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1.
Int J Mol Sci ; 21(19)2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33036230

RESUMO

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Virais Reguladoras e Acessórias/química , Sítios de Ligação , Cristalografia por Raios X , Ligação Proteica , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Homologia de Sequência , Proteínas Virais Reguladoras e Acessórias/metabolismo , Dedos de Zinco
2.
Cell Prolif ; 53(11): e12891, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33030764

RESUMO

Autophagy is a mechanism that enables cells to maintain cellular homeostasis by removing damaged materials and mobilizing energy reserves in conditions of starvation. Although nutrient availability strongly impacts the process of autophagy, the specific metabolites that regulate autophagic responses have not yet been determined. Recent results indicate that S-adenosylmethionine (SAM) represents a critical inhibitor of methionine starvation-induced autophagy. SAM is primarily involved in four key metabolic pathways: transmethylation, transsulphuration, polyamine synthesis and 5'-deoxyadenosyl 5'-radical-mediated biochemical transformations. SAM is the sole methyl group donor involved in the methylation of DNA, RNA and histones, modulating the autophagic process by mediating epigenetic effects. Moreover, the metabolites of SAM, such as homocysteine, glutathione, decarboxylated SAM and spermidine, also exert important influences on the regulation of autophagy. From our perspective, nuclear-cytosolic SAM is a conserved metabolic inhibitor that connects cellular metabolic status and the regulation of autophagy. In the future, SAM might be a new target of autophagy regulators and be widely used in the treatment of various diseases.


Assuntos
Autofagia , S-Adenosilmetionina/metabolismo , Animais , Metilação de DNA , Epigênese Genética , Humanos , Redes e Vias Metabólicas
3.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
4.
Nat Commun ; 11(1): 3718, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709886

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.


Assuntos
Metiltransferases/química , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Betacoronavirus , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Difração de Raios X
5.
In Vivo ; 34(3 Suppl): 1593-1596, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: covidwho-536995

RESUMO

The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Liases de Carbono-Enxofre/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Metionina/metabolismo , Pneumonia Viral/tratamento farmacológico , Capuzes de RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , Administração Oral , Antivirais/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/uso terapêutico , Betacoronavirus/fisiologia , Liases de Carbono-Enxofre/administração & dosagem , Ensaios Clínicos como Assunto , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina/prevenção & controle , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Metanálise como Assunto , Metilação/efeitos dos fármacos , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Pseudomonas putida/enzimologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , S-Adenosilmetionina/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Replicação Viral/efeitos dos fármacos
6.
Nucleic Acids Res ; 48(13): 7545-7556, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32520325

RESUMO

While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem-loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen-Watson-Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.


Assuntos
Dobramento de RNA , Riboswitch , S-Adenosil-Homocisteína/química , S-Adenosilmetionina/química , Pareamento de Bases , Transferência Ressonante de Energia de Fluorescência , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo
7.
In Vivo ; 34(3 Suppl): 1593-1596, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32503816

RESUMO

The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Liases de Carbono-Enxofre/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Metionina/metabolismo , Pneumonia Viral/tratamento farmacológico , Capuzes de RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , Administração Oral , Antivirais/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/uso terapêutico , Betacoronavirus/fisiologia , Liases de Carbono-Enxofre/administração & dosagem , Ensaios Clínicos como Assunto , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina/prevenção & controle , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Metanálise como Assunto , Metilação/efeitos dos fármacos , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Pseudomonas putida/enzimologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , S-Adenosilmetionina/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Replicação Viral/efeitos dos fármacos
8.
Nat Commun ; 11(1): 2794, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493973

RESUMO

All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.


Assuntos
RNA de Transferência de Metionina/metabolismo , Riboswitch , S-Adenosilmetionina/metabolismo , Sequência de Bases , Loci Gênicos , Modelos Biológicos , Conformação de Ácido Nucleico , Óperon/genética , Biossíntese de Proteínas , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genética
9.
J Virol ; 94(16)2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32461321

RESUMO

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.


Assuntos
Exorribonucleases/metabolismo , Interferon Tipo I/biossíntese , Interferons/biossíntese , Vírus da Diarreia Epidêmica Suína/fisiologia , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Exorribonucleases/genética , Expressão Gênica , Guanina/metabolismo , Imunidade Inata , Metilação , Mutação , Vírus da Diarreia Epidêmica Suína/enzimologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Suínos , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral
10.
Nucleic Acids Res ; 48(8): 4081-4099, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32187373

RESUMO

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT's activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Saccharomycetales/genética , Engenharia Celular , Centrômero , Cromatina/química , DNA (Citosina-5-)-Metiltransferases/genética , Técnicas de Introdução de Genes , Genoma Fúngico , Humanos , Redes Neurais de Computação , S-Adenosilmetionina/metabolismo , Saccharomycetales/metabolismo , Estresse Fisiológico/genética , Telômero , Transcrição Genética
11.
Mol Cell ; 78(2): 210-223.e8, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32208170

RESUMO

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress.


Assuntos
Metilação de DNA/genética , Heterocromatina/genética , Metaboloma/genética , S-Adenosilmetionina/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Citoplasma/genética , Citoplasma/metabolismo , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Células HCT116 , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Metionina/genética , Camundongos , Processamento de Proteína Pós-Traducional/genética , Proteômica/métodos
12.
J Mol Biol ; 432(9): 3018-3032, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32135193

RESUMO

The methyltransferases that belong to the SpoU-TrmD family contain trefoil knots in their backbone fold. Recent structural dynamic and binding analyses of both free and bound homologs indicate that the knot within the polypeptide backbone plays a significant role in the biological activity of the molecule. The knot loops form the S-adenosyl-methionine (SAM)-binding pocket as well as participate in SAM binding and catalysis. Knots contain both at once a stable core as well as moving parts that modulate long-range motions. Here, we sought to understand allosteric effects modulated by the knotted topology. Uncovering the residues that contribute to these changes and the functional aspects of these protein motions are essential to understanding the interplay between the knot, activation of the methyltransferase, and the implications in RNA interactions. The question we sought to address is as follows: How does the knot, which constricts the backbone as well as forms the SAM-binding pocket with its three distinctive loops, affect the binding mechanism? Using a minimally tied trefoil protein as the framework for understanding the structure-function roles, we offer an unprecedented view of the conformational mechanics of the knot and its relationship to the activation of the ligand molecule. Focusing on the biophysical characterization of the knot region by NMR spectroscopy, we identify the SAM-binding region and observe changes in the dynamics of the loops that form the knot. Importantly, we also observe long-range allosteric changes in flanking helices consistent with winding/unwinding in helical propensity as the knot tightens to secure the SAM cofactor.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Sítio Alostérico , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína
13.
Chem Commun (Camb) ; 56(22): 3317-3320, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32077874

RESUMO

In this work, the preparation of new S-adenosyl-l-methionine (SAM) analogues for sequence specific DNA labeling is evaluated. These non-natural analogues, comprising cysteine rather than the natural homolog, were obtained in near quantitative conversions from readily available starting materials without relying on using an excess amount of labor intensive molecules. The synthetic strategy was used to generate fluorescent cofactors, with colours spanning the whole visible spectrum, and their applicability in methyltransferase based optical mapping is shown.


Assuntos
DNA/metabolismo , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , DNA/química , Corantes Fluorescentes/química , Plasmídeos/genética , Plasmídeos/metabolismo , S-Adenosilmetionina/análogos & derivados
14.
Biochem J ; 477(5): 1033-1047, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32091571

RESUMO

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer in adults. Among the altered pathways leading to HCC, an increasing role is attributed to abnormal epigenetic regulation. Members of the Heterochromatin Protein (HP1) 1 family are key players in chromatin organisation, acting as docking sites for chromatin modifiers. Here, we inactivated HP1α in HepG2 human liver carcinoma cells and showed that HP1α participated in cell proliferation. HP1α-depleted cells have a global decrease in DNA methylation and consequently a perturbed chromatin organisation, as exemplified by the reactivation of transcription at centromeric and pericentromeric regions, eventhough the protein levels of chromatin writers depositing methylation marks, such as EZH2, SETDB1, SUV39H1, G9A and DNMT3A remained unaltered. This decrease was attributed mainly to a low S-Adenosyl Methionine (SAM) level, a cofactor involved in methylation processes. Furthermore, we showed that this decrease was due to a modification in the Methionine adenosyl transferase 2A RNA (MAT2A) level, which modifies the ratio of MAT1A/MAT2A, two enzymes that generate SAM. Importantly, HP1α reintroduction into HP1α-depleted cells restored the MAT2A protein to its initial level. Finally, we demonstrated that this transcriptional deregulation of MAT2A in HP1α-depleted cells relied on a lack of recruitment of HP1ß and HP1γ to MAT2A promoter where an improper non-CpG methylation site was promoted in the vicinity of the transcription start site where HP1ß and HP1γ bound. Altogether, these results highlight an unanticipated link between HP1 and the SAM synthesis pathway, and emphasise emerging functions of HP1s as sensors of some aspects of liver cell metabolism.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas Cromossômicas não Histona/deficiência , Neoplasias Hepáticas/metabolismo , S-Adenosilmetionina/metabolismo , Vias Biossintéticas/fisiologia , Células HEK293 , Células Hep G2 , Humanos
15.
J Agric Food Chem ; 68(8): 2516-2527, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32050067

RESUMO

In the Gram-negative bacterium Aeromonas hydrophila, N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS) influences pathogenicity, protein secretion, and motility. However, the catalytic mechanism of AHL biosynthesis and the structural basis and substrate specificity for AhyI members remain unclear. In this study, we cloned the ahyI gene from the isolate A. hydrophila HX-3, and the overexpressed AhyI protein was confirmed to produce six types of AHLs by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, contrasting with previous reports that AhyI only produces N-butanoyl-l-homoserine lactone (C4-HSL) and N-hexanoyl-l-homoserine lactone (C6-HSL). The results of an in vitro biosynthetic assay showed that purified AhyI can catalyze the formation of C4-HSL using S-adenosyl-l-methionine (SAM) and butyryl-acyl carrier protein (ACP) as substrates and indicated that the fatty acyl substrate used in AhyI-mediated AHL synthesis is derived from acyl-ACP rather than acyl-CoA. The kinetic data of AhyI using butyryl-ACP as an acyl substrate indicated that the catalytic efficiency of the A. hydrophila HX-3 AhyI enzyme is within an order of magnitude compared to other LuxI homologues. In this study, for the first time, the tertiary structural modeling results of AhyI and those of molecular docking and structural and functional analyses showed the importance of several crucial residues, as well as the secondary structure with respect to acylation. A Phe125-Phe152 clamp grasps the terminal methyl group to assist in stabilizing the long acyl chains in a putative binding pocket. The stacking interactions within a strong hydrophobic environment, a hydrogen-bonding network, and a ß bulge presumably stabilize the ACP acyl chain for the attack of the SAM α-amine toward the thioester carbon, offering a relatively reasonable explanation for how AhyI can synthesize AHLs with diverse acyl-chain lengths. Moreover, Trp34 participates in forming the binding pocket for C4-ACP and becomes ordered upon SAM binding, providing a good basis for catalysis. The novel finding that AhyI can produce both short- and long-chain AHLs enhances current knowledge regarding the variety of AHLs produced by this enzyme. These structural data are expected to serve as a molecular rationale for AHL synthesis by AhyI.


Assuntos
Aeromonas hydrophila/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Acil-Butirolactonas/química , Acil-Butirolactonas/metabolismo , Aeromonas hydrophila/química , Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/genética , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , Espectrometria de Massas em Tandem
16.
Enzyme Microb Technol ; 134: 109475, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044022

RESUMO

Quorum sensing is a population density-dependent gene expression regulation mechanism in bacteria. The substrate specificity of RhlI, an enzyme in the RhlI-RhlR quorum sensing system of Pseudomonas aeruginosa, was explored by directed evolution to gain insight into the molecular mechanisms of quorum sensing. RhlI catalyzes S-adenosyl methionine and butanoyl or hexanoyl acyl carrier protein to form N-butanoyl homoserine lactone (BHL) and or N-hexanoyl homoserine lactone (HHL), respectively, none of which contain 3-oxo groups. We developed high-throughput genetic screening and selection methods to identify RhlI mutants via four rounds of directed evolution and identified RhlI-4M1 as the mutant that generated new catalytic activity and synthesized 3-oxo-hexanoyl homoserine lactone (OHHL) containing the 3-oxo group in Escherichia coli. Additionally, the synthesizing activities of BHL and HHL were improved by 3.98- and 3.01-fold, respectively. RhlI-4M1 contains five amino acid substitutions (A15D, K31R, T92S, Y129N, and L184Q) and one stop codon (Q193*) mutations. The deletion of nine amino acids in the C-terminus was crucial for OHHL production by RhlI mutants. This work demonstrates that the genetic screen/selection should be useful in the development of applications involving the manipulation of bacterial quorum sensing. The new catalytic activity of these RhlI mutants will prove beneficial in elucidating the mechanistic understanding of bacterial quorum sensing and similarly, may prove beneficial in the development of new drugs including antimicrobial compounds.


Assuntos
Evolução Molecular Direcionada/métodos , Ligases/genética , Pseudomonas aeruginosa/genética , Percepção de Quorum , Fatores de Transcrição/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ensaios de Triagem em Larga Escala , Ligases/metabolismo , Mutação , Pseudomonas aeruginosa/enzimologia , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismo
17.
J Biotechnol ; 309: 100-106, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31926980

RESUMO

S-adenosyl-l-methionine (SAM) has been attracting increasing attention because of its significance in the pharmaceutical industry; however, the high cost of this compound limits its application. Tofu yellow serofluid exhibits high nutritional value and is not costly; therefore, it can be utilized as a substrate in the fermentation industry. In the current study, Saccharomyces cerevisiae was cultured in the tofu yellow serofluid fermentation medium for the SAM biosynthesis. The optimum tofu yellow serofluid fermentation medium contained 70 g/L of glucose, 30 % of yellow serofluid, 20 g/L of l-methionine, and 2.5 g/L of ammonium citrate. Under these conditions, the optimum feeding strategy was established. The results revealed that the dry cell weight (DCW) reached 123.1 g/L, the maximum production of SAM was 16.14 g/L, the highest SAM productivity reached 1.048 g/L/h, and SAM content was determined at 131.1 mg/g DCW. Furthermore, addition of tofu yellow serofluid reduced the average cost of SAM by 31.9 % to compare with the culture process without addition of tofu yellow serofluid. Thus, the tofu yellow serofluid fermentation medium improved the production of SAM and significantly reduced the production costs.


Assuntos
Fermentação , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Alimentos de Soja , Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Ácido Cítrico/metabolismo , Meios de Cultura/química , Etanol/metabolismo , Metionina/metabolismo , Compostos de Amônio Quaternário/metabolismo
18.
PLoS One ; 15(1): e0227647, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940410

RESUMO

mRNA modification by N6-methyladenosine (m6A) is involved in many post-transcriptional regulation processes including mRNA stability, splicing and promotion of translation. Accordingly, the recently identified mRNA methylation complex containing METTL3, METTL14, and WTAP has been the subject of intense study. However, METTL16 (METT10D) has also been identified as an RNA m6A methyltransferase that can methylate both coding and noncoding RNAs, but its biological role remains unclear. While global studies have identified many potential RNA targets of METTL16, only a handful, including the long noncoding RNA MALAT1, the snRNA U6, as well as the mRNA MAT2A have been verified and/or studied to any great extent. In this study we identified/verified METTL16 targets by immunoprecipitation of both endogenous as well as exogenous FLAG-tagged protein. Interestingly, exogenously overexpressed METTL16 differed from the endogenous protein in its relative affinity for RNA targets which prompted us to investigate METTL16's localization within the cell. Surprisingly, biochemical fractionation revealed that a majority of METTL16 protein resides in the cytoplasm of a number of cells. Furthermore, siRNA knockdown of METTL16 resulted in expression changes of a few mRNA targets suggesting that METTL16 may play a role in regulating gene expression. Thus, while METTL16 has been reported to be a nuclear protein, our findings suggest that METTL16 is also a cytoplasmic methyltransferase that may alter its RNA binding preferences depending on its cellular localization. Future studies will seek to confirm differences between cytoplasmic and nuclear RNA targets in addition to exploring the physiological role of METTL16 through long-term knockdown.


Assuntos
Metiltransferases/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Humanos , Metionina Adenosiltransferase/genética , Metilação , Proteínas Nucleares/genética , Processamento de RNA/genética , Estabilidade de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , S-Adenosilmetionina/metabolismo
19.
J Biosci ; 452020.
Artigo em Inglês | MEDLINE | ID: mdl-31965988

RESUMO

S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) are involved in diverse cellular functions. These enzymes show little sequence conservation but have a conserved structural fold. The DNA MTases have characteristic motifs that are involved in AdoMet binding, DNA target recognition and catalysis. Motif III of these MTases have a highly conserved acidic residue, often an aspartate, whose functional significance is not clear. Here, we report a mutational study of the residue in the ß family MTase of the Type III restriction-modification enzyme EcoP15I. Replacement of this residue by alanine affects its methylation activity. We propose that this residue contributes to the affinity of the enzyme for AdoMet. Analysis of the structures of DNA, RNA and protein MTases reveal that the acidic residue is conserved in all of them, and interacts with N6 of the adenine moiety of AdoMet. Interestingly, in the SET-domain protein lysine MTases, which have a fold different from other AdoMet-dependent MTases, N6 of the adenine moiety is hydrogen bonded to the main chain carbonyl group of the histidine residue of the highly conserved motif III. Our study reveals the evolutionary conservation of a carbonyl group in DNA, RNA and protein AdoMet-dependent MTases for specific interaction by hydrogen bond with AdoMet, despite the lack of overall sequence conservation.


Assuntos
DNA/genética , Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/ultraestrutura , Proteínas Repressoras/ultraestrutura , DNA Metiltransferases Sítio Específica (Adenina-Específica)/ultraestrutura , Sequência de Aminoácidos/genética , Sequência Conservada/genética , DNA/ultraestrutura , Metilação de DNA/genética , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/ultraestrutura , Humanos , Ligação de Hidrogênio , Metiltransferases/ultraestrutura , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Conformação Proteica , Conformação Proteica em Folha beta/genética , Dobramento de Proteína , Proteína-Arginina N-Metiltransferases/genética , RNA/genética , RNA/ultraestrutura , Proteínas Repressoras/genética , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética
20.
J Biosci ; 452020.
Artigo em Inglês | MEDLINE | ID: mdl-31965999

RESUMO

In mammals, DNA methyltransferases transfer a methyl group from S-adenosylmethionine to the 5 position of cytosine in DNA. The product of this reaction, 5-methylcytosine (5mC), has many roles, particularly in suppressing transposable and repeat elements in DNA. Moreover, in many cellular systems, cell lineage specification is accompanied by DNA demethylation at the promoters of genes expressed at high levels in the differentiated cells. However, since direct cleavage of the C-C bond connecting the methyl group to the 5 position of cytosine is thermodynamically disfavoured, the question of whether DNA methylation was reversible remained unclear for many decades. This puzzle was solved by our discovery of the TET (Ten- Eleven Translocation) family of 5-methylcytosine oxidases, which use reduced iron, molecular oxygen and the tricarboxylic acid cycle metabolite 2-oxoglutarate (also known as a-ketoglutarate) to oxidise the methyl group of 5mC to 5-hydroxymethylcytosine (5hmC) and beyond. TET-generated oxidised methylcytosines are intermediates in at least two pathways of DNA demethylation, which differ in their dependence on DNA replication. In the decade since their discovery, TET enzymes have been shown to have important roles in embryonic development, cell lineage specification, neuronal function and cancer. We review these findings and discuss their implications here.


Assuntos
Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/genética , Dioxigenases/genética , 5-Metilcitosina/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Humanos , Oxirredução , S-Adenosilmetionina/metabolismo
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