RESUMEN

It has been hypothesized that --Parkinson's disease (PD) may be initiated in the gastrointestinal tract, before manifesting in the central nervous system. In this respect, it was demonstrated that lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, accelerates the in vitro formation of α-synuclein (aSyn) fibrils, whose intracellular deposits is a histological hallmark of the degeneration of dopaminergic neurons in PD. Herein, N-terminal acetylation and missense mutations of aSyn (A30P, A53T, E46K, H50Q and G51D) linked to rare, early-onset forms of familial PD were investigated regarding their effect on aSyn aggregation stimulated by either LPS or small unilamellar lipid vesicles (SUVs). Our findings indicated that LPS as well as SUVs induce the fibrillation of N-terminally acetylated wild-type aSyn (Ac-aSyn-WT) more remarkably than the non-acetylated protein, while the LPS-free protein alone did not undergo fibrillation under our assay conditions. In addition, with the exception of A30P, PD mutations increased the fibrillation of Ac-aSyn in the presence of LPS compared with Ac-aSyn-WT. The most pronounced effect of LPS was noticed for A53T, as observed when either Thioflavin-T or JC-1 were used as fluorescent probes for fibrils. Overall, our results suggest for the first time the existence of a synergy between LPS and PD mutations/N-terminal acetylation toward aSyn fibrillation.

Asunto(s)

Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/patología , Lipopolisacáridos , Acetilación , Mutación

RESUMEN

Intestinal dysbiosis seems to play a role in neurodegenerative pathologies. Parkinson's disease (PD) patients have an altered gut microbiota. Moreover, mice treated orally with the gut microbe Proteus mirabilis developed Parkinson's-like symptoms. Here, the possible involvement of P. mirabilis urease (PMU) and its B subunit (PmUreß) in the pathogenesis of PD was assessed. Purified proteins were given to mice intraperitoneally (20 µg/animal/day) for one week. Behavioral tests were conducted, and brain homogenates of the treated animals were subjected to immunoassays. After treatment with PMU, the levels of TNF-α and IL-1ß were measured in Caco2 cells and cellular permeability was assayed in Hek 293. The proteins were incubated in vitro with α-synuclein and examined via transmission electron microscopy. Our results showed that PMU treatment induced depressive-like behavior in mice. No motor deficits were observed. The brain homogenates had an increased content of caspase-9, while the levels of α-synuclein and tyrosine hydroxylase decreased. PMU increased the pro-inflammatory cytokines and altered the cellular permeability in cultured cells. The urease, but not the PmUreß, altered the morphology of α-synuclein aggregates in vitro, forming fragmented aggregates. We concluded that PMU promotes pro-inflammatory effects in cultured cells. In vivo, PMU induces neuroinflammation and a depressive-like phenotype compatible with the first stages of PD development.

RESUMEN

INTRODUCTION: Since the discovery of the presynaptic protein α-synuclein (aSyn) as a central player in Parkinson's disease (PD), several key questions on the function of the protein in neurodegeneration processes remain unclear, including: is there a synergy between dopamine metabolism and the formation of toxic aSyn species in neurons? What is the role of aSyn in the immunological system? AREAS COVERED: Herein, the authors revisit the intricate pathways related to dopamine metabolism and how it impacts on aSyn aggregation/function. Additionally, they discuss the importance of aSyn in the immune response to viral infections as well as the current findings on the possible protective role of certain virus vaccines against PD and other neuropathologies. EXPERT OPINION: The physiological function of aSyn seems to cover different pathways, such as immune response against infections and a neuroprotective role, besides the already-established regulation of synaptic vesicle trafficking. Clinical studies with monoclonal antibodies against aSyn aggregates have shown disappointing results in patients with early-stage PD. Alternatively, we could consider, as immunological target, specific neurotoxic oligomers of aSyn formed in the presence of dopamine metabolites, such as DOPAL. Nevertheless, the crucial question remains as to whether removing these protein deposits will affect the clinical course of the disease.

Asunto(s)

Enfermedad de Parkinson , Virosis , Humanos , alfa-Sinucleína , Enfermedad de Parkinson/metabolismo , Dopamina , Neuronas/metabolismo , Virosis/patología

RESUMEN

The misfolding and aggregation of the protein α-synuclein (aSyn) into potentially neurotoxic oligomers is believed to play a pivotal role in the neuropathogenesis of Parkinson's disease (PD). Herein, we explore how apomorphine (Apo), a nonselective dopamine D1 and D2 receptor agonist utilized in the therapy for PD, affects the aggregation and toxicity of aSyn in vitro. Our data indicated that Apo inhibits aSyn fibrillation leading to the formation of large oligomeric species (Apo-aSyn-O), which exhibit remarkable toxicity in mesencephalic dopaminergic neurons in primary cultures. Interestingly, purified Apo-aSyn-O, even at very low concentrations, seems to be capable of converting unmodified aSyn monomer into neurotoxic species. Collectively, our findings warn for a possible dangerous effect of Apo on aSyn misfolding/aggregation pathway.

Asunto(s)

alfa-Sinucleína

RESUMEN

The current pandemic of coronavirus disease 2019 (COVID-19) has gained increased attention in the neuroscience community, especially taking into account the neuroinvasive potential of its causative agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the impact of its infection on the structure and function of the brain. Apart from the neurotropic properties of SARS-CoV-2, it is likewise important the observation that virus infection may perturb specific cellular processes that are believed to play an important role in the pathogenesis of diverse neurological disorders, particularly in Parkinson's disease (PD). In this scenario, viral infection-induced colon inflammation, gut microbial imbalance, and α-synuclein upregulation are of particular interest with regard to the interplay between the gastrointestinal tract and the central nervous system (microbiome-gut-brain axis). In this Perspective, we present a critical view on the different hypotheses that are recently being raised by neuroscientists about the relationship between SARS-CoV-2 infection and long-lasting neurodegenerative disorders, opening the question of whether COVID-19 might represent a risk factor for the development of PD.

Asunto(s)

Disbiosis/virología , Enfermedades Neurodegenerativas/virología , SARS-CoV-2/patogenicidad , Virosis/virología , Encéfalo/patología , Encéfalo/virología , Disbiosis/complicaciones , Microbioma Gastrointestinal/fisiología , Humanos , Enfermedades Neurodegenerativas/patología , Virosis/complicaciones , Virosis/patología

Asunto(s)

COVID-19/complicaciones , Disbiosis/complicaciones , Microbioma Gastrointestinal , Enfermedad de Parkinson/etiología , SARS-CoV-2/patogenicidad , Traslocación Bacteriana/inmunología , COVID-19/microbiología , Sistema Nervioso Entérico/fisiopatología , Humanos , Inflamación , Lipopolisacáridos/metabolismo , Lipopolisacáridos/toxicidad , Microglía/inmunología , Microglía/metabolismo , Modelos Biológicos , Neuroinmunomodulación , Enfermedad de Parkinson/microbiología , Agregación Patológica de Proteínas , Factores de Riesgo , Nervio Vago/fisiopatología , alfa-Sinucleína/metabolismo

RESUMEN

Identifying the mechanisms by which the presynaptic protein α-synuclein (aSyn) is associated with neurodegeneration of dopamine neurons is a major priority in the Parkinson's disease (PD) field. Studies indicate that DOPAL (3,4-dihydroxyphenylacetaldehyde), an aldehyde generated from the enzymatic oxidation of dopamine, may convert aSyn monomer into a neurotoxin via formation of covalently stabilized toxic oligomers. Herein we investigated the role of N-terminal acetylation and familial aSyn mutations (A30P, A53T, E46K, G51D, and H50Q) on DOPAL-induced oligomerization of the protein. Our results indicate that the wild-type (WT) N-terminally acetylated aSyn (Ac-aSyn) is less prone to form oligomers upon incubation with DOPAL than the non-N-terminally acetylated protein. On the other hand, familial mutants from Ac-aSyn, particularly A53T, E46K, and H50Q increased the formation of DOPAL-derived aSyn oligomers, especially large oligomers. Binding of aSyn to synaptic-like small unilamellar vesicles (SUVs) protected distinctive aSyn variants against the effects of DOPAL. While N-terminal acetylation increased the protective action of SUVs against DOPAL-induced aSyn oligomerization, A53T, A30P, and H50Q mutations in Ac-aSyn had an opposite effect. This means that PD-linked mutations may not only perturb the affinity of aSyn for membranes but also influence the formation of DOPAL-mediated oligomers. Overall, our findings provide important evidence for the existence of a connection between familial mutations of aSyn, their distinct affinity to lipid membranes, and the formation of potentially toxic oligomers of the protein mediated by DOPAL.

Asunto(s)

Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Neuronas Dopaminérgicas/efectos de los fármacos , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Ácido 3,4-Dihidroxifenilacético/farmacología , Acetilación/efectos de los fármacos , Dopamina/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Mutación/genética , Oxidación-Reducción/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , alfa-Sinucleína/metabolismo

RESUMEN

The formation of neurotoxic oligomers of the presynaptic protein α-Synuclein (aSyn) is suggested to be associated with Parkinson's disease neurodegeneration. In this respect, it was demonstrated that the aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), a product from the enzymatic oxidation of dopamine, is capable of stabilizing potentially toxic aSyn oligomers via formation of covalent adducts with Lys residues of the protein. In addition, DOPAL-induced production of reactive oxygen species (ROS) leads to the oxidation of aSyn's Met residues to Met-sulfoxide. Recently, our group pointed out that the pre-oxidation of all-four Met residues of aSyn, upon treatment with H2O2, decreases the formation of large aSyn-DOPAL oligomers, which are suggested to be more toxic to neurons than the corresponding small oligomers (Carmo-Gonçalves et al., Biochem. Biophys. Res. Comm. 505, 295-301. 2018). By using a series of Met to Val mutants of aSyn, we demonstrated that the ability of aSyn to scavenge ROS/H2O2 generated from DOPAL oxidation is primarily dependent on Met residues located at the C-terminal domain of the protein, which contrasts with the reactivity of aSyn against H2O2 itself in which N-terminal Met residues (notably Met5) were more readily oxidized. Interestingly, the substitution of C-terminal Met residues (particularly Met127) by Val increased the formation of DOPAL-induced large oligomers in comparison with the wild-type protein. In this context, we demonstrated that the hydrophobicity of aSyn monomer, which is affected distinctively by the oxidation of N- versus C-terminal methionines, is correlated with the formation of large (but not small) oligomers of aSyn mediated by DOPAL.

Asunto(s)

Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Peróxido de Hidrógeno/química , Metionina/química , Valina/química , alfa-Sinucleína/química , Ácido 3,4-Dihidroxifenilacético/química , Ácido 3,4-Dihidroxifenilacético/metabolismo , Sustitución de Aminoácidos , Naftalenosulfonatos de Anilina/química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Metionina/metabolismo , Mutación , Oxidación-Reducción , Dominios Proteicos , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Valina/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo

RESUMEN

BACKGROUND: Salsolinol (SALSO), a product from the reaction of dopamine (DA) with acetaldehyde, is found increased in dopaminergic neurons of Parkinson's disease (PD) patients. The administration of SALSO in rats causes myenteric neurodegeneration followed by the formation of deposits of the protein α-synuclein (aS), whose aggregation is intimately associated to PD. METHODS: NMR, isothermal titration calorimetry and MS were used to evaluate the interaction of SALSO with aS. The toxicity of SALSO and in vitro-produced aS-SALSO species was evaluated on mesencephalic primary neurons from mice. RESULTS: SALSO, under oxidative conditions, stabilizes the monomeric state besides a minor population of oligomers of aS, resulting in a strong inhibition of the fibrillation process. SALSO does not promote any chemical modification of the protein. Instead, the interaction of SALSO with aS seems to occur via hydrophobic effect, likely mediated by the NAC (non-amyloid component) domain of the protein. aS-SALSO species were found to be innocuous on primary neurons, while SALSO alone induces apoptosis via caspase-3 activation. Importantly, exogenous aS monomer was capable of protecting neurons against SALSO toxicity irrespective whether the protein was co-administered with SALSO or added until 2 h after SALSO, as evidenced by DAPI and cleaved-caspase 3 assays. Similar protective action of aS was found by pre-incubating neurons with aS before the administration of SALSO. CONCLUSIONS: Interaction of SALSO with aS leads to the formation of fibril-incompetent and innocuous adducts. SALSO toxicity is attenuated by aS monomer. SIGNIFICANCE: aS could exhibit a protective role against the neurotoxic effects of SALSO in dopaminergic neuron.

Asunto(s)

Neuronas Dopaminérgicas/efectos de los fármacos , Isoquinolinas/toxicidad , Sinapsis/metabolismo , alfa-Sinucleína/fisiología , Animales , Apoptosis/efectos de los fármacos , Calorimetría , Caspasa 3/metabolismo , Células Cultivadas , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Humanos , Espectrometría de Masas , Ratones , Oxidación-Reducción , Ratas , Espectrometría de Fluorescencia , alfa-Sinucleína/metabolismo

RESUMEN

The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.

Asunto(s)

Ácido 3,4-Dihidroxifenilacético/farmacología , Metionina/química , Neuronas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/toxicidad , Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Peróxido de Hidrógeno/farmacología , Metionina/genética , Ratones , Mutación , Neuronas/citología , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , alfa-Sinucleína/química , alfa-Sinucleína/genética

RESUMEN

Epidemiological data have suggested that exposure to environmental toxins might be associated with the etiology of Parkinson's disease (PD). In this context, certain agrochemicals are able to induce Parkinsonism in different animal models via the inhibition of mitochondrial complex I, which leads to an increase in both oxidative stress and the death of nigrostriatal neurons. Additionally, in vitro experiments have indicated that pesticides are capable of accelerating the fibrillation of the presynaptic protein α-synuclein (aS) by binding directly to the protein. However, the molecular details of these interactions are poorly understood. In the present work we demonstrate that paraquat and rotenone, two agrochemicals that lead to a Parkinsonian phenotype in vivo, bind to aS via solvent effects rather than through specific interactions. In fact, these compounds produced no significant effects on aS fibrillation under physiological concentrations of NaCl. NMR data suggest that paraquat interacts with the C-terminal domain of the disordered aS monomer. This interaction was markedly reduced in the presence of NaCl, presumably due to the disruption of electrostatic interactions between the protein and paraquat. Interestingly, the effects produced by short-term incubation of paraquat with aS on the protein conformation resembled those produced by incubating the protein with NaCl alone. Taken together, our data indicate that the effects of these agrochemicals on PD cannot be explained via direct interactions with aS, reinforcing the idea that the role of these compounds in PD is limited to the inhibition of mitochondrial complex I and/or the up-regulation of aS.

Asunto(s)

Paraquat/farmacología , Plaguicidas/farmacología , Rotenona/farmacología , Regulación hacia Arriba/efectos de los fármacos , alfa-Sinucleína/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/efectos de los fármacos , Radioisótopos/farmacocinética , Cloruro de Sodio/farmacología , alfa-Sinucleína/química , alfa-Sinucleína/ultraestructura

RESUMEN

The degeneration of dopaminergic neurons in Parkinson's disease (PD) is suggested to be associated with the generation of cytotoxic products from dopamine (DA) metabolism and the formation of fibrillar inclusions of the protein α-synuclein (AS). Despite of the role of AS in the pathogenesis of PD is not completely understood, the stabilization of nontoxic aggregates could represent a potential therapeutic route. In respect to the DA metabolism, a well-established strategy is the inhibition of the enzyme monoamine oxidase, which is responsible to catalyze the major route of inactivation of neurotransmitters. Although pharmacological strategies to treat different aspects of the parkinsonian condition are under investigation, the development of multifunctional molecules that act simultaneously on different targets associated to PD has gained attention only recently. In this work we examine the biochemical properties of synthetic and natural molecules that are capable of interfering on both DA system (via monoamine oxidase inhibition) and AS fibrillation.

Asunto(s)

Inhibidores de la Monoaminooxidasa/uso terapéutico , Monoaminooxidasa/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , alfa-Sinucleína/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Inhibidores de la Monoaminooxidasa/farmacología , Enfermedad de Parkinson/metabolismo

RESUMEN

Thioflavin-T (ThT) is a cationic benzothiazole dye that displays enhanced fluorescence upon binding to amyloid fibrils. This property makes ThT the current reagent of choice for the quantification of amyloid fibrils. Herein, we investigate the main pitfalls associated with the use of ThT-based assays to monitor the fibrillation of α-synuclein (α-syn), a protein linked to Parkinson's disease and other α-synucleinopathies. We demonstrated for the first time that ThT interacts with α-syn disordered monomer and accelerates the protein fibrillation in vitro. As a consequence, misleading conclusions may arise from the use of ThT-based real-time assays in the evaluation of anti-fibrillogenic compounds. Interestingly, NMR experiments indicated that C-terminal domain of α-syn is the main region perturbed by ThT interaction, similarly to that found for the pesticide paraquat, a well-documented accelerator of α-syn fibrillation. Moreover, we demonstrated that certain potent inhibitors of α-syn fibrillation, such as oxidized catecholamines and polyphenols, undergo spontaneous oxidation in aqueous solution, generating compounds that strongly quench ThT fluorescence. In light of these findings, we alert for possible artifacts associated to the measure of the anti-fibrillogenic activity based only on ThT fluorescence approach.

Asunto(s)

Amiloide/análisis , Amiloide/efectos de los fármacos , Tiazoles/química , Tiazoles/farmacología , alfa-Sinucleína/efectos de los fármacos , alfa-Sinucleína/metabolismo , Amiloide/química , Amiloide/metabolismo , Artefactos , Benzotiazoles , Humanos , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Tiazoles/análisis , Tiazoles/metabolismo , alfa-Sinucleína/química

RESUMEN

BACKGROUND: Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent ß-hairpin motif consistent with either neurotoxicity or pore formation. METHODS: Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the ß-hairpin motif (residues 61-74), JbtxΔ-ß; 2) a peptide corresponding the N-terminal half (residues 1-44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45-93), Jbtx C-ter. RESULTS: 1) JbtxΔ-ß disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the ß-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure-function analysis. MAJOR CONCLUSIONS: The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the ß-hairpin motif. Although the ß-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx. GENERAL SIGNIFICANCE: Jbtx represents a new type of insecticidal and membrane-active peptide.

Asunto(s)

Canavalia/enzimología , Insecticidas/farmacología , Ureasa/farmacología , Secuencia de Aminoácidos , Animales , Cucarachas , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Neuromuscular/efectos de los fármacos , Proteínas de Plantas , Isoformas de Proteínas , Proteínas Recombinantes/farmacología , Relación Estructura-Actividad , Ureasa/química

RESUMEN

Monoamine oxidase (MAO) action has been involved in the regulation of neurotransmitters levels, cell signaling, cellular growth, and differentiation as well as in the balance of the intracellular polyamine levels. Although so far obscure, MAO inhibitors are believed to have some effect on tumors progression. 1,4-naphthoquinone (1,4-NQ) has been pointed out as a potential pharmacophore for inhibition of both MAO and DNA topoisomerase activities, this latter associated with antitumor activity. Herein, we demonstrated that certain antitumor 1,4-NQs, including spermidine-1,4-NQ, lapachol, and nor-lapachol display inhibitory activity on human MAO-A and MAO-B. Kinetic studies indicated that these compounds are reversible and competitive MAO inhibitors, being the enzyme selectivity greatly affected by substitutions on 1,4-NQ ring. Molecular docking studies suggested that the most potent MAO inhibitors are capable to bind to the MAO active site in close proximity of flavin moiety. Furthermore, ability to inhibit both MAO-A and MAO-B can be potentialized by the formation of hydrogen bonds between these compounds and FAD and/or the residues in the active site. Although spermidine-1,4-NQs exhibit antitumor action primarily by inhibiting topoisomerase via DNA intercalation, our findings suggest that their effect on MAO activity should be taken into account when their application in cancer therapy is considered.

Asunto(s)

Inhibidores de la Monoaminooxidasa/síntesis química , Inhibidores de la Monoaminooxidasa/farmacología , Naftoquinonas/síntesis química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Dominio Catalítico , Sistemas de Liberación de Medicamentos , Activación Enzimática/efectos de los fármacos , Humanos , Cinética , Simulación del Acoplamiento Molecular , Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/química , Naftoquinonas/química , Naftoquinonas/farmacología , Isoformas de Proteínas

RESUMEN

Fibrillization of the protein α-synuclein (α-syn) is a hallmark of Parkinson's disease and other α-synucleinopathies. The well-established idea that α-syn is a natively disordered monomer prone to forming fibrils was recently challenged by data showing that the protein mostly exists in vitro and in vivo as helically folded tetramers that are resistant to fibrillization. These apparently conflicting findings may be reconciled by the idea that α-syn exists as a disordered monomer in equilibrium with variable amounts of dynamic oligomeric species. In this context, varying the approaches used for protein purification, such as the method used to lyse cells or the inclusion of denaturing agents, could dramatically perturb this equilibrium and hence alter the relative abundance of the disordered monomer. In the present study, we investigated how the current methods for α-syn purification affect the structure and oligomeric state of the protein, and we discuss the main pitfalls associated with the production of recombinant α-syn in Escherichia coli. We demonstrate that α-syn was expressed in E. coli as a disordered monomer independent of both the cell lysis method and the use of heating/acidification for protein purification. In addition, we provide convincing evidence that the disordered monomer exists in equilibrium with a dynamic dimer, which is not an artefact of the cross-linking protocol as previously suggested. Unlike the helically folded tetramer, α-syn dimer is prone to fibrillate and thus it may be an interesting target for anti-fibrillogenic molecules.

Asunto(s)

Escherichia coli/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Dicroismo Circular , Escherichia coli/genética , Espectroscopía de Resonancia Magnética , Multimerización de Proteína

RESUMEN

In the last decades, a series of compounds, including quinones and polyphenols, has been described as having anti-fibrillogenic action on α-synuclein (α-syn) whose aggregation is associated to the pathogenesis of Parkinson's disease (PD). Most of these molecules act as promiscuous anti-amyloidogenic agents, interacting with the diverse amyloidogenic proteins (mostly unfolded) through non-specific hydrophobic interactions. Herein we investigated the effect of the vitamins K (phylloquinone, menaquinone and menadione), which are 1,4-naphthoquinone (1,4-NQ) derivatives, on α-syn aggregation, comparing them with other anti-fibrillogenic molecules such as quinones, polyphenols and lipophilic vitamins. Vitamins K delayed α-syn fibrillization in substoichiometric concentrations, leading to the formation of short, sheared fibrils and amorphous aggregates, which are less prone to produce leakage of synthetic vesicles. In seeding conditions, menadione and 1,4-NQ significantly inhibited fibrils elongation, which could be explained by their ability to destabilize preformed fibrils of α-syn. Bidimensional NMR experiments indicate that a specific site at the N-terminal α-syn (Gly31/Lys32) is involved in the interaction with vitamins K, which is corroborated by previous studies suggesting that Lys is a key residue in the interaction with quinones. Together, our data suggest that 1,4-NQ, recently showed up by our group as a potential scaffold for designing new monoamine oxidase inhibitors, is also capable to modulate α-syn fibrillization in vitro.

Asunto(s)

Antifibrinolíticos , Neurofibrillas/efectos de los fármacos , Quinonas/farmacología , Vitamina K/farmacología , alfa-Sinucleína/metabolismo , Núcleo Celular/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , Naftoquinonas/farmacología , Vitamina K/análogos & derivados , Vitamina K/química , Vitamina K 1/farmacología , Vitamina K 2/farmacología , Vitamina K 3/farmacología , alfa-Sinucleína/genética

RESUMEN

BACKGROUND: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. METHODOLOGY/PRINCIPAL FINDINGS: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. CONCLUSIONS/SIGNIFICANCE: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

Asunto(s)

Lipoproteínas/química , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Bovinos , Equinococosis/parasitología , Electroforesis , Humanos , Lipoproteínas/aislamiento & purificación , Espectrometría de Masas , Microscopía , Datos de Secuencia Molecular , Subunidades de Proteína/química , Homología de Secuencia de Aminoácido

RESUMEN

Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic and exogenous amines and its inhibitors have therapeutic value for several conditions including affective disorders, stroke, neurodegenerative diseases and aging. The discovery of 2,3,6-trimethyl-1,4-naphthoquinone (TMN) as a nonselective and reversible inhibitor of MAO, has suggested 1,4-naphthoquinone (1,4-NQ) as a potential scaffold for designing new MAO inhibitors. Combining molecular modeling tools and biochemical assays we evaluate the kinetic and molecular details of the inhibition of human MAO by 1,4-NQ, comparing it with TMN and menadione. Menadione (2-methyl-1,4-naphthoquinone) is a multitarget drug that acts as a precursor of vitamin K and an inducer of mitochondrial permeability transition. Herein we show that MAO-B was inhibited competitively by 1,4-NQ (K(i)=1.4 µM) whereas MAO-A was inhibited by non-competitive mechanism (K(i)=7.7 µM). Contrasting with TMN and 1,4-NQ, menadione exhibited a 60-fold selectivity for MAO-B (K(i)=0.4 µM) in comparison with MAO-A (K(i)=26 µM), which makes it as selective as rasagiline. Fluorescence and molecular modeling data indicated that these inhibitors interact with the flavin moiety at the active site of the enzyme. Additionally, docking studies suggest the phenyl side groups of Tyr407 and Tyr444 (for MAO-A) or Tyr398 and Tyr435 (for MAO-B) play an important role in the interaction of the enzyme with 1,4-NQ scaffold through forces of dispersion as verified for menadione, TMN and 1,4-NQ. Taken together, our findings reveal the molecular details of MAO inhibition by 1,4-NQ scaffold and show for the first time that menadione acts as a competitive and reversible inhibitor of human MAO.

Asunto(s)

Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Naftoquinonas/farmacología , Vitamina K 3/farmacología , Vitaminas/farmacología , Animales , Humanos , Cinética , Modelos Moleculares , Monoaminooxidasa/química , Naftoquinonas/química , Unión Proteica , Vitamina K 3/química , Vitaminas/química

RESUMEN

The Ebola fusion peptide (EBO16) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO16 and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO16 to induce lipid mixing. On the other hand, EBO16 was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO16. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO16 and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

Asunto(s)

Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/etiología , Fusión de Membrana , Microdominios de Membrana/metabolismo , Proteínas Virales de Fusión/metabolismo , Colesterol/metabolismo , Unión Proteica , Proteínas del Envoltorio Viral , Virosis