RESUMEN
Limb-girdle muscular dystrophy R3 (LGMDR3) is caused by mutations in the SGCA gene coding for α-sarcoglycan (SG). Together with ß- γ- and δ-SG, α-SG forms a tetramer embedded in the dystrophin associated protein complex crucial for protecting the sarcolemma from mechanical stresses elicited by muscle contraction. Most LGMDR3 cases are due to missense mutations, which result in non-properly folded, even though potentially functional α-SG. These mutants are prematurely discarded by the cell quality control. Lacking one subunit, the SG-complex is disrupted. The resulting loss of function leads to sarcolemma instability, muscle fiber damage and progressive limb muscle weakness. LGMDR3 is severely disabling and, unfortunately, still incurable. Here, we propose the use of small molecules, belonging to the class of cystic fibrosis transmembrane regulator (CFTR) correctors, for recovering mutants of α-SG defective in folding and trafficking. Specifically, CFTR corrector C17 successfully rerouted the SG-complex containing the human R98H-α-SG to the sarcolemma of hind-limb muscles of a novel LGMDR3 murine model. Notably, the muscle force of the treated model animals was fully recovered. To our knowledge, this is the first time that a compound designated for cystic fibrosis is successfully tested in a muscular dystrophy and may represent a novel paradigm of treatment for LGMDR3 as well as different other indications in which a potentially functional protein is prematurely discarded as folding-defective. Furthermore, the use of small molecules for recovering the endogenous mutated SG has an evident advantage over complex procedures such as gene or cell transfer.
Asunto(s)
Fibrosis Quística , Distrofia Muscular de Cinturas , Distrofias Musculares , Animales , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Ratones , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Distrofia Muscular de Cinturas/genética , Sarcoglicanos/genética , Sarcoglicanos/metabolismoRESUMEN
Vaccines are the most effective agents to control infections. In addition to the pathogen antigens, vaccines contain adjuvants that are used to enhance protective immune responses. However, the molecular mechanism of action of most adjuvants is ill-known, and a better understanding of adjuvanticity is needed to develop improved adjuvants based on molecular targets that further enhance vaccine efficacy. This is particularly important for tuberculosis, malaria, AIDS, and other diseases for which protective vaccines do not exist. Release of endogenous danger signals has been linked to adjuvanticity; however, the role of extracellular ATP during vaccination has never been explored. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminum hydroxide, calcium phosphate, incomplete Freund's adjuvant, and the oil-in-water emulsion MF59. We found that intramuscular injection is always associated with a weak transient release of ATP, which was greatly enhanced by the presence of MF59 but not by all other adjuvants tested. Local injection of apyrase, an ATP-hydrolyzing enzyme, inhibited cell recruitment in the muscle induced by MF59 but not by alum or incomplete Freund's adjuvant. In addition, apyrase strongly inhibited influenza-specific T-cell responses and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link extracellular ATP with an enhanced response to vaccination.
Asunto(s)
Adenosina Trifosfato/metabolismo , Adyuvantes Inmunológicos/farmacología , Linfocitos T CD4-Positivos/inmunología , Músculo Esquelético/metabolismo , Polisorbatos/farmacología , Escualeno/farmacología , Vacunación/métodos , Hidróxido de Aluminio/inmunología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Fosfatos de Calcio/inmunología , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Adyuvante de Freund/inmunología , Lípidos/inmunología , Mediciones Luminiscentes , Ratones , Ratones Endogámicos BALB C , Organismos Libres de Patógenos Específicos , Escualeno/inmunologíaRESUMEN
The venom of the snake Bothrops asper causes muscle necrosis, pain and inflammation. This venom contains myotoxins which cause an increase in intracellular Ca(2+) concentration and release of K(+) and ATP from myotubes. ATP is a key danger molecule that triggers a variety of reactions, including activation of the innate immune response. Here, using ATP-luciferase bioluminescence imaging technique, we show for the first time in vivo, that the purified myotoxins induce rapid release of ATP, whilst the complete venom of B. asper does at a very small extent. This apparent contradiction is explained by the finding that the venom contains powerful nucleotidases that in vivo convert ATP into ADP, AMP and Adenosine. These findings indicate that high concentrations of adenosine are generated by the double action of the venom and provide the experimental basis to the suggestion that in situ generated adenosine plays an important role in envenomation via its hypotensive, paralyzing and anti-coagulant activities.
