RESUMO
BACKGROUND: Richmond agitation-sedation scale (RASS) is a simple and widely used tool for evaluating sedation and agitation in adult ICU patients. Early deep sedation has been shown to be an important independent predictor of death, however, studies on the role of RASS in the prognostic assessment of neurocritical patients are lacking. The purpose of this study was to investigate the relationship between RASS and in-hospital mortality in neurocritical patients, and to develop and validate an effective predictive model based on this. METHODS: This was a retrospective study of neurocritical patients from a large clinical database. A total of 2651 patients were collected, including general demographic characteristics, past medical history, biochemical test data and physical examination within 24 h of admission, and related medical records. Univariate and multivariate logistic regression analyses were used to screen out significant variables. Finally, 11 significant predictors were included into the logistic regression to establish the nomogram. RESULTS: The area under the curve (AUC) of the nomogram was 0.9087(0.8950-0.9224) and the corrected c index was 0.9043, which gave the model better discriminatory ability compared with critical care related scales, such as SOFA and SAPSII scores. Besides, tools including calibration curve, decision curve analysis (DCA), and clinical impact curve (CIC) were used to verify that the model had good discrimination, calibration, and clinical applicability. CONCLUSIONS: RASS score was an independent prognostic predictor of in-hospital death in neurocritical patients, and patients who are deeply sedated have a worse prognosis. RASS-related nomogram could be applied to predict the prognosis of neurocritical patients and to take effective intervention measures in early stage.
Assuntos
Nomogramas , Adulto , Humanos , Estudos Retrospectivos , Mortalidade Hospitalar , Prognóstico , Modelos LogísticosRESUMO
The neurotransmitter acetylcholine (ACh) acts in part through a family of nicotinic ACh receptors (nAChRs), which mediate diverse physiological processes including muscle contraction, neurotransmission, and sensory transduction. Pharmacologically, nAChRs are responsible for tobacco addiction and are targeted by medicines for hypertension and dementia. Nicotinic AChRs were the first ion channels to be isolated. Recent studies have identified molecules that control nAChR biogenesis, trafficking, and function. These nAChR accessories include protein and chemical chaperones as well as auxiliary subunits. Whereas some factors act on many nAChRs, others are receptor specific. Discovery of these regulatory mechanisms is transforming nAChR research in cells and tissues ranging from central neurons to spinal ganglia to cochlear hair cells. Nicotinic AChR-specific accessories also enable drug discovery on high-confidence targets for psychiatric, neurological, and auditory disorders.
Assuntos
Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Membrana Celular/metabolismo , Descoberta de Drogas , Retículo Endoplasmático/metabolismo , Humanos , Ligantes , Músculo Esquelético/metabolismo , Neurofarmacologia , Nicotina/metabolismo , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/químicaRESUMO
The α6ß4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6ß4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6ß4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6ß4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6ß4 receptor assembly. Effects on α6ß4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6ß4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6ß4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6ß4 receptor.
Assuntos
Agonistas Colinérgicos/farmacologia , Endorribonucleases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Colinérgicos/biossíntese , Animais , Endorribonucleases/genética , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA/efeitos dos fármacos , Ratos , Receptores Colinérgicos/genética , Proteína 1 de Ligação a X-Box/genéticaRESUMO
Auditory hair cells receive olivocochlear efferent innervation, which refines tonotopic mapping, improves sound discrimination, and mitigates acoustic trauma. The olivocochlear synapse involves α9α10 nicotinic acetylcholine receptors (nAChRs), which assemble in hair cells only coincident with cholinergic innervation and do not express in recombinant mammalian cell lines. Here, genome-wide screening determined that assembly and surface expression of α9α10 require ligand binding. Ion channel function additionally demands an auxiliary subunit, which can be transmembrane inner ear (TMIE) or TMEM132e. Both of these single-pass transmembrane proteins are enriched in hair cells and underlie nonsyndromic human deafness. Inner hair cells from TMIE mutant mice show altered postsynaptic α9α10 function and retain α9α10-mediated transmission beyond the second postnatal week associated with abnormally persistent cholinergic innervation. Collectively, this study provides a mechanism to link cholinergic input with α9α10 assembly, identifies unexpected functions for human deafness genes TMIE/TMEM132e, and enables drug discovery for this elusive nAChR implicated in prevalent auditory disorders.
Assuntos
Surdez/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas de Membrana/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Cóclea/metabolismo , Surdez/genética , Humanos , Ligantes , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Receptores Nicotínicos/genética , Sinapses/metabolismoRESUMO
The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Glicosilação , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Ratos , Transdução de SinaisRESUMO
Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4ß2, but not α6 subunits. Polyamines are classically studied as regulators of ion channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology.
Assuntos
Canais Iônicos/metabolismo , Poliaminas/metabolismo , Receptores Nicotínicos/metabolismo , Acetiltransferases , Animais , Biocatálise , DNA Complementar/genética , Elementos Facilitadores Genéticos/genética , Fluorescência , Genoma Humano , Células HEK293 , Humanos , Ativação do Canal Iônico , Camundongos , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores Nicotínicos/químicaRESUMO
Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs. Leveraging the robust recombinant expression of α7 nAChRs with NACHO, we utilized genome-wide cDNA library screening and discovered that several anti-apoptotic Bcl-2 family proteins further upregulate receptor assembly and cell surface expression. These effects are mediated by an intracellular motif on α7 that resembles the BH3 binding domain of pro-apoptotic Bcl-2 proteins, and can be blocked by BH3 mimetic Bcl-2 inhibitors. Overexpression of Bcl-2 member Mcl-1 in neurons enhanced surface expression of endogenous α7 nAChRs, while a combination of chemotherapeutic Bcl2-inhibitors suppressed neuronal α7 receptor assembly. These results demonstrate that Bcl-2 proteins link α7 nAChR assembly to cell survival pathways.
Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neurônios/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Motivos de Aminoácidos/genética , Animais , Benzotiazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células HEK293 , Humanos , Isoquinolinas/farmacologia , Chaperonas Moleculares/metabolismo , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Ratos , Transmissão Sináptica/efeitos dos fármacos , Tiofenos/farmacologia , Regulação para Cima , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Acetylcholine gates a large family of nicotinic receptor cation channels that control neuronal excitation and neurotransmitter release. These receptors are key targets for neuropsychiatric disorders; however, difficulties in expressing nicotinic acetylcholine (nACh) receptors hamper elaboration of their pharmacology and obscure elucidation of their biological functions. Particularly intriguing are α6-containing nACh receptors, which mediate nicotine-induced dopamine release in striatum-nucleus accumbens. Using genome-wide cDNA screening, we identify three accessory proteins, ß-anchoring and -regulatory protein (BARP), lysosomal-associated membrane protein 5 (LAMP5), and SULT2B1, that complement the nACh receptor chaperone NACHO to reconstitute α6ß2ß3 channel function. Whereas NACHO mediates α6ß2ß3 assembly, BARP primarily enhances channel gating and LAMP5 and SULT2B1 promote receptor surface trafficking. BARP knockout mice show perturbations in presynaptic striatal nACh receptors that are consistent with BARP modulation of receptor desensitization. These studies unravel the molecular complexity of α6ß2ß3 biogenesis and enable physiological studies of this crucial neuropharmacological target.
Assuntos
Corpo Estriado , Núcleo Accumbens/metabolismo , Multimerização Proteica , Receptores Nicotínicos/metabolismo , Transmissão Sináptica , Acetilcolina/genética , Acetilcolina/metabolismo , Animais , Corpo Estriado/metabolismo , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Compostos Orgânicos , Ratos , Receptores Nicotínicos/genéticaRESUMO
Targeted therapy for neuropsychiatric disorders requires selective modulation of dysfunctional neuronal pathways. Receptors relevant to CNS disorders typically have associated proteins discretely expressed in specific neuronal pathways; these accessory proteins provide a new dimension for drug discovery. Recent studies show that targeting a TARP auxiliary subunit of AMPA receptors selectively modulates neuronal excitability in specific forebrain pathways relevant to epilepsy. Other medicinally important ion channels, gated by glutamate, γ-aminobutyric acid (GABA), and acetylcholine, also have associated proteins, which may be druggable. This emerging pharmacology of receptor-associated proteins provides a new approach for improving drug efficacy while mitigating side effects.
Assuntos
Neurofarmacologia , Receptores de Neurotransmissores/efeitos dos fármacos , Animais , Humanos , Proteínas do Tecido Nervoso/efeitos dos fármacosRESUMO
Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and ß subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4ß2-, and α6-containing nAChRs, respectively. By contrast, GABAA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.
Assuntos
Encéfalo/metabolismo , Chaperonas Moleculares/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Bungarotoxinas/química , Bungarotoxinas/metabolismo , Linhagem Celular , Disfunção Cognitiva/patologia , Conotoxinas/química , Conotoxinas/metabolismo , Humanos , Radioisótopos do Iodo/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Neurônios/metabolismo , Nicotina/química , Nicotina/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Piridinas/química , Piridinas/metabolismo , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Receptores Nicotínicos/genéticaRESUMO
Cellular signaling is in part regulated by the composition and subcellular localization of a series of protein interactions that collectively form a signaling complex. Using the α7 nicotinic acetylcholine receptor (α7nAChR) as a proof-of-concept target, we developed a platform to identify functional modulators (or auxiliary proteins) of α7nAChR signaling. The Broad cDNA library was transiently cotransfected with α7nAChR cDNA in HEK293T cells in a high-throughput fashion. Using this approach in combination with a functional assay, we identified positive modulators of α7nAChR activity. We identified known positive modulators/auxiliary proteins present in the cDNA library that regulate α7nAChR signaling, in addition to identifying novel modulators of α7nAChR signaling. These included NACHO, SPDYE11, TCF4, and ZC3H12A, all of which increased PNU-120596-mediated nicotine-dependent calcium flux. Importantly, these auxiliary proteins did not modulate GluR1(o)-mediated Ca flux. To elucidate a possible mechanism of action, we employed an α7nAChR-HA surface staining assay. NACHO enhanced α7nAChR surface expression; however, the mechanism responsible for the SPDYE11-, TCF4-, and ZC3H12A-dependent modulation of α7nAChR has yet to be defined. This report describes the development and validation of a high-throughput, genome-wide cDNA screening platform coupled to FLIPR functional assays in order to identify functional modulators of α7nAChR signaling.
Assuntos
Genoma Humano/genética , Fatores de Transcrição/genética , Receptor Nicotínico de Acetilcolina alfa7/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Ribonucleases/genética , Transdução de Sinais , Fator de Transcrição 4/genéticaRESUMO
Nicotine exerts its behavioral and additive actions through a family of brain nicotinic acetylcholine receptors (nAChRs). Enhancing α7-type nAChR signaling improves symptoms in Alzheimer's disease and schizophrenia. The pharmaceutical study of α7 receptors is hampered because these receptors do not form their functional pentameric structure in cell lines, and mechanisms that underlie α7 receptor assembly in neurons are not understood. Here, a genomic screening strategy solves this long-standing puzzle and identifies NACHO, a transmembrane protein of neuronal endoplasmic reticulum that mediates assembly of α7 receptors. NACHO promotes α7 protein folding, maturation through the Golgi complex, and expression at the cell surface. Knockdown of NACHO in cultured hippocampal neurons or knockout of NACHO in mice selectively and completely disrupts α7 receptor assembly and abolishes α7 channel function. This work identifies NACHO as an essential, client-specific chaperone for nAChRs and has implications for physiology and disease associated with these widely distributed neurotransmitter receptors.
Assuntos
Hipocampo/metabolismo , Neurônios/fisiologia , Subunidades Proteicas/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Calnexina/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/farmacologia , Células HEK293 , Hipocampo/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoxazóis/farmacologia , Masculino , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Subunidades Proteicas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serotonina/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH.
Assuntos
Toxinas Botulínicas/química , Neurotoxinas/química , Toxinas Botulínicas/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Neurotoxinas/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
How botulinum neurotoxins (BoNTs) cross the host intestinal epithelial barrier in foodborne botulism is poorly understood. Here, we present the crystal structure of a clostridial hemagglutinin (HA) complex of serotype BoNT/A bound to the cell adhesion protein E-cadherin at 2.4 angstroms. The HA complex recognizes E-cadherin with high specificity involving extensive intermolecular interactions and also binds to carbohydrates on the cell surface. Binding of the HA complex sequesters E-cadherin in the monomeric state, compromising the E-cadherin-mediated intercellular barrier and facilitating paracellular absorption of BoNT/A. We reconstituted the complete 14-subunit BoNT/A complex using recombinantly produced components and demonstrated that abolishing either E-cadherin- or carbohydrate-binding of the HA complex drastically reduces oral toxicity of BoNT/A complex in vivo. Together, these studies establish the molecular mechanism of how HAs contribute to the oral toxicity of BoNT/A.
Assuntos
Toxinas Botulínicas Tipo A/química , Caderinas/química , Hemaglutininas/química , Animais , Toxinas Botulínicas Tipo A/genética , Caderinas/genética , Cristalografia por Raios X , Técnicas de Silenciamento de Genes , Células HT29 , Hemaglutininas/genética , Humanos , Camundongos , Estrutura Secundária de Proteína , Proteínas Recombinantes/químicaRESUMO
Botulinum neurotoxins (BoNTs) are potent bacterial toxins. The high oral toxicity of BoNTs is largely attributed to the progenitor toxin complex (PTC), which is assembled from BoNT and nontoxic neurotoxin-associated proteins (NAPs) that are produced together with BoNT in bacteria. Here, we performed ex vivo studies to examine binding of the highly homogeneous recombinant NAPs to mouse small intestine. We also carried out the first comprehensive glycan array screening with the hemagglutinin (HA) component of NAPs. Our data confirmed that intestinal binding of the PTC is partly mediated by the HA moiety through multivalent interactions between HA and host carbohydrates. The specific HA-carbohydrate recognition could be inhibited by receptor-mimicking saccharides.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Hemaglutininas/metabolismo , Polissacarídeos/metabolismo , Animais , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a â¼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.
Assuntos
Toxinas Botulínicas , Botulismo , Complexos Multiproteicos , Animais , Toxinas Botulínicas/química , Toxinas Botulínicas/genética , Toxinas Botulínicas/toxicidade , Clostridium botulinum/genética , Clostridium botulinum/metabolismo , Feminino , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/toxicidade , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, but paradoxically, BoNT-containing medicines and cosmetics have been used with great success in the clinic. Accidental BoNT poisoning mainly occurs through oral ingestion of food contaminated with Clostridium botulinum. BoNTs are naturally produced in the form of progenitor toxin complexes (PTCs), which are high molecular weight (up to ~900 kDa) multiprotein complexes composed of BoNT and several non-toxic neurotoxin-associated proteins (NAPs). NAPs protect the inherently fragile BoNTs against the hostile environment of the gastrointestinal (GI) tract and help BoNTs pass through the intestinal epithelial barrier before they are released into the general circulation. These events are essential for ingested BoNTs to gain access to motoneurons, where they inhibit neurotransmitter release and cause muscle paralysis. In this review, we discuss the structural basis for assembly of NAPs and BoNT into the PTC that protects BoNT and facilitate its delivery into the bloodstream.
Assuntos
Toxinas Botulínicas/metabolismo , Clostridium botulinum/metabolismo , Complexos Multiproteicos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico , Toxinas Botulínicas/genética , Toxinas Botulínicas/toxicidade , Clostridium botulinum/genética , Clostridium botulinum/patogenicidade , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Hemaglutininas/metabolismo , Interações Hospedeiro-Patógeno , Concentração de Íons de Hidrogênio , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Neurotoxinas/genética , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Relação Estrutura-AtividadeRESUMO
Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. NMJ formation requires neural agrin, a motoneuron-derived protein, which interacts with LRP4 (low-density lipoprotein receptor-related protein 4) to activate the receptor tyrosine kinase MuSK (muscle-specific kinase). However, little is known of how signals are transduced from agrin to MuSK. Here, we present the first crystal structure of an agrin-LRP4 complex, consisting of two agrin-LRP4 heterodimers. Formation of the initial binary complex requires the z8 loop that is specifically present in neuronal, but not muscle, agrin and that promotes the synergistic formation of the tetramer through two additional interfaces. We show that the tetrameric complex is essential for neuronal agrin-induced acetylcholine receptor (AChR) clustering. Collectively, these results provide new insight into the agrin-LRP4-MuSK signaling cascade and NMJ formation and represent a novel mechanism for activation of receptor tyrosine kinases.
Assuntos
Agrina/química , Agrina/metabolismo , Modelos Moleculares , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de LDL/química , Receptores de LDL/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Linhagem Celular , Ativação Enzimática , Camundongos , Neurônios/metabolismo , Estrutura Quaternária de Proteína , RatosRESUMO
Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostridium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial nontoxic nonhemagglutinin (NTNHA) protein at 2.7 angstroms. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication and of delivery vehicles for oral administration of biologics.
Assuntos
Proteínas de Bactérias/química , Toxinas Botulínicas Tipo A/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Toxinas Botulínicas Tipo A/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutagênese , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de ProteínaRESUMO
The AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subfamily of iGluRs (ionotropic glutamate receptors) is essential for fast excitatory neurotransmission in the central nervous system. The malfunction of AMPARs (AMPA receptors) has been implicated in many neurological diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The active channels of AMPARs and other iGluR subfamilies are tetramers formed exclusively by assembly of subunits within the same subfamily. It has been proposed that the assembly process is controlled mainly by the extracellular ATD (N-terminal domain) of iGluR. In addition, ATD has also been implicated in synaptogenesis, iGluR trafficking and trans-synaptic signalling, through unknown mechanisms. We report in the present study a 2.5 Å (1 Å=0.1 nm) resolution crystal structure of the ATD of GluA1. Comparative analyses of the structure of GluA1-ATD and other subunits sheds light on our understanding of how ATD drives subfamily-specific assembly of AMPARs. In addition, analysis of the crystal lattice of GluA1-ATD suggests a novel mechanism by which the ATD might participate in inter-tetramer AMPAR clustering, as well as in trans-synaptic protein-protein interactions.