RESUMO
Ionic liquid (IL) cationic species have recently captivated the attention of pharmacists, biochemists, and biomedical scientists as promising antibacterial agents to deal with the multidrug resistance bacteria crisis. The structure and functional groups of ILs influence their physiochemical properties and biological activities. However, a comprehensive study is required to fully understand the details of the antibacterial activity of ILs carrying various functional groups. Herein, dicationic ILs (DCILs) are reported based on imidazolium rings as efficient antibacterial agents. The DCILs carried various functionalities such as 2-hydroxybutyl (DCIL-1), 2-hydroxy-3-isopropoxypropyl (DCIL-2), 2-hydroxy-3-(methacryloyloxy)propyl (DCIL-3), 2-hydroxy-2-phenylethyl (DCIL-4), and 2-hydroxy-3-phenoxypropyl (DCIL-5). The structure-antibacterial activity relationships of the DCILs against Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) were comprehensively studied through antibacterial tests, morphology analysis, and adhesion tests. The experimental assays revealed an antibacterial efficacy order of DCIL-5 > DCIL-1 > DCIL-4 > DCIL-2 > DCIL-3. The all-atom molecular dynamics (MD) simulation showed a deep permeation of the hydrophobic -OPh functional group of DCIL-5 through the E. coli membrane model in agreement with the experimental observations. Current findings assist scientists in designing new task-specific DCILs for effective interactions with biological membranes for different applications.
Assuntos
Líquidos Iônicos , Líquidos Iônicos/farmacologia , Líquidos Iônicos/química , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/química , Relação Estrutura-Atividade , Cátions/químicaRESUMO
Green tea polyphenols (GTPs), particularly epigallocatechin-3-gallate, stand out among natural small molecules screened for their ability to target protein aggregates due to their potent anti-amyloidogenic and neuroprotective activities against various disease-related peptides and proteins. However, the clinical applications of GTPs in amyloid-related diseases have been greatly limited by drawbacks such as poor chemical stability and low bioavailability. To address these limitations, this study utilized an Iranian green tea polyphenolic extract as a reducing agent to neutralize silver ions and facilitate the formation of silver nanoparticle capped by GTPs (GTPs-capped AgNPs). The results obtained from this study demonstrate that GTPs-capped AgNPs are more effective than free GTPs at inhibiting amyloid fibrillation and reducing cytotoxicity induced by amyloid fibrils of human insulin and α-synuclein (α-syn). This improved efficacy is attributed to the increased surface/volume ratio of GTPs-capped AgNPs, which can enhance their binding affinity to amyloidogenic species and boosts their antioxidant activity. The mechanism by which GTPs-capped AgNPs inhibit amyloid fibrillation appears to vary depending on the target protein. For structured protein human insulin, GTPs-capped AgNPs hinder fibrillation by constraining the protein in its native-like state. In contrast, GTPs-capped AgNPs modulate fibrillation of intrinsically disordered proteins like α-syn by redirecting the aggregation pathway towards the formation of non-toxic off-pathway oligomers or amorphous aggregates. These findings highlight polyphenol-functionalized nanoparticles as a promising strategy for targeting protein aggregates associated with neurodegenerative diseases.
Assuntos
Nanopartículas Metálicas , alfa-Sinucleína , Humanos , Prata/farmacologia , Prata/química , Agregados Proteicos , Antioxidantes , Irã (Geográfico) , Amiloide/metabolismo , Polifenóis/farmacologia , Proteínas Amiloidogênicas , Insulina , Chá/químicaRESUMO
The interplay between α-synuclein (α-syn) and catechols plays a central role in Parkinson's disease. This may be related to the modulating effects of catechols on the various aspects of α-syn fibrillization. Some of these effects may be attributed to the membrane-binding properties of the protein. In this work, we compare the effect of some catechols, including dopamine, epinephrine, DOPAL, and levodopa in micromolar concentrations, on the in vitro cytotoxicity of α-syn fibrils on human neuroblastoma SH-SY5Y cells. The study was followed by comparing the interactions of resulting structures with rat brain mitochondria used as an in vitro biological model. The obtained results demonstrate that catechols-induced structures have lost their cytotoxicity mimicking apoptotic cell death mediated by α-syn aggregates in different proportions. Moreover, α-syn fibrils-induced mitochondrial dysfunction, evaluated by a range of biochemical assays, was modulated by catechols-modified α-syn oligomers in different manners, as levodopa and DOPAL demonstrated the maximal and minimal effects, respectively. The plausible mechanism causing the inhibition of α-syn cytotoxic fibrillization and mitochondrial dysfunction by catechols is discussed. Taken together, we propose that catechols can prevent the cytotoxic assembly of α-syn and its destructive effects on mitochondria at various stages, suggesting that decreased levels of catechols in dopaminergic neurons might accelerate the α-syn cytotoxicity and mitochondrial dysfunction implicating Parkinson's disease.
Assuntos
Neuroblastoma , Doença de Parkinson , Humanos , alfa-Sinucleína/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Levodopa , Catecóis/farmacologia , Amiloide/metabolismo , Proteínas AmiloidogênicasRESUMO
Considering the central role of oxidative stress in the onset and progress of Parkinson's diseases (PD), search for compounds with antioxidant properties has attracted a growing body of attention. Here, we compare the neuroprotective effect of bulk and nano forms of the polyphenolic fraction of propolis (PFP) against rotenone-induced cellular and animal models of PD. Mass spectrometric analysis of PFP confirmed the presence of multiple polyphenols including kaempferol, naringenin, coumaric acid, vanillic acid, and ferulic acid. In vitro cellular experiments indicate the improved efficiency of the nano form, compared to the bulk form, of PFP in attenuating rotenone-induced cytotoxicity characterized by a decrease in cell viability, release of lactate dehydrogenase, increased ROS generation, depolarization of the mitochondrial membrane, decreased antioxidant enzyme activity, and apoptosis induction. In vivo experiments revealed that while no significant neuroprotection was observed relating to the bulk form, PFP nanosheets were very effective in protecting animals, as evidenced by the improved behavioral and neurochemical parameters, including decreased lipid peroxidation, increased GSH content, and antioxidant enzyme activity enhancement. We suggest that improved neuroprotective effects of PFP nanosheets may be attributed to their increased water solubility and enrichment with oxygen-containing functional groups (such as OH and COOH), leading to increased antioxidant activity of these compounds.
Assuntos
Fármacos Neuroprotetores , Doença de Parkinson , Própole , Animais , Doença de Parkinson/tratamento farmacológico , Rotenona/toxicidade , Fármacos Neuroprotetores/farmacologia , Própole/farmacologia , Antioxidantes/farmacologia , Polifenóis/farmacologia , Estresse Oxidativo , Modelos Animais de DoençasRESUMO
Parkinson's disease (PD) is the most common neurological movement disorder characterized by the selective and irreversible loss of dopaminergic neurons in substantia nigra pars compacta resulting in dopamine deficiency in the striatum. While most cases are sporadic or environmental, about 10% of patients have a positive family history with a genetic cause. The misfolding and aggregation of α-synuclein (α-syn) as a casual factor in the pathogenesis of PD has been supported by a great deal of literature. Extensive studies of mechanisms underpinning degeneration of the dopaminergic neurons induced by α-syn dysfunction suggest a complex process that involves multiple pathways, including mitochondrial dysfunction and increased oxidative stress, impaired calcium homeostasis through membrane permeabilization, synaptic dysfunction, impairment of quality control systems, disruption of microtubule dynamics and axonal transport, endoplasmic reticulum/Golgi dysfunction, nucleus malfunction, and microglia activation leading to neuroinflammation. Among them mitochondrial dysfunction has been considered as the most primary target of α-syn-induced toxicity, leading to neuronal cell death in both sporadic and familial forms of PD. Despite reviewing many aspects of PD pathogenesis related to mitochondrial dysfunction, a systemic study on how α-syn malfunction/aggregation damages mitochondrial functionality and leads to neurodegeneration is missing in the literature. In this review, we give a detailed molecular overview of the proposed mechanisms by which α-syn, directly or indirectly, contributes to mitochondrial dysfunction. This may provide valuable insights for development of new therapeutic approaches in relation to PD. Antioxidant-based therapy as a potential strategy to protect mitochondria against oxidative damage, its challenges, and recent developments in the field are discussed.
Assuntos
Doença de Parkinson , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Antioxidantes/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/terapiaRESUMO
Multidrug resistance of bacteria and persistent infections related to biofilms, as well as the low availability of new antibacterial drugs, make it urgent to develop new antibiotics. Here, we evaluate the antibacterial and anti-biofilm properties of ticlopidine (TP), an anti-platelet aggregation drug, TP showed antibacterial activity against both gram-positive (MRSA) and gram-negative (E. coli, and P. aeruginosa) bacteria over a long treatment period. TP significantly reduced the survival of gram-negative bacteria in human blood though impact on gram-positives was more limited. TP may cause death in MRSA by inhibiting staphyloxanthin pigment synthesis, leading to oxidative stress, while scanning electron microscopy imaging indicate a loss of membrane integrity, damage, and consequent death due to lysis in gram-negative bacteria. TP showed good anti-biofilm activity against P. aeruginosa and MRSA, and a stronger biofilm degradation activity on P. aeruginosa compared to MRSA. Measuring fluorescence of the amyloid-reporter Thioflavin T (ThT) in biofilm implicated inhibition of amyloid formation as part of TP activity. This was confirmed by assays on the purified protein in P. aeruginosa, FapC, whose fibrillation kinetics was inhibited by TP. TP prolonged the lag phase of aggregation and reduced the subsequent growth rate and prolonging the lag phase to very long times provides ample opportunity to exert TP's antibacterial effect. We conclude that TP shows activity as an antibiotic against both gram-positive and gram-negative bacteria thanks to a broad range of activities, targeting bacterial metabolic processes, cellular structures and the biofilm matrix.
Assuntos
Antibacterianos , Escherichia coli , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas , BiofilmesRESUMO
Natural compounds with anti-aggregation capacity are increasingly recognized as viable candidates against neurodegenerative diseases. Recently, the polyphenolic fraction of propolis (PFP), a complex bee product, has been shown to inhibit amyloid aggregation of a model protein especially in the nanosheet form. Here, we examine the aggregation-modulating effects of the PFP nanosheets on α-synuclein (α-syn), an intrinsically disordered protein involved in the pathogenesis of Parkinson's disease. Based on a range of biophysical data including intrinsic and extrinsic fluorescence, circular dichroism (CD) data, and nuclear magnetic resonance spectroscopy, we propose a model for the interaction of α-syn with PFP nanosheets, where the positively charged N-terminal and the middle non-amyloid component regions of α-syn act as the main binding sites with the negatively charged PFP nanosheets. The Thioflavin T (ThT) fluorescence, Congo red absorbance, and CD data reveal a prominent dose-dependent inhibitory effect of PFP nanosheets on α-syn amyloid aggregation, and the microscopy images and MTT assay data suggest that the PFP nanosheets redirect α-syn aggregation toward nontoxic off-pathway oligomers. When preformed α-syn amyloid fibrils are present, fluorescence images show co-localization of PFP nanosheets and ThT, further confirming the binding of PFP nanosheets with α-syn amyloid fibrils. Taken together, our results demonstrate the binding and anti-aggregation activity of PFP nanosheets in a disease-related protein system and propose them as potential nature-based tools for probing and targeting pathological protein aggregates in neurodegenerative diseases.
Assuntos
Própole , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Própole/farmacologia , Polifenóis/farmacologia , Agregados ProteicosRESUMO
Poor water solubility and low bioavailability are considered as two main factors restricting therapeutic applications of natural polyphenols in relation to various disorders including amyloid-related diseases. Among various strategies developed to overcome these limitations, nanonization has attracted considerable attention. Herein, we compared the potency of bulk and nano forms of the polyphenolic fraction of pomegranate seed (PFPS) for modulating Hen Egg White Lysozyme (HEWL) amyloid fibril formation. Prepared PFPS nanosheets using direct oxidative pyrolysis were characterized by employing a range of spectroscopic and microscopic techniques. We found that the nano form can inhibit the assembly process and disintegrate preformed fibrils of HEWL much more effective than the bulk form of PFPS. Moreover, MTT-based cell viability and hemolysis assays showed the capacity of both bulk and nano forms of PFPS in attenuating HEWL amyloid fibril-induced toxicity, where the nano form was more effective. On the basis of thioflavin T results, a delay in the initiation of amyloid fibril assembly of HEWL appears to be the mechanism of action of PFPS nanosheets. We suggest that the improved efficiency of PFPS nanosheets in modulating the HEWL fibrillation process may be attributed to their increased surface area in accord with the surface-assistance model. Our results may present polyphenol-based nanosheets as a powerful approach for drug design against amyloid-related diseases.
RESUMO
Alpha-synuclein (α-syn) aggregation and mitochondrial dysfunction are considered as two of the main factors associated with Parkinson's disease (PD). In the present investigation, the effectiveness of the amyloid fibrils obtained from α-syn with those of hen egg white lysozyme (HEWL), as disease-related and-unrelated proteins, to damage rat brain and rat liver mitochondria have been investigated. This was extended by looking at SH-SY5Y human neuroblastoma cells and erythrocytes, thereby investigating the significance of structural characteristics of amyloid fibrils related to their interactions with biomembranes obtained from various sources. Results presented clearly demonstrate substantial differences in the response of tested biomembranes to toxicity induced by α-syn/HEWL amyloid fibrils, highlighting a structure-function relationship. We found that fibrillar aggregates of α-syn, but not HEWL, caused a significant increase in mitochondrial ROS, loss of membrane potential, and mitochondrial swelling, in a dose-dependent manner. Toxicity was found to be more pronounced in brain mitochondria, as compared to liver mitochondria. For SH-SY5Y cells and erythrocytes, however, both α-syn and HEWL amyloid fibrils showed the capacity to induce toxicity. Taken together, these results may suggest selective toxicity of α-syn amyloid fibrils to mitochondria mediated likely by their direct interaction with the outer mitochondrial membrane, indicating a correlation between specific structural characteristics of α-syn fibrils and an organelle strongly implicated in PD pathology.
Assuntos
Amiloide/química , Encéfalo/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , alfa-Sinucleína/química , Amiloide/farmacologia , Animais , Encéfalo/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Galinhas , Clara de Ovo/química , Eritrócitos/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Muramidase/química , Muramidase/farmacologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ratos , Relação Estrutura-Atividade , alfa-Sinucleína/genéticaRESUMO
Among common strategies for amyloid fibrillation inhibition, the use of naturally occurring polyphenols as an efficient therapeutic approach has attracted a growing body of attention. However, the poor water solubility and low bioavailability of these compounds have greatly restricted their clinical application in amyloid-related diseases. Thus, different types of formulations have been developed to overcome these limitations; among them, nanonization appears to be one of the most notable approaches. Herein, we show that the polyphenolic fraction of propolis (PFP), in the nanosheet form (PFP nanosheet), exhibits an improved capacity for amyloid fibrillation inhibition as well as clearance of preformed fibrils of bovine insulin. This increased efficiency is suggested to be related to the aqueous solubility and surface area enhancement as well as surface modifications upon undergoing the nanonization process, which can lead to strong binding with and trapping of protein at the surface of the nanosheets. On the basis of thioflavin T results, it is suggested that although PFP may modulate the fibrillation process via shortening of the lag phase, prolongation of the nucleation phase through interaction with and stabilizing monomeric species is the mechanism of action of PFP nanosheets. We propose that nanonization of natural small molecules can be considered as a powerful approach to improve their anti-amyloidogenic properties and overcome obstacles originating from poor water solubility and low bioavailability of drug candidates relating to neurodegenerative diseases. Taken together, the obtained results may suggest PFP nanosheets as a potential candidate for use against neurological disorders.
Assuntos
Amiloide/antagonistas & inibidores , Materiais Biocompatíveis/farmacologia , Insulina/química , Nanopartículas/química , Polifenóis/farmacologia , Própole/química , Amiloide/metabolismo , Animais , Materiais Biocompatíveis/química , Bovinos , Insulina/metabolismo , Teste de Materiais , Tamanho da Partícula , Polifenóis/químicaRESUMO
There are many reports demonstrating that various derivatives of carbon nanoparticles are effective inhibitors of protein aggregation. As surface structural features of nanoparticles play a key role on modulating amyloid fibrillation process, in the present in vitro study, bovine insulin and hen egg white lysozyme (HEWL) were selected as two model proteins to investigate the reducing effect of graphene oxide quantum dots (GOQDs) on their assembly under amyloidogenic conditions. GOQDs were prepared through direct pyrolysis of citric acid, and the reduction step was carried out using ascorbic acid. The prepared nanoparticles were characterized by UV-Vis, X-ray photoelectron, and FT-IR spectroscopies, transmission electron and atomic force microscopies, zeta potential measurement, and Nile red fluorescence assay. They showed the tendencies to modulate the assembly of the proteins through different mechanisms. While GOQDs appeared to have the capacity to inhibit fibrillation, the presence of reduced GOQDs (rGOQDs) was found to promote protein assembly via shortening the nucleation phase, as suggested by ThT fluorescence data. Moreover, the structures produced in the presence of GOQDs or rGOQDs were totally nontoxic. We suggest that surface properties of these particles may be part of the differences in their mechanism(s) of action.
Assuntos
Grafite/química , Grafite/metabolismo , Oxigênio/metabolismo , Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/química , Amiloidose/metabolismo , Animais , Bovinos , Insulina/química , Modelos Biológicos , Muramidase/química , Nanopartículas/química , Oxigênio/fisiologia , Agregados Proteicos/efeitos dos fármacos , Agregados Proteicos/fisiologia , Pontos Quânticos/química , Propriedades de Superfície/efeitos dos fármacosRESUMO
Extensive research has shown that assembling of α-synuclein amyloid aggregates on mitochondria is an important mechanistic feature of Parkinson's disease (PD) and other Lewy body disorders. However, the molecular mechanism(s) of its neuronal toxicity remain unclear. Type 1 Hexokinase (HKI), a key enzyme in the control of brain glucose metabolism, plays an important role in protecting against mitochondrially-regulated apoptosis through reducing generation of reactive oxygen species (ROS). The release of mitochondrially-bound HKI causes a significant decrease in enzyme activity and triggers oxidative stress. Here, we have investigated the potency of amyloid fibrillation products arising from α-synuclein and hen egg white lysozyme (HEWL) for the release of HKI and ROS content enhancement in mitochondria isolated from rat brain. Results clearly indicate the capacity of the fibrillation products of α-synuclein, and not HEWL, to trigger release of HKI from the Type A binding site of mitochondria for the enzyme and to induce mitochondrial ROS enhancement in a dose-dependent manner. Moreover, we found that curcumin was very effective in preventing mitochondrial HKI release and ROS enhancement induced by α-synuclein fibrillation products. The pathophysiological significance of mitochondrial HKI activity and localization in pathogenesis of neurodegenerative disorders including PD are discussed. Taken together, these results may offer insight into a possible mechanism by which disease-related peptides and proteins may exert their neuronal toxicity.
Assuntos
Amiloide/toxicidade , Curcumina/farmacologia , Hexoquinase/metabolismo , Mitocôndrias/metabolismo , Doença de Parkinson/etiologia , alfa-Sinucleína/química , Amiloide/biossíntese , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Galinhas , Humanos , Muramidase , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , alfa-Sinucleína/toxicidadeRESUMO
In this study, we formulated silymarin-HSA nanoplex and assayed its ability to reduce LPS-induced toxicity in vitro and in vivo. Silymarin molecules were encapsulated into HSA nanoplex and the loading efficiency and characterization of fabricated nanoplex were performed by using HPLC, TEM, SEM, DLS, FTIR analysis, and theoretical studies. Afterwards, their protective effect against LPS (20⯵g/ml) -induced toxicity in SH-SY5Y cells was investigated by MTT, ROS, and apoptosis assays. For in vivo experiments, rats were pre-treated with either silymarin or silymarin -HSA nanoplex (200â¯mg/kg) orally for 3â¯days and at third day received LPS by IP at a dose of 0.5â¯mg/kg, 150â¯min before scarification followed by SOD and CAT activity assay. The formulation of silymarin-HSA nanoplex showed a spherical shape with an average diameter between 50â¯nm and 150â¯nm, hydrodynamic radius of 188.3â¯nm, zeta potential of -26.6â¯mV, and a drug loading of 97.3%. In LPS-treated cells, pretreatments with silymarin-HSA noncomplex recovered the cell viability and decreased the ROS level and corresponding apoptosis more significantly than free silymarin. In rats, it was also depicted that, silymarin-HSA noncomplex can increase the SOD and CAT activity in brain tissue at LPS-triggered oxidative stress model more significantly than the free counterpart. Therefore, nanoformulation of silymarin improved its capability to reduce LPS-induced oxidative stress by restoring cell viability and elevation of SOD and CAT activity in vitro and in vivo, respectively. In conclusion, formulation of silymarin may hold a great promise in the development of antioxidant agents.
Assuntos
Antioxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Albumina Sérica Humana/química , Silimarina/farmacologia , Animais , Antioxidantes/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lipopolissacarídeos/toxicidade , Masculino , Neuroblastoma/patologia , Tamanho da Partícula , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Silimarina/administração & dosagemRESUMO
A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from α-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.
Assuntos
Amiloide/metabolismo , Encéfalo/citologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Membrana Celular/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Modelos Biológicos , Muramidase/metabolismo , Muramidase/farmacologia , Ratos , Espécies Reativas de Oxigênio , alfa-Sinucleína/metabolismo , alfa-Sinucleína/farmacologiaRESUMO
A growing body of evidence suggests that secretion and assembly of insulin to amyloid fibrils reduce its efficacy in treating type II diabetes and may lead to dysfunctioning of several organs. The research presented here explores the effects of silibinin on the in vitro amyloid fibrillation and cytotoxicity of bovine insulin fibrils on SH-SY5Y human neuroblastoma cells. Interaction of the resulting structures with rat brain mitochondria was also investigated. Using a range of methods for amyloid detection we showed that insulin fibrillation was significantly inhibited by silibinin in a dose-dependent fashion. Moreover, we found that silibinin was very effective in attenuating insulin fibril-induced neuronal toxicity characterized by decrease of cell viability, the release of lactate dehydrogenase, intracellular reactive oxygen species enhancement, morphological alterations, and apoptotic cell death induction. While insulin fibrillation products showed the capacity to damage mitochondria, the resultant structures produced in the presence of silibinin were totally ineffective. Together, results demonstrate the capacity of insulin fibrils to cause SH-SY5Y cell death by inducing necrosis/apoptosis changes and suggest how silibinin may afford protection. It is concluded that elucidation of such protection may provide important insights into the development of preventive and therapeutic agents for amyloid-related diseases.
Assuntos
Amiloide/química , Amiloide/toxicidade , Insulina/química , Insulina/toxicidade , Membranas Mitocondriais/efeitos dos fármacos , Agregados Proteicos , Silibina/farmacologia , Animais , Bovinos , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Membranas Mitocondriais/metabolismoRESUMO
In the present study, we have investigated the effects of protein concentration and stirring on the in vitro assembly of Hen Egg White Lysozyme (HEWL), particularly with regard to the aggregate morphology and anti-amyloidogenic properties of two naturally occurring polyphenols, taxifolin and silibinin. The results obtained clearly demonstrated that applying stirring and concentration enhancement alter the amount as well as morphology of amyloid fibrils formed. Additionally, latter aggregates exhibited higher affinity for amyloid-specific dyes. The second part of the present investigation was devoted to studies involving anti-amyloidogenic properties of selected polyphenols. Importantly, we found that the potency of polyphenols to inhibit HEWL amyloid fibrillation and related toxicity is strongly dependent on the amyloidogenic conditions in which amyloid fibrils are produced. Based on obtained data, under condition where the rate of protein assembly is high (higher protein concentration and stirring), the capacity of polyphenols to inhibit HEWL fibrillogenesis and related cytotoxicity may dramatically decrease. Similar results were obtained when we used taxifolin to inhibit bovine insulin amyloid fibrillation. Additionally, amyloidogenic conditions may also affect the mechanism by which these molecules inhibit HEWL fibrillation. The possible mechanism by which selected polyphenols exert their inhibitory effects, under various experimental conditions, is also discussed.
Assuntos
Amiloide/química , Muramidase/química , Polifenóis/farmacologia , Agregados Proteicos/efeitos dos fármacos , Amiloide/toxicidade , Animais , Bovinos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Muramidase/toxicidade , Propriedades de SuperfícieRESUMO
Post-translational modifications play important roles in conformational properties and aggregation propensities of different peptides and proteins. In the present study, we have investigated the effects of acetylation of lysine residues on the structure and aggregation properties of apomyoglobin (apoMb).All of the 19 lysine residues were modified. Far-, near-UV CD, intrinsic and acrylamide quenching fluorescence studies indicated that acetylation significantly influences conformation of apoMb by altering both its secondary and tertiary structures. A considerable decrease in ANS fluorescence intensity was observed, which also suggested disruption of the heme pocket. Dynamic light scattering indicated partial compaction of protein structure as a consequence of the shielding effect of acetylation. While the presence of well-defined mature fibrils was detected in solutions of native apoMb, acetylation promoted formation of non-toxic amorphous aggregates, with low ß-sheets content and decreased affinity for Thioflavin T, an amyloid-specific dye. Results are discussed in terms of the role of surface charge in conformational alterations of proteins and how small changes in ionic networks may affect aggregation pathways and morphology of the resulting aggregates. The physiological significance of the modification process in controlling cytotoxicity of the aggregated species is also discussed.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Apoproteínas/metabolismo , Heme/metabolismo , Lisina/metabolismo , Mioglobina/metabolismo , Agregados Proteicos , Processamento de Proteína Pós-Traducional , Acetilação , Proteínas Amiloidogênicas/química , Animais , Apoproteínas/química , Benzotiazóis , Sobrevivência Celular/efeitos dos fármacos , Heme/química , Cavalos , Lisina/química , Mioglobina/química , Células PC12 , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Espectrometria de Fluorescência , Eletricidade Estática , Tiazóis/químicaRESUMO
Among therapeutic approaches for amyloid-related diseases, attention has recently turned to the use of natural products as effective anti-aggregation compounds. Although a wealth of in vitro and in vivo evidence indicates some common inhibitory activity of these compounds, they don't generally suggest the same mechanism of action. Here, we show that taxifolin, a ubiquitous bioactive constituent of foods and herbs, inhibits formation of HEWL amyloid fibrils and their related toxicity by causing formation of very large globular, chain-like aggregates. A range of amyloid-specific techniques were employed to characterize this process. We found that taxifolin exerts its effect by binding to HEWL prefibrillar species, rather than by stabilizing the molecule in its native-like state. Furthermore, it's binding results in diverting the amyloid pathway toward formation of very large globular, chain-like aggregates with low ß-sheet content and reduced solvent-exposed hydrophobic patches. ThT fluorescence measurements show that the binding capacity of taxifolin is significantly reduced, upon generation of large protofibrillar aggregates at the end of growth phase. We believe these results may help design promising inhibitors of protein aggregation for amyloid-related diseases.
Assuntos
Neurofibrilas/efeitos dos fármacos , Quercetina/análogos & derivados , Sítios de Ligação , Linhagem Celular , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Simulação de Acoplamento Molecular , Neurofibrilas/metabolismo , Quercetina/metabolismo , Quercetina/farmacologia , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Increasing body of evidence suggests that polyphenols frequently interacting with amyloid aggregates and/or interfering with aggregate species to bind biomembranes may serve as a therapeutic approach for the treatment of amyloid-related diseases. Hence, in the present study, the possible effects of three naturally occurring polyphenols including Curcumin, Quercetin, and Resveratrol on mitochondrial membrane permeabilization induced by Hen Egg White Lysozyme (HEWL) oligomers were investigated. Our results indicated that pre-incubation of mitochondrial homogenate with polyphenols considerably inhibit membrane permeabilization in a concentration dependent manner. In parallel, HEWL oligomers, which were co-incubated with the polyphenols, showed less effectiveness on membrane permeabilization, suggesting that toxicity of oligomers was hindered. Using a range of techniques including fluorescence quenching, Nile red binding assay, zeta potential and size measurements, CD (far- and near-UV) spectroscopy, and molecular docking, we found that the polyphenols, structure-dependently, interact with and induce conformational changes in HEWL oligomers, thereby inhibit their toxicity. We proposed a mechanism by which selected polyphenols induce their protective effects through binding to mitochondria and interfering with HEWL oligomer-membrane interactions and/or by direct interaction with HEWL oligomers, induction of conformational changes, and generating far less toxic species. However, additional studies are needed to elucidate the detailed mechanisms involved.
Assuntos
Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Muramidase/química , Polifenóis/farmacologia , Multimerização Proteica , Animais , Cinética , Simulação de Acoplamento Molecular , Muramidase/metabolismo , Permeabilidade/efeitos dos fármacos , Polifenóis/metabolismo , Estrutura Quaternária de Proteína , RatosRESUMO
An increasing number of studies conducted under in vitro and in vivo conditions, have concluded that polyphenols, compounds frequently occurring in many herbs with antioxidant properties, prevent and reverse amyloid fibril formation. However, the mechanisms by which these natural products modulate the protein aggregation process are poorly understood. Herein, a range of techniques including thioflavin T (ThT) and ANS fluorescence assays, electron microscopy and circular dichroism have been employed to determine the efficacy of rosmarinic acid (RA) and resveratrol (Res) on the inhibition/reversion of fibrillogenesis and hindering cytotoxicity induced by protofibrils and amyloid fibrils of hen egg white lysozyme (HEWL). Results demonstrated that both polyphenols effectively inhibit fibrillogenesis and destabilize preformed fibrils of HEWL in a concentration-dependent manner. Cytotoxicity protection on PC12 cells was also observed using the MTT assay, ROS production assay, and phase-contrast microscopy. It is suggested that the mechanism underlying the inhibitory effects of RA and Res is to prevent hydrophobic interactions between HEWL amyloidogenic prefibrillar species, although additional studies is needed to elucidate the detailed mechanisms involved. A combination of antioxidative and anti-amyloidogenic properties of these molecules may provide them with the described neuroprotective capacities.