RESUMEN
The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.
Asunto(s)
Fusión de Membrana/efectos de los fármacos , Proteínas SNARE/antagonistas & inhibidores , alfa-Sinucleína/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dopamina/metabolismo , Dopamina/farmacología , Evaluación Preclínica de Medicamentos , Liposomas , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Mutación Missense , Fragmentos de Péptidos/farmacología , Mutación Puntual , Unión Proteica , Multimerización de Proteína , Proteolípidos/química , Proteínas Recombinantes de Fusión/farmacología , Proteínas SNARE/fisiología , Proteína 2 de Membrana Asociada a Vesículas/antagonistas & inhibidores , Proteína 2 de Membrana Asociada a Vesículas/fisiología , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadRESUMEN
The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders are unclear. Here, we apply a genetic approach, using isogenic human induced pluripotent stem cells, to evaluate the effects of schizophrenia (SZ)-associated common variants predicted to function as SZ expression quantitative trait loci (eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ eQTL (FURIN rs4702) and four top-ranked SZ eQTL genes (FURIN, SNAP91, TSNARE1 and CLCN3), our platform resolves pre- and postsynaptic neuronal deficits, recapitulates genotype-dependent gene expression differences and identifies convergence downstream of SZ eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.
Asunto(s)
Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Células Madre Pluripotentes Inducidas/patología , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Esquizofrenia/genética , Esquizofrenia/patología , Sistemas CRISPR-Cas , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Femenino , Furina/antagonistas & inhibidores , Furina/genética , Furina/metabolismo , Edición Génica , Estudio de Asociación del Genoma Completo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Proteínas de Ensamble de Clatrina Monoméricas/antagonistas & inhibidores , Proteínas de Ensamble de Clatrina Monoméricas/genética , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
Assembly of neuronal SNARE protein complexes is essential for fusion of synaptic vesicles with the presynaptic plasma membrane, which releases neurotransmitters into the synaptic cleft and mediates neurotransmission. However, despite the potential of pharmacological regulation of this process for the treatment of various neurological disorders, only a few reagents, including botulinum neurotoxins, are currently available. Here, we report that buforin-1, an antimicrobial peptide from the Asian toad Bufo gargarizans, inhibits neuronal SNARE complex assembly, resulting in neuronal SNARE-mediated membrane fusion in vitro via its direct association with neuronal t-SNAREs syntaxin-1 and SNAP-25. Consistently, buforin-1 significantly inhibited neuronal-SNARE-mediated exocytosis in PC-12â¯cells. Thus, buforin-1 has potential for the treatment of neurological disorders caused by dysregulated neurotransmission.
Asunto(s)
Fusión de Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Proteínas/farmacología , Proteínas SNARE/antagonistas & inhibidores , Animales , Bufonidae , Línea Celular , Exocitosis/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Ratas , Proteínas SNARE/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismoRESUMEN
Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca2+-triggered synaptic vesicle fusion. We speculate that Ca2+-triggered fusion commences with the release of inhibition by Ca2+ binding to synaptotagmin C2 domains. Subsequently, fusion is assisted by SNARE complex zippering and by active membrane remodeling properties of synaptotagmin. This additional, inhibitory role of synaptotagmin may be a general principle since other recent studies suggest that Ca2+ binding to extended synaptotagmin C2 domains enables lipid transport by releasing an inhibited state of the system, and that Munc13 may nominally be in an inhibited state, which is released upon Ca2+ binding to one of its C2 domains.
Asunto(s)
Calcio/farmacología , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Dominios C2/efectos de los fármacos , Calcio/metabolismo , Humanos , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/metabolismo , Sinaptotagmina I/antagonistas & inhibidores , Sinaptotagmina I/metabolismoRESUMEN
Astrocytes are implicated in modulation of neuronal excitability and synaptic function, but it remains unknown if these glial cells can directly control activities of motor circuits to influence complex behaviors in vivo. This study focused on the vital respiratory rhythm-generating circuits of the preBötzinger complex (preBötC) and determined how compromised function of local astrocytes affects breathing in conscious experimental animals (rats). Vesicular release mechanisms in astrocytes were disrupted by virally driven expression of either the dominant-negative SNARE protein or light chain of tetanus toxin. We show that blockade of vesicular release in preBötC astrocytes reduces the resting breathing rate and frequency of periodic sighs, decreases rhythm variability, impairs respiratory responses to hypoxia and hypercapnia, and dramatically reduces the exercise capacity. These findings indicate that astrocytes modulate the activity of CNS circuits generating the respiratory rhythm, critically contribute to adaptive respiratory responses in conditions of increased metabolic demand and determine the exercise capacity.
Asunto(s)
Astrocitos/fisiología , Tronco Encefálico/fisiología , Periodicidad , Condicionamiento Físico Animal/fisiología , Respiración , Potenciales de Acción/fisiología , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Tronco Encefálico/citología , Calcio/metabolismo , Femenino , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hipercapnia/metabolismo , Hipercapnia/fisiopatología , Hipoxia/metabolismo , Hipoxia/fisiopatología , Masculino , Bulbo Raquídeo/citología , Bulbo Raquídeo/fisiología , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
Synaptotagmin 1 (Syt1) is the Ca2+ sensor protein with an essential role in neurotransmitter release. Since the wrinkle formation is due to the excessive muscle fiber stimulation in the face, a helpful stratagem to diminish the wrinkle line intenseness is to weaken the innervating neuron activity through Syt1 inhibition which is one of the possible therapeutic strategies against wrinkles. Recently, experimental evidence showed that botox-like peptides, which are typically used as SNARE modulators, may inhibit Syt1. In this work, we applied molecular modeling to (1) characterize the structural framework and (2) define the atomistic information of the factors for the inhibition mechanism. The modeling identified the plausible binding cleft able to efficiently bind all botox-like peptides. The MD simulations revealed that all peptides induced significant Syt1 rigidity by binding in the cleft of the C2A-C2B interface. The consequence of this binding event is the suppression of the protein motion associated with conformational change of Syt1 from the closed form to the open form. On this basis, this finding may therefore be of subservience for the advancement of novel botox-like molecules for the therapeutic treatment of wrinkle, targeting and modulating the function of Syt1.
Asunto(s)
Simulación del Acoplamiento Molecular , Péptidos/química , Proteínas SNARE , Sinaptotagmina I/química , Humanos , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/químicaRESUMEN
Regulated exocytosis can be split into a sequence of steps ending with the formation and the dilation of a fusion pore, a neck-like connection between the vesicle and the plasma membrane. Each of these steps is precisely controlled to achieve the optimal spatial and temporal profile of the release of signalling molecules. At the level of the fusion pore, tuning of the exocytosis can be achieved by preventing its formation, by stabilizing the unproductive narrow fusion pore, by altering the speed of fusion pore expansion and by completely closing the fusion pore. The molecular structure and dynamics of fusion pores have become a major focus of cell research, especially as a promising target for therapeutic strategies. Electrophysiological, optical and electrochemical methods have been used extensively to illuminate how cells regulate secretion at the level of a single fusion pore. Here, we describe recent advances in the structure and mechanisms of the initial fusion pore formation and the progress in therapeutic strategies with the focus on exocytosis.
Asunto(s)
Exocitosis/fisiología , Fusión de Membrana/fisiología , Proteínas SNARE/metabolismo , Animales , Clostridium botulinum/metabolismo , Exocitosis/efectos de los fármacos , Ketamina/farmacología , Fusión de Membrana/efectos de los fármacos , Neurotoxinas/metabolismo , Neurotoxinas/farmacología , Proteínas SNARE/antagonistas & inhibidoresRESUMEN
Alzheimer's disease (AD) and Parkinson's disease (PD) are caused by ß-amyloid (Aß) and α-synuclein (αS), respectively. Ample evidence suggests that these two pathogenic proteins are closely linked and have a synergistic effect on eliciting neurodegenerative disorders. However, the pathophysiological consequences of Aß and αS coexistence are still elusive. Here, we show that large-sized αS oligomers, which are normally difficult to form, are readily generated by Aß42-seeding and that these oligomers efficiently hamper neuronal SNARE-mediated vesicle fusion. The direct binding of the Aß-seeded αS oligomers to the N-terminal domain of synaptobrevin-2, a vesicular SNARE protein, is responsible for the inhibition of fusion. In contrast, large-sized Aß42 oligomers (or aggregates) or the products of αS incubated without Aß42 have no effect on vesicle fusion. These results are confirmed by examining PC12 cell exocytosis. Our results suggest that Aß and αS cooperate to escalate the production of toxic oligomers, whose main toxicity is the inhibition of vesicle fusion and consequently prompts synaptic dysfunction.
Asunto(s)
Péptidos beta-Amiloides/fisiología , Vesículas Citoplasmáticas/fisiología , Fusión de Membrana , Proteínas SNARE/antagonistas & inhibidores , alfa-Sinucleína/fisiología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Exocitosis/genética , Humanos , Fusión de Membrana/genética , Células PC12 , Unión Proteica/genética , Multimerización de Proteína/fisiología , Ratas , Proteínas SNARE/metabolismo , Sinapsis/genética , Sinapsis/metabolismo , Transfección , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
CONTEXT: Targeted secretion inhibitors (TSIs), a new class of recombinant biotherapeutic proteins engineered from botulinum toxin, represent a novel approach for treating diseases with excess secretion. They inhibit hormone secretion from targeted cell types through cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) proteins. qGHRH-LH(N)/D is a TSI targeting pituitary somatotroph through binding to the GHRH-receptor and cleavage of the vesicle-associated membrane protein (VAMP) family of SNARE proteins. OBJECTIVE: Our objective was to study SNARE protein expression in pituitary adenomas and to inhibit GH secretion from somatotropinomas using qGHRH-LH(N)/D. DESIGN: We analyzed human pituitary adenoma analysis for SNARE expression and response to qGHRH-LH(N)/D treatment. SETTING: The study was conducted in University Hospitals. PATIENTS: We used pituitary adenoma samples from 25 acromegaly and 47 nonfunctioning pituitary adenoma patients. OUTCOME: Vesicle-SNARE (VAMP1-3), target-SNARE (syntaxin1, SNAP-23, and SNAP-25), and GHRH-receptor detection with RT-qPCR, immunocytochemistry, and immunoblotting. Assessment of TSI catalytic activity on VAMPs and release of GH from adenoma cells. RESULTS: SNARE proteins were variably expressed in pituitary samples. In vitro evidence using recombinant GFP-VAMP2&3 or pituitary adenoma lysates suggested sufficient catalytic activity of qGHRH-LH(N)/D to degrade VAMPs, but was unable to inhibit GH secretion in somatotropinoma cell cultures. CONCLUSIONS: SNARE proteins are present in human pituitary somatotroph adenomas that can be targeted by TSIs to inhibit GH secretion. qGHRH-LH(N)/D was unable to inhibit GH secretion from human somatotroph adenoma cells. Further studies are required to understand how the SNARE proteins drive GH secretion in human somatotrophs to allow the development of novel TSIs with a potential therapeutic benefit.
Asunto(s)
Adenoma/tratamiento farmacológico , Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Adenoma Hipofisario Secretor de Hormona del Crecimiento/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Hipófisis/efectos de los fármacos , Proteínas SNARE/antagonistas & inhibidores , Vías Secretoras/efectos de los fármacos , Acromegalia/etiología , Acromegalia/prevención & control , Adenoma/metabolismo , Adenoma/patología , Antineoplásicos/química , Toxinas Botulínicas/química , Toxinas Botulínicas/genética , Toxinas Botulínicas/farmacología , Diseño de Fármacos , Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/farmacología , Adenoma Hipofisario Secretor de Hormona del Crecimiento/metabolismo , Adenoma Hipofisario Secretor de Hormona del Crecimiento/patología , Hormona de Crecimiento Humana/antagonistas & inhibidores , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/metabolismo , Humanos , Ligandos , Terapia Molecular Dirigida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Hipófisis/metabolismo , Hipófisis/patología , Neoplasias Hipofisarias/tratamiento farmacológico , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Receptores LHRH/antagonistas & inhibidores , Receptores LHRH/genética , Receptores LHRH/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Células Tumorales CultivadasRESUMEN
Atrial myocytes are continuously exposed to mechanical forces including shear stress. However, in atrial myocytes, the effects of shear stress are poorly understood, particularly with respect to its effect on ion channel function. Here, we report that shear stress activated a large outward current from rat atrial myocytes, with a parallel decrease in action potential duration. The main ion channel underlying the increase in current was found to be Kv1.5, the recruitment of which could be directly observed by total internal reflection fluorescence microscopy, in response to shear stress. The effect was primarily attributable to recruitment of intracellular pools of Kv1.5 to the sarcolemma, as the response was prevented by the SNARE protein inhibitor N-ethylmaleimide and the calcium chelator BAPTA. The process required integrin signaling through focal adhesion kinase and relied on an intact microtubule system. Furthermore, in a rat model of chronic hemodynamic overload, myocytes showed an increase in basal current despite a decrease in Kv1.5 protein expression, with a reduced response to shear stress. Additionally, integrin beta1d expression and focal adhesion kinase activation were increased in this model. This data suggests that, under conditions of chronically increased mechanical stress, the integrin signaling pathway is overactivated, leading to increased functional Kv1.5 at the membrane and reducing the capacity of cells to further respond to mechanical challenge. Thus, pools of Kv1.5 may comprise an inducible reservoir that can facilitate the repolarization of the atrium under conditions of excessive mechanical stress.
Asunto(s)
Atrios Cardíacos/citología , Canal de Potasio Kv1.5/metabolismo , Miocitos Cardíacos/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Análisis de Varianza , Animales , Fenómenos Biomecánicos , Western Blotting , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Etilmaleimida/farmacología , Técnica del Anticuerpo Fluorescente , Integrina beta1/metabolismo , Masculino , Microscopía Fluorescente , Modelos Biológicos , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Proteínas SNARE/antagonistas & inhibidores , Sarcolema/metabolismo , Resistencia al CorteRESUMEN
RATIONALE: Astrocytes play an integral role in modulating synaptic transmission and plasticity, both key mechanisms underlying addiction. However, while astrocytes are capable of releasing chemical transmitters that can modulate neuronal function, the role of these gliotransmitters in mediating behaviors associated with drugs of abuse has been largely unexplored. OBJECTIVES: The objective of the present study was to utilize mice with astrocytes that lack the ability to release chemical transmitters to evaluate the behavioral consequence of impaired gliotransmission on cocaine-related behaviors. These mice have previously been used to examine the role of gliotransmission in sleep homeostasis; however, no studies to date have utilized them in the study of addictive behaviors. METHODS: Mice expressing a dominant-negative SNARE protein selectively in astrocytes (dnSNARE mice) were tested in a variety of behavioral paradigms examining cocaine-induced behavioral plasticity. These paradigms include locomotor sensitization, conditioned place preference followed by cocaine-induced reinstatement of CPP, and cocaine self-administration followed by cue-induced reinstatement of cocaine-seeking behavior. RESULTS: Wild-type and dnSNARE mice demonstrated no significant differences in the development or maintenance of locomotor sensitization. While there were non-significant trends for reduced CPP following a low dose of cocaine, drug-induced reinstatement of CPP is completely blocked in dnSNARE mice. Similarly, while dnSNARE mice demonstrated a non-significant trend toward reduced cocaine self-administration compared with wild-type mice, dnSNARE mice do not demonstrate cue-induced reinstatement in this paradigm. CONCLUSIONS: Gliotransmission is necessary for reinstatement of drug-seeking behaviors by cocaine or associated cues.
Asunto(s)
Astrocitos , Conducta Animal/efectos de los fármacos , Cocaína/administración & dosificación , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Proteínas SNARE/antagonistas & inhibidores , Transmisión Sináptica/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Cocaína/efectos adversos , Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/psicología , Condicionamiento Operante/efectos de los fármacos , Extinción Psicológica/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Endogámicos , Actividad Motora/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Esquema de Refuerzo , Proteínas SNARE/genética , AutoadministraciónRESUMEN
CONTEXT: Botulinum neurotoxins (BoNTs) are popularly used to treat various diseases and for cosmetic purposes. They act by blocking neurotransmission through specific cleavage of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Recently, several polyphenols were shown to interfere with SNARE complex formation by wedging into the hydrophobic core interface, thereby leading to reduced neuroexocytosis. OBJECTIVE: In order to find industrially-viable plant extract that functions like BoNT, 71 methanol extracts of flowers were screened and BoNT-like activity of selected extract was evaluated. MATERIALS AND METHODS: After evaluating the inhibitory effect of 71 flower methanol extracts on SNARE complex formation, seven candidates were selected and they were subjected to SNARE-driven membrane fusion assay. Neurotransmitter release from neuronal PC12 cells and SNARE complex formation inside the cell was also evaluated. Finally, the effect of one selected extract on muscle contraction and digit abduction score was determined. RESULTS: The extract of Potentilla chinensis Ser. (Rosaceae)(Chinese cinquefoil) flower inhibited neurotransmitter release from neuronal PC12 cells by approximately 90% at a concentration of 10 µg/mL. The extract inhibited neuroexocytosis by interfering with SNARE complex formation inside cells. It reduced muscle contraction of phrenic nerve-hemidiaphragm by approximately 70% in 60 min, which is comparable to the action of the Ca²âº-channel blocker verapamil and BoNT type A. DISCUSSION AND CONCLUSION: While BoNT blocks neuroexocytosis by cleaving SNARE proteins, the Potentilla chinensis extract exhibited the same activity by inhibiting SNARE complex formation. The extract paralyzed muscle as efficiently as BoNT, suggesting the potential versatility in cosmetics and therapeutics.
Asunto(s)
Fusión de Membrana/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Fármacos Neuromusculares/farmacología , Neuronas/efectos de los fármacos , Extractos Vegetales/farmacología , Potentilla/química , Proteínas SNARE/antagonistas & inhibidores , Animales , Toxinas Botulínicas/efectos adversos , Toxinas Botulínicas/farmacología , Descubrimiento de Drogas , Exocitosis/efectos de los fármacos , Femenino , Flores/química , Extremidad Inferior , Ratones , Ratones Endogámicos ICR , Músculo Esquelético/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuromusculares/efectos adversos , Neuronas/metabolismo , Norepinefrina/metabolismo , Células PC12 , Extractos Vegetales/efectos adversos , Ratas , Proteínas SNARE/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions.
Asunto(s)
Toxinas Botulínicas Tipo A/química , Ingeniería de Proteínas/métodos , Proteínas SNARE/antagonistas & inhibidores , Toxinas Botulínicas , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/uso terapéutico , Endopeptidasas/metabolismo , Modelos Moleculares , Neurotoxinas , Péptido Hidrolasas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Most cosmetic and therapeutic applications of Clostridium botulinum neurotoxin (BoNT) are related to muscle paralysis caused by the blocking of neurotransmitter release at the neuromuscular junction. BoNT specifically cleaves SNARE proteins at the nerve terminal and impairs neuroexocytosis. Recently, we have shown that several polyphenols inhibit neurotransmitter release from neuronal PC12 cells by interfering with SNARE complex formation. Based on our previous result, we report here that myricetin, delphinidin, and cyanidin indeed paralyze muscle by inhibiting acetylcholine release at the neuromuscular junction. While the effect of myricetin on muscle paralysis was modest compared to BoNT/A, myricetin exhibited a shorter response time than BoNT/A. Intraperitoneally-injected myricetin at an extreme dose of 1000 mg/kg did not induce death of mice, alleviating the safety issue. Thus, these polyphenols might be useful in treating various human hypersecretion diseases for which BoNT/A has been the only option of choice.
Asunto(s)
Toxinas Botulínicas Tipo A/farmacología , Cosméticos/farmacología , Bloqueantes Neuromusculares/farmacología , Polifenoles/farmacología , Proteínas SNARE/antagonistas & inhibidores , Animales , Antocianinas/farmacología , Femenino , Flavonoides/farmacología , Humanos , Ratones , Fitoterapia , Extractos Vegetales/farmacología , Proteínas SNARE/metabolismoRESUMEN
The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT-SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT-SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT-SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT-SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT-SNAP-23 inhibited the increase in plasma membrane expression of gp91(phox) in TNF-α-primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.
Asunto(s)
Gránulos Citoplasmáticos/inmunología , Exocitosis/inmunología , Activación Neutrófila/inmunología , Estallido Respiratorio/inmunología , Apoptosis/genética , Apoptosis/inmunología , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Exocitosis/genética , Productos del Gen tat/antagonistas & inhibidores , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , VIH-1/inmunología , Humanos , Activación Neutrófila/genética , Fagocitosis/genética , Fagocitosis/inmunología , Factor de Activación Plaquetaria/fisiología , Estructura Terciaria de Proteína/genética , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Estallido Respiratorio/genética , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Botulinum toxin type A (BTX-A) is a potent neurotoxin that blocks acetylcholine release from presynaptic nerve terminals by cleaving the SNARE complex. BTX-A has been reported to have analgesic effects independent of its action on muscle tone. The most robust results have been observed in patients with neuropathic pain. Neuropathic pain due to peripheral lesions has been the most widely studied. BTX-A has shown its efficacy on pain and allodynia in various animal models of inflammatory neuropathic pain. The only randomized, double-blind, placebo-controlled trial in patients with focal painful neuropathies due to nerve trauma or postherpetic neuralgia demonstrated significant effects on average pain intensity from 2 weeks after the injections to 14 weeks. Most patients reported pain during the injections, but there were no further local or systemic side effects. The efficacy of BTX-A in painful peripheral neuropathies has been more recently studied. Results were positive in the only study in an animal model of peripheral neuropathy. One study in patients with diabetic painful peripheral neuropathy demonstrated a significant decrease in Visual Analog Scale. In conclusion, one session of multiple intradermal injection of BTX-A produces long-lasting analgesic effects in patients with focal painful neuropathies and diabetic neuropathic pain, and is particularly well tolerated. The findings are consistent with a reduction of peripheral sensitisation, the place of a possible central effect remaining to define. Further studies are needed to assess some important issues, i.e. BTX-A efficacy in patients with small fiber neuropathies and the relevance of early and repeated injections. Future studies could also provide valuable insights into pathophysiology of neuropathic pain.
Asunto(s)
Toxinas Botulínicas Tipo A/uso terapéutico , Neuralgia/tratamiento farmacológico , Animales , Toxinas Botulínicas Tipo A/farmacología , Ensayos Clínicos Controlados como Asunto , Neuropatías Diabéticas/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Humanos , Inyecciones Intradérmicas , Neuritis/tratamiento farmacológico , Ratas , Proteínas SNARE/antagonistas & inhibidoresRESUMEN
Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent.
Asunto(s)
Aparato de Golgi/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas Qa-SNARE/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Ratones Desnudos , Neovascularización Fisiológica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Qa-SNARE/antagonistas & inhibidores , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/fisiología , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiología , Transfección , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
VLDLs (very-low-density lipoproteins) are synthesized in the liver and play an important role in the pathogenesis of atherosclerosis. Following their biogenesis in hepatic ER (endoplasmic reticulum), nascent VLDLs are exported to the Golgi which is a physiologically regulatable event. We have previously shown that a unique ER-derived vesicle, the VTV (VLDL-transport vesicle), mediates the targeted delivery of VLDL to the Golgi lumen. Because VTVs are different from other ER-derived transport vesicles in their morphology and biochemical composition, we speculated that a distinct set of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins would form a SNARE complex which would eventually facilitate the docking/fusion of VTVs with Golgi. Our results show that Sec22b is concentrated in VTVs as compared with the ER. Electron microscopic results show that Sec22b co-localizes with p58 and Sar1 on the VTV surface. Pre-treatment of VTV with antibodies against Sec22b inhibited VTV-Golgi fusion, indicating its role as a v-SNARE (vesicle SNARE). To isolate the SNARE complex, we developed an in vitro docking assay in which VTVs were allowed to dock with the Golgi, but fusion was prevented to stabilize the SNARE complex. After the docking reaction, VTV-Golgi complexes were collected, solubilized in 2% Triton X-100 and the SNARE complex was co-immunoprecipitated using anti-Sec22b or GOS28 antibodies. A approximately 110 kDa complex was identified in non-boiled samples that was dissociated upon boiling. The components of the complex were identified as Sec22b, syntaxin 5, rBet1 and GOS28. Antibodies against each SNARE component significantly inhibited VTV-Golgi fusion. We conclude that the SNARE complex required for VTV-Golgi fusion is composed of Sec22b, syntaxin 5, rBet1 and GOS28.
Asunto(s)
Aparato de Golgi/metabolismo , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Proteínas SNARE/metabolismo , Animales , Transporte Biológico Activo , Retículo Endoplásmico/metabolismo , Técnicas In Vitro , Fusión de Membrana/fisiología , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratas , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/inmunología , Vesículas Transportadoras/metabolismoRESUMEN
Glycogen synthase kinase-3 (GSK-3), a Ser/Thr protein kinase abundantly expressed in neurons, plays diverse functions in physiological and neurodegenerative conditions. Our recent study shows that upregulation of GSK-3 suppresses long-term potentiation and presynaptic release of glutamate; however, the underlying mechanism is elusive. Here, we show that activation of GSK-3beta retards the synaptic vesicle exocytosis in response to membrane depolarization. Using calcium imaging, whole-cell patch-clamp, as well as specific Ca(2+) channel inhibitors, we demonstrate that GSK-3beta phosphorylates the intracellular loop-connecting domains II and III (L(II-III)) of P/Q-type Ca(2+) channels, which leads to a decrease of intracellular Ca(2+) rise through the P/Q-type voltage-dependent calcium channel. To further illustrate the mechanisms of GSK-3beta's action, we show that activation of GSK-3beta interferes with the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex through: (1) weakening the association of synaptobrevin with SNAP25 and syntaxin; (2) reducing the interactions among the phosphorylated L(II-III) and synaptotagmin, SNAP25, and syntaxin; and (3) inhibiting dissociation of synaptobrevin from synaptophysin I. These results indicate that GSK-3beta negatively regulates synaptic vesicle fusion events via interfering with Ca(2+)-dependent SNARE complex formation.
Asunto(s)
Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/metabolismo , Glucógeno Sintasa Quinasa 3/fisiología , Terminales Presinápticos/metabolismo , Proteínas SNARE/antagonistas & inhibidores , Vesículas Sinápticas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Exocitosis/fisiología , Glucógeno Sintasa Quinasa 3/biosíntesis , Glucógeno Sintasa Quinasa 3 beta , Datos de Secuencia Molecular , Inhibición Neural/fisiología , Fosforilación , Ratas , Proteínas SNARE/biosíntesisRESUMEN
The soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor (SNARE) protein syntaxin 1A forms nano-sized clusters (membrane rafts) on the plasma membrane (PM) that are in equilibrium with freely diffusing syntaxin molecules. SNARE-complex formation between syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa) on the PM and synaptobrevin 2 on the vesicles (trans-SNAREs) is crucial for vesicle priming and fusion. This process might be impeded by the spontaneous accumulation of non-fusogenic cis-SNARE complexes formed when all three SNARE proteins reside on the PM. We investigated the kinetics of cis-SNARE complex assembly and disassembly and both exhibited biphasic behavior. The experimental measurements were analyzed through integration of differential rate equations pertinent to the reaction mechanism and through the application of a heuristic search for time constants and concentrations using a genetic algorithm. Reconstruction of the measurements necessitated the partitioning of syntaxin into two phases that might represent the syntaxin clusters and free syntaxin outside the clusters. The analysis suggests that most of the syntaxin in the clusters is concentrated in a nonreactive form. Consequently, cis-SNARE complex assembly in the clusters is substantially slower than outside the rafts. Interestingly, the clusters also mediate efficient disassembly of cis-SNARE complexes possibly attributable to the high local concentration of complexes in the clusters area that allows efficient disassembly by the enzymatic reaction of NSF.