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1.
Cancer Sci ; 110(11): 3520-3532, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31505062

RESUMO

Cancer stem cells (CSC) are a subpopulation of tumor cells with properties of high tumorigenicity and drug resistance, which lead to recurrence and poor prognosis. Although a better understanding of CSC is essential for developing cancer therapies, scarcity of the CSC population has hindered such analyses. The aim of the present study was to elucidate whether the E-cadherin-Fc chimera protein (E-cad-Fc) enhances cancer stem-like properties because studies show that soluble E-cadherin stimulates human epithelial growth factor receptor (EGFR) and downstream signaling pathways that are reported to play a crucial role in CSC. For this purpose, we used ornithine decarboxylase (ODC)-degron-transduced (Degron(+)) KM12SM cells as a CSC model that retains relatively low CSC properties. Compared to cultures without E-cad-Fc treatment, we found that E-cad-Fc treatment further suppressed proteasome activity and largely enhanced cancer stem-like properties of ODC-degron-transduced KM12SM cells. These results include increased expression of stem cell markers Lgr5, Bmi-1, SOX9, CD44, and CD44v9, aldehyde dehydrogenase (ALDH), and enhancement of robust spheroid formation, and chemoresistance to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP). These effects could be attributed to activation of the EGFR pathway as identified by extensive phosphorylation of EGFR, ERK, PI3K, AKT, and mTOR. In SW480 cells, E-cad-Fc matrix induced some CSC markers such as CD44v9 and ALDH. We also found that E-cad-Fc matrix showed high efficiency of inducing mesenchymal changes in colon cancer cells. Our data suggest that the E-cad-Fc matrix may enhance CSC properties such as enhancement of chemoresistance and sphere formation.


Assuntos
Neoplasias do Colo/patologia , Receptores ErbB , Fragmentos Fc das Imunoglobulinas , Células-Tronco Neoplásicas/patologia , Proteínas Recombinantes de Fusão/metabolismo , Aldeído Desidrogenase/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Humanos , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Ornitina Descarboxilase , Oxaliplatina/farmacologia , Complexo Repressor Polycomb 1/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição SOX9/metabolismo , Esferoides Celulares
2.
Nucleic Acids Res ; 47(16): 8913-8925, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392336

RESUMO

The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.


Assuntos
DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Genes Dev ; 33(17-18): 1191-1207, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371435

RESUMO

The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.


Assuntos
Aminoácidos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Recombinases/química , Recombinases/metabolismo , Recombinação Genética/genética , Aminoácidos/genética , Animais , Pareamento Incorreto de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Mutação , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
5.
Nat Commun ; 10(1): 2947, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270320

RESUMO

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.


Assuntos
Epitopos/metabolismo , Sondas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula , Animais , Linhagem Celular Tumoral , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/metabolismo , Coloração e Rotulagem , Peixe-Zebra/embriologia
6.
Curr Microbiol ; 76(10): 1161-1167, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31278426

RESUMO

The haloalkane dehalogenase DhaA can degrade sulfur mustard (2,2'-dichlorethyl sulfide; also known by its military designation HD) in a rapid and environmentally safe manner. However, DhaA is sensitive to temperature and pH, which limits its applications in natural or harsh environments. Spore surface display technology using resistant spores as a carrier to ensure enzymatic activity can reduce production costs and extend the range of applications of DhaA. To this end, we cloned recombinant Bacillus subtilis spores pHY300PLK-cotg-dhaa-6his/DB104(FH01) for the delivery of DhaA from Rhodococcus rhodochrous NCIMB 13064. A dot blotting showed that the fusion protein CotG-linker-DhaA accounted for 0.41% ± 0.03% (P < 0.01) of total spore coat proteins. Immunofluorescence analyses confirmed that DhaA was displayed on the spore surface. The hydrolyzing activity of DhaA displayed on spores towards the HD analog 2-chloroethyl ethylsulfide was 1.74 ± 0.06 U/mL (P < 0.01), with a specific activity was 0.34 ± 0.04 U/mg (P < 0.01). This is the first demonstration that DhaA displayed on the surface of B. subtilis spores retains enzymatic activity, which suggests that it can be used effectively in real-world applications including bioremediation of contaminated environments.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Esporos Bacterianos/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Estabilidade Enzimática , Expressão Gênica , Hidrolases/genética , Gás de Mostarda/análogos & derivados , Gás de Mostarda/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rhodococcus/enzimologia , Rhodococcus/genética , Esporos Bacterianos/genética , Especificidade por Substrato
7.
Food Chem ; 299: 125037, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31279128

RESUMO

Immobilization of enzymes is an essential strategy with outstanding prospects in biocatalytic processes. Nontoxic, inexpensive immobilized enzyme approach is especially important for food enzymes. We here demonstrate that a carbohydrate-binding module family 56 domain (CBM56-Tag) mediates the immobilization of fusion enzymes with the curdlan (ß-1,3-glucan) particle support, thereby enabling the one-step immobilization-purification of target enzymes. CBM56-Tag exhibits an immunoglobulin-like ß-sandwich fold, which can be adsorbed by curdlan via hydrogen bond-mediated binding. The maximum adsorption capacity of a fusion chitosanase (CBM56-GsCsn46A) on curdlan is 50.72 mg/g. The immobilized enzyme could be directly used in the packed-bed reactor. This immobilization strategy utilizes a natural polysaccharide without any treatment, avoiding the negative environmental effects. Moreover, the one step immobilization-purification simplifies the purification step, which reduces the use of chemicals. Our study provides a nontoxic and inexpensive immobilization strategy for the biocatalytic reaction in food industry.


Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/isolamento & purificação , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biocatálise , Enzimas Imobilizadas/metabolismo , Glicosídeo Hidrolases/metabolismo , Ligações de Hidrogênio , beta-Glucanas/química
8.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 871-879, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223005

RESUMO

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.


Assuntos
Proteínas de Escherichia coli , Peptídeos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
9.
Nat Chem ; 11(7): 605-614, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209296

RESUMO

Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns in fractal-shaped branched objects, such as trees, lungs and sponges, results in a high surface area to volume ratio, which provides key functional advantages including molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies could provide access to functional biomaterials. Here we describe a computational design approach for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Guided by atomic-resolution models, we develop fusions of Src homology 2 (SH2) domain or a phosphorylatable SH2-binding peptide, respectively, to two symmetric, homo-oligomeric proteins. Mixing the two designed components resulted in a variety of dendritic, hyperbranched and sponge-like topologies that are phosphorylation-dependent and self-similar over three decades (~10 nm-10 µm) of length scale, in agreement with models from multiscale computational simulations. Designed assemblies perform efficient phosphorylation-dependent capture and release of cargo proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Fractais , Agregados Proteicos , Proteínas Recombinantes de Fusão/metabolismo , Algoritmos , Proteínas de Bactérias/genética , Escherichia coli/química , Humanos , Modelos Químicos , Modelos Moleculares , Fosforilação , Engenharia de Proteínas/métodos , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Domínios de Homologia de src/genética , Quinases da Família src/metabolismo
10.
Nat Neurosci ; 22(7): 1089-1098, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235908

RESUMO

Pericytes are positioned between brain capillary endothelial cells, astrocytes and neurons. They degenerate in multiple neurological disorders. However, their role in the pathogenesis of these disorders remains debatable. Here we generate an inducible pericyte-specific Cre line and cross pericyte-specific Cre mice with iDTR mice carrying Cre-dependent human diphtheria toxin receptor. After pericyte ablation with diphtheria toxin, mice showed acute blood-brain barrier breakdown, severe loss of blood flow, and a rapid neuron loss that was associated with loss of pericyte-derived pleiotrophin (PTN), a neurotrophic growth factor. Intracerebroventricular PTN infusions prevented neuron loss in pericyte-ablated mice despite persistent circulatory changes. Silencing of pericyte-derived Ptn rendered neurons vulnerable to ischemic and excitotoxic injury. Our data demonstrate a rapid neurodegeneration cascade that links pericyte loss to acute circulatory collapse and loss of PTN neurotrophic support. These findings may have implications for the pathogenesis and treatment of neurological disorders that are associated with pericyte loss and/or neurovascular dysfunction.


Assuntos
Proteínas de Transporte/fisiologia , Citocinas/fisiologia , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/patologia , Pericitos/fisiologia , Choque/fisiopatologia , Animais , Isquemia Encefálica/fisiopatologia , Capilares/fisiopatologia , Proteínas de Transporte/uso terapêutico , Células Cultivadas , Circulação Cerebrovascular/fisiologia , Citocinas/deficiência , Citocinas/uso terapêutico , Células Endoteliais/citologia , Feminino , Genes Reporter , Infusões Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/tratamento farmacológico , Neuroglia/metabolismo , Neurônios/metabolismo , Neurotoxinas/toxicidade , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Choque/metabolismo , Choque/patologia
11.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180049

RESUMO

We examined interactions between the 83 kDa heat-shock protein (Hsp83) and hsrω long noncoding RNAs (lncRNAs) in hsrω66 Hsp90GFP homozygotes, which almost completely lack hsrω lncRNAs but over-express Hsp83. All +/+; hsrω66 Hsp90GFP progeny died before the third instar. Rare Sp/CyO; hsrω66 Hsp90GFP reached the third instar stage but phenocopied l(2)gl mutants, becoming progressively bulbous and transparent with enlarged brain and died after prolonged larval life. Additionally, ventral ganglia too were elongated. However, hsrω66 Hsp90GFP/TM6B heterozygotes, carrying +/+ or Sp/CyO second chromosomes, developed normally. Total RNA sequencing (+/+, +/+; hsrω66/hsrω66, Sp/CyO; hsrω66/ hsrω66, +/+; Hsp90GFP/Hsp90GFP and Sp/CyO; hsrω66 Hsp90GFP/hsrω66 Hsp90GFP late third instar larvae) revealed similar effects on many genes in hsrω66 and Hsp90GFP homozygotes. Besides additive effect on many of them, numerous additional genes were affected in Sp/CyO; hsrω66 Hsp90GFP larvae, with l(2)gl and several genes regulating the central nervous system being highly down-regulated in surviving Sp/CyO; hsrω66 Hsp90GFP larvae, but not in hsrω66 or Hsp90GFP single mutants. Hsp83 and several omega speckle-associated hnRNPs were bioinformatically found to potentially bind with these gene promoters and transcripts. Since Hsp83 and hnRNPs are also known to interact, elevated Hsp83 in an altered background of hnRNP distribution and dynamics, due to near absence of hsrω lncRNAs and omega speckles, can severely perturb regulatory circuits with unexpected consequences, including down-regulation of tumoursuppressor genes such as l(2)gl.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico/genética , Larva/genética , RNA Longo não Codificante/genética , Mutações Sintéticas Letais , Animais , Animais Geneticamente Modificados , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Gânglios dos Invertebrados/crescimento & desenvolvimento , Gânglios dos Invertebrados/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Heterozigoto , Homozigoto , Larva/crescimento & desenvolvimento , Larva/metabolismo , Fenótipo , RNA Longo não Codificante/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcriptoma
12.
Artigo em Inglês | MEDLINE | ID: mdl-31176867

RESUMO

In rice field eel (Monopterus albus), germ cell development in the developing gonad has been revealed in detail. However, it is unclear how primordial germ cells (PGCs) migrate to the somatic part of the gonad (genital ridge). This study visualized PGC migration by injecting a chimeric mRNA containing a fluorescent protein fused to the 3' untranslated region (3'UTR) of three different genes, nanos3 of zebrafish (Danio rerio) and dead end (dnd) and vasa of rice field eel. The mRNAs were injected either alone or in pairs into embryos at the one-cell stage. The results showed that mRNAs containing nanos3 and dnd 3'UTRs labeled PGCs over a wider time frame than those containing vasa 3'UTR, suggesting that nanos3 and dnd 3'UTRs are suitable for visualizing PGCs in rice field eel. Using this direct visualization method, the normal migration route of PGCs was observed from the 50%-epiboly stage to hatching stage for the first time, and the ectopic PGCs were also visualized during this period in rice field eel. These findings extend our knowledge of germ cell development, and lay a foundation for further research on the relationship between PGCs and sex differentiation, and on incubation conditions for embryos in rice field eel.


