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1.
Phys Chem Chem Phys ; 23(10): 5852-5863, 2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33688867

RESUMO

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP analogues because it not only terminates the polymerization activity of RNA-dependent RNA polymerase (RdRp), but also inhibits the proofreading activity of intrinsic exoribonuclease (ExoN). Even though the static structure of Remdesivir binding to RdRp has been solved and biochemical experiments have suggested it to be a "delayed chain terminator", the underlying molecular mechanisms is not fully understood. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 24 microseconds to elucidate the inhibitory mechanism of Remdesivir on nucleotide addition and proofreading. We found that when Remdesivir locates at an upstream site in RdRp, the 1'-cyano group experiences electrostatic interactions with a salt bridge (Asp865-Lys593), which subsequently halts translocation. Our findings can supplement the current understanding of the delayed chain termination exerted by Remdesivir and provide an alternative molecular explanation about Remdesivir's inhibitory mechanism. Such inhibition also reduces the likelihood of Remdesivir to be cleaved by ExoN acting on 3'-terminal nucleotides. Furthermore, our study also suggests that Remdesivir's 1'-cyano group can disrupt the cleavage site of ExoN via steric interactions, leading to a further reduction in the cleavage efficiency. Our work provides plausible and novel mechanisms at the molecular level of how Remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Cianetos/química , Nucleotídeos/metabolismo , /fisiologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Alanina/uso terapêutico , /patologia , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , Ribose/química , /metabolismo , Eletricidade Estática , Replicação Viral/efeitos dos fármacos
2.
Nat Commun ; 12(1): 731, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531504

RESUMO

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3' to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.


Assuntos
Nucleotídeos/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Mutação/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética
3.
Nat Commun ; 12(1): 548, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483497

RESUMO

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Nucleotídeos/metabolismo , Actinas/química , Algoritmos , Animais , Polarização de Fluorescência , Corantes Fluorescentes/química , Hidrólise , Cinética , Modelos Teóricos , Proteínas Musculares/química , Nucleotídeos/química , Ligação Proteica , Coelhos , Termodinâmica
4.
J Med Chem ; 64(1): 782-796, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33356231

RESUMO

Nucleotide analogues are used for treating viral infections such as HIV, hepatitis B, hepatitis C, influenza, and SARS-CoV-2. To become polymerase substrates, a nucleotide analogue must be phosphorylated by cellular kinases which is rate-limiting. The goal of this study is to develop dNTP/NTP analogues directly from nucleotides. Tenofovir (TFV) analogues were synthesized by conjugating with amino acids. We demonstrate that some conjugates act as dNTP analogues and HIV-1 reverse transcriptase (RT) catalytically incorporates the TFV part as the chain terminator. X-ray structures in complex with HIV-1 RT/dsDNA showed binding of the conjugates at the polymerase active site, however, in different modes in the presence of Mg2+ versus Mn2+ ions. The adaptability of the compounds is seemingly essential for catalytic incorporation of TFV by RT. 4d with a carboxyl sidechain demonstrated the highest incorporation. 4e showed weak incorporation and rather behaved as a dNTP-competitive inhibitor. This result advocates the feasibility of designing NTP/dNTP analogues by chemical substitutions to nucleotide analogues.


Assuntos
Aminoácidos/química , Nucleotídeos/química , Tenofovir/química , Viroses/tratamento farmacológico , Sítios de Ligação , /virologia , Domínio Catalítico , Desenho de Fármacos , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Humanos , Magnésio/química , Simulação de Acoplamento Molecular , Nucleotídeos/metabolismo , Fosforilação , Especificidade por Substrato , Viroses/virologia
5.
Biochem Biophys Res Commun ; 533(4): 1470-1476, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33333712

RESUMO

Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2-/-) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2-/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.


Assuntos
Nucleotídeos/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Embrião não Mamífero/anormalidades , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Larva/genética , Larva/fisiologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Proteína Básica da Mielina/genética , Nucleotídeos/genética , Sirolimo/farmacologia , Peixe-Zebra/embriologia
6.
PLoS Biol ; 18(12): e3001015, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33332391

RESUMO

Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.


Assuntos
Capsídeo/metabolismo , HIV-1/metabolismo , Replicação Viral/fisiologia , Capsídeo/química , Capsídeo/fisiologia , Proteínas do Capsídeo/genética , Replicação do DNA/fisiologia , DNA Viral/metabolismo , Células HEK293 , HIV-1/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Simulação de Dinâmica Molecular , Nucleotídeos/metabolismo , Permeabilidade , Ácido Fítico/análise , Ácido Fítico/metabolismo , Vírion/genética , Montagem de Vírus/fisiologia , Replicação Viral/genética
7.
Nat Commun ; 11(1): 5388, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097692

RESUMO

The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5'-monophosphate-3'-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp.


Assuntos
Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Nucleotídeos de Guanina/metabolismo , Nucleotídeos/metabolismo , Bacillus/genética , Bacillus anthracis/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligação Proteica , Biossíntese de Proteínas
8.
J Phys Chem Lett ; 11(21): 9408-9414, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33104327

RESUMO

Chemical similarity-based approaches employed to repurpose or develop new treatments for emerging diseases, such as COVID-19, correlates molecular structure-based descriptors of drugs with those of a physiological counterpart or clinical phenotype. We propose novel descriptors based on a COSMO-RS (short for conductor-like screening model for real solvents) σ-profiles for enhanced drug screening enabled by machine learning (ML). The descriptors' performance is hereby illustrated for nucleotide analogue drugs that inhibit the ribonucleic acid-dependent ribonucleic acid polymerase, key to viral transcription and genome replication. The COSMO-RS-based descriptors account for both chemical reactivity and structure, and are more effective for ML-based screening than fingerprints based on molecular structure and simple physical/chemical properties. The descriptors are evaluated using principal component analysis, an unsupervised ML technique. Our results correlate with the active monophosphate forms of the leading drug remdesivir and the prospective drug EIDD-2801 with nucleotides, followed by other promising drugs, and are superior to those from molecular structure-based descriptors and molecular docking. The COSMO-RS-based descriptors could help accelerate drug discovery for the treatment of emerging diseases.


Assuntos
Aprendizado de Máquina , Nucleotídeos/química , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Simulação de Acoplamento Molecular , Nucleotídeos/metabolismo , Nucleotídeos/uso terapêutico , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Análise de Componente Principal , Teoria Quântica , RNA Viral/química , RNA Viral/metabolismo , /genética , /metabolismo
9.
PLoS One ; 15(9): e0238592, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32877448

RESUMO

DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows. Golden Gate assembly is a frequently employed DNA assembly methodology that utilizes a Type IIS restriction enzyme and a DNA ligase to generate recombinant DNA constructs from smaller DNA fragments. However, the utility of this methodology has been limited by a lack of resources to guide experimental design. For example, selection of the DNA sequences at fusion sites between fragments is based on broad assembly guidelines or pre-vetted sets of junctions, rather than being customized for a particular application or cloning project. To facilitate the design of robust assembly reactions, we developed a high-throughput DNA sequencing assay to examine reaction outcomes of Golden Gate assembly with T4 DNA ligase and the most commonly used Type IIS restriction enzymes that generate three-base and four-base overhangs. Next, we incorporated these findings into a suite of webtools that design assembly reactions using the experimental data. These webtools can be used to create customized assemblies from a target DNA sequence or a desired number of fragments. Lastly, we demonstrate how using these tools expands the limits of current assembly systems by carrying out one-pot assemblies of up to 35 DNA fragments. Full implementation of the tools developed here enables direct expansion of existing assembly standards for modular cloning systems (e.g. MoClo) as well as the formation of robust new high-fidelity standards.


Assuntos
DNA/metabolismo , Biologia Sintética/métodos , DNA Ligases/metabolismo , Enzimas de Restrição do DNA/metabolismo , Nucleotídeos/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(35): 21740-21746, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817533

RESUMO

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel is essential for epithelial salt-water balance. CFTR mutations cause cystic fibrosis, a lethal incurable disease. In cells CFTR is activated through the cAMP signaling pathway, overstimulation of which during cholera leads to CFTR-mediated intestinal salt-water loss. Channel activation is achieved by phosphorylation of its regulatory (R) domain by cAMP-dependent protein kinase catalytic subunit (PKA). Here we show using two independent approaches--an ATP analog that can drive CFTR channel gating but is unsuitable for phosphotransfer by PKA, and CFTR mutants lacking phosphorylatable serines--that PKA efficiently opens CFTR channels through simple binding, under conditions that preclude phosphorylation. Unlike when phosphorylation happens, CFTR activation by PKA binding is completely reversible. Thus, PKA binding promotes release of the unphosphorylated R domain from its inhibitory position, causing full channel activation, whereas phosphorylation serves only to maintain channel activity beyond termination of the PKA signal. The results suggest two levels of CFTR regulation in cells: irreversible through phosphorylation, and reversible through R-domain binding to PKA--and possibly also to other members of a large network of proteins known to interact with the channel.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ânions/metabolismo , Fenômenos Biofísicos , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Ativação do Canal Iônico/fisiologia , Mutagênese Sítio-Dirigida , Nucleotídeos/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Fosforilação , Ligação Proteica/fisiologia , Serina/metabolismo , Canais de Ânion Dependentes de Voltagem/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
11.
Mol Cell ; 79(5): 846-856.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755594

RESUMO

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.


