RESUMO
OBJECTIVES: Our aim was to investigate pathophysiology of pseudoaneurysm of the thoracic aorta, an acute or chronic pathology, respectively, secondary to blunt thoracic trauma and aortitis, or complicating a deep penetrating aortic ulcer, intraparietal hematoma, aortic aneurysm, and even aortic graft, often with atherosclerosis as a common background. METHODS: Given the relative rarity of this disease, an "inductive" retrospective method made it possible to retrieve clinical, radiological, and histopathological elements, which were mutually compared and validated through a "deductive" process of reinterpretation. RESULTS: We have identified three main structural constituents of this disease: a cavity, a single blood entry port, communicating with the aortic lumen, and a pseudocapsule. It is often caused by a chronic degenerative pathology of the intima and medial layers of the aorta, typically involving elastic fibers and smooth muscle cells, with possible intermediate stages of deep aortic ulcer or intraparietal hematoma. Otherwise, the acute onset may be secondary to acute aortitis or aortic injury. CONCLUSIONS: Today, thanks to the current angiographic tools represented by 3-D high resolution multidetector CT and MRI angiography, the diagnosis of thoracic aortic pseudoaneurysm is easier, as well as its surgical indications.
RESUMO
The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.
Assuntos
Arildialquilfosfatase , Proteômica , Arildialquilfosfatase/metabolismo , Arildialquilfosfatase/química , Humanos , Proteômica/métodos , Redobramento de Proteína , Pseudomonas aeruginosa/enzimologia , Estabilidade Enzimática , Biofilmes , Processamento de Proteína Pós-TraducionalRESUMO
Pseudomonas aeruginosa is one of the six antimicrobial-resistant pathogens known as "ESKAPE" that represent a global threat to human health and are considered priority targets for the development of novel antimicrobials and alternative therapeutics. The virulence of P. aeruginosa is regulated by a four-chemicals communication system termed quorum sensing (QS), and one main class of QS signals is termed acylhomoserine lactones (acyl-HSLs), which includes 3-Oxo-dodecanoil homoserine lactone (3-Oxo-C12-HSL), which regulates the expression of genes implicated in virulence and biofilm formation. Lactonases, like Paraoxonase 2 (PON2) from humans and the phosphotriesterase-like lactonases (PLLs) from thermostable microorganisms, are able to hydrolyze acyl-HSLs. In this work, we explored in vitro and in an animal model the effect of some lactonases on the production of Pseudomonas virulence factors. This study presents a model of chronic infection in which bacteria were administered by feeding, and Drosophila adults were treated with enzymes and the antibiotic tobramycin, alone or in combination. In vitro, we observed significant effects of lactonases on biofilm formation as well as effects on bacterial motility and the expression of virulence factors. The treatment in vivo by feeding with the lactonase SacPox allowed us to significantly increase the biocidal effect of tobramycin in chronic infection.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Animais , Drosophila melanogaster/metabolismo , Infecção Persistente , Biofilmes , Percepção de Quorum , Fatores de Virulência/genética , Lactonas/farmacologia , Bactérias/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Tobramicina/farmacologiaRESUMO
The widespread use of pesticides in the last decades and their accumulation into the environment gave rise to major environmental and human health concerns. To address this topic, the scientific community pointed out the need to develop methodologies to detect and measure the presence of pesticides in different matrices. Biosensors have been recently explored as fast, easy, and sensitive methods for direct organophosphate pesticides monitoring. Thus, the present work aimed at designing and testing a 3D printed adapter useful on different equipment, and a membrane support to immobilize the esterase-2 from Alicyclobacillus acidocaldarius (EST2) bioreceptor. The latter is labelled with the IAEDANS, a bright fluorescent probe. EST2 was selected since it shows a high specificity toward paraoxon. Our results showed good stability and replicability, with an increasing linear fluorescent intensity recorded from 15 to 150 pmol of labelled EST2. Linearity of data was also observed when using the immobilized labelled EST2 to detect increasing amounts of paraoxon, with a limit of detection (LOD) of 0.09 pmol. This LOD value reveals the high sensitivity of our membrane support when mounted on the 3D adapter, comparable to modern methods using robotic workstations. Notably, the use of an independent support significantly simplified the manipulation of the membrane during experimental procedures and enabled it to match the specificities of different systems. In sum, this work emphasizes the advantages of using 3D printed accessories adapted to respond to the newest research needs.
Assuntos
Enzimas Imobilizadas/metabolismo , Esterases/metabolismo , Compostos Organofosforados/análise , Praguicidas/análise , Impressão Tridimensional , FluorescênciaRESUMO
Surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry is a variant of the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. It is used in many cases especially for the analysis of protein profiling and for preliminary screening of biomarkers in complex samples. Unfortunately, these analyses are time consuming and protein identification is generally strictly limited. SELDI-TOF analysis of mass spectra (SELYMATRA) is a web application (WA) developed to reduce these limitations by (i) automating the identification processes and (ii) introducing the possibility to predict proteins in complex mixtures from cells and tissues. The WA architectural pattern is the model-view-controller, commonly used in software development. The WA compares the mass value between two mass spectra (sample vs. control) to extract differences, and, according to the set parameters, it queries a local database to predict most likely proteins based on their masses and different expression amplification. The WA was validated in a cellular model overexpressing a tagged NURR1 receptor, being able to recognize the tagged protein in the profiling of transformed cells. A help page, including a description of parameters for WA use, is available on the website.
Assuntos
Análise Serial de Proteínas , Proteínas , Análise Serial de Proteínas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas/análise , Biomarcadores/análise , SoftwareRESUMO
The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser-His-Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.
Assuntos
Técnicas Biossensoriais , Inseticidas , Praguicidas , Ecossistema , Transferência Ressonante de Energia de Fluorescência , Paraoxon/toxicidade , Praguicidas/análiseRESUMO
The DING proteins are ubiquitous in the three domains of life, from mesophiles to thermo- and hyperthermophiles. They belong to a family of more than sixty members and have a characteristic N-terminus, DINGGG, which is considered a "signature" of these proteins. Structurally, they share a highly conserved phosphate binding site, and a three dimensional organization resembling the "Venus Flytrap", both reminding the ones of PstS proteins. They have unusually high sequence conservation, even between distantly related species. Nevertheless despite that the genomes of most of these species have been sequenced, the DING gene has not been reported for all the relative characterized DING proteins. Identity of known DING proteins has been confirmed immunologically and, in some cases, by N-terminal sequence analysis. Only a few of the DING proteins have been purified and biochemically characterized. DING proteins are heterogeneous for their wide range of biological activities and some show different activities not always correlated with each other. Most of them have been originally identified for different biological properties, or rather for binding to phosphate and also to other ligands. Their involvement in pathologies is described. This review is an update of the most recent findings on old and new DING proteins.
Assuntos
Extremófilos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Sequência de Aminoácidos , Archaea/metabolismo , Eucariotos/metabolismo , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/genéticaRESUMO
Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.
Assuntos
Técnicas Biossensoriais/métodos , Organofosfatos/química , Compostos Organofosforados/química , Robótica/métodos , Alicyclobacillus/química , Bioensaio/métodos , Monitoramento Ambiental/métodos , Esterases/química , Fluorescência , Limite de Detecção , Praguicidas/químicaRESUMO
Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M-1 s-1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.
Assuntos
Arildialquilfosfatase/química , Arildialquilfosfatase/metabolismo , Proteínas Mutantes/metabolismo , Multimerização Proteica , Sulfolobus solfataricus/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Dicroísmo Circular , Evolução Molecular Direcionada , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Íons , Metais/química , Modelos Moleculares , Agentes Neurotóxicos/química , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , TemperaturaRESUMO
Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL-1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.
Assuntos
Paraoxon/urina , Técnicas Biossensoriais/métodos , Ensaios Enzimáticos/métodos , Fluorescência , Humanos , Inseticidas/urina , Nitrofenóis/urina , Organofosfatos/urina , Compostos Organofosforados/urina , Praguicidas/urinaRESUMO
BACKGROUND & AIMS: Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. METHODS: Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. RESULTS: Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. CONCLUSION: PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure. LAY SUMMARY: Acute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival.
Assuntos
Isocumarinas/farmacologia , L-Lactato Desidrogenase/metabolismo , Falência Hepática Aguda , Fígado , Complexo Piruvato Desidrogenase/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Falência Hepática Aguda/tratamento farmacológico , Falência Hepática Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. RESULTS: Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L- 1 and 47.1 U·L- 1·h- 1 for SacPox and to 8700 U·L- 1 and 180.6 U·L- 1·h- 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications. CONCLUSIONS: In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.
Assuntos
Hidrolases de Triester Fosfórico/metabolismo , Proteínas Recombinantes/isolamento & purificação , Sulfolobus acidocaldarius/enzimologia , Sulfolobus solfataricus/enzimologia , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Técnicas de Cultura Celular por Lotes/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Biodegradação Ambiental , Precipitação Química , Cromatografia em Gel/métodos , Estabilidade Enzimática , Escherichia coli/genética , Fermentação , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfolobus acidocaldarius/genética , Sulfolobus solfataricus/genética , Ultrafiltração/métodosRESUMO
The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability, and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membranes seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer, and to provide a microenvironment with tuned physicochemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme ( SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other classes of enzymes and related applications.
Assuntos
Reatores Biológicos , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Hidrolases de Triester Fosfórico/química , Sulfolobus solfataricus/enzimologia , Biocatálise , Enzimas Imobilizadas/metabolismo , Desenho de Equipamento , Interações Hidrofóbicas e Hidrofílicas , Cinética , Membranas Artificiais , Hidrolases de Triester Fosfórico/metabolismo , Polivinil/química , Sulfolobus solfataricus/química , Sulfolobus solfataricus/metabolismoRESUMO
DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.
Assuntos
Proteínas Arqueais/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Pseudomonas fluorescens/enzimologia , Sulfolobus solfataricus/enzimologia , Proteínas Arqueais/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Poli(ADP-Ribose) Polimerases/química , Domínios Proteicos , Homologia de SequênciaRESUMO
Thermostable phosphotriesterase-like lactonases (PLLs) from extremophile archaea, like SsoPox from Sulfolobus solfataricus, are attractive biotechnological tools with industrial applications as organophosphate decontaminants, but their manufacturing still remains an unresolved issue because of the high costs and the low production yields. In this paper, for the first time, an efficient biotechnological process for the production and purification of a recombinant, engineered PLL, SsoPox W263F, expressed in E. coli, has been set up by studying new induction strategies, by designing high cell density cultivations and a new membrane-based downstream process. In fed batches, the enzyme production was boosted of 69-fold up to 4660.0 U L-1 using galactose as inducer in the replacement of IPTG; the process was scalable from 2.5 up to 150 L. By coupling a single thermo-precipitation step and an ultrafiltration process, a total enzyme recovery of 77% with a purity grade of almost 80% was reached.
Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Genes Arqueais , Organofosfatos/química , Hidrolases de Triester Fosfórico/biossíntese , Sulfolobus solfataricus/genética , Técnicas de Cultura Celular por Lotes , Meios de Cultura/química , Descontaminação , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Microbiologia Industrial , Microrganismos Geneticamente Modificados , Engenharia de Proteínas , Sulfolobus solfataricus/metabolismoRESUMO
In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1). The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces.
Assuntos
Descontaminação/métodos , Praguicidas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Sulfolobus solfataricus/enzimologia , Sequência de Aminoácidos , Biotransformação , Evolução Molecular Direcionada , Hidrólise , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Conformação Proteica , Sulfolobus solfataricus/genéticaRESUMO
The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two α-D-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl ß-D-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3',5'-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity.
