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1.
Int J Nanomedicine ; 14: 8627-8645, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31806961

RESUMO

Background and purpose: Systemic lupus erythematous (SLE) is an autoimmune disease caused by many factors. Lupus nephritis (LN) is a common complication of SLE and represents a major cause of morbidity and mortality. Previous studies have shown the advantages of multi-targeted therapy for LN and that TLR4 signaling is a target of anti-LN drugs. High-mobility group box 1 (HMGB1), a nuclear protein with a proinflammatory cytokine activity, binds specifically to TLR4 to induce inflammation. We aimed to develop PEGylated TAT peptide-cationic liposomes (TAT-CLs) to deliver anti-HMGB1 siRNA and dihydroartemisinin (DHA) to increase LN therapeutic efficiency and explore their treatment mechanism. Methods: We constructed the TAT-CLs-DHA/siRNA delivery system using the thin film hydration method. The uptake and localization of Cy3-labeled siRNA were detected by confocal microscopy and flow cytometry. MTT assays were used to detect glomerular mesangial cell proliferation. Real-time PCR, Western blot analysis, and ELISA evaluated the anti-inflammatory mechanism of TAT-CLs-DHA/siRNA. Results: We constructed the TAT-CLs-DHA/siRNA delivery system measuring approximately 140 nm with superior storage and serum stabilities. In vitro, it showed significantly greater uptake compared with unmodified liposomes and significant inhibition of glomerular mesangial cell proliferation. TAT-CLs-DHA/siRNA inhibited NF-κB activation in a concentration-dependent manner. Real-time PCR and Western blot analysis showed that TAT-CLs-DHA/siRNA downregulated expression of HMGB1 mRNA and protein. TAT-CLs-DHA/siRNA markedly diminished Toll-like receptor 4 (TLR4) expression and subsequent activation of MyD88, IRAK4, and NF-κB. Conclusion: TAT-CLs-DHA/siRNA may have the potential for treatment of inflammatory diseases such as LN mediated by the TLR4 signaling pathway.


Assuntos
Artemisininas/administração & dosagem , Produtos do Gene tat/genética , Proteína HMGB1/genética , Lipossomos/administração & dosagem , Nefrite Lúpica/terapia , RNA Interferente Pequeno/administração & dosagem , Receptor 4 Toll-Like/metabolismo , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipossomos/química , Lipossomos/farmacologia , Nefrite Lúpica/metabolismo , NF-kappa B/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
2.
Int J Mol Sci ; 20(19)2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31590403

RESUMO

G-protein-coupled receptors associate into dimers/oligomers whose function is not well understood. One approach to investigate this issue is to challenge oligomerization by peptides replicating transmembrane domains and to study their effect on receptor functionality. The disruptor peptides are typically delivered by means of cell-penetrating vectors such as the human immunodeficiency virus (HIV) transcription trans-activation protein Tat. In this paper we report a cyclic, Tat-like peptide that significantly improves its linear analogue in targeting interreceptor sequences in the transmembrane space. The same cyclic Tat-like vector fused to a transmembrane region not involved in receptor oligomerization was totally ineffective. Besides higher efficacy, the cyclic version has enhanced proteolytic stability, as shown by trypsin digestion experiments.


Assuntos
Produtos do Gene tat/metabolismo , Peptídeos Cíclicos/metabolismo , Receptor A2A de Adenosina/metabolismo , Produtos do Gene tat/genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Peptídeos Cíclicos/genética , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica
3.
EcoSal Plus ; 8(2)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31215506

RESUMO

The Tat pathway for protein translocation across bacterial membranes stands out for its selective handling of fully folded cargo proteins. In this review, we provide a comprehensive summary of our current understanding of the different known Tat components, their assembly into different complexes, and their specific roles in the protein translocation process. In particular, this overview focuses on the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Bacillus subtilis. Using these organisms as examples, we discuss structural features of Tat complexes alongside mechanistic models that allow for the Tat pathway's unique protein proofreading and transport capabilities. Finally, we highlight recent advances in exploiting the Tat pathway for biotechnological benefit, the production of high-value pharmaceutical proteins.


Assuntos
Arginina/metabolismo , Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Produtos do Gene tat/metabolismo , Transporte Proteico , Bacillus subtilis/genética , Escherichia coli/genética , Produtos do Gene tat/genética , Redes e Vias Metabólicas , Sinais Direcionadores de Proteínas
4.
Exp Cell Res ; 372(1): 73-82, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30244178

RESUMO

The process of sealing ring formation requires major actin filament reorganization. We previously demonstrated that an actin-bundling protein L-plastin has a role in the cross-linking of actin filaments into tight bundles and forms actin aggregates (denoted as nascent sealing zones). These nascent sealing zones mature into fully functional sealing rings. We have shown here that TNF-alpha signaling regulates the phosphorylation of serine-5 and -7 in L-plastin which increases the actin bundling capacity of L-plastin and hence the formation of nascent sealing zones in mouse osteoclasts. Using the TAT-mediated transduction method, we confirmed the role of L-plastin in nascent sealing zones formation at the early phase of the sealing ring assembly. Transduction of TAT-fused full-length L-plastin peptide significantly increases the number of nascent sealing zones and therefore sealing rings. But, transduction of amino-terminal L-plastin peptides consisting of the serine-5 and -7 reduces the formation of both nascent sealing zones and sealing rings. Therefore, bone resorption in vitro was reduced considerably. The decrease was associated with the selective inhibition of cellular L-plastin phosphorylation by the transduced peptides. Neither the formation of podosomes nor the migration was affected in these osteoclasts. Phosphorylation of L- plastin on serine 5 and -7 residues increases the F-actin bundling capacity. The significance of our studies stands on laying the groundwork for a better understanding of L-plastin as a potential regulator at the early phase of sealing ring formation and could be a new therapeutic target to treat bone loss.


Assuntos
Citoesqueleto de Actina/metabolismo , Reabsorção Óssea/genética , Osteoclastos/metabolismo , Fosfoproteínas/genética , Serina/metabolismo , Fator de Necrose Tumoral alfa/genética , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Actinas/metabolismo , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Proteínas do Citoesqueleto , Fêmur/citologia , Fêmur/metabolismo , Regulação da Expressão Gênica , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Peptídeos/genética , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Podossomos/metabolismo , Podossomos/ultraestrutura , Cultura Primária de Células , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transdução Genética , Fator de Necrose Tumoral alfa/metabolismo
5.
BMB Rep ; 51(7): 362-367, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29936932

RESUMO

A major feature of type 1 diabetes mellitus (T1DM) is hyperglycemia and dysfunction of pancreatic ß-cells. In a previous study, we have shown that Tat-DJ-1 protein inhibits pancreatic RINm5F ß-cell death caused by oxidative stress. In this study, we examined effects of Tat-DJ-1 protein on streptozotocin (STZ)-induced diabetic mice. Wild type (WT) Tat-DJ-1 protein transduced into pancreas where it markedly inhibited pancreatic ß-cell destruction and regulated levels of serum parameters including insulin, alkaline phosphatase (ALP), and free fatty acid (FFA) secretion. In addition, transduced WT Tat-DJ-1 protein significantly inhibited the activation of NF-κB and MAPK (ERK and p38) expression as well as expression of COX-2 and iNOS in STZ exposed pancreas. In contrast, treatment with C106A mutant Tat-DJ-1 protein showed no protective effects. Collectively, our results indicate that WT Tat-DJ-1 protein can significantly ameliorate pancreatic tissues in STZ-induced diabetes in mice. [BMB Reports 2018; 51(7): 362-367].


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Produtos do Gene tat/genética , Substâncias Protetoras/uso terapêutico , Proteína Desglicase DJ-1/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Fosfatase Alcalina/sangue , Animais , Ciclo-Oxigenase 2/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácidos Graxos não Esterificados/sangue , Insulina/sangue , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Nat Commun ; 9(1): 2511, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29955037

RESUMO

RNA-protein interactions permeate biology. Transcription, translation, and splicing all hinge on the recognition of structured RNA elements by RNA-binding proteins. Models of RNA-protein interactions are generally limited to short linear motifs and structures because of the vast sequence sampling required to access longer elements. Here, we develop an integrated approach that calculates global pairwise interaction scores from in vitro selection and high-throughput sequencing. We examine four RNA-binding proteins of phage, viral, and human origin. Our approach reveals regulatory motifs, discriminates between regulated and non-regulated RNAs within their native genomic context, and correctly predicts the consequence of mutational events on binding activity. We design binding elements that improve binding activity in cells and infer mutational pathways that reveal permissive versus disruptive evolutionary trajectories between regulated motifs. These coupling landscapes are broadly applicable for the discovery and characterization of protein-RNA recognition at single nucleotide resolution.


Assuntos
Produtos do Gene tat/química , RNA Nucleotidiltransferases/química , Proteínas de Ligação a RNA/química , RNA/química , Proteínas Virais Reguladoras e Acessórias/química , Sequência de Aminoácidos , Bacteriófago lambda/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Vírus da Imunodeficiência Bovina/química , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , RNA/genética , RNA/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de RNA , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
7.
J Virol ; 92(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29743356

RESUMO

Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5'ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs, and splicing at this site has to be tightly regulated to allow the balanced production of all viral RNAs and proteins. We demonstrate that the viral Tat protein, which is known to activate viral transcription, also stimulates splicing at the major 5'ss. As for the transcription effect, Tat requires the viral long terminal repeat promoter and the trans-acting responsive RNA hairpin for splicing regulation. These results indicate that HIV-1 transcription and splicing are tightly coupled processes through the coordinated action of the essential Tat protein.IMPORTANCE The HIV-1 proviral DNA encodes a single RNA transcript that is used as RNA genome and packaged into newly assembled virus particles. This full-length RNA is also used as mRNA for the production of structural and enzymatic proteins. Production of other essential viral proteins depends on alternative splicing of the primary transcript, which yields a large set of differentially spliced mRNAs. Optimal virus replication requires a balanced production of all viral RNAs, which means that the splicing process has to be strictly regulated. We show that the HIV-1 Tat protein, a factor that is well known for its transcription activating function, also stimulates splicing. Thus, Tat controls not only the level of the viral RNA but also the balance between spliced and unspliced RNAs.


Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene tat/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Processamento de RNA , RNA Viral/genética , Produtos do Gene tat/genética , Células HEK293 , HIV-1/isolamento & purificação , Humanos , Replicação Viral
8.
J Cell Mol Med ; 22(3): 1601-1613, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29265583

RESUMO

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA.


Assuntos
Trifosfato de Adenosina/biossíntese , Fibroblastos/enzimologia , Produtos do Gene tat/genética , Metilmalonil-CoA Mutase/genética , Mitocôndrias/enzimologia , Proteínas Recombinantes de Fusão/genética , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Sistemas CRISPR-Cas , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/patologia , Expressão Gênica , Produtos do Gene tat/metabolismo , Terapia Genética/métodos , Células Hep G2 , Humanos , Fígado/enzimologia , Fígado/patologia , Potencial da Membrana Mitocondrial , Ácido Metilmalônico/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Doenças Mitocondriais/terapia , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Engenharia de Proteínas/métodos , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
9.
Colloids Surf B Biointerfaces ; 162: 326-334, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29223647

RESUMO

We developed a high-efficiency nucleus-targeted co-delivery vector that delivers genes and drugs directly into the nucleus of cancer cells. The system is based on grafted poly-(N-3-carbobenzyloxy-lysine) (CPCL) with transactivator of transcription (TAT)- chitosan on the surface. It is designed to perform highly efficient nucleus- targeted gene and drug co-delivery. Confocal laser scanning microscopy (CLSM) revealed that more TAT-CPCL entered the nucleus than does CPCL alone. The TAT-modified vector serves as a gene and drug co-delivery mechanism to achieve high gene transfection efficiency, high apoptosis and low viability in HeLa cells. TAT-CPCL may become a vector for cancer gene treatment and a template for designing better co-deliver systems.


Assuntos
Núcleo Celular/efeitos dos fármacos , Quitosana/química , Portadores de Fármacos , Produtos do Gene tat/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/química , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Doxorrubicina/farmacologia , Produtos do Gene tat/genética , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Micelas , Tamanho da Partícula , Polilisina/química , Propriedades de Superfície
10.
Nature ; 552(7685): 415-420, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29236688

RESUMO

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a 'blank slate' to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at 'top-down' modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary 'bottom-up' approach with considerable advantages in programmability and control.


Assuntos
Bioengenharia , Evolução Molecular Direcionada , Genoma Viral , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Montagem de Vírus , Animais , Sistemas de Liberação de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Aptidão Genética , Terapia Genética , Vírus da Imunodeficiência Bovina/química , Vírus da Imunodeficiência Bovina/genética , Camundongos , Modelos Moleculares , Nucleocapsídeo/química , RNA Mensageiro/metabolismo , Seleção Genética
11.
Cell Death Dis ; 8(10): e3075, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28981094

RESUMO

In the present study, we searched for possible candidates that can prevent ischemic damage in the rabbit spinal cord. For this study, we used two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, in sham- and ischemia-operated animals. As the level of protein disulfide-isomerase A3 (PDIA3) significantly decreased 3 h after ischemia/reperfusion, we further investigated its possible role against ischemic damage using an in vitro spinal cord cell line and in vivo spinal cord ischemic model. The administration of Tat-PDIA3 significantly reduced the hydrogen peroxide-induced formation of reactive oxygen species and cell death, based on terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling and a colorimetric WST-1 assay. Further, Tat-PDIA3 significantly ameliorated the ischemia-induced deficits in motor function, based on Tarlov's criteria, 24-72 h after ischemia/reperfusion, as well as the degeneration of motor neurons in the ventral horn 72 h after ischemia/reperfusion. Tat-PDIA3 administration also reduced the ischemia-induced activation of microglia and lipid peroxidation in the motor neurons 72 h after ischemia/reperfusion. PDIA3 also potentially ameliorated the ischemia-induced increase in oxidative markers in serum and decreased the activity of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase in spinal cord homogenates, 24 h and 72 h after ischemia/reperfusion. These results suggest that Tat-PDIA3 could be used to protect spinal cord neurons from ischemic damage, due to its modulatory action on the oxidative/anti-oxidative balance. Tat-PDIA3 could be applicable to protects neurons from the ischemic damage induced by thoracoabdominal aorta obstruction.


Assuntos
Produtos do Gene tat/genética , Isomerases de Dissulfetos de Proteínas/genética , Traumatismo por Reperfusão/genética , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Modelos Animais de Doenças , Produtos do Gene tat/administração & dosagem , Glutationa Peroxidase/genética , Humanos , Peróxido de Hidrogênio/química , Peroxidação de Lipídeos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Neurônios Motores/química , Neurônios Motores/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/administração & dosagem , Coelhos , Espécies Reativas de Oxigênio , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/terapia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Superóxido Dismutase/genética
12.
Biotechnol Bioeng ; 114(12): 2828-2836, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28842980

RESUMO

Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L-1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Produtos do Gene tat/genética , Melhoramento Genético/métodos , Hormônio do Crescimento/biossíntese , Periplasma/metabolismo , Via Secretória/genética , Hormônio do Crescimento/genética , Hormônio do Crescimento/isolamento & purificação , Humanos , Redes e Vias Metabólicas/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
13.
Phytopathology ; 107(9): 1011-1021, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28699375

RESUMO

Xanthomonas oryzae pv. oryzae, an economically important bacterium, causes a serious disease in rice production worldwide called bacterial leaf blight. How X. oryzae pv. oryzae infects rice and causes symptoms remains incompletely understood. Our earlier works demonstrated that the twin-arginine translocation (Tat) pathway plays an vital role in X. oryzae pv. oryzae fitness and virulence but the underlying mechanism is unknown. In this study, we used strain PXO99A as a working model, and identified 15 potential Tat-dependent translocation proteins (TDTP) by using comparative proteomics and bioinformatics analyses. Combining systematic mutagenesis, phenotypic characterization, and gene expression, we found that multiple TDTP play key roles in X. oryzae pv. oryzae adaption or virulence. In particular, four TDTP (PXO_02203, PXO_03477, PXO_02523, and PXO_02951) were involved in virulence, three TDTP (PXO_02203, PXO_03477, and PXO_02523) contributed to colonization in planta, one TDTP (PXO_02671) had a key role in attachment to leaf surface, four TDTP (PXO_02523, PXO_02951, PXO_03132, and PXO_03841) were involved in tolerance to multiple stresses, and two TDTP (PXO_02523 and PXO_02671) were required for full swarming motility. These findings suggest that multiple TDTP may have differential contributions to involvement of the Tat pathway in X. oryzae pv. oryzae adaption, physiology, and pathogenicity.


Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Bactérias/metabolismo , Estresse Fisiológico/fisiologia , Xanthomonas/metabolismo , Xanthomonas/patogenicidade , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Movimento , Oryza/microbiologia , Doenças das Plantas , Transporte Proteico , Virulência
14.
Int J Gynecol Cancer ; 27(6): 1082-1087, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28604448

RESUMO

BACKGROUND: Paclitaxel is recommended as a first-line chemotherapeutic agent against ovarian cancer, but drug resistance becomes a major limitation. The key molecule or mechanism associated with paclitaxel resistance in ovarian cancer still remains unclear. Recent studies have revealed an association between autophagy and drug resistance. METHODS: We previously synthesized a MAPK kinase-recombinant fusion protein, MAP2K6-FP, that contains 3 domains: a protein transduction domain TAT, a human ovarian cancer HO8910 cell-specific binding peptide, and a potential antitumor effector domain MKK6(E). In this study, we investigated the effect of MAP2K6-FP on HO8910 cells treated with paclitaxel. RESULTS: The IC50 (concentration by which 50% cell growth was inhibited) was 20 µM for paclitaxel alone, 1.5 µg/mL for MAP2K6-FP alone, and 0.3 µg/mL for MAP2K6-FP and 15 µM for paclitaxel if combined, respectively. In addition, immunohistochemistry assay demonstrated that tumor tissues from ovarian cancer patients showed higher expression of LC-3, the autophagy-related protein, compared with normal ovarian tissues. MAP2K6-FP (0, 2.5, 5, 10, 20, and 40 µg/mL) dose-dependently increased the LC-3 expression in HO8910 cells. Immunofluorescence assay showed that paclitaxel alone increased the expression of LC-3 in HO8910 cells, which was further enhanced by the combination with MAP2K6-FP. Downregulation of LC-3 expression using LC-3 small interfering RNA inhibited the cytotoxicity effect of MAP2K6-FP. Furthermore, either MAP2K6-FP alone or in combination with paclitaxel increased the ratio of expressions of Beclin-1/Bcl-2, another autophagy-related markers, compared with paclitaxel alone. CONCLUSIONS: MAP2K6-FP enhanced the sensitiveness of paclitaxel for ovarian cancer via inducing autophagy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , MAP Quinase Quinase 6/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Produtos do Gene tat/genética , Produtos do Gene tat/farmacologia , Humanos , MAP Quinase Quinase 6/genética , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Domínios Proteicos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética
15.
Int Immunopharmacol ; 48: 118-125, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28501765

RESUMO

Allergen-specific sublingual immunotherapy (SLIT) is well known as an effective and non-invasive route to induce allergy desensitization. The goal of this study was to investigate whether a TAT-fused recombinant allergen could enhance SLIT efficacy. BALB/c mice sensitized to the main allergen (Che a 3) of Chenopodium album pollen were treated sublingually either with rChe a 3 (100µg/dose) or rTAT-Che a 3 (100µg/dose), two times per week for eight weeks. SLIT with rTAT-Che a 3 led to significantly greater allergen-specific IgG2a than rChe a 3; however, neither rTAT-Che a 3 nor rChe a 3 affected allergen-specific IgE or IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in re-stimulated splenocytes from the rTAT-Che a 3 mice were significantly lower than in those from the rChe a 3 mice, while interferon-γ (IFN-γ) was significantly greater in the rChe a 3 mice than in the rTAT-Che a 3 mice. Furthermore, sublingual administration of rTAT-Che a 3 induced significantly greater TGF-ß secretion in re-stimulated splenocytes than administration of rChe a 3. Accordingly, SLIT with rTAT-Che a 3 led to significantly greater expression of TGF-ß- and Foxp3-specific mRNAs in the splenocytes than in those from the rChe a 3 mice. Our findings demonstrate that TAT-fused rChe a 3 suppressed the allergic response through preferential enhancement of systemic regulatory T-cell (Treg)-mediated immunity responses, likely by facilitating allergen capture and presentation by sublingual Langerhans-like dendritic cells.


Assuntos
Alérgenos/administração & dosagem , Antígenos de Plantas/administração & dosagem , Proteínas de Ligação ao Cálcio/administração & dosagem , Produtos do Gene tat/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Rinite Alérgica Sazonal/terapia , Imunoterapia Sublingual , Animais , Antígenos de Plantas/genética , Proteínas de Ligação ao Cálcio/genética , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/genética , Produtos do Gene tat/genética , Camundongos , Camundongos Endogâmicos BALB C , Rinite Alérgica Sazonal/imunologia , Baço/citologia , Baço/imunologia , Proteínas com Domínio T/genética , Linfócitos T/imunologia
16.
J Control Release ; 255: 1-11, 2017 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-28385674

RESUMO

The cytosolic delivery of therapeutic proteins (e.g., antibodies or enzymes) by cell-penetrating peptides (CPPs), such as a human immunodeficiency virus-derived TAT peptide, is facilitated by fusogenic peptides (FPs). For instance, we recently demonstrated that an FP, B18, which is derived from a sea urchin gamete fusion protein, promotes endosomal escape of an enhanced green fluorescent protein (eGFP)-TAT fusion protein directly conjugated to it. However, the potential clinical use of FPs raises concerns because all conventional FPs are non-human-derived. To solve this problem, we have attempted to identify novel human-derived FPs from two human proteins, including a human sperm protein, IZUMO1, which is involved in gamete recognition and fusion, and a human endogenous retroviral envelope protein, Syncytin1, which is involved in placental morphogenesis. Partial peptides from the core domains of the abovementioned proteins were chosen as candidates to generate human-derived FPs. We prepared fusion proteins of these peptides with eGFP and TAT in Escherichia coli and observed the localization of these fusion proteins in HeLa cells using confocal microscopy. Our results suggested that a 19-residues peptide of Syncytin1 (positions 322-340), named S19, possessed strong intracellular uptake activities with no detectable cytotoxicity. In addition, we estimated the number of molecules that escaped from endosomes using a nuclear localization signal, suggesting that the S19 peptide stimulated the intracellular delivery of TAT-fused eGFP by ~90-fold. Furthermore, we confirmed that S19 promoted the intracellular delivery of eGFP to various human cell lines, including HeLa, A431, HepG2, and SK-N-SH. In addition, we demonstrated that not only eGFP but also SNAP-tag and ß-galactosidase were delivered efficiently and retained their activities.


Assuntos
Sistemas de Liberação de Medicamentos , Produtos do Gene env/química , Produtos do Gene tat/administração & dosagem , Proteínas de Fluorescência Verde/administração & dosagem , Imunoglobulinas/química , Proteínas de Membrana/química , Peptídeos/administração & dosagem , Proteínas da Gravidez/química , Linhagem Celular , Linhagem Celular Tumoral , Produtos do Gene tat/genética , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química
17.
Rinsho Ketsueki ; 57(10): 2250-2258, 2016.
Artigo em Japonês | MEDLINE | ID: mdl-27795537

