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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.
BMC Complement Med Ther ; 21(1): 41, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478471

RESUMO

BACKGROUND: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today. METHODS: Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (- 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings. RESULTS: Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules. CONCLUSION: Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.


Assuntos
Alérgenos/química , Alérgenos/imunologia , /imunologia , Odorantes , Perfumes/química , /imunologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alérgenos/administração & dosagem , Alérgenos/uso terapêutico , Benzopiranos/química , Benzopiranos/metabolismo , Compostos de Benzil/química , Compostos de Benzil/metabolismo , Cinamatos/química , Cinamatos/metabolismo , /metabolismo , /metabolismo , Diterpenos/química , Diterpenos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Perfumes/administração & dosagem , Perfumes/uso terapêutico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 12(1): 92, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397920

RESUMO

Telomere maintenance is a universal hallmark of cancer. Most tumors including low-grade oligodendrogliomas use telomerase reverse transcriptase (TERT) expression for telomere maintenance while astrocytomas use the alternative lengthening of telomeres (ALT) pathway. Although TERT and ALT are hallmarks of tumor proliferation and attractive therapeutic targets, translational methods of imaging TERT and ALT are lacking. Here we show that TERT and ALT are associated with unique 1H-magnetic resonance spectroscopy (MRS)-detectable metabolic signatures in genetically-engineered and patient-derived glioma models and patient biopsies. Importantly, we have leveraged this information to mechanistically validate hyperpolarized [1-13C]-alanine flux to pyruvate as an imaging biomarker of ALT status and hyperpolarized [1-13C]-alanine flux to lactate as an imaging biomarker of TERT status in low-grade gliomas. Collectively, we have identified metabolic biomarkers of TERT and ALT status that provide a way of integrating critical oncogenic information into non-invasive imaging modalities that can improve tumor diagnosis and treatment response monitoring.


Assuntos
Neoplasias Encefálicas/genética , Homeostase do Telômero , Telômero/metabolismo , Alanina/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Isótopos de Carbono/metabolismo , Linhagem Celular Tumoral , Engenharia Genética , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Ácido Láctico/metabolismo , Masculino , Metaboloma , Modelos Biológicos , Gradação de Tumores , Proteínas de Neoplasias/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Nus , Telomerase/genética , Telomerase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Commun ; 12(1): 150, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420011

RESUMO

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase-DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.


Assuntos
Antibacterianos/farmacologia , Cloro/metabolismo , DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Alanina/química , Alanina/metabolismo , Antibacterianos/química , Cristalografia por Raios X , DNA Girase/química , DNA Topoisomerases Tipo II , DNA de Cadeia Simples/metabolismo , Desenho de Fármacos , Canal de Potássio ERG1/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Quinolinas/química , Quinolinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/química
5.
J Neurosci ; 41(4): 797-810, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33334867

RESUMO

Although Tau accumulation is clearly linked to pathogenesis in Alzheimer's disease and other Tauopathies, the mechanism that initiates the aggregation of this highly soluble protein in vivo remains largely unanswered. Interestingly, in vitro Tau can be induced to form fibrillar filaments by oxidation of its two cysteine residues, generating an intermolecular disulfide bond that promotes dimerization and fibrillization. The recently solved structures of Tau filaments revealed that the two cysteine residues are not structurally equivalent since Cys-322 is incorporated into the core of the fibril, whereas Cys-291 projects away from the core to form the fuzzy coat. Here, we examined whether mutation of these cysteines to alanine affects differentially Tau mediated toxicity and dysfunction in the well-established Drosophila Tauopathy model. Experiments were conducted with both sexes, or with either sex. Each cysteine residue contributes differentially to Tau stability, phosphorylation status, aggregation propensity, resistance to stress, learning, and memory. Importantly, our work uncovers a critical role of Cys-322 in determining Tau toxicity and dysfunction.SIGNIFICANCE STATEMENT Cysteine-291 and Cysteine-322, the only two cysteine residues of Tau present in only 4-Repeat or all isoforms, respectively, have competing functions: as the key residues in the catalytic center, they enable Tau auto-acetylation; and as residues within the microtubule-binding repeat region are important not only for Tau function but also instrumental in the initiation of Tau aggregation. In this study, we present the first in vivo evidence that their substitution leads to differential consequences on Tau's physiological and pathophysiological functions. These differences raise the possibility that cysteine residues play a potential role in determining the functional diversity between isoforms.


Assuntos
Cisteína/metabolismo , Tauopatias/metabolismo , Tauopatias/patologia , Proteínas tau/metabolismo , Envelhecimento/metabolismo , Alanina/metabolismo , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Comportamento Animal , Drosophila , Epitopos , Feminino , Humanos , Masculino , Microtúbulos/metabolismo , Mutação/genética , Fosforilação , Proteínas tau/química , Proteínas tau/toxicidade
6.
PLoS One ; 15(12): e0239269, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315887

RESUMO

The integral membrane zinc metalloprotease ZMPSTE24 plays a key role in the proteolytic processing of farnesylated prelamin A, the precursor of the nuclear scaffold protein lamin A. Failure of this processing step results in the accumulation of permanently farnesylated forms of prelamin A which cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS), as well as related progeroid disorders, and may also play a role in physiological aging. ZMPSTE24 is an intriguing and unusual protease because its active site is located inside of a closed intramembrane chamber formed by seven transmembrane spans with side portals in the chamber permitting substrate entry. The specific features of prelamin A that make it the sole known substrate for ZMPSTE24 in mammalian cells are not well-defined. At the outset of this work it was known that farnesylation is essential for prelamin A cleavage in vivo and that the C-terminal region of prelamin A (41 amino acids) is sufficient for recognition and processing. Here we investigated additional features of prelamin A that are required for cleavage by ZMPSTE24 using a well-established humanized yeast system. We analyzed the 14-residue C-terminal region of prelamin A that lies between the ZMPSTE24 cleavage site and the farnesylated cysteine, as well 23-residue region N-terminal to the cleavage site, by generating a series of alanine substitutions, alanine additions, and deletions in prelamin A. Surprisingly, we found that there is considerable flexibility in specific requirements for the length and composition of these regions. We discuss how this flexibility can be reconciled with ZMPSTE24's selectivity for prelamin A.


Assuntos
Lamina Tipo A/metabolismo , Membranas/metabolismo , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Zinco/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cisteína/metabolismo , Proteínas de Membrana/metabolismo , Prenilação/fisiologia , Leveduras/metabolismo
7.
PLoS One ; 15(12): e0241576, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362225

RESUMO

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Alanina/genética , Alanina/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/isolamento & purificação , Lisina/genética , Lisina/metabolismo , Membranas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Estabilidade Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Ubiquitinação/fisiologia
8.
Nature ; 585(7826): 530-537, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32968259

RESUMO

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature1,2. Although synthetic protein functionalization strategies allow mimicry of PTMs3,4, as well as formation of unnatural protein variants with diverse potential functions, including drug carrying5, tracking, imaging6 and partner crosslinking7, the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C• radicals that form the native (ß-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C• radicals that form equivalent ß-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF2-labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized 'alkylator proteins' with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function.


Assuntos
Luz , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteínas/química , Proteínas/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Sítios de Ligação , Carbono/química , Carbono/metabolismo , Enzimas/química , Enzimas/metabolismo , Ésteres/síntese química , Ésteres/química , Células HeLa , Humanos , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/metabolismo , Indicadores e Reagentes/química , Oxirredução , Processos Fotoquímicos/efeitos da radiação , Domínios e Motivos de Interação entre Proteínas
9.
Mol Genet Genomics ; 295(6): 1529-1535, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32894358

RESUMO

Lanthipeptides are a subgroup of ribosomally encoded and post-translationally modified peptides (RiPPs) which frequently possess potent biological activity. Here we provide the first comprehensive bioinformatic analysis of the lanthipeptide-producing capability of the Salinispora genus, a marine actinomycete. One hundred twenty-two Salinispora arenicola, tropica, and pacifica genomic sequences were analyzed for lanthipeptide gene clusters, and the resulting 182 clusters were divided into seven groups based on sequence similarities. Group boundaries were defined based on LanB and LanM sequences with greater than 80% similarity within groups. Of the seven groups, six are predicted to encode class I lanthipeptides while only one group is predicted to encode class II lanthipeptides. Leader and core peptides were predicted for each cluster along with the number of possible lanthionine bridges. Notably, all of the predicted products of these clusters would represent novel lanthipeptide scaffolds. Of the 122 Salinispora genomes analyzed in this study, 92% contained at least one lanthipeptide gene cluster suggesting that Salinispora is a rich, yet untapped, source of lanthipeptides.


Assuntos
Alanina/análogos & derivados , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Micromonosporaceae/metabolismo , Fragmentos de Peptídeos/metabolismo , Sulfetos/metabolismo , Alanina/isolamento & purificação , Alanina/metabolismo , Proteínas de Bactérias/genética , Genômica , Micromonosporaceae/genética , Micromonosporaceae/crescimento & desenvolvimento , Fragmentos de Peptídeos/isolamento & purificação , Sulfetos/isolamento & purificação
10.
Antimicrob Agents Chemother ; 64(11)2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32868327

RESUMO

Remdesivir has reported efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and in vivo Drug-drug interactions limit therapeutic options in transplant patients. Remdesivir and its metabolite GS-441524 are excreted principally in urine. In intensive care unit (ICU) settings, in which multiple-organ dysfunctions can occur rapidly, hemodialysis may be a viable option for maintaining remdesivir treatment, while improving tolerance, by removing both remdesivir's metabolite (GS-441524) and sulfobutylether ß-cyclodextrin sodium (SEBCD). Additional studies may prove informative, particularly in the evaluations of therapeutic options for coronavirus disease 2019 (COVID-19).


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/administração & dosagem , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/terapia , Furanos/urina , Pneumonia Viral/terapia , Pirróis/urina , Triazinas/urina , beta-Ciclodextrinas/urina , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/efeitos adversos , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/administração & dosagem , Alanina/efeitos adversos , Alanina/química , Alanina/metabolismo , Antivirais/efeitos adversos , Antivirais/química , Antivirais/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/cirurgia , Infecções por Coronavirus/virologia , Interações Medicamentosas , Furanos/efeitos adversos , Furanos/química , Humanos , Unidades de Terapia Intensiva , Transplante de Pulmão , Insuficiência de Múltiplos Órgãos , Pandemias , Pneumonia Viral/cirurgia , Pneumonia Viral/virologia , Pirróis/efeitos adversos , Pirróis/química , Diálise Renal , Transplantados , Triazinas/efeitos adversos , Triazinas/química , beta-Ciclodextrinas/efeitos adversos , beta-Ciclodextrinas/química
11.
PLoS Pathog ; 16(8): e1008512, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776984

RESUMO

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that the presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. The deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteAΔA503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, the production of Bp BteAΔA503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response.


Assuntos
Alanina/metabolismo , Proteínas de Bactérias/metabolismo , Bordetella pertussis/fisiologia , Regulação Bacteriana da Expressão Gênica , Coqueluche/patologia , Alanina/genética , Animais , Proteínas de Bactérias/genética , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Virulência , Coqueluche/genética , Coqueluche/microbiologia
12.
Ecotoxicol Environ Saf ; 205: 111102, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32836152

RESUMO

The increased production and environmental release of graphene nanoparticles has raised concerns about its environmental impact, but the effects of graphene on living organisms at the metabolic level remain unknown. In this study, we used matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)-based untargeted metabolomics to investigate the metabolic response of juvenile earthworms (Eisenia fetida) to graphene exposure in soil tests for the first time. Our results reveal that graphene-exposure significantly disturbs earthworm metabolome, and graphene toxicity on earthworm shows non-concentration-dependent effect. Alanine, phenylalanine, proline, glutamate, arginine, histidine, maltose, glucose, malate, succinate, myo-inositol, and spermidine were successfully screened as significantly change compounds in earthworms for the exposure of graphene. The heterogeneous distributions of these metabolites in earthworm were also clearly imaged by MALDI-MSI. Our MSI results fully showed that the metabolite expression levels in juvenile earthworms significantly changed (up-/down-regulation) after exposure to graphene nanoparticles. This work improves our understanding of graphene nanoparticle toxicity to juvenile earthworms and also enables the continued progression of MALDI-MSI-based metabolomics as an emerging, reliable, and rapid ecotoxicological tool for assessing contaminant toxicity.


Assuntos
Grafite/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Alanina/metabolismo , Animais , Grafite/metabolismo , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Oligoquetos/efeitos dos fármacos , Solo/química , Poluentes do Solo/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
ACS Chem Neurosci ; 11(15): 2137-2144, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32639711

RESUMO

Now, it has been evidenced that Covid19 (SARS-CoV-2) infects the brain tissues. Along with this, a challenge has been raised for research professionals to find effective drugs for its treatment since the recent spread of this virus from Wuhan, China. Targeting the treatment of brain infection, it has also been a challenge that the clinical drug should have good CNS penetration ability to cross the blood-brain barrier.


Assuntos
Betacoronavirus , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/metabolismo , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/metabolismo , Antivirais/administração & dosagem , Antivirais/metabolismo , Betacoronavirus/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/virologia , Encéfalo/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/administração & dosagem , Fármacos do Sistema Nervoso Central/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Metilprednisolona/administração & dosagem , Metilprednisolona/metabolismo , Pandemias , Resultado do Tratamento
14.
J Phys Chem B ; 124(32): 6955-6962, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32521159

RESUMO

Starting from late 2019, the coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures, and supporting apparatuses. Recently, because of its positive results in clinical trials, remdesivir was approved by the Food and Drug Administration to treat COVID-19 through Emergency Use Authorization. Here, we used molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). We first constructed the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identity of 95.8%). We then built a putative preinsertion binding structure by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp without the RNA template. The putative binding structure was further optimized with molecular dynamics simulations. The resulting stable preinsertion state of remdesivir appeared to form hydrogen bonds with the RNA template when aligned with the newly solved cryo-EM structure of SARS-CoV-2 RdRp. The relative binding free energy between remdesivir and ATP was calculated to be -2.80 ± 0.84 kcal/mol, where remdesivir bound much stronger to SARS-CoV-2 RdRp than the natural substrate ATP. The ∼100-fold improvement in the Kd from remdesivir over ATP indicates an effective replacement of ATP in blocking of the RdRp preinsertion site. Key residues D618, S549, and R555 are found to be the contributors to the binding affinity of remdesivir. These findings suggest that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA replication, with key residues also identified for future lead optimization and/or drug resistance studies.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/metabolismo , Betacoronavirus/enzimologia , Inibidores Enzimáticos/metabolismo , /metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Antivirais/química , Sítios de Ligação , Inibidores Enzimáticos/química , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Ligação Proteica , Termodinâmica , Proteínas não Estruturais Virais/química
16.
Proc Natl Acad Sci U S A ; 117(25): 14552-14560, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513689

RESUMO

Both inorganic fertilizer inputs and crop yields have increased globally, with the concurrent increase in the pollution of water bodies due to nitrogen leaching from soils. Designing agroecosystems that are environmentally friendly is urgently required. Since agroecosystems are highly complex and consist of entangled webs of interactions between plants, microbes, and soils, identifying critical components in crop production remain elusive. To understand the network structure in agroecosystems engineered by several farming methods, including environmentally friendly soil solarization, we utilized a multiomics approach on a field planted with Brassica rapa We found that the soil solarization increased plant shoot biomass irrespective of the type of fertilizer applied. Our multiomics and integrated informatics revealed complex interactions in the agroecosystem showing multiple network modules represented by plant traits heterogeneously associated with soil metabolites, minerals, and microbes. Unexpectedly, we identified soil organic nitrogen induced by soil solarization as one of the key components to increase crop yield. A germ-free plant in vitro assay and a pot experiment using arable soils confirmed that specific organic nitrogen, namely alanine and choline, directly increased plant biomass by acting as a nitrogen source and a biologically active compound. Thus, our study provides evidence at the agroecosystem level that organic nitrogen plays a key role in plant growth.


Assuntos
Brassica rapa/crescimento & desenvolvimento , Produção Agrícola , Produtos Agrícolas/crescimento & desenvolvimento , Nitrogênio/metabolismo , Solo/química , Alanina/química , Alanina/metabolismo , Biomassa , Brassica rapa/metabolismo , Colina/química , Colina/metabolismo , Produtos Agrícolas/metabolismo , Conjuntos de Dados como Assunto , Redes e Vias Metabólicas/efeitos da radiação , Metabolômica , Microbiota/fisiologia , Microbiota/efeitos da radiação , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Rizosfera , Microbiologia do Solo , Luz Solar
17.
Cell ; 182(2): 417-428.e13, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32526208

RESUMO

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.


Assuntos
Betacoronavirus/química , Betacoronavirus/enzimologia , Proteínas não Estruturais Virais/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Modelos Químicos , Modelos Moleculares , RNA Viral/metabolismo , Transcrição Genética , Replicação Viral
18.
PLoS One ; 15(6): e0233961, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479512

RESUMO

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Biblioteca de Peptídeos , Alanina/genética , Alanina/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Técnicas de Visualização da Superfície Celular/economia , Análise Custo-Benefício , Citometria de Fluxo/economia , Citometria de Fluxo/métodos , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Mutação , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
19.
Science ; 368(6498): 1499-1504, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32358203

RESUMO

The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/química , Betacoronavirus/enzimologia , /química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Domínio Catalítico , Microscopia Crioeletrônica , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , RNA Viral/química , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
20.
J Ind Microbiol Biotechnol ; 47(4-5): 395-402, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32303871

RESUMO

The biodesulfurization 4S pathway can specifically desulfurize an aromatic S heterocyclic compound (which is difficult to desulfurize by hydrodesulfurization) and maintain the integrity of its combustion value. The four Dsz enzymes in the pathway convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). DszC is the first enzyme in the 4S pathway and is subject to feedback inhibition and substrate inhibition. This study is the first attempt to further modify the DszC mutant AKWC to improve its tolerance to DBT. Alanine scanning was performed on the dimeric surface of the DszC mutant AKWC, and the HBP yield of the BAD (AKWCP413A) strain was increased compared to the BAD (AKWC) strain. Site-directed saturation mutagenesis was performed on the 413th amino acid of AKWC, and the substrate inhibition parameter KI value of the mutant AKWCPI was 5.6 times higher than that of AKWC. When the DBT concentration was 0.25 mM, the HBP production of the recombinant strain overexpressing AKWCPI was increased by approximately 1.4-fold compared to the BL21(DE3)/BADC*+C* strain. The protein engineering of DszC further improved the substrate tolerance after overcoming the feedback inhibition, which provided a reference for the analysis of the inhibition mechanism of DszC substrate. Overexpression of DszC-beneficial mutants also greatly improved the efficiency of desulfurization.


Assuntos
Alanina/metabolismo , Proteínas de Bactérias/metabolismo , Mutagênese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares , Estrutura Terciária de Proteína , Tiofenos/farmacologia
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