Asunto(s)
Adenosina Trifosfato/metabolismo , Venenos de Crotálidos/enzimología , Fosfolipasas A2 Grupo II/farmacología , Nucleotidasas/farmacología , Proteínas de Reptiles/farmacología , Adenosina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Fosfolipasas A2 Grupo II/química , Fosfolipasas A2 Grupo II/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL , Nucleotidasas/química , Nucleotidasas/aislamiento & purificación , Proteínas de Reptiles/química , Proteínas de Reptiles/aislamiento & purificaciónRESUMEN
Myotoxins play a major role in the pathogenesis of the envenomations caused by snake bites in large parts of the world where this is a very relevant public health problem. We show here that two myotoxins that are major constituents of the venom of Bothrops asper, a deadly snake present in Latin America, induce the release of large amounts of K(+) and ATP from skeletal muscle. We also show that the released ATP amplifies the effect of the myotoxins, acting as a "danger signal," which spreads and causes further damage by acting on purinergic receptors. In addition, the release of ATP and K(+) well accounts for the pain reaction characteristic of these envenomations. As Bothrops asper myotoxins are representative of a large family of snake myotoxins with phospholipase A(2) structure, these findings are expected to be of general significance for snake bite envenomation. Moreover, they suggest potential therapeutic approaches for limiting the extent of muscle tissue damage based on antipurinergic drugs.
Asunto(s)
Adenosina Trifosfato/metabolismo , Bothrops , Venenos de Crotálidos/metabolismo , Músculo Esquelético/metabolismo , Potasio/metabolismo , Animales , Dolor/etiología , Fosfolipasas A2 , Receptores PurinérgicosRESUMEN
Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape.
Asunto(s)
Bioimpresión , Organoides , Hidrogeles/química , Ingeniería de Tejidos/métodos , Polaridad Celular , PulmónRESUMEN
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation-transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers.
Asunto(s)
Calcio , Receptores Nicotínicos , Calcio/metabolismo , Inositol , Receptores de Inositol 1,4,5-Trifosfato , Músculo Esquelético/metabolismo , Unión NeuromuscularRESUMEN
The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects.
RESUMEN
Lysophosphatidylcholine (LPC) is a major bioactive lipid that is enzymatically generated by phospholipase A(2) (PLA(2)). Previously, we showed that LPC is present in the saliva of the blood-sucking hemipteran Rhodnius prolixus and modulates cell-signaling pathways involved in vascular biology, which aids blood feeding. Here, we show that the saliva of the predator insect Belostoma anurum contains a large number of lipids with LPC accounting for 25% of the total phospholipids. A PLA(2) enzyme likely to be involved in LPC generation was characterized. The activity of this enzyme is 5-fold higher in Belostoma saliva than in other studied hemipterans, suggesting a close association with the predator feeding habits of this insect. Belostoma employs extra-oral digestion, which allows for ingestion of larger prey than itself, including small vertebrates such as amphibians and fish. Therefore, prey immobilization during digestion is essential, and we show here that Belostoma saliva and B. anurum saliva purified LPC have paralytic activity in zebrafish. This is the first evidence that lysophospholipids might play an important role in prey immobilization, in addition to contributing to blood feeding, and might have been an evolutionary acquisition that occurred long before the appearance of hematophagy in this animal group.
Asunto(s)
Heterópteros/metabolismo , Heterópteros/patogenicidad , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/toxicidad , Conducta Predatoria/fisiología , Animales , Células Cultivadas , Inmovilización , Técnicas In Vitro , Ratones , Neuronas/efectos de los fármacos , Parálisis/inducido químicamente , Fosfolipasas A2/metabolismo , Nervio Frénico/efectos de los fármacos , Ratas , Saliva/metabolismo , Especificidad de la Especie , Pez CebraRESUMEN
Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three-dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models, including nervous system and neuromuscular junctional homing. Since the nervous system plays pivotal roles during skeletal muscle regeneration and in tissue homeostasis, support of reinnervation is a crucial aspect to be considered. However, the effect of decellularized muscles on reinnervation and on neuronal axon growth has been poorly investigated. Here, we characterized residual protein composition of decellularized muscles by mass spectrometry and we show that scaffolds preserve structural proteins of the ECM of both skeletal muscle and peripheral nervous system. To investigate whether decellularized scaffolds could per se attract neural axons, organotypic sections of spinal cord were cultured three dimensionally in vitro, in presence or in absence of decellularized muscles. We found that neural axons extended from the spinal cord are attracted by the decellularized muscles and penetrate inside the scaffolds upon 3D coculture. These results demonstrate that decellularized scaffolds possess intrinsic neurotrophic properties, supporting their potential use for the treatment of clinical cases where extensive functional regeneration of the muscle is required.
Asunto(s)
Matriz Extracelular/metabolismo , Imagenología Tridimensional/métodos , Músculo Esquelético/metabolismo , Proteómica/métodos , Ingeniería de Tejidos/métodos , Animales , Femenino , Humanos , Masculino , RatasRESUMEN
Surgical repair of large muscular defects requires the use of autologous graft transfer or prosthetic material. Naturally derived matrices are biocompatible materials obtained by tissue decellularization and are commonly used in clinical practice. Despite promising applications described in the literature, the use of acellular matrices to repair large defects has been only partially successful, highlighting the need for more efficient constructs. Scaffold recellularization by means of tissue engineering may improve not only the structure of the matrix, but also its ability to functionally interact with the host. The development of such a complex construct is challenging, due to the complexity of the native organ architecture and the difficulties in recreating the cellular niche with both proliferative and differentiating potential during growth or after damage. In this study, we tested a mouse decellularized diaphragmatic extracellular matrix (ECM) previously described by our group, for the generation of a cellular skeletal muscle construct with functional features. The decellularized matrix was stored using different conditions to mimic the off-the-shelf clinical need. Pediatric human muscle precursors were seeded into the decellularized scaffold, demonstrating proliferation and differentiation capability, giving rise to a functioning three-dimensional skeletal muscle structure. Furthermore, we exposed the engineered construct to cardiotoxin injury and demonstrated its ability to activate a regenerative response in vitro promoting cell self-renewal and a positive ECM remodeling. Functional reconstruction of an engineered skeletal muscle with maintenance of a stem cell pool makes this a promising tool toward future clinical applications in diaphragmatic regeneration. Stem Cells Translational Medicine 2019;8:858&869.
Asunto(s)
Autorrenovación de las Células , Diafragma/citología , Mioblastos/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Diferenciación Celular , Células Cultivadas , Matriz Extracelular/química , Humanos , Ratones , Ratones Endogámicos C57BL , Mioblastos/fisiologíaRESUMEN
Congenital diaphragmatic hernia (CDH) is a neonatal defect in which the diaphragm muscle does not develop properly, thereby raising abdominal organs into the thoracic cavity and impeding lung development and function. Large diaphragmatic defects require correction with prosthetic patches to close the malformation. This treatment leads to a consequent generation of unwelcomed mechanical stress in the repaired diaphragm and hernia recurrences, thereby resulting in high morbidity and significant mortality rates. We proposed a specific diaphragm-derived extracellular matrix (ECM) as a scaffold for the treatment of CDH. To address this strategy, we developed a new surgical CDH mouse model to test the ability of our tissue-specific patch to regenerate damaged diaphragms. Implantation of decellularized diaphragmatic ECM-derived patches demonstrated absence of rejection or hernia recurrence, in contrast to the performance of a commercially available synthetic material. Diaphragm-derived ECM was able to promote the generation of new blood vessels, boost long-term muscle regeneration, and recover host diaphragmatic function. In addition, using a GFPâ¯+â¯Schwann cell mouse model, we identified re-innervation of implanted patches. These results demonstrated for the first time that implantation of a tissue-specific biologic scaffold is able to promote a regenerating diaphragm muscle and overcome issues commonly related to the standard use of prosthetic materials. STATEMENT OF SIGNIFICANCE: Large diaphragmatic hernia in paediatric patients require application of artificial patches to close the congenital defect. The use of a muscle-specific decellularized scaffold in substitution of currently used synthetic materials allows new blood vessel growth and nerve regeneration inside the patch, supporting new muscle tissue formation. Furthermore, the presence of a tissue-specific scaffold guaranteed long-term muscle regeneration, improving diaphragm performance to almost complete functional recovery. We believe that diaphragm-derived scaffold will be key player in future pre-clinical studies on large animal models.
Asunto(s)
Matriz Extracelular/trasplante , Hernia Diafragmática/cirugía , Músculo Esquelético , Regeneración , Andamios del Tejido , Aloinjertos , Animales , Femenino , Hernia Diafragmática/metabolismo , Hernia Diafragmática/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/inervación , Músculo Esquelético/fisiologíaRESUMEN
The neuromuscular junction (NMJ) is the specialized synapse by which peripheral motor neurons innervate muscle fibers and control skeletal muscle contraction. The NMJ is the target of several xenobiotics, including chemicals, plant, animal and bacterial toxins, as well as of autoantibodies raised against NMJ antigens. Depending on their biochemical nature, the site they target (either the nerve or the muscle) and their mechanism of action, substances affecting NMJ produce very specific alterations of neuromuscular functionality. Here we provide a detailed protocol to isolate the diaphragmatic muscle from mice and to set up two autonomously innervated hemidiaphragms. This preparation can be used to study bioactive substances like toxins, venoms and neuroactive molecules of various origin, or to measure the force of skeletal muscle contraction. The 'mouse phrenic nerve hemidiaphragm assay' (MPN) is an established model of ex vivo NMJ and recapitulates the complexity of neuromuscular transmission in a system easy to control and to manipulate, thus representing a valuable tool to study both NMJ physiology and the mechanism of action of toxins and other molecules acting at this synapse.
RESUMEN
The study of the effect of snake presynaptic neurotoxins with phospholipase A2 activity on nerve terminals has recently unveiled the inhibitory action of a lysophosphatidylcholine (LysoPC)/fatty acid mixture. We report here that these neurotoxins have no activity on Drosophila melanogaster nerve terminals. However, a 1:1 mixture of LysoPC and oleic acid induces an early increase, followed by an inhibition of both evoked and spontaneous neurotransmitter release. This effect is also induced by LysoPC alone. The present findings provide an indirect evidence that the lipid hemifusion-to-pore transition is a key event in neuroexocytosis in Drosophila. Moreover, these findings substantiate the use of LysoPC as a general agonist of membrane fusion at nerve terminals. This novel tool could contribute to the unraveling of the molecular steps involved in neuroexocytosis, particularly in Drosophila, where it is straightforward to combine it with electrophysiology and genetics.
Asunto(s)
Endocitosis/efectos de los fármacos , Lisofosfatidilcolinas/farmacología , Unión Neuromuscular/efectos de los fármacos , Neurotransmisores/metabolismo , Ácido Oléico/farmacología , Animales , Drosophila melanogaster , Electrofisiología , Potenciales Evocados/efectos de los fármacos , Exocitosis/efectos de los fármacos , Larva/efectos de los fármacos , Larva/fisiología , Fusión de Membrana/efectos de los fármacos , Unión Neuromuscular/fisiología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Lysophosphatidylcholine rapidly paralyses the neuromuscular junction (NMJ), similarly to snake phospholipase A2 neurotoxins, implicating a lipid hemifusion-pore transition in neuroexocytosis. The mode and kinetics of NMJ paralysis of different lysophospholipids (lysoPLs) in high or low [Mg2+] was investigated. The following order of potency was found: lysophosphatidylcholine>lysophosphatidylethanolamine>lysophosphatidic acid>lysophosphatidylserine>lysophosphatidylglycerol. The latter two lysoPLs closely mimic the profile of paralysis caused by the toxins in high [Mg2+]. This paralysis is fully reversed by albumin washing. These findings provide novel insights on the mode of action of snake neurotoxins and qualify lysoPLs as novel agents to study neuroexocytosis.
Asunto(s)
Diafragma/metabolismo , Exocitosis/efectos de los fármacos , Lisofosfolípidos/toxicidad , Unión Neuromuscular/metabolismo , Neurotoxinas/toxicidad , Parálisis/inducido químicamente , Nervio Frénico/metabolismo , Albúminas/farmacología , Animales , Magnesio/farmacología , Ratones , Técnicas de Cultivo de Órganos , Fosfolipasas A/toxicidad , Fosfolipasas A2 , Venenos de Serpiente/toxicidadRESUMEN
Synaptotagmin is a synaptic vesicle membrane protein which changes conformation upon Ca(2+) binding and triggers the fast neuroexocytosis that takes place at synapses. We have synthesized a series of peptides corresponding to the sequence of the cytosolic juxtamembrane domain of synaptotagmin, which is highly conserved among different isoforms and animal species, with or without either a hexyl hydrophobic chain or the hexyl group plus a fluorescein moiety. We show that these peptides inhibit neurotransmitter release, that they localize on the presynaptic membrane of the motor axon terminal at the neuromuscular junction and that they bind monophosphoinositides in a Ca(2+)-independent manner. Based on these findings, we propose that the juxtamembrane cytosolic domain of synaptotagmin binds the cytosolic layer of the presynaptic membrane at rest. This binding brings synaptic vesicles and plasma membrane in a very close apposition, favouring the formation of hemifusion intermediates that enable rapid vesicle fusion.
RESUMEN
Tetanus and botulinum neurotoxins (TeNT and BoNTs) block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such activity is exerted by the N-terminal 50 kDa light chain (L) domain, which is a zinc-dependent endopeptidase. TeNT, BoNT/B, /D, /F and /G cleave vesicle associated membrane protein (VAMP), a protein of the neurotransmitter-containing small synaptic vesicles, at different single peptide bonds. Since the proteolytic activity of these metalloproteases is higher on native VAMP inserted in synaptic vesicles than on recombinant VAMP, we have investigated the influence of liposomes of different lipid composition on this activity. We found that the rate of VAMP cleavage with all neurotoxins tested here is strongly enhanced by negatively charged lipid mixtures. This effect is at least partially due to the binding of the metalloprotease to the lipid membranes, with electrostatic interactions playing an important role.
Asunto(s)
Toxinas Botulínicas/metabolismo , Liposomas/química , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Toxina Tetánica/metabolismo , Cinética , Liposomas/metabolismo , Proteínas R-SNARE , Electricidad EstáticaRESUMEN
Tetanus neurotoxin (TeNT) is among the most poisonous substances on Earth and a major cause of neonatal death in nonvaccinated areas. TeNT targets the neuromuscular junction (NMJ) with high affinity, yet the nature of the TeNT receptor complex remains unknown. Here, we show that the presence of nidogens (also known as entactins) at the NMJ is the main determinant for TeNT binding. Inhibition of the TeNT-nidogen interaction by using small nidogen-derived peptides or genetic ablation of nidogens prevented the binding of TeNT to neurons and protected mice from TeNT-induced spastic paralysis. Our findings demonstrate the direct involvement of an extracellular matrix protein as a receptor for TeNT at the NMJ, paving the way for the development of therapeutics for the prevention of tetanus by targeting this protein-protein interaction.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Metaloendopeptidasas/uso terapéutico , Neuronas Motoras/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Toxina Tetánica/uso terapéutico , Tétanos/prevención & control , Animales , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Ratones , Ratones Endogámicos , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismo , Péptidos/farmacología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Toxina Tetánica/antagonistas & inhibidores , Toxina Tetánica/químicaRESUMEN
Notexin (Ntx) is a group I phospholipase A2 (PLA2) protein, main component of the Australian snake Notechis scutatus scutatus venom. It is both a presynaptic neurotoxin and a myotoxin. In this work, for the first time, a method for the production and folding of recombinant Ntx was developed. Ntx was produced with wild type sequence (rNtx), with an extra peptide (T7-Ntx) or a methionine (M-Ntx) before Asn-1, and with Asn-1 substituted by alanine (Ntx-A1) or by serine (Ntx-S1). The proteins were analyzed for their catalytic and toxic activities. rNtx activity resulted to be comparable to that of the venom extracted protein. The Ntx N-terminus was found to have a major influence on both the catalytic and toxic activities of the protein. The first amino acid of snake venom PLA2s is highly conserved: it is an asparagine in about all group I PLA2s, while in most (>70%) of group II PLA2s it is a serine or an asparagine. Interestingly, Ntx-S1 resulted to be, for both enzymatic and toxic activities, the mutant most similar to the wild type protein. The role of the catalytic activity of Ntx in its toxicity was investigated by replacing the aspartic acid 49, involved in the coordination of the cofactor calcium ion, by a lysine. The obtained mutant (Ntx-K49) is deprived of catalytic activity but possesses a residual toxicity.
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Venenos Elapídicos/biosíntesis , Escherichia coli/genética , Mutación , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Venenos Elapídicos/química , Venenos Elapídicos/genética , Venenos Elapídicos/aislamiento & purificación , Venenos Elapídicos/toxicidad , Ratones , Datos de Secuencia Molecular , Fosfolipasas A2/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Bothrops snakes are the major cause of ophidian envenomings in Latin America. Their venom contains myotoxins that cause prominent muscle damage, which may lead to permanent disability. These toxins include myotoxins Mt-I and Mt-II, which share the phospholipase A2 (PLA2) fold, but Mt-II lacks enzymatic activity because the essential active site Asp49 is replaced by Lys. Both myotoxins cause sarcolemma alterations, with Ca²âº entry and loss of ATP and K⺠from muscle cells, but the molecular lesions at the basis of their cellular action are not known, particularly the role of phospholipid hydrolysis. Here we tested their PLA2 activity in vivo, and evaluated the hypothesis that Ca²âº-activated endogenous PLA2s may be involved in the action of Mt-II. The time course of phospholipid hydrolysis by Mt-I and Mt-II in myotubes in culture and in tibialis anterior mouse muscles was determined. Mt-I rapidly hydrolyzed phosphatidylcholine and phosphatidylethanolamine but not phosphatidylserine, but no phospho-lipids were hydrolyzed in the presence of Mt-II. Whole Bothrops asper venom induced a higher extent of phospholipid hydrolysis than Mt-I alone. These results demonstrate in vivo PLA2 activity of Mt-I for the first time, and indicate that it acts only on the external monolayer of the sarcolemma. They also exclude activation of endogenous PLA2s in the action of Mt-II, implying that plasma membrane disruption by this toxin does not depend on phospholipid hydrolysis. Therefore, both Bothrops myotoxins induce Ca²âº entry and release of ATP and cause myonecrosis, but through different biochemical mechanisms.
Asunto(s)
Bothrops , Venenos de Crotálidos/enzimología , Fosfolipasas A2 Grupo II/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fosfolípidos/metabolismo , Proteínas de Reptiles/metabolismo , Animales , Señalización del Calcio , Dominio Catalítico , Línea Celular , Venenos de Crotálidos/metabolismo , Fosfolipasas A2 Grupo II/química , Cinética , Lipólisis , Masculino , Ratones , Ratones Endogámicos ICR , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteínas de Reptiles/química , Sarcolema/metabolismo , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
OBJECTIVE: Botulinum neurotoxins act on nerve endings and block neurotransmitter release. Their potency is due to their enzymatic activity and high affinity binding to neurons. Botulinum toxin type A is used in the treatment of human diseases characterized by hyperactivity of peripheral cholinergic nerve terminals, but some patients are or become resistant to it. This can be overcome by using other botulinum toxins, and studies have been performed with different toxin serotypes. Botulinum neurotoxin type D has never been tested in humans in vivo, and, therefore, we investigated the action of this toxin in mouse and human muscles. METHODS: Botulinum toxin type D potency was determined on mouse hemidiaphragm and on rat neuronal cultures. From these experiments, doses to be injected in human volunteers were decided. The compound muscle action potential of toxin-injected Extensor Digitorum Brevis muscle was measured at different times points after injection in human volunteers. RESULTS: Botulinum toxin type D is poorly effective in inducing human skeletal muscle paralysis. CONCLUSIONS: Botulinum toxin type D is very potent in mice and almost ineffective in humans in vivo. SIGNIFICANCE: The results shed new light on the mechanism of toxin type D binding to the neuronal surface receptors.