Assuntos
Células Germinativas/metabolismo , Smegmamorpha/embriologia , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Células Germinativas/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Smegmamorpha/genética , Smegmamorpha/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
13.
Ecotoxicol Environ Saf ; 181: 301-307, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202929

RESUMO

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis and heavy metal detoxification. In our previous study, a novel full length MT cDNA was successfully cloned from the freshwater crab (Sinopotamon henanense). In the present study, tandem repeats of two and three copies of the crab MT gene were integrated by overlap extension PCR (SOE-PCR) and expressed in Escherichia coli. The SUMO fusion expression system was adopted to increase the stability and solubility of the recombinant MT proteins. The recombinant proteins were purified and their metal-binding abilities were further analyzed by the ultraviolet absorption spectral scan. Furthermore, the metal tolerance and bioaccumulation of E. coli cells expressing oligomeric MTs were determined. Results showed that the recombinant plasmids pET28a-SUMO-2MT and pET28a-SUMO-3MT were successfully constructed. SDS-PAGE analysis showed that the SUMO-2MT and SUMO-3MT were expressed mainly in the soluble forms. Oligomeric MTs expression significantly enhanced Cu, Cd or Zn tolerance and accumulation in E. coli in the order: SUMO-3MT˃SUMO-2MT˃SUMO-MT˃control. Cells harboring pET28a-SUMO -3MT exhibited the highest Cu, Cd or Zn bioaccumulation at 5.8-fold, 3.1-fold or 6.7-fold higher than that of the control cells. Our research could lay a foundation for large-scale preparation of MTs and provide a scientific basis for bioremediation of heavy metal pollution by oligomeric MTs.


Assuntos
Escherichia coli/genética , Metalotioneína/genética , Metais Pesados/metabolismo , Animais , Braquiúros/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Metalotioneína/metabolismo , Metais Pesados/toxicidade , Proteínas Recombinantes de Fusão/metabolismo
14.
PLoS Pathog ; 15(5): e1007759, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116791

RESUMO

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.


Assuntos
Hepacivirus/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Anticorpos Neutralizantes/metabolismo , Células HEK293 , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Conformação Proteica , Multimerização Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus
15.
Int J Nanomedicine ; 14: 3129-3143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118627

RESUMO

Background: Bacillus Calmette-Guérin, the attenuated strain of Mycobacterium bovis, remains the only available vaccine against tuberculosis (TB). However, its ineffectiveness in adults against pulmonary TB and varied protective efficacy (0-80%) speak to an urgent need for the development of an improved and efficient TB vaccine. In this milieu, poly(lactic-co-glycolic acid) (PLGA), is a preferential candidate, due to such properties as biocompatibility, targeted delivery, sustained antigen release, and atoxic by-products. Methods: In this study, we formulated PLGA nanoparticles (NPs) encapsulating the bivalent H1 antigen, a fusion of Mycobacterium tuberculosis (Mtb) Ag85B and ESAT6 proteins, and investigated its role in immunomodulation and protection against Mtb challenge. Using the classical water-oil-water solvent-evaporation method, H1-NPs were prepared, with encapsulation efficiency of 86.1%±3.2%. These spherical NPs were ~244.4±32.6 nm in diameter, with a negatively charged surface (ζ-potential -4±0.6 mV). Results: Under physiological conditions, NPs degraded slowly and the encapsulated H1 antigen was released over a period of weeks. As a proof-of-concept vaccine candidate, H1 NPs were efficiently internalized by the THP-1 human macrophages. Six weeks after a single-dose vaccination, H1 NP-immunized C57BL/6J mice showed significant increase in the production of total serum IgG (P<0.0001) and its isotypes compared to H1 alone, IgG2a being the predominant one, followed by IgG1. Further, the cytokine-release profile of antigen-stimulated splenocyteculture supernatant indicated a strong TH1-biased immunoresponse in H1 NP-vaccinated mice, with ~6.03- and ~2.8-fold increase in IFNγ and TNFα cytokine levels, and ~twofold and 1.6 fold increase in IL4 and IL10 cytokines, respectively, compared to H1 alone-immunized mice. In protection studies, H1 NP-vaccinated mice displayed significant reductions in lung and spleen bacillary load (P<0.05) at 5-week post-Mtb H37Rv challenge and prolonged survival, with a mean survival time of 177 days, compared to H1 alone-vaccinated mice (mean survival time 80 days). Conclusion: Altogether, our findings highlight the significance of the H1-PLGA nanoformulation in terms of providing long-term protection in mice with a single dose.


Assuntos
Imunidade , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas Recombinantes de Fusão/metabolismo , Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Citocinas/metabolismo , Relação Dose-Resposta Imunológica , Liberação Controlada de Fármacos , Endocitose , Epitopos , Feminino , Humanos , Imunidade Humoral , Imunização , Imunoglobulina G/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , Baço/citologia , Baço/imunologia , Análise de Sobrevida , Células THP-1 , Células Th1/imunologia , Vacinas contra a Tuberculose/imunologia , Vacinação
16.
Cell Mol Life Sci ; 76(20): 4165-4178, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31076805

RESUMO

Efficient cell-to-cell transfer of Listeria monocytogenes (L. monocytogenes) requires the proper formation of actin-rich membrane protrusions. To date, only the host proteins ezrin, the binding partner of ezrin, CD44, as well as cyclophilin A (CypA) have been identified as crucial components for L. monocytogenes membrane protrusion stabilization and, thus, efficient cell-to-cell movement of the microbes. Here, we examine the classical binding partner of CypA, CD147, and find that this membrane protein is also hijacked by the bacteria for their cellular dissemination. CD147 is enriched at the plasma membrane surrounding the membrane protrusions as well as the resulting invaginations generated in neighboring cells. In cells depleted of CD147, these actin-rich structures appear similar to those generated in CypA depleted cells as they are significantly shorter and more contorted as compared to their straighter counterparts formed in wild-type control cells. The presence of malformed membrane protrusions hampers the ability of L. monocytogenes to efficiently disseminate from CD147-depleted cells. Our findings uncover another important host protein needed for L. monocytogenes membrane protrusion formation and efficient microbial dissemination.


Assuntos
Basigina/genética , Membrana Celular/microbiologia , Interações Hospedeiro-Patógeno/genética , Listeria monocytogenes/fisiologia , Shigella flexneri/fisiologia , Células A549 , Actinas/genética , Actinas/metabolismo , Animais , Basigina/antagonistas & inibidores , Basigina/metabolismo , Células CACO-2 , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Ciclofilina A/deficiência , Ciclofilina A/genética , Endocitose , Fibroblastos/microbiologia , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/ultraestrutura , Camundongos , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Shigella flexneri/patogenicidade , Shigella flexneri/ultraestrutura , Transdução de Sinais
17.
J Ovarian Res ; 12(1): 45, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092272

RESUMO

BACKGROUND: While tumor suppressor p53 functions primarily as a transcription factor in the nucleus, cellular stress can cause p53 to translocate to the mitochondria and directly trigger a rapid apoptotic response. We have previously shown that fusing p53 (or its DNA binding domain, DBD, alone) to the mitochondrial targeting signal (MTS) from Bak or Bax can target p53 to the mitochondria and induce apoptosis in gynecological cancer cell lines including cervical cancer cells (HeLa; wt p53), ovarian cancer cells (SKOV-3; p53 267del non-expressing), and breast cancer cells (T47D; L194F p53 mutation). However, p53 with Bak or Bax MTSs have not been previously tested in cancers with strong dominant negative (DN) mutant p53 which are capable of inactivating wt p53 by homo-oligomerization. Since p53-Bak or Bax MTS constructs act as monomers, they are not subject to DN inhibition. For this study, the utility of p53-Bak or p53-Bax MTS constructs was tested for ovarian cancers which are known to have varying p53 statuses, including a strong DN contact mutant p53 (Ovcar-3 cells), a p53 DN structural mutant (Kuramochi cells), and a p53 wild type, low expressing cells (ID8). RESULTS: Our mitochondrial p53 constructs were tested for their ability to localize to the mitochondria in both mutant non-expressing p53 (Skov-3) and p53 structural mutant (Kuramochi) cell lines using fluorescence microscopy and a nuclear transcriptional activity assay. The apoptotic activity of these mitochondrial constructs was determined using a mitochondrial outer membrane depolarization assay (TMRE), caspase assay, and a late stage cell death assay (7-AAD). We also tested the possibility of using our constructs with paclitaxel, the current standard of care in ovarian cancer treatment. Our data indicates that our mitochondrial p53 constructs are able to effectively localize to the mitochondria in cancer cells with structural mutant p53 and induce apoptosis in many ovarian cancer cell lines with different p53 statuses. These constructs can also be used in combination with paclitaxel for an increased apoptotic effect. CONCLUSIONS: The results suggest that targeting p53 to mitochondria can be a new strategy for ovarian cancer treatment.


Assuntos
Mitocôndrias/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular , Linhagem Celular Tumoral , Feminino , Humanos , Mutação , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Domínios Proteicos , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
18.
Plant Sci ; 284: 117-126, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084864

RESUMO

Previously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression of CHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.


Assuntos
Antígenos de Protozoários/metabolismo , Proteínas de Choque Térmico/metabolismo , Leishmania infantum/metabolismo , Fotossíntese , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/metabolismo , Clorofila/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Temperatura Alta , Imunoprecipitação , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/parasitologia , Tabaco
19.
J Biotechnol ; 300: 32-39, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31085201

RESUMO

Epidermis-specific promoters are necessary for ectopic expression of specific functional genes such as the cuticle-related genes. Previous studies indicated that both ECERIFERUM 6 (AtCER6) and MERISTEM L1 LAYER (ATML1) promoters from Arabidopsis thaliana can drive gene expression specifically in the epidermis of shoot apical meristems (SAMs) and leaves. However, the epidermis-specific promoters from legume plants have not been reported. Here, we cloned a 5' flanking sequence from the upstream -2150 bp to the translational start ATG codon of MtML1 gene of legume model plant Medicago truncatula. PlantCARE analysis indicated that this sequence matches the characteristics of a promoter, having TATA box and CAAT box, as well as contains some conserved elements of epidermis-specific promoters like AtCER6 and ATML1 promoters. The ß-glucuronidase (GUS) histochemical analysis showed that MtML1 promoter can drive GUS gene expression in transiently transformed Nicotiana tabacum leaves under non-inducing condition. Furthermore, it can also control GUS expression in leaves and siliques rather than roots of the stably transformed Arabidopsis. More importantly, the leaf cross-section observations indicated that MtML1 exclusively expressed in the epidermis of leaves. These results suggested that MtML1 promoter performed the epidermis-specific in plant shoot. Our study establishes the foundation for driving the cuticle-related gene to express in epidermis, which may be very useful in genetic engineering of legume plants.


Assuntos
Medicago truncatula/genética , Epiderme Vegetal/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Clonagem Molecular , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Medicago truncatula/metabolismo , Especificidade de Órgãos , Folhas de Planta/metabolismo , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tabaco/genética , Tabaco/metabolismo
20.
Talanta ; 201: 397-405, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122440

RESUMO

This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL-1, with a limit of detection (LOD) of 20 and 2 ng mL-1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based.


Assuntos
Ração Animal/análise , Contaminação de Alimentos/análise , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Zearalenona/análise , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Humanos , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Triticum/química , Zea mays/química , Zearalenona/imunologia , Zearalenona/metabolismo
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