RESUMO
Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental in vivo and in vitro studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the in vitro validation of our findings to improve therapeutic efficacy and safety of p5.
Assuntos
Exocitose , Simulação de Dinâmica Molecular , Proteínas Munc18 , Neurônios , Proteínas Munc18/metabolismo , Proteínas Munc18/química , Proteínas Munc18/genética , Exocitose/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Humanos , Quinase 5 Dependente de Ciclina/metabolismo , Quinase 5 Dependente de Ciclina/química , Ligação Proteica , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , AnimaisRESUMO
The compartmentalization of eukaryotic cells is reliant on the fidelity of vesicle-mediated intracellular transport. Vesicles deliver their cargo via membrane fusion, a process requiring membrane tethers, Sec1/Munc18 (SM) proteins, and SNAREs. These components function in concert to ensure that membrane fusion is efficient and accurate, but the mechanisms underlying their cooperative action are still in many respects mysterious. In this brief review, we highlight recent progress toward a more integrative understanding of the vesicle fusion machinery. We focus particular attention on cryo-electron microscopy structures of intact multisubunit tethers in complex with SNAREs or SM proteins, as well as a structure of an SM protein bound to multiple SNAREs. The insights gained from this work emphasize the advantages of studying the fusion machinery intact and in context.
Assuntos
Fusão de Membrana , Proteínas SNARE , Microscopia Crioeletrônica , Proteínas SNARE/metabolismo , Proteínas Munc18/química , Proteínas Munc18/metabolismoRESUMO
As the prototype of Sec1/Munc18 (SM) family proteins, Munc18-1 can manipulate the distinct conformations of syntaxin-1 for controlling intracellular membrane fusion. The Munc18-1-interacting domain of Mint1 (Mint1-MID) binds to Munc18-1 together with syntaxin-1 to form a Mint1-Munc18-1-syntaxin-1 complex, but the mechanism underlying the complex assembly remains unclear. Here, we determine the structure of the Mint1-MID-Munc18-1-syntaxin-1 complex. Unexpectedly, Munc18-1 recognizes Mint1-MID and syntaxin-1 simultaneously via two opposite sites. The canonical central cavity between domains 1 and 3a of Munc18-1 embraces closed syntaxin-1, whereas the non-canonical basic pocket in domain 3b captures the acidic Mint1-MID helix. The domain 3b-mediated recognition of an acidic-helical motif is distinct from other target-recognition modes of Munc18-1. Mutations in the interface between domain 3b and Mint1-MID disrupt the assembly of the Mint1-Munc18-1-syntaxin-1 complex. This work reveals a non-canonical target-binding site in Munc18-1 domain 3b for assembling the Mint1-Munc18-1-syntaxin-1 complex.
Assuntos
Proteínas Munc18 , Proteínas SNARE , Proteínas Qa-SNARE/metabolismo , Sítios de Ligação , Proteínas Munc18/genética , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Sintaxina 1/química , Domínios Proteicos , Ligação Proteica , Proteínas SNARE/metabolismoRESUMO
Fusion of transmitter-containing vesicles with plasma membranes at the synaptic and neuromuscular junctions mediates neurotransmission and muscle contractions, respectively, thereby underlying all thoughts and actions. The fusion process is driven by the coupled folding and assembly of three synaptic SNARE proteins--syntaxin-1 and SNAP-25 on the target plasma membrane (t-SNAREs) and VAMP2 on the vesicular membrane (v-SNARE) into a four-helix bundle. Their assembly is chaperoned by Munc18-1 and many other proteins to achieve the speed and accuracy required for neurotransmission. However, the physiological pathway of SNARE assembly and its coupling to membrane fusion remains unclear. Here, we review recent progress in understanding SNARE assembly and membrane fusion, with a focus on results obtained by single-molecule manipulation approaches and electric recordings of single fusion pores. We describe two pathways of synaptic SNARE assembly, their associated intermediates, energetics, and kinetics. Assembly of the three SNAREs in vitro begins with the formation of a t-SNARE binary complex, on which VAMP2 folds in a stepwise zipper-like fashion. Munc18-1 significantly alters the SNARE assembly pathway: syntaxin-1 and VAMP2 first bind on the surface of Munc18-1 to form a template complex, with which SNAP-25 associates to conclude SNARE assembly and displace Munc18-1. During membrane fusion, multiple trans-SNARE complexes cooperate to open a dynamic fusion pore in a manner dependent upon their copy number and zippering states. Together, these results demonstrate that stepwise and cooperative SNARE assembly drive stagewise membrane fusion.
Assuntos
Fusão de Membrana , Proteínas SNARE , Cinética , Fusão de Membrana/fisiologia , Proteínas Munc18/química , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.
Assuntos
Proteínas SNARE/química , Proteínas SNARE/metabolismo , Doença/genética , Humanos , Fusão de Membrana , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Mutação , Pinças Ópticas , Fosforilação , Domínios Proteicos , Dobramento de Proteína , Proteínas SNARE/genéticaRESUMO
Neonatal hemophagocytic lymphohistiocytosis (HLH) is a medical emergency that can be associated with significant morbidity and mortality. Often these patients present with familial HLH (f-HLH), which is caused by gene mutations interfering with the cytolytic pathway of cytotoxic T-lymphocytes (CTLs) and natural killer cells. Here we describe a male newborn who met the HLH diagnostic criteria, presented with profound cholestasis, and carried a maternally inherited heterozygous mutation in syntaxin-binding protein-2 [STXBP2, c.568C>T (p.Arg190Cys)] in addition to a severe pathogenic variant in glucose 6-phosphate dehydrogenase [G6PD, hemizygous c.1153T>C (Cys385Arg)]. Although mutations in STXBP2 gene are associated with f-HLH type 5, the clinical and biological relevance of the p.Arg190Cys mutation identified in this patient was uncertain. To assess its role in disease pathogenesis, we performed functional assays and biochemical and microscopic studies. We found that p.Arg190Cys mutation did not alter the expression or subcellular localization of STXBP2 or STX11, neither impaired the STXBP2/STX11 interaction. In contrast, forced expression of the mutated protein into normal CTLs strongly inhibited degranulation and reduced the cytolytic activity outcompeting the effect of endogenous wild-type STXBP2. Interestingly, arginine 190 is located in a structurally conserved region of STXBP2 where other f-HLH-5 mutations have been identified. Collectively, data strongly suggest that STXBP2-R190C is a deleterious variant that may act in a dominant-negative manner by probably stabilizing non-productive interactions between STXBP2/STX11 complex and other still unknown factors such as the membrane surface or Munc13-4 protein and thus impairing the release of cytolytic granules. In addition to the contribution of STXBP2-R190C to f-HLH, the accompanied G6PD mutation may have compounded the clinical symptoms; however, the extent by which G6PD deficiency has contributed to HLH in our patient remains unclear.
Assuntos
Exocitose/genética , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Deficiência de Glucosefosfato Desidrogenase/genética , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/genética , Proteínas Munc18/genética , Mutação , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Apoptose/genética , Apoptose/imunologia , Biomarcadores , Citotