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1.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366154

RESUMO

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.


Assuntos
Grafite/química , Proteínas de Fluorescência Verde/química , Hidrogéis/química , Proteínas Imobilizadas/química , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Alginatos/química , Técnicas Biossensoriais , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metacrilatos/química , Nanopartículas/química , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Processos Fotoquímicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sefarose/química
2.
Appl Microbiol Biotechnol ; 103(11): 4443-4453, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989251

RESUMO

The availability of preimmune libraries of antibody fragments allows for the fast generation of binders which can be expressed in both eukaryotic and prokaryotic systems. We exploited the recombinant nature of antibody fragments to demonstrate the possibility of expressing them as functional proteins displayed on the surface of Escherichia coli and by such a way to generate living reagents ready-to-use for diagnostics. Such immunoreagents were effectively exploited without the necessity of any purification step to prepare immunocapture surfaces suitable for the diagnostic of both cancer cells and toxic microalgae. The same nanobody-displaying bacteria were also engineered to coexpress GFP in their cytoplasm. Suspensions of such living fluorescent immunoreagents effectively bound to eukaryotic cells making them visible and quantifiable by flow cytometry analysis and using 96-well plate readers. The collected data showed the suitability of such living immunoreagents for reproducible and inexpensive diagnostic applications.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Técnicas Citológicas/métodos , Escherichia coli/metabolismo , Proteínas Imobilizadas/metabolismo , Fatores Imunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/metabolismo , Aderência Bacteriana , Escherichia coli/genética , Proteínas Imobilizadas/genética , Imunoensaio/métodos , Fatores Imunológicos/genética , Proteínas Recombinantes/genética , Anticorpos de Domínio Único/genética , Coloração e Rotulagem/métodos
3.
Int J Biol Macromol ; 133: 614-623, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31002900

RESUMO

Microbial cell surface display technology is a powerful tool for displaying proteins on the surfaces of cells. However, few anchoring proteins can be employed for the display of target proteins on the cell surface of the environmentally benign Gram-positive bacterium Bacillus subtilis. In this study, bioinformatics tools were used to screen all of the encoded proteins of B. subtilis for potential anchoring proteins. A green fluorescent protein (eGFP) reporter system was constructed to evaluate the cell-display efficiency of the selected membrane proteins and their promoters. The anchoring protein SpoIIIJ demonstrated the strongest anchoring activity of all of the selected anchoring proteins from Bacillus spp. cells. A linker was designed to link the anchoring protein SpoIIIJ and eGFP, which had the ability to increase the expression of the fusion protein by 58.32%. Two bio-remediated related proteins (the organophosphorus hydrolase OPHC2 and the metal binding protein CadR) were successfully expressed on the cell surfaces of Bacillus spp. using this system. Therefore, our results suggest that this microbial surface display system may be useful for the expression of target proteins on the cell surfaces and has potential applications in the bioremediation of environmental pollution.


Assuntos
Bacillus/genética , Proteínas de Bactérias/genética , Técnicas de Visualização da Superfície Celular/métodos , Arildialquilfosfatase/genética , Biodegradação Ambiental , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética
4.
Protein Expr Purif ; 153: 131-137, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30240632

RESUMO

This work describes a novel strategy for the integrated expression and purification of recombinant proteins in Pichia pastoris cultures. Hydrophobins can be used as fusion tags, proteins fused to them alter their hydrophobicity and can be purified by aqueous two-phase systems (ATPS) based on non-ionic surfactants. Here, the consensus dengue virus envelope protein domain III fused to hydrophobin I of Trichoderma reesei was expressed in Pichia pastoris cultures and an in situ product removal by an ATPS using a non-ionic detergent, (Triton X-114) was performed. The protein was produced and purified directly from the yeast culture supernatant both efficiently and with no loss. The purified protein was properly immobilized by adsorption in solid phase and recognized by anti-dengue antibodies, showing its potential for the development of an indirect immunoassay for dengue virus.


Assuntos
Clonagem Molecular/métodos , Vírus da Dengue/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Imobilizadas/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas do Envelope Viral/isolamento & purificação , Sequência de Aminoácidos , Sequência Consenso , Meios de Cultura/química , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Pichia/genética , Pichia/metabolismo , Polietilenoglicóis/química , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Microextração em Fase Sólida/métodos , Trichoderma/genética , Trichoderma/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
5.
Langmuir ; 35(5): 1266-1272, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29801414

RESUMO

Development of antifouling films which selectively capture or target proteins of interest is essential for controlling interactions at the "bio/nano" interface. However, in order to synthesize biofunctional films from synthetic polymers that incorporate chemical "motifs" for surface immobilization, antifouling, and oriented biomolecule attachment, multiple reaction steps need to be carried out at the solid/liquid interface. EKx is a zwitterionic peptide that has previously been shown to have excellent antifouling properties. In this study, we recombinantly expressed EKx peptides and genetically encoded both surface attachment and antibody-binding motifs, before characterizing the resultant biopolymers by traditional methods. These peptides were then immobilized to organosilica nanoparticles for binding IgG, and subsequently capturing dengue NS1 as a model antigen from serum-containing solution. We found that a mixed layer of a short peptide (4.9 kDa) "backfilled" with a longer peptide terminated with an IgG-binding Z-domain (18 kDa) demonstrated selective capture of dengue NS1 protein down to ∼10 ng mL-1 in either PBS or 20% serum.


Assuntos
Incrustação Biológica/prevenção & controle , Imunoglobulina G/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Vírus da Dengue/química , Escherichia coli/genética , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Imunoglobulina G/química , Nanopartículas/química , Peptídeos/genética , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Dióxido de Silício/química , Proteínas não Estruturais Virais/metabolismo
6.
J Phys Chem B ; 122(35): 8330-8342, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30109934

RESUMO

Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification. The Cytc variants were thoroughly characterized with respect to possible structural perturbations due to labeling. For the proteins in solution, the relative hydrogen bond occupancy and the calculated electric fields, both obtained from molecular dynamics (MD) simulations, and the experimental nitrile stretching frequencies were used to develop a relationship for separating hydrogen-bonding and non-hydrogen-bonding electric field effects. This relationship provides an excellent description for the stable Cytc variants in solution. For the proteins bound to Au electrodes coated with charged self-assembled monolayers (SAMs), the underlying MD simulations can only account for the electric field changes Δ Eads due to the formation of the electrostatic SAM-Cytc complexes but not for the additional contribution, Δ Eint, representing the consequences of the potential drops over the electrode/SAM/protein interfaces. Both Δ Eads and Δ Eint, determined at distances between 20 and 30 Å with respect to the SAM surface, are comparable in magnitude to the non-hydrogen-bonding electric field in the unbound protein. This long-range modulation of the internal electric field may be of functional relevance for proteins in complexes with partner proteins (Δ Eads) and attached to membranes (Δ Eads + Δ Eint).


Assuntos
Citocromos c/química , Campos Eletromagnéticos , Animais , Citocromos c/genética , Técnicas Eletroquímicas , Eletrodos , Ouro/química , Cavalos , Ligação de Hidrogênio , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Simulação de Dinâmica Molecular , Mutação , Nitrilos/química , Eletricidade Estática
7.
PLoS One ; 13(5): e0198375, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29851992

RESUMO

The conformational conversion of pentameric C-reactive protein (pCRP) to monomeric CRP (mCRP) has been shown to play important roles in the action of CRP in inflammation regulation. In vivo studies revealed the origin of mCRP and provided insights into how pCRP dissociation affected its functions. However, the interplay and exact bioactivities of CRP isoforms still remain uncertain due to the rapid conformational conversion and complex milieu in vivo. Herein, we have used surface-immobilization of pCRP to generate a preservable intermediate with dual antigenicity expression of both pCRP and mCRP. The intermediate has been further shown to exhibit modified bioactivities, such as a high affinity with solution-phase pCRP and an enhanced capacity of complement interaction. These results thus not only provide the conformational conversion details of CRP, but also propose a simple way in vitro to study how the functions of CRP are tuned by distinct isoforms.


Assuntos
Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Proteína C-Reativa/genética , Proteína C-Reativa/imunologia , Regulação da Expressão Gênica , Humanos , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/imunologia , Inflamação/metabolismo , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína
8.
Bioconjug Chem ; 29(5): 1756-1762, 2018 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-29648798

RESUMO

Thermoresponsive magnetic nanoparticles (MNPs) were synthesized using a magnetosome display system. An elastin-like polypeptide decamer of VPGVG (ELP10), which is hydrophobic above the transition temperature ( Tt) and can form an insoluble aggregation, was immobilized on biogenic MNPs in the magnetotactic bacterium, Magnetospirillum magneticum AMB-1. It was suggested that hydrophobicity of the MNP surface increased at 60 °C compared with 20 °C by the immobilization of ELP10. Size distribution analysis indicated that the immobilization of ELP10 onto MNPs induced the increased hydrophobicity with increasing temperatures up to 60 °C, promoting aggregation of the particles by hydrophobic and magnetic interactions. These results suggest that the acceleration of magnetic collection at 60 °C was caused by particle aggregation promoted by hydrophobic interaction between ELP-MNPs. Furthermore, the immobilization of ELP on MNPs gave a quick magnetic collection at 60 °C by external magnetic field. The thermoresponsive properties will further expand the utility of biotechnological applications of biogenic MNPs.


Assuntos
Elastina/química , Nanopartículas de Magnetita/química , Magnetossomos/química , Magnetospirillum/química , Peptídeos/química , Elastina/genética , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Campos Magnéticos , Magnetossomos/genética , Magnetossomos/metabolismo , Magnetospirillum/genética , Magnetospirillum/metabolismo , Peptídeos/genética , Temperatura , Transformação Genética , Temperatura de Transição
9.
Biotechnol J ; 13(7): e1700739, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29485238

RESUMO

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization).


Assuntos
Extratos Celulares/química , Proteínas Imobilizadas/química , Proteínas Recombinantes/química , Azidas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Biochim Biophys Acta Proteins Proteom ; 1866(1): 52-59, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28870733

RESUMO

Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.


Assuntos
Ácido 3-Hidroxibutírico/química , Bacillus megaterium/genética , Biotecnologia/métodos , Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Proteínas Imobilizadas/biossíntese , Proteínas Mitocondriais/biossíntese , Ácido 3-Hidroxibutírico/biossíntese , Animais , Bacillus megaterium/enzimologia , Biocatálise , Bovinos , Colesterol/química , Colesterol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Grânulos Citoplasmáticos/química , Liofilização , Expressão Gênica , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Pregnenolona/biossíntese , Pregnenolona/química , Refrigeração , Transgenes
11.
J Biol Chem ; 292(35): 14617-14624, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28710276

RESUMO

Using the energy of ATP hydrolysis, ABC transporters catalyze the trans-membrane transport of molecules. In bacteria, these transporters partner with a high-affinity substrate-binding protein (SBP) to import essential micronutrients. ATP binding by Type I ABC transporters (importers of amino acids, sugars, peptides, and small ions) stabilizes the interaction between the transporter and the SBP, thus allowing transfer of the substrate from the latter to the former. In Type II ABC transporters (importers of trace elements, e.g. vitamin B12, heme, and iron-siderophores) the role of ATP remains debatable. Here we studied the interaction between the Yersinia pestis ABC heme importer (HmuUV) and its partner substrate-binding protein (HmuT). Using real-time surface plasmon resonance experiments and interaction studies in membrane vesicles, we find that in the absence of ATP the transporter and the SBP tightly bind. Substrate in excess inhibits this interaction, and ATP binding by the transporter completely abolishes it. To release the stable docked SBP from the transporter hydrolysis of ATP is required. Based on these results we propose a mechanism for heme acquisition by HmuUV-T where the substrate-loaded SBP docks to the nucleotide-free outward-facing conformation of the transporter. ATP binding leads to formation of an occluded state with the substrate trapped in the trans-membrane translocation cavity. Subsequent ATP hydrolysis leads to substrate delivery to the cytoplasm, release of the SBP, and resetting of the system. We propose that other Type II ABC transporters likely share the fundamentals of this mechanism.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Modelos Moleculares , Yersinia pestis/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/química , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Dimerização , Heme/química , Proteínas Ligantes de Grupo Heme , Hemeproteínas/química , Hemeproteínas/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Hidrólise , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície
12.
J Biol Chem ; 292(32): 13381-13390, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28655759

RESUMO

In eukaryotes, precursor mRNA (pre-mRNA) splicing removes non-coding intron sequences to produce mature mRNA. This removal is controlled in part by RNA-binding proteins that regulate alternative splicing decisions through interactions with the splicing machinery. RNA binding motif protein 25 (RBM25) is a putative splicing factor strongly conserved across eukaryotic lineages. However, the role of RBM25 in global splicing regulation and its cellular functions are unknown. Here we show that RBM25 is required for the viability of multiple human cell lines, suggesting that it could play a key role in pre-mRNA splicing. Indeed, transcriptome-wide analysis of splicing events demonstrated that RBM25 regulates a large fraction of alternatively spliced exons throughout the human genome. Moreover, proteomic analysis indicated that RBM25 interacts with components of the early spliceosome and regulators of alternative splicing. Previously, we identified an RBM25 species that is mono-methylated at lysine 77 (RBM25K77me1), and here we used quantitative mass spectrometry to show that RBM25K77me1 is abundant in multiple human cell lines. We also identified a region of RBM25 spanning Lys-77 that binds with high affinity to serine- and arginine-rich splicing factor 2 (SRSF2), a crucial protein in exon definition, but only when Lys-77 is unmethylated. Together, our findings uncover a pivotal role for RBM25 as an essential regulator of alternative splicing and reveal a new potential mechanism for regulation of pre-mRNA splicing by lysine methylation of a splicing factor.


Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Spliceossomos/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Éxons , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Imunoprecipitação , Lisina/metabolismo , Metilação , Domínios e Motivos de Interação entre Proteínas , Proteômica/métodos , Precursores de RNA/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Processamento de Serina-Arginina/química , Fatores de Processamento de Serina-Arginina/genética
13.
J Biol Chem ; 292(32): 13402-13414, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28652405

RESUMO

Dectin-2, a C-type lectin on macrophages and other cells of the innate immune system, functions in response to pathogens, particularly fungi. The carbohydrate-recognition domain (CRD) in dectin-2 is linked to a transmembrane sequence that interacts with the common Fc receptor γ subunit to initiate immune signaling. The molecular mechanism by which dectin-2 selectively binds to pathogens has been investigated by characterizing the CRD expressed in a bacterial system. Competition binding studies indicated that the CRD binds to monosaccharides with modest affinity and that affinity was greatly enhanced for mannose-linked α1-2 or α1-4 to a second mannose residue. Glycan array analysis confirmed selective binding of the CRD to glycans that contain Manα1-2Man epitopes. Crystals of the CRD in complex with a mammalian-type high-mannose Man9GlcNAc2 oligosaccharide exhibited interaction with Manα1-2Man on two different termini of the glycan, with the reducing-end mannose residue ligated to Ca2+ in a primary binding site and the nonreducing terminal mannose residue occupying an adjacent secondary site. Comparison of the binding sites in DC-SIGN and langerin, two other pathogen-binding receptors of the innate immune system, revealed why these two binding sites accommodate only terminal Manα1-2Man structures, whereas dectin-2 can bind Manα1-2Man in internal positions in mannans and other polysaccharides. The specificity and geometry of the dectin-2-binding site provide the molecular mechanism for binding of dectin-2 to fungal mannans and also to bacterial lipopolysaccharides, capsular polysaccharides, and lipoarabinomannans that contain the Manα1-2Man disaccharide unit.


Assuntos
Dissacarídeos/metabolismo , Imunidade Inata , Lectinas Tipo C/metabolismo , Manose/metabolismo , Modelos Moleculares , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Sítios de Ligação , Configuração de Carboidratos , Cristalografia por Raios X , Dissacarídeos/química , Epitopos/química , Epitopos/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Corpos de Inclusão/metabolismo , Cinética , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ligantes , Manose/química , Oligossacarídeos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Filogenia , Polissacarídeos/química , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
14.
J Biol Chem ; 292(32): 13345-13360, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28637873