Assuntos
Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resveratrol/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Resistência a Medicamentos/genética , Humanos , Hidroxiureia/farmacologia , Células Jurkat , Nucleotídeos/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologia
12.
Mol Cell ; 79(5): 758-767.e6, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755596

RESUMO

During proteotoxic stress, bacteria maintain critical processes like DNA replication while removing misfolded proteins, which are degraded by the Lon protease. Here, we show that in Caulobacter crescentus Lon controls deoxyribonucleoside triphosphate (dNTP) pools during stress through degradation of the transcription factor CcrM. Elevated dNTP/nucleotide triphosphate (NTP) ratios in Δlon cells protects them from deletion of otherwise essential deoxythymidine triphosphate (dTTP)-producing pathways and shields them from hydroxyurea-induced loss of dNTPs. Increased dNTP production in Δlon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limited protease and that Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth. Because Lon recognizes misfolded proteins regardless of the stress, this mechanism allows for response to a variety of unanticipated conditions.


Assuntos
Caulobacter crescentus/metabolismo , Nucleotídeos/metabolismo , Protease La/metabolismo , Dobramento de Proteína , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/enzimologia , Elementos de DNA Transponíveis , Didesoxinucleosídeos/metabolismo , Regulação Bacteriana da Expressão Gênica , Nucleotídeo Desaminases/genética , Nucleotídeo Desaminases/metabolismo , Ribonucleotídeo Redutases/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Regulação para Cima
13.
J Vis Exp ; (161)2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32716367

RESUMO

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in direct sequencing RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides (pseudouridine (Ψ) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (<35 nt) when using a standard high-resolution LC-MS system, and can also be used for sequence verification of modified therapeutic RNA oligonucleotides. In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.


Assuntos
Cromatografia Líquida/métodos , Nucleotídeos/metabolismo , RNA/genética , Análise de Sequência de RNA/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos
14.
PLoS One ; 15(7): e0232072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645038

RESUMO

The vasculature within a tumor is highly disordered both structurally and functionally. Endothelial cells that comprise the vasculature are poorly connected causing vessel leakage and exposing the endothelium to a hypoxic microenvironment. Therefore, most anti-angiogenic therapies are generally inefficient and result in acquired resistance to increased hypoxia due to elimination of the vasculature. Recent studies have explored the efficacy of targeting metabolic pathways in tumor cells in combination with anti-angiogenic therapy. However, the metabolic alterations of endothelial cells in response to hypoxia have been relatively unexplored. Here, we measured polar metabolite levels in microvascular endothelial cells exposed to short- and long-term hypoxia with the goal of identifying metabolic vulnerabilities that can be targeted to normalize tumor vasculature and improve drug delivery. We found that many amino acid-related metabolites were altered by hypoxia exposure, especially within alanine-aspartate-glutamate, serine-threonine, and cysteine-methionine metabolism. Additionally, there were significant changes in de novo pyrimidine synthesis as well as glutathione and taurine metabolism. These results provide key insights into the metabolic alterations that occur in endothelial cells in response to hypoxia, which serve as a foundation for future studies to develop therapies that lead to vessel normalization and more efficient drug delivery.


Assuntos
Hipóxia Celular , Células Endoteliais/metabolismo , Redes e Vias Metabólicas , Aminoácidos/metabolismo , Ácido Aspártico/metabolismo , Linhagem Celular , Cisteína/metabolismo , Células HEK293 , Humanos , Microvasos/metabolismo , Nucleotídeos/metabolismo
15.
PLoS Comput Biol ; 16(6): e1007903, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584811

RESUMO

Nucleotides comprise small molecules that perform critical signaling roles in biological systems. Adenosine-based nucleotides, including adenosine tri-, di-, and mono-phosphate, are controlled through their rapid degradation by diphosphohydrolases and ecto-nucleotidases (NDAs). The interplay between nucleotide signaling and degradation is especially important in synapses formed between cells, which create signaling 'nanodomains'. Within these 'nanodomains', charged nucleotides interact with densely-packed membranes and biomolecules. While the contributions of electrostatic and steric interactions within such nanodomains are known to shape diffusion-limited reaction rates, less is understood about how these factors control the kinetics of nucleotidase activity. To quantify these factors, we utilized reaction-diffusion numerical simulations of 1) adenosine triphosphate (ATP) hydrolysis into adenosine monophosphate (AMP) and 2) AMP into adenosine (Ado) via two representative nucleotidases, CD39 and CD73. We evaluate these sequentially-coupled reactions in nanodomain geometries representative of extracellular synapses, within which we localize the nucleotidases. With this model, we find that 1) nucleotidase confinement reduces reaction rates relative to an open (bulk) system, 2) the rates of AMP and ADO formation are accelerated by restricting the diffusion of substrates away from the enzymes, and 3) nucleotidase co-localization and the presence of complementary (positive) charges to ATP enhance reaction rates, though the impact of these contributions on nucleotide pools depends on the degree to which the membrane competes for substrates. As a result, these contributions integratively control the relative concentrations and distributions of ATP and its metabolites within the junctional space. Altogether, our studies suggest that CD39 and CD73 nucleotidase activity within junctional spaces can exploit their confinement and favorable electrostatic interactions to finely control nucleotide signaling.


Assuntos
Adenosina Trifosfatases/metabolismo , Adenosina/metabolismo , Nucleotídeos/metabolismo , Cinética , Transdução de Sinais , Propriedades de Superfície
16.
Sci Adv ; 6(25): eabb5813, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32596474

RESUMO

The COVID-19 outbreak has become a global health risk, and understanding the response of the host to the SARS-CoV-2 virus will help to combat the disease. RNA editing by host deaminases is an innate restriction process to counter virus infection, but it is not yet known whether this process operates against coronaviruses. Here, we analyze RNA sequences from bronchoalveolar lavage fluids obtained from coronavirus-infected patients. We identify nucleotide changes that may be signatures of RNA editing: adenosine-to-inosine changes from ADAR deaminases and cytosine-to-uracil changes from APOBEC deaminases. Mutational analysis of genomes from different strains of Coronaviridae from human hosts reveals mutational patterns consistent with those observed in the transcriptomic data. However, the reduced ADAR signature in these data raises the possibility that ADARs might be more effective than APOBECs in restricting viral propagation. Our results thus suggest that both APOBECs and ADARs are involved in coronavirus genome editing, a process that may shape the fate of both virus and patient.


Assuntos
Betacoronavirus/genética , Betacoronavirus/metabolismo , Infecções por Coronavirus/genética , Interações Hospedeiro-Patógeno/genética , Pneumonia Viral/genética , Edição de RNA/genética , Transcriptoma , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Sequência de Bases/genética , Líquido da Lavagem Broncoalveolar/virologia , Infecções por Coronavirus/virologia , Genoma Viral/genética , Humanos , Taxa de Mutação , Nucleotídeos/genética , Nucleotídeos/metabolismo , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Replicação Viral/genética
17.
Nat Commun ; 11(1): 3214, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32587247

RESUMO

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.


Assuntos
Regulação Neoplásica da Expressão Gênica , Nucleotídeos/metabolismo , RNA Longo não Codificante/genética , Ribonucleosídeo Difosfato Redutase , Proteína 1 de Ligação a Y-Box , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo
18.
J Med Chem ; 63(11): 6003-6027, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32421343

RESUMO

We disclose a study on nucleoside triphosphate (NTP) analogues in which the γ-phosphate is covalently modified by two different biodegradable masking units and d4T as nucleoside analogue that enable the delivery of d4TTP with high selectivity in phosphate buffer (pH 7.3) and by enzyme-triggered reactions in human CD4+ T-lymphocyte CEM cell extracts. This allows the bypass of all steps normally needed in the intracellular phosphorylation. These TriPPPro-nucleotides comprising an acyloxybenzyl (AB; ester) or an alkoxycarbonyloxybenzyl (ACB; carbonate) in combination with an ACB moiety are described as NTP delivery systems. The introduction of these two different groups led to the selective formation of γ-(ACB)-d4TTPs by chemical hydrolysis and in particular by cell extract enzymes. γ-(AB)-d4TTPs are faster cleaved than γ-(ACB)-d4TTPs. In antiviral assays, the compounds are highly active against HIV-1 and HIV-2 in wild-type CEM/O cells and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK-).


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Nucleotídeos/química , Pró-Fármacos/química , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacocinética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Esterases/metabolismo , Meia-Vida , Humanos , Hidrólise , Fígado/enzimologia , Nucleotídeos/metabolismo , Nucleotídeos/farmacologia , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Suínos
19.
Nat Chem Biol ; 16(8): 834-840, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32393900

RESUMO

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.


Assuntos
Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Sequência de Aminoácidos , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Regulação Bacteriana da Expressão Gênica/genética , Genes rel/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Ligases/fisiologia , Nucleotídeos/metabolismo , Ribossomos/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/metabolismo
20.
J Vis Exp ; (159)2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32449708

RESUMO

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5' hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.


Assuntos
Bioensaio/métodos , Nucleotídeos/metabolismo , Fosforilação/genética
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