Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 15.624
Filtrar
1.
Vaccine ; 38(42): 6487-6499, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32907757

RESUMO

The many carbohydrate chains on Covid-19 coronavirus SARS-CoV-2 and its S-protein form a glycan-shield that masks antigenic peptides and decreases uptake of inactivated virus or S-protein vaccines by APC. Studies on inactivated influenza virus and recombinant gp120 of HIV vaccines indicate that glycoengineering of glycan-shields to present α-gal epitopes (Galα1-3Galß1-4GlcNAc-R) enables harnessing of the natural anti-Gal antibody for amplifying vaccine efficacy, as evaluated in mice producing anti-Gal. The α-gal epitope is the ligand for the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. Upon administration of vaccines presenting α-gal epitopes, anti-Gal binds to these epitopes at the vaccination site and forms immune complexes with the vaccines. These immune complexes are targeted for extensive uptake by APC as a result of binding of the Fc portion of immunocomplexed anti-Gal to Fc receptors on APC. This anti-Gal mediated effective uptake of vaccines by APC results in 10-200-fold higher anti-viral immune response and in 8-fold higher survival rate following challenge with a lethal dose of live influenza virus, than same vaccines lacking α-gal epitopes. It is suggested that glycoengineering of carbohydrate chains on the glycan-shield of inactivated SARS-CoV-2 or on S-protein vaccines, for presenting α-gal epitopes, will have similar amplifying effects on vaccine efficacy. α-Gal epitope synthesis on coronavirus vaccines can be achieved with recombinant α1,3galactosyltransferase, replication of the virus in cells with high α1,3galactosyltransferase activity as a result of stable transfection of cells with several copies of the α1,3galactosyltransferase gene (GGTA1), or by transduction of host cells with replication defective adenovirus containing this gene. In addition, recombinant S-protein presenting multiple α-gal epitopes on the glycan-shield may be produced in glycoengineered yeast or bacteria expression systems containing the corresponding glycosyltransferases. Prospective Covid-19 vaccines presenting α-gal epitopes may provide better protection than vaccines lacking this epitope because of increased uptake by APC.


Assuntos
Antígenos Virais/genética , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/genética , Trissacarídeos/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Engenharia Genética , Proteína do Núcleo p24 do HIV/química , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Imunogenicidade da Vacina , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Trissacarídeos/química , Vacinas Virais/administração & dosagem , Vacinas Virais/biossíntese , Vacinas Virais/genética
2.
PLoS One ; 15(8): e0237295, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756606

RESUMO

We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with a fragment of the receptor-binding domain (RBD) of the Spike Protein S of the SARS-CoV-2 virus, via atomistic molecular dynamics simulations. We see that some ACE2 glycans interact with the S fragments, and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.


Assuntos
Betacoronavirus/metabolismo , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/química , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Glicosilação , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
Nature ; 584(7820): 291-297, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728216

RESUMO

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.


Assuntos
Espaço Extracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Antígenos CD/metabolismo , Apolipoproteína E4/metabolismo , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Receptores ErbB/metabolismo , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Camundongos , Domínios Proteicos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Solubilidade , Especificidade por Substrato
4.
Proc Natl Acad Sci U S A ; 117(31): 18832-18839, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32709746

RESUMO

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.


Assuntos
Proteínas NLR , Imunidade Vegetal , Proteínas de Plantas , Proteínas Recombinantes de Fusão , Transdução de Sinais , Animais , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Mamíferos , Proteínas NLR/química , Proteínas NLR/genética , Proteínas NLR/imunologia , Proteínas NLR/metabolismo , Imunidade Vegetal/genética , Imunidade Vegetal/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
5.
Proc Natl Acad Sci U S A ; 117(28): 16313-16323, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601209

RESUMO

Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.


Assuntos
Peroxirredoxinas/química , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(30): 17757-17763, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669430

RESUMO

Vaccination has been used to control the spread of seasonal flu; however, the virus continues to evolve and escape from host immune response through mutation and increasing glycosylation. Efforts have been directed toward development of a universal vaccine with broadly protective activity against multiple influenza strains and subtypes. Here we report the design and evaluation of various chimeric vaccines based on the most common avian influenza H5 and human influenza H1 sequences. Of these constructs, the chimeric HA (cHA) vaccine with consensus H5 as globular head and consensus H1 as stem was shown to elicit broadly protective CD4+ and CD8+ T cell responses. Interestingly, the monoglycosylated cHA (cHAmg) vaccine with GlcNAc on each glycosite induced more stem-specific antibodies, with higher antibody-dependent cellular cytotoxicity (ADCC), and better neutralizing and stronger cross-protection activities against H1, H3, H5, and H7 strains and subtypes. Moreover, the cHAmg vaccine combined with a glycolipid adjuvant designed for class switch further enhanced the vaccine efficacy with more IFN-γ, IL-4, and CD8+ memory T cells produced.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteção Cruzada/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Orthomyxoviridae/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Influenza Humana/virologia , Camundongos , Modelos Moleculares , Testes de Neutralização , Orthomyxoviridae/classificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Relação Estrutura-Atividade , Vacinação
7.
Arch Biochem Biophys ; 688: 108401, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32376316

RESUMO

HIV-1 glycoprotein 41 (gp41) mediates fusion between virus and target cells by folding into a fusion active state, in which the C-terminal heptad repeat (CHR) regions associate externally to the N-terminal heptad repeat (NHR) trimer and form a very stable six-helix bundle coiled-coil structure. Therefore, interfering with the NHR-CHR interaction of gp41 is a promising therapeutic approach against HIV-1. However, a full understanding of the molecular and mechanistic details of this interaction is still incomplete. Here, we use single-chain, chimeric proteins (named covNHR) that reproduce accurately the CHR-NHR interactions to analyze the binding thermodynamics of several peptides with different length from the CHR region. The results indicate that cooperative binding involving two or more pockets of the NHR groove is necessary to obtain relevant affinities and that the binding energy is broadly distributed along the interface, underlining a crucial role of a middle pocket to achieve tight binding. In contrast, targeting only the deep hydrophobic pocket is insufficient to achieve significant affinity. Moreover, calorimetry experiments in combination with limited proteolysis performed using a mutant with occluded binding in the N-terminal pocket reveal the existence of an allosteric communication between the different regions. This study is the first detailed thermodynamic dissection of the NHR-CHR interaction in gp41 and contributes therefore to a better understanding of HIV fusion. These results are relevant for the development of potential fusion inhibitors.


Assuntos
Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/química , Fragmentos de Peptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Calorimetria , Proteína gp41 do Envelope de HIV/química , Fragmentos de Peptídeos/química , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Termodinâmica
8.
Nucleic Acids Res ; 48(11): 6053-6067, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374866

RESUMO

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.


Assuntos
Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Fluorescência , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/química , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Resposta SOS em Genética
9.
Science ; 368(6492)2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32409444

RESUMO

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo-designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.


Assuntos
Anticorpos Neutralizantes/biossíntese , Biologia Computacional/métodos , Epitopos Imunodominantes/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Vacinas contra Vírus Sincicial Respiratório/química , Vírus Sincicial Respiratório Humano/imunologia , Motivos de Aminoácidos , Humanos , Epitopos Imunodominantes/imunologia , Conformação Proteica , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia
10.
Mol Immunol ; 123: 88-96, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32447084

RESUMO

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.


Assuntos
Bacillus subtilis/metabolismo , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Vacinas Bacterianas/uso terapêutico , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/imunologia , Animais , Bacillus subtilis/genética , Toxinas Bacterianas/metabolismo , Vacinas Bacterianas/química , Vacinas Bacterianas/genética , Infecções por Clostridium/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Vacinação/métodos , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/uso terapêutico
11.
PLoS One ; 15(5): e0226472, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379828

RESUMO

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.


Assuntos
Proteínas de Bactérias/metabolismo , Centrômero/química , Centrômero/metabolismo , DNA Bacteriano/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Simportadores/genética , Simportadores/metabolismo
12.
Parasitol Res ; 119(6): 1753-1765, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32363442

RESUMO

RbAp46/RBBP7 and RbAp48/RBBP4 are WD40-repeat histone chaperones and chromatin adaptors that reside in multiple complexes involved in maintenance of chromatin structure. RbAp48 is the essential subunit of the chromatin assembly factor-1 (CAF-1) complex, therefore also named as CAF-1C. A detailed in silico sequence and structure analysis of homologs of RbAp46/48 in Plasmodium falciparum (PF3D7_0110700 and PF3D7_1433300) exhibited conservation of characteristic features in both the protein-seven-bladed WD40 ß-propeller conformation and different binding interfaces. A comparative structural analysis highlighted species-specific features of the parasite, yeast, drosophila, and human RbAp46/48. In the present study, we report cloning, expression, and characterization of P. falciparum PF3D7_0110700, a putative RbAp46/48 (PfRbAp46/48). PfRbAp46/48 was cloned into pTEM11 vector in fusion with 6xHistidine tag and over-expressed in Escherichia coli B834 cells. The protein was purified by Ni-NTA followed by gel permeation chromatography. The protein expressed in all the three asexual blood stages and exhibited nuclear localization. We showed direct interaction of the purified rPfRbAp46/48 with the histone H4. These findings further our understanding of RbAp46/48 proteins and role of these proteins in the parasite biology.


