RESUMO
Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.
Assuntos
Íntrons , Metionina Adenosiltransferase/genética , Metiltransferases/metabolismo , Splicing de RNA , S-Adenosilmetionina/metabolismo , Animais , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Sequências Repetidas Invertidas , Metionina Adenosiltransferase/química , Metilação , Metiltransferases/química , Schizosaccharomyces/metabolismoRESUMO
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.
Assuntos
Adenosina/análogos & derivados , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Biossíntese de Proteínas , S-Adenosilmetionina/metabolismo , Adenosina/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Feminino , Células HEK293 , Células HeLa , Humanos , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Metilação , Camundongos Nus , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
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.
Assuntos
Epigênese Genética , Formaldeído , Metionina Adenosiltransferase , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Humanos , Carbono/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genéticaRESUMO
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.
Assuntos
Metionina Adenosiltransferase , S-Adenosilmetionina , Camundongos , Animais , S-Adenosilmetionina/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Mucosa Intestinal/metabolismo , Metionina , Suplementos NutricionaisRESUMO
Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.
Assuntos
Astrócitos/patologia , Sistema Nervoso Central/patologia , Inflamação/patologia , Fator de Transcrição MafG/genética , Proteínas Repressoras/genética , Animais , Antioxidantes/metabolismo , Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , Metilação de DNA , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Inflamação/genética , Masculino , Metionina Adenosiltransferase/genética , Camundongos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Fator 2 Relacionado a NF-E2/genética , Análise de Sequência de RNA , Transdução de Sinais , Transcrição GênicaRESUMO
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.
Assuntos
Desoxiadenosinas , Metionina Adenosiltransferase , Neoplasias , Proteína-Arginina N-Metiltransferases , Purina-Núcleosídeo Fosforilase , S-Adenosilmetionina , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxiadenosinas/antagonistas & inibidores , Desoxiadenosinas/genética , Desoxiadenosinas/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Neoplasias/genética , Neoplasias/fisiopatologia , Neoplasias/terapia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/farmacologia , Pirrolidinas/uso terapêutico , S-Adenosilmetionina/metabolismoRESUMO
Alternative splicing of pre-mRNAs can regulate gene expression levels by coupling with nonsense-mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS-NMD) in an organism, we performed long-read RNA sequencing of poly(A)+ RNAs from an NMD-deficient mutant strain of Caenorhabditis elegans, and obtained full-length sequences for mRNA isoforms from 259 high-confidence AS-NMD genes. Among them are the S-adenosyl-L-methionine (SAM) synthetase (sams) genes sams-3 and sams-4. SAM synthetase activity autoregulates sams gene expression through AS-NMD in a negative feedback loop. We furthermore find that METT-10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation inâ£vivo, and specifically methylates the invariant AG dinucleotide at the distal 3' splice site (3'SS) inâ£vitro. Direct RNA sequencing coupled with machine learning confirms m6 A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m6 A modification at the 3'SS of the sams genes.
Assuntos
Processamento Alternativo/genética , Homeostase/genética , Ligases/genética , Metionina Adenosiltransferase/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , S-Adenosilmetionina/metabolismo , Animais , Caenorhabditis elegans/genética , Metiltransferases/genética , Precursores de RNA/genéticaRESUMO
S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events, so cells need to carefully control SAM levels. MAT2A encodes the only SAM synthetase expressed in the majority of human cells, and its 3'-UTR has six conserved regulatory hairpins (hp1-6) that can be methylated by the N6-methyladenosine methyltransferase METTL16. Hp1 begins 8 nt from the stop codon, whereas hp2-6 are clustered further downstream (â¼800 nt). These hairpins have been proposed to regulate MAT2A mRNA levels in response to intracellular SAM levels by regulating intron detention of the last intron of MAT2A and by modulating the stability of the fully spliced mRNA. However, a dissection of these two posttranscriptional mechanisms has not been previously reported. Using a modular reporter system, we show that hp1 functions primarily when the detained intron is included in the reporter and when that intron has a suboptimal polypyrimidine tract. In contrast, the hp2-6 cluster modulates mRNA stability independent of the detained intron, although hp1 may make a minor contribution to the regulation of decay as well. Taken with previously published reports, these data support a two-tiered model for MAT2A posttranscriptional regulation by METTL16 through its interactions with hp1 and hp2-6. In the upstream tier, hp1 and METTL16 control MAT2A intron detention, whereas the second tier involves METTL16-dependent methylation of hp2-6 to control MAT2A mRNA stability. Thus, cells use a similar set of molecular factors to achieve considerable complexity in the posttranscriptional regulation of SAM homeostasis.
Assuntos
Adenosina , Regulação da Expressão Gênica , Humanos , Metilação , Adenosina/metabolismo , S-Adenosilmetionina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Proteínas Cromossômicas não Histona/genética , Metionina Adenosiltransferase/genética , Metiltransferases/genéticaRESUMO
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.
Assuntos
Hepatopatias Alcoólicas , Metionina Adenosiltransferase , Sumoilação , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/genética , Animais , Camundongos , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/genética , Humanos , Mitocôndrias Hepáticas/metabolismo , Masculino , Hepatócitos/metabolismo , Fígado/metabolismoRESUMO
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.
Assuntos
Cucurbita , Frutas , Regulação da Expressão Gênica de Plantas , Metionina Adenosiltransferase , RNA Longo não Codificante , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Cucurbita/genética , Cucurbita/crescimento & desenvolvimento , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Etilenos/metabolismoRESUMO
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.
Assuntos
Caenorhabditis elegans , Metiltransferases , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Homeostase/genética , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Metilação , Metiltransferases/química , Precursores de RNARESUMO
Methylation, a widely occurring natural modification serving diverse regulatory and structural functions, is carried out by a myriad of S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). The AdoMet cofactor is produced from l-methionine (Met) and ATP by a family of multimeric methionine adenosyltransferases (MAT). To advance mechanistic and functional studies, strategies for repurposing the MAT and MTase reactions to accept extended versions of the transferable group from the corresponding precursors have been exploited. Here, we used structure-guided engineering of mouse MAT2A to enable biocatalytic production of an extended AdoMet analogue, Ado-6-azide, from a synthetic methionine analogue, S-(6-azidohex-2-ynyl)-l-homocysteine (N3-Met). Three engineered MAT2A variants showed catalytic proficiency with the extended analogues and supported DNA derivatization in cascade reactions with M.TaqI and an engineered variant of mouse DNMT1 both in the absence and presence of competing Met. We then installed two of the engineered variants as MAT2A-DNMT1 cascades in mouse embryonic stem cells by using CRISPR-Cas genome editing. The resulting cell lines maintained normal viability and DNA methylation levels and showed Dnmt1-dependent DNA modification with extended azide tags upon exposure to N3-Met in the presence of physiological levels of Met. This for the first time demonstrates a genetically stable system for biosynthetic production of an extended AdoMet analogue, which enables mild metabolic labeling of a DNMT-specific methylome in live mammalian cells.
Assuntos
Metilação de DNA , Metionina Adenosiltransferase , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/química , Animais , Camundongos , Engenharia de Proteínas , Epigenoma , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , HumanosRESUMO
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.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/metabolismo , Neoplasias Colorretais/genética , Neoplasias Hepáticas/patologia , Metaloproteinase 7 da Matriz/genética , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Proibitinas , Microambiente TumoralRESUMO
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.
Assuntos
Apoptose , Carcinoma Pulmonar de Células não Pequenas , Sinergismo Farmacológico , Neoplasias Pulmonares , Metionina Adenosiltransferase , Metiltransferases , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/genética , Metiltransferases/metabolismo , Metiltransferases/antagonistas & inibidores , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Mieloma Múltiplo , S-Adenosilmetionina , Humanos , S-Adenosilmetionina/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Bortezomib/farmacologia , Prognóstico , Serina-Treonina Quinases TOR , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismoRESUMO
The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.
Assuntos
Metionina Adenosiltransferase , S-Adenosilmetionina , Animais , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/metabolismo , Masculino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ratos , Metionina/metabolismo , Ratos Sprague-Dawley , Purina-Núcleosídeo Fosforilase/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , MultiômicaRESUMO
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.
Assuntos
Criptosporidiose , Cryptosporidium , Humanos , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/química , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/metabolismo , Projetos Piloto , Cryptosporidium/metabolismo , Escherichia coli/genéticaRESUMO
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.
Assuntos
Metionina Adenosiltransferase , S-Adenosilmetionina , Animais , Humanos , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Metilação , RNA Mensageiro/metabolismo , S-Adenosilmetionina/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Ferroptose , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Fatores de Transcrição , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Histona Desacetilase 2/metabolismoRESUMO
BACKGROUND AND AIMS: Methionine adenosyltransferase 1A (MAT1A) is responsible for S-adenosylmethionine (SAMe) biosynthesis in the liver. Mice lacking Mat1a have hepatic SAMe depletion and develop NASH and HCC spontaneously. Several kinases are activated in Mat1a knockout (KO) mice livers. However, characterizing the phospho-proteome and determining whether they contribute to liver pathology remain open for study. Our study aimed to provide this knowledge. APPROACH AND RESULTS: We performed phospho-proteomics in Mat1a KO mice livers with and without SAMe treatment to identify SAMe-dependent changes that may contribute to liver pathology. Our studies used Mat1a KO mice at different ages treated with and without SAMe, cell lines, in vitro translation and kinase assays, and human liver specimens. We found that the most striking change was hyperphosphorylation and increased content of La-related protein 1 (LARP1), which, in the unphosphorylated form, negatively regulates translation of 5'-terminal oligopyrimidine (TOP)-containing mRNAs. Consistently, multiple TOP proteins are induced in KO livers. Translation of TOP mRNAs ribosomal protein S3 and ribosomal protein L18 was enhanced by LARP1 overexpression in liver cancer cells. We identified LARP1-T449 as a SAMe-sensitive phospho-site of cyclin-dependent kinase 2 (CDK2). Knocking down CDK2 lowered LARP1 phosphorylation and prevented LARP1-overexpression-mediated increase in translation. LARP1-T449 phosphorylation induced global translation, cell growth, migration, invasion, and expression of oncogenic TOP-ribosomal proteins in HCC cells. LARP1 expression is increased in human NASH and HCC. CONCLUSIONS: Our results reveal a SAMe-sensitive mechanism of LARP1 phosphorylation that may be involved in the progression of NASH to HCC.