Assuntos
Alicyclobacillus/enzimologia , Proteínas de Bactérias/química , Esterases/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Esterases/metabolismo , Glucose/análogos & derivados , Glucose/química , Temperatura Alta , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Especificidade por SubstratoRESUMO
Pyruvate dehydrogenase complex (PDHC) is a key enzyme in metabolism linking glycolysis to tricarboxylic acid cycle and its activity is tightly regulated by phosphorylation catalyzed by four pyruvate dehydrogenase kinase (PDK) isoforms. PDKs are pharmacological targets for several human diseases including cancer, diabetes, obesity, heart failure, and inherited PDHC deficiency. We investigated the inhibitory activity of phenylbutyrate toward PDKs and found that PDK isoforms 1-to-3 are inhibited whereas PDK4 is unaffected. Moreover, docking studies revealed putative binding sites of phenylbutyrate on PDK2 and 3 that are located on different sites compared to dichloroacetate (DCA), a previously known PDK inhibitor. Based on these findings, we showed both in cells and in mice that phenylbutyrate combined to DCA results in greater increase of PDHC activity compared to each drug alone. These results suggest that therapeutic efficacy can be enhanced by combination of drugs increasing PDHC enzyme activity.
Assuntos
Ácido Dicloroacético/farmacologia , Fenilbutiratos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Complexo Piruvato Desidrogenase/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Ácido Dicloroacético/química , Ácido Dicloroacético/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenilbutiratos/química , Fenilbutiratos/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Complexo Piruvato Desidrogenase/antagonistas & inibidores , Complexo Piruvato Desidrogenase/química , Doença da Deficiência do Complexo de Piruvato Desidrogenase/metabolismoRESUMO
Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.
Assuntos
Técnicas Biossensoriais , Substâncias para a Guerra Química/isolamento & purificação , Esterases/química , Organofosfatos/isolamento & purificação , Bacillus/enzimologia , Substâncias para a Guerra Química/toxicidade , Humanos , Inseticidas , Organofosfatos/toxicidade , Paraoxon , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Automatic exposure control (AEC) plays a crucial role in mammography by determining the exposure conditions needed to achieve specific image quality based on the absorption characteristics of compressed breasts. This study aimed to characterize the behavior of AEC for digital mammography (DM), digital breast tomosynthesis (DBT), and low-energy (LE) and high-energy (HE) acquisitions used in contrast-enhanced mammography (CEM) for three mammography systems from two manufacturers. METHODS: Using phantoms simulating various breast thicknesses, 363 studies were acquired using all available AEC modes 165 DM, 132 DBT, and 66 LE-CEM and HE-CEM. AEC behaviors were compared across systems and modalities to assess the impact of different technical components and manufacturers' strategies on the resulting mean glandular doses (MGDs) and image quality metrics such as contrast-to-noise ratio (CNR). RESULTS: For all systems and modalities, AEC increased MGD for increasing phantom thicknesses and decreased CNR. The median MGD values (interquartile ranges) were 1.135 mGy (0.772-1.668) for DM, 1.257 mGy (0.971-1.863) for DBT, 1.280 mGy (0.937-1.878) for LE-CEM, and 0.630 mGy (0.397-0.713) for HE-CEM. Medians CNRs were 14.2 (7.8-20.2) for DM, 4.91 (2.58-7.20) for a single projection in DBT, 11.9 (8.0-18.2) for LE-CEM, and 5.2 (3.6-9.2) for HE-CEM. AECs showed high repeatability, with variations lower than 5% for all modes in DM, DBT, and CEM. CONCLUSIONS: The study revealed substantial differences in AEC behavior between systems, modalities, and AEC modes, influenced by technical components and manufacturers' strategies, with potential implications in radiation dose and image quality in clinical settings. RELEVANCE STATEMENT: The study emphasized the central role of automatic exposure control in DM, DBT, and CEM acquisitions and the great variability in dose and image quality among manufacturers and between modalities. Caution is needed when generalizing conclusions about differences across mammography modalities. KEY POINTS: ⢠AEC plays a crucial role in DM, DBT, and CEM. ⢠AEC determines the "optimal" exposure conditions needed to achieve specific image quality. ⢠The study revealed substantial differences in AEC behavior, influenced by differences in technical components and strategies.