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1.
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
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.
Cancer Immunol Immunother ; 68(8): 1379-1389, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31338557

RESUMO

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide and epidermal growth factor receptor (EGFR) is overexpressed in greater than 90% of patient tumors. Cetuximab is a monoclonal antibody that binds to EGFR and can activate immune cells, such as natural killer (NK) cells, that express receptors for the Fc (constant region) of immunoglobulin G. IL-15 (interleukin-15) is a critical factor for the development, proliferation and activation of effector NK cells. A novel IL-15 compound known as ALT-803 that consists of genetically modified IL-15 plus the IL-15 receptor alpha protein (IL15Rα) fused to the Fc portion of IgG1 has recently been developed. We hypothesized that treatment with ALT-803 would increase NK cell-mediated cytotoxicity of cetuximab-coated head and neck squamous cells. CD56+ NK cells from normal healthy donors were treated overnight with ALT-803 and tested for their ability to lyse cetuximab-coated tumor cells. Cytotoxicity was greater following NK cell ALT-803 activation, as compared to controls. ALT-803-treated NK cells secreted significantly higher levels of IFN-γ than control conditions. Additionally, NK cells showed increased levels of phospho-ERK and phospho-STAT5 when co-cultured with cetuximab-coated tumors and ALT-803. Administration of both cetuximab and ALT-803 to mice harboring Cal27 SCCHN tumors resulted in significantly decreased tumor volume when compared to controls and compared to single-agent treatment alone. Overall, the present data suggest that cetuximab treatment in combination with ALT-803 in patients with EGFR-positive SCCHN may result in significant NK cell activation and have important anti-tumor activity.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Cetuximab/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Proteínas/uso terapêutico , Animais , Carcinoma de Células Escamosas/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-15/genética , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária , Camundongos , Proteínas/genética , Receptores de Interleucina-15/genética , Proteínas Recombinantes de Fusão/genética , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Neuron ; 103(4): 583-597.e8, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31272828

RESUMO

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.


Assuntos
Técnicas de Introdução de Genes/métodos , Genômica/métodos , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos/genética , Humanos , Imunoquímica/métodos , Inteínas , Camundongos , Mutagênese Insercional , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteômica , RNA Guia/genética , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência do Ácido Nucleico
5.
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
6.
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
7.
Neuron ; 103(4): 642-657.e7, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31255486

RESUMO

Neuronal subtypes show diverse injury responses, but the molecular underpinnings remain elusive. Using transgenic mice that allow reliable visualization of axonal fate, we demonstrate that intrinsically photosensitive retinal ganglion cells (ipRGCs) are both resilient to cell death and highly regenerative. Using RNA sequencing (RNA-seq), we show genes that are differentially expressed in ipRGCs and that associate with their survival and axon regeneration. Strikingly, thrombospondin-1 (Thbs1) ranked as the most differentially expressed gene, along with the well-documented injury-response genes Atf3 and Jun. THBS1 knockdown in RGCs eliminated axon regeneration. Conversely, RGC overexpression of THBS1 enhanced regeneration in both ipRGCs and non-ipRGCs, an effect that was dependent on syndecan-1, a known THBS1-binding protein. All structural domains of the THBS1 were not equally effective; the trimerization and C-terminal domains promoted regeneration, while the THBS type-1 repeats were dispensable. Our results identify cell-type-specific induction of Thbs1 as a novel gene conferring high regenerative capacity.


Assuntos
Regeneração Nervosa/fisiologia , Células Ganglionares da Retina/fisiologia , Trombospondina 1/fisiologia , Animais , Apoptose , Axônios/metabolismo , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Genes Reporter , Fator de Crescimento Insulin-Like I/deficiência , Fator de Crescimento Insulin-Like I/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Compressão Nervosa , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/fisiopatologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Opsinas de Bastonetes/deficiência , Opsinas de Bastonetes/fisiologia , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/fisiologia , Trombospondina 1/biossíntese , Trombospondina 1/genética , Transcrição Genética
8.
Appl Microbiol Biotechnol ; 103(15): 6071-6079, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31175428

RESUMO

Single-chain variable fragment (scFv) has great prospect in medical therapies and diagnostic applications due to its binding affinity and low immunogenicity. However, the application of scFv is limited by its heterologous expression facing challenges of insoluble aggregation. sfGFP has been developed as fusion tag to facilitate the solubility of fusion partner in Escherichia coli. We designed fusion protein of anti-influenza PB2 scFv at C-terminus of sfGFP and successfully obtained soluble expression of sfGFP-scFv-His in Escherichia coli. The expression level of sfGFP-scFv-His reached at 20 mg/L of bacterial culture when the culture was induced with 0.1 mM IPTG at 18 °C for 16 h. And 6 mg scFv-His was obtained from the cleavage of 10 mg pure sfGFP-scFv-His with TEV protease. In addition, we found that sfGFP-scFv-His was more stable than scFv-His in chicken serum, suggesting that sfGFP not only facilitated the solubility of scFv in Escherichia coli, but also promoted the stability of scFv. The immunologic activity of sfGFP-scFv-His was confirmed by Western blot and ELISA; the results showed that anti-PB2 sfGFP-scFv-His exhibited specific binding to PB2. Hemagglutination and comparative real-time RT-PCR analysis indicated that sfGFP-scFv-His and scFv-His inhibited the replication of H1N1 influenza virus in the infected A549 cells. These results further develop the application of scFv as an agent, such as anti-influenza. Furthermore, soluble expression of scFv using sfGFP as fusion partner provide a cost-effective preparation model for manufacturing scFv against pandemic disease.


Assuntos
Anticorpos Antivirais/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Anticorpos de Cadeia Única/biossíntese , Células A549 , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Biotecnologia/métodos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Cultura de Vírus , Replicação Viral/efeitos dos fármacos
9.
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
10.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(6): 295-302, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31189782

RESUMO

The herb Ruta chalepensis L. exhibits medical effects, such as anti-inflammatory, central nervous system depressant, and antipyretic activities. However, a genetic transformation method has not yet been developed for this species. In this paper, a simple and efficient tissue culture and genetic transformation system for R. chalepensis is reported. An amyloid ß-peptide (Aß) gene, which is considered to be a causative agent of Alzheimer's disease (AD), fused with green-fluorescent protein (GFP), was introduced into R. chalepensis. When the leaves of R. chalepensis expressing Aß-GFP were administered orally to C57BL/6J mice, serum anti-Aß antibody titers of several mice were elevated without the use of an adjuvant. These results indicated that an oral vaccine against AD using R. chalepensis may be feasible. R. chalepensis is rich in bioactive compounds that may have synergistic effects with the vaccine for AD. Plant-derived vaccines are safer and cheaper than those produced from animal cells or microbes, because plants can serve as biofactories at low cost and with high biosynthetic capacity.


Assuntos
Peptídeos beta-Amiloides/genética , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/genética , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Ruta/genética , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Animais , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Transformação Genética , Vacinas/genética , Vacinas/imunologia
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.
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
13.
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
14.
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
15.
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
16.
Nat Commun ; 10(1): 2212, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101808

RESUMO

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Engenharia Genética/métodos , Rad51 Recombinase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reparo de DNA por Recombinação , Proteína 9 Associada à CRISPR/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Células HEK293 , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas , Células K562 , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos
17.
Microb Pathog ; 132: 275-281, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31078709

RESUMO

Toxoplasma gondii is an obligate intracellular parasite that causes one of the most common parasitic infections in humans and other warm-blooded animals. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the development of a vaccine to protect against toxoplasmosis is an attractive option for avoiding infection. The aim of this study was to design an epitope-based vaccine for T. gondii. In the present study, an in silico approach was used to predict and analyze B-cell and T-cell epitopes and the transmembrane domain of proteins SAG1, MIC3, and ROP8. We also predicted the antigenicity, allergenicity, secondary and tertiary structures, and physicochemical characteristics of a chimeric protein. Next, codon optimization and mRNA structure prediction were conducted using bioinformatics tools, and the designed construct was chemically synthesized and cloned into the pET28a vector. SAG1 (amino acid positions 85-235), MIC3 (30-180), and ROP8 (85-185) were found to have several strong immunodominant epitopes that were joined with a rigid linker A(EAAAK)2A. Although the resultant protein called MRS (MIC3, ROP8, and SAG1) did not turn out to be an allergen, its antigenicity was estimated to be 0.7983. Additionally, MRS was selected as the best vaccine candidate on the basis of its secondary and tertiary structures. The number of amino acids, molecular weight, and numbers of negatively and positively charged residues of MRS were 427 and 45,661.31 Da, 45, and 50, respectively. ΔG of the best-predicted structure was -413.0 kcal/mol, and the first nucleotides at the 5' end did not form a stable hairpin or pseudoknot. Finally, successful expression and verification of the expressed MRS protein showed that in silico analysis was almost accurate. This vaccine candidate selected by in silico tools should be validated in experimental studies.


Assuntos
Antígenos de Protozoários/imunologia , Imunogenicidade da Vacina/imunologia , Vacinas Protozoárias/imunologia , Proteínas Recombinantes de Fusão/imunologia , Toxoplasma/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Biologia Computacional , Simulação por Computador , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Expressão Gênica , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Toxoplasmose/imunologia , Toxoplasmose/prevenção & controle
18.
Microb Cell Fact ; 18(1): 91, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133014

RESUMO

BACKGROUND: Self-assembling amphipathic peptides (SAPs) may improve protein production or induce the formation of inclusion bodies by fusing them to the N-terminus of proteins. However, they do not function uniformly well with all target enzymes and systematic research on how the composition of SAPs influence the production of fusion protein is still limited. RESULTS: To improve the efficiency of SAPs, we studied factors that might be involved in SAP-mediated protein production using S1 (AEAEAKAK)2 as the original SAP and green fluorescent protein (GFP) as the reporter. The results indicate that hydrophobicity and net charges of SAPs play a key role in protein expression. As hydrophobicity regulation tend to cause the formation of insoluble inclusion bodies of protein, an expression tag library composed of SAPs, which varied in net charge (from + 1 to + 20), was constructed based on the random amplification of S1nv1 (ANANARAR)10. The efficiency of the library was validated by polygalacturonate lyase (PGL), lipoxygenase (LOX), L-asparaginase (ASN) and transglutaminase (MTG). To accelerate preliminary screening, each enzyme was fused at the C-terminus with GFP. Among the four enzyme fusions, the SAPs with + 2 - + 6 net charges were optimal for protein expression. Finally, application of the library improved the expression of PGL, LOX, ASN, and MTG by 8.3, 3.5, 2.64, and 3.68-fold relative to that of the corresponding wild-type enzyme, respectively. CONCLUSIONS: This is the first report to study key factors of SAPs as an expression tag to enhance recombinant enzyme production. The SAP library could be used as a novel plug-and-play protein-engineering method to screen for enzymes or proteins with enhanced production.


Assuntos
Escherichia coli/genética , Biblioteca Gênica , Peptídeos/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas de Fluorescência Verde/química , Ensaios de Triagem em Larga Escala , Interações Hidrofóbicas e Hidrofílicas , Corpos de Inclusão/metabolismo
19.
Mol Med Rep ; 20(1): 671-677, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115584

RESUMO

Preeclampsia is one of the complications of pregnancy. Often presenting as new­onset hypertension and proteinuria after 20 weeks of gestation in a previously normotensive patient, it can progress rapidly to serious complications, and is a leading cause of maternal and perinatal mortality. The present study involved the construction of a fusion protein consisting of a single­chain antibody variable fragment (scFv) and the retinol­binding protein 4 (RBP4). and investigated the function of this protein. The MaxCodon™ Optimization Program (v13) was used to optimize the amino acid sequence of the scFv­RBP4 fusion protein and full­length splice primers were designed by Detai Bio Tech. The scFv­RBP4 gene was inserted into a proEM expression vector using double digestion, and the accuracy of the final expression vector was confirmed by restriction enzyme digestion and sequencing. The plasmid was transfected into DH5α competent cells and the plasmid was extracted from cells using a transfection reagent. The plasmid and scFv­RBP4 fusion protein were purified by nickel­iminodiacetic acid affinity chromatography. Cell proliferation was determined using the Cell Counting Kit­8 assay and cell invasion was measured using a Transwell invasion assay. The results from the digestion and sequencing showed that the scFv­RBP4 fusion protein was constructed correctly and that the purity of the target protein was >90%. The scFv­RBP4 fusion protein was stably expressed in 293T cells. The scFv­RBP4 fusion protein was extracted from the 293T cells and functional studies were carried out. The scFv­RBP4 fusion protein significantly increased the invasion, but not the proliferation, of HTR8/SVneo cells.


Assuntos
Pré-Eclâmpsia/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Plasmáticas de Ligação ao Retinol/genética , Anticorpos de Cadeia Única/genética , Proliferação de Células/genética , Feminino , Expressão Gênica/genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Pré-Eclâmpsia/patologia , Gravidez , Transfecção
20.
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
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