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1.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802775

RESUMO

Silver nanoparticles (AgNPs) are the one of the most extensively used nanomaterials. The strong antimicrobial properties of AgNPs have led to their use in a wide range of medical and consumer products. Although the neurotoxicity of AgNPs has been confirmed, the molecular mechanisms have not been extensively studied, particularly in immature organisms. Based on information gained from previous in vitro studies, in the present work, we examine whether ionotropic NMDA glutamate receptors contribute to AgNP-induced neurotoxicity in an animal model of exposure. In brains of immature rats subjected to a low dose of AgNPs, we identified ultrastructural and molecular alterations in the postsynaptic region of synapses where NMDA receptors are localized as a multiprotein complex. We revealed decreased expression of several NMDA receptor complex-related proteins, such as GluN1 and GluN2B subunits, scaffolding proteins PSD95 and SynGAP, as well as neuronal nitric oxide synthase (nNOS). Elucidating the changes in NMDA receptor-mediated molecular mechanisms induced by AgNPs, we also identified downregulation of the GluN2B-PSD95-nNOS-cGMP signaling pathway which maintains LTP/LTD processes underlying learning and memory formation during development. This observation is accompanied by decreased density of NMDA receptors, as assessed by a radioligand binding assay. The observed effects are reversible over the post-exposure time. This investigation reveals that NMDA receptors in immature rats are a target of AgNPs, thereby indicating the potential health hazard for children and infants resulting from the extensive use of products containing AgNPs.


Assuntos
Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Nanopartículas Metálicas/toxicidade , Receptores de N-Metil-D-Aspartato/metabolismo , Prata/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/ultraestrutura , GMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Ligantes , Masculino , Nanopartículas Metálicas/ultraestrutura , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Subunidades Proteicas/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/ultraestrutura
2.
Sci Adv ; 7(16)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33863729

RESUMO

The COVID-19 (coronavirus disease 2019) pandemic underwent a rapid transition with the emergence of a dominant viral variant (from the "D-form" to the "G-form") that carried an amino acid substitution D614G in its "Spike" protein. The G-form is more infectious in vitro and is associated with increased viral loads in the upper airways. To gain insight into the molecular-level underpinnings of these characteristics, we used microsecond all-atom simulations. We show that changes in the protein energetics favor a higher population of infection-capable states in the G-form through release of asymmetry present in the D-form inter-protomer interactions. Thus, the increased infectivity of the G-form is likely due to a higher rate of profitable binding encounters with the host receptor. It is also predicted to be more neutralization sensitive owing to enhanced exposure of the receptor binding domain, a key target region for neutralizing antibodies. These results are critical for vaccine design.


Assuntos
/genética , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , /metabolismo , Anticorpos Neutralizantes/imunologia , /virologia , Glicosilação , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , /isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
3.
Biochemistry (Mosc) ; 86(3): 257-261, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33838638

RESUMO

The imbalance of the renin-angiotensin system is currently considered as a potentially important factor of the pathogenesis of COVID-19 disease. It has been shown previously in the murine model, that the expression of angiotensin-converting enzyme 2 (ACE2) on the cell surface is downregulated in response to the infection by SARS-CoV virus or recombinant spike protein (S protein) alone. In the case of natural infection, circulation of the S protein in a soluble form is unlikely. However, in SARS-CoV-2, a large fraction of S protein trimers is pre-processed during virion morphogenesis due to the presence of furin protease cleavage site between the S1 and S2 subunits. Therefore, S protein transition into the fusion conformation may be accompanied by the separation of the S1 subunits carrying the receptor-binding domains from the membrane-bound S2 subunits. The fate of the S1 particles shed due to the spontaneous "firing" of some S protein trimers exposed on the virions and on the surface of infected cells has been never investigated. We hypothesize that the soluble S1 subunits of the SARS-CoV-2 S protein shed from the infected cells and from the virions in vivo may bind to the ACE2 and downregulate cell surface expression of this protein. The decrease in the ACE2 activity on the background of constant or increased ACE activity in the lungs may lead to the prevalence of angiotensin II effects over those of angiotensin (1-7), thus promoting thrombosis, inflammation, and pulmonary damage. This hypothesis also suggests the association between less pronounced shedding of the S1 particles reported for the S protein carrying the D614G mutation (vs. the wild type D614 protein), and lack of increased severity of the COVID-19 infection caused by the mutant (D614G) SARS-CoV-2 strain, despite its higher infectivity and higher in vivo viral load.


Assuntos
/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , /imunologia , Animais , /patologia , Humanos , Camundongos , Modelos Moleculares , Mutação , Multimerização Proteica , Subunidades Proteicas , Sistema Renina-Angiotensina , /patogenicidade , Glicoproteína da Espícula de Coronavírus/genética
4.
Nat Commun ; 12(1): 2333, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879791

RESUMO

Acaryochloris marina is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of A. marina photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll d and its epimer chlorophyll d'. The primary electron acceptor is pheophytin a, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Complexo de Proteína do Fotossistema I/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clorofila/química , Clorofila/metabolismo , Microscopia Crioeletrônica , Cianobactérias/genética , Cianobactérias/metabolismo , Transporte de Elétrons , Luz , Modelos Moleculares , Oxigênio/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas , Eletricidade Estática
5.
Nat Commun ; 12(1): 2249, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33883550

RESUMO

The RNA chaperone Hfq, acting as a hexamer, is a known mediator of post-transcriptional regulation, expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA, is a functional partner of Hfq that facilitates Hfq-mediated sRNA-mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine-Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA.


Assuntos
Proteínas de Escherichia coli/metabolismo , GTP Pirofosfoquinase/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/química , GTP Pirofosfoquinase/genética , Fator Proteico 1 do Hospedeiro/química , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , RNA Bacteriano/química , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Deleção de Sequência
6.
Nat Commun ; 12(1): 2154, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846324

RESUMO

Transient receptor potential (TRP) channels are polymodal molecular sensors involved in numerous physiological processes and implicated in a variety of human diseases. Several structures of the founding member of the TRP channel family, TRPV1, are available, all of which were determined for the protein missing the N- and C-termini and the extracellular S5-P-loop. Here, we present structures of the full-length thirteen-lined ground squirrel TRPV1 solved by cryo-EM. Our structures resolve the extracellular cap domain formed by the S5-P-loops and the C-terminus that wraps around the three-stranded ß-sheet connecting elements of the TRPV1 intracellular skirt. The cap domain forms a dome above the pore's extracellular entrance, with four portals leading to the ion conductance pathway. Deletion of the cap increases the TRPV1 average conductance, reduces the open probability and affects ion selectivity. Our data show that both the termini and the cap domain are critical determinants of TRPV1 function.


Assuntos
Espaço Extracelular/química , Ativação do Canal Iônico , Canais de Cátion TRPV/metabolismo , Animais , Células HEK293 , Humanos , Ligantes , Lipídeos/química , Domínios Proteicos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Sciuridae , Canais de Cátion TRPV/química
7.
Nat Commun ; 12(1): 2159, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846330

RESUMO

N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is installed by a multi-component writer complex; however, the exact roles of each component remain poorly understood. Here we show that a potential E3 ubiquitin ligase Hakai colocalizes and interacts with other m6A writer components, and Hakai mutants exhibit typical m6A pathway defects in Drosophila, such as lowered m6A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects. Hakai, Vir, Fl(2)d and Flacc form a stable complex, and disruption of either Hakai, Vir or Fl(2)d led to the degradation of the other three components. Furthermore, MeRIP-seq indicates that the effective m6A modification is mostly distributed in 5' UTRs in Drosophila, in contrast to the mammalian system. Interestingly, we demonstrate that m6A modification is deposited onto the Sxl mRNA in a sex-specific fashion, which depends on the m6A writer. Together, our work not only advances the understanding of mechanism and regulation of the m6A writer complex, but also provides insights into how Sxl cooperate with the m6A pathway to control its own splicing.


Assuntos
Adenosina/análogos & derivados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regiões 5' não Traduzidas/genética , Adenosina/metabolismo , Processamento Alternativo/genética , Animais , Sequência de Bases , Comportamento Animal , Códon de Iniciação/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Proteínas de Fluorescência Verde/metabolismo , Masculino , Metilação , Mutação/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Asas de Animais/metabolismo
8.
Nat Commun ; 12(1): 2162, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846343

RESUMO

Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of diguanylate cyclases. Full-length crystal structures reveal that BeF3- pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many diguanylate cyclases with N-terminal coiled-coil linkers.


Assuntos
Proteínas de Escherichia coli/metabolismo , Leptospira/enzimologia , Fósforo-Oxigênio Liases/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Berílio/química , Ativação Enzimática , Proteínas de Escherichia coli/química , Retroalimentação Fisiológica , Fluoretos/química , Cinética , Modelos Moleculares , Fósforo-Oxigênio Liases/química , Fosforilação , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rotação
9.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799739

RESUMO

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos/genética , Fatores de Transcrição/genética , Transcrição Genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Hibridização In Situ/métodos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional
11.
Nat Struct Mol Biol ; 28(3): 310-318, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33712813

RESUMO

Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.


Assuntos
Celulose/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Biocatálise , Microscopia Crioeletrônica , Proteínas de Escherichia coli/ultraestrutura , Modelos Moleculares , Complexos Multienzimáticos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes
12.
Molecules ; 26(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33673017

RESUMO

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.


Assuntos
Farmacorresistência Viral/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Subunidades Proteicas/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Proteínas Virais/antagonistas & inibidores , Cristalografia por Raios X , Vírus da Influenza A/efeitos dos fármacos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Teoria Quântica , /química , Termodinâmica , Proteínas Virais/química , Proteínas Virais/metabolismo
13.
Science ; 372(6537): 52-56, 2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33707221

RESUMO

Eukaryotic transcription requires the assembly of a multisubunit preinitiation complex (PIC) composed of RNA polymerase II (Pol II) and the general transcription factors. The coactivator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at a resolution below 4 angstroms. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.


Assuntos
Complexo Mediador/química , RNA Polimerase II/química , Fatores Genéricos de Transcrição/química , Iniciação da Transcrição Genética , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Humanos , Complexo Mediador/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fatores Genéricos de Transcrição/metabolismo
14.
Front Immunol ; 12: 637651, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33767706

RESUMO

As COVID-19 cases continue to rise, it is imperative to learn more about antibodies and T-cells produced against the causative virus, SARS-CoV-2, in order to guide the rapid development of therapies and vaccines. While much of the current antibody and vaccine research focuses on the receptor-binding domain of S1, a less-recognized opportunity is to harness the potential benefits of the more conserved S2 subunit. Similarities between the spike proteins of both SARS-CoV-2 and HIV-1 warrant exploring S2. Possible benefits of employing S2 in therapies and vaccines include the structural conservation of S2, extant cross-reactive neutralizing antibodies in populations (due to prior exposure to common cold coronaviruses), the steric neutralization potential of antibodies against S2, and the stronger memory B-cell and T-cell responses. More research is necessary on the effect of glycans on the accessibility and stability of S2, SARS-CoV-2 mutants that may affect infectivity, the neutralization potential of antibodies produced by memory B-cells, cross-reactive T-cell responses, antibody-dependent enhancement, and antigen competition. This perspective aims to highlight the evidence for the potential advantages of using S2 as a target of therapy or vaccine design.


Assuntos
/uso terapêutico , /imunologia , Glicoproteína da Espícula de Coronavírus/uso terapêutico , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , /virologia , Reações Cruzadas , Epitopos , Interações Hospedeiro-Patógeno , Humanos , Imunogenicidade da Vacina , Subunidades Proteicas , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/uso terapêutico
15.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669532

RESUMO

Although understanding of the biomedical basis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is growing, the underlying pathological mechanisms remain uncertain. We recently reported a reduction in the proportion of basal oxygen consumption due to ATP synthesis by Complex V in ME/CFS patient-derived lymphoblast cell lines, suggesting mitochondrial respiratory inefficiency. This was accompanied by elevated respiratory capacity, elevated mammalian target of rapamycin complex 1 (mTORC1) signaling activity and elevated expression of enzymes involved in the TCA cycle, fatty acid ß-oxidation and mitochondrial transport. These and other observations led us to hypothesise the dysregulation of pathways providing the mitochondria with oxidisable substrates. In our current study, we aimed to revisit this hypothesis by applying a combination of whole-cell transcriptomics, proteomics and energy stress signaling activity measures using subsets of up to 34 ME/CFS and 31 healthy control lymphoblast cell lines from our growing library. While levels of glycolytic enzymes were unchanged in accordance with our previous observations of unaltered glycolytic rates, the whole-cell proteomes of ME/CFS lymphoblasts contained elevated levels of enzymes involved in the TCA cycle (p = 1.03 × 10-4), the pentose phosphate pathway (p = 0.034, G6PD p = 5.5 × 10-4), mitochondrial fatty acid ß-oxidation (p = 9.2 × 10-3), and degradation of amino acids including glutamine/glutamate (GLS p = 0.034, GLUD1 p = 0.048, GOT2 p = 0.026), branched-chain amino acids (BCKDHA p = 0.028, BCKDHB p = 0.031) and essential amino acids (FAH p = 0.036, GCDH p = 0.006). The activity of the major cellular energy stress sensor, AMPK, was elevated but the increase did not reach statistical significance. The results suggest that ME/CFS metabolism is dysregulated such that alternatives to glycolysis are more heavily utilised than in controls to provide the mitochondria with oxidisable substrates.


Assuntos
Síndrome de Fadiga Crônica/metabolismo , Linfócitos/metabolismo , Mitocôndrias/metabolismo , Adulto , Idoso , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Oxirredução , Fosforilação Oxidativa , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato , Transcriptoma/genética
16.
Nat Commun ; 12(1): 1715, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731724

RESUMO

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Betacoronavirus/imunologia , Epitopos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Betacoronavirus/classificação , Camelus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reações Cruzadas , Epitopos/química , Epitopos/genética , Humanos , Camundongos , Conformação Proteica , Subunidades Proteicas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
17.
Science ; 372(6541): 525-530, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33727252

RESUMO

Substitution for aspartic acid (D) by glycine (G) at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. Here, we report cryo-electron microscopy structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations that differ primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer-effectively increasing the number of functional spikes and enhancing infectivity-and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.


Assuntos
/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Substituição de Aminoácidos , /metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Microscopia Crioeletrônica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , /metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
18.
Nat Commun ; 12(1): 1395, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654088

RESUMO

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.


Assuntos
DNA/metabolismo , Neoplasias/metabolismo , Polissacarídeos/biossíntese , Antígenos Glicosídicos Associados a Tumores/metabolismo , Toxina da Cólera/metabolismo , Gangliosídeo G(M1)/metabolismo , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Análise de Sequência com Séries de Oligonucleotídeos , Polissacarídeos/química , Subunidades Proteicas/metabolismo
19.
Sci Adv ; 7(10)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658206

RESUMO

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses, with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing a pandemic in 2020. Coronaviral non-structural proteins (nsps) form the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complexes has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations, SARS-CoV-2 nsp7+8 consists primarily of heterotetramers. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model proposed.


Assuntos
Alphacoronavirus/metabolismo , Betacoronavirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Reagentes para Ligações Cruzadas/química , Modelos Moleculares , Multimerização Proteica , Subunidades Proteicas/metabolismo , Espalhamento de Radiação , Espalhamento a Baixo Ângulo , Especificidade da Espécie , Proteínas não Estruturais Virais/química , Difração de Raios X
20.
Am J Physiol Renal Physiol ; 320(3): F485-F491, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33522411

RESUMO

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the γ subunit. Here, we investigated the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in Fisher rat thyroid cells by coexpressing the apical membrane-bound protease channel-activating protease 1 (CAP1; prostasin). To compare the peptides produced in the heterologous system with those in the mouse kidney, we treated both lysates with PNGaseF to remove N-linked glycosylation. The apparent molecular mass of the smallest COOH-terminal fragment of γENaC (52 kDa) was indistinguishable from that of the CAP1-induced species in Fisher rat thyroid cells. Similar cleaved peptides were observed in total and cell surface fractions of the rat kidney. This outcome suggests that most of the subunits at the surface have been processed by extracellular proteases. This was confirmed using nonreducing gels, in which the NH2- and COOH-terminal fragments of γENaC are linked by a disulfide bond. Under these conditions, the major cleaved form in the rat kidney had an apparent molecular mass of 56 kDa, ∼4 kDa lower than that of the full-length form, consistent with excision of a short peptide by two proteolytic events. We conclude that the most abundant γENaC species in the apical membrane of rat and mouse kidneys on a low-Na diet is the twice-cleaved, presumably activated form.NEW & NOTEWORTHY We have identified the major aldosterone-dependent cleaved form of the epithelial Na channel (ENaC) γ subunit in the kidney as a twice-cleaved peptide. This form appears to be identical in size with a subunit cleaved in vitro by the extracellular protease channel-activating protease 1 (prostasin). In the absence of reducing agents, it has an overall molecular mass less than that of the intact subunit, consistent with the excision of an inhibitory domain.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Serina Endopeptidases/metabolismo , Sódio/metabolismo , Aldosterona/metabolismo , Animais , Dieta Hipossódica/métodos , Camundongos , Subunidades Proteicas/metabolismo , Proteólise , Ratos
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