RESUMO

Adult T-cell leukemia/lymphoma (ATL) is a HTLV-1 induced T-cell malignancy with an extremely poor prognosis. There is a long latency period between HTLV-1 infection and the onset of ATL, which indicates the existence of multistep mechanisms of leukemogenesis in the infected cells. Tax, which is encoded by the HTLV-1 pX region, plays a crucial role in HTLV-1 leukemogenesis and is a major target of CTL. We developed an anti-ATL therapeutic vaccine consisting of autologous dendritic cells that is pulsed with Tax peptides (Tax-DC). The vaccination protocol was completed with three injections at a 2-week interval, within one month. Good quality of life and long-term treatment-free survival were observed for more than 3 years in two of the three patients enrolled in the pilot study. Furthermore, the proviral load remained mostly around the carrier level, with minor fluctuation, after vaccination. Tax-specific proliferative CTL responses were observed in all cases and sporadically augmented responses were also subsequently detected. The Tax-DC vaccine might be a well-tolerated and long-lasting maintenance therapy that is acceptable even for elderly patients. Based on the encouraging results, we are now conducting a clinical trial of Tax-DC vaccine combined with anti-CCR4 antibody to enhance the efficacy of the vaccine as next-generation immunotherapy.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano/imunologia , Imunoterapia , Leucemia-Linfoma de Células T do Adulto/terapia , Células Dendríticas/imunologia , Produtos do Gene tat/genética , Produtos do Gene tat/imunologia , Genoma Viral , Humanos , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucemia-Linfoma de Células T do Adulto/virologia , Vacinação
18.
BMB Rep ; 49(11): 617-622, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27616357

RESUMO

Oxidative stress is closely associated with various diseases and is considered to be a major factor in ischemia. NAD(P)H:quinone oxidoreductase 1 (NQO1) protein is a known antioxidant protein that plays a protective role in various cells against oxidative stress. We therefore investigated the effects of cell permeable Tat-NQO1 protein on hippocampal HT-22 cells, and in an animal ischemia model. The Tat-NQO1 protein transduced into HT-22 cells, and significantly inhibited against hydrogen peroxide (H2O2)-induced cell death and cellular toxicities. Tat-NQO1 protein inhibited the Akt and mitogen activated protein kinases (MAPK) activation as well as caspase-3 expression levels, in H2O2 exposed HT-22 cells. Moreover, Tat-NQO1 protein transduced into the CA1 region of the hippocampus of the animal brain and drastically protected against ischemic injury. Our results indicate that Tat-NQO1 protein exerts protection against neuronal cell death induced by oxidative stress, suggesting that Tat-NQO1 protein may potentially provide a therapeutic agent for neuronal diseases. [BMB Reports 2016; 49(11): 617-622].


Assuntos
Produtos do Gene tat/genética , NAD(P)H Desidrogenase (Quinona)/genética , Estresse Oxidativo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Produtos do Gene tat/metabolismo , Gerbillinae , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia
19.
Neuropharmacology ; 109: 205-215, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27316905

RESUMO

Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals.


Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Produtos do Gene tat/biossíntese , HIV-1 , Memória/fisiologia , Tempo de Reação/fisiologia , Recompensa , Animais , Relação Dose-Resposta a Droga , Expressão Gênica , Produtos do Gene tat/genética , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tempo de Reação/efeitos dos fármacos , Fatores de Tempo
20.
Cryo Letters ; 37(2): 103-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27224521

RESUMO

BACKGROUND: The discovery of proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. OBJECTIVE: We investigated the effect of TAT-EGFP (trans-activator of transcription-enhanced green fluorescent protein) intra-cellular delivery on the survival and development of mature porcine oocytes after cryopreservasion. MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6 mm in diameter in abattoir-derived oocytesries of prepubertal gilts were on vitro matured (IVM). After IVM, the oocytes were used for TAT-EGFP delivery test and cryopreservation with and without TAT-EGFP supplementation. Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were parthened and cultured in vitro, to assess their ability to be activated and to therefore develop. RESULTS: The results show that the TAT-EGFP was well delivered into the nuclear of the Hela cell and oocytes also. In the medium toxic test, the proportion of viable oocytes in seven groups showed no significance. In vitrification experiments, the viability of oocytes in group supplemented with TAT-EGFP was significantly higher than that in the without TAT-EGFP group and the control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, the developmental abilities of oocytes in the supplement TAT-EGFP, EGFP and Control groups revealed that the vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%, respectively). CONCLUSION: the supplement of TAT-EGFP protein into vitrification medium does not affect the viability of the oocytes whereas it improved the viability and developmental potential of oocytes after it was vitrified.


Assuntos
Criopreservação/veterinária , Produtos do Gene tat/genética , Proteínas de Fluorescência Verde/genética , Oócitos , Sus scrofa/fisiologia , Animais , Permeabilidade da Membrana Celular , Criopreservação/métodos , Feminino , Produtos do Gene tat/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Vitrificação
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