RESUMO
Carboxylesterase 1 (CES1) is the predominant carboxylesterase in the human liver, involved in metabolism of both xenobiotics and endogenous substrates. Genetic or epigenetic factors that alter CES1 activity or expression are associated with changes in drug response, lipid, and glucose homeostasis. However, the transcriptional regulation of CES1 in the human liver remains uncertain. By applying both the random forest and Sobol's Sensitivity Indices (SSI) to analyze existing liver RNA expression microarray data (GSE9588), we identified nuclear receptor subfamily 1 group H member 3 (NR1H3) liver X receptor (LXR)α as a key factor regulating constitutive CES1 expression. This model prediction was validated using small interfering RNA (siRNA) knockdown and CRISPR-mediated transcriptional activation of NR1H3 in Huh7 and HepG2 cells. We found that NR1H3's activation of CES1 is splice isoform-specific, namely that increased expression of the NR1H3-211 isoform increased CES1 expression whereas NR1H3-201 did not. Also, in human liver samples, expression of NR1H3-211 and CES1 are correlated, whereas NR1H3-201 and CES1 are not. This trend also occurs during differentiation of induced pluripotent stem cells (iPSCs) to hepatocytes, where only expression of the NR1H3-211 isoform parallels expression of CES1 Moreover, we found that treatment with the NR1H3 agonist T0901317 in HepG2 cells had no effect on CES1 expression. Overall, our results demonstrate a key role of NR1H3 in maintaining the constitutive expression of CES1 in the human liver. Furthermore, our results support that the effect of NR1H3 is splice isoform-specific and appears to be ligand independent. SIGNIFICANCE STATEMENT: Despite the central role of carboxylesterase 1 (CES1) in metabolism of numerous medications, little is known about its transcriptional regulation. This study identifies nuclear receptor subfamily 1 group H member 3 as a key regulator of constitutive CES1 expression and therefore is a potential target for future studies to understand interperson variabilities in CES1 activity and drug metabolism.
Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Receptores X do Fígado/genética , Receptores X do Fígado/fisiologia , Fígado/enzimologia , Idoso , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Isoenzimas/genética , Isoenzimas/metabolismo , Receptores X do Fígado/agonistas , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno , Ativação Transcricional/genéticaRESUMO
Melatonin has been indicated to ameliorate tau hyperphosphorylation in the pathogenesis of tau diseases, but the role of melatonin-receptor signal transduction has not been clearly discovered. In this study, we found intensive tau hyperphosphorylation in melatonin receptor knockout mice. Bielschowsky silver staining showed ghostlike neurofibrillary tangles in melatonin receptor-2 knockout (MT2KO) as well as melatonin receptors-1 and -2 knockout (DKO) mice, and an argyrophilic substance was deposited in melatonin receptor-1 knockout (MT1KO) mice. Furthermore, we found significantly decreased activity of protein phosphatase 2A (PP2A) by Western blot and enzyme-linked immunosorbent assay (ELISA), which was partly due to the overexpression of protein phosphatase methylesterase-1 (PME-1), but not glycogen synthase kinase-3ß (GSK-3ß), cyclin-dependent kinase 5 (CDK5) or protein kinase B (Akt). Finally, we observed a significant increase in cyclic adenosine monophosphate (cAMP) and a decrease in miR-125b-5p levels in MT1KO, MT2KO and DKO mice. Using a luciferase reporter assay, we discovered that miR-125b-5p largely decreased the expression of firefly luciferase by interfering with the 3'UTR of PME-1. Furthermore, miR-125b-5p mimics significantly decreased the expression of PME-1, while miR-125b-5p inhibitor induced tau hyperphosphorylation. These results show that melatonin-receptor signal transduction plays an important role in tau hyperphosphorylation and tangle formation.
Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Regulação Enzimológica da Expressão Gênica , MicroRNAs/metabolismo , Receptores de Melatonina/deficiência , Proteínas tau/metabolismo , Animais , Hidrolases de Éster Carboxílico/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Fosforilação , Receptores de Melatonina/metabolismo , Proteínas tau/genéticaRESUMO
Cholinesterases act as bio scavengers to clear organophosphorus (OP) compounds and prodrugs. The butyrylcholinesterase (BChE) gene has been found in several types of teleost fish but this gene has yet to be identified in cyprinid fish. Indeed, BChE homologs have not been found in the zebrafish (Danio rerio) genomic database. Here, we demonstrate that BChE activity is present in zebrafish, in line with other groups' findings. Using in-gel native-PAGE enzymatic activity staining and LC-MS/MS technique, an atypical BChE-like protein was identified in zebrafish. The si:ch211-93f2.1 gene was cloned, and His-tagged recombinant protein was expressed using the Pichia yeast system. The purified protein (molecular weight ~ 180 kDa) showed BChE activity, and degraded acetylcholinesterase (ACh) at a higher rate than BCh. However, phylogram analysis shows that this novel cholinesterase shared an evolutionary origin with carboxylic esterase rather than BChE. The zebrafish BChE-like protein shares structural characteristics with cholinesterase and carboxylesterase. The 2-arachidonoylglycerol (2-AG), nicosulfuron, and triacetin exhibited a higher binding affinity to the zebrafish BChE-like protein than BCh and ACh. With the identification of BChE-like protein in zebrafish, this study could shed light on the origin of BChE and may contribute towards the development of a BChE knockout zebrafish model for sensitive drug or toxin screening.
Assuntos
Hidrolases de Éster Carboxílico , Clonagem Molecular , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Domínios Proteicos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
The current study heterologously expressed a cutinase from Fusarium verticillioides by Pichia pastoris and investigated its properties and effects on the hydrolysis of rice straw. The optimal pH and temperature for F. verticillioides cutinase were 8.0 and 50 °C, respectively. F. verticillioides cutinase had poor thermal stability and could be inhibited by some metal ions, inhibitors, and detergents (5 mM), including Ni2+, Zn2+, Cu2+, Ca2+, Mn2+, sodium dodecyl sulfate, EDTA, and Tween-20. F. verticillioides cutinase could tolerate 15% methanol and dimethyl sulfoxide but was significantly repressed by 15% ethanol and acetone with 48% and 63% residual activity, respectively. F. verticillioides cutinase could degrade the cuticle of rice straw with palmitic acid and stearic acid as the main products. However, the dissolving sugars released from the rice straw treated with F. verticillioides cutinase were significantly reduced by 29.2 µg/mL compared with the control (107.9 µg/mL). Similarly, the reducing sugars produced from the cellulase hydrolysis of rice straw pretreated with F. verticillioides cutinase were reduced by 63.5 µg/mL relative to the control (253.6 µg/mL). Scanning electron microscopy results showed that numerous tuberculate or warty protrusions were present nearly everywhere on the surface of rice straw treated with F. verticillioides cutinase, and some protrusions even covered and blocked the stomata of the rice straw surface. Current limited data indicate that F. verticillioides cutinase might not be an appropriate choice for improving the utilization of agricultural straws.
Assuntos
Hidrolases de Éster Carboxílico/farmacologia , Proteínas Fúngicas/farmacologia , Fusarium/enzimologia , Oryza , Caules de Planta/efeitos dos fármacos , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/genética , Celulase/farmacologia , Detergentes/farmacologia , Ácidos Graxos/isolamento & purificação , Fermentação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Hidrólise , Microbiologia Industrial/métodos , Metais/farmacologia , Oryza/química , Caules de Planta/química , Proteínas Recombinantes/farmacologia , Solventes/farmacologia , Açúcares/isolamento & purificaçãoRESUMO
Aureobasidium pullulans has been observed as one of the most abundant species in freshly pressed grape juice. Despite this, little is known about the consequences for the wine-making process associated with the presence and proliferation of this fungus, including its interaction with other ferment-derived microorganisms and impact on the composition of the resulting wine. In this study, the physiology of abundant A. pullulans grape juice isolates was investigated through lab scale fermentation trials, demonstrating the ability of this species to survive in grape juice while producing polysaccharides, polymers of malic acid (poly ß-malic acid) and enzymes with pectinase, ß - glucosidase and tannase activity. A possible antagonistic effect against yeast through competition for metals including Fe and Zn was also observed. Overall, the data suggests this abundant species could have important implications for wine production and quality.
Assuntos
Ascomicetos/fisiologia , Fermentação , Sucos de Frutas e Vegetais/análise , Sucos de Frutas e Vegetais/microbiologia , Vitis/microbiologia , Ascomicetos/enzimologia , Hidrolases de Éster Carboxílico/biossíntese , Polissacarídeos Fúngicos/biossíntese , Ferro/metabolismo , Poligalacturonase/biossíntese , Vinho/microbiologia , Zinco/metabolismo , beta-Glucosidase/biossínteseRESUMO
BACKGROUND: Gallic acid has received a significant amount of interest for its biological properties. Thus, there have been recent attempts to apply this substance in various industries and in particular the feed industry. As opposed to yeasts, fungi and bacteria and their tannases have been well documented for their potential bioconversion and specifically for the biotransformation of tannic acid to gallic acid. In this research, Sporidiobolus ruineniae A45.2 is introduced as a newly pigment-producing and tannase-producing yeast that has gained great interest for its use as an additive in animal feed. However, there is a lack of information on the efficacy of gallic acid production from tannic acid and the relevant tannase properties. The objective of this research study is to optimize the medium composition and conditions for the co-production of gallic acid from tannic acid and tannase with a focus on developing an integrated production strategy for its application as a feed additive. RESULTS: Tannase produced by S. ruineniae A45.2 has been classified as a cell-associated tannase (CAT). Co-production of gallic acid obtained from tannic acid and CAT by S. ruineniae A45.2 was optimized using response surface methodology and then validated with the synthesis of 11.2 g/L gallic acid from 12.3 g/L tannic acid and the production of 31.1 mU/mL CAT after 48 h of cultivation in a 1-L stirred tank fermenter. Tannase was isolated from the cell wall, purified and characterized in comparison with its native form (CAT). The purified enzyme (PT) revealed the same range of pH and temperature optima (pH 7) as CAT but was distinctively less stable. Specifically, CAT was stable at up to 70 °C for 60 min, and active under its optimal conditions (40 °C) at up to 8 runs. CONCLUSION: Co-production of gallic acid and CAT is considered an integrated and green production strategy. S. ruineniae biomass could be promoted as an alternative source of carotenoids and tannase. Thus, the biomass, in combination with gallic acid that was formed in the fermentation medium, could be directly used as a feed additive. On the other hand, gallic acid could be isolated and purified for food and pharmaceutical applications. This paper is the first of its kind to report that the CAT obtained from yeast can be resistant to high temperatures of up to 70 °C.
Assuntos
Basidiomycota/metabolismo , Hidrolases de Éster Carboxílico/biossíntese , Ácido Gálico/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Fermentação , Ácido Gálico/químicaRESUMO
Beta-amyloid (Aß) is thought to play a critical role in Alzheimer's disease (AD), and application of soluble oligomeric forms of Aß produces AD-like impairments in cognition and synaptic plasticity in experimental systems. We found previously that transgenic overexpression of the PP2A methylesterase, PME-1, or the PP2A methyltransferase, LCMT-1, altered the sensitivity of mice to Aß-induced impairments, suggesting that PME-1 inhibition may be an effective approach for preventing or treating these impairments. To explore this possibility, we examined the behavioral and electrophysiological effects of acutely applied synthetic Aß oligomers in male and female mice heterozygous for either a PME-1 KO or an LCMT-1 gene-trap mutation. We found that heterozygous PME-1 KO mice were resistant to Aß-induced impairments in cognition and synaptic plasticity, whereas LCMT-1 gene-trap mice showed increased sensitivity to Aß-induced impairments. The heterozygous PME-1 KO mice produced normal levels of endogenous Aß and exhibited normal electrophysiological responses to picomolar concentrations of Aß, suggesting that reduced PME-1 expression in these animals protects against Aß-induced impairments without impacting normal physiological Aß functions. Together, these data provide additional support for roles for PME-1 and LCMT-1 in regulating sensitivity to Aß-induced impairments, and suggest that inhibition of PME-1 may constitute a viable therapeutic approach for selectively protecting against the pathologic actions of Aß in AD.SIGNIFICANCE STATEMENT Elevated levels of ß-amyloid (Aß) in the brain are thought to contribute to the cognitive impairments observed in Alzheimer's disease patients. Here we show that genetically reducing endogenous levels of the PP2A methylesterase, PME-1, prevents the cognitive and electrophysiological impairments caused by acute exposure to pathologic concentrations of Aß without impairing normal physiological Aß function or endogenous Aß production. Conversely, reducing endogenous levels of the PP2A methyltransferase, LCMT-1, increases sensitivity to Aß-induced impairments. These data offer additional insights into the molecular factors that control sensitivity to Aß-induced impairments, and suggest that inhibiting PME-1 may constitute a viable therapeutic avenue for preventing Aß-related impairments in Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Hidrolases de Éster Carboxílico/biossíntese , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/enzimologia , Proteína O-Metiltransferase/biossíntese , Animais , Hidrolases de Éster Carboxílico/genética , Disfunção Cognitiva/fisiopatologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos/fisiologia , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Proteína O-Metiltransferase/genéticaRESUMO
There are numerous reports on poly-ß-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30°C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg-1mL-1). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the natural soil condition is the result of the activity of soil microbes that complemented the biodegradation of PHB by Stenotrophomonas sp. RZS7.
Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Hidroxibutiratos/química , Plásticos/química , Poliésteres/química , Poluentes do Solo/química , Stenotrophomonas/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Biodegradação Ambiental , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/isolamento & purificação , Cromatografia de Afinidade , Meios de Cultura/química , Humanos , Hidrólise , Peso Molecular , Proibitinas , Solo/química , Resíduos Sólidos , Stenotrophomonas/químicaRESUMO
Feruloyl esterases (FAEs) have great potential applications in paper and breeding industry. A new thermo-stable feruloyl esterase gene, TtfaeB was identified from the thermophilic fungus Thielavia terrestris h408. Deduced protein sequence shares the identity of 67% with FAEB from Neurospora crassa. The expression vector pPIC9K-TtfaeB was successfully constructed and electro-transformed into GS115 strain of Pichia pastoris. One transformant with high feruloyl esterase yield was obtained through plate screening and named TtFAEB1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TtFAEB1 showed a distinct protein band appearing at the position of about 35-kDa, indicating that TtfaeB gene has been successfully expressed in P. pastoris. The recombinant TtFAEB was purified by affinity chromatography and the specific activity of purified TtFAEB was 6.06 ± 0.72 U/mg. The optimal temperature and pH for purified recombinant TtFAEB was 60 °C and 7.0, respectively. TtFAEB was thermostable, retaining 96.89 and 84.16% of the maximum activity after being treated for 1 h at 50 °C and 60 °C, respectively. Additionally, the enzyme was stable in the pH range 4.5-8.0. The homology model of TtFAEB showed that it consists of a single domain adopting a typical α/ß-hydrolase fold and contains a catalytic triad formed by Ser117, Asp201, and His260. TtFAEB in association with xylanase from Trichoderma reesei could release 77.1% of FA from destarched wheat bran. The present results indicated that the recombinant TtFAEB with excellent enzymatic properties is a promising candidate for potential applications in biomass deconstruction and biorefinery.
Assuntos
Hidrolases de Éster Carboxílico , Clonagem Molecular , Proteínas Fúngicas , Sordariales , Biomassa , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sordariales/enzimologia , Sordariales/genéticaRESUMO
BACKGROUND: Remimazolam is an ultra-short acting benzodiazepine under development for procedural sedation and general anesthesia. It is hydrolyzed by CES1 to an inactive metabolite (CNS7054). PURPOSE: In this study, the effect of continuous remimazolam exposure on its metabolism and on CES1 expression was investigated in a dynamic 3-D bioreactor culture model inoculated with primary human hepatocytes. METHODS: Remimazolam was continuously infused into bioreactors for 5 days at a final concentration of 3,000 ng/ml (6.8 µM). In parallel, 2-D cultures were run with cells from the same donors, but with discontinuous exposure to remimazolam. RESULTS: Daily measurement of clinical chemistry parameters (glucose, lactate, urea, ammonia, and liver enzymes) in culture supernatants indicated no noxious effect of remimazolam on hepatocyte integrity as compared to untreated controls. Concentrations of remimazolam reached steady-state values of around 250 ng/ml within 8 hours in 3-D bioreactors whereas in 2-D cultures remimazolam concentrations declined to almost zero within the same time frame. Levels of CNS7054 showed an inverse time-course reaching average values of 1,350 ng/ml in perfused 3-D bioreactors resp. 2,800 ng/ml in static 2-D cultures. Analysis of mRNA expression levels of CES1 indicated no changes in gene expression over the culture period. CONCLUSION: The results indicated a stable metabolism of remimazolam during 5 days of continuous exposure to clinically relevant concentrations of the drug. Moreover, there was no evidence for a harmful effect of remimazolam exposure on the integrity and metabolic activity of in vitro cultivated primary human hepatocytes.
Assuntos
Benzodiazepinas/metabolismo , Reatores Biológicos , Hepatócitos/metabolismo , Benzodiazepinas/administração & dosagem , Benzodiazepinas/farmacologia , Hidrolases de Éster Carboxílico/biossíntese , Hepatócitos/efeitos dos fármacos , HumanosRESUMO
Coenzyme CoQ10 (CoQ10), a ubiquinone compound, has been reported to inhibit tyrosinase activity and melanin production in melanoma B16F10 cells. However, the molecular mechanism underlying this inhibitory effect is poorly understood. In this paper we aimed to investigate the molecular mechanisms involved in the anti-melanogenic activity of CoQ10 (1-2⯵M) in UVA (5â¯J/cm2)-irradiated keratinocyte HaCaT cells and α-MSH stimulated B16-F10 cells. It was observed that CoQ10 suppressed p53/POMC, α-MSH production as well as inhibited ROS generation in UVA-irradiated keratinocyte HaCaT cells. CoQ10 down-regulated the melanin synthesis in α-MSH-stimulated B16-F10 cells by suppressing the MITF expression by down regulating the cAMP mediated CREB signaling cascades. Furthermore, in vivo evidence demonstrated the inhibitory effect of CoQ10 on endogenous pigmentation in zebrafish. Increased nuclear Nrf2 translocation accompanied by the induction of HO-1 and γ-GCLC genes were observed in CoQ10 treated keratinocyte HaCaT cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished CoQ10-mediated anti-melanogenic activity, as evidenced by impaired antioxidant HO-1 gene, uncontrolled ROS generation, and α-MSH production following UVA irradiation. To conclude, CoQ10 is an effective de-pigmention or skin-whitening agent and could be used in cosmetics for topical application.
Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/biossíntese , Preparações Clareadoras de Pele/farmacologia , Ubiquinona/análogos & derivados , Raios Ultravioleta , alfa-MSH/metabolismo , Animais , Antioxidantes/farmacologia , Hidrolases de Éster Carboxílico/genética , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Melanoma Experimental/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Ubiquinona/farmacologia , Peixe-Zebra , alfa-MSH/antagonistas & inibidoresRESUMO
Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP)2-CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP)2-CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105⯱â¯7â¯h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP)2-CocH3 has a ~21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP)2-CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.
Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Fragmentos Fc das Imunoglobulinas/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Animais , Células CHO , Hidrolases de Éster Carboxílico/genética , Cricetulus , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genéticaRESUMO
In the present study, it was presented a strategy to maximize the cutinase production by solid-state fermentation from different microorganisms and substrates. The best results were observed using Fusarium verticillioides, rice bran being the main substrate. Maximum yield of cutinase obtained by the strain was 16.22 U/g. For concentration, ethanol precipitation was used, and the purification factor was 2.4. The optimum temperature and pH for enzyme activity were 35 °C and 6.5, respectively. The enzyme was stable at a wide range of temperature and at all pH values tested. The concentrated cutinase was used as an adjuvant in a formulation containing cutinase + bioherbicide. The use of enzyme increased the efficiency of bioherbicide, since cutinase was responsible to remove/degrade the cutin that recovery the weed leaves and difficult the bioherbicide absorption. Cutinase showed to be a promising product to be used in formulation of bioherbicides.
Assuntos
Hidrolases de Éster Carboxílico , Proteínas Fúngicas , Fusarium/enzimologia , Herbicidas/metabolismo , Controle Biológico de Vetores , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Herbicidas/química , Concentração de Íons de Hidrogênio , Oryza/químicaRESUMO
The present investigation evaluates the protective effect of vorinostat on neuronal cells in the traumatic brain injury (TBI) and also postulates the possible mechanism of its action. Marmarou's weight-drop model was used to induce the TBI. Further, animals were treated with vorinostat 100 mg/kg intraperitoneally 30 min before the TBI induction. Neurological score and brain water content were determined in all the groups and thereafter oxidative stress parameters and adenosine triphosphate (ATP) content were determined in the neuronal tissues of TBI mice. Western blot assay and reverse transcription polymerase chain reaction (RT-PCR) was performed for the determination of the expression of several proteins in the neuronal tissues. Moreover, immunohistochemical staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was also done on the neuronal tissues of TBI mice. Data of the study reveal that treatment with vorinostat significantly reduces the altered level of grip test scores and water content in the brain of traumatic injured mice. Moreover, the altered level of oxidative stress parameters, translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and ATP content was attenuated by treating TBI mice with vorinostat. Also treatment with vorinostat ameliorates the altered expression of p-Akt, NF-ï«B, iNOS and caspase by the western blot assay in the neuronal tissue of TBI mice and mRNA level of HO-1 and NQO-1 significantly enhanced in vorinostat compared to the negative control group. Furthermore, the TUNEL assay also reveals that the apoptosis of neuronal cells was significantly (p < 0.01) reduced in the vorinostat-treated group compared to the negative control group. The present study concludes that vorinostat protects the neuronal injury in TBI mice by reducing the altered level of oxidative stress and inflammatory response.
Assuntos
Lesões Encefálicas Traumáticas/patologia , Encéfalo/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vorinostat/farmacologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas Traumáticas/metabolismo , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/efeitos dos fármacosRESUMO
Production of tannase was performed in packed bed reactor filled with an inert support polyurethane foam (PUF) using Bacillus gottheilii M2S2. The influence of process parameters such as fermentation time (24-72 h), tannic acid concentration (0.5-2.5% w/v), inoculum size (7-12% v/v), and aeration rate (0-0.2 L/min) on tannase production with PUF were analyzed using one variable at a time (OVAT) approach. The outcome of OVAT was optimized by central composite design. Based on the statistical investigation, the proposed mathematical model recommends 1% (w/v) of tannic acid, 10% (v/v) of inoculum size and 0.13 L/min of aeration rate for maximum production (76.57 ± 1.25 U/L). The crude enzyme was purified using ammonium sulfate salt precipitation method followed by dialysis. The biochemical properties of partially purified tannase were analyzed and found the optimum pH (4.0), temperature (40 °C) for activity, and Km (1.077 mM) and Vmax (1.11 µM/min) with methyl gallate as a substrate. Based on the SDS-PAGE analysis, tannase exhibited two bands with molecular weights of 57.5 and 42.3 kDa. Briefly, the partially purified tannase showed 4.2 fold increase (63 ± 1.60 U/L) in comparison to the submerged fermentation and the production of tannase was validated by using NMR spectrometer.
Assuntos
Bacillus/metabolismo , Proteínas de Bactérias/biossíntese , Hidrolases de Éster Carboxílico/biossíntese , Técnicas de Cultura de Células/métodos , Fermentação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Taninos/química , TemperaturaRESUMO
A novel tannase and gallic acid-producing Penicillium rolfsii (CCMB 714) was isolated from cocoa leaves from the South of Bahia. The influence of nutritional sources and the simultaneous effect of parameters involved in the fermentation process were available. Tannase (9.97 U mL-1) and gallic acid (9 mg mL-1) production were obtained in 48 h by submerged fermentation in non-optimized conditions. Among the carbon sources, tested gallic acid and tannic acid showed the highest tannase production (p<.05) when compared with methyl gallate and glucose. After optimization using the temperature and tannic acid concentration as variables with the Central Compound Rotational Design (CCRD), the maximal tannase production (25.6 U mL-1) was obtained at 29.8 °C and 12.7%, respectively, which represents an increase of 2.56 times in relation to the initial activity. The parameters optimized for the maximum production of gallic acid (21.51 mg mL-1) were 30 °C and 10% tannic acid. P. rolfsii CCMB 714 is a new strain with a high tannase and gallic acid production and the gallic acid produced is very important, mainly for its applications in the food and pharmaceutical industry.
Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Proteínas Fúngicas/biossíntese , Ácido Gálico/metabolismo , Penicillium/metabolismo , Penicillium/isolamento & purificaçãoRESUMO
N-Acyl Homoserine Lactones (N-AHLs) are an important group of small quorum-sensing molecules generated and released into the surroundings by Gram-negative bacteria. N-AHLs play a crucial role in various infection-related biological processes of marine Vibrio species, including survival, colonization, invasion, and pathogenesis. With the increasing problem of antibiotic abuse and subsequently the emergence of drug-resistant bacteria, studies on AHLs are therefore expected to bring potential new breakthroughs for the prevention and treatment of Vibrio infections. This article starts from AHLs generation in marine Vibrio, and then discusses the advantages, disadvantages, and trends in the future development of various detection methods for AHLs characterization. In addition to a detailed classification of the various marine Vibrio-derived AHL types that have been reported over the years, the regulatory mechanisms of AHLs and their roles in marine Vibrio biofilms, pathogenicity and interaction with host cells are also highlighted. Intervention measures for AHLs in different stages are systematically reviewed, and the prospects of their future development and application are examined.
Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Percepção de Quorum/fisiologia , Vibrio/fisiologia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biofilmes , Hidrolases de Éster Carboxílico/biossíntese , Interações Hospedeiro-Patógeno , Humanos , Oceanos e Mares , Vibrio/patogenicidade , Vibrioses/microbiologiaRESUMO
Feruloyl esterase (FAE)-encoding genes AnfaeA and AnfaeB were isolated from Aspergillus niger 0913. For overexpression of the two genes in Trichoderma reesei, constitutive and inductive expression plasmids were constructed based on parental plasmid pAg1-H3. The constructed plasmids contained AnfaeA or AnfaeB gene under the control of glyceraldehyde-3-phosphate dehydrogenase A gene (gpdA) promoter (from A. nidulans) or cellobiohydrolases I (cbh I) gene promoter (from T. reesei), and cbh I terminator from T. reesei. The target plasmids were transferred into T. reesei D-86271 (Rut-C30) by Agrobacterium tumefaciens-mediated transformation (ATMT), respectively. A high level of feruloyl esterase was produced by the recombinant fungal strains under solid-state fermentation, and the cbh I promoter was more efficient than the gpdA promoter in the expression of AnfaeA. The optimum temperatures and pH values were 50 °C and 5.0 for AnFAEA, and 35 °C and 6.0 for AnFAEB. The maximum production levels were 20.69 U/gsd for AnFAEA and 15.08 U/gsd for AnFAEB. The recombinant fungal enzyme systems could release 62.9% (for AnFAEA) and 52.2% (for AnFAEB) of total ferulic acids from de-starched wheat bran, which was higher than the 46.3% releasing efficiency of A. niger 0913. The supplement of xylanase from T. longibrachiatum in the enzymatic hydrolysis led to a small increment of the ferulic acids release.
Assuntos
Aspergillus niger/genética , Hidrolases de Éster Carboxílico , Ácidos Cumáricos/metabolismo , Fibras na Dieta , Proteínas Fúngicas , Expressão Gênica , Trichoderma/enzimologia , Aspergillus niger/enzimologia , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Trichoderma/genéticaRESUMO
Parkinson's disease (PD) is an aging-associated neurodegenerative disease affecting millions worldwide. Misfolding, oligomerization and accumulation of the human α-synuclein protein is a key pathological hallmark of PD and is associated with the progressive loss of dopaminergic neurons over the course of aging. Lifespan extension via the suppression of IGF-1/insulin-like signaling (IIS) offers a possibility to retard disease onset through induction of metabolic changes that provide neuroprotection. The nceh-1 gene of Caenorhabditis elegans encodes an ortholog of neutral cholesterol ester hydrolase 1 (NCEH-1), an IIS downstream protein that was identified in a screen as a modulator of α-synuclein accumulation in vivo. The mechanism whereby cholesterol metabolism functionally impacts neurodegeneration induced by α-synuclein is undefined. Here we report that NCEH-1 protects dopaminergic neurons from α-synuclein-dependent neurotoxicity in C. elegans via a mechanism that is independent of lifespan extension. We discovered that the presence of cholesterol, LDLR-mediated cholesterol endocytosis, and cholesterol efflux are all essential to NCEH-1-mediated neuroprotection. In protecting from α-synuclein neurotoxicity, NCEH-1 also stimulates cholesterol-derived neurosteroid formation and lowers cellular reactive oxygen species in mitochondria. Collectively, this study augments our understanding of how cholesterol metabolism can modulate a neuroprotective mechanism that attenuates α-synuclein neurotoxicity, thereby pointing toward regulation of neuronal cholesterol turnover as a potential therapeutic avenue for PD.
Assuntos
Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Colesterol/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Hidrolases de Éster Carboxílico/biossíntese , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Humanos , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/enzimologia , Doença de Parkinson/terapia , Transdução de Sinais , Esterol EsteraseRESUMO
Cutinase as a promising biocatalyst has been intensively studied and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory.