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1.
Am J Physiol Regul Integr Comp Physiol ; 321(5): R802-R811, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34612088

RESUMO

Hospitalized preterm infants experience painful medical procedures. Oral sucrose is the nonpharmacological standard of care for minor procedural pain relief. Infants are treated with numerous doses of sucrose, raising concerns about potential long-term effects. The objective of this study was to determine the long-term effects of neonatal oral sucrose treatment on growth and liver metabolism in a mouse model. Neonatal female and male mice were randomly assigned to one of two oral treatments (n = 7-10 mice/group/sex): sterile water or sucrose. Pups were treated 10 times/day for the first 6 days of life with 0.2 mg/g body wt of respective treatments (24% solution; 1-4 µL/dose) to mimic what is given to preterm infants. Mice were weaned at age 3 wk onto a control diet and fed until age 16 wk. Sucrose-treated female and male mice gained less weight during the treatment period and were smaller at weaning than water-treated mice (P ≤ 0.05); no effect of sucrose treatment on body weight was observed at adulthood. However, adult sucrose-treated female mice had smaller tibias and lower serum insulin-like growth factor-1 than adult water-treated female mice (P ≤ 0.05); these effects were not observed in males. Lower liver S-adenosylmethionine, phosphocholine, and glycerophosphocholine were observed in adult sucrose-treated compared with water-treated female and male mice (P ≤ 0.05). Sucrose-treated female, but not male, mice had lower liver free choline and higher liver betaine compared with water-treated female mice (P < 0.01). Our findings suggest that repeated neonatal sucrose treatment has long-term sex-specific effects on growth and liver methionine and choline metabolism.


Assuntos
Analgésicos/toxicidade , Colina/metabolismo , Glucocorticoides/metabolismo , Fígado/efeitos dos fármacos , Sacarose/toxicidade , Tíbia/efeitos dos fármacos , Ganho de Peso/efeitos dos fármacos , Administração Oral , Fatores Etários , Analgésicos/administração & dosagem , Animais , Animais Recém-Nascidos , Betaína/metabolismo , Feminino , Glicerilfosforilcolina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilcolina/metabolismo , S-Adenosilmetionina/metabolismo , Fatores Sexuais , Sacarose/administração & dosagem , Tíbia/crescimento & desenvolvimento
2.
Molecules ; 26(20)2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34684735

RESUMO

In continuation of our previous effort, different in silico selection methods were applied to 310 naturally isolated metabolites that exhibited antiviral potentialities before. The applied selection methods aimed to pick the most relevant inhibitor of SARS-CoV-2 nsp10. At first, a structural similarity study against the co-crystallized ligand, S-Adenosyl Methionine (SAM), of SARS-CoV-2 nonstructural protein (nsp10) (PDB ID: 6W4H) was carried out. The similarity analysis culled 30 candidates. Secondly, a fingerprint study against SAM preferred compounds 44, 48, 85, 102, 105, 182, 220, 221, 282, 284, 285, 301, and 302. The docking studies picked 48, 182, 220, 221, and 284. While the ADMET analysis expected the likeness of the five candidates to be drugs, the toxicity study preferred compounds 48 and 182. Finally, a density-functional theory (DFT) study suggested vidarabine (182) to be the most relevant SARS-Cov-2 nsp10 inhibitor.


Assuntos
Antivirais/química , Produtos Biológicos/química , SARS-CoV-2/metabolismo , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , Produtos Biológicos/metabolismo , Produtos Biológicos/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/patologia , Teoria da Densidade Funcional , Humanos , Ligantes , Simulação de Acoplamento Molecular , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/isolamento & purificação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Vidarabina/química , Vidarabina/metabolismo , Vidarabina/uso terapêutico , Proteínas Virais Reguladoras e Acessórias/metabolismo
3.
Biochemistry ; 60(38): 2865-2874, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34506710

RESUMO

Adenosylhopane is a crucial precursor of C35 hopanoids, which are believed to modulate the fluidity and permeability of bacterial cell membranes. Adenosylhopane is formed by a crosslinking reaction between diploptene and a 5'-deoxyadenosyl radical that is generated by the radical S-adenosyl-L-methionine (SAM) enzyme HpnH. We previously showed that HpnH from Streptomyces coelicolor A3(2) (ScHpnH) converts diploptene to (22R)-adenosylhopane. However, the mechanism of the stereoselective C-C bond formation was unclear. Thus, here, we performed biochemical and mutational analysis of another HpnH, from the ethanol-producing bacterium Zymomonas mobilis (ZmHpnH). Similar to ScHpnH, wild-type ZmHpnH afforded (22R)-adenosylhopane. Conserved cysteine and tyrosine residues were suggested as possible hydrogen sources to quench the putative radical reaction intermediate. A Cys106Ala mutant of ZmHpnH had one-fortieth the activity of the wild-type enzyme and yielded both (22R)- and (22S)-adenosylhopane along with some related byproducts. Radical trapping experiments with a spin-trapping agent supported the generation of a radical intermediate in the ZmHpnH-catalyzed reaction. We propose that the thiol of Cys106 stereoselectively reduces the radical intermediate generated at the C22 position by the addition of the 5'-deoxadenosyl radical to diploptene, to complete the reaction.


Assuntos
Adenosina/análogos & derivados , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Adenosina/biossíntese , Adenosina/genética , Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Catálise , Cisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Triterpenos/química , Zymomonas/metabolismo
4.
Life Sci ; 285: 119943, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34516992

RESUMO

Glycine betaine (N, N, N-trimethyl amine) is an osmolyte accumulated in cells that is key for cell volume and turgor regulation, is the principal methyl donor in the methionine cycle and is a DNA and proteins stabilizer. In humans, glycine betaine is synthesized from choline and can be obtained from some foods. Glycine betaine (GB) roles are illustrated in chemical, metabolic, agriculture, and clinical medical studies due to its chemical and physiological properties. Several studies have extensively described GB role and accumulation related to specific pathologies, focusing mainly on analyzing its positive and negative role in these pathologies. However, it is necessary to explain the relationship between glycine betaine and different pathologies concerning its role as an antioxidant, ability to methylate DNA, interact with transcription factors and cell receptors, and participate in the control of homocysteine concentration in liver, kidney and brain. This review summarizes the most important findings and integrates GB role in neurodegenerative, cardiovascular, hepatic, and renal diseases. Furthermore, we discuss GB impact on other dysfunctions as inflammation, oxidative stress, and glucose metabolism, to understand their cross-talks and provide reliable data to establish a base for further investigations.


Assuntos
Betaína/metabolismo , Doenças Cardiovasculares/metabolismo , Nefropatias/metabolismo , Hepatopatias/metabolismo , Doenças Neurodegenerativas/metabolismo , Tamanho Celular , Humanos , Hiper-Homocisteinemia/metabolismo , Concentração Osmolar , S-Adenosilmetionina/metabolismo
5.
Enzyme Microb Technol ; 150: 109881, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489034

RESUMO

Natural fluorinated products are rare and attract great attention. The de novo fluorometabolites biosynthetic pathway in microbes has been studied. It is revealed that the carbon-fluorine (C-F) bond is formed by an exotic enzyme called fluorinase (FLA) when using fluorine ions and S-adenosyl-l-methionine (SAM) as substrates. However, the resource of the precursor SAM is still elusive. To solve this, a novel methionine adenosyltransferase from Streptomyces xinghaiensis (SxMAT) was identified and characterized. We proved that SAM was enzymatically synthesized by SxMAT, an enzyme that mediated the reaction between adenosine triphosphate (ATP) and l-methionine (l-Met) with 99% diastereoisomeric excess (d.e.) and 80% yield. Such high diastereoselectivity had never been reported before. SxMAT was a Co2+-dependent metalloenzyme. The results showed that the metal cobalt ion contributes to the activity and selectivity of SxMAT. Molecular docking was performed to reveal its catalytic mechanism. The optimal temperature and pH were 55 °C and 8.5, respectively. Lastly, a two-step tandem enzymatic reaction using SxMAT and FLA both from S. xinghaiensis to generate 5'-fluoro-deoxyadenosine (5'-FDA) was performed. This implied that SxMAT may be present in this fluorometabolites biosynthetic route. These results suggested that SxMAT could be a useful biocatalyst for the synthesis of optically pure (S)-S-adenosyl-l-methionine, an important nutraceutical. In addition, SxMAT will probably play an important role in the biosynthetic pathway of fluorinated natural products in bacteria.


Assuntos
Metionina Adenosiltransferase , S-Adenosilmetionina , Vias Biossintéticas , Metionina/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Simulação de Acoplamento Molecular , S-Adenosilmetionina/metabolismo , Streptomyces
6.
Curr Protoc ; 1(8): e213, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370893

RESUMO

Protein methyltransferases (PMTs) regulate many aspects of normal and disease processes through substrate methylation, with S-adenosyl-L-methionine (SAM) as a cofactor. It has been challenging to elucidate cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live-cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ-synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analogue cofactors can only be processed by the engineered PMTs-and not native PMTs-to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome-wide methylation and validate specific methylation events. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live-cell labeling of substrates of protein methyltransferases GLP1 and PRMT1 with lcBPPM-feasible enzymes and SAM analogue precursors Support Protocol: Gram-scale synthesis of Hey-Met Basic Protocol 2: Click labeling of lcBPPM cell lysates with a biotin-azide probe Alternate Protocol: Click labeling of small-scale lcBPPM cell lysates with a TAMRA-azide dye for in-gel fluorescence visualization Basic Protocol 3: Enrichment of biotinylated lcBPPM proteome with streptavidin beads Basic Protocol 4: Proteome-wide identification of lcBPPM targets with mass spectrometry Basic Protocol 5: Validation of individual lcBPPM targets by western blot.


Assuntos
Metionina , S-Adenosilmetionina , Humanos , Metilação , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteoma/metabolismo , Proteínas Repressoras , S-Adenosilmetionina/metabolismo
7.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Metiltransferases , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
8.
Angew Chem Int Ed Engl ; 60(36): 19957-19964, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34164914

RESUMO

Aminomalonate (Ama) is a widespread structural motif in Nature, whereas its biosynthetic route is only partially understood. In this study, we show that a radical S-adenosylmethionine (rSAM) enzyme involved in cyclophane biosynthesis exhibits remarkable catalytic promiscuity. This enzyme, named three-residue cyclophane forming enzyme (3-CyFE), mainly produces cyclophane in vivo, whereas it produces formylglycine (FGly) as a major product and barely produce cyclophane in vitro. Importantly, the enzyme can further oxidize FGly to produce Ama. Bioinformatic study revealed that 3-CyFEs have evolved from a common ancestor with anaerobic sulfatase maturases (anSMEs), and possess a similar set of catalytic residues with anSMEs. Remarkably, the enzyme does not need leader peptide for activity and is fully active on a truncated peptide containing only 5 amino acids of the core sequence. Our work discloses the first ribosomal path towards Ama formation, providing a possible hint for the rich occurrence of Ama in Nature.


Assuntos
Malonatos/metabolismo , Peptídeos/metabolismo , S-Adenosilmetionina/metabolismo , Sulfatases/metabolismo , Radicais Livres/química , Radicais Livres/metabolismo , Malonatos/química , Estrutura Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina/química , Sulfatases/química
9.
Biochemistry ; 60(27): 2179-2185, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34184886

RESUMO

TYW1 is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine-containing tRNAPhe, forming 4-demethylwyosine-containing tRNAPhe. Homologues of TYW1 are found in both archaea and eukarya; archaeal homologues consist of a single domain, while eukaryal homologues contain a flavin binding domain in addition to the radical SAM domain shared with archaeal homologues. In this study, TYW1 from Saccharomyces cerevisiae (ScTYW1) was heterologously expressed in Escherichia coli and purified to homogeneity. ScTYW1 is purified with 0.54 ± 0.07 and 4.2 ± 1.9 equiv of flavin mononucleotide (FMN) and iron, respectively, per mole of protein, suggesting the protein is ∼50% replete with Fe-S clusters and FMN. While both NADPH and NADH are sufficient for activity, significantly more product is observed when used in combination with flavin nucleotides. ScTYW1 is the first example of a radical SAM flavoenzyme that is active with NAD(P)H alone.


Assuntos
Oxirredutases/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Mononucleotídeo de Flavina/metabolismo , NADP/metabolismo
10.
EMBO J ; 40(14): e106434, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34152017

RESUMO

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 in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3' splice site (3'SS) in␣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/genética
11.
Biochem J ; 478(18): 3429-3444, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34133721

RESUMO

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from the catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase (PfLPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP- targeting approach, PfLPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of PfLPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that PfLPL20 is not essential for the parasite. However, in PfLPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with up-regulation of phosphocholine and SAM levels; these results show up-regulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights the presence of alternate pathways for lipid homeostasis/membrane-biogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite.


Assuntos
Eritrócitos/metabolismo , Lisofosfolipase/genética , Fosfatidilcolinas/biossíntese , Fosforilcolina/metabolismo , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Carboxiliases/genética , Carboxiliases/metabolismo , Colina/metabolismo , Eritrócitos/parasitologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Homeostase/genética , Humanos , Estágios do Ciclo de Vida/genética , Metabolismo dos Lipídeos/genética , Lisofosfatidilcolinas/metabolismo , Lisofosfolipase/deficiência , Metiltransferases/genética , Metiltransferases/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , S-Adenosilmetionina/metabolismo , Serina/metabolismo
12.
Nat Commun ; 12(1): 3877, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162884

RESUMO

Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.


Assuntos
Conformação de Ácido Nucleico , Nucleosídeo Q/química , RNA/química , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Cinética , Metilação , Estrutura Molecular , Nucleosídeo Q/metabolismo , RNA/genética , RNA/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
13.
Sci Signal ; 14(689)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34131072

RESUMO

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.


Assuntos
COVID-19/virologia , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Manganês/metabolismo , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Capuzes de RNA/química , Capuzes de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , Transdução de Sinais , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
14.
Elife ; 102021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33949310

RESUMO

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.


Assuntos
Regulação da Expressão Gênica , Homeostase/genética , Metionina Adenosiltransferase/genética , Splicing de RNA , S-Adenosilmetionina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Regiões 3' não Traduzidas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células HEK293 , Humanos , Íntrons/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
15.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33972410

RESUMO

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.


Assuntos
Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , Cristalografia , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/química , RNA Mensageiro/química , RNA Viral/química , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Síncrotrons , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo
16.
Nucleic Acids Res ; 49(W1): W207-W215, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34019643

RESUMO

Transcriptome profiling is essential for gene regulation studies in development and disease. Current web-based tools enable functional characterization of transcriptome data, but most are restricted to applying gene-list-based methods to single datasets, inefficient in leveraging up-to-date and species-specific information, and limited in their visualization options. Additionally, there is no systematic way to explore data stored in the largest transcriptome repository, NCBI GEO. To fill these gaps, we have developed eVITTA (easy Visualization and Inference Toolbox for Transcriptome Analysis; https://tau.cmmt.ubc.ca/eVITTA/). eVITTA provides modules for analysis and exploration of studies published in NCBI GEO (easyGEO), detailed molecular- and systems-level functional profiling (easyGSEA), and customizable comparisons among experimental groups (easyVizR). We tested eVITTA on transcriptomes of SARS-CoV-2 infected human nasopharyngeal swab samples, and identified a downregulation of olfactory signal transducers, in line with the clinical presentation of anosmia in COVID-19 patients. We also analyzed transcriptomes of Caenorhabditis elegans worms with disrupted S-adenosylmethionine metabolism, confirming activation of innate immune responses and feedback induction of one-carbon cycle genes. Collectively, eVITTA streamlines complex computational workflows into an accessible interface, thus filling the gap of an end-to-end platform capable of capturing both broad and granular changes in human and model organism transcriptomes.


Assuntos
Visualização de Dados , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Internet , Transcriptoma/genética , Animais , COVID-19/genética , COVID-19/virologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Humanos , Imunidade Inata , Nasofaringe/virologia , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Especificidade da Espécie , Fluxo de Trabalho
17.
J Biol Chem ; 296: 100621, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33811856

RESUMO

5-Deoxyadenosine (5dAdo) is the byproduct of many radical S-adenosyl-l-methionine enzyme reactions in all domains of life. 5dAdo is also an inhibitor of the radical S-adenosyl-l-methionine enzymes themselves, making it necessary for cells to construct pathways to recycle or dispose of this toxic metabolite. However, the specific pathways involved have long remained unexplored. Recent research demonstrated a growth advantage in certain organisms by using 5dAdo or intermediates as a sole carbon source and elucidated the corresponding salvage pathway. We now provide evidence using supernatant analysis by GC-MS for another 5dAdo recycling route. Specifically, in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus), the activity of promiscuous enzymes leads to the synthesis and excretion first of 5-deoxyribose and subsequently of 7-deoxysedoheptulose. 7-Deoxysedoheptulose is an unusual deoxy-sugar, which acts as an antimetabolite of the shikimate pathway, thereby exhibiting antimicrobial and herbicidal activity. This strategy enables organisms with small genomes and lacking canonical gene clusters for the synthesis of secondary metabolites, like S. elongatus, to produce antimicrobial compounds from primary metabolism and enzymatic promiscuity. Our findings challenge the view of bioactive molecules as sole products of secondary metabolite gene clusters and expand the range of compounds that microorganisms can deploy to compete for their ecological niche.


Assuntos
Proteínas de Bactérias/metabolismo , Desoxiadenosinas/metabolismo , Hidrolases/metabolismo , S-Adenosilmetionina/metabolismo , Metabolismo Secundário , Synechococcus/metabolismo , Proteínas de Bactérias/genética , Hidrolases/genética , Synechococcus/crescimento & desenvolvimento
18.
J Med Chem ; 64(8): 4430-4449, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33829783

RESUMO

The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is adjacent to the CDKN2A tumor suppressor and codeleted with CDKN2A in approximately 15% of all cancers. Previous attempts to target MAT2A with small-molecule inhibitors identified cellular adaptations that blunted their efficacy. Here, we report the discovery of highly potent, selective, orally bioavailable MAT2A inhibitors that overcome these challenges. Fragment screening followed by iterative structure-guided design enabled >10 000-fold improvement in potency of a family of allosteric MAT2A inhibitors that are substrate noncompetitive and inhibit release of the product, S-adenosyl methionine (SAM), from the enzyme's active site. We demonstrate that potent MAT2A inhibitors substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors. These data supported progressing AG-270 into current clinical studies (ClinicalTrials.gov NCT03435250).


Assuntos
Inibidores Enzimáticos/química , Metionina Adenosiltransferase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/genética , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Homozigoto , Humanos , Metionina Adenosiltransferase/metabolismo , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Purina-Núcleosídeo Fosforilase/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade
19.
Chem Phys Lipids ; 237: 105086, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33930379

RESUMO

The endogenous molecule, S-adenosyl-l-methionine (SAMe) is a key factor due to its role in the methylation cycle and modulation of monoaminergic neurotransmission. Since many mental disorders have linked to the monoaminergic system, the level of SAMe in blood and cerebrospinal fluid is important in the treatment of major depression. In this study, solid lipid nanoparticles (SLN) were prepared in order to increase the limited oral bioavailability of SAMe, and SLN based nanocomposite particles (SAMe-SLN-NC) were further developed using an enteric polymer for passive targeting of intestinal lymphatic system. In this manner, it was also aimed to protect SAMe loaded SLN from harsh gastric environment as well as hepatic first-pass metabolism. Dynamic light scattering (DLS) analysis of SLN was performed, drug content was measured, SAMe release patterns were examined and the permeation ability of SAMe was investigated by the Parallel Artificial Membrane Permeability Assay (PAMPA) to characterize SAMe loaded SLN formulation. According to the PAMPA results, SAMe-SLN with the average particle size of 242 nm showed enhanced SAMe permeability in comparison to pure drug. Delayed drug release obtained by SLN nanocomposite particles indicated the protection of drug-loaded SLN in the acidic gastric medium and their intact presence in the intestine. SAMe solution or particle suspensions were prepared using 0.45 (w/v) hydroxypropyl methylcellulose aqueous solution to be applied to groups of animals for pharmacokinetic studies. In vivo pharmacokinetic parameters revealed enhancement in relative bioavailability of SAMe upon oral administration of SLN based formulations. This was attributed to intact absorption of lipid matrix through lymphatic path. A statistically significant increase in SAMe plasma levels was obtained at 15th and 30th minutes with SAMe-SLN and at 2nd and 4th hours with SAMe-SLN-NC. Overall results suggest that SLN is a promising carrier to passive lymphatic targeting of SAMe and novel SLN nanocomposite particles which presented efficient oral bioavailability is a potential way for oral delivery of SAMe and treatment of major depression.


Assuntos
Lipídeos/química , Nanocompostos/química , S-Adenosilmetionina/metabolismo , Administração Oral , Animais , Disponibilidade Biológica , Varredura Diferencial de Calorimetria , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Meia-Vida , Nanopartículas/química , Tamanho da Partícula , Ratos , S-Adenosilmetionina/sangue , S-Adenosilmetionina/química , S-Adenosilmetionina/farmacocinética
20.
Front Immunol ; 12: 648913, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912173

RESUMO

The excessive M1 polarization of macrophages drives the occurrence and development of inflammatory diseases. The reprogramming of macrophages from M1 to M2 can be achieved by targeting metabolic events. Taurine promotes for the balance of energy metabolism and the repair of inflammatory injury, preventing chronic diseases and complications. However, little is known about the mechanisms underlying the action of taurine modulating the macrophage polarization phenotype. In this study, we constructed a low-dose LPS/IFN-γ-induced M1 polarization model to simulate a low-grade pro-inflammatory process. Our results indicate that the taurine transporter TauT/SlC6A6 is upregulated at the transcriptional level during M1 macrophage polarization. The nutrient uptake signal on the membrane supports the high abundance of taurine in macrophages after taurine supplementation, which weakens the status of methionine metabolism, resulting in insufficient S-adenosylmethionine (SAM). The low availability of SAM is directly sensed by LCMT-1 and PME-1, hindering PP2Ac methylation. PP2Ac methylation was found to be necessary for M1 polarization, including the positive regulation of VDAC1 and PINK1. Furthermore, its activation was found to promote the elimination of mitochondria by macrophages via the mitophagy pathway for metabolic adaptation. Mechanistically, taurine inhibits SAM-dependent PP2Ac methylation to block PINK1-mediated mitophagy flux, thereby maintaining a high mitochondrial density, which ultimately hinders the conversion of energy metabolism to glycolysis required for M1. Our findings reveal a novel mechanism of taurine-coupled M1 macrophage energy metabolism, providing novel insights into the occurrence and prevention of low-grade inflammation, and propose that the sensing of taurine and SAM availability may allow communication to inflammatory response in macrophages.


Assuntos
Glicólise/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , S-Adenosilmetionina/metabolismo , Taurina/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos/classificação , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Metilação/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Células THP-1 , Taurina/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 1 Dependente de Voltagem/metabolismo
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