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1.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614187

RESUMO

Ladderane lipids (found in the membranes of anaerobic ammonium-oxidizing [anammox] bacteria) have unique ladder-like hydrophobic groups, and their highly strained exotic structure has attracted the attention of scientists. Although enzymes encoded in type II fatty acid biosynthesis (FASII) gene clusters in anammox bacteria, such as S-adenosyl-l-methionine (SAM)-dependent enzymes, have been proposed to construct a ladder-like structure using a substrate connected to acyl carrier protein from anammox bacteria (AmxACP), no experimental evidence to support this hypothesis was reported to date. Here, we report the crystal structure of a SAM-dependent methyltransferase from anammox bacteria (AmxMT1) that has a substrate and active site pocket between a class I SAM methyltransferase-like core domain and an additional α-helix inserted into the core domain. Structural comparisons with homologous SAM-dependent C-methyltransferases in polyketide synthase, AmxACP pull-down assays, AmxACP/AmxMT1 complex structure predictions by AlphaFold, and a substrate docking simulation suggested that a small compound connected to AmxACP could be inserted into the pocket of AmxMT1, and then the enzyme transfers a methyl group from SAM to the substrate to produce branched lipids. Although the enzymes responsible for constructing the ladder-like structure remain unknown, our study, for the first time, supports the hypothesis that biosynthetic intermediates connected to AmxACP are processed by SAM-dependent enzymes, which are not typically involved in the FASII system, to produce the ladder-like structure of ladderane lipids in anammox bacteria.


Assuntos
Metionina , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Metionina/metabolismo , Proteína de Transporte de Acila/metabolismo , Metiltransferases/metabolismo , Oxidação Anaeróbia da Amônia , Bactérias/metabolismo , Racemetionina/metabolismo , Lipídeos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
2.
Nature ; 613(7943): 383-390, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36599982

RESUMO

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


Assuntos
Microscopia Crioeletrônica , Proteínas de Ligação ao GTP , Metilação , Metiltransferases , Processamento Pós-Transcricional do RNA , RNA de Transferência , Humanos , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/ultraestrutura , RNA de Transferência/química , RNA de Transferência/metabolismo , RNA de Transferência/ultraestrutura , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , Biocatálise
3.
Nutrients ; 15(2)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36678154

RESUMO

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


Assuntos
Leite Humano , S-Adenosilmetionina , Adulto , Criança , Lactente , Feminino , Humanos , S-Adenosilmetionina/metabolismo , Cromatografia Líquida , Leite Humano/metabolismo , Carbono , Espectrometria de Massas em Tandem , Metionina/metabolismo , Racemetionina , S-Adenosil-Homocisteína/metabolismo , Homocisteína
4.
Biotechnol Lett ; 45(2): 255-262, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36550338

RESUMO

S-Adenosylmethionine (SAM) is a crucial small-molecule metabolite widely used in food and medicine. The development of high-throughput biosensors for SAM biosynthesis can significantly improve the titer of SAM. This paper constructed a synthetic transcription factor (TF)-based biosensor for SAM detecting in Saccharomyces cerevisiae. The synthetic TF, named MetJ-hER-VP16, consists of an Escherichia coli-derived DNA-binding domain MetJ, GS linker, the human estrogen receptor binding domain hER, and the viral activation domain VP16. The synthetic biosensor is capable of sensing SAM in a dose-dependent manner with fluorescence as the output. Additionally, it is tightly regulated by the inducer SAM and ß-estradiol, which means that the fluorescence output is only available when both are present together. The synthetic SAM biosensor could potentially be applied for high-throughput metabolic engineering and is expected to improve SAM production.


Assuntos
Técnicas Biossensoriais , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/metabolismo , S-Adenosilmetionina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Etoposídeo/metabolismo , Escherichia coli/metabolismo
5.
Biochemistry ; 62(2): 494-506, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36584291

RESUMO

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.


Assuntos
RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Metilação , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , RNA Mensageiro/metabolismo , Splicing de RNA , Conformação de Ácido Nucleico , Metionina Adenosiltransferase/metabolismo
6.
FASEB J ; 37(1): e22698, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36520012

RESUMO

Folate plays an important role in the modulation of one-carbon metabolism and DNA methylation through a complex biosynthesis pathway. Folate deficiency during pregnancy has been associated with an increased risk for birth defects. This study investigates the extent to which the availability of folate and S-Adenosylmethionine (SAM) affects placental DNA methylation. We hypothesized that maintaining sufficient levels of folate and SAM is particularly important in individuals carrying the MTHFR C677T polymorphism. Maternal- and cord blood was analyzed to genotype the MTHFR rs1801133 SNP. Red blood cell (RBC) folate, vitamin B12, SAM, and S-Adenosylhomocysteine (SAH) were analyzed in cord blood. Epigenome-wide methylation analyses were performed on 90 placenta tissue samples isolated from the fetal side of the placenta; 45 originating from mother-infant dyads homozygous for the MTHFR C677T variant and 45 originating from mother-infant dyads with the homozygous wild type MTHFR677 genotype. Verification of the results was performed using pyrosequencing assays. Genome-wide placental DNA methylation patterns were relatively stable and not significantly affected by levels of one-carbon metabolites. MTHFR genotype was associated with DNA methylation of several loci, including a locus in the MTHFR region. RBC folate and particularly the SAM:SAH ratio did affect overall CpG DNA methylation in some CpG regions when the loci were split according to their CpG island relation. This was most evident in participants carrying the MTHFR C677T variant suggesting a stronger influence of the biosynthesis pathway on the overall placental DNA methylation in MTHFR TT individuals than in MTHFR CC individuals.


Assuntos
Metilação de DNA , Ácido Fólico , Metilenotetra-Hidrofolato Redutase (NADPH2) , Placenta , Feminino , Humanos , Lactente , Gravidez , Carbono/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Ácido Fólico/metabolismo , Ácido Fólico/farmacologia , Ácido Fólico/uso terapêutico , Genótipo , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Placenta/efeitos dos fármacos , Placenta/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo
7.
J Med Chem ; 66(1): 934-950, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36581322

RESUMO

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 µM and KD ∼ 0.2 µM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.


Assuntos
Clostridioides difficile , Metiltransferases , Animais , Humanos , Metiltransferases/química , Adenosina/metabolismo , Adenina/farmacologia , Adenina/metabolismo , S-Adenosilmetionina/metabolismo , DNA/metabolismo , Proteína-Arginina N-Metiltransferases , Mamíferos/metabolismo
8.
Comput Biol Chem ; 102: 107796, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36495748

RESUMO

Epigenetic mechanisms leading to transcriptional regulation, including DNA methylation, are frequently dysregulated in diverse cancers. Interfering with aberrant DNA methylation performed by DNA cytosine methyltransferases (DNMTs) is a clinically validated approach. In particular, the selective inhibition of the de novo DNMT3A and DNMT3B enzymes, whose expression is limited to early embryogenesis, adult stem cells, and in cancers, is particularly attractive; such selectivity is likely to attenuate the dose limiting toxicity shown by current, non-selective DNMT inhibitors. We use molecular dynamics (MD) based computational analysis to study known small molecule binders of DNMT3A, then propose reversible, tight binding, and selective inhibitors that exploit the Asn1192/Arg688 difference between the maintenance DNMT1 and DNMT3A near the active site. A similar strategy exploiting the presence of a unique active site cysteine Cys666 is used to propose DNMT3A-selective irreversible inhibitors. We report our results of relative binding energies of the known and proposed compounds estimated using MM/GBSA and umbrella sampling (US) techniques, and our evaluation of other end-point binding free energy calculation methods for these receptors. These calculations offer insight into the potential for small molecules to selectively target the active site of DNMT3A.


Assuntos
DNA Metiltransferase 3A , Neoplasias , Adulto , Humanos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A/antagonistas & inibidores , Metionina/genética , Metionina/metabolismo , Neoplasias/genética , Racemetionina/metabolismo , S-Adenosilmetionina/metabolismo
9.
Enzyme Microb Technol ; 164: 110189, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36586225

RESUMO

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15-33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g-1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L-1L-met and 0.60 g L-1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L-1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L-1 to 10.1 g L-1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.


Assuntos
S-Adenosilmetionina , Saccharomyces cerevisiae , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Metionina/metabolismo , Fermentação
10.
Int J Mol Sci ; 23(24)2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-36555116

RESUMO

The circadian rhythm of melatonin secretion in the pineal gland is highly conserved in vertebrates. Melatonin levels are always elevated at night. Acetylserotonin O-methyltransferase (ASMT) is the last enzyme in the regulation of melatonin biosynthesis (N-acetyl-5-hydroxytryptamine-melatonin). S-adenosylmethionine (SAM) is an important methyl donor in mammals and can be used as a substrate for the synthesis of melatonin. Methionine adenosyltransferase (MAT) catalyzes the synthesis of SAM from methionine and ATP and has a circadian rhythm. CircRNA is an emerging type of endogenous noncoding RNA with a closed loop. Whether circRNAs in the pineal gland can participate in the regulation of melatonin synthesis by binding miRNAs to target mat2a as part of the circadian rhythm is still unclear. In this study, we predicted the targeting relationship of differentially expressed circRNAs, miRNAs and mRNAs based on the results of rat pineal RNA sequencing. Mat2a siRNA transfection confirmed that mat2a is involved in the synthesis of melatonin. Circ-ERC2 and miR-125a-5p were screened out by software prediction, dual-luciferase reporter experiments, cell transfection, etc. Finally, we constructed a rat superior cervical ganglionectomy model (SCGx), and the results showed that circ-ERC2 could participate in the synthesis of melatonin through the miR-125a-5p/MAT2A axis. The results of the study revealed that circ-ERC2 can act as a molecular sponge of miR-125a-5p to regulate the synthesis of melatonin in the pineal gland by targeting mat2a. This experiment provides a basis for research on the circadian rhythm of noncoding RNA on pineal melatonin secretion.


Assuntos
Melatonina , Metionina Adenosiltransferase , MicroRNAs , Glândula Pineal , RNA Circular , Animais , Ratos , Ritmo Circadiano/genética , Melatonina/metabolismo , Metionina Adenosiltransferase/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Glândula Pineal/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , S-Adenosilmetionina/metabolismo
11.
Genes (Basel) ; 13(12)2022 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-36553579

RESUMO

The RNA methylation of adenosine at the N6-position (m6A) has attracted significant attention because of its abundance and dynamic nature. It accounts for more than 80% of all RNA modifications present in bacteria and eukaryotes and regulates crucial aspects of RNA biology and gene expression in numerous biological processes. The majority of m6A found in mammals is deposited by a multicomponent complex formed between methyltransferase-like (METTL) proteins METTL3 and METTL14. In the last few years, the list of m6A writers has grown, resulting in an expansion of our understanding of the importance of m6A and the methylation machinery. The characterization of the less familiar family member METTL16 has uncovered a new function of the m6A methylation apparatus, namely the fine-tuning of the cellular levels of the major methyl donor S-adenosylmethionine (SAM). METTL16 achieves this by adjusting the levels of the enzyme that synthesizes SAM in direct response to fluctuations in the SAM availability. This review summarizes recent progress made in understanding how METTL16 can sense and relay metabolic information and considers the wider implications. A brief survey highlights similarities and differences between METTL16 and the better-known METTL3/14 complex, followed by a discussion of the target specificity, modes of action and potential roles of METTL16.


Assuntos
RNA , S-Adenosilmetionina , Animais , RNA/metabolismo , S-Adenosilmetionina/metabolismo , RNA Mensageiro/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Homeostase/genética , Expressão Gênica , Mamíferos/genética
12.
Molecules ; 27(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36500483

RESUMO

Folate (vitamin B9) is involved in one-carbon transfer reactions and plays a significant role in nucleic acid synthesis and control of cellular proliferation, among other key cellular processes. It is now recognized that the role of folates in different stages of carcinogenesis is complex, and more research is needed to understand how folate reactions become dysregulated in cancers and the metabolic consequences that occur as a result. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism expressed in many tissues, is ubiquitously downregulated in cancers and is not expressed in cancer cell lines. The RT4 cell line (derived from papillary bladder cancer) which expresses high levels of ALDH1L1 represents an exception, providing an opportunity to explore the metabolic consequences of the loss of this enzyme. We have downregulated this protein in RT4 cells (shRNA driven knockdown or CRISPR driven knockout) and compared metabolomes of ALDH1L1-expressing and -deficient cells to determine if metabolic changes linked to the loss of this enzyme might provide proliferative and/or survival advantages for cancer cells. In this study, cell extracts were analyzed using Ultra High Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-HR-MS). A total of 13,339 signals were identified or annotated using an in-house library and public databases. Supervised and unsupervised multivariate analysis revealed metabolic differences between RT4 cells and ALDH1L1-deficient clones. Glycine (8-fold decrease) and metabolites derived from S-adenosylmethionine utilizing pathways were significantly decreased in the ALDH1L1-deficient clones, compared with RT4 cells. Other changes linked to ALDH1L1 downregulation include decreased levels of amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid. While the ALDH1L1-catalyzed reaction is directly linked to glycine biosynthesis and methyl group flux, its overall effect on cellular metabolism extends beyond immediate metabolic pathways controlled by this enzyme.


Assuntos
Ácido Fólico , Neoplasias , Humanos , Ácido Fólico/metabolismo , Glicina/metabolismo , Retinal Desidrogenase/metabolismo , Metilação , Família Aldeído Desidrogenase 1/metabolismo , S-Adenosilmetionina/metabolismo , Metabolômica
13.
Insect Biochem Mol Biol ; 151: 103863, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36341863

RESUMO

Juvenile hormone acid methyltransferase (JHAMT) is a rate-limiting enzyme of juvenile hormone (JH) biosynthesis in insects. It transfers the methyl group of S-adenosyl methionine to either the carboxyl group of JH acids or farnesoic acid to produce JH. Six JHAMT paralogues have been identified in the silkworm (Bombyx mori); among them, JHAMT1 and JHAMT2 display a methyltransferase activity. Here, the three-dimensional crystal structure of inactive JHAMT3 and the binary complex of JHAMT3 with its cofactor S-adenosyl-l-homocysteine were determined through X-ray crystallization. Comparative structural analysis revealed that JHAMT3 adopted a similar structural pattern to that of functional JHAMT2, which comprised one core Rossmann fold domain and one substrate-binding domain. Similar to JHAMT2, JHAMT3 underwent a conformational change at the Rossmann fold domain because of cofactor binding, which promoted ligand accommodation. However, it exhibited a relatively rigid substrate-binding pocket compared with that of JHAMT2. JHAMT3 was also highly expressed in the silk gland of fourth- and fifth-instar B. mori larvae. The results of expression profiling combined with activity analysis suggested that JHAMT3 might function as a binding protein of JH acids for the regulation of JH acid titers. These findings provide a structural basis for enhancing the understanding of the physiological function of JHAMT3 and a rational framework for the development of potent and specific inhibitors of JHAMT family members.


Assuntos
Bombyx , Hormônios Juvenis , Animais , Hormônios Juvenis/metabolismo , Bombyx/metabolismo , Metiltransferases/metabolismo , Sequência de Aminoácidos , S-Adenosilmetionina/metabolismo
14.
Antiviral Res ; 208: 105460, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36334638

RESUMO

Usutu virus (USUV), is a mosquito-borne flavivirus currently spreading outside the African continent producing substantial avian mortality. In contrast, infected humans could exhibit mild neurological symptoms or remain asymptomatic. As in other flaviviruses, the capped USUV genome encodes three structural and seven non-structural (NS) proteins. Among the NS proteins, NS5 plays crucial roles in virus replication, harbouring the capping and methyltransferase (MTase) activities in its N-terminal domain and the RNA-dependent RNA polymerase (RdRP) activity at the C-terminus. In this work, we present the first structural and functional characterization of the USUV MTase domain. The first structure of the USUV MTase has been determined in complex with its natural ligands (S-adenosyl-L-methionine [SAM]) and S-adenosyl-L-homocysteine [SAH]) at 2.2 Å resolution, showing a molecular dimer in the crystal asymmetric unit. One molecule is bound to the methyl donor SAM while the second is bound to the reaction by-product SAH. Both molecules are almost identical and also show a high structural similarity to the MTase domains of other flaviviruses. The structure of the USUV MTase bound to the inhibitor sinefungin at 1.8 Å resolution is also described. Careful comparisons of the interactions in the SAM-binding cavity prompt us to hypothesize about the strength and weakness of the structure-based design of antivirals directed to the SAM/SAH binding site that could be effective to deal with this threat.


Assuntos
Flavivirus , Metiltransferases , Flavivirus/genética , Flavivirus/metabolismo , Metiltransferases/química , RNA Polimerase Dependente de RNA/genética , S-Adenosilmetionina/metabolismo , Proteínas não Estruturais Virais/química
15.
Cell Rep ; 41(5): 111568, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36323249

RESUMO

Gene-environment interactions can perturb the epigenome, triggering network alterations that participate in cancer pathogenesis. Integrating epigenomics, transcriptomics, and metabolic analyses with functional perturbation, we show that the tumor suppressor p53 preserves genomic integrity by empowering adequate levels of the universal methyl donor S-adenosylmethionine (SAM). In p53-deficient cells, perturbation of DNA methylation promotes derepression of heterochromatin, massive loss of histone H3-lysine 9 methylation, and consequent upregulation of satellite RNAs that triggers R-loop-associated replication stress and chromosomal aberrations. In p53-deficient cells, the inadequate SAM level underlies the inability to respond to perturbation because exogenous reintroduction of SAM represses satellite elements and restores the ability to cope with stress. Mechanistically, p53 transcriptionally controls genes involved in one-carbon metabolism, including Slc43a2, the methionine uptake transporter that is critical for SAM synthesis. Supported by clinical data, our findings shed light on the role of p53-mediated metabolism in preventing unscheduled R-loop-associated genomic instability.


Assuntos
Estruturas R-Loop , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , S-Adenosilmetionina/metabolismo , Metilação de DNA , Instabilidade Genômica
16.
Biochemistry ; 61(23): 2643-2647, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36326713

RESUMO

The radical S-adenosyl-l-methionine (SAM) enzyme TYW1 catalyzes the condensation of C-2 and C-3 atoms of pyruvate with N-methylguanosine containing tRNAPhe to form 4-demethylwyosine (imG-14) modified tRNAPhe. The fate of C-1 is not known, and either formate or carbon dioxide (CO2) has been proposed. In this study, a coupled assay that transforms either CO2 or formate to oxaloacetate (OAA) was used to determine the fate of C-1. In the presence of [1-13C1]-pyruvate, 13C-enriched OAA was observed in a process that is concomitant with the formation of imG-14, under conditions that preferentially transform CO2 and not formate to OAA. These findings are discussed in the context of the cofactor content of TYW1 and a new role for the auxiliary cluster in catalyzing the oxidative cleavage of C-1-C-2 bond of pyruvate in the catalytic cycle of TYW1.


Assuntos
Proteínas Ferro-Enxofre , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , RNA de Transferência de Fenilalanina/química , Ácido Pirúvico/química , Dióxido de Carbono , Metionina , Proteínas Ferro-Enxofre/química , RNA de Transferência/metabolismo , Catálise , Estresse Oxidativo
17.
Cells ; 11(22)2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36429035

RESUMO

Stem cells are a population of undifferentiated cells with self-renewal and differentiation capacities. Normal and cancer stem cells share similar characteristics in relation to their stemness properties. One-carbon metabolism (OCM), a network of interconnected reactions, plays an important role in this dependence through its role in the endogenous synthesis of methionine and S-adenosylmethionine (SAM), the universal donor of methyl groups in eukaryotic cells. OCM genes are differentially expressed in stem cells, compared to their differentiated counterparts. Furthermore, cultivating stem cells in methionine-restricted conditions hinders their stemness capacities through decreased SAM levels with a subsequent decrease in histone methylation, notably H3K4me3, with a decrease in stem cell markers. Stem cells' reliance on methionine is linked to several mechanisms, including high methionine flux or low endogenous methionine biosynthesis. In this review, we provide an overview of the recent discoveries concerning this metabolic dependence and we discuss the mechanisms behind them. We highlight the influence of SIRT1 on SAM synthesis and suggest a role of PGC-1α/PPAR-α in impaired stemness produced by methionine deprivation. In addition, we discuss the potential interest of methionine restriction in regenerative medicine and cancer treatment.


Assuntos
Metionina , Neoplasias , Metionina/metabolismo , Sirtuína 1 , PPAR alfa , Racemetionina , S-Adenosilmetionina/metabolismo , Células-Tronco Neoplásicas/metabolismo
18.
Int J Mol Sci ; 23(22)2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36430451

RESUMO

Methyltransferases (MTases) enzymes, responsible for RNA capping into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are emerging important targets for the design of new anti-SARS-CoV-2 agents. Here, analogs of S-adenosylmethionine (SAM), obtained from the bioisosteric substitution of the sulfonium and amino acid groups, were evaluated by rigorous computational modeling techniques such as molecular dynamics (MD) simulations followed by relative binding free analysis against nsp16/nsp10 complex from SARS-CoV-2. The most potent inhibitor (2a) shows the lowest binding free energy (-58.75 Kcal/mol) and more potency than Sinefungin (SFG) (-39.8 Kcal/mol), a pan-MTase inhibitor, which agrees with experimental observations. Besides, our results suggest that the total binding free energy of each evaluated SAM analog is driven by van der Waals interactions which can explain their poor cell permeability, as observed in experimental essays. Overall, we provide a structural and energetic analysis for the inhibition of the nsp16/nsp10 complex involving the evaluated SAM analogs as potential inhibitors.


Assuntos
S-Adenosilmetionina , Humanos , S-Adenosilmetionina/farmacologia , S-Adenosilmetionina/metabolismo , SARS-CoV-2 , Proteínas não Estruturais Virais/metabolismo , Metiltransferases/metabolismo
19.
BMC Biol ; 20(1): 228, 2022 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-36209095

RESUMO

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.


Assuntos
Sulfeto de Hidrogênio , Ácidos Nucleicos , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Carbono/metabolismo , Ditiotreitol/metabolismo , Ácido Fólico/metabolismo , Homocisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Ligases/metabolismo , Lipídeos , Mercaptoetanol/metabolismo , Metionina/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Oxigênio/metabolismo , Substâncias Redutoras/metabolismo , S-Adenosilmetionina/metabolismo , Compostos de Sulfidrila/metabolismo , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Vitaminas/metabolismo
20.
J Microbiol Biotechnol ; 32(11): 1447-1453, 2022 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-36310362

RESUMO

Prohibitin 1 (Phb1) is a pleiotropic protein, located mainly in the mitochondrial inner membrane and involved in the regulation of cell proliferation and the stabilization of mitochondrial protein. Acetaminophen (APAP) is one of the most commonly used over-the-counter analgesics worldwide. However, at high dose, the accumulation of N-acetyl-p-benzoquinone imine (NAPQI) can lead to APAP-induced hepatotoxicity. In this study, we sought to understand the regulation of mRNA expression in relation to APAP and GSH metabolism by Phb1 in normal mouse AML12 hepatocytes. We used two different Phb1 silencing levels: high-efficiency (HE, >90%) and low-efficiency (LE, 50-60%). In addition, the siRNA-transfected cells were further pretreated with 0.5 mM of S-adenosylmethionine (SAMe) for 24 h before treatment with APAP at different doses (1-2 mM) for 24 h. The expression of APAP metabolism-related and antioxidant genes such as Cyp2e1 and Ugt1a1 were increased during SAMe pretreatment. Moreover, SAMe increased intracellular GSH concentration and it was maintained after APAP treatment. To sum up, Phb1 silencing and APAP treatment impaired the metabolism of APAP in hepatocytes, and SAMe exerted a protective effect against hepatotoxicity by upregulating antioxidant genes.


Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Camundongos , Animais , Acetaminofen/toxicidade , Acetaminofen/metabolismo , Proibitinas , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Antioxidantes/farmacologia , Fígado , Camundongos Endogâmicos C57BL
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