RESUMEN
Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to the cytosol through a self-formed channel. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localized Golgi in a retromer-dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by the toxin, revealing a new pathway mediating BoNT/A cellular toxicity.
Asunto(s)
Retículo Endoplásmico , Neuronas , Transporte de Proteínas , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Animales , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/toxicidad , Toxinas Botulínicas Tipo A/genética , Ratas , Aparato de Golgi/metabolismo , Línea Celular , Citosol/metabolismoRESUMEN
Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.
Asunto(s)
Toxinas Botulínicas Tipo A , Exocitosis , Animales , Humanos , Ratas , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/aislamiento & purificación , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Masculino , Sinaptosomas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/genética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/genética , Toxinas Botulínicas/química , Toxinas Botulínicas/aislamiento & purificaciónRESUMEN
The emergence of multidrug-resistant pathogens has outpaced the development of new antibiotics, leading to renewed interest in endolysins. Endolysins have been investigated as novel biocontrol agents for Gram-positive bacteria. However, their efficacy against Gram-negative species is limited by the barrier presented by their outer membrane, which prevents endolysin access to the peptidoglycan substrate. Here, we used the translocation domain of botulinum neurotoxin to deliver endolysin across the outer membrane of Gram-negative bacteria. The translocation domain selectively interacts with and penetrates membranes composed of anionic lipids, which have been used in nature to deliver various proteins into animal cells. In addition to the botulinum neurotoxin translocation domain, we have fused bacteriophage-derived receptor binding protein to endolysins. This allows the attached protein to efficiently bind to a broad spectrum of Gram-negative bacteria. By attaching these target-binding and translocation machineries to endolysins, we aimed to develop an engineered endolysin with broad-spectrum targeting and enhanced antibacterial activity against Gram-negative species. To validate our strategy, we designed engineered endolysins using two well-known endolysins, T5 and LysPA26, and tested them against 23 strains from six species of Gram-negative bacteria, confirming that our machinery can act broadly. In particular, we observed a 2.32 log reduction in 30 min with only 0.5 µM against an Acinetobacter baumannii isolate. We also used the SpyTag/SpyCatcher system to easily attach target-binding proteins, thereby improving its target-binding ability. Overall, our newly developed endolysin engineering strategy may be a promising approach to control multidrug-resistant Gram-negative bacterial strains.
Asunto(s)
Antibacterianos , Membrana Externa Bacteriana , Endopeptidasas , Bacterias Gramnegativas , Endopeptidasas/farmacología , Endopeptidasas/metabolismo , Endopeptidasas/química , Bacterias Gramnegativas/efectos de los fármacos , Antibacterianos/farmacología , Membrana Externa Bacteriana/efectos de los fármacos , Membrana Externa Bacteriana/metabolismo , Pruebas de Sensibilidad Microbiana , Transporte de Proteínas , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/metabolismo , Acinetobacter baumannii/efectos de los fármacosRESUMEN
The trafficking of aquaporin 5 (AQP5) is critical for salivary secretion. Synaptosomal-associated protein 23 (SNAP23) is an important regulator in the process of membrane fusion. However, the role of SNAP23 on AQP5 trafficking has not been explored. Botulinum toxin type A (BoNT/A) is a bacterial toxin that effectively treats sialorrhea. We previously reported that BoNT/A induced AQP5 redistribution in cultured acinar cells, but the mechanism remained unclear. In this study, SNAP23 was predominantly localized to the plasma membrane of acinar cells in the rat submandibular gland (SMG) and colocalized with AQP5 at the apical membrane of acinar cells. In stable GFP-AQP5-transfected SMG-C6 cells, the acetylcholine receptor agonist carbachol (CCh) induced trafficking of AQP5 from intracellular vesicles to the apical membrane. Furthermore, SNAP23 knockdown by siRNA significantly inhibited CCh-induced AQP5 trafficking, whereas this inhibitory effect was reversed by SNAP23 re-expression, indicating that SNAP23 was essential in AQP5 trafficking. More importantly, BoNT/A inhibited salivary secretion from SMGs, and the underlying mechanism involved that BoNT/A blocked CCh-triggered AQP5 trafficking by decreasing SNAP23 in acinar cells. Taken together, these results identified a crucial role for SNAP23 in AQP5 trafficking and provided new insights into the mechanism of BoNT/A in treating sialorrhea and thereby a theoretical basis for clinical applications.
Asunto(s)
Toxinas Botulínicas Tipo A , Sialorrea , Ratas , Animales , Toxinas Botulínicas Tipo A/farmacología , Toxinas Botulínicas Tipo A/metabolismo , Acuaporina 5/genética , Acuaporina 5/metabolismo , Células Acinares , Sialorrea/metabolismo , Glándula Submandibular/metabolismoRESUMEN
Botulinum neurotoxin A (BoNT) is being shown to have anticancer action as a potential adjuvant treatment. The transient receptor potential (TRP) melastatin 2 (TRPM2) stimulator action of BoNT was reported in glioblastoma cells, but not in colorectal cancer (HT29) cells. By activating TRPM2, we evaluated the impacts of BoNT and oxaliplatin (OXA) incubations on oxidant and apoptotic values within the HT29 cells. Control, BoNT (5 IU for 24 h), OXA (50 µM for 24 h) and their combinations were induced. We found that TRPM2 protein is upregulated and mediates enhanced BoNT and OXA-induced Ca2+ entry in cells as compared to control cells. The increase of free reactive oxygen species (ROS), but the decrease of glutathione is the main ROS responsible for TRPM2 activation on H29 exposure to oxidative stress. BoNT and OXA-mediated Ca2+ entry through TRPM2 stimulation in response to H2 O2 results in mitochondrial Ca2+ overload, followed by mitochondrial membrane depolarization, apoptosis and caspase-3/-8/-9, although they were diminished in the TRPM2 antagonist groups (N-(p-amylcinnamoyl)anthranilic acid and carvacrol). In conclusion, by increasing the susceptibility of HT29 tumour cells to oxidative stress and apoptosis, the combined administration of BoNT and OXA via the targeting of TRPM2 may offer a different approach to kill the tumour cells.
Asunto(s)
Toxinas Botulínicas Tipo A , Neoplasias Colorrectales , Canales Catiónicos TRPM , Humanos , Oxaliplatino/farmacología , Especies Reactivas de Oxígeno/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Regulación hacia Arriba , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Muerte Celular , Estrés Oxidativo/fisiología , Apoptosis/fisiología , Neoplasias Colorrectales/tratamiento farmacológico , Calcio/metabolismoRESUMEN
Botulinum toxin is a protein toxin secreted by Clostridium botulinum that is strongly neurotoxic. Due to its characteristics of being super toxic, quick acting, and difficult to prevent, the currently reported antiviral studies focusing on monoclonal antibodies have limited effectiveness. Therefore, for the sake of effectively prevention and treatment of botulism and to maintain country biosecurity as well as the health of the population, in this study, we intend to establish a single chain antibody (scFv) targeting the carboxyl terminal binding functional domain of the botulinum neurotoxin heavy chain (BONT/AHc) of botulinum neurotoxin type A, and explore the value of a new passive immune method in antiviral research which based on adeno-associated virus (AAV) mediated vector immunoprophylaxis (VIP) strategy. The scFv small-molecular single-chain antibody sequenced, designed, constructed, expressed and purified by hybridoma has high neutralising activity and affinity level, which can lay a good foundation for the modification and development of antibody engineering drugs. In vivo experiments, AAV-mediated scFv engineering drug has good anti-BONT/A toxin neutralisation ability, has advantages of simple operation, stable expression and good efficacy, and may be one of the effective treatment strategies for long-term prevention and protection of BONT/A botulinum neurotoxin.
Asunto(s)
Toxinas Botulínicas Tipo A , Botulismo , Clostridium botulinum , Humanos , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/uso terapéutico , Botulismo/tratamiento farmacológico , Botulismo/prevención & control , Clostridium botulinum/metabolismo , Anticuerpos Monoclonales , Antivirales/uso terapéuticoRESUMEN
BACKGROUND AND OBJECTIVES: Botulinum toxin type A (BTA) is often used for wrinkles and muscle convulsive diseases due to its blocking of the transmission of nerve impulses. Stromal vascular fraction gel (SVF-gel) prepared from adipose tissue has novel effects on skin depression and poor texture. Both BTA and SVF-gel are proved to possess anti-scar potential. This study aimed to assess and compare their therapeutic effects on hypertrophic scars. MATERIALS AND METHODS: The rabbit ear scar model was established and treated with BTA and SVF-gel, alone or in combination. Gross evaluation using Manchester Scar Scale (MSS) was conducted immediately, 4 and 8 weeks after initial treatment. After tissue sample harvest, histological and Western blot analyses were performed. RESULTS: All the treatments alleviated scar hyperplasia in different degrees by inhibiting fibroblast activation (Ki-67, α-SMA), tissue inflammation (CD45, IL-1ß) and the transforming growth factor-ß1 (TGF-ß1)/Smad3 pathway. Despite an excellent anti-inflammatory effect, improvement of scar appearance and pathological characteristics in SVF-gel-contained groups was not as good as that in BTA-only group, which might be related to the retention of M2-type macrophages (CD163 +) and partial maintenance of TGF-ß1 expression. CONCLUSION: Our data suggest that BTA has better anti-scar efficacy than SVF-gel, and the combination of these two treatments shows no obvious combinatorial effect.
Asunto(s)
Toxinas Botulínicas Tipo A , Quemaduras , Cicatriz Hipertrófica , Animales , Conejos , Cicatriz Hipertrófica/patología , Toxinas Botulínicas Tipo A/farmacología , Toxinas Botulínicas Tipo A/uso terapéutico , Toxinas Botulínicas Tipo A/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Fracción Vascular Estromal , Quemaduras/complicaciones , Quemaduras/terapia , Quemaduras/metabolismo , FibroblastosRESUMEN
Botulinum toxin type A (BoNT/A) has exhibited efficacy in postherpetic neuralgia (PHN) treatment, and this study aims to uncover its underlying mechanisms. Resiniferatoxin (RTX)-induced PHN rats were given BoNT/A. Rat postoperative pain behaviors were assessed by Von Frey test. Cleaved-synaptosomal protein 25 kDa (cl-SNAP-25) or cathelicidin antimicrobial peptide (CAMP) expression in rat dorsal root ganglia (DRG) was detected by immunofluorescence or immunohistochemistry. Healthy rat-derived DRG neurons were transfected, incubated with lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) to stimulate pyroptosis and treated with BoNT/A. The CCK-8, Western blot, ELISA, and qRT-PCR were used to assess the viability, levels of pyroptosis-related proteins proinflammatory cytokine levels, as well as CAMP and ELANE mRNA levels. BoNT/A (30 U/kg) promoted cl-SNAP-25 expression in rat DRG and reversed RTX-induced decrease of rat paw withdrawal thresholds and CAMP expression and increase of pyroptosis-associated protein and inflammatory factor expression in rat DRG. CAMP interacted with ELANE in rat DRG neurons. BoNT/A attenuated LPS/ATP-stimulated inhibition of viability and CAMP expression and upregulation of inflammatory mediators, pyroptosis-related proteins, and ELANE expression in rat DRG neurons, which was counteracted by CAMP silencing. However, ELANE knockdown offset the effect of CAMP silencing in LPS/ATP/BoNT/A-treated rat DRG neurons. On the whole, BoNT/A alleviates rat DRG neuron pyroptosis during PHN by upregulating CAMP to inhibit ELANE.
Asunto(s)
Toxinas Botulínicas Tipo A , Neuralgia Posherpética , Ratas , Animales , Toxinas Botulínicas Tipo A/farmacología , Toxinas Botulínicas Tipo A/metabolismo , Neuralgia Posherpética/metabolismo , Catelicidinas/metabolismo , Catelicidinas/farmacología , Elastasa de Leucocito/metabolismo , Elastasa de Leucocito/farmacología , Ganglios Espinales/metabolismo , Lipopolisacáridos/farmacología , Piroptosis , Neuronas , Adenosina Trifosfato/metabolismoRESUMEN
In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We identify DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography reveals that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and ß-exosite. This DARPin does not block substrate cleavage of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, results in a loss of inhibition of substrate cleavage. In contrast to the in vitro results, DARPin-F5 promotes faster substrate cleavage of BoNT/A1 in primary neurons and muscle tissue by increasing toxin translocation. Our findings could have important implications for the application of BoNT/A1 in therapeutic areas requiring faster onset of toxin action combined with long persistence.
Asunto(s)
Toxinas Botulínicas Tipo A , Toxinas Botulínicas , Clostridium botulinum , Proteínas de Repetición de Anquirina Diseñadas , Toxinas Botulínicas Tipo A/metabolismo , Clostridium botulinum/genéticaRESUMEN
Botulinum toxin A (BoNT-A) has emerged as a treatment option for temporomandibular disorder (TMD). By injecting BoNT-A into the masseter muscle, it is possible to reduce mechanical loading on the temporomandibular joint (TMJ). However, numerous prior studies have indicated excessive reduction in mechanical loading can have detrimental effects on TMJ cartilage. This study proposes that autophagy, a process influenced by mechanical loading, could play a role in BoNT-A-induced mandibular condyle cartilage degeneration. To explore this hypothesis, we employed both BoNT-A injection and an excessive biting model to induce variations in mechanical loading on the condyle cartilage of C57BL/6 mice, thereby simulating an increase and decrease in mechanical loading, respectively. Results showed a significant reduction in cartilage thickness and downregulation of Runt-related transcription factor 2 (Runx2) expression in chondrocytes following BoNT-A injection. In vitro experiments demonstrated that the reduction of Runx2 expression in chondrocytes is associated with autophagy, possibly dependent on decreased YAP expression induced by low mechanical loading. This study reveals the potential involvement of the YAP/LC3/Runx2 signaling pathway in BoNT-A mediated mandibular condylar cartilage degeneration.
Asunto(s)
Toxinas Botulínicas Tipo A , Cartílago Articular , Ratones , Animales , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/farmacología , Ratones Endogámicos C57BL , Cóndilo Mandibular/metabolismo , Condrocitos/metabolismo , AutofagiaRESUMEN
Tetanus toxin (TeNT) and botulinum neurotoxins (BoNTs) are neuroprotein toxins, with the latter being the most toxic known protein. They are structurally similar and contain three functional domains: an N-terminal catalytic domain (light chain), an internal heavy-chain translocation domain (HN domain), and a C-terminal heavy chain receptor binding domain (Hc domain or RBD). In this study, fusion functional domain molecules consisting of the TeNT RBD (THc) and the BoNT/A RBD (AHc) (i.e., THc-Linker-AHc and AHc-Linker-THc) were designed, prepared, and identified. The interaction of each Hc domain and the ganglioside receptor (GT1b) or the receptor synaptic vesicle glycoprotein 2 (SV2) was explored in vitro. Their immune response characteristics and protective efficacy were investigated in animal models. The recombinant THc-linker-AHc and AHc-linker-THc proteins with the binding activity had the correct size and structure, thus representing novel subunit vaccines. THc-linker-AHc and AHc-linker-THc induced high levels of specific neutralizing antibodies, and showed strong immune protective efficacy against both toxins. The high antibody titers against the two novel fusion domain molecules and against individual THc and AHc suggested that the THc and AHc domains, as antigens in the fusion functional domain molecules, do not interact with each other and retain their full key epitopes responsible for inducing neutralizing antibodies. Thus, the recombinant THc-linker-AHc and AHc-linker-THc molecules are strong and effective bivalent biotoxin vaccines, protecting against two biotoxins simultaneously. Our experimental design will be valuable to develop recombinant double-RBD fusion molecules as potent bivalent subunit vaccines against bio-toxins. KEY POINTS: ⢠Double-RBD fusion molecules from two toxins had the correct structure and activity. ⢠THc-linker-AHc and AHc-linker-THc efficiently protected against both biotoxins. ⢠Such bivalent biotoxin vaccines based on the RBD are a valuable experimental design.
Asunto(s)
Toxinas Botulínicas Tipo A , Toxina Tetánica , Animales , Toxina Tetánica/genética , Toxina Tetánica/metabolismo , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/metabolismo , Unión Proteica , Anticuerpos Neutralizantes , Vacunas de Subunidad/genéticaRESUMEN
OBJECTIVE: To determine whether Botulinum neurotoxin type A (BoNT-A) ameliorates the effects of interleukin 1 (IL-1) on equine articular cartilage, or exerts negative effects on normal equine articular cartilage homeostasis in vitro. SAMPLE: Articular cartilage explants from 6 healthy femoropatellar joints of 3 adult horses. METHODS: Explants were allocated to the IL-1 challenged or unchallenged group, then exposed to 1 of 6 concentrations of BoNT-A (0, 1, 10, 50, 100, or 500 pg/mL) for 96 hours. To assess BoNT-A's effects on inflammation, prostaglandin E2 (PGE2) was measured in media via ELISA. Matrix degradation was determined as the percentage of sulfated glycosaminoglycans (sGAG) released from explants via dimethylmethylene blue assay. Aggrecan synthesis was estimated using CS846 ELISA and collagen type II degradation was estimated using C2C ELISA on media. Chondrocyte apoptosis was assessed via in-situ TUNEL assay. Generalized linear mixed models were fitted to determine treatment effects using α = 0.05. RESULTS: The challenge with IL-1 resulted in increased concentrations of PGE2 and CS846 in media and increased release of sGAG from explants. BoNT-A did not significantly impact PGE2 or CS846 concentration in media, percentage of sGAG released, or chondrocyte apoptosis in IL-1 challenged or unchallenged cartilage explants. The concentration of C2C in media was below the quantifiable limit of the ELISA in all samples. CLINICAL RELEVANCE: BoNT-A did not show chondroprotective effects or have negative effects on cartilage homeostasis in vitro at the concentrations tested. While chondroprotective effects were not observed, BoNT-A may be safe for intraarticular use. In vivo testing is warranted before clinical use.
Asunto(s)
Toxinas Botulínicas Tipo A , Cartílago Articular , Caballos , Animales , Cartílago Articular/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Toxinas Botulínicas Tipo A/metabolismo , Proteoglicanos/metabolismo , Dinoprostona/farmacología , Dinoprostona/metabolismo , Glicosaminoglicanos/metabolismo , Interleucina-1/metabolismo , Interleucina-1/farmacologíaRESUMEN
Skeletal muscle and bone interact at the level of mechanical loading through the application of force by muscles to the skeleton and more recently focus has been placed on molecular/biochemical coupling of these two tissues. We sought to determine if muscle and muscle-derived factors were essential to the osteocyte response to loading. Botox® induced muscle paralysis was used to investigate the role of muscle contraction during in vivo tibia compression loading. 5-6 month-old female TOPGAL mice had their right hindlimb muscles surrounding the tibia injected with either BOTOX® or saline. At four days post injections when muscle paralysis peaked, the right tibia was subjected to a single session of in vivo compression loading at â¼2600 µÎµ. At 24 h post-load we observed a 2.5-fold increase in ß-catenin signaling in osteocytes in the tibias of the saline injected mice, whereas loading of tibias from Botox® injected mice failed to active ß-catenin signaling in osteocytes. This suggests that active muscle contraction produces a factor(s) that is necessary for or conditions the osteocyte's ability to respond to load. To further investigate the role of muscle derived factors, MLO-Y4 osteocyte-like cells and a luciferase based ß-catenin reporter (TOPflash-MLO-Y4) cell line we developed were treated with conditioned media (CM) from C2C12 myoblasts (MB) and myotubes (MT) and ex vivo contracted Extensor Digitorum Longus (EDL) and Soleus (Sol) muscles under static or loading conditions using fluid flow shear stress (FFSS). 10 % C2C12 myotube CM, but not myoblast or NIH3T3 fibroblast cells CM, induced a rapid activation of the Akt signaling pathway, peaking at 15 min and returning to baseline by 1-2 h under static conditions. FFSS applied to MLO-Y4 cells for 2 h in the presence of 10 % MT-CM resulted in a 6-8 fold increase in pAkt compared to a 3-4 fold increase under control or when exposed to 10 % MB-CM. A similar response was observed in the presence of 10 % EDL-CM, but not in the presence of 10 % Sol-CM. TOPflash-MLO-Y4 cells were treated with 10 ng/ml Wnt3a in the presence or absence of MT-CM. While MT-CM resulted in a 2-fold activation and Wnt3a produced a 10-fold activation, the combination of MT-CM + Wnt3a resulted in a 25-fold activation of ß-catenin signaling, implying a synergistic effect of factors in MT-CM with Wnt3a. These data provide clear evidence that specific muscles and myotubes produce factors that alter important signaling pathways involved in the response of osteocytes to mechanical load. These data strongly suggest that beyond mechanical loading there is a molecular coupling of muscle and bone.
Asunto(s)
Toxinas Botulínicas Tipo A , Osteocitos , Femenino , Animales , Ratones , Osteocitos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , beta Catenina/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Células 3T3 NIH , Músculo Esquelético/metabolismo , Parálisis/metabolismoRESUMEN
Joensuu and colleagues have recently shown that botulinum neurotoxin (BoNT) type A exploits a heterotrimeric complex in the presynaptic membrane to bind to and enter neurons using a Trojan horse-like mechanism. Similar processes may be relevant to the neuronal entry of different botulinum toxin serotypes and other neuropathogens.
Asunto(s)
Toxinas Botulínicas Tipo A , Toxinas Botulínicas Tipo A/metabolismo , Vesículas Sinápticas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismoRESUMEN
Botulinum toxin type A (BoNT/A) induces direct analgesic effects in neuropathic pain by inhibiting the release of substance P, calcitonin gene-related peptide (CGRP) and glutamate. Vesicular nucleotide transporter (VNUT) was responsible for the storage and release of ATP in vivo, and one of the mechanisms underlying neuropathic pain is VNUT-dependent release of extracellular ATP from dorsal horn neurons. However, the analgesic effect of BoNT/A by affecting the expression of VNUT remained largely unknown. Thus, in this study, we aimed to elucidate the antinociceptive potency and analgesic mechanism of BoNT/A in chronic constriction injury of the sciatic nerve (CCI) induced neuropathic pain. Our results showed that a single intrathecal injection of 0.1 U BoNT/A seven days after CCI surgery produced significant analgesic activity and decreased the expression of VNUT in the spinal cord of CCI rats. Similarly, BoNT/A inhibited the CCI-induced increase in ATP content in the rat spinal cord. Overexpression of VNUT in the spinal cord of CCI-induced rats markedly reversed the antinociceptive effect of BoNT/A. Furthermore, 33 U/mL BoNT/A dramatically reduced the expression of VNUT in pheochromocytoma (PC12) cells but overexpressing SNAP-25 increased VNUT expression in PC12 cells. Our current study is the first to demonstrate that BoNT/A is involved in neuropathic pain by regulating the expression of VNUT in the spinal cord in rats.
Asunto(s)
Toxinas Botulínicas Tipo A , Neuralgia , Ratas , Animales , Toxinas Botulínicas Tipo A/uso terapéutico , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Nucleótidos/metabolismo , Nucleótidos/farmacología , Constricción , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Médula Espinal/metabolismo , Nervio Ciático , Analgésicos/uso terapéutico , Analgésicos/farmacología , Adenosina Trifosfato/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismoRESUMEN
The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.
Asunto(s)
Toxinas Botulínicas Tipo A , Toxinas Botulínicas Tipo A/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Animales , RatasRESUMEN
BACKGROUND: To have a better and longer effect, botulinum neurotoxin (BoNT) is injected several times in a treatment course, which could increase side effects and cost. Some of the most cutting-edge strategies being investigated for proteins to their physiologic targets involve the reformulation of BoNT based on peptide-based delivery systems. For this purpose, cell-penetrating peptides (CPPs) are of particular interest because of their capacity to cross the biological membranes. OBJECTIVES: A short and simple CPP sequence was used as a carrier to create nanocomplex particles from BoNT/A, with the purpose of increasing toxin entrapment by target cells, reducing diffusion, and increasing the duration of the effect. METHOD: CPP-BoNT/A nanocomplexes were formed by polyelectrolyte complex (PEC) method, considering the anionic structure of botulinum toxin and the cationic CPP sequence. The cellular toxicity, and absorption profile of the complex nanoparticles were evaluated, and the digit abduction score (DAS) was used to assess the local muscle weakening efficacy of BoNT/A and CPP-BoNT/A. RESULTS: The provided optimized polyelectrolyte complex nanoparticles had a 244 ± 20 nm particle size and 0.28 ± 0.04 PdI. In cellular toxicity, CPP-BoNT/A nanocomplexes as extended-release formulations of BoNT/A showed that nanocomplexes had a more toxic effect than BoNT/A. Furthermore, the comparison of weakening effectiveness on muscle was done among nanoparticles and free toxin on mice based on the digit abduction score (DAS) method, and nanocomplexes had a slower onset effect and a longer duration of action than toxin. CONCLUSION: Using PEC method allowed us to form nanocomplex from proteins, and peptides without a covalent bond and harsh conditions. The muscle-weakening effect of toxin in CPP-BoNT/A nanocomplexes showed acceptable efficacy and extended-release pattern.
Asunto(s)
Toxinas Botulínicas Tipo A , Péptidos de Penetración Celular , Animales , Ratones , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Péptidos de Penetración Celular/farmacología , PolielectrolitosRESUMEN
Chronic pain affects one in five people across human societies, with few therapeutic options available. Botulinum neurotoxin (BoNT) can provide long-lasting pain relief by inhibiting local release of neuropeptides and neurotransmitters, but its highly paralytic nature has limited its analgesic potential. Recent advances in protein engineering have raised the possibility of synthesising non-paralysing botulinum molecules for translation to pain sufferers. However, the synthesis of these molecules, via several synthetic steps, has been challenging. Here, we describe a simple platform for safe production of botulinum molecules for treating nerve injury-induced pain. We produced two versions of isopeptide-bonded BoNT from separate botulinum parts using an isopeptide bonding system. Although both molecules cleaved their natural substrate, SNAP25, in sensory neurons, the structurally elongated iBoNT did not cause motor deficit in rats. We show that the non-paralytic elongated iBoNT targets specific cutaneous nerve fibres and provides sustained pain relief in a rat nerve injury model. Our results demonstrate that novel botulinum molecules can be produced in a simple and safe manner and be useful for treating neuropathic pain.
Asunto(s)
Toxinas Botulínicas Tipo A , Dolor Crónico , Neuralgia , Ratas , Humanos , Animales , Dolor Crónico/tratamiento farmacológico , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Toxinas Botulínicas Tipo A/uso terapéutico , Analgésicos/farmacología , Analgésicos/uso terapéutico , Células Receptoras Sensoriales/metabolismoRESUMEN
Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (HCE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.
Asunto(s)
Toxinas Botulínicas Tipo A , Vesículas Sinápticas , Humanos , Vesículas Sinápticas/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Unión ProteicaRESUMEN
The ability to deliver proteins and peptides across the plasma membrane into the cytosol of living mammalian cells would be highly impactful for both basic science and medicine. Natural cell-penetrating protein toxins have shown promise as protein delivery platforms, but existing approaches are limited by immunogenicity, lack of cell-type-specificity, or their multi-component nature. Here we explore inactivated botulinum neurotoxin (BoNT) as a protein delivery platform. Using split luciferase reconstitution in the cytosol as a readout for endosomal escape and cytosolic delivery, we showed that BoNT chimeras with nanobodies replacing their natural receptor binding domain can be selectively targeted to cells expressing nanobody-matched surface markers. We used chimeric BoNTs to deliver a range of cargo from 1.3 to 55 kDa in size, and demonstrated selective delivery of orthogonal cargoes to distinct cell populations within a mixed culture. These explorations suggest that BoNT may be a versatile platform for targeted protein and peptide delivery into mammalian cells.