA-dependent control of protein synthesis.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient, growth, and oncogenic signals. We found that mTORC1 stimulates the synthesis of the major methyl donor, S-adenosylmethionine (SAM), through the control of methionine adenosyltransferase 2 alpha (MAT2A) expression. The transcription factor c-MYC, downstream of mTORC1, directly binds to intron 1 of MAT2A and promotes its expression. Furthermore, mTORC1 increases the protein abundance of Wilms' tumor 1-associating protein (WTAP), the positive regulatory subunit of the human N6-methyladenosine (m6A) RNA methyltransferase complex. Through the control of MAT2A and WTAP levels, mTORC1 signaling stimulates m6A RNA modification to promote protein synthesis and cell growth. A decline in intracellular SAM levels upon MAT2A inhibition decreases m6A RNA modification, protein synthesis rate, and tumor growth. Thus, mTORC1 adjusts m6A RNA modification through the control of SAM and WTAP levels to prime the translation machinery for anabolic cell growth.

Formaldehyde regulates one-carbon metabolism and epigenetics.

Recently, Pham et al. used an array of model systems to uncover a role for the enzyme methionine adenosyltransferase (MAT)-1A, which is mainly expressed in liver, in both sensing formaldehyde and regulating transcriptional responses that protect against it. This provides a new lens for understanding the effects of formaldehyde on gene regulation.

Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery.

The factors that sequester transcriptionally repressed heterochromatin at the nuclear periphery are currently unknown. In a genome-wide RNAi screen, we found that depletion of S-adenosylmethionine (SAM) synthetase reduces histone methylation globally and causes derepression and release of heterochromatin from the nuclear periphery in Caenorhabditis elegans embryos. Analysis of histone methyltransferases (HMTs) showed that elimination of two HMTs, MET-2 and SET-25, mimics the loss of SAM synthetase, abrogating the perinuclear attachment of heterochromatic transgenes and of native chromosomal arms rich in histone H3 lysine 9 methylation. The two HMTs target H3K9 in a consecutive fashion: MET-2, a SETDB1 homolog, mediates mono- and dimethylation, and SET-25, a previously uncharacterized HMT, deposits H3K9me3. SET-25 colocalizes with its own product in perinuclear foci, in a manner dependent on H3K9me3, but not on its catalytic domain. This colocalization suggests an autonomous, self-reinforcing mechanism for the establishment and propagation of repeat-rich heterochromatin.

S-adenosyl-L-methionine supplementation alleviates damaged intestinal epithelium and inflammatory infiltration caused by Mat2a deficiency.

Methionine is important for intestinal development and homeostasis in various organisms. However, the underlying mechanisms are poorly understood. Here, we demonstrate that the methionine adenosyltransferase gene Mat2a is essential for intestinal development and that the metabolite S-adenosyl-L-methionine (SAM) plays an important role in intestinal homeostasis. Intestinal epithelial cell (IEC)-specific knockout of Mat2a exhibits impaired intestinal development and neonatal lethality. Mat2a deletion in the adult intestine reduces cell proliferation and triggers IEC apoptosis, leading to severe intestinal epithelial atrophy and intestinal inflammation. Mechanistically, we reveal that SAM maintains the integrity of differentiated epithelium and protects IECs from apoptosis by suppressing the expression of caspases 3 and 8 and their activation. SAM supplementation improves the defective intestinal epithelium and reduces inflammatory infiltration sequentially. In conclusion, our study demonstrates that methionine metabolism and its intermediate metabolite SAM play essential roles in intestinal development and homeostasis in mice.

Structural Basis for Regulation of METTL16, an S-Adenosylmethionine Homeostasis Factor.

S-adenosylmethionine (SAM) is an essential metabolite that acts as a cofactor for most methylation events in the cell. The N6-methyladenosine (m6A) methyltransferase METTL16 controls SAM homeostasis by regulating the abundance of SAM synthetase MAT2A mRNA in response to changing intracellular SAM levels. Here we present crystal structures of METTL16 in complex with MAT2A RNA hairpins to uncover critical molecular mechanisms underlying the regulated activity of METTL16. The METTL16-RNA complex structures reveal atomic details of RNA substrates that drive productive methylation by METTL16. In addition, we identify a polypeptide loop in METTL16 near the SAM binding site with an autoregulatory role. We show that mutations that enhance or repress METTL16 activity in vitro correlate with changes in MAT2A mRNA levels in cells. Thus, we demonstrate the structural basis for the specific activity of METTL16 and further suggest the molecular mechanisms by which METTL16 efficiency is tuned to regulate SAM homeostasis.

Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition.

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

Sumoylation of methionine adenosyltransferase alpha 1 promotes mitochondrial dysfunction in alcohol-associated liver disease.

BACKGROUND AND AIMS: Methionine adenosyltransferase alpha1 (MATα1) is responsible for the biosynthesis of S-adenosylmethionine in normal liver. Alcohol consumption enhances MATα1 interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), which blocks MATα1 mitochondrial targeting, resulting in lower mitochondrial MATα1 content and mitochondrial dysfunction in alcohol-associated liver disease (ALD) in part through upregulation of cytochrome P450 2E1. Conversely, alcohol intake enhances SUMOylation, which enhances cytochrome P450 2E1 expression. MATα1 has potential SUMOylation sites, but whether MATα1 is regulated by SUMOylation in ALD is unknown. Here, we investigated if MATα1 is regulated by SUMOylation and, if so, how it impacts mitochondrial function in ALD. APPROACH AND RESULTS: Proteomics profiling revealed hyper-SUMOylation of MATα1, and prediction software identified lysine 48 (K48) as the potential SUMOylation site in mice (K47 in humans). Experiments with primary hepatocytes, mouse, and human livers revealed that SUMOylation of MAT1α by SUMO2 depleted mitochondrial MATα1. Furthermore, mutation of MATα1 K48 prevented ethanol-induced mitochondrial membrane depolarization, MATα1 depletion, and triglyceride accumulation. Additionally, CRISPR/CRISPR associated protein 9 gene editing of MATα1 at K48 hindered ethanol-induced MATα1-PIN1 interaction, degradation, and phosphorylation of MATα1 in vitro. In vivo, CRISPR/CRISPR associated protein 9 MATα1 K48 gene-edited mice were protected from ethanol-induced fat accumulation, liver injury, MATα1-PIN1 interaction, mitochondrial MATα1 depletion, mitochondrial dysfunction, and low S-adenosylmethionine levels. CONCLUSIONS: Taken together, our findings demonstrate an essential role for SUMOylation of MATα1 K48 for interaction with PIN1 in ALD. Preventing MATα1 K48 SUMOylation may represent a potential treatment strategy for ALD.

A long noncoding RNA functions in pumpkin fruit development through S-adenosyl-L-methionine synthetase.

Long noncoding RNAs (lncRNAs) play important roles in various biological processes. However, the regulatory roles of lncRNAs underlying fruit development have not been extensively studied. The pumpkin (Cucurbita spp.) is a preferred model for understanding the molecular mechanisms regulating fruit development because of its variable shape and size and large inferior ovary. Here, we performed strand-specific transcriptome sequencing on pumpkin (Cucurbita maxima "Rimu") fruits at 6 developmental stages and identified 5,425 reliably expressed lncRNAs. Among the 332 lncRNAs that were differentially expressed during fruit development, the lncRNA MSTRG.44863.1 was identified as a negative regulator of pumpkin fruit development. MSTRG.44863.1 showed a relatively high expression level and an obvious period-specific expression pattern. Transient overexpression and silencing of MSTRG.44863.1 significantly increased and decreased the content of 1-aminocyclopropane carboxylic acid (a precursor of ethylene) and ethylene production, respectively. RNA pull-down and microscale thermophoresis assays further revealed that MSTRG.44863.1 can interact with S-adenosyl-L-methionine synthetase (SAMS), an enzyme in the ethylene synthesis pathway. Considering that ethylene negatively regulates fruit development, these results indicate that MSTRG.44863.1 plays an important role in the regulation of pumpkin fruit development, possibly through interacting with SAMS and affecting ethylene synthesis. Overall, our findings provide a rich resource for further study of fruit-related lncRNAs while offering insights into the regulation of fruit development in plants.

Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis.

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

Hepatic prohibitin 1 and methionine adenosyltransferase α1 defend against primary and secondary liver cancer metastasis.

BACKGROUND & AIMS: The liver is a common site of cancer metastasis, most commonly from colorectal cancer, and primary liver cancers that have metastasized are associated with poor outcomes. The underlying mechanisms by which the liver defends against these processes are largely unknown. Prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) are highly expressed in the liver. They positively regulate each other and their deletion results in primary liver cancer. Here we investigated their roles in primary and secondary liver cancer metastasis. METHODS: We identified common target genes of PHB1 and MAT1A using a metastasis array, and measured promoter activity and transcription factor binding using luciferase reporter assays and chromatin immunoprecipitation, respectively. We examined how PHB1 or MAT1A loss promotes liver cancer metastasis and whether their loss sensitizes to colorectal liver metastasis (CRLM). RESULTS: Matrix metalloproteinase-7 (MMP-7) is a common target of MAT1A and PHB1 and its induction is responsible for increased migration and invasion when MAT1A or PHB1 is silenced. Mechanistically, PHB1 and MAT1A negatively regulate MMP7 promoter activity via an AP-1 site by repressing the MAFG-FOSB complex. Loss of MAT1A or PHB1 also increased MMP-7 in extracellular vesicles, which were internalized by colon and pancreatic cancer cells to enhance their oncogenicity. Low hepatic MAT1A or PHB1 expression sensitized to CRLM, but not if endogenous hepatic MMP-7 was knocked down first, which lowered CD4+ T cells while increasing CD8+ T cells in the tumor microenvironment. Hepatocytes co-cultured with colorectal cancer cells express less MAT1A/PHB1 but more MMP-7. Consistently, CRLM raised distant hepatocytes' MMP-7 expression in mice and humans. CONCLUSION: We have identified a PHB1/MAT1A-MAFG/FOSB-MMP-7 axis that controls primary liver cancer metastasis and sensitization to CRLM. IMPACT AND IMPLICATIONS: Primary and secondary liver cancer metastasis is associated with poor outcomes but whether the liver has underlying defense mechanism(s) against metastasis is unknown. Here we examined the hypothesis that hepatic prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) cooperate to defend the liver against metastasis. Our studies found PHB1 and MAT1A form a complex that suppresses matrix metalloproteinase-7 (MMP-7) at the transcriptional level and loss of either PHB1 or MAT1A sensitizes the liver to metastasis via MMP-7 induction. Strategies that target the PHB1/MAT1A-MMP-7 axis may be a promising approach for the treatment of primary and secondary liver cancer metastasis.

The combination of methionine adenosyltransferase 2A inhibitor and methyltransferase like 3 inhibitor promotes apoptosis of non-small cell lung cancer cells and produces synergistic anti-tumor activity.

Methionine adenosyltransferase 2 A (MAT2A) mediates the synthesis of methyl donor S-Adenosylmethionine (SAM), providing raw materials for methylation reactions in cells. MAT2A inhibitors are currently used for the treatment of tumors with methylthioadenosine phosphorylase (MTAP) deficiency in clinical research. Methyltransferase like 3 (METTL3) catalyzes N6-methyladenosine (m6A) modification of mRNA in mammalian cells using SAM as the substrate which has been shown to affect the tumorigenesis of non-small cell lung cancer (NSCLC) from multiple perspectives. MAT2A-induced SAM depletion may have the potential to inhibit the methyl transfer function of METTL3. Therefore, in order to expand the applicability of inhibitors, improve anti-tumor effects and reduce toxicity, the combinational effect of MAT2A inhibitor AG-270 and METTL3 inhibitor STM2457 was evaluated in NSCLC. The results showed that this combination induced cell apoptosis rather than cell cycle arrest, which was non-tissue-specific and was independent of MTAP expression status, resulting in a significant synergistic anti-tumor effect. We further elucidated that the combination-induced enhanced apoptosis was associated with the decreased m6A level, leading to downregulation of PI3K/AKT protein, ultimately activating the apoptosis-related proteins. Unexpectedly, although combination therapy resulted in metabolic recombination, no significant change in methionine metabolic metabolites was found. More importantly, the combination also exerted synergistic effects in vivo. In summary, the combination of MAT2A inhibitor and METTL3 inhibitor showed synergistic effects both in vivo and in vitro, which laid a theoretical foundation for expanding the clinical application research of the two types of drugs.

S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is incurable because of the inevitable development of drug resistance. Methionine adenosyltransferase 2α (MAT2A) is the primary producer of the methyl donor S-adenosylmethionine (SAM) and several studies have documented MAT2A deregulation in different solid cancers. As the role of MAT2A in MM has not been investigated yet, the aim of this study was to clarify the potential role and underlying molecular mechanisms of MAT2A in MM, exploring new therapeutic options to overcome drug resistance. By analyzing publicly available gene expression profiling data, MAT2A was found to be more highly expressed in patient-derived myeloma cells than in normal bone marrow plasma cells. The expression of MAT2A correlated with an unfavorable prognosis in relapsed patients. MAT2A inhibition in MM cells led to a reduction in intracellular SAM levels, which resulted in impaired cell viability and proliferation, and induction of apoptosis. Further mechanistic investigation demonstrated that MAT2A inhibition inactivated the mTOR-4EBP1 pathway, accompanied by a decrease in protein synthesis. MAT2A targeting in vivo with the small molecule compound FIDAS-5 was able to significantly reduce tumor burden in the 5TGM1 model. Finally, we found that MAT2A inhibition can synergistically enhance the anti-MM effect of the standard-of-care agent bortezomib on both MM cell lines and primary human CD138+ MM cells. In summary, we demonstrate that MAT2A inhibition reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, our findings suggest that the MAT2A inhibitor FIDAS-5 could be a novel compound to improve bortezomib-based treatment of MM.

Discovery of novel MAT2A inhibitors by an allosteric site-compatible fragment growing approach.

The methionine adenosyltransferase MAT2A catalyzes the synthesis ofthe methyl donor S-adenosylmethionine (SAM) and thereby regulates critical aspects of metabolism and transcription. Aberrant MAT2A function can lead to metabolic and transcriptional reprogramming of cancer cells, and MAT2A has been shown to promote survival of MTAP-deficient tumors, a genetic alteration that occurs in âˆ¼ 13 % of all tumors. Thus, MAT2A holds great promise as a novel anticancer target. Here, we report a novel series of MAT2A inhibitors generated by a fragment growing approach from AZ-28, a low-molecular weight MAT2A inhibitor with promising pre-clinical properties. X-ray co-crystal structure revealed that compound 7 fully occupies the allosteric pocket of MAT2A as a single molecule mimicking MAT2B. By introducing additional backbone interactions and rigidifying the requisite linker extensions, we generated compound 8, which exhibited single digit nanomolar enzymatic and sub-micromolar cellular inhibitory potency for MAT2A.

A growth-based assay using fluorescent protein emission to screen for S-adenosylmethionine synthetase inhibitors.

The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l-methionine S-adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S-adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species-selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog-selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum.

Elucidating the Kinetic Mechanism of Human METTL16.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 µM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 µM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 µM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.

MAT1A Suppression by the CTBP1/HDAC1/HDAC2 Transcriptional Complex Induces Immune Escape and Reduces Ferroptosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) remains a significant health burden globally due to its high prevalence and morbidity. C-terminal-binding protein 1 (CTBP1) is a transcriptional corepressor that modulates gene transcription by interacting with transcription factors or chromatin-modifying enzymes. High CTBP1 expression has been associated with the progression of various human cancers. In this study, bioinformatics analysis suggested the existence of a CTBP1/histone deacetylase 1 (HDAC1)/HDAC2 transcriptional complex that regulates the expression of methionine adenosyltransferase 1A (MAT1A), whose loss has been associated with ferroptosis suppression and HCC development. Thus, this study aims to investigate the interactions between the CTBP1/HDAC1/HDAC2 complex and MAT1A and their roles in HCC progression. First, high expression of CTBP1 was observed in HCC tissues and cells, where it promoted HCC cell proliferation and mobility while inhibiting cell apoptosis. CTBP1 interacted with HDAC1 and HDAC2 to suppress the MAT1A transcription, and silencing of either HDAC1 or HDAC2 or overexpression of MAT1A led to the inhibition of cancer cell malignancy. In addition, MAT1A overexpression resulted in increased S-adenosylmethionine levels, which promoted ferroptosis of HCC cells directly or indirectly by increasing CD8+ T-cell cytotoxicity and interferon-γ production. In vivo, MAT1A overexpression suppressed growth of CTBP1-induced xenograft tumors in mice while enhancing immune activity and inducing ferroptosis. However, treatment with ferrostatin-1, a ferroptosis inhibitor, blocked the tumor-suppressive effects of MAT1A. Collectively, this study reveals that the CTBP1/HDAC1/HDAC2 complex-induced MAT1A suppression is liked to immune escape and reduced ferroptosis of HCC cells.

Inhibition of MAT2A suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss.

Methionine adenosyltransferase II alpha (MAT2A) is the key enzyme to transform methionine and adenosine-triphosphate (ATP) to S-adenosylmethionine (SAM), a general methyl-group donor in vitro. MAT2A has been reported to participate in the NF-κB pathway and maintain the methylated modification, which also affects osteoclastogenesis. In this study, we found the expression of MAT2A was increased upon RANKL stimulation. Pharmacological inhibition of MAT2A by its selective inhibitor AG-270 or genetic silencing by MAT2A-shRNA suppressed osteoclast formation and function in vitro. In vivo treatment with the inhibitor AG-270 also prevented OVX-induced bone loss. Further study revealed that the inhibition of MAT2A affected osteoclast differentiation mainly by suppressing crucial transcription factors and reactive oxygen species induced by RANKL. A quasi-targeted metabolomics assay performed by LC-MS/MS indicated that SAM was reduced by MAT2A knockdown, and the administration of SAM partly rescued the effects of MAT2A inhibition on osteoclastogenesis. These findings revealed that MAT2A is crucial for osteoclastogenesis and might be a potential target for the treatment of osteoporosis attributed to osteoclast dysfunction.

The SMYD3-dependent H3K4me3 status of IGF2 intensifies local Th2 differentiation in CRSwNP via positive feedback.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous and common upper airway disease divided into various inflammatory endotypes. Recent epidemiological findings showed a T helper 2 (Th2)-skewed dominance in CRSwNP patients. Histone modification alterations can regulate transcriptional and translational expression, resulting in abnormal pathogenic changes and the occurrence of diseases. Trimethylation of histone H3 lysine 4 (H3K4me3) is considered an activator of gene expression through modulation of accessibility for transcription, which is closely related to CRSwNP. H3K4me3 levels in the human nasal epithelium may change under Th2-biased inflammatory conditions, resulting in exaggerated local nasal Th2 responses via the regulation of naïve CD4+ T-cell differentiation. Here, we revealed that the level of SET and MYND domain-containing protein 3 (SMYD3)-mediated H3K4me3 was increased in NPs from Th2 CRSwNP patients compared with those from healthy controls. We demonstrated that SMYD3-mediated H3K4me3 is increased in human nasal epithelial cells under Th2-biased inflammatory conditions via S-adenosyl-L-methionine (SAM) production and further found that the H3K4me3high status of insulin-like growth factor 2 (IGF2) produced in primary human nasal epithelial cells could promote naïve CD4+ T-cell differentiation into Th2 cells. Moreover, we found that SAM production was dependent on the c-Myc/methionine adenosyltransferase 2A (MAT2A) axis in the nasal epithelium. Understanding histone modifications in the nasal epithelium has immense potential utility in the development of novel classes of therapeutics targeting Th2 polarization in Th2 CRSwNP. Video Abstract.

Enhanced selenocysteine biosynthesis for seleno-methylselenocysteine production in Bacillus subtilis.

Seleno-methylselenocysteine (SeMCys) is an effective component for selenium supplementation with anti-carcinogenic potential and can ameliorate neuropathology and cognitive deficits. In this study, we aimed to engineer Bacillus subtilis 168 for the microbial production of SeMCys. First, the accumulation of intracellular selenocysteine (SeCys) as the precursor of SeMCys was enhanced through overexpression of serine O-acetyltransferase, which was desensitized against feedback inhibition by cysteine. Next, the S-adenosylmethionine (SAM) synthetic pathway was optimized to improve methyl donor availability through expression of S-adenosylmethionine synthetase. Further, SeMCys was successfully produced through expression of the selenocysteine methyltransferase in SeCys and SAM-producing strain. The increased expression level of selenocysteine methyltransferase benefited the SeMCys production. Finally, all the heterologous genes were integrated into the genome of B. subtilis, and the strain produced SeMCys at a titer of 18.4 µg/L in fed-batch culture. This is the first report on the metabolic engineering of B. subtilis for microbial production of SeMCys and provides a good starting point for future pathway engineering to achieve the industrial-grade production of SeMCys. KEY POINTS: • Expression of the feedback-insensitive serine O-acetyltransferase provided B. subtilis the ability of accumulating SeCys. • SAM production was enhanced through expressing S-adenosylmethionine synthetase in B. subtilis. • Expression of selenocysteine methyltransferase in SeCys and SAM-accumulating strain facilitated SeMCys production.