Assuntos
Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Expressão Gênica , Chaperonas de Histonas/genética , Histonas/metabolismo , Estágios do Ciclo de Vida/genética , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
13.
Nat Commun ; 11(1): 2070, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: covidwho-116533

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização , Peptidil Dipeptidase A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Betacoronavirus/metabolismo , Ligação Competitiva/efeitos dos fármacos , Reações Cruzadas , Desenho de Fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/farmacocinética , Peptidil Dipeptidase A/farmacologia , Domínios Proteicos/genética , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
14.
Nat Commun ; 11(1): 2070, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332765

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização , Peptidil Dipeptidase A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Betacoronavirus/metabolismo , Ligação Competitiva/efeitos dos fármacos , Reações Cruzadas , Desenho de Fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/farmacocinética , Peptidil Dipeptidase A/farmacologia , Domínios Proteicos/genética , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
15.
Biotechnol Bioeng ; 117(7): 1990-2007, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32297972

RESUMO

High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the "self" RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation "universal" influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/química , Infecções por Coronavirus/diagnóstico , Hibridomas/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Chaperonas Moleculares , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Antígenos Virais/imunologia , Infecções por Coronavirus/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Vacinas contra Influenza , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Testes Sorológicos , Solubilidade
16.
Nat Commun ; 11(1): 1529, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251274

RESUMO

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.


Assuntos
Biotecnologia/métodos , Biblioteca Gênica , Inteínas/genética , Engenharia de Proteínas/métodos , Processamento de Proteína , Clonagem Molecular , Estudos de Viabilidade , Fluorescência , Genes Reporter/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Peptídeos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/genética , Transformação Bacteriana
17.
Viruses ; 12(3)2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32204464

RESUMO

Cats are becoming more popular as household companions and pets, forming close relationships with humans. Although feline viral diseases can pose serious health hazards to pet cats, commercialized preventative vaccines are lacking. Interferons (IFNs), especially type I IFNs (IFN-α, IFN-ß, and interferon omega (IFN-ω)), have been explored as effective therapeutic drugs against viral diseases in cats. Nevertheless, there is limited knowledge regarding feline IFN-ω (feIFN-ω), compared to IFN-α and IFN-ß. In this study, we cloned the genes encoding feIFN-ωa and feIFN-ωb from cat spleen lymphocytes. Homology and phylogenetic tree analysis revealed that these two genes belonged to new subtypes of feIFN-ω. The recombinant feIFN-ωa and feIFN-ωb proteins were expressed in their soluble forms in Escherichia coli, followed by purification. Both proteins exhibited effective anti-vesicular stomatitis virus (VSV) activity in Vero, F81 (feline kidney cell), Madin-Darby bovine kidney (MDBK), Madin-Darby canine kidney (MDCK), and porcine kidney (PK-15) cells, showing broader cross-species antiviral activity than the INTERCAT IFN antiviral drug. Furthermore, the recombinant feIFN-ωa and feIFN-ωb proteins demonstrated antiviral activity against VSV, feline coronavirus (FCoV), canine parvovirus (CPV), bovine viral diarrhea virus (BVDV), and porcine epidemic diarrhea virus (PEDV), indicating better broad-spectrum antiviral activity than the INTERCAT IFN. The two novel feIFN-ω proteins (feIFN-ωa and feIFN-ωb) described in this study show promising potential to serve as effective therapeutic agents for treating viral infections in pet cats.


Assuntos
Antivirais/farmacologia , Interferon Tipo I/genética , Interferon Tipo I/farmacologia , Sequência de Aminoácidos , Animais , Antivirais/isolamento & purificação , Sequência de Bases , Gatos , Bovinos , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Cães , Escherichia coli/genética , Escherichia coli/metabolismo , Interferon Tipo I/química , Interferon Tipo I/metabolismo , Filogenia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Suínos , Células Vero , Vírus/classificação , Vírus/efeitos dos fármacos
18.
PLoS One ; 15(3): e0230344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214330

RESUMO

In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01- Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.


Assuntos
Técnicas Biossensoriais/instrumentação , Medições Luminescentes/instrumentação , Proteínas Recombinantes de Fusão/química , Fator A de Crescimento do Endotélio Vascular/análise , Animais , Córnea/patologia , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/patologia , Transferência de Energia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Luciferases de Renilla/química , Luciferases de Renilla/genética , Degeneração Macular/diagnóstico , Degeneração Macular/patologia , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retina/patologia , Transfecção , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
19.
Nat Commun ; 11(1): 1542, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210238

RESUMO

Natural photosynthesis can be divided between the chlorophyll-containing plants, algae and cyanobacteria that make up the oxygenic phototrophs and a diversity of bacteriochlorophyll-containing bacteria that make up the anoxygenic phototrophs. Photosynthetic light harvesting and reaction centre proteins from both kingdoms have been exploited for solar energy conversion, solar fuel synthesis and sensing technologies, but the energy harvesting abilities of these devices are limited by each protein's individual palette of pigments. In this work we demonstrate a range of genetically-encoded, self-assembling photosystems in which recombinant plant light harvesting complexes are covalently locked with reaction centres from a purple photosynthetic bacterium, producing macromolecular chimeras that display mechanisms of polychromatic solar energy harvesting and conversion. Our findings illustrate the power of a synthetic biology approach in which bottom-up construction of photosystems using naturally diverse but mechanistically complementary components can be achieved in a predictable fashion through the encoding of adaptable, plug-and-play covalent interfaces.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Bactérias/química , Bacterioclorofilas/química , Complexos de Proteínas Captadores de Luz/química , Energia Solar , Biologia Sintética/métodos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Bacterioclorofilas/genética , Bacterioclorofilas/efeitos da radiação , Carotenoides/química , Carotenoides/efeitos da radiação , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/efeitos da radiação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/efeitos da radiação , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/efeitos da radiação , Luz Solar
20.
J Chromatogr A ; 1620: 461003, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32156458

RESUMO

The enormous growth in drug discovery paradigm has necessitated continuous exploration of new methods for drug-protein interaction analysis. To enhance the role of these methodologies in designing rational drugs, this work extended an immobilized angiotensin II type I receptor (AT1R) based affinity chromatography in antihypertensive compound identification. We fused haloalkane dehalogenase at C-terminus of AT1R and expressed the fusion receptor in E. coli. The expressed receptor was covalently immobilized onto 8.0 µm microspheres by mixing the cell lysate with 6-chlorocaproic acid-modified amino polystyrene microspheres. The immobilized AT1R was utilized for thermodynamic and kinetic interaction analysis between the receptor and four specific ligands. Following confirmation of these interactions by molecular docking, we identified puerarin and rosmarinic acid by determining their binding to the receptor. Azilsartan, candesartan, valsartan and olmesartan displayed two kinds of binding sites to AT1R by injection amount-dependent method. By molecular docking, we recognize the driving forces of the interaction as electrostatic interaction, hydrogen bonds and van der Waals force. The dissociation rate constants (kd) of azilsartan, candesartan, valsartan and olmesartan to AT1R were 0.01138 ± 0.003, 0.05142 ± 0.003, 0.07547 ± 0.004 and 0.01310 ± 0.005 min-1 by peak profiling assay. Comparing with these parameters, puerarin and rosmarinic acid presented lower affinity (KA: 0.12 × 104 and 1.5 × 104/M) and slower kinetics (kd: 0.6864 ± 0.03 and 0.3005 ± 0.01 min-1) to the receptor. These results, taking together, indicated that the immobilized AT1R has the capacity to probe antihypertensive compounds.


Assuntos
Anti-Hipertensivos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Receptor Tipo 1 de Angiotensina/metabolismo , Anti-Hipertensivos/química , Benzimidazóis/química , Benzimidazóis/metabolismo , Sítios de Ligação , Cromatografia de Afinidade , Cinamatos/metabolismo , Depsídeos/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Isoflavonas/metabolismo , Cinética , Ligantes , Simulação de Acoplamento Molecular , Oxidiazóis/química , Oxidiazóis/metabolismo , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Tetrazóis/química , Tetrazóis/metabolismo , Termodinâmica , Valsartana/química , Valsartana/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA