Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 19.196
Filtrar
1.
Nucleic Acids Res ; 48(4): 1985-1999, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31875230

RESUMO

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which ß-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the ß-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.


Assuntos
Peptídeos/genética , Biossíntese de Proteínas/genética , Proteínas Ribossômicas/química , Ribossomos/genética , Sequência de Aminoácidos/genética , Arabidopsis/química , Arabidopsis/genética , Sistema Livre de Células , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Peptídeos/química , Genética Reversa , Proteínas Ribossômicas/genética , Ribossomos/química
2.
Sheng Wu Gong Cheng Xue Bao ; 35(12): 2269-2283, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31880135

RESUMO

Cell-free synthetic biology system can perform biological transcription and translation process in vitro. Because of its advanced features, such as flexible openness, easy control, short expression time and high tolerance to cytotoxicity, this systemhas been successfully used to synthesize proteins that are difficult to express in cells. With the continuous development of cell-free biosensing technology and the lyophilization technology, its applications have widely expanded into many biomedical fields. This review discusses the current research progress of cell-free synthetic biology system in on-demand biopharmaceutical synthesis, portable diagnostics, and others. Further development of the system can lead to even more complicated synthesis of therapeutic proteins with post-translational modifications and evolution of different cell-free biosensors with high sensitivity. Cell-free synthetic biology as an emerging engineering strategy can be a better means applied to high-throughput screening of pharmaceutical proteins, detection of new pathogens, and other important health-care fields in the future.


Assuntos
Técnicas Biossensoriais , Biologia Sintética , Sistema Livre de Células , Indústrias
3.
Enzyme Microb Technol ; 131: 109392, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615678

RESUMO

Rosmarinic acid (RA), as a hydroxycinnamic acid ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid (3,4-DHPL), is a phenylpropanoid-derived plant natural product and has diverse biological activities. This work acts as a modular platform for microbial production using a two-cofactor (ATP and CoA) regeneration system to product RA based on a cell-free biosynthetic approach. Optimal activity of the reaction system was pH 8 and 30 °C. Total turnover number for ATP and CoA was 820.60 ±â€¯28.60 and 444.50 ±â€¯9.65, respectively. Based on the first hour data, the RA productivity reached 320.04 mg L-1 h-1 (0.889 mM L-1 h-1).


Assuntos
Trifosfato de Adenosina/metabolismo , Anti-Inflamatórios não Esteroides/metabolismo , Cinamatos/metabolismo , Coenzima A/metabolismo , Depsídeos/metabolismo , Sistema Livre de Células , Concentração de Íons de Hidrogênio , Temperatura Ambiente
4.
Anal Chim Acta ; 1088: 137-143, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31623709

RESUMO

Here, we report a novel fluorescence method for the highly selective and sensitive detection of RNase H by combining the use of a dual-pyrene-labeled DNA/RNA duplex with supramolecular inclusion-enhanced fluorescence. Initially, the probe is in the "off" state due to the rigidness of the double-stranded duplex, which separates the two pyrene units. In the presence of RNase H, the RNA strand of the DNA/RNA duplex will be hydrolyzed, and the DNA strand transforms into a hairpin structure, bringing close the two pyrene units which in turn enter the hydrophobic cavity of a γ-cyclodextrin. As a result, the pyrene excimer emission is greatly enhanced, thereby realizing the detection of RNase H activity. Under optimal conditions, RNase H detection can be achieved in the range from 0.08 to 4 U/mL, with a detection limit of 0.02 U/mL.


Assuntos
Técnicas Biossensoriais/métodos , Ciclodextrinas/química , Limite de Detecção , Pirenos/química , Ribonuclease H/análise , Sequência de Bases , Linhagem Celular Tumoral , Sistema Livre de Células/enzimologia , Sondas de DNA/química , Sondas de DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Sondas RNA/química , Sondas RNA/genética , Ribonuclease H/sangue
5.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510048

RESUMO

Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential 'scanning' of the 5' untranslated regions (UTRs). Scanning through the 5'UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5'UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured ß-globin sequences in the 5'UTR but not that of an mRNA with a poly(A) sequence as the 5'UTR. By contrast, the nonhydrolysable ATP analogue ß,γ-imidoadenosine 5'-triphosphate (AMP-PNP) inhibited translation irrespective of the 5'UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA ('toeprinting'), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This '-8 nt toeprint' was seen with mRNA 5'UTRs of different length, sequence and structure potential. Importantly, the '-8 nt toeprint' was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5'UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5'UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.


Assuntos
Regiões 5' não Traduzidas/genética , Biossíntese de Proteínas , Capuzes de RNA/genética , RNA Mensageiro/genética , Subunidades Ribossômicas Menores/genética , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Animais , Linhagem Celular Tumoral , Sistema Livre de Células , Fator de Iniciação 4F em Eucariotos/metabolismo , Camundongos , Modelos Genéticos , RNA Helicases/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
6.
BMC Biol ; 17(1): 64, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395057

RESUMO

Cell-free systems (CFS) have recently evolved into key platforms for synthetic biology applications. Many synthetic biology tools have traditionally relied on cell-based systems, and while their adoption has shown great progress, the constraints inherent to the use of cellular hosts have limited their reach and scope. Cell-free systems, which can be thought of as programmable liquids, have removed many of these complexities and have brought about exciting opportunities for rational design and manipulation of biological systems. Here we review how these simple and accessible enzymatic systems are poised to accelerate the rate of advancement in synthetic biology and, more broadly, biotechnology.


Assuntos
Biotecnologia/métodos , Sistema Livre de Células , Biologia Sintética/métodos
7.
Oxid Med Cell Longev ; 2019: 6528106, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396304

RESUMO

In the cold environments of the interstellar medium, a variety of molecules in which a hydrogen (H) atom has been replaced by its heavier isotope deuterium (D) can be found. From its emergence, life had to counteract the toxic action of many agents, which posed a constant threat to its development and propagation. Oxygen-reactive species are archaic toxicants that lead to protein damage and genomic instability. Most of the oxidative lesions involve cleavage of C-H bonds and H abstraction. According to free radical chemistry principles, the substitution of D for H in oxidation-sensitive positions of cellular components should confer protection against the oxidative attack without compromising the chemical identity of the compounds. Here, we show that deuterated nucleosides and proteins protect from oxidative damage. Our data suggest a new, subtle but likely role of D in terrestrial life's evolution in that its inclusion in critical biomolecules might have facilitated their resistance during the infinite generations of life entities, cells, and organisms.


Assuntos
Deutério/química , Estresse Oxidativo , Sobrevivência Celular/efeitos dos fármacos , Sistema Livre de Células , Dano ao DNA/efeitos dos fármacos , Radicais Livres/química , Produtos Finais de Glicação Avançada/análise , Humanos , Células Jurkat , Nucleosídeos/química , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica , Proteínas/química , Proteínas/metabolismo
8.
Appl Microbiol Biotechnol ; 103(19): 8009-8019, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31396682

RESUMO

Cysteine is a commercially valuable amino acid with an increasing demand in the food, cosmetic, and pharmaceutical industries. Although cysteine is conventionally manufactured by extraction from animal proteins, this method has several problems, such as troublesome waste-water treatment and incompatibility with some dietary restrictions. Fermentative production of cysteine from plant-derived substrates is a promising alternative for the industrial production of cysteine. However, it often suffers from low product yield as living organisms are equipped with various regulatory systems to control the intracellular cysteine concentration at a moderate level. In this study, we constructed an in vitro cysteine biosynthetic pathway by assembling 11 thermophilic enzymes. The in vitro pathway was designed to be insensitive to the feedback regulation by cysteine and to balance the intra-pathway consumption and regeneration of cofactors. A kinetic model for the in vitro pathway was built using rate equations of individual enzymes and used to optimize the loading ratio of each enzyme. Consequently, 10.5 mM cysteine could be produced from 20 mM glucose through the optimized pathway. However, the observed yield and production rate of the assay were considerably lower than those predicted by the model. Determination of cofactor concentrations in the reaction mixture indicated that the inconsistency between the model and experimental assay could be attributed to the depletion of ATP and ADP, likely due to host-derived, thermo-stable enzyme(s). Based on these observations, possible approaches to improve the feasibility of cysteine production through an in vitro pathway have been discussed.


Assuntos
Vias Biossintéticas/genética , Sistema Livre de Células , Cisteína/metabolismo , Glucose/metabolismo , Enzimas/genética , Enzimas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
Biosci Biotechnol Biochem ; 83(12): 2213-2219, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31362590

RESUMO

5-Aminolevulinic acid (ALA) is an important cellular metabolic intermediate that has broad agricultural and medical applications. Previously, attempts have been made to synthesize ALA by multiple enzymes in cell free systems. Here we report the development of a semi-permeable system for ALA production using stable enzymes. Glucose, sodium polyphosphate, ATP, tRNA, glutamate and NADPH were used as substrates for ALA synthesis by a total of nine enzymes: adenylate kinase, polyphosphate kinase, glucose-6-phosphate dehydrogenase, phosphogluconolactonase, 6-phosphogluconate dehydrogenase, glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase from E. coli, hexokinase from yeast, as well as glutamyl-tRNA reductase and its stimulator protein glutamyl-tRNA reductase binding protein (GBP) from Arabidopsis in a semi-permeable system. After reaction for 48 h, the glutamate conversion reached about 95%. This semi-permeable system facilitated the reuse of enzymes, and was helpful for the separation and purification of the product. The ALA production could be further improved by process optimization and enzyme engineering.Abbreviations: PPK: polyphosphate kinase; ADK: adenylate kinase; ALA: 5-Aminolevulinic acid; HK: hexokinase; ZWF: glucose-6-phosphatedehydrogenase; PGL: phosphogluconolactonase; GND: 6-phosphogluconate dehydrogenase; GTS: glutamyl-tRNA synthetase; GTR: glutamyl-tRNA reductase; GBP: GTR binding protein; GSAAT: glutamate-1-semialdehyde aminotransferase.


Assuntos
Trifosfato de Adenosina/metabolismo , Ácido Aminolevulínico/metabolismo , NADP/metabolismo , Sistema Livre de Células , Enzimas/isolamento & purificação , Enzimas/metabolismo , Escherichia coli/metabolismo , Permeabilidade
10.
Infect Immun ; 87(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31427447

RESUMO

Coxiella burnetii is an obligate intracellular Gram-negative bacterium which causes human Q fever. An acidified citrate cysteine medium (ACCM-2) has been developed which mimics the intracellular replicative niche of C. burnetii and allows axenic growth of the bacteria. To determine if C. burnetii cultured in ACCM-2 retains immunogenicity, we compared the protective efficacies of formalin-inactivated C. burnetii Nine Mile phase I (PIV) and phase II (PIIV) vaccines derived from axenic culture 7, 14, and 28 days postvaccination. PIV conferred significant protection against virulent C. burnetii as early as 7 days postvaccination, which suggests that ACCM-2-derived PIV retains immunogenicity and protectivity. We analyzed the cellular immune response in spleens from PIV- and PIIV-vaccinated mice by flow cytometry at 7 and 14 days postvaccination and found significantly more granulocytes in PIV-vaccinated mice than in PIIV-vaccinated mice. Interestingly, we found these infiltrating granulocytes to be SSChigh CD11b+ CD125+ Siglec-F+ (where SSChigh indicates a high side scatter phenotype) eosinophils. There was no change in the number of eosinophils in PIV-vaccinated CD4-deficient mice compared to the level in controls, which suggests that eosinophil accumulation is CD4+ T cell dependent. To evaluate the importance of eosinophils in PIV-mediated protection, we vaccinated and challenged eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice had significantly worse disease than their wild-type counterparts when challenged 7 days postvaccination, while no significant difference was seen at 28 days postvaccination. Nevertheless, ΔdblGATA mice had elevated serum IgM with decreased IgG1 and IgG2a whether mice were challenged at 7 or 28 days postvaccination. These results suggest that eosinophils may play a role in early vaccine protection against C. burnetii and contribute to antibody isotype switching.


Assuntos
Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/imunologia , Coxiella burnetii/imunologia , Eosinófilos/fisiologia , Switching de Imunoglobulina/imunologia , Febre Q/prevenção & controle , Animais , Sistema Livre de Células , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/citologia , Vacinação
11.
Talanta ; 205: 120092, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450435

RESUMO

VEGF mRNA, as an important biomarker for disease diagnosis and therapeutics, has received extensive attention. However, how to monitor its mRNA levels rapidly and sensitively remains a challenge. Herein, a strategy was designed for facile and efficient detection of VEGF mRNA and imaging in living cells using a collaborative system of a fluorophore-labeled single-stranded probe (P), reduced graphene oxide (rGO) and double-specific nuclease (DSN). The combination of strong fluorophore-quenching ability of rGO with DSN assisted signal amplification contributes to the superior sensitivity of the assay for VEGF mRNA, which was reflected by the lower limit of mRNA detection of 100 fM obtained using dual signal amplification manner. Furthermore, the developed sensor was directly used for intracellular mRNA imaging in vitro without the assistance of transfection reagent. In summary, the simple, ultra-sensitive and cost-effective mRNA assay system, which provided a general analysis strategy for other mRNAs assay by replacing the sequence of the probe, is hopeful for applying on the clinical diagnosis and therapy.


Assuntos
Grafite/química , Imagem Molecular/métodos , RNA Mensageiro/análise , Fator A de Crescimento do Endotélio Vascular/genética , Sistema Livre de Células , Sondas de DNA/química , Sondas de DNA/genética , DNA de Cadeia Simples/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Imagem Molecular/instrumentação , Sensibilidade e Especificidade
12.
Yakugaku Zasshi ; 139(7): 989-992, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31257257

RESUMO

The molecular basis underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) has not been fully elucidated. The protein-misfolding cyclic amplification (PMCA) technique, which can amplify PrPSc in vitro with the use of intermittent sonication, mimics the process of in vivo PrPSc replication. Accumulating evidence suggests that co-factors other than PrP may play a crucial role in the faithful replication of PrPSc. In conventional PMCA, brain homogenates (BHs) from normal animals are used as the PrPC substrate. Since BHs contain many impurities, it is difficult to identify the co-factors using conventional PMCA. Thus, we developed a modified PMCA system using baculovirus and insect cell-derived recombinant PrP as a substrate (insect cell PMCA; iPMCA). We demonstrated that nucleic acids and glycosaminoglycans (GAGs) such as heparan sulfate (HS) or its analogue heparin (HP) are critical for PrPSc amplification in iPMCA. Of note, the addition of HS or HP restored the conversion efficiency in iPMCA under nucleic acid-depleted conditions. Moreover, the iPMCA products were infectious and preserved the strain properties of the input seed PrPSc. These data suggest that not only nucleic acids but also some GAGs play an important role in facilitating faithful replication of prions, at least in vitro.


Assuntos
Baculoviridae/genética , Insetos/genética , Proteínas Priônicas/química , Animais , Sistema Livre de Células , Glicosaminoglicanos , Heparina , Heparitina Sulfato , Técnicas In Vitro , Ácidos Nucleicos , Proteínas Recombinantes/química
13.
Int J Biol Macromol ; 137: 62-68, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31255626

RESUMO

Differences between the levan obtained from bacteria and from cell-free systems were studied in this work. Results showed that both polymers are non-porous solids (type II isotherm with 20 m2/g) with a main thermal decomposition at 200 °C and a negligible value of protein adsorption. Microbial levan produced nanoparticles of 90 nm in diameter whereas nanoparticles of 110 nm were obtained with the polymer obtained from a cell-free system. Both polymers behave as aggregates depending on the critical aggregation concentration. At the same time, that concentration depends on the technique used for the polymer synthesis. Cell-free system aggregation concentration is 0.24 mg/mL whereas a concentration of 0.05 mg/mL was found for the microbial system. In both cases, the average molecular weight of the aggregate is higher than 2000 kDa. These results highlight the existence of aggregation equilibrium for both polymers that has to be taken into account for future applications.


Assuntos
Acinetobacter/metabolismo , Sistema Livre de Células/metabolismo , Frutanos/biossíntese , Frutanos/química , Nanopartículas/química , Tamanho da Partícula
14.
Biochim Biophys Acta Gene Regul Mech ; 1862(9): 194411, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31356988

RESUMO

Conserved ribosomal protein uS3 contains a decapeptide fragment in positions 55-64 (human numbering), which has a very specific ability to cross-link to various RNA derivatives bearing aldehyde groups, likely provided by K62. It has been shown that during translation in the cell-free protein-synthesizing system, uS3 becomes accessible for such cross-linking only after eIF3j leaves the mRNA binding channel of the 40S ribosomal subunit. We studied the functional role of K62 and its nearest neighbors in the ribosomal assembly and translation with the use of HEK293T-derived cell cultures capable of producing FLAG-tagged uS3 (uS3FLAG) or its mutant form with amino acid residues at positions 60-63 replaced with alanines. Analysis of polysome profiles from the respective cells and cytosol lysates showed that the mutation significantly affected the uS3 ability to participate in the assembly of 40S subunits, but it was not essential for their maturation and did not prevent the binding of mRNAs to 40S subunits during translation initiation. The most striking effect of the replacement of amino acid residues in the above uS3 positions was that it almost completely deprived the 40S subunits of their ability to form 80S ribosomes, suggesting that the 48S pre-initiation complexes assembled on these subunits were defective in the binding of 60S subunits. Thus, our results revealed the previously unknown crucial role of the uS3 tetrapeptide 60GEKG63 in translation initiation related to maintaining the proper structure of the 48S complex, most likely via the prevention of premature mRNA loading into the ribosomal channel.


Assuntos
Peptídeos/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Aminoácidos/química , Aminoácidos/genética , Sistema Livre de Células , Células HEK293 , Humanos , Peptídeos/química , Polirribossomos/química , Polirribossomos/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/química
15.
Curr Opin Biotechnol ; 60: 221-229, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31207555

RESUMO

Conversion of biological feedstocks into value-added chemicals is mostly performed via microbial fermentation. An emerging alternative approach is the use of cell-free systems, consisting of purified enzymes and cofactors. Unfortunately, the in vivo and in vitro research communities rarely interact, which leads to oversimplifications and exaggerations that do not permit fair comparison of the two strategies and impede synergistic interactions. Here, we provide a comprehensive account for the advantages and drawbacks associated with each strategy, and further discuss recent research efforts that aim to breach the limits of cellular and cell-free production. We also explore emerging hybrid solutions that integrate the benefits of both worlds and could expand the boundaries of biosynthesis.


Assuntos
Sistema Livre de Células , Fermentação , Engenharia Metabólica
16.
Biotechnol Adv ; 37(7): 107406, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31200016

RESUMO

Saccharides have recently attracted considerable attention because of their biological functions and potential applications in the pharmaceutical, cosmetic and food industries. Over the decades, a large amount of enzymes involved in saccharide synthesis have been discovered and characterised with the aid of available genome sequences. The advancement of metabolic engineering and synthetic biology strategies facilitated the artificial pathway design and construction for production of multiple sugars in vitro and in vivo based on those characterized enzymes. This review presented a panoramic view of enzymes related to saccharide synthesis and gave the detailed information. Furthermore, we provide an extensive overview of the recent advances in the construction of cell-free reaction systems and engineering of microbial cells for the production of natural or unnatural saccharides. In addition, the future trends in the synthesis of sugars with high structural diversity through the combination of multiple pathways are presented and evaluated.


