RESUMEN
The misfolding and aggregation of the presynaptic protein α-synuclein (α-syn) is a pathological hallmark of Parkinson's disease (PD). Targeting α-syn has emerged as a promising therapeutic strategy for PD. Emerging in vitro evidence supports a dual action of epigallocatechin-3-gallate (EGCG) against amyloid neurotoxicity. EGCG can halt the formation of toxic aggregates by redirecting the amyloid fibril aggregation pathway toward non-toxic aggregates and remodeling the existing toxic fibrils into non-toxic aggregates. Moreover, EGCG oxidation can enhance fibril's remodeling by forming Schiff bases, leading to crosslinking of the fibril. However, this covalent modification is not required for amyloid remodeling, and establishing non-specific hydrophobic interactions with sidechains seems to be the main driver of amyloid remodeling by EGCG. Thioflavin (ThT) is a gold standard probe to detect amyloid fibrils in vitro, and oxidized EGCG competes with ThT for amyloid fibrils' binding sites. In this work, we performed docking and molecular dynamics (MD) simulations to gain insights into the intermolecular interactions of oxidized EGCG and ThT with a mature α-syn fibril. We find that oxidized EGCG moves within lysine-rich sites within the hydrophobic core of the α-syn fibril, forming aromatic and hydrogen-bonding (H-bond) interactions with different residues during the whole MD simulation time. In contrast, ThT, which does not remodel amyloid fibrils, was docked to the same sites but only via aromatic interactions. Our findings suggest that non-covalent interactions play a role in oxidized EGCG binding into the hydrophobic core, including H-bond and aromatic interactions with some residues in the amyloid remodeling processes. These interactions would ultimately lead to a disturbance of structural features as determinants for stabilizing this fibril into a compact and pathogenic Greek key topology.
Asunto(s)
Catequina , Enfermedad de Parkinson , Humanos , alfa-Sinucleína/química , Amiloide/química , Enfermedad de Parkinson/metabolismo , Proteínas Amiloidogénicas , Catequina/química , Agregado de ProteínasRESUMEN
Previous work has suggested that highly positively charged protein segments coded by rare codons or poly (A) stretches induce ribosome stalling and translational arrest through electrostatic interactions with the negatively charged ribosome exit tunnel, leading to inefficient elongation. This arrest leads to the activation of the Ribosome Quality Control (RQC) pathway and results in low expression of these reporter proteins. However, the only endogenous yeast proteins known to activate the RQC are Rqc1, a protein essential for RQC function, and Sdd1, a protein with unknown function, both of which contain polybasic sequences. To explore the generality of this phenomenon, we investigated whether the RQC complex controls the expression of other proteins with polybasic sequences. We showed by ribosome profiling data analysis and western blot that proteins containing polybasic sequences similar to, or even more positively charged than those of Rqc1 and Sdd1, were not targeted by the RQC complex. We also observed that the previously reported Ltn1-dependent regulation of Rqc1 is posttranslational, independent of the RQC activity. Taken together, our results suggest that RQC should not be regarded as a general regulatory pathway for the expression of highly positively charged proteins in yeast.
Asunto(s)
Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Expresión Génica , Unión Proteica , Saccharomyces cerevisiae/genéticaRESUMEN
A molecular hallmark in Parkinson's disease (PD) pathogenesis are α-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of α-synuclein. Here, we investigated the effects of CDNF on α-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with α-synuclein with a KD = 23 ± 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of α-synuclein fibrils and induces the formation of insoluble phosphorylated α-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with α-synuclein fibrils, though it did not reduce the number of phosphorylated α-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with α-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.
Asunto(s)
Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/metabolismo , Enfermedad de Parkinson/fisiopatología , Sustancia Negra/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Sitios de Unión , Línea Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Modelos Moleculares , Enfermedad de Parkinson/metabolismo , Fosforilación , Cultivo Primario de Células , Agregado de Proteínas , Unión Proteica , Conformación Proteica , RatasRESUMEN
We investigated the gene-expression variation among humans by analysing previously published mRNA-seq and ribosome footprint profiling of heart left-ventricles from healthy donors. We ranked the genes according to their coefficient of variation values and found that the top 5% most variable genes had special features compared to the rest of the genome, such as lower mRNA levels and shorter half-lives coupled to increased translation efficiency. We observed that these genes are mostly involved with immune response and have a pleiotropic effect on disease phenotypes, indicating that asymptomatic conditions contribute to the gene expression diversity of healthy individuals.
Asunto(s)
Biología Computacional/métodos , Redes Reguladoras de Genes , Miocardio/química , Bases de Datos Genéticas , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia de ARNRESUMEN
Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Secuencia de Bases , Codón , Humanos , Poli A/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
The codon stabilization coefficient (CSC) is derived from the correlation between each codon frequency in transcripts and mRNA half-life experimental data. In this work, we used this metric as a reference to compare previously published Saccharomyces cerevisiae mRNA half-life datasets and investigate how codon composition related to protein levels. We generated CSCs derived from nine studies. Four datasets produced similar CSCs, which also correlated with other independent parameters that reflected codon optimality, such as the tRNA abundance and ribosome residence time. By calculating the average CSC for each gene, we found that most mRNAs tended to have more non-optimal codons. Conversely, a high proportion of optimal codons was found for genes coding highly abundant proteins, including proteins that were only transiently overexpressed in response to stress conditions. We also used CSCs to identify and locate mRNA regions enriched in non-optimal codons. We found that these stretches were usually located close to the initiation codon and were sufficient to slow ribosome movement. However, in contrast to observations from reporter systems, we found no position-dependent effect on the mRNA half-life. These analyses underscore the value of CSCs in studies of mRNA stability and codon bias and their relationships with protein expression.
Asunto(s)
Codón/genética , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Bases , Conjuntos de Datos como Asunto , Genes Fúngicos/genética , Genoma Fúngico/genética , Semivida , Biosíntesis de Proteínas/genética , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Familial amyloid polyneuropathy (FAP) or ATTRv (amyloid TTR variant) amyloidosis is a fatal hereditary disease characterized by the deposition of amyloid fibrils composed of transthyretin (TTR). The current diagnosis of ATTRv relies on genetic identification of TTR mutations and on Congo Red-positive amyloid deposits, which are absent in most ATTRv patients that are asymptomatic or early symptomatic, supporting the need for novel biomarkers to identify patients in earlier disease phases allowing disease control. METHODS: In an effort to search for new markers for ATTRv, our group searched for nine inflammation markers in ATTRv serum from a cohort of 28 Brazilian ATTRv patients. RESULTS: We found that the levels of six markers were increased (TNF-α, IL-1ß, IL-8, IL-33, IFN-ß and IL-10), one had decreased levels (IL-12) and two of them were unchanged (IL-6 and cortisol). Interestingly, asymptomatic patients already presented high levels of IL-33, IL-1ß and IL-10, suggesting that inflammation may take place before fibril deposition. CONCLUSIONS: Our findings shed light on a new, previously unidentified aspect of ATTRv, which might help define new criteria for disease management, as well as provide additional understanding of ATTRv aggressiveness.
Asunto(s)
Neuropatías Amiloides Familiares/sangre , Neuropatías Amiloides Familiares/inmunología , Biomarcadores/sangre , Inflamación/sangre , Inflamación/inmunología , Brasil , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Translation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5' UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occur. For some genes, translation elongation also affects the rate of protein synthesis. With a GFP library containing nearly all possible combinations of nucleotides from the 3rd to the 5th codon positions in the protein coding region of the mRNA, it was previously demonstrated that some nucleotide combinations increased GFP expression up to four orders of magnitude. While it is clear that the codon region from positions 3 to 5 can influence protein expression levels of artificial constructs, its impact on endogenous proteins is still unknown. Through bioinformatics analysis, we identified the nucleotide combinations of the GFP library in Escherichia coli genes and examined the correlation between the expected levels of translation according to the GFP data with the experimental measures of protein expression. We observed that E. coli genes were enriched with the nucleotide compositions that enhanced protein expression in the GFP library, but surprisingly, it seemed to affect the translation efficiency only marginally. Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli, suggesting an evolutionary pressure towards the conservation of short translational enhancer sequences.
Asunto(s)
Codón/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Iniciación de la Cadena Peptídica Traduccional , Regiones no Traducidas 5' , Secuencia de Bases , Evolución Biológica , Codón/química , Biología Computacional/métodos , Elementos de Facilitación Genéticos , Escherichia coli/metabolismo , Biblioteca de Genes , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Sistemas de Lectura Abierta , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
As proteins are synthesized, the nascent polypeptide must pass through a negatively charged exit tunnel. During this stage, positively charged stretches can interact with the ribosome walls and slow the translation. Therefore, charged polypeptides may be important factors that affect protein expression. To determine the frequency and distribution of positively and negatively charged stretches in different proteomes, the net charge was calculated for every 30 consecutive amino acid residues, which corresponds to the length of the ribosome exit tunnel. The following annotated and reviewed proteins in the UniProt database (Swiss-Prot) were analyzed: 551,705 proteins from different organisms and a total of 180 million protein segments. We observed that there were more negative than positive stretches and that super-charged positive sequences (i.e., net charges ≥ 14) were underrepresented in the proteomes. Overall, the proteins were more positively charged at their N-termini and C-termini, and this feature was present in most organisms and subcellular localizations. To investigate whether the N-terminal charges affect the elongation rates, previously published ribosomal profiling data obtained from S. cerevisiae, without translation-interfering drugs, were analyzed. We observed a nonlinear effect of the charge on the ribosome occupancy in which values ≥ +5 and ≤ -6 showed increased and reduced ribosome densities, respectively. These groups also showed different distributions across 80S monosomes and polysomes. Basic polypeptides are more common within short proteins that are translated by monosomes, whereas negative stretches are more abundant in polysome-translated proteins. These findings suggest that the nascent peptide charge impacts translation and can be one of the factors that regulate translation efficiency and protein expression.
Asunto(s)
Aminoácidos/química , Biosíntesis de Proteínas/fisiología , Proteínas/química , Proteoma/química , Ribosomas/metabolismo , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation.
Asunto(s)
Amiloide/farmacología , Trampas Extracelulares/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Vía de Pentosa Fosfato , Acetato de Tetradecanoilforbol/farmacología , Amiloide/ultraestructura , Trampas Extracelulares/metabolismo , Fructosa/metabolismo , Fructosa/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Inmunohistoquímica , Microscopía Confocal , Microscopía Electrónica de Transmisión , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.
Asunto(s)
Biopolímeros/metabolismo , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/fisiología , Fármacos Neuroprotectores , alfa-Sinucleína/metabolismo , Animales , Línea Celular , Cristalografía por Rayos X , Humanos , Ratones , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Relación Estructura-ActividadRESUMEN
It has been proposed that polybasic peptides cause slower movement of ribosomes through an electrostatic interaction with the highly negative ribosome exit tunnel. Ribosome profiling data-the sequencing of short ribosome-bound fragments of mRNA-is a powerful tool for the analysis of mRNA translation. Using the yeast Saccharomyces cerevisiae as a model, we showed that reduced translation efficiency associated with polybasic protein sequences could be inferred from ribosome profiling. However, an increase in ribosome density at polybasic sequences was evident only when the commonly used translational inhibitors cycloheximide and anisomycin were omitted during mRNA isolation. Since ribosome profiling performed without inhibitors agrees with experimental evidence obtained by other methods, we conclude that cycloheximide and anisomycin must be avoided in ribosome profiling experiments.
Asunto(s)
ARN Mensajero/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ARN/métodos , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
We investigated the folding of the 70 kDa human cytosolic inducible protein (Hsp70) in vitro using high hydrostatic pressure as a denaturing agent. We followed the structural changes in Hsp70 induced by high hydrostatic pressure using tryptophan fluorescence, molecular dynamics, circular dichroism, high-performance liquid chromatography gel filtration, dynamic light scattering, ATPase activity, and chaperone activity. Although monomeric, Hsp70 is very sensitive to hydrostatic pressure; after pressure had been removed, the protein did not return to its native sate but instead formed oligomeric species that lost chaperone activity but retained ATPase activity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Presión Hidrostática , Chaperonas Moleculares/metabolismo , Conformación Proteica , Dicroismo Circular , Proteínas HSP70 de Choque Térmico/química , Humanos , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
Protein aggregation and oxidative stress have gained significant research attention due to their association with a group of diseases known as amyloidosis. Among the strategies developed to prevent amyloidosis, utilization of polyphenols stands out as one of the most commonly employed approaches. Scutellaria baicalensis is renowned as one of the foremost herbal sources of polyphenols. In this study, we employed a direct oxidative pyrolysis method for polymerizing S. baicalensis's polyphenols (SBPPs) after their extraction, resulting in the formation of novel SBPPs nanoparticles. Upon polymerization, SBPPs nanoparticles showed remarkable properties including heightened water solubility, increased surface area, modified surface functional groups, and enhanced stability. As a result of these diverse factors, there was a considerable enhancement in the anti-amyloidogenic properties and antioxidant effects of SBPPs nanoparticles compared to its bulk form. The fibrillation kinetics, AFM images, and cytotoxicity assays strongly indicate that SBPPs nanoparticles are more effective than SBPPs at preventing amyloid fibril formation and associated cell toxicity. Additionally, SBPPs nanoparticles demonstrated more effective prevention of reactive oxygen species (ROS) production. In conclusion, the use of SBPPs in nanoparticle form presents a promising strategy to enhance anti-amyloidogenic properties, mitigate oxidative stress, and offer potential therapeutic benefits for amyloidosis-related diseases.
Asunto(s)
Amiloidosis , Nanopartículas , Antioxidantes/farmacología , Scutellaria baicalensis , Polifenoles/farmacologíaRESUMEN
During protein synthesis, organisms detect translation defects that induce ribosome stalling and result in protein aggregation. The Ribosome-associated Quality Control (RQC) complex, comprising TCF25, LTN1, and NEMF, is responsible for identifying incomplete protein products from unproductive translation events, targeting them for degradation. Although RQC disruption causes adverse effects on vertebrate neurons, data regarding mRNA/protein expression and regulation across tissues are lacking. Employing high-throughput methods, we analyzed public datasets to explore RQC gene expression and phenotypes. Our findings revealed widespread expression of RQC components in human tissues; however, silencing of RQC yielded only mild negative effects on cell growth. Notably, TCF25 exhibited elevated mRNA levels that were not reflected in the protein content. We experimentally demonstrated that this disparity arose from post-translational protein degradation by the proteasome. Additionally, we observed that cellular aging marginally influenced RQC expression, leading to reduced mRNA levels in specific tissues. Our results suggest the necessity of RQC expression in all mammalian tissues. Nevertheless, when RQC falters, alternative mechanisms seem to compensate, ensuring cell survival under nonstress conditions.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Biosíntesis de Proteínas , Ubiquitinación , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Mamíferos/metabolismoRESUMEN
The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.
Asunto(s)
Amiloide/fisiología , Cromatina/metabolismo , Neutrófilos/patología , Acetofenonas/farmacología , Amiloide/química , Amiloide/genética , Neuropatías Amiloides Familiares/enzimología , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/patología , Amiloidosis/enzimología , Amiloidosis/metabolismo , Amiloidosis/patología , Animales , Biomarcadores/metabolismo , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Cromatina/enzimología , Cricetinae , Espacio Extracelular/enzimología , Espacio Extracelular/metabolismo , Células Hep G2 , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Pulmón/enzimología , Pulmón/metabolismo , Pulmón/patología , Mutación Missense , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Neutrófilos/metabolismo , Compuestos Onio/farmacología , Elastasa Pancreática , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/fisiología , Prealbúmina/química , Prealbúmina/genética , Prealbúmina/fisiología , Estructura Cuaternaria de Proteína , Proteolisis , Especies Reactivas de Oxígeno/metabolismo , Piel/enzimología , Piel/metabolismo , Piel/patología , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/fisiologíaRESUMEN
Protein misfolding and/or aggregation has been implicated as the cause of several human diseases, such as Alzheimer's and Parkinson's diseases and familial amyloid polyneuropathy. These maladies are referred to as amyloid diseases, named after the cross-ß-sheet amyloid fibril aggregates or deposits common to these disorders. Epigallocatechin-3-gallate (EGCG), the principal polyphenol present in green tea, has been shown to be effective at preventing aggregation and is able to remodel amyloid fibrils comprising different amyloidogenic proteins, although the mechanistic underpinnings are unclear. Herein, we work toward an understanding of the molecular mechanism(s) by which EGCG remodels mature amyloid fibrils made up of Aß(1-40), IAPP(8-24), or Sup35NM(7-16). We show that EGCG amyloid remodeling activity in vitro is dependent on auto-oxidation of the EGCG. Oxidized and unoxidized EGCG binds to amyloid fibrils, preventing the binding of thioflavin T. This engagement of the hydrophobic binding sites in Aß(1-40), IAPP(8-24), or Sup35NM(Ac7-16) YâF amyloid fibrils seems to be sufficient to explain the majority of the amyloid remodeling observed by EGCG treatment, although how EGCG oxidation drives remodeling remains unclear. Oxidized EGCG molecules react with free amines within the amyloid fibril through the formation of Schiff bases, cross-linking the fibrils, which may prevent dissociation and toxicity, but these aberrant post-translational modifications do not appear to be the major driving force for amyloid remodeling by EGCG treatment. These insights into the molecular mechanism of action of EGCG provide boundary conditions for exploring amyloid remodeling in more detail.
Asunto(s)
Amiloide/química , Catequina/análogos & derivados , Amiloide/metabolismo , Catequina/síntesis química , Catequina/química , Catequina/farmacología , Células HEK293 , Humanos , Modelos Moleculares , Estructura MolecularRESUMEN
The overproduction of reactive oxygen species (ROS) induces oxidative stress, a well-known process associated with aging and several human pathologies, such as cancer and neurodegenerative diseases. A large number of synthetic compounds have been described as antioxidant enzyme mimics, capable of eliminating ROS and/or reducing oxidative damage. In this study, we investigated the antioxidant activity of a water-soluble 1,10-phenantroline-octanediaoate Mn2+-complex on cells under oxidative stress, and assessed its capacity to attenuate alpha-synuclein (aSyn) toxicity and aggregation, a process associated with increased oxidative stress. This Mn2+-complex exhibited a significant antioxidant potential, reducing intracelular oxidation and increasing oxidative stress resistance in S. cerevisiae cells and in vivo, in G. mellonella, increasing the activity of the intracellular antioxidant enzymes superoxide dismutase and catalase. Strikingly, the Mn2+-complex reduced both aSyn oligomerization and aggregation in human cell cultures and, using NMR and DFT/molecular docking we confirmed its interaction with the C-terminal region of aSyn. In conclusion, the Mn2+-complex appears as an excellent lead for the design of new phenanthroline derivatives as alternative compounds for preventing oxidative damages and oxidative stress - related diseases.
Asunto(s)
Antioxidantes , Manganeso , Fenantrolinas , alfa-Sinucleína , Antioxidantes/farmacología , Manganeso/farmacología , Simulación del Acoplamiento Molecular , Fenantrolinas/farmacología , Especies Reactivas de Oxígeno , Saccharomyces cerevisiae , AguaRESUMEN
Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.