RESUMEN
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.
Asunto(s)
Azul de Evans/farmacología , Fármacos Neuroprotectores/farmacología , Receptores Purinérgicos P2X4/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Infarto Encefálico/complicaciones , Infarto Encefálico/patología , Muerte Celular/efectos de los fármacos , Línea Celular , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Ácido Yodoacético , Masculino , Ratones , Modelos Biológicos , Simulación del Acoplamiento Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteínas Amiloidogénicas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Proteínas Amiloidogénicas/aislamiento & purificación , Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/aislamiento & purificación , Humanos , Deficiencias en la Proteostasis/metabolismo , alfa-Sinucleína/aislamiento & purificación , Proteínas tau/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Péptidos beta-Amiloides/química , Indoles/farmacología , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Simulación de Dinámica Molecular , Neuronas/efectos de los fármacosRESUMEN
We have previously discovered an amine-containing flavonoid monomer FM04 as a potent P-glycoprotein (P-gp) inhibitor (EC50 = 83 nM). Here, a series of photoactive FM04 analogues were synthesized and used together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the FM04-binding sites on P-gp. Point mutations around the photo-crosslinked sites were made for verification. Together with the results from mutational studies, molecular docking, and molecular dynamics simulations, it was found that FM04 can interact with Q1193 and I1115 in the nucleotide-binding domain 2 (NBD2) of human P-gp. It was proposed that FM04 can inhibit P-gp in 2 novel mechanisms. FM04 can either bind to (1) Q1193, followed by interacting with the functionally critical residues H1195 and T1226 or (2) I1115 (a functionally critical residue itself), disrupting the R262-Q1081-Q1118 interaction pocket and uncoupling ICL2-NBD2 interaction and thereby inhibiting P-gp. Q1118 would subsequently be pushed to the ATP-binding site and stimulate ATPase.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Flavonoides , Humanos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Sitios de Unión , Cromatografía Liquida , Flavonoides/farmacología , Flavonoides/química , Simulación del Acoplamiento Molecular , Nucleótidos/metabolismo , Espectrometría de Masas en TándemRESUMEN
Misfolding and the subsequent self-assembly of amyloid-ß protein (Aß) is very important in the occurrence of Alzheimer's disease (AD). Thus, inhibition of Aß aggregation is currently an effective method to alleviate and treat AD. Herein, a carboxylated single-walled carbon nanotube (SWCNT-COOH) was rationally designed based on the hydrophobic binding-electrostatic repulsion (HyBER) mechanism. The inhibitory effect of SWCNT-COOH on Aß fibrillogenesis was first studied. Based on the results of thioflavin T fluorescence and atomic force microscopy imaging assays, it was shown that SWCNT-COOH can not only effectively inhibit Aß aggregation, but also depolymerize the mature fibrils of Aß. In addition, its inhibitory action will be affected by the content of carboxyl groups. Moreover, the influence of SWCNT-COOH on cytotoxicity induced by Aß was investigated by the MTT method. It was found that SWCNT-COOH can produce an anti-Aß neuroprotective effect in vitro. Molecular dynamics simulations showed that SWCNT-COOH significantly destroyed the overall and internal structural stability of an Aß40 trimer. Moreover, SWCNT-COOH interacted strongly with the N-terminal region, turn region and C-terminal region of the Aß40 trimer via hydrogen bonds, salt bridges and π-π interactions, which triggered a large structural disturbance of the Aß40 trimer, reduced the ß-sheet content of the Aß40 trimer and led to more disorder in these regions. All the above data not only reveal the suppressive effect of SWCNT-COOH on Aß aggregation, but also reveal its inhibitory mechanism, which provides a useful clue to exploit anti-Aß drugs in the future.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Materiales Biocompatibles/farmacología , Nanotubos de Carbono/química , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Supervivencia Celular/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Simulación de Dinámica Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Células PC12 , Tamaño de la Partícula , Agregado de Proteínas/efectos de los fármacos , Ratas , Electricidad EstáticaRESUMEN
α-Synuclein (α-syn) aggregates into cytotoxic amyloid fibrils, which are recognized as the defining neuropathological feature of Parkinson's disease (PD). Therefore, inhibiting α-syn fibrillogenesis and disrupting the preformed fibrils are both considered attractive strategies to cure PD. We discovered that a safe food additive, fast green FCF, is capable of inhibiting α-synuclein fibrillogenesis and reducing the related cytotoxicity. Thioflavin T fluorescence assays demonstrated that fast green FCF could inhibit the fibrillogenesis α-synuclein. In the presence of 100 µM fast green FCF, amorphous aggregates were formed and observed by atomic force microscopy. Toxicity assays in cell cultures revealed that fast green FCF significantly reduced the cytotoxicity of α-syn. Molecular dynamics simulations revealed the potential mechanism of the interactions between fast green FCF and α-synuclein. Fast green FCF greatly disrupted the α-synuclein pentamer and reduced the ß-sheet content by reducing both nonpolar and polar interactions. Furthermore, two binding sites were identified, named region I (Y39-K45) and region II (H50-Q62). Our data reveal that electrostatic interactions, hydrogen bonds, and π-π interactions synergistically contribute to the binding of fast green FCF to the α-synuclein pentamer. These results indicate that fast green FCF is a candidate prototype for the development of drugs against the aggregation of amyloid fibrils in PD.
Asunto(s)
Amiloide/efectos adversos , Aditivos Alimentarios/farmacología , Colorantes Verde de Lisamina/química , Colorantes Verde de Lisamina/farmacología , Síndromes de Neurotoxicidad/tratamiento farmacológico , alfa-Sinucleína/química , alfa-Sinucleína/efectos de los fármacos , Animales , Benzotiazoles , Supervivencia Celular/efectos de los fármacos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Neuronas , Células PC12 , Enfermedad de Parkinson/metabolismo , Sustancias Protectoras , Agregado de Proteínas , Ratas , Electricidad EstáticaRESUMEN
Previous studies have indicated that 5-hydroxycyclopenicillone (HCP), an active compound derived from marine sponge, could inhibit oligomerization of amyloid ß-protein (Aß). However, the molecular basis for the interaction between HCP and Aß remains unclear. Herein, all-atom molecular dynamics (MD) simulations were used to explore the conformational conversion of an Aß40 monomer at different concentrations (0-40 mM) of HCP at the atomic level. It is confirmed that the conformational transition of the Aß40 monomer is prevented by HCP in a concentration-dependent manner in silico. In 40 mM HCP solution, the initial α-helix-rich conformation of Aß40 monomer is kept under the action of HCP. The intra-peptide hydrophobic collapse and D23-K28 salt bridge are prevented by HCP. Moreover, it is indicated that the non-polar binding energy dominates the binding between HCP and Aß40 monomer as evaluated by molecular mechanics Poisson-Boltzmann surface area method. And, the residues of F4, Y10, V12, L17 and L34 in Aß40 might contribute to the binding energy in HCP-Aß40 complex. All these results elucidate the molecular mechanism underlying the inhibitory effects of HCP against the conformational transformation of Aß40, providing a support that HCP may be developed as a potential anti-Aß compound for the treatment of Aß-related diseases.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Humanos , Conformación Molecular , Simulación de Dinámica Molecular , Fragmentos de PéptidosRESUMEN
Previous work has shown that edaravone inhibits fibrillogenesis of amyloid-ß protein (Aß). However, the detailed mechanism by which edaravone inhibits the conformational transition of the Aß42 monomer is not known at the molecular level. Here, explicit-solvent molecular dynamics (MD) simulations were coupled with molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method to address the issue. MD simulations confirmed that edaravone inhibits the conformational transition of the Aß42 monomer in a dose-dependent manner. It was found that the direct interactions between edaravone and Aß42 are responsible for its inhibiting effects. The analysis of binding free energy using the MM-PBSA method demonstrated that the nonpolar interactions provide favourable contributions (about -71.7 kcal/mol). Conversely, the polar interactions are unfavourable for the binding process. A total of 14 residues were identified as greatly contributing to the binding free energy between edaravone and the Aß42 monomer. In addition, the intra-peptide hydrophobic interactions were weakened and the salt bridge D23-K28 was interrupted by edaravone. Therefore, the conformational transition was inhibited. Our studies provide molecular-level insights into how edaravone molecules inhibit the conformational transition of the Aß42 monomer, which may be useful for designing amyloid inhibitors.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Péptidos beta-Amiloides/química , Edaravona/química , Simulación de Dinámica Molecular , Fragmentos de Péptidos/químicaRESUMEN
Alzheimer's disease (AD) is mainly caused by the fibrillogenesis of amyloid-ß protein (Aß). Therefore, the development of effective inhibitors against Aß fibrillogenesis offers great hope for the treatment of AD. Cyanidin-3-O-glucoside (Cy-3G) is a commonly found anthocyanin that is mainly present in fruits, with established neuroprotective effects in situ. However, it remains unknown if Cy-3G can prevent Aß fibrillogenesis and alleviate the corresponding cytotoxicity. In this study, extensive biochemical, biophysical, biological and computational experiments were combined to address this issue. It was found that Cy-3G significantly inhibits Aß40 fibrillogenesis and disintegrates mature Aß fibrils, and its inhibitory capacity is dependent on the Cy-3G concentration. The circular dichroism results showed that Cy-3G and Aß40 at a molar ratio of 3 : 1 slightly prevents the structural transformation of Aß40 from its initial random coil to the ß-sheet-rich structure. Co-incubation of Aß40 with Cy-3G significantly reduced the production of intracellular reactive oxygen species induced by Aß40 fibrillogenesis and thus reduced Aß40-induced cytotoxicity. Molecular dynamics simulations revealed that Cy-3G disrupted the ß-sheet structure of the Aß40 trimer. Cy-3G was found to mainly interact with the N-terminal region, the central hydrophobic cluster and the ß-sheet region II via hydrophobic and electrostatic interactions. The ten hot spot residues D7, Y10, E11, F19, F20, E22, I31, I32, M35 and V40 were also identified. These findings not only enable a comprehensive understanding of the inhibitory effect of Cy-3G on Aß40 fibrillogenesis, but also allow the identification of a valuable dietary ingredient that possesses great potential to be developed into functional foods to alleviate AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Antocianinas/farmacología , Supervivencia Celular/efectos de los fármacos , Glucósidos/farmacología , Animales , Modelos Moleculares , Simulación de Dinámica Molecular , Fármacos Neuroprotectores , Células PC12 , Fragmentos de Péptidos , Unión Proteica , Conformación Proteica , Ratas , Especies Reactivas de OxígenoRESUMEN
To enhance the biomass conversion efficiency, the R17L mutant of the lytic polysaccharide monooxygenase (LPMO) MtC1LPMO with improved catalytic efficiency was constructed via rational point mutation based on the HotSpot Wizard 3.0 and dezyme web servers. Compared with the wild-type (WT) MtC1LPMO, R17L exhibited a 1.8-fold increase of specific activity and 1.92-fold increase of catalytic efficiency (kcat/Km). The degree of increase of the reducing sugar yield from microcrystalline cellulose and three plant biomass materials during synergistic hydrolysis using cellulase in combination with R17L was about 2 times higher than with the WT. Molecular dynamics simulations revealed that the R17L mutation reduced the stability of the region R18-I36, which then weakened the direct interactions between region N24-V31 and the substrate cellohexaose. Consequently, the deflection time of the cellohexaose conformation in R17L was prolonged compared to the WT, which enhanced its catalytic efficiency.
Asunto(s)
Simulación de Dinámica Molecular , Mutación Puntual , Biomasa , LigninaRESUMEN
To enhance the thermostability of phospholipase D (PLD), error-prone polymerase chain reaction method was used to create mutants of PLD (PLDsh) from Streptomyces halstedii. One desirable mutant (S163F) with Ser to Phe substitution at position 163 was screened with high-throughput assay. S163F exhibited a 10 °C higher optimum temperature than wild-type (WT). Although WT exhibited almost no activity after incubating at 50 °C for 40 min, S163F still displayed 27% of its highest activity after incubating at 50 °C for 60 min. Furthermore, the half-life of S163F at 50 °C was 3.04-fold higher than that of WT. The analysis of molecular dynamics simulation suggested that the Ser163Phe mutation led to the formation of salt bridge between Lys300 and Glu314 and a stronger hydrophobic interaction of Phe163 with Pro341, Leu342, and Trp460, resulting in an increased structural rigidity and overall enhanced stability at high temperature. This study provides novel insights on PLD tolerance to high temperature by investigating the structure-activity relationship. In addition, it provides strong theoretical foundation and preliminary information on the engineering of PLD with improved characteristics to meet industrial demand.
Asunto(s)
Sustitución de Aminoácidos , Fosfolipasa D/química , Fosfolipasa D/metabolismo , Streptomyces/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Evolución Molecular Dirigida , Estabilidad de Enzimas , Semivida , Calor , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulación de Dinámica Molecular , Fosfolipasa D/genética , Estructura Terciaria de Proteína , Streptomyces/genética , Relación Estructura-ActividadRESUMEN
Amyloid self-assembly is pathologically linked to many neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). While many inhibitors have been developed individually for specific amyloid proteins, there are a few effective platforms to screen on a large scale general amyloid inhibitors against different amyloid proteins. Herein, we developed a new class of amyloid inhibitor probes by site-specific conjugation of aggregation-induced emission (AIE) molecules with amyloid proteins (i.e., AIE@amyloid probes) to realize a high-throughput screening of small-molecule inhibitors. Optimization of site-specific AIE conjugation with two amyloid proteins, amyloid-ß protein (Aß) and α-synuclein (αSN), enabled us to retain their high amyloidogenic properties; i.e., AIE-amyloid probes alone exhibited strong fluorescence due to amyloid-like aggregation, but they showed no fluorescence in the presence of amyloid inhibitors to prevent amyloid aggregation. From integration of AIE@amyloid probes and computational virtual screening from a large drug database, it was found that tolcapone possessed a dual inhibition against the aggregation and cytotoxicity of both Aß and αSN. More importantly, tolcapone significantly improved the spatial cognition and recognition of Aß-treated mice. This work represents an innovative attempt to design an AIE-based anti-amyloid drug platform for identifying new small-molecule inhibitors against amyloidogenesis in both AD and PD or other amyloid diseases.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Tolcapona/uso terapéutico , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Proteínas Amiloidogénicas/metabolismo , Animales , Ratones , Simulación de Dinámica Molecular , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismoRESUMEN
The fibrillogenesis of amyloid-ß protein (Aß) is considered a crucial factor in the pathogenesis of Alzheimer's disease (AD). Hence, inhibiting Aß fibrillogenesis is regarded as the primary therapeutic strategy for the prevention and treatment of AD. However, the development of effective inhibitors against Aß fibrillogenesis has faced significant challenges. Previous studies have shown that pristine single-walled carbon nanotubes (SWNTs) can inhibit fibrillogenesis of some amyloid proteins. However, the poor dispersibility of SWNTs in an aqueous environment greatly hinders their inhibitory efficacy. Here, we examined the inhibitory activity of hydroxylated SWNTs (SWNT-OH) on the aggregation and cytotoxicity of Aß42 using thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), cellular viability assays, and molecular dynamics (MD) simulations. ThT and AFM results showed that SWNT-OH inhibits Aß42 fibrillogenesis and disaggregates preformed amyloid fibrils in a dose-dependent manner. Furthermore, the ratio of hydroxyl groups in SWNT-OH is crucial for their effect against Aß42 aggregation. SWNT-OH exerted cytoprotective effects against Aß42 fibrillation-induced cytotoxicity. The results of free-energy decomposition studies based on MD simulations revealed that nonpolar interactions, and especially van der Waals forces, contributed most of the free energy of binding in the SWNT-OH-Aß complex. Two regions of the Aß pentamer were identified to interact with SWNT-OH, spanning H13-Q15 and V36-G38. The findings presented here will contribute to a comprehensive understanding of the inhibitory effect of hydroxylated nanoparticles against Aß fibrillogenesis, which is critical for the search for more effective agents that can counteract amyloid-mediated pathologies.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Amiloide/metabolismo , Fragmentos de Péptidos/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Simulación de Dinámica Molecular , Nanotubos de Carbono , Sustancias Protectoras/farmacologíaRESUMEN
Aggregation of amyloid ß peptide (Aß) is closely associated with the occurrence and development of Alzheimer's disease (AD). Reproducible and detailed studies on the aggregation kinetics and structure of various aggregates have been conducted using recombinant Aß peptides. While the His6-tag is commonly used in the purification of recombinant proteins due to its great simplicity and affinity, there is little information on the aggregation of His6-tagged Aß and its corresponding cytotoxicity. Moreover, it is also unclear whether there is an effect of the His6-tag on the amyloidogenicity and cytotoxicity of recombinant Aß1-42. Herein, a method to express and purify a mutant C-terminally His6-tagged Aß1-42 (named as Aß1-42-His6) from Escherichia coli was described. Aß1-42-His6 aggregated into ß-sheet-rich fibrils as shown by thioflavin T fluorescence, atomic force microscopy and circular dichroism spectroscopy. Moreover, the fibrillar recombinant Aß1-42-His6 showed strong toxicity toward PC12 cells in vitro. Molecular dynamics simulations revealed that the His6-tag contributed little to the secondary structure and intermolecular interactions, including hydrophobic interactions, salt bridges, and hydrogen bonding of the fibrillar pentamer of Aß1-42. This highlights the biological importance of modification on the molecular structure of Aß. Thus, the easily purified high-quality Aß1-42-His6 offers great advantages for screening aggregation inhibitors or in vitro confirmation of rationally designed drugs for the treatment of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Supervivencia Celular , Fragmentos de Péptidos/metabolismo , Agregación Patológica de Proteínas/metabolismo , Péptidos beta-Amiloides/química , Animales , Supervivencia Celular/fisiología , Escherichia coli , Simulación de Dinámica Molecular , Células PC12 , Fragmentos de Péptidos/química , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Fibrillogenesis of α-synuclein (αSN) is associated with the onset and progression of Parkinson's disease (PD). Dihydromyricetin (DHM), a natural flavonoid compound extracted from Ampelopsis grossedentata, has proven antioxidative, antineuroinflammatory, and neuroprotective effects in dementia. However, it remains unclear if DHM can impede αSN fibrillogenesis and attenuate the corresponding cytotoxicity. Herein, we found that DHM could inhibit αSN fibrillogenesis and destabilize mature αSN fibrils in a dose-dependent manner. Moreover, DHM protected against αSN-induced cytotoxicity by improving the cell viability by 34.73 ± 3.68% at a 1:1 molar ratio of αSN to DHM. Molecular dynamics simulations showed that DHM interacts with the αSN trimer mainly via nonpolar mechanisms. The key residues by which αSN interacts with DHM were hydrophobic, and their side chains and main chains showed a synergistic effect via hydrophobic and hydrogen-bonding interactions. These findings suggest that DHM possesses great potential to be developed into a new aggregation inhibitor for αSN.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Medicamentos Herbarios Chinos/farmacología , Flavonoles/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/química , Ampelopsis/química , Péptidos beta-Amiloides/química , Animales , Apoptosis/efectos de los fármacos , Medicamentos Herbarios Chinos/química , Flavonoles/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Neuronas/citología , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Células PC12 , Enfermedad de Parkinson/genética , Agregado de Proteínas/efectos de los fármacos , Ratas , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
The inhibitory effect of brazilin against α-synuclein (α-syn) fibrillogenesis, disruption effect against mature fibrils, and the following cytotoxicity were examined by systematical biochemical, biophysical, cellular biological, and molecular simulation experiments. It is found that brazilin inhibited α-syn fibrillogenesis and disrupted the performed fibrils with a concentration-dependent manner. Moreover, cellular experimental data showed that brazilin effectively reduced the cytotoxicity induced by α-syn aggregates. Finally, molecular dynamics simulations were performed to explore the interactions between brazilin and α-syn pentamer. It is found that brazilin directly interacts with α-syn pentamer, and the hydrophobic interactions are favorable for brazilin binding with the α-syn pentamer, while the electrostatic part provides adverse effects. Three binding regions were identified to inhibit α-syn fibrillogenesis or disrupt the preformed aggregates. Furthermore, six important residues (i.e., G51, V52, A53, E61, V66, and K80) of α-syn were also identified. We expected that brazilin is an effective agent against α-syn fibrillogenesis and associated cytotoxicity.
Asunto(s)
Amiloide/química , Benzopiranos/química , Sustancias Protectoras/química , alfa-Sinucleína/química , Secuencias de Aminoácidos , Amiloide/metabolismo , Amiloide/toxicidad , Animales , Benzopiranos/metabolismo , Línea Celular , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Células PC12 , Agregado de Proteínas , Unión Proteica , Ratas , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidadRESUMEN
Misfolding and fibrillogenesis of amyloid-ß protein (Aß) play a key role in the onset and progression of Alzheimer's disease (AD). Screening for inhibitors against Aß amyloidogenesis is helpful for rational designing and developing new anti-AD drugs and therapeutic strategies. Dihydromyricetin, a natural flavonoid extracted from a Chinese herb, Ampelopsis grossedentata, has been proven with antioxidative, anti-inflammatory, and neuroprotective effects against neurodegenerative disease. Herein, we found that dihydromyricetin could inhibit Aß40 aggregation, impede the protofibril formation, disassemble preformed Aß40 fibrils, and protect PC12 cells from the Aß40-induced cytotoxicity using a series of biochemical and biophysical assays, including thioflavin T fluorescence, atomic force microscopy, and cell toxicity assays. Circular dichroism spectroscopy data proved that dihydromyricetin delayed the Aß40 conformational conversion. In addition, the results of molecular dynamics simulations indicated that the interaction between dihydromyricetin and Aß40 trimer is mainly nonpolar interactions. Key residues (i.e., V18, A21, and D23) of the Aß40 interacting with dihydromyricetin were also identified. This study suggested that dihydromyricetin shows great potential to be developed as a novel Aß40 inhibitor.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Amiloidosis/metabolismo , Citotoxinas/antagonistas & inhibidores , Flavonoides/metabolismo , Flavonoles/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Animales , Citotoxinas/toxicidad , Relación Dosis-Respuesta a Droga , Flavonoides/farmacología , Flavonoles/farmacología , Células PC12 , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Estructura Secundaria de Proteína , RatasRESUMEN
To improve the thermostability of the lipase LIP2 from Yarrowia lipolytica, molecular dynamics (MD) simulations at various temperatures were used to investigate the common fluctuation sites of the protein, which are considered to be thermally weak points. Two of these residues were selected for mutations to improve the enzyme's thermostability, and the variants predicted by MD simulations to have improved thermostability were expressed in Pichia pastoris GS115 for further investigations. According to the proline rule, the high fluctuation site S115 or V213 was replaced with proline residue, the two lipase mutants S115P and V213P were obtained. The mutant V213P exhibited evidently enhanced thermostability with an approximately 70% longer half-life at 50⯰C than that of the parent LIP2 expressed in P. pastoris. The temperature optimum of V213P was 42⯰C, which was about 5.0⯰C higher than that of the parent LIP2, while its specific catalytic activity was comparable to that of the parent and reached 876.5â¯U/mg. The improved thermostability of V213P together with its high catalytic efficiency indicated that the rational design strategy employed here can be efficiently applied for structure optimization of industrially important enzymes.
Asunto(s)
Lipasa/química , Lipasa/genética , Ingeniería de Proteínas , Temperatura , Yarrowia/enzimología , Biocatálisis , Estabilidad de Enzimas/genética , Calor , Lipasa/metabolismo , Simulación de Dinámica Molecular , Mutación , Conformación ProteicaRESUMEN
ß-Glucosidases hydrolyze terminal, non-reducing ß-d-glucosyl residues and thereby release ß-d-glucose. They have applications in the production of biofuels, beverages and pharmaceuticals. In this study, a ß-glucosidase derived from Aspergillus aculeatus (BGLA) was expressed, characterized, and the molecular mechanism of its acid denaturation was comprehensively probed. BGLA exhibited maximal activity at pHâ¯5.0-6.0. Its optimal temperature was 70⯰C. Its enzyme activity was enhanced by Mg2+, Ca2+ and Ba2+, while Cu2+, Mn2+, Zn2+, Fe2+ and Fe3+ had a negative effect. BGLA showed activity on a broad range of substrates including salicin, cellobiose, arbutin, geniposide and polydatin. Finally, the acid-denaturation mechanism of BGLA was probed using molecular dynamics (MD) simulations. The results of simulation at pHâ¯2.0 imply that the contact number, solvent accessible surface area and number of hydrogen bonds in BGLA decreased greatly. Moreover, the distance between the residues Asp280 and Glu509 that are part of the active site increased, which eventually destroyed the enzyme's catalytic activity. These MD results explain the molecular mechanism of acid denaturation of BGLA, which will greatly benefit the rational design of more acid-stable ß-glucosidase variants in the future.