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1.
PLoS One ; 16(6): e0252507, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34061896

RESUMO

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Assuntos
Teste para COVID-19/normas , COVID-19/diagnóstico , COVID-19/epidemiologia , Testes Diagnósticos de Rotina/normas , Indicadores e Reagentes/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/genética , COVID-19/virologia , Teste para COVID-19/métodos , Camarões/epidemiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Gana/epidemiologia , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Indicadores e Reagentes/provisão & distribuição , Técnicas de Diagnóstico Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Biologia Sintética/métodos , Transformação Bacteriana , Reino Unido/epidemiologia
2.
Molecules ; 26(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072087

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Descoberta de Drogas , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/genética , Animais , Antivirais/uso terapêutico , Ácido Aurintricarboxílico/farmacologia , Ácido Aurintricarboxílico/uso terapêutico , COVID-19/virologia , Chlorocebus aethiops , Ácido Elágico/farmacologia , Ácido Elágico/uso terapêutico , Heparina/farmacologia , Heparina/uso terapêutico , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos
3.
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
4.
Nat Commun ; 12(1): 3214, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088904

RESUMO

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.


Assuntos
Proteínas Arqueais/metabolismo , Divisão Celular/fisiologia , Methanobrevibacter/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular , Divisão Celular/genética , Sequência Conservada , Cristalografia por Raios X , Evolução Molecular , Methanobrevibacter/genética , Methanobrevibacter/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
5.
Nat Commun ; 12(1): 3384, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099674

RESUMO

Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational "anchor extension" methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.


Assuntos
Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacologia , Peptídeos Cíclicos/farmacologia , Relação Estrutura-Atividade , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Ensaios Enzimáticos , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/isolamento & purificação , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/ultraestrutura , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/isolamento & purificação , Desacetilase 6 de Histona/ultraestrutura , Inibidores de Histona Desacetilases/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/ultraestrutura
6.
Nat Commun ; 12(1): 2987, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016967

RESUMO

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC's ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Parede Celular/ultraestrutura , Sequência Conservada/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Mutagênese , Filogenia , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Domínios Proteicos/genética , Multimerização Proteica , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
7.
Nat Commun ; 12(1): 2962, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016969

RESUMO

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.


Assuntos
DNA Topoisomerases Tipo II/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , Regulação Alostérica , Animais , Domínio Catalítico , Linhagem Celular , Microscopia Crioeletrônica , DNA/metabolismo , DNA/ultraestrutura , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/isolamento & purificação , DNA Topoisomerases Tipo II/metabolismo , Humanos , Mesocricetus , Modelos Moleculares , Nucleoproteínas , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/isolamento & purificação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
8.
Nat Commun ; 12(1): 2970, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016972

RESUMO

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infecções por Cardiovirus/imunologia , Infecções por Respirovirus/imunologia , Ubiquitina Tiolesterase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Animais , Infecções por Cardiovirus/virologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Vírus da Encefalomiocardite/imunologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional/imunologia , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Infecções por Respirovirus/virologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/isolamento & purificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/imunologia
9.
Nat Commun ; 12(1): 3172, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039996

RESUMO

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.


Assuntos
Proteínas do Tecido Nervoso/ultraestrutura , Neuropilina-1/ultraestrutura , Receptores de Superfície Celular/ultraestrutura , Semaforina-3A/ultraestrutura , Animais , Células COS , Chlorocebus aethiops , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-1/genética , Neuropilina-1/isolamento & purificação , Neuropilina-1/metabolismo , Ligação Proteica/genética , Domínios Proteicos/genética , Multimerização Proteica/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Semaforina-3A/genética , Semaforina-3A/isolamento & purificação , Semaforina-3A/metabolismo
10.
Nat Commun ; 12(1): 3231, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050142

RESUMO

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.


Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação Alostérica/genética , Animais , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma Fúngico , Histonas/genética , Histonas/isolamento & purificação , Humanos , Larva/genética , Larva/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
11.
Nat Commun ; 12(1): 3239, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050165

RESUMO

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.


Assuntos
Proteases Dependentes de ATP/ultraestrutura , Proteínas Mitocondriais/ultraestrutura , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Envelhecimento/metabolismo , Bortezomib/farmacologia , Domínio Catalítico/efeitos dos fármacos , Microscopia Crioeletrônica , Ensaios Enzimáticos , Humanos , Hidrólise , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Proteólise , Proteostase , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
12.
Bioresour Technol ; 335: 125286, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34022479

RESUMO

Endo-ß-1,3-glucanase with high specific activity is a prerequisite for enzymatic preparation of valuable ß-oligoglucosides. Heterologous expression in Pichia pastoris GS115 with error-prone PCR technology was implemented, and the mutant strain 7 N12 was obtained. The mutant endo-ß-1,3-glucanase showed efficient specific activities for degrading curdlan (366 U mg-1) and scleroglucan (274.5 U mg-1). Thereafter, one-step production of functional branched oligoglucosides was established with coupled fermentation of Pichia pastoris and Sclerotium rolfsii. During the fermentation process, the endo-ß-1,3-glucanase secreted by Pichia pastoris GS115 can efficiently hydrolyse scleroglucan metabolized by Sclerotium rolfsii WSH-G01. The maximum yields of ß-oligoglucosides in the shake flasks and 7-L bioreactor reached 1.73 g L-1 and 12.71 g L-1, respectively, with polymerization degrees of 2-17. The successful implementation of heterologous expression with error-prone PCR and the coupled fermentation simplified the multi-step enzymatic ß-oligoglucoside preparation procedures, which makes it a potential strategy for industrial production of functional oligosaccharides.


Assuntos
Pichia , Saccharomycetales , Basidiomycota , Fermentação , Pichia/genética , Proteínas Recombinantes/genética
13.
Front Immunol ; 12: 641360, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054808

RESUMO

Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows its role in immune surveillance against pathogens. Higher levels of serum SP-D have been reported in the patients with severe acute respiratory syndrome coronavirus (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro, in vivo and ex vivo. In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing human angiotensin converting enzyme 2 (hACE2). The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following treatment with rfhSP-D (10 µg/ml). These results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merit pre-clinical studies in animal models.


Assuntos
COVID-19/prevenção & controle , Vírus da Influenza A/fisiologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Mucosa Respiratória/fisiologia , Vírus Sinciciais Respiratórios/fisiologia , Vírion/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Ligação Proteica , Proteína D Associada a Surfactante Pulmonar/genética , Proteínas Recombinantes/genética , Mucosa Respiratória/virologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
14.
Nat Commun ; 12(1): 2791, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990582

RESUMO

Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin-membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.


Assuntos
Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Animais , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Variação Genética , Inseticidas/química , Inseticidas/farmacologia , Lipossomos/química , Modelos Moleculares , Controle Biológico de Vetores , Domínios Proteicos , Estrutura Quaternária de Proteína , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Homologia Estrutural de Proteína
15.
Nat Commun ; 12(1): 2942, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011988

RESUMO

The association between reduced myofilament force-generating capacity (Fmax) and heart failure (HF) is clear, however the underlying molecular mechanisms are poorly understood. Here, we show impaired Fmax arises from reduced BAG3-mediated sarcomere turnover. Myofilament BAG3 expression decreases in human HF and positively correlates with Fmax. We confirm this relationship using BAG3 haploinsufficient mice, which display reduced Fmax and increased myofilament ubiquitination, suggesting impaired protein turnover. We show cardiac BAG3 operates via chaperone-assisted selective autophagy (CASA), conserved from skeletal muscle, and confirm sarcomeric CASA complex localization is BAG3/proteotoxic stress-dependent. Using mass spectrometry, we characterize the myofilament CASA interactome in the human heart and identify eight clients of BAG3-mediated turnover. To determine if increasing BAG3 expression in HF can restore sarcomere proteostasis/Fmax, HF mice were treated with rAAV9-BAG3. Gene therapy fully rescued Fmax and CASA protein turnover after four weeks. Our findings indicate BAG3-mediated sarcomere turnover is fundamental for myofilament functional maintenance.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Insuficiência Cardíaca/fisiopatologia , Miócitos Cardíacos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Modelos Animais de Doenças , Feminino , Terapia Genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcômeros/metabolismo
16.
mSphere ; 6(3)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980675

RESUMO

Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43.IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.


Assuntos
Coronavirus Humano OC43/fisiologia , Efeito Citopatogênico Viral , Receptores Virais/metabolismo , Serina Endopeptidases/metabolismo , Carga Viral/métodos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Coronavirus Humano OC43/isolamento & purificação , Humanos , Técnicas Imunoenzimáticas , Receptores Virais/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Células Vero/virologia , Cultura de Vírus , Internalização do Vírus , Replicação Viral
17.
Nat Commun ; 12(1): 2702, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976201

RESUMO

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Iniciação da Transcrição Genética , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elongação da Transcrição Genética
18.
Sheng Wu Gong Cheng Xue Bao ; 37(4): 1368-1375, 2021 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-33973449

RESUMO

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.


Assuntos
Toxina Diftérica , Escherichia coli , Proteínas de Bactérias , Toxina Diftérica/genética , Escherichia coli/genética , Humanos , Chaperonas Moleculares/genética , Proteínas Recombinantes/genética
19.
Nat Commun ; 12(1): 2971, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016973

RESUMO

The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.


Assuntos
Hipoglicemiantes/química , Receptores do Leucotrieno B4/ultraestrutura , Animais , Sítios de Ligação/genética , Cristalografia por Raios X , Diabetes Mellitus Tipo 2 , Células HEK293 , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Ligantes , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Receptores do Leucotrieno B4/agonistas , Receptores do Leucotrieno B4/antagonistas & inibidores , Receptores do Leucotrieno B4/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera , Relação Estrutura-Atividade
20.
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
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