Assuntos
Carboidratos/biossíntese , Engenharia Metabólica , Biologia Sintética , Sistema Livre de Células
17.
Methods Mol Biol ; 1988: 71-81, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147933

RESUMO

In the endoplasmic reticulum (ER), MHC class I molecules associate with several specialized proteins, forming a large macromolecular complex referred to as the "peptide-loading complex" (PLC). In the PLC, antigenic peptides undergo a stringent selection process that determines which antigen becomes part of the repertoire presented by MHC class I molecules. This ensures that the immune system elicits robust CD8+ T-cell responses to viruses and solid tumors. The ability to reconstitute in vitro MHC class I molecules in association with key proteins of the PLC provides a mean for studying at the molecular level how antigenic peptides are selected for presentation to CD8+ T-cells. Here, we describe practical procedures for generating a cell-free system made up of MHC class I molecules and tapasin that can be used for mechanistic studies of peptide loading and exchange.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Biologia Molecular/métodos , Peptídeos/metabolismo , Sistema Livre de Células , Corpos de Inclusão/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Solubilidade , Microglobulina beta-2/metabolismo
18.
Methods Mol Biol ; 1982: 75-101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172467

RESUMO

The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear. Undoubtedly cis-AA participates in the synergistic multi-partner assembly that can be hardly studied at the molecular level in vivo due to cellular complexity. The capacity of this anionic amphiphilic fatty acid to activate the oxidase is mainly explained by its ability to disrupt intramolecular bonds, mimicking phosphorylation events in cell signaling and therefore allowing protein-protein interactions. Interestingly the geometric isomerism of the fatty acid and its purity are crucial for optimal superoxide production in cell-free assays. Indeed, optimal NADPH oxidase assembly was hampered by the substitution of the cis form by the trans forms of AA isomers (Souabni et al., BBA-Biomembranes 1818:2314-2324, 2012). Structural analysis of the changes induced by these two compounds, by circular dichroism and by biochemical methods, revealed differences in the interaction between subunits. We describe how the specific geometry of AA plays an important role in the activation of the NOX2 complex.


Assuntos
Ácido Araquidônico/metabolismo , NADPH Oxidases/metabolismo , Fagócitos/enzimologia , Ácido Araquidônico/química , Fracionamento Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Sistema Livre de Células , Colorimetria , Ativação Enzimática , Isomerismo , Estrutura Molecular , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/química , NADPH Oxidases/isolamento & purificação , Neutrófilos/enzimologia , Fagócitos/imunologia , Proteínas Recombinantes de Fusão , Análise Espectral
19.
Methods Mol Biol ; 1982: 103-111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172468

RESUMO

NADPH oxidases (NOX) are a family of transmembrane enzymes, which catalyze the formation of O2 ˙- and H2O2. Membrane fractions of leukocytes are highly enriched in the phagocyte NOX isoform (NOX2). This feat has allowed the development of a complex NOX2 cell-free assay, which has been a key tool for the understanding of the mode of action of NOX2, its biochemistry, pharmacology, and identification of NOX2-specific inhibitors. In addition to NOX2, there are six other NOX isoforms in humans, but cell-free assays of non-phagocytic oxidases are infrequently used, and their specificity has recently been debated. Here we describe a NOX5 cell-free assay. We present a method to purify the membranous component of cells stably transduced with NOX5 and to measure O2 ˙- in a high-throughput format (96-w or 384-w plates). The experimental description allows high-throughput screening of small molecules with limited cost.


Assuntos
Sistema Livre de Células , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , NADPH Oxidase 5/antagonistas & inibidores , Fracionamento Celular , Membrana Celular/enzimologia , Sistema Livre de Células/enzimologia , Descoberta de Drogas , Inibidores Enzimáticos/química , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , NADPH Oxidase 5/química , NADPH Oxidase 5/genética , NADPH Oxidase 5/metabolismo , Oxirredução , Espécies Reativas de Oxigênio , Bibliotecas de Moléculas Pequenas , Análise Espectral
20.
Elife ; 82019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31172942

RESUMO

Applying pre-steady state kinetics to an Escherichia-coli-based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.


Assuntos
Antibacterianos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Viomicina/farmacologia , Sistema Livre de Células
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA