RESUMO
In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex.
Assuntos
Proteínas Munc18/fisiologia , Neurônios/fisiologia , Sintaxina 1/química , Animais , Exocitose , Transferência Ressonante de Energia de Fluorescência , Cinética , Fusão de Membrana , Mutação , Nanotecnologia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RatosRESUMO
Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network in T. gondii respectively.
Assuntos
Proteínas Munc18/fisiologia , Organelas/metabolismo , Proteínas de Protozoários/fisiologia , Toxoplasma/metabolismo , Fibroblastos , Células HEK293 , HumanosRESUMO
Munc18-1 and Munc13-1 orchestrate assembly of the SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, allowing exquisite regulation of neurotransmitter release. Non-regulated neurotransmitter release might be prevented by αSNAP, which inhibits exocytosis and SNARE-dependent liposome fusion. However, distinct mechanisms of inhibition by αSNAP were suggested, and it is unknown how such inhibition is overcome. Using liposome fusion assays, FRET and NMR spectroscopy, here we provide a comprehensive view of the mechanisms underlying the inhibitory functions of αSNAP, showing that αSNAP potently inhibits liposome fusion by: binding to syntaxin-1, hindering Munc18-1 binding; binding to syntaxin-1-SNAP-25 heterodimers, precluding SNARE complex formation; and binding to trans-SNARE complexes, preventing fusion. Importantly, inhibition by αSNAP is avoided only when Munc18-1 binds first to syntaxin-1, leading to Munc18-1-Munc13-1-dependent liposome fusion. We propose that at least some of the inhibitory activities of αSNAP ensure that neurotransmitter release occurs through the highly-regulated Munc18-1-Munc13-1 pathway at the active zone.
Assuntos
Proteínas Munc18/fisiologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Bovinos , Cricetulus , Escherichia coli/genética , Fusão de Membrana , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Conformação Proteica , Ratos , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismoRESUMO
Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. MCs store immunoregulatory compounds in their large cytoplasmic granules and, upon stimulation, secrete them via regulated exocytosis. Exocytosis in many cells requires the participation of Munc18 proteins (also known as syntaxin-binding proteins), and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3. To study their functions in MC effector responses and test the role of MC degranulation in anaphylaxis, we used conditional knockout (cKO) mice in which each Munc18 protein was deleted exclusively in MCs. Using recordings of plasma membrane capacitance for high-resolution analysis of exocytosis in individual MCs, we observed an almost complete absence of exocytosis in Munc18-2-deficient MCs but intact exocytosis in MCs lacking Munc18-1 or Munc18-3. Stereological analysis of EM images of stimulated MCs revealed that the deletion of Munc18-2 also abolishes the homotypic membrane fusion required for compound exocytosis. We confirmed the severe defect in regulated exocytosis in the absence of Munc18-2 by measuring the secretion of mediators stored in MC granules. Munc18-2 cKO mice had normal morphology, development, and distribution of their MCs, indicating that Munc18-2 is not essential for the migration, retention, and maturation of MC-committed progenitors. Despite that, we found that Munc18-2 cKO mice were significantly protected from anaphylaxis. In conclusion, MC-regulated exocytosis is required for the anaphylactic response, and Munc18-2 is the sole Munc18 isoform that mediates membrane fusion during MC degranulation.
Assuntos
Exocitose/fisiologia , Mastócitos/metabolismo , Proteínas Munc18/fisiologia , Anafilaxia/fisiopatologia , Animais , Degranulação Celular , Deleção de Genes , Mastócitos/ultraestrutura , Fusão de Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Proteínas Munc18/genética , Técnicas de Patch-ClampAssuntos
Mutação com Ganho de Função/genética , Mutação com Ganho de Função/fisiologia , Proteínas Munc18/genética , Proteínas Munc18/fisiologia , Mutação/genética , Mutação/fisiologia , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Humanos , Neurotransmissores/metabolismo , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
Genetic studies point to a major role of de novo mutations in neurodevelopmental disorders of intellectual disability, autism spectrum disorders, and epileptic encephalopathy. The STXBP1 gene encodes the syntaxin-binding protein 1 (Munc18-1) that critically controls synaptic vesicle exocytosis and synaptic transmission. This gene harbors a high frequency of de novo mutations, which may play roles in these neurodevelopmental disorders. However, the system and behavioral-level pathophysiological changes caused by these genetic defects remain poorly understood. Constitutional (Stxbp1+/-), dorsal-telencephalic excitatory (Stxbp1fl/+/Emx), or global inhibitory neuron-specific (Stxbp1fl/+/Vgat) mice were subjected to a behavioral test battery examining locomotor activity, anxiety, fear learning, and social interactions including aggression. Furthermore, measurements of local field potentials in multiple regions of the brain were performed. Stxbp1+/- male mice exhibited enhanced aggressiveness and impaired fear learning associated with elevated gamma activity in several regions of the brain including the prefrontal cortex. Stxbp1fl/+/Emx mice showed fear-learning deficits, but neither Stxbp1fl/+/Emx nor Stxbp1fl/+/Vgat mice showed increased aggressiveness. Pharmacological potentiation of the excitatory transmission at active synapses via the systemic administration of ampakine CX516, which enhances the excitatory postsynaptic function, ameliorated the aggressive phenotype of Stxbp1+/- mice. These findings suggest that synaptic impairments of the dorsal telencephalic and subcortical excitatory neurons cause learning deficits and enhanced aggression in Stxbp1+/- mice, respectively. Additionally, normalizing the excitatory synaptic transmission is a potential therapeutic option for managing aggressiveness in patients with STXBP1 mutations.
Assuntos
Proteínas Munc18/metabolismo , Transmissão Sináptica/fisiologia , Agressão/fisiologia , Animais , Encéfalo/metabolismo , Dioxóis/farmacocinética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Haploinsuficiência , Deficiência Intelectual/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Munc18/genética , Proteínas Munc18/fisiologia , Transtornos do Neurodesenvolvimento/metabolismo , Neurônios/metabolismo , Piperidinas/farmacocinética , Sinapses/metabolismoRESUMO
Recent studies suggest revisions to the SNARE paradigm of membrane fusion. Membrane tethers and/or SNAREs recruit proteins of the Sec 1/Munc18 family to catalyze SNARE assembly into trans-complexes. SNARE-domain zippering draws the bilayers into immediate apposition and provides a platform to position fusion triggers such as Sec 17/α-SNAP and/or synaptotagmin, which insert their apolar "wedge" domains into the bilayers, initiating the lipid rearrangements of fusion.
Assuntos
Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , Animais , Humanos , Lipídeos , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Munc18/metabolismo , Proteínas Munc18/fisiologia , Ligação Proteica , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaRESUMO
Synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of â¼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.
Assuntos
Proteínas SNARE/química , Sequência de Aminoácidos , Animais , Fusão de Membrana , Camundongos , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Proteínas Munc18/química , Proteínas Munc18/fisiologia , Pinças Ópticas , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Estabilidade Proteica , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/fisiologia , Proteínas SNARE/genética , Proteínas SNARE/fisiologia , Transmissão Sináptica/fisiologia , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/fisiologia , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/fisiologiaRESUMO
Schizophrenia is a serious psychiatric illness which is experienced by about 1 % of individuals worldwide and has a debilitating impact on perception, cognition, and social function. Over the years, several models/hypotheses have been developed which link schizophrenia to dysregulations of the dopamine, glutamate, and serotonin receptor pathways. An important segment of these pathways that have been extensively studied for the pathophysiology of schizophrenia is the presynaptic neurotransmitter release mechanism. This set of molecular events is an evolutionarily well-conserved process that involves vesicle recruitment, docking, membrane fusion, and recycling, leading to efficient neurotransmitter delivery at the synapse. Accumulated evidence indicate dysregulation of this mechanism impacting postsynaptic signal transduction via different neurotransmitters in key brain regions implicated in schizophrenia. In recent years, after ground-breaking work that elucidated the operations of this mechanism, research efforts have focused on the alterations in the messenger RNA (mRNA) and protein expression of presynaptic neurotransmitter release molecules in schizophrenia and other neuropsychiatric conditions. In this review article, we present recent evidence from schizophrenia human postmortem studies that key proteins involved in the presynaptic release mechanism are dysregulated in the disorder. We also discuss the potential impact of dysfunctional presynaptic neurotransmitter release on the various neurotransmitter systems implicated in schizophrenia.
Assuntos
Encéfalo/fisiopatologia , Esquizofrenia/fisiopatologia , Psicologia do Esquizofrênico , Vesículas Sinápticas/fisiologia , Animais , Encéfalo/patologia , Humanos , Proteínas Munc18/fisiologia , Neurotransmissores/metabolismo , Proteínas Qa-SNARE/fisiologia , Proteínas R-SNARE/fisiologia , RNA Mensageiro/genética , Receptores Pré-Sinápticos/fisiologia , Proteínas SNARE/fisiologia , Esquizofrenia/patologia , Transdução de Sinais/fisiologia , Sinapsinas/fisiologia , Vesículas Sinápticas/genética , Sinaptofisina/fisiologia , Proteína 25 Associada a Sinaptossoma/fisiologiaRESUMO
Mast cell exocytosis, which includes compound degranulation and vesicle-associated piecemeal degranulation, requires multiple Q- and R- SNAREs. It is not clear how these SNAREs pair to form functional trans-SNARE complexes and how these trans-SNARE complexes are selectively regulated for fusion. Here we undertake a comprehensive examination of the capacity of two Q-SNARE subcomplexes (syntaxin3/SNAP-23 and syntaxin4/SNAP-23) to form fusogenic trans-SNARE complexes with each of the four granule-borne R-SNAREs (VAMP2, 3, 7, 8). We report the identification of at least six distinct trans-SNARE complexes under enhanced tethering conditions: i) VAMP2/syntaxin3/SNAP-23, ii) VAMP2/syntaxin4/SNAP-23, iii) VAMP3/syntaxin3/SNAP-23, iv) VAMP3/syntaxin4/SNAP-23, v) VAMP8/syntaxin3/SNAP-23, and vi) VAMP8/syntaxin4/SNAP-23. We show for the first time that Munc18a operates synergistically with SNAP-23-based non-neuronal SNARE complexes (i to iv) in lipid mixing, in contrast to Munc18b and c, which exhibit no positive effect on any SNARE combination tested. Pre-incubation with Munc18a renders the SNARE-dependent fusion reactions insensitive to the otherwise inhibitory R-SNARE cytoplasmic domains, suggesting a protective role of Munc18a for its cognate SNAREs. Our findings substantiate the recently discovered but unexpected requirement for Munc18a in mast cell exocytosis, and implicate post-translational modifications in Munc18b/c activation.
Assuntos
Degranulação Celular/fisiologia , Proteínas Munc18/fisiologia , Proteínas SNARE/fisiologia , Animais , HumanosRESUMO
Numerous neurotransmitters have been implicated in neurodevelopmental processes. In addition, developing neurons show an abundance of vesicles in the growth cones, and express proteins of the SNARE complex early on. This has led to propose a role for vesicular fusion machinery in axonal growth and synapse formation. However, as the molecular machinery of vesicular fusion started to unveil, and knockouts for the major proteins of this complex were generated, it came as a surprise that none of these proteins was essential for the construction of brain architecture, although they were crucial for vital functions of the organism, leading to early mortality of exocytosis mutants. Because of this early death, conditional ablation of these genes in well-defined neuronal populations was necessary to study their role at later stages of neural circuit development, when activity-dependent mechanisms are best defined. Early studies showed that mutants of Munc18-1, a gene essential for both constitutive and calcium triggered release, were required for target dependent cell survival but not for axon growth or early refinement of topographic targeting, at least in the retinotectal system. Conditional knockout of the Rim1 and Rim2 genes allowed to interrogate more specifically the role of calcium-triggered release. Rims (rab interacting molecules) play a key role in the assembly of calcium channels and their coupling to the SNARE complex alters calcium-triggered release with little effect on constitutive release. When Rim1/Rim2 genes were ablated in the thalamus, layer IV neurons failed to organize into barrel structures, and to form the characteristic asymmetric distribution of their dendrites. More surprisingly, thalamocortical axons still organized in precise topographic maps and formed well differentiated synapses despite considerable reduction of calcium-induced synaptic release. However, this reduction in release probability altered axon targeting in the visual system where axons from both eyes compete for the same target. Thus, genetic tools targeting the exocytosis machinery are allowing to dissect more precisely the contribution of synaptic and non-synaptic mechanisms to activity-dependent circuit wiring.
Assuntos
Sistema Nervoso/crescimento & desenvolvimento , Neurotransmissores/fisiologia , Sinapses/fisiologia , Animais , Axônios/fisiologia , Exocitose/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/fisiologia , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Proteínas Munc18/genética , Proteínas Munc18/fisiologia , Mutação , Neurônios/fisiologia , Retina/ultraestrutura , Proteínas SNARE/genética , Proteínas SNARE/fisiologia , Sensação , Vesículas Sinápticas/fisiologia , Tálamo , Visão OcularRESUMO
MicroRNAs (miRNAs) have been implicated in a variety of physiological processes, however, the function of miRNAs in insulin secretion and type 2 diabetes is still unclear. Stxbp1 plays an essential role in exocytosis, and is crucial for insulin secretion. In this study, we focused on the molecular mechanism of Stxbp1 in insulin secretion by identifying its upstream regulators: miR-218 and miR-322. The expression of Stxbp1 was significantly increased in isolated mouse islets exposed to high levels of glucose within 1âh; while two of its predicted upstream miRNAs were found to be downregulated. Further study found that miR-218 and miR-322 directly interact with Stxbp1 by targeting the 3'UTR of its mRNA. MIN6 cells overexpressing the two miRNAs showed a sharp decline in insulin secretion and a decreased sensitivity to glucose; while the inhibition of the two miRNAs promoted insulin secretion. However, islets treated with prolonged high levels of glucose, which is known as glucolipotoxicity, displayed relatively high expression of miR-218 and miR-322, and a reduced level of expression of Stxbp1 accompanied by the blocking of insulin secretion. In summary, this study identified a pathway consisting of miR-218/322 and Stxbp1 in insulin secretion, contributing to a network of ß-cell function involving miRNA.
Assuntos
MicroRNAs/genética , Proteínas Munc18/fisiologia , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Expressão Gênica , Glucose/fisiologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Interferência de RNARESUMO
Insulin resistance, a hallmark of impaired glucose tolerance and type 2 diabetes (T2D), arises from dysfunction of insulin action and subsequent glucose uptake by peripheral tissues, predominantly skeletal muscle and fat. Exocytosis of glucose transporter (GLUT4)-containing vesicles facilitated by soluble NSF (N-ethylmaleimide-sensitive factor) attachment receptor (SNARE) protein isoforms, and Munc18c (mammalian homolog of Unc-18c) mediates this glucose uptake. Emerging evidences, including recent human clinical studies, point to pivotal roles for Munc18c in peripheral insulin action in adipose and skeletal muscle. Intriguing new advances are also initiating debates regarding the molecular mechanism(s) controlling Munc18c action. The objective of this review is therefore to present a balanced perspective of new continuities and controversies surrounding the regulation and requirement for Munc18c in the regulation of peripheral insulin action.
Assuntos
Insulina/metabolismo , Insulina/farmacologia , Proteínas Munc18/fisiologia , Tecido Adiposo/metabolismo , Animais , Glucose/metabolismo , Humanos , Resistência à Insulina/genética , Complexos Multiproteicos/metabolismo , Músculo Esquelético/metabolismo , Proteínas SNARE/metabolismoRESUMO
Proprioceptive feedback derived from specialized receptors in skeletal muscle is critical in forming an accurate map of limb position in space, and is used by the central nervous system to plan future movements and to determine accuracy of executed movements. Knockout mouse strains for genes expressed by proprioceptive sensory neurons have been generated that result in generalized motor deficits, but these deficits have not been quantitatively characterized. Here we characterize a conditional knockout mouse model wherein proprioceptive sensory neuron synaptic transmission has been blocked by selective ablation of munc18-1, a synaptic vesicle associated protein required for fusion of synaptic vesicles with the plasma membrane. Proprioceptive munc18-1 conditional mutants are impaired in surface righting--a dynamic postural adjustment task--and display several specific deficits in pivoting, an early locomotor behavior. Before the emergence of forward locomotion during postnatal development, animals explore their surroundings through pivoting, or rotating the upper torso around the relatively immobile base of the hind limbs. 3-D kinematic analysis was used to quantitatively describe this pivoting behavior at postnatal days 5 and 8 in control and munc18-1 conditional mutants. Mutant animals also pivot, but demonstrate alterations in movement strategy and in postural placement of the forelimbs during pivoting when compared to controls. In addition, brief forelimb stepping movements associated with pivoting are altered in mutant animals. Step duration and step height are increased in mutant animals. These results underscore the importance of proprioceptive feedback even at early stages in postnatal development.
Assuntos
Animais Recém-Nascidos/fisiologia , Retroalimentação Fisiológica/fisiologia , Locomoção/fisiologia , Propriocepção/genética , Propriocepção/fisiologia , Animais , Comportamento Animal/fisiologia , Fenômenos Biomecânicos , Membro Anterior/fisiologia , Mecanorreceptores/fisiologia , Camundongos , Camundongos Mutantes Neurológicos , Proteínas Munc18/genética , Proteínas Munc18/fisiologia , Músculo Esquelético/fisiologia , Equilíbrio Postural/fisiologia , Postura/fisiologia , Software , Gravação em VídeoRESUMO
Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.
Assuntos
Fusão de Membrana , Proteínas Munc18/fisiologia , Proteínas SNARE/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Humanos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Proteínas Munc18/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas SNARE/metabolismoRESUMO
Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet ß-cells is coincident with type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1(+/-) and ß-cell specific Munc18-1(-/-) knock-out mice, we establish that Munc18-1 is required for the first phase of insulin secretion. Conversely, human islets expressing elevated levels of Munc18-1 elicited significant potentiation of only first-phase insulin release. Insulin secretory changes positively correlated with insulin granule number at the plasma membrane: Munc18-1-deficient cells lacked 35% of the normal component of pre-docked insulin secretory granules, whereas cells with elevated levels of Munc18-1 exhibited a â¼20% increase in pre-docked granule number. Pre-docked syntaxin 1-based SNARE complexes bound by Munc18-1 were detected in ß-cell lysates but, surprisingly, were reduced by elevation of Munc18-1 levels. Paradoxically, elevated Munc18-1 levels coincided with increased binding of syntaxin 4 to VAMP2 at the plasma membrane. Accordingly, syntaxin 4 was a requisite for Munc18-1 potentiation of insulin release. Munc18c, the cognate SM isoform for syntaxin 4, failed to bind SNARE complexes. Given that Munc18-1 does not pair with syntaxin 4, these data suggest a novel indirect role for Munc18-1 in facilitating syntaxin 4-mediated granule pre-docking to support first-phase insulin exocytosis.
Assuntos
Insulina/metabolismo , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/metabolismo , Vesículas Secretórias/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Glucose/metabolismo , Glucose/fisiologia , Haploinsuficiência , Homeostase , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Munc18/genética , Proteínas Munc18/fisiologia , Isoformas de Proteínas/metabolismo , Proteínas Qa-SNARE/genética , Ratos , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Via Secretória , Vesículas Secretórias/ultraestrutura , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Munc18-1 was originally described as an essential docking factor in chromaffin cells. Recent findings showed that Munc18-1 has an additional role in the regulation of the cortical F-actin network, which is thought to function as a physical barrier preventing secretory vesicles from access to their release sites under resting conditions. In our review, we discuss whether this function is evolutionarily conserved in all Sec1/Munc18-like (SM) proteins. In addition, we introduce a new quantification method that improves the analysis of cortical filamentous actin (F-actin) in comparison with existing methods. Since the docking process is highly evolutionarily conserved in the SM protein superfamily, we use our novel quantification method to investigate whether the F-actin-regulating function is similarly conserved among SM proteins. Our preliminary data suggest that the regulation of cortical F-actin is a shared function of SM proteins, and we propose a way to gain more insight in the molecular mechanism underlying the Munc18-1-mediated cortical F-actin regulation.
Assuntos
Actinas/fisiologia , Células Cromafins/fisiologia , Proteínas Munc18/fisiologia , Vesículas Secretórias/fisiologia , Animais , Células Cultivadas , Células Cromafins/citologia , Técnicas Citológicas/métodos , Camundongos , Camundongos Knockout , Proteínas Munc18/genética , Vesículas Secretórias/genéticaRESUMO
Most cells contain various transport vesicles that target to different destinations. The underlying molecular mechanisms are highly conserved in evolution. Sec1/Munc-18 (SM) proteins play an important role on regulating vesicle transport by interacting with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) at each vesicle fusion sites. SM proteins interact with syntaxin, an important component in SNARE complex, to regulate the assembly of SNARE complex, and promote overall membrane fusion process together with SNARE complex. This review summaries new research progresses of structure and function of SM protein.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Proteínas Munc18/fisiologia , Animais , Transporte Biológico , Proteínas de Ligação ao Cálcio/fisiologia , Humanos , Proteínas Munc18/química , Proteínas do Tecido Nervoso/fisiologia , Proteínas R-SNARE/fisiologia , Proteínas SNARE/fisiologiaRESUMO
Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission.
Assuntos
Proteínas Munc18/fisiologia , Proteínas SNARE/fisiologia , Transmissão Sináptica/fisiologia , Animais , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Mutação Puntual , Ligação Proteica , Vesículas SinápticasRESUMO
Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically determined hyperinflammatory syndrome caused by uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, and macrophages. STXBP2 mutations have recently been associated with FHL5. To better characterize the genetic and clinical spectrum of FHL5, we analyzed a cohort of 185 patients with suspected FHL for mutations in STXBP2. We detected biallelic mutations in 37 patients from 28 families of various ethnic origins. Missense mutations and mutations affecting 1 of the exon 15 splice sites were the predominant changes detectable in this cohort. Patients with exon 15 splice-site mutations (n = 13) developed clinical manifestations significantly later than patients with other mutations (median age, 4.1 year vs 2 months) and showed less severe impairment of degranulation and cytotoxic function of NK cells and CTLs. Patients with FHL5 showed several atypical features, including sensorineural hearing deficit, abnormal bleeding, and, most frequently, severe diarrhea that was only present in early-onset disease. In conclusion, we report the largest cohort of patients with FHL5 so far, describe an extended disease spectrum, and demonstrate for the first time a clear genotype-phenotype correlation.