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1.
Int J Mol Sci ; 22(13)2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34281258

RESUMO

Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures' supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1ß and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1ß. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.


Assuntos
Proteus mirabilis/enzimologia , Proteus mirabilis/patogenicidade , Urease/metabolismo , Urease/toxicidade , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/microbiologia , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/microbiologia , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Sinais de Localização Nuclear , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Urease/química , Virulência/fisiologia
2.
Sci Rep ; 11(1): 7832, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837264

RESUMO

Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that serves as an effective therapeutic for several neuromuscular disorders via induction of temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells is mediated by its binding domain (HC/A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant HC/A was also shown to bind to FGFR3. As FGFR dimerization is an indirect measure of ligand-receptor binding, an FCS & TIRF receptor dimerization assay was developed to measure rHC/A-induced dimerization of fluorescently tagged FGFR subtypes (FGFR1-3) in cells. rHC/A dimerized FGFR subtypes in the rank order FGFR3c (EC50 ≈ 27 nM) > FGFR2b (EC50 ≈ 70 nM) > FGFR1c (EC50 ≈ 163 nM); rHC/A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC50 ≈ 18 nM). Mutating the ganglioside binding site in HC/A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A. Overall, the results suggest that the FCS & TIRF receptor dimerization assay can assess FGFR dimerization with known and novel ligands and support a model wherein HC/A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Clostridium botulinum/enzimologia , Neurotoxinas/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sorogrupo , Transdução de Sinais/genética , Animais , Sítios de Ligação , Toxinas Botulínicas Tipo A/química , Membrana Celular/metabolismo , Dimerização , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gangliosídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurotoxinas/química , Células PC12 , Ligação Proteica , Domínios Proteicos , Ratos , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transfecção
3.
Biomed Khim ; 67(1): 51-65, 2021 Jan.
Artigo em Russo | MEDLINE | ID: mdl-33645522

RESUMO

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.


Assuntos
Isatina , Neurotoxinas , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Isatina/metabolismo , Isatina/farmacologia , Camundongos , Mitocôndrias/metabolismo , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Proteômica
4.
Toxins (Basel) ; 13(2)2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33546362

RESUMO

In contrast to the clearly documented evolution of venom in many animal lineages, the origin of reptilian venom is highly debated. Historically, venom has been theorised to have evolved independently in snakes and lizards. However, some of the recent works have argued for the common origin of venom in "Toxicofera" reptiles, which include the order Serpentes (all snakes), and Anguimorpha and Iguania lizards. Nevertheless, in both these contrasting hypotheses, the lizards of the family Scincidae are considered to be harmless and devoid of toxic venoms. Interestingly, an unusual clinical case claiming neurotoxic envenoming by a scincid lizard was recently reported in Southern India. Considering its potentially significant medicolegal, conservation and evolutionary implications, we have summarised the scientific evidence that questions the validity of this clinical report. We argue that the symptoms documented in the patient are likely to have resulted from krait envenomation, which is far too frequent in these regions.


Assuntos
Mordeduras e Picadas/metabolismo , Lagartos/metabolismo , Neurotoxinas/metabolismo , Peçonhas/metabolismo , Animais , Evolução Molecular , Lagartos/genética , Neurotoxinas/efeitos adversos , Neurotoxinas/genética , Filogenia , Peçonhas/efeitos adversos , Peçonhas/genética
5.
Toxins (Basel) ; 13(2)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567660

RESUMO

The genus Calliophis is the most basal branch of the family Elapidae and several species in it have developed highly elongated venom glands. Recent research has shown that C. bivirgatus has evolved a seemingly unique toxin (calliotoxin) that produces spastic paralysis in their prey by acting on the voltage-gated sodium (NaV) channels. We assembled a transcriptome from C. bivirgatus to investigate the molecular characteristics of these toxins and the venom as a whole. We find strong confirmation that this genus produces the classic elapid eight-cysteine three-finger toxins, that δδ-elapitoxins (toxins that resemble calliotoxin) are responsible for a substantial portion of the venom composition, and that these toxins form a distinct clade within a larger, more diverse clade of C. bivirgatus three-finger toxins. This broader clade of C. bivirgatus toxins also contains the previously named maticotoxins and is somewhat closely related to cytotoxins from other elapids. However, the toxins from this clade that have been characterized are not themselves cytotoxic. No other toxins show clear relationships to toxins of known function from other species.


Assuntos
Venenos Elapídicos/genética , Elapidae/genética , Evolução Molecular , Neurotoxinas/genética , Proteínas de Répteis/genética , Transcriptoma , Animais , Venenos Elapídicos/metabolismo , Elapidae/metabolismo , Perfilação da Expressão Gênica , Neurotoxinas/metabolismo , Filogenia , Proteínas de Répteis/metabolismo
6.
Toxins (Basel) ; 13(2)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572266

RESUMO

Envenomation resulted from sea snake bite is a highly lethal health hazard in Southeast Asia. Although commonly caused by sea snakes of Hydrophiinae, each species is evolutionarily distinct and thus, unveiling the toxin gene diversity within individual species is important. Applying next-generation sequencing, this study investigated the venom-gland transcriptome of Hydrophis curtus (spine-bellied sea snake) from Penang, West Malaysia. The transcriptome was de novo assembled, followed by gene annotation and sequence analyses. Transcripts with toxin annotation were only 96 in number but highly expressed, constituting 48.18% of total FPKM in the overall transcriptome. Of the 21 toxin families, three-finger toxins (3FTX) were the most abundantly expressed and functionally diverse, followed by phospholipases A2. Lh_FTX001 (short neurotoxin) and Lh_FTX013 (long neurotoxin) were the most dominant 3FTXs expressed, consistent with the pathophysiology of envenomation. Lh_FTX001 and Lh_FTX013 were variable in amino acid compositions and predicted epitopes, while Lh_FTX001 showed high sequence similarity with the short neurotoxin from Hydrophis schistosus, supporting cross-neutralization effect of Sea Snake Antivenom. Other toxins of low gene expression, for example, snake venom metalloproteinases and L-amino acid oxidases not commonly studied in sea snake venom were also identified, enriching the knowledgebase of sea snake toxins for future study.


Assuntos
Venenos Elapídicos/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Hydrophiidae/genética , Neurotoxinas/genética , Proteínas de Répteis/genética , Transcriptoma , Estruturas Animais , Animais , Bases de Dados Genéticas , Venenos Elapídicos/imunologia , Venenos Elapídicos/metabolismo , Venenos Elapídicos/toxicidade , Epitopos , Evolução Molecular , Hydrophiidae/anatomia & histologia , Hydrophiidae/imunologia , Hydrophiidae/metabolismo , Malásia , Neurotoxinas/imunologia , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Filogenia , Proteínas de Répteis/imunologia , Proteínas de Répteis/metabolismo , Proteínas de Répteis/toxicidade
7.
J Neurochem ; 158(6): 1244-1253, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33629408

RESUMO

Tetanus is a deadly but preventable disease caused by a protein neurotoxin produced by Clostridium tetani. Spores of C. tetani may contaminate a necrotic wound and germinate into a vegetative bacterium that releases a toxin, termed tetanus neurotoxin (TeNT). TeNT enters the general circulation, binds to peripheral motor neurons and sensory neurons, and is transported retroaxonally to the spinal cord. It then enters inhibitory interneurons and blocks the release of glycine or GABA causing a spastic paralysis. This review attempts to correlate the metalloprotease activity of TeNT and its trafficking and localization into the vertebrate body to the nature and sequence of appearance of the symptoms of tetanus.


Assuntos
Encéfalo/metabolismo , Nervos Periféricos/metabolismo , Medula Espinal/metabolismo , Toxina Tetânica/metabolismo , Tétano/metabolismo , Animais , Encéfalo/microbiologia , Humanos , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/metabolismo , Nervos Periféricos/microbiologia , Medula Espinal/microbiologia , Tétano/prevenção & controle , Toxina Tetânica/antagonistas & inibidores , Toxoide Tetânico/administração & dosagem , Toxoide Tetânico/metabolismo
8.
Life Sci ; 270: 119012, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33422543

RESUMO

AIMS: Memantine is a non-competitive antagonist of glutamatergic NMDA receptor that is mainly used in the treatment of Alzheimer's disease. The excitatory toxicity mediated by glutamate via glutamatergic receptor signals is considered to be one of the mechanisms mediating neuronal injury and cognitive impairment after exposure to a hypoxic environment at a high altitude. Therefore, in this study, we hypothesized that inhibiting glutamate signaling using memantine could alleviate neuronal injury and cognitive impairment in rats exposed to chronic hypoxia. MAIN METHODS: we made animal models in the natural environment of the Qinghai-Tibet Plateau at an altitude of 4300 m, and used animal behavior, morphology, molecular biology and other methods to evaluate the impact of chronic hypoxia exposure on cognitive function and the neuroprotective effect of Memantine. KEY FINDINGS: Our results showed that the expression of NMDA receptors increased, while the expression of AMPA receptors decreased, after 4 weeks of chronic hypoxia exposure. Concomitantly, apoptotic neuronal cell death in the hippocampus and frontal cortex was significantly increased, along with levels of oxidative stress, whereas innate ability to inhibit free radicals decreased. Moreover, after 8 weeks of hypoxia exposure, learning, memory, and space exploration abilities were significantly decreased. Notably, after treatment with memantine, apoptotic neuronal cell death, oxidative stress, and free radical levels decreased, and the cognitive function of the animals improved. SIGNIFICANCE: Present study shows that chronic hypoxia can produce the excitatory toxicity leading to neural injury and cognitive impairment that can be suppressed with memantine treatment by inhibiting excitatory toxicity.


Assuntos
Doença da Altitude/metabolismo , Disfunção Cognitiva/tratamento farmacológico , Memantina/farmacologia , Altitude , Doença da Altitude/tratamento farmacológico , Doença de Alzheimer/tratamento farmacológico , Animais , Morte Celular/efeitos dos fármacos , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hipóxia/metabolismo , Masculino , Memantina/metabolismo , Memória/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo
9.
Mol Biol Rep ; 48(2): 1475-1483, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33492574

RESUMO

Rutin is an important flavonoid consumed in the daily diet. It is also known as vitamin P and has been extensively investigated due to its pharmacological properties. On the other hand, neuronal death induced by glutamate excitotoxicity is present in several diseases including neurodegenerative diseases. The neuroprotective properties of rutin have been under investigation, although its mechanism of action is still poorly understood. We hypothesized that the mechanisms of neuroprotection of rutin are associated with the increase in glutamate metabolism in astrocytes. This study aimed to evaluate the protective effects of rutin with a focus on the modulation of glutamate detoxification. We used brain organotypic cultures from post-natal Wistar rats (P7-P9) treated with rutin to evaluate neural cell protection and levels of proteins involved in the glutamate metabolism. Moreover, we used cerebral cortex slices from adult Wistar rats to evaluate glutamate uptake. We showed that rutin inhibited the cell death and loss of glutamine synthetase (GS) induced by glutamate that was associated with an increase in glutamate-aspartate transporter (GLAST) in brain organotypic cultures from post-natal Wistar rats. Additionally, it was observed that rutin increased the glutamate uptake in cerebral cortex slices from adult Wistar rats. We conclude that rutin is a neuroprotective agent that prevents glutamate excitotoxicity and thereof suggest that this effect involves the regulation of astrocytic metabolism.


Assuntos
Morte Celular/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Neurônios/efeitos dos fármacos , Rutina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Transportador 1 de Aminoácido Excitatório , Glutamato-Amônia Ligase/genética , Ácido Glutâmico/toxicidade , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Ratos , Ratos Wistar
10.
Mol Neurobiol ; 58(1): 34-54, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32894500

RESUMO

In Alzheimer's disease (AD), excessive amounts of quinolinic acid (QUIN) accumulate within the brain parenchyma and dystrophic neurons. QUIN also regulates glutamate uptake into neurons, which may be due to modulation of Na+-dependent excitatory amino acid transporters (EAATs). To determine the biological relationships between QUIN and glutamate dysfunction, we first quantified the functionality and kinetics of [3H]QUIN uptake in primary human neurons using liquid scintillation. We then measured changes in the protein expression of the glutamate transporter EAAT3 and EAAT1b in primary neurons treated with QUIN and the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (2,4-PDC) using western blotting and immunohistochemistry. Immunohistochemistry was further used to elucidate intracellular transport of exogenous QUIN and the lysosomal-associated membrane protein 2 (LAMP2). Structural insights into the binding between QUIN and EAAT3 were further investigated using molecular docking techniques. We report significant temperature-dependent high-affinity transport leading to neuronal uptake of [3H]QUIN with a Km of 42.2 µM, and a Vmax of 9.492 pmol/2 min/mg protein, comparable with the uptake of glutamate. We also found that QUIN increases expression of the EAAT3 monomer while decreasing the functional trimer. QUIN uptake into primary neurons was shown to involve EAAT3 as uptake was significantly attenuated following EAAT inhibition. We also demonstrated that QUIN increases the expression of aberrant EAAT1b protein in neurons further implicating QUIN-induced glutamate dysfunction. Furthermore, we demonstrated that QUIN is metabolised exclusively in lysosomes. The involvement of EAAT3 as a modulator for QUIN uptake was further confirmed using molecular docking. This study is the first to characterise a mechanism for QUIN uptake into primary human neurons involving EAAT3, opening potential targets to attenuate QUIN-induced excitotoxicity in neuroinflammatory diseases.


Assuntos
Endocitose , Neurônios/metabolismo , Neurotoxinas/metabolismo , Ácido Quinolínico/metabolismo , Células Cultivadas , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/química , Transportador 3 de Aminoácido Excitatório/metabolismo , Feto/metabolismo , Humanos , Cinética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Modelos Moleculares , Ácido Quinolínico/química , Fatores de Tempo
11.
Molecules ; 25(24)2020 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-33322668

RESUMO

Manganese (Mn) is an essential trace element, serving as a cofactor for several key enzymes, such as glutamine synthetase, arginase, pyruvate decarboxylase, and mitochondrial superoxide dismutase. However, its chronic overexposure can result in a neurological disorder referred to as manganism, presenting symptoms similar to those inherent to Parkinson's disease. The pathological symptoms of Mn-induced toxicity are well-known, but the underlying mechanisms of Mn transport to the brain and cellular toxicity leading to Mn's neurotoxicity are not completely understood. Mn's levels in the brain are regulated by multiple transporters responsible for its uptake and efflux, and thus, dysregulation of these transporters may result in Mn accumulation in the brain, causing neurotoxicity. Its distribution and subcellular localization in the brain and associated subcellular toxicity mechanisms have also been extensively studied. This review highlights the presently known Mn transporters and their roles in Mn-induced neurotoxicity, as well as subsequent molecular and cellular dysregulation upon its intracellular uptakes, such as oxidative stress, neuroinflammation, disruption of neurotransmission, α-synuclein aggregation, and amyloidogenesis.


Assuntos
Encéfalo/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Intoxicação por Manganês/metabolismo , Manganês/metabolismo , Neurotoxinas/metabolismo , Animais , Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Inflamação/metabolismo , Neurotransmissores/metabolismo , Estresse Oxidativo , Fatores de Transcrição/metabolismo , Transferrina/metabolismo , alfa-Sinucleína/metabolismo
12.
Cell Death Dis ; 11(10): 939, 2020 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-33130818

RESUMO

The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), has been widely utilized to establish experimental models of Parkinson disease and to reveal the critical molecules and pathway underlying neuronal death. The profile of gene expression changes following 6-OHDA treatment of MN9D dopaminergic neuronal cells was investigated using a TwinChip Mouse-7.4K microarray. Functional clustering of altered sets of genes identified RING-finger protein 166 (RNF166). RNF166 is composed of an N-terminal RING domain and C-terminal ubiquitin interaction motif. RNF166 localized in the cytosol and nucleus. At the tissue level, RNF166 was widely expressed in the central nervous system and peripheral organs. In the cerebral cortex, its expression decreased over time. In certain conditions, overexpression of RNF166 accelerates the naturally occurring neuronal death and 6-OHDA-induced MN9D cell death as determined by TUNEL and annexin-V staining, and caspase activation. Consequently, 6-OHDA-induced apoptotic cell death was attenuated in RNF166-knockdown cells. In an attempt to elucidate the mechanism underlying this pro-apoptotic activity, binding protein profiles were assessed using the yeast two-hybrid system. Among several potential binding candidates, RNF166 was shown to interact with the cytoplasmic X-linked inhibitor of apoptosis (XIAP), inducing ubiquitin-dependent degradation of XIAP and eventually accelerating caspase activation following 6-OHDA treatment. RNF166's interaction with and resulting inhibition of the XIAP anti-caspase activity was further enhanced by XIAP-associated factor-1 (XAF-1). Consequently, depletion of RNF166 suppressed 6-OHDA-induced caspase activation and apoptotic cell death, which was reversed by XIAP knockdown. In summary, our data suggest that RNF166, a novel E3 ligase, plays a pro-apoptotic role via caspase activation in neuronal cells.


Assuntos
Neurotoxinas/metabolismo , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Humanos , Camundongos , Transfecção
13.
Exp Cell Res ; 396(2): 112322, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-33068559

RESUMO

Meningothelial cells (MECs) are the cellular component of the meninges that provide physical protection to the central nervous system (CNS). Their main function is the formation of a barrier enclosing the brain including the cerebrospinal fluid (CSF). Further, MECs are involved in maintaining CSF homeostasis by clearing CSF from bacteria and apoptotic cells. Furthermore, secretion of pro- and anti-inflammatory cytokines and chemokines involves MECs in immunological processes in the CNS. We demonstrated that meningothelial Ben-Men-1 cells ingest neurotoxic peptides amyloid-ß (Aß1-40) and protein α-synuclein up to about 10-fold more efficiently compared to neuronal-like SH-SY5Y cells. Aß1-40 and α-synuclein are mainly taken up via macropinocytosis. Caveolar endocytosis in addition contributes to α-synuclein ingestion. Upon uptake, both are trafficked towards lysosomal degradation. While production of reactive oxygen species (ROS) following exposure to Aß25-35 and α-synuclein was similar between Ben-Men-1 and SH-SY5Y cells, mitochondrial function in Ben-Men-1 was significantly more robust to Aß25-35 treatment compared to neuronal-like SHSY5Y cells. Similarly, Ben-Men-1 were significantly less susceptible to Aß25-35-induced cell death than neuronal-like cells. Furthermore, co-culture with Ben-Men-1 offered significant protection to neuronal-like cells against Aß25-35-induced apoptosis. This study reveals for the first time the function of MECs as scavengers of neurotoxic Aß and α-synuclein, thereby connecting these cells to neuroprotective processes and suggesting a new mechanism and pathway for clearing neurotoxic substances from the CSF.


Assuntos
Células Epiteliais/metabolismo , Meninges/citologia , Neurotoxinas/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular Tumoral , Endocitose , Humanos , Mitocôndrias/metabolismo , Neuroproteção , Frações Subcelulares/metabolismo , alfa-Sinucleína/metabolismo
14.
Toxins (Basel) ; 12(10)2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33023159

RESUMO

Venomous snakes are important subjects of study in evolution, ecology, and biomedicine. Many venomous snakes have alpha-neurotoxins (α-neurotoxins) in their venom. These toxins bind the alpha-1 nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction, causing paralysis and asphyxia. Several venomous snakes and their predators have evolved resistance to α-neurotoxins. The resistance is conferred by steric hindrance from N-glycosylated asparagines at amino acids 187 or 189, by an arginine at position 187 that has been hypothesized to either electrostatically repulse positively charged neurotoxins or sterically interfere with α-neurotoxin binding, or proline replacements at positions 194 or 197 of the nAChR ligand-binding domain to inhibit α-neurotoxin binding through structural changes in the receptor. Here, we analyzed this domain in 148 vertebrate species, and assessed its amino acid sequences for resistance-associated mutations. Of these sequences, 89 were sequenced de novo. We find widespread convergent evolution of the N-glycosylation form of resistance in several taxa including venomous snakes and their lizard prey, but not in the snake-eating birds studied. We also document new lineages with the arginine form of inhibition. Using an in vivo assay in four species, we provide further evidence that N-glycosylation mutations reduce the toxicity of cobra venom. The nAChR is of crucial importance for normal neuromuscular function and is highly conserved throughout the vertebrates as a result. Our research shows that the evolution of α-neurotoxins in snakes may well have prompted arms races and mutations to this ancient receptor across a wide range of sympatric vertebrates. These findings underscore the inter-connectedness of the biosphere and the ripple effects that one adaption can have across global ecosystems.


Assuntos
Resistência a Medicamentos , Evolução Molecular , Junção Neuromuscular/efeitos dos fármacos , Neurotoxinas/toxicidade , Antagonistas Nicotínicos/toxicidade , Receptores Nicotínicos/efeitos dos fármacos , Mordeduras de Serpentes/metabolismo , Venenos de Serpentes/toxicidade , Serpentes/metabolismo , Animais , Sítios de Ligação , Resistência a Medicamentos/genética , Glicosilação , Mutação , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Neurotoxinas/metabolismo , Antagonistas Nicotínicos/metabolismo , Filogenia , Ligação Proteica , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Mordeduras de Serpentes/fisiopatologia , Venenos de Serpentes/metabolismo , Especificidade da Espécie
15.
Toxicon ; 187: 86-92, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32889025

RESUMO

Acetylcholine binding proteins (AChBPs), structural and functional surrogates of the extracellular binding domain of nicotinic acetylcholine receptor (nAChRs), in complex with various antagonists and agonists have provided detailed insights into the neurotransmitter binding site of nAChRs. The classical long-chain α-neurotoxins bungarotoxin (44-fold) and cobratoxin (7-fold) bind to Lymnaea stagnalis (Ls)-AChBP with higher affinity compared to Aplysia californica (Ac)-AChBP. In this study, we describe a novel long chain α-neurotoxin Drysdalin, which has higher binding affinity (7-fold) to Ac-AChBP when compared to Ls-AChBP. This suggests an involvement of different regions or modes of interaction of drysdalin, when compared to the bungarotoxin and cobratoxin. We also found that the C-terminal 24-amino acid residues of drysdalin are critical for the binding to Ac-AChBP and its removal caused ~90-fold reduction in affinity. Further to understand the interaction of drysdalin with Ac-AChBP, we studied the role of three non-conserved amino acid residues of drysdalin, namely Arg30, Leu34 and Ala37. Substitution of Arg30 with the conserved Phe residue caused a ~100-fold reduction, Leu34 with conserved Arg caused a ~6-fold reduction, whereas substitution of Ala37 with conserved Arg enhanced the binding by 3-fold. The dramatic influence of this carboxyl terminal sequence enriched in arginine and proline residues suggests that the toxin binding pose is influenced primarily by this extended sequence.


Assuntos
Acetilcolina/metabolismo , Aplysia , Lymnaea , Neurotoxinas/toxicidade , Venenos de Serpentes/toxicidade , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico , Bungarotoxinas , Proteínas de Transporte , Modelos Moleculares , Neurotoxinas/metabolismo , Conformação Proteica , Receptores Nicotínicos , Venenos de Serpentes/metabolismo , Serpentes
16.
Toxins (Basel) ; 12(9)2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32947868

RESUMO

Three-finger toxins (3FTX) are a group of peptides that affect multiple receptor types. One group of proteins affected by 3FTX are nicotinic acetylcholine receptors (nAChR). Structural information on how neurotoxins interact with nAChR is limited and is confined to a small group of neurotoxins. Therefore, in silico methods are valuable in understanding the interactions between 3FTX and different nAChR subtypes, but there are no established protocols to model 3FTX-nAChR interactions. We followed a homology modeling and protein docking protocol to address this issue and tested its success on three different systems. First, neurotoxin peptides co-crystallized with acetylcholine binding protein (AChBP) were re-docked to assess whether Rosetta protein-protein docking can reproduce the native poses. Second, experimental data on peptide binding to AChBP was used to test whether the docking protocol can qualitatively distinguish AChBP-binders from non-binders. Finally, we docked eight peptides with known α7 and muscle-type nAChR binding properties to test whether the protocol can explain the differential activities of the peptides at the two receptor subtypes. Overall, the docking protocol predicted the qualitative and some specific aspects of 3FTX binding to nAChR with reasonable success and shed light on unknown aspects of 3FTX binding to different receptor subtypes.


Assuntos
Simulação de Acoplamento Molecular , Neurotoxinas/metabolismo , Receptores Nicotínicos/metabolismo , Neurotoxinas/química , Ligação Proteica , Conformação Proteica , Receptores Nicotínicos/química , Software , Relação Estrutura-Atividade
17.
Adv Biosyst ; 4(10): e2000139, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32869522

RESUMO

Excitotoxicity is a cellular phenomenon that comprises the consequences of toxic actions of excitatory neurotransmitters, such as glutamate. This process is usually related to overproduction of reactive oxygen species (ROS) and ammonia (NH4 + ) toxicity. Platinum nanoparticle (Pt-NP)-based microreactors able to degrade hydrogen peroxide (H2 O2 ) and NH4 + , are previously described as a novel therapeutical approach against excitotoxicity, conferring protection to neuroblasts. Now, it is demonstrated that these microreactors are compatible with rat primary cortical neurons, show high levels of neuronal membrane interaction, and are able to improve cell survival and neuronal activity when neurons are exposed to H2 O2 or NH4 + . Additionally, more complex microreactors are assembled, including enzyme-loaded liposomes containing glutamate dehydrogenase and glutathione reductase, in addition to Pt-NP. The in vitro activity of these microreactors is characterized and they are compared to the Pt-NP-based microreactors in terms of biological activity, concluding that they enhance cell viability similarly or more extensively than the latter. Extracellular electrophysiological recordings demonstrate that these microreactors rescue neuronal functionality lost upon incubation with H2 O2 or NH4 + . This study provides more evidence for the potential application of these microreactors in a biomedical context with more complex cellular environments.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Neurônios , Fármacos Neuroprotetores , Estresse Oxidativo/efeitos dos fármacos , Amônia/metabolismo , Animais , Células Cultivadas , Peróxido de Hidrogênio/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/metabolismo , Ratos , Ratos Sprague-Dawley
18.
Proc Natl Acad Sci U S A ; 117(40): 24920-24928, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958636

RESUMO

Australian funnel-web spiders are infamous for causing human fatalities, which are induced by venom peptides known as δ-hexatoxins (δ-HXTXs). Humans and other primates did not feature in the prey or predator spectrum during evolution of these spiders, and consequently the primate lethality of δ-HXTXs remains enigmatic. Funnel-web envenomations are mostly inflicted by male spiders that wander from their burrow in search of females during the mating season, which suggests a role for δ-HXTXs in self-defense since male spiders rarely feed during this period. Although 35 species of Australian funnel-web spiders have been described, only nine δ-HXTXs from four species have been characterized, resulting in a lack of understanding of the ecological roles and molecular evolution of δ-HXTXs. Here, by profiling venom-gland transcriptomes of 10 funnel-web species, we report 22 δ-HXTXs. Phylogenetic and evolutionary assessments reveal a remarkable sequence conservation of δ-HXTXs despite their deep evolutionary origin within funnel-web spiders, consistent with a defensive role. We demonstrate that δ-HXTX-Ar1a, the lethal toxin from the Sydney funnel-web spider Atrax robustus, induces pain in mice by inhibiting inactivation of voltage-gated sodium (NaV) channels involved in nociceptive signaling. δ-HXTX-Ar1a also inhibited inactivation of cockroach NaV channels and was insecticidal to sheep blowflies. Considering their algogenic effects in mice, potent insecticidal effects, and high levels of sequence conservation, we propose that the δ-HXTXs were repurposed from an initial insecticidal predatory function to a role in defending against nonhuman vertebrate predators by male spiders, with their lethal effects on humans being an unfortunate evolutionary coincidence.


Assuntos
Evolução Molecular , Neurotoxinas/genética , Poliaminas/química , Aranhas/genética , Sequência de Aminoácidos/genética , Animais , Austrália , Sequência Conservada/genética , Feminino , Humanos , Masculino , Camundongos , Neurotoxinas/química , Neurotoxinas/metabolismo , Peptídeos/genética , Filogenia , Poliaminas/metabolismo , Comportamento Sexual Animal/fisiologia , Venenos de Aranha/genética , Aranhas/patogenicidade , Transcriptoma/genética , Vertebrados/genética , Vertebrados/fisiologia
19.
Proc Natl Acad Sci U S A ; 117(38): 23815-23822, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900920

RESUMO

Prions are infectious agents which cause rapidly lethal neurodegenerative diseases in humans and animals following long, clinically silent incubation periods. They are composed of multichain assemblies of misfolded cellular prion protein. While it has long been assumed that prions are themselves neurotoxic, recent development of methods to obtain exceptionally pure prions from mouse brain with maintained strain characteristics, and in which defined structures-paired rod-like double helical fibers-can be definitively correlated with infectivity, allowed a direct test of this assertion. Here we report that while brain homogenates from symptomatic prion-infected mice are highly toxic to cultured neurons, exceptionally pure intact high-titer infectious prions are not directly neurotoxic. We further show that treatment of brain homogenates from prion-infected mice with sodium lauroylsarcosine destroys toxicity without diminishing infectivity. This is consistent with models in which prion propagation and toxicity can be mechanistically uncoupled.


Assuntos
Neurotoxinas , Doenças Priônicas , Príons , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Química Encefálica , Modelos Animais de Doenças , Camundongos , Neurônios/efeitos dos fármacos , Neurotoxinas/isolamento & purificação , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Doenças Priônicas/metabolismo , Doenças Priônicas/fisiopatologia , Príons/isolamento & purificação , Príons/metabolismo , Príons/patogenicidade
20.
Toxins (Basel) ; 12(8)2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823543

RESUMO

Produced by cyanobacteria and some plants, BMAA is considered as an important environmental factor in the occurrence of some neurodegenerative diseases. Neither the underlying mechanism of its toxicity, nor its biosynthetic or metabolic pathway in cyanobacteria is understood. Interestingly, BMAA is found to be toxic to some cyanobacteria, making it possible to dissect the mechanism of BMAA metabolism by genetic approaches using these organisms. In this study, we used the cyanobacterium Anabaena PCC 7120 to isolate BMAA-resistant mutants. Following genomic sequencing, several mutations were mapped to two genes involved in amino acids transport, suggesting that BMAA was taken up through amino acid transporters. This conclusion was supported by the protective effect of several amino acids against BMAA toxicity. Furthermore, targeted inactivation of genes encoding different amino acid transport pathways conferred various levels of resistance to BMAA. One mutant inactivating all three major amino acid transport systems could no longer take up BMAA and gained full resistance to BMAA toxicity. Therefore, BMAA is a substrate of amino acid transporters, and cyanobacteria are interesting models for genetic analysis of BMAA transport and metabolism.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Diamino Aminoácidos/metabolismo , Aminoácidos/metabolismo , Anabaena/genética , Anabaena/metabolismo , Diamino Aminoácidos/farmacologia , Anabaena/efeitos dos fármacos , Genoma Bacteriano , Mutação , Neurotoxinas/metabolismo
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