RESUMO

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of ES PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on ES Conversely, PspCN boosted the CA, on ES, of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Ativação do Complemento , Degeneração Macular/genética , Mutação , Substituição de Aminoácidos , Animais , Síndrome Hemolítico-Urêmica Atípica/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , C3 Convertase da Via Alternativa do Complemento/química , C3 Convertase da Via Alternativa do Complemento/genética , C3 Convertase da Via Alternativa do Complemento/metabolismo , Complemento C3d/química , Complemento C3d/genética , Complemento C3d/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Fator I do Complemento/química , Fator I do Complemento/genética , Fator I do Complemento/metabolismo , Eritrócitos/química , Hemólise , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Degeneração Macular/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Carneiro Doméstico , Solubilidade , Streptococcus pneumoniae/metabolismo , Propriedades de Superfície
15.
Methods Mol Biol ; 1586: 337-344, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28470616

RESUMO

Site-specific biotinylation of proteins is often the method of choice to enable efficient immobilization of a protein on a surface without interfering with protein folding. The tight interaction of biotin and streptavidin is frequently used to immobilize an antigen during phage display selections of binders. Here we describe a method of in vivo biotinylation of proteins during expression in E. coli, by tagging the protein with the short biotin acceptor peptide sequence, Avi tag, and co-expression of the E. coli biotin ligase (BirA) resulting in precise biotinylation of a specific lysine residue in the tag.


Assuntos
Antígenos/química , Antígenos/genética , Escherichia coli/genética , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Animais , Biotina/química , Biotinilação , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/genética , Clonagem Molecular/métodos , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Humanos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Estreptavidina/química
16.
J Biotechnol ; 252: 55-64, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28506931

RESUMO

The mismatch binding protein MutS is responsible for the recognition of mispaired and unpaired bases, which is the initial step in DNA repair. Among the MutS proteins most extensively studied in vitro are those derived from Thermus thermophilus, Thermus aquaticus and Escherichia coli. Here, we present the first report on the in vitro examination of DNA mismatch binding activity of MutS protein from Deinococcus radiodurans and confront this with the properties of those from E. coli and T. thermophilus. The analyses which included mobility gel-shift assay, colorimetric and qPCR estimation of MutS-bound DNA clearly showed that D. radiodurans MutS exhibited much higher affinity towards mismatched DNA in vitro than its counterparts from E. coli and T. thermophilus. In addition, D. radiodurans MutS displayed a significantly higher specificity of DNA mismatch binding than the two other orthologues. The specificity expressed as the ratio of mismatched to fully complementary DNA bound reached over 4 and 20-fold higher values for D. radiodurans than for T. thermophilus and E. coli MutS, respectively. The results demonstrate mainly the biotechnological potential of D. radiodurans MutS but the in vitro characteristics of the MutS orthologues could reflect substantial differences in DNA mismatch binding activities existing in vivo.


Assuntos
DNA Bacteriano/metabolismo , Deinococcus/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Pareamento Incorreto de Bases , DNA Bacteriano/genética , Escherichia coli/genética , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Ligação Proteica , Thermus thermophilus/genética
17.
Proc Natl Acad Sci U S A ; 114(9): 2319-2324, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28193885

RESUMO

Chemokines control the migration of a large array of cells by binding to specific receptors on cell surfaces. The biological function of chemokines also depends on interactions between nonreceptor binding domains and proteoglycans, which mediate chemokine immobilization on cellular or extracellular surfaces and formation of fixed gradients. Chemokine gradients regulate synchronous cell motility and integrin-dependent cell adhesion. Of the various chemokines, CXCL12 has a unique structure because its receptor-binding domain is distinct and does not overlap with the immobilization domains. Although CXCL12 is known to be essential for the germinal center (GC) response, the role of its immobilization in biological functions has never been addressed. In this work, we investigated the unexplored paradigm of CXCL12 immobilization during the germinal center reaction, a fundamental process where cellular traffic is crucial for the quality of humoral immune responses. We show that the structure of murine germinal centers and the localization of GC B cells are impaired when CXCL12 is unable to bind to cellular or extracellular surfaces. In such mice, B cells carry fewer somatic mutations in Ig genes and are impaired in affinity maturation. Therefore, immobilization of CXCL12 is necessary for proper trafficking of B cells during GC reaction and for optimal humoral immune responses.


Assuntos
Linfócitos B/imunologia , Quimiocina CXCL12/imunologia , Centro Germinativo/imunologia , Proteínas Imobilizadas/imunologia , Imunidade Humoral , Imunoglobulinas/genética , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos B/citologia , Movimento Celular , Quimiocina CXCL12/genética , Eritrócitos/química , Eritrócitos/imunologia , Expressão Gênica , Regulação da Expressão Gênica , Centro Germinativo/citologia , Proteínas Imobilizadas/genética , Imunização , Imunoglobulinas/metabolismo , Camundongos , Camundongos Transgênicos , Ovinos , Hipermutação Somática de Imunoglobulina
18.
Microbiol Res ; 196: 17-25, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28164787

RESUMO

Factor H (FH), a regulatory protein of the complement system, can bind specifically to factor H-binding proteins (FHBPs) of Streptococcus suis serotype 2 (SS2), which contribute to evasion of host innate immune defenses. In the present study, we aimed to identify novel FHBPs and characterize the biological functions of FH in SS2 pathogenesis. Here, a method that combined proteomics and Far-western blotting was developed to identify the surface FHBPs of SS2. With this method, fourteen potential novel FHBPs were identified among SS2 surface proteins. We selected eight newly identified proteins and further confirmed their binding activity to FH. The binding of SS2 to immobilized FH decreased dramatically after pre-incubation with anti-FHBPs polyclonal antibodies. We showed for the first time that SS2 also interact specifically with mouse FH. Furthermore, we found that FH play an important role in adherence and invasion of SS2 to HEp-2 cells. Additionally, using a mouse model of intraperitoneal challenge, we confirmed that SS2 pre-incubated with FH enhanced bacteremia and brain invasion, compared with SS2 not pretreated with FH. Taken together, this study provides a useful method to characterize the host-bacteria interactions. These results first indicated that binding of FH to the cell surface improved the adherence and invasion of SS2 to HEp-2 cells, promoting SS2 to resist killing and leading to enhance virulence.


Assuntos
Antígenos de Bactérias/imunologia , Fator H do Complemento/imunologia , Streptococcus suis/imunologia , Streptococcus suis/patogenicidade , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Aderência Bacteriana/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Fator H do Complemento/farmacologia , Escherichia coli/genética , Feminino , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/imunologia , Proteínas Imobilizadas/metabolismo , Evasão da Resposta Imune , Imunidade Inata , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Streptococcus suis/metabolismo , Virulência
19.
J Am Soc Mass Spectrom ; 28(3): 479-485, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27966173

RESUMO

To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts. Graphical Abstract ᅟ.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas/métodos , Receptor X Retinoide alfa/metabolismo , Amilose/química , Amilose/metabolismo , Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Ligantes , Magnetismo , Proteínas Ligantes de Maltose/metabolismo , Espectrometria de Massas/instrumentação , Microesferas , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Receptor X Retinoide alfa/genética , Software , Ultrafiltração
20.
Colloids Surf B Biointerfaces ; 149: 233-242, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27768913

RESUMO

The regeneration of bone via a tissue engineering approach involves components from the macroscopic to the nanoscopic level, including appropriate 3D scaffolds, cells and growth factors. In this study, hexagonal scaffolds of different diagonals were fabricated by Direct Laser Writing using a photopolymerizable hybrid material. The proliferation of bone marrow (BM) mesenchymal stem cells (MSCs) cultured on structures with various diagonals, 50, 100, 150 and 200µm increased significantly after 10days in culture, however without significant differences among them. Next, recombinant human bone morphogenetic protein 2 (rhBMP-2) was immobilized onto the hybrid material both via covalent binding and physical adsorption. Both immobilization types exhibited similar high releaseate bioactivity profiles and a sustained delivery of rhBMP-2. The collagen and calcium levels produced in the extracellular matrix (ECM) were significantly elevated for the samples functionalized with BMP-2 compared to those in the osteogenic medium. Furthermore, significant upregulation of gene expression in both types of BMP-2 immobilized scaffolds was observed for alkaline phosphatase (ALPL) and osteocalcin (BGLAP) at days 7, 14, and 21, for RUNX2 at day 21, and for osteonectin (SPARC) at days 7 and 14. The results suggest that the release of bioactive rhBMP-2 from the hybrid scaffolds enhance the control over the osteogenic differentiation during cell culture.


Assuntos
Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Tecidos Suporte , Adsorção , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno/genética , Colágeno/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacologia , Lasers , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteonectina/genética , Osteonectina/metabolismo , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Engenharia Tecidual
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