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1.
Bioengineered ; 12(1): 2836-2850, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34227905

RESUMO

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/etiologia , Proteínas de Membrana/genética , SARS-CoV-2 , Serina Endopeptidases/genética , Tupaiidae/genética , Tupaiidae/metabolismo , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Bioengenharia , COVID-19/enzimologia , COVID-19/genética , Biologia Computacional , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Homologia Estrutural de Proteína , Distribuição Tecidual , Tupaiidae/virologia
2.
Sci Signal ; 14(690)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230209

RESUMO

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Polifosfatos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Administração por Inalação , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/administração & dosagem , Antivirais/química , COVID-19/metabolismo , COVID-19/virologia , Células CACO-2 , Chlorocebus aethiops , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Citocinas/metabolismo , Células HEK293 , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Técnicas In Vitro , Modelos Biológicos , Simulação de Acoplamento Molecular , Nebulizadores e Vaporizadores , Polifosfatos/administração & dosagem , Polifosfatos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteólise/efeitos dos fármacos , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
3.
Molecules ; 26(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063759

RESUMO

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission at neuromuscular and autonomic ganglionic synapses in the peripheral nervous system. The postsynaptic localization of muscle ((α1)2ß1γδ) and neuronal ((α3ß4)2ß4) nicotinic receptors at these synapses is mediated by interactions between the nAChR intracellular domains and cytoplasmic scaffolding proteins. Recent high resolution structures and functional studies provide new insights into the molecular determinants that mediate these interactions. Surprisingly, they reveal that the muscle nAChR binds 1-3 rapsyn scaffolding molecules, which dimerize and thereby form an interconnected lattice between receptors. Moreover, rapsyn binds two distinct sites on the nAChR subunit cytoplasmic loops; the MA-helix on one or more subunits and a motif specific to the ß subunit. Binding at the latter site is regulated by agrin-induced phosphorylation of ßY390, and increases the stoichiometry of rapsyn/AChR complexes. Similarly, the neuronal nAChR may be localized at ganglionic synapses by phosphorylation-dependent interactions with 14-3-3 adaptor proteins which bind specific motifs in each of the α3 subunit cytoplasmic loops. Thus, postsynaptic localization of nAChRs is mediated by regulated interactions with multiple scaffolding molecules, and the stoichiometry of these complexes likely helps regulate the number, density, and stability of receptors at the synapse.


Assuntos
Receptores Nicotínicos/metabolismo , Sinapses/metabolismo , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Animais , Músculos/metabolismo , Neurônios/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptores Nicotínicos/química , Homologia de Sequência de Aminoácidos
4.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: covidwho-1253936

RESUMO

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


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
5.
Arch Virol ; 166(8): 2337-2341, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34091784

RESUMO

Anemone mosaic virus (AnMV) and ranunculus mild mosaic virus (RanMMV) infect anemone plants, which exhibit characteristic mosaic patterns on their leaves. Employing next-generation sequencing of plant material imported from the Netherlands, the complete genome sequences of these two viruses were determined for the first time. AnMV and RanMMV have 9698 and 9537 nucleotides (nt), respectively, excluding the poly(A) tail. They share 80.0%/82.0% and 98.0%/97.0% nt/amino acid (aa) sequence identity, which is above the species demarcation value, in the previously reported AnMV and RanMMV coat protein sequences, but they share 69.0%/70.0% nt/aa sequence identity or less with other potyviruses in all 10 mature protein coding regions of the genome. Additionally, phylogenetic analysis confirmed the relationship of the AnMV and RanMMV genome sequences to previously reported partial sequences and placed them within the genus Potyvirus. These results show that these two viruses represent separate species within the genus Potyvirus.


Assuntos
Anemone/virologia , Potyvirus/classificação , Sequenciamento Completo do Genoma/métodos , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Japão , Países Baixos , Fases de Leitura Aberta , Filogenia , Filogeografia , Potyvirus/genética , Potyvirus/isolamento & purificação , Homologia de Sequência de Aminoácidos
6.
Nat Commun ; 12(1): 3856, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158470

RESUMO

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.


Assuntos
Motivos de Aminoácidos , DNA Helicases/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
7.
Nat Commun ; 12(1): 3855, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158477

RESUMO

Human voltage-gated proton channels (hHv1) extrude protons from cells to compensate for charge and osmotic imbalances due metabolism, normalizing intracellular pH and regulating protein function. Human albumin (Alb), present at various levels throughout the body, regulates oncotic pressure and transports ligands. Here, we report Alb is required to activate hHv1 in sperm and neutrophils. Dose-response studies reveal the concentration of Alb in semen is too low to activate hHv1 in sperm whereas the higher level in uterine fluid yields proton efflux, allowing capacitation, the acrosomal reaction, and oocyte fertilization. Likewise, Alb activation of hHv1 in neutrophils is required to sustain production and release of reactive oxygen species during the immune respiratory burst. One Alb binds to both voltage sensor domains (VSDs) in hHv1, enhancing open probability and increasing proton current. A computational model of the Alb-hHv1 complex, validated by experiments, identifies two sites in Alb domain II that interact with the VSDs, suggesting an electrostatic gating modification mechanism favoring the active "up" sensor conformation. This report shows how sperm are triggered to fertilize, resolving how hHv1 opens at negative membrane potentials in sperm, and describes a role for Alb in physiology that will operate in the many tissues expressing hHv1.


Assuntos
Albuminas/metabolismo , Mediadores da Inflamação/metabolismo , Canais Iônicos/metabolismo , Neutrófilos/metabolismo , Capacitação Espermática/fisiologia , Reação Acrossômica/fisiologia , Albuminas/química , Sequência de Aminoácidos , Fertilização/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Canais Iônicos/genética , Masculino , Potenciais da Membrana/fisiologia , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Prótons , Sêmen/citologia , Sêmen/metabolismo , Homologia de Sequência de Aminoácidos , Espermatozoides/fisiologia , Eletricidade Estática
8.
Nat Commun ; 12(1): 3867, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162839

RESUMO

Enzymes can evolve new catalytic activity when environmental changes present them with novel substrates. Despite this seemingly straightforward relationship, factors other than the direct catalytic target can also impact adaptation. Here, we characterize the catalytic activity of a recently evolved bacterial methyl-parathion hydrolase for all possible combinations of the five functionally relevant mutations under eight different laboratory conditions (in which an alternative divalent metal is supplemented). The resultant adaptive landscapes across this historical evolutionary transition vary in terms of both the number of "fitness peaks" as well as the genotype(s) at which they are found as a result of genotype-by-environment interactions and environment-dependent epistasis. This suggests that adaptive landscapes may be fluid and molecular adaptation is highly contingent not only on obvious factors (such as catalytic targets), but also on less obvious secondary environmental factors that can direct it towards distinct outcomes.


Assuntos
Adaptação Fisiológica/genética , Bactérias/genética , Proteínas de Bactérias/genética , Epistasia Genética , Hidrolases/genética , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Evolução Molecular , Interação Gene-Ambiente , Genótipo , Hidrolases/química , Hidrolases/metabolismo , Cinética , Metais/química , Metais/metabolismo , Metil Paration/química , Metil Paration/metabolismo , Mutação , Domínios Proteicos , Homologia de Sequência de Aminoácidos
9.
Nat Commun ; 12(1): 3883, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162887

RESUMO

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.


Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , DNA Fúngico/genética , Complexo de Reconhecimento de Origem/genética , Origem de Replicação/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

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


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
11.
Nat Commun ; 12(1): 3292, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078910

RESUMO

Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9-MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.


Assuntos
Autofagia/genética , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Cadeias Pesadas de Miosina/genética , Quinases Relacionadas a NIMA/genética , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Cílios/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Quinases Relacionadas a NIMA/deficiência , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
12.
Nucleic Acids Res ; 49(W1): W277-W284, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33978743

RESUMO

The InterEvDock3 protein docking server exploits the constraints of evolution by multiple means to generate structural models of protein assemblies. The server takes as input either several sequences or 3D structures of proteins known to interact. It returns a set of 10 consensus candidate complexes, together with interface predictions to guide further experimental validation interactively. Three key novelties were implemented in InterEvDock3 to help obtain more reliable models: users can (i) generate template-based structural models of assemblies using close and remote homologs of known 3D structure, detected through an automated search protocol, (ii) select the assembly models most consistent with contact maps from external methods that implement covariation-based contact prediction with or without deep learning and (iii) exploit a novel coevolution-based scoring scheme at atomic level, which leads to significantly higher free docking success rates. The performance of the server was validated on two large free docking benchmark databases, containing respectively 230 unbound targets (Weng dataset) and 812 models of unbound targets (PPI4DOCK dataset). Its effectiveness has also been proven on a number of challenging examples. The InterEvDock3 web interface is available at http://bioserv.rpbs.univ-paris-diderot.fr/services/InterEvDock3/.


Assuntos
Simulação de Acoplamento Molecular , Conformação Proteica , Software , Subunidades Proteicas/química , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína
13.
Nat Commun ; 12(1): 2976, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016977

RESUMO

The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we used a 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transtorno do Espectro Autista/genética , Códon de Iniciação , Códon de Terminação , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/isolamento & purificação , Técnicas de Inativação de Genes , Glutarredoxinas/genética , Humanos , Mutação , Fases de Leitura Aberta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
14.
Nat Commun ; 12(1): 2675, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976148

RESUMO

Developing molecules that emulate the properties of naturally occurring ice-binding proteins (IBPs) is a daunting challenge. Rather than relying on the (limited) existing structure-property relationships that have been established for IBPs, here we report the use of phage display for the identification of short peptide mimics of IBPs. To this end, an ice-affinity selection protocol is developed, which enables the selection of a cyclic ice-binding peptide containing just 14 amino acids. Mutational analysis identifies three residues, Asp8, Thr10 and Thr14, which are found to be essential for ice binding. Molecular dynamics simulations reveal that the side chain of Thr10 hydrophobically binds to ice revealing a potential mechanism. To demonstrate the biotechnological potential of this peptide, it is expressed as a fusion ('Ice-Tag') with mCherry and used to purify proteins directly from cell lysate.


Assuntos
Proteínas Anticongelantes/genética , Técnicas de Visualização da Superfície Celular/métodos , Mutação , Peptídeos Cíclicos/genética , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas Anticongelantes/química , Proteínas Anticongelantes/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Cristalização , Interações Hidrofóbicas e Hidrofílicas , Gelo , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
15.
Nat Commun ; 12(1): 2947, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011950

RESUMO

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.


Assuntos
Evolução Molecular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Sistemas de Secreção Tipo II/genética , Sistemas de Secreção Tipo II/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Eucariotos/classificação , Eucariotos/genética , Eucariotos/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Modelos Moleculares , Naegleria/classificação , Naegleria/genética , Naegleria/metabolismo , Peroxissomos/metabolismo , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Sistemas de Secreção Tipo II/classificação
16.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947016

RESUMO

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher's) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name "B. belcheri EAS/AOS" (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.


Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Anfioxos/enzimologia , Alcadienos/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Peróxido de Hidrogênio/metabolismo , Cinética , Anfioxos/genética , Oxilipinas/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato
17.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947021

RESUMO

Despite increasing reports on the function of CCCH zinc finger proteins in plant development and stress response, the functions and molecular aspects of many non-tandem CCCH zinc finger (non-TZF) proteins remain uncharacterized. AtC3H59/ZFWD3 is an Arabidopsis non-TZF protein and belongs to the ZFWD subfamily harboring a CCCH zinc finger motif and a WD40 domain. In this study, we characterized the biological and molecular functions of AtC3H59, which is subcellularly localized in the nucleus. The seeds of AtC3H59-overexpressing transgenic plants (OXs) germinated faster than those of wild type (WT), whereas atc3h59 mutant seeds germinated slower than WT seeds. AtC3H59 OX seedlings were larger and heavier than WT seedlings, whereas atc3h59 mutant seedlings were smaller and lighter than WT seedlings. Moreover, AtC3H59 OX seedlings had longer primary root length than WT seedlings, whereas atc3h59 mutant seedlings had shorter primary root length than WT seedlings, owing to altered cell division activity in the root meristem. During seed development, AtC3H59 OXs formed larger and heavier seeds than WT. Using yeast two-hybrid screening, we isolated Desi1, a PPPDE family protein, as an interacting partner of AtC3H59. AtC3H59 and Desi1 interacted via their WD40 domain and C-terminal region, respectively, in the nucleus. Taken together, our results indicate that AtC3H59 has pleiotropic effects on seed germination, seedling development, and seed development, and interacts with Desi1 in the nucleus via its entire WD40 domain. To our knowledge, this is the first report to describe the biological functions of the ZFWD protein and Desi1 in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Sementes/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Contagem de Células , Núcleo Celular/metabolismo , Sequência Consenso , Germinação , Meristema/citologia , Família Multigênica , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Mapeamento de Interação de Proteínas , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
18.
Commun Biol ; 4(1): 475, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: covidwho-1180282

RESUMO

COVID-19 is a respiratory illness caused by a novel coronavirus called SARS-CoV-2. The viral spike (S) protein engages the human angiotensin-converting enzyme 2 (ACE2) receptor to invade host cells with ~10-15-fold higher affinity compared to SARS-CoV S-protein, making it highly infectious. Here, we assessed if ACE2 polymorphisms can alter host susceptibility to SARS-CoV-2 by affecting this interaction. We analyzed over 290,000 samples representing >400 population groups from public genomic datasets and identified multiple ACE2 protein-altering variants. Using reported structural data, we identified natural ACE2 variants that could potentially affect virus-host interaction and thereby alter host susceptibility. These include variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R that were predicted to increase susceptibility, while variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y were predicted to be protective variants that show decreased binding to S-protein. Using biochemical assays, we confirmed that K31R and E37K had decreased affinity, and K26R and T92I variants showed increased affinity for S-protein when compared to wildtype ACE2. Consistent with this, soluble ACE2 K26R and T92I were more effective in blocking entry of S-protein pseudotyped virus suggesting that ACE2 variants can modulate susceptibility to SARS-CoV-2.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto/genética , Polimorfismo Genético , Receptores Virais/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
19.
EMBO J ; 40(11): e102277, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: covidwho-1194823

RESUMO

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Regulação Viral da Expressão Gênica , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vírus da SARS/fisiologia , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Cromatografia em Gel , Proteases Semelhantes à Papaína de Coronavírus/química , Cristalografia por Raios X , Genes Reporter , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes , Modelos Moleculares , Fatores de Iniciação de Peptídeos/química , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , RNA Viral/genética , Proteínas de Ligação a RNA/química , RNA Polimerase Dependente de RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Subunidades Ribossômicas/metabolismo , Vírus da SARS/genética , SARS-CoV-2/genética , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Difração de Raios X
20.
Plant Cell ; 33(2): 358-380, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793852

RESUMO

Phycobilisomes (PBSs), the principal cyanobacterial antenna, are among the most efficient macromolecular structures in nature, and are used for both light harvesting and directed energy transfer to the photosynthetic reaction center. However, under unfavorable conditions, excess excitation energy needs to be rapidly dissipated to avoid photodamage. The orange carotenoid protein (OCP) senses light intensity and induces thermal energy dissipation under stress conditions. Hence, its expression must be tightly controlled; however, the molecular mechanism of this regulation remains to be elucidated. Here, we describe the discovery of a posttranscriptional regulatory mechanism in Synechocystis sp. PCC 6803 in which the expression of the operon encoding the allophycocyanin subunits of the PBS is directly and in an inverse fashion linked to the expression of OCP. This regulation is mediated by ApcZ, a small regulatory RNA that is derived from the 3'-end of the tetracistronic apcABC-apcZ operon. ApcZ inhibits ocp translation under stress-free conditions. Under most stress conditions, apc operon transcription decreases and ocp translation increases. Thus, a key operon involved in the collection of light energy is functionally connected to the expression of a protein involved in energy dissipation. Our findings support the view that regulatory RNA networks in bacteria evolve through the functionalization of mRNA 3'-UTRs.


Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Luz , RNA Bacteriano/metabolismo , Synechocystis/metabolismo , Synechocystis/efeitos da radiação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Modelos Biológicos , Mutação/genética , Óperon/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Synechocystis/genética
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