RESUMO
BACKGROUND: Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer's disease. The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity. METHODS AND RESULTS: Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tauMBD) were performed. The tauMBD was heterologously expressed in E. coli. Ni-chelating affinity chromatography was then performed to purify the target protein. Thereafter, tauMBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tauMBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments. CONCLUSIONS: The final yield of the recombinant tauMBD was ~ 20 mg L-1. It is shown that TauMBD, in the absence of an inducer, self-assembled into the typical fibrils at a faster rate than wild-type tau. Finally, the in vitro cytotoxicity of tauMBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.
Assuntos
Escherichia coli , Tauopatias , Animais , Ratos , Escherichia coli/genética , Tauopatias/genética , Citoesqueleto , Emaranhados Neurofibrilares , MicrotúbulosRESUMO
Indole monooxygenases (IMOs) are enzymes from the family of Group E monooxygenases, requiring flavin adenine dinucleotide (FAD) for their activities. IMOs play important roles in both sulfoxidation and epoxidation reactions. The broad substrate range and high selectivity of IMOs make them promising biocatalytic tools for synthesizing chiral compounds. In the present study, quantum chemical calculations using the cluster approach were performed to investigate the reaction mechanism and the enantioselectivity of the IMO from Variovorax paradoxus EPS (VpIndA1). The sulfoxidation of methyl phenyl sulfide (MPS) and the epoxidation of indene were chosen as the representative reactions. The calculations confirmed that the FADOOH intermediate is the catalytic species in the VpIndA1 reactions. The oxidation of MPS adopts a one-step mechanism involving the direct oxygen-transfer from FADOOH to the substrate and the proton transfer from the -OH group back to FAD, while the oxidation of indene follows a stepwise mechanism involving a carbocation intermediate. It was computationally predicted that VpIndA1 prefers the formation of (S)-product for the MPS sulfoxidation and (1S,2R)-product for the indene epoxidation, consistent with the experimental observations. Importantly, the factors controlling the stereo-preference of the two reactions are identified. The findings in the present study provide valuable insights into the VpIndA1-catalyzed reactions, which are essential for the rational design of this enzyme and other IMOs for industrial applications. It is also worth emphasizing that the quantum chemical cluster approach is again demonstrated to be powerful in studying the enantioselectivity of enzymatic reactions.
Assuntos
Oxigenases de Função Mista , Oxirredução , Estereoisomerismo , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/química , Teoria Quântica , Sulfetos/química , Sulfetos/metabolismo , Indóis/química , Indóis/metabolismo , Modelos Químicos , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Modelos MolecularesRESUMO
Enzymes are commonly used as biocatalysts for various biological and chemical processes. However, some major drawbacks of free enzymes (e.g. poor reusability and instability) significantly restrict their industrial practices. How to overcome these weaknesses remain considerable challenges. Enzyme immobilization is one of the most effective ways to improve the reusability and stability of enzymes. Cross-linked enzyme aggregates (CLEAs) has been known as a novel and versatile carrier-free immobilization method. CLEAs is attractive due to its simplicity and robustness, without purification. It generally shows: high catalytic specificity and selectivity, good operational and storage stabilities, and good reusability. Moreover, co-immobilization of different kinds of enzymes can be acquired. These CLEAs advantages provide opportunities for further industrial applications. Herein, the preparation parameters of CLEAs were first summarized. Next, characterization of structural and catalytic properties, stability and reusability are also proposed. Finally, some important applications of this technique in: environmental protection, industrial chemistry, food industry, and pharmaceutical synthesis and delivery are introduced. Potential challenges and future research directions, such as improving cross-linking efficiency and internal mass transfer efficiency, are also presented. This implies that CLEAs provide an efficient and feasible technique to improve the properties of enzymes for use in the industry.
Assuntos
Enzimas Imobilizadas , Estabilidade Enzimática , Reagentes de Ligações Cruzadas/química , Enzimas Imobilizadas/química , CatáliseRESUMO
Lytic polysaccharide monooxygenases (LPMOs) can oxidatively break the glycosidic bonds of crystalline cellulose, providing more actionable sites for cellulase to facilitate the conversion of cellulose to cello-oligosaccharides, cellobiose and glucose. In this work, a bioinformatics analysis of BaLPMO10 revealed that it is a hydrophobic, stable and secreted protein. By optimizing the fermentation conditions, the highest protein secretion level was found at a IPTG concentration of 0.5 mM and 20 h of fermentation at 37 °C, with a yield of 20 mg/L and purity > 95%. The effect of metal ions on the enzyme activity of BaLPMO10 was measured, and it was found that 10 mM Ca2+ and Na+ increased the enzyme activity by 47.8% and 98.0%, respectively. However, DTT, EDTA and five organic reagents inhibited the enzyme activity of BaLPMO10. Finally, BaLPMO10 was applied in biomass conversion. The degradation of corn stover pretreated with different steam explosions was performed. BaLPMO10 and cellulase had the best synergistic degradation effect on corn stover pretreated at 200 °C for 12 min, improving reducing sugars by 9.2% compared to cellulase alone. BaLPMO10 was found to be the most efficient for ethylenediamine-pretreated Caragana korshinskii by degrading three different biomasses, increasing the content of reducing sugars by 40.5% compared to cellulase alone following co-degradation with cellulase for 48 h. The results of scanning electron microscopy revealed that BaLPMO10 disrupted the structure of Caragana korshinskii, making its surface coarse and poriferous, which increased the accessibility of other enzymes and thus promoted the process of conversion. These findings provide guidance for improving the efficiency of enzymatic digestion of lignocellulosic biomass.
Assuntos
Celulase , Oxigenases de Função Mista , Oxigenases de Função Mista/metabolismo , Biomassa , Polissacarídeos/metabolismo , Celulose/metabolismo , Celulase/metabolismo , Celobiose , HidróliseRESUMO
The main pathological features of ischemic stroke include neuronal damage and blood-brain barrier (BBB) dysfunction. Previous studies have shown that Evans Blue, a dye used to probe BBB integrity, could enter the brain only during the pathological status of ischemic stroke, indicating the potential pathologically activated therapeutic use of this chemical to treat ischemic stroke. In this study, we have reported that Evans Blue could produce in vitro neuroprotective effects against iodoacetic acid (IAA)-induced hypoxia neuronal death in HT22 cells. We further found that P2X purinoreceptor 4 (P2X4R), a subtype of ATP-gated cation channel, was expressed in HT22 cells. Evans Blue could prevent IAA-induced increase of P2X4R mRNA and protein expression. Interestingly, shRNA of P2X4R could protect against IAA-induced activation of p38, and SB203580, a specific inhibitor of p38, could reverse IAA-induced neurotoxicity, indicating that p38 is a downstream signaling molecule of P2X4R. Molecular docking analysis further demonstrated the possible interaction between Evans Blue and the ATP binding site of P2X4R. Most importantly, pre-treatment of Evans Blue could largely reduce neurological and behavioral abnormity, and decrease brain infarct volume in middle cerebral artery occlusion/reperfusion (MCAO) rats. All these results strongly suggested that Evans Blue could exert neuroprotective effects via inhibiting the P2X4R/p38 pathway, possibly by acting on the ATP binding site of P2X4R, indicating that Evans Blue might be further developed as a pathologically activated therapeutic drug against ischemic stroke.
Assuntos
Azul Evans/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores Purinérgicos P2X4/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Infarto Encefálico/complicações , Infarto Encefálico/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Ácido Iodoacético , Masculino , Camundongos , Modelos Biológicos , Simulação de Acoplamento Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Alzheimer's disease (AD) is a prevalent neurodegenerative disorder that has multiple causes. Therefore, multiple-target-directed ligands (MTDLs), which act on multiple targets, have been developed as a novel strategy for AD therapy. In this study, novel drug candidates were designed and synthesized by the covalent linkings of tacrine, a previously used anti-AD acetylcholinesterase (AChE) inhibitor, and dipicolylamine, an ß-amyloid (Aß) aggregation inhibitor. Most tacrine-dipicolylamine dimers potently inhibited AChE and Aß1-42 aggregation in vitro, and 13a exhibited nanomolar level inhibition. Molecular docking analysis suggested that 13a could interact with the catalytic active sites and the peripheral anion site of AChE, and bind to Aß1-42 pentamers. Moreover, 13a effectively attenuated Aß1-42 oligomers-induced cognitive dysfunction in mice by activating the cAMP-response element binding protein/brain-derived neurotrophic factor signaling pathway, decreasing tau phosphorylation, preventing synaptic toxicity, and inhibiting neuroinflammation. The safety profile of 13a in mice was demonstrated by acute toxicity experiments. All these results suggested that novel tacrine-dipicolylamine dimers, especially 13a, have multi-target neuroprotective and cognitive-enhancing potentials, and therefore might be developed as MTDLs to combat AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Aminas/farmacologia , Inibidores da Colinesterase/farmacologia , Desenho de Fármacos , Fármacos Neuroprotetores/farmacologia , Ácidos Picolínicos/farmacologia , Tacrina/farmacologia , Acetilcolinesterase/metabolismo , Doença de Alzheimer/metabolismo , Aminas/química , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Humanos , Ligantes , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Ácidos Picolínicos/química , Agregados Proteicos/efeitos dos fármacos , Relação Estrutura-Atividade , Tacrina/químicaRESUMO
Misfolding and accumulation of amyloidogenic proteins into various forms of aggregated intermediates and insoluble amyloid fibrils is associated with more than 50 human diseases. Large amounts of high-quality amyloid proteins are required for better probing of their aggregation and neurotoxicity. Due to their intrinsic hydrophobicity, it is a challenge to obtain amyloid proteins with high yield and purity, and they have attracted the attention of researchers from all over the world. The rapid development of bioengineering technology provides technical support for obtaining large amounts of recombinant amyloidogenic proteins. This review discusses the available expression and purification methods for three amyloid proteins including amyloid ß-protein, tau, and α-synuclein in microbial expression systems, especially Escherichia coli, and discusses the advantages and disadvantages of these methods. Importantly, these protocols can also be referred to for the expression and purification of other hydrophobic proteins.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Proteínas Amiloidogênicas/isolamento & purificação , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Humanos , Deficiências na Proteostase/metabolismo , alfa-Sinucleína/isolamento & purificação , Proteínas tau/isolamento & purificaçãoRESUMO
OBJECTIVE: To investigate the role of the pannexin-1 (Panx1) protein in the invasion and migration of testicular cancer Tcam-2 cells and its possible action mechanism. METHODS: Tcam-2 cells were treated with carbenoxolone (CBX) at 100 µmol/L and probenecid (PBN) 200 µmol/L. Then the intercellular fluorescence transmission was assessed by real-time fluorescence assay, the extracellular ATP concentration measured by chemi-luminescence immunoassay, the invasive and migratory abilities of the Tcam-2 cells detected by Transwell assay, and the expressions of the proteins Panx1, p-ERK1/2, ERK1/2, vimentin, MMP-9 and E-cadherin in the TM3 Leydig cells and testicular cancer Tcam-2 cells determined by Western blot. RESULTS: Western blot showed that the expression of the Panx1 protein was significantly higher in the testicular cancer Tcam-2 cells than in the TM3 Leydig cells (2.79 ± 0.17 vs 1.00 ± 0.06, P<0.05). The rates of intercellular fluorescence transmission in the Tcam-2 cells treated with CBX and PBN were markedly decreased as compared with the blank control group (ï¼»61.54 ± 3.30ï¼½% and ï¼»68.06 ± 4.03ï¼½% vs ï¼»99.50 ± 3.12ï¼½%, P<0.01), and so were the extracellular ATP concentrations (ï¼»57.06 ± 5.80ï¼½% and ï¼»56.42 ± 7.70ï¼½% vs ï¼»110 ± 8.16ï¼½%, P<0.01). The numbers of migrated Tcam-2 cells in the CBX and PBN groups were significantly reduced in comparison with that in the control (11.5 ± 1.11 and 8.25 ± 1.23 vs 331.00 ± 30.80, P<0.05), and so were those of the invaded ones (11.75 ± 3.77 and 11.5 ± 3.5 vs 89.00 ± 13.09, P<0.01). CBX and PBN significantly down-regulated the expression of p-ERK1/2 as compared with that in the blank control group (0.538 ± 0.05 and 0.476 ± 0.02 vs 0.98 ± 0.03, P<0.05), as well as those of vimentin (0.541 ± 0.09 and 0.705 ± 0.07, P<0.01) and MMP-9 (0.439 ± 0.08 and 0.557 ± 0.065, P<0.01) but up-regulated that of E-cadherin (3.896 ± 0.06 and 3.551 ± 0.04, P<0.01). CONCLUSIONS: The Panx1 protein is highly expressed in testicular cancer Tcam-2 cells. CBX and PBN can inhibit the function of the panneixn1 channel and reduce the invasive and migratory abilities of the Tcam-2 cells, which is associated with the decreased expression of the p-ERK1/2 protein.
Assuntos
Movimento Celular , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Testiculares/patologia , Carbenoxolona/farmacologia , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Metaloproteinase 9 da Matriz , Probenecid/farmacologiaRESUMO
The pumping of antitumor drugs by P-glycoprotein (P-gp) causes multidrug resistance (MDR) and consequent failure of chemotherapy. However, the understanding on the molecular mechanism of P-gp for transporting substrates is still far from adequate. Herein, the transport of a typical antitumor drug, doxorubicin, by P-gp is investigated using targeted molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis. The MM-PBSA analysis identifies the driving forces for the transport of doxorubicin toward the extracellular space as electrostatic repulsions in the initial stage, which are contributed by positively charged residues (R148, K181, K189, K285, K291, K734, R789, K826, K934, and K1000) and then hydrophobic interactions provided by hydrophobic residues (L65, M69, F336, I340, F343, Y953, V982, F983, and M986). The contributions of these residues are further validated by targeted MD simulations, which shows blocked pumping after the mutation of these important residues to glycine. The MM-PBSA and minimum distance analyses of each residue during the transport reveal that the positively charged residues promote the transport of doxorubicin through long-range electrostatic repulsions and the hydrophobic residues provide a pathway through continuous hydrophobic interactions to maintain the transport. The results have thus provided molecular insights into the function of P-gp and would be beneficial in the design of potent P-gp inhibitors against MDR in the medication of cancers.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Doxorrubicina/metabolismo , Simulação de Dinâmica Molecular , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Humanos , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
ß-Amyloid (Aß) is regarded as an important pathogenic target for Alzheimer's disease (AD), the most prevalent neurodegenerative disease. Aß can assemble into oligomers and fibrils, and produce neurotoxicity. Therefore, Aß aggregation inhibitors may have anti-AD therapeutic efficacies. It was found, here, that the marine-derived alkaloid, fascaplysin, inhibits Aß fibrillization in vitro. Moreover, the new analogue, 9-methylfascaplysin, was designed and synthesized from 5-methyltryptamine. Interestingly, 9-methylfascaplysin is a more potent inhibitor of Aß fibril formation than fascaplysin. Incubation of 9-methylfascaplysin with Aß directly reduced Aß oligomer formation. Molecular dynamics simulations revealed that 9-methylfascaplysin might interact with negatively charged residues of Aß42 with polar binding energy. Hydrogen bonds and πâ»π interactions between the key amino acid residues of Aß42 and 9-methylfascaplysin were also suggested. Most importantly, compared with the typical Aß oligomer, Aß modified by nanomolar 9-methylfascaplysin produced less neuronal toxicity in SH-SY5Y cells. 9-Methylfascaplysin appears to be one of the most potent marine-derived compounds that produces anti-Aß neuroprotective effects. Given previous reports that fascaplysin inhibits acetylcholinesterase and induces P-glycoprotein, the current study results suggest that fascaplysin derivatives can be developed as novel anti-AD drugs that possibly act via inhibition of Aß aggregation along with other target mechanisms.
Assuntos
Peptídeos beta-Amiloides/química , Indóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Doença de Alzheimer/tratamento farmacológico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Neurônios/efeitos dos fármacosRESUMO
BACKGROUND: Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor. RESULTS: Recombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively). CONCLUSIONS: The successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.
Assuntos
Cetosteroides/metabolismo , Mutagênese Sítio-Dirigida/métodos , Oxirredutases/metabolismo , Biotransformação , Especificidade por SubstratoRESUMO
The oligomer of ß-amyloid (Aß) is considered the main neurotoxin in Alzheimer's disease (AD). Therefore, the inhibition of the formation of Aß oligomer could be a target for AD therapy. In this study, with the help of the dot blotting assay and transmission electronic microscopy, it was have discovered that 5-hydroxycyclopenicillone, a cyclopentenone recently isolated from a sponge-associated fungus, effectively reduced the formation of Aß oligomer from Aß peptide in vitro. Molecular dynamics simulations suggested hydrophobic interactions between 5-hydroxycyclopenicillone and Aß peptide, which might prevent the conformational transition and oligomerization of Aß peptide. Moreover, Aß oligomer pre-incubated with 5-hydroxycyclopenicillone was less toxic when added to neuronal SH-SY5Y cells compared to the normal Aß oligomer. Although 5-hydroxycyclopenicillone is not bioavailable in the brain in its current form, further modification or encapsulation of this chemical might improve the penetration of 5-hydroxycyclopenicillone into the brain. Based on the current findings and the anti-oxidative stress properties of 5-hydroxycyclopenicillone, it is suggested that 5-hydroxycyclopenicillone may have potential therapeutic efficacy in treating AD.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Ciclopentanos/química , Ciclopentanos/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Doença de Alzheimer/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fungos/química , Humanos , Simulação de Dinâmica Molecular , Neurônios/patologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/uso terapêuticoRESUMO
Cross-sequence interactions between different amyloid peptides are important not only for the fundamental understanding of amyloid aggregation and polymorphism mechanisms, but also for probing a potential molecular link between different amyloid diseases. Here, we computationally modeled and simulated a series of hybrid hIAPP (human islet amyloid polypeptide)-rIAPP (rat islet amyloid polypeptide) assemblies and probed their structural stability, lateral association, and interfacial interactions using combined peptide-packing search, molecular dynamics (MD) simulations, and the Monte Carlo sampling method. We then identified a number of stable and highly populated hIAPP-rIAPP assemblies at the lowest energy states, in which hIAPP and rIAPP oligomers were stacked laterally on top of each other to form supramolecular ß-sheet double layers in an antiparallel fashion. These hIAPP-rIAPP assemblies adopted different interfaces formed by C-terminal ß-sheets of hIAPP and rIAPP oligomers (hCCr), N-terminal ß-sheets of hIAPP and rIAPP oligomers (hNNr), and alternative N-terminal/C-terminal ß-sheets of hIAPP and rIAPP oligomers (hNCr and hCNr). Different interfaces along with distinct interfacial residue packings provided different driving interfacial forces to laterally associate two ß-sheet layers of hIAPP and rIAPP together for forming polymorphic hIAPP-rIAPP assemblies. Such lateral association between hIAPP and rIAPP not only explained the experimentally observed cross-seeding behavior of hIAPP and rIAPP, but also demonstrated the co-existence of polymorphic amyloid cross-seeding species. A cross-seeding mechanism for hIAPP and rIAPP aggregation was proposed on the basis of our simulated models and experimental data. This work provides a better understanding of cross-seeding aggregation and polymorphism mechanisms of amyloidogenesis.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Simulação de Dinâmica Molecular , Multimerização Proteica , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Método de Monte Carlo , Estabilidade Proteica , Estrutura Secundária de Proteína , RatosRESUMO
Ulva polysaccharides present several physiological activities including antiviral, antitumor and anti-plasmodial effects. However, current processing usually results in low yields and high prices, thus lacking commercialization potential. The aim of this study was to develop an efficient method for the extraction of Ulva polysaccharides with high biological activity. The effect of cell wall-degrading enzymes including cellulase, hemicellulase, pectinase and protease on Ulva polysaccharide extraction was studied by statistical mixing design. Using the most effective enzyme preparations as the basic components, the optimal proportions of the enzyme mixture were determined as follows: cellulase 35.3%, pectinase 34.5%, alkaline protease 30.2%, which increased the polysaccharide yield from 6.43% in the absence of enzymes to 26.68%. Subsequently, through response surface analysis, the optimal conditions were determined: enzyme concentration of 1.5%, enzymatic time of 1.1 h, ultrasonic time of 90 min and enzymatic temperature of 60 °C. Under the optimal extraction conditions, the extraction yield of Ulva polysaccharides could be increased to 30.14%. Moreover, extracted polysaccharides exhibit strong antioxidant properties in DPPH, ABTS, hydroxyl radical, superoxide radical and H2O2-induced cellular damage models. This study laid a solid foundation for the use and development of Ulva polysaccharides.
RESUMO
Thermal stability is one of the most important properties of ulvan lyases for their application in algae biomass degradation. The Knowledge gaining directed eVolution (KnowVolution) protein engineering strategy could be employed to improve thermostability of ulvan lyase with less screening effort. Herein, the unfolding free energies (ΔΔG) of the loop region were calculated using FoldX and four sites (D103, G104, T113, Q229) were selected for saturation mutagenesis, resulting in the identification of a favorable single-site mutant Q229M. Subsequently, iteration mutation was carried out with the mutant N57P (previously obtained by our group) to further enhance the performance of ulvan lyase. The results showed that the most beneficial variant N57P/Q229M exhibited a 1.67-fold and 2-fold increase in residual activity compared to the wild type after incubation at 40 °C and 50 °C for 1 h, respectively. In addition, the variant produced 1.06 mg/mL of reducing sugar in 2 h, which was almost four times as much as the wild type. Molecular dynamics simulations revealed that N57P/Q229M mutant enhanced the structural rigidity by augmenting intramolecular hydrogen bonds. Meanwhile, the shorter proton transmission distance between the general base of the enzyme and the substrate contributed to the glycosidic bond breakage. Our research showed that in silico saturation mutagenesis using position scan module in FoldX allowed for faster screening of mutants with improved thermal stability, and combining it with KnowVolution enabled a balanced effect of thermal stability and enzyme activity in protein engineering.
Assuntos
Polissacarídeos , Engenharia de Proteínas , Polissacarídeos/metabolismo , Mutação , Mutagênese , Estabilidade EnzimáticaRESUMO
The aggregation of ß-amyloid (Aß) peptides to form amyloid plaques is one of the primary hallmarks for Alzheimer's disease (AD). Dietary flavonoid supplements containing hesperetin have an ability to decline the risk of developing AD, but the molecular mechanism is still unclear. In this work, hesperetin, a flavanone abundant in citrus fruits, has been proven to prevent the formation of Aß aggregates and depolymerized preformed fibrils in a concentration-dependent fashion. Hesperetin inhibited the conformational conversion from the natural structure to a ß-sheet-rich conformation. It was found that hesperetin significantly reduced the cytotoxicity and relieved oxidative stress eventuated by Aß aggregates in a concentration-dependent manner. Additionally, the beneficial effects of hesperetin were confirmed in Caenorhabditis elegans, including the inhibition of the formation and deposition of Aß aggregates and extension of their lifespan. Finally, the results of molecular dynamics simulations showed that hesperetin directly interacted with an Aß42 pentamer mainly through strong non-polar and electrostatic interactions, which destroyed the structural stability of the preformed pentamer. To summarize, hesperetin exhibits great potential as a prospective dietary supplement for preventing and improving AD.
Assuntos
Peptídeos beta-Amiloides , Caenorhabditis elegans , Hesperidina , Hesperidina/farmacologia , Hesperidina/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/química , Animais , Caenorhabditis elegans/efeitos dos fármacos , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/prevenção & controle , Amiloide/metabolismo , Simulação de Dinâmica Molecular , Estresse Oxidativo/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacosRESUMO
The fibrillogenesis of amyloid ß-protein (Aß) gradually accumulates to form neurotoxic Aß aggregates in the human brain, which is the direct cause of Alzheimer's disease (AD) related symptoms. There are currently no effective therapies for AD. Brazilin, a natural polyphenol, inhibits Aß fibrillogenesis, disrupts the mature fibrils and alleviates the corresponding cytotoxicity, but it also has the high toxic. Therefore, brazilin-7-2-butenoate (B-7-2-B), a brazilin derivative, was designed and synthesized. B-7-2-B exhibited lower toxicity and stronger inhibitory effect on Aß aggregation than brazilin. B-7-2-B could prevent the formation of Aß fibrils and oligomers, and depolymerize pre-formed aggregates in a dose-dependent manner. Furthermore, B-7-2-B prominently alleviated the cytotoxicity and the oxidative stress induced by Aß aggregates in PC12 cells. The protective impacts of B-7-2-B were further demonstrated by using the Caenorhabditis elegans model, including decreasing the extent of Aß aggregation, improving the motility and sensation disorders. Eventually, B-7-2-B was proven to be no apparent damage to worms. In summarize, it can be concluded that B-7-2-B has the potential as a drug for treating AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Animais , Ratos , Humanos , Peptídeos beta-Amiloides/toxicidade , Caenorhabditis elegans , Benzopiranos/farmacologia , Células PC12 , Doença de Alzheimer/tratamento farmacológico , AmiloideRESUMO
In the food industry, sulfides are commonly used as preservatives and flavor regulators. However, long-term excessive intake of sulfides can lead to serious health problems. Therefore, developing efficient sulfide detection methods is particularly important. Here, we have effectively synthesized a novel bifunctional copper hydroxide nitrate (Cu2(OH)3NO3) nanozyme with outstanding peroxidase-like and laccase-like behaviors in basic deep eutectic solvents (DES). Because the various types of sulfides have diverse regulatory effects on the two catalytic behaviors of Cu2(OH)3NO3, a two channel nanozyme sensor array based on the peroxidase-like and laccase-like behaviors of Cu2(OH)3NO3 was constructed and successfully used for the identification of six kinds of sulfides (Na2S, Na2S2O3, Na2SO3, Na2SO4, NaHSO3, and Na2S2O8). Remarkably, the sensor array has achieved successful discrimination among six sulfides present in wine, egg, and milk samples. Finally, the sensor array has successfully distinguished and differentiated three actual samples (wine, egg, and milk). This study is of great significance in promoting the efficient construction of array units and improving the effective identification of sulfides in complex food samples.
Assuntos
Técnicas Biossensoriais , Cobre , Análise de Alimentos , Sulfetos , Sulfetos/química , Cobre/química , Técnicas Biossensoriais/métodos , Análise de Alimentos/instrumentação , Nitratos/análise , Nitratos/química , Leite/química , Vinho/análise , Animais , Hidróxidos/química , Nanoestruturas/químicaRESUMO
In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YNDG236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YNDG236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.
RESUMO
The identification of phosphates holds significant importance in many physiological processes and disease diagnosis, and traditional detection techniques struggle to simultaneously detect and distinguish phosphates. The complexity of synthesizing sensing units restricts the construction of sensor arrays as well. In this study, a bifunctional dicopper chloride trihydroxide (Cu2Cl(OH)3) nanozyme with conspicuous laccase- and peroxidase-like activities has been synthesized in basic deep eutectic solvents (DES). Exploiting the various regulatory impacts of multiple phosphates on the dual-enzyme mimicking activities, the sensor array based on the laccase mimic and peroxidase mimic properties of Cu2Cl(OH)3 was designed, which has been successfully harnessed for the identification of eight phosphates (ATP, ADP, AMP, PPi, Pi, GTP, GDP, and GMP). This approach streamlines the creation of sensor arrays. Besides, the three simulated actual samples (healthy individuals, moderately ill patients, and severely ill patients) have been accurately distinguished. This work makes a substantial contribution to enhancing the highly effective construction of array channels and promoting discrimination of phosphates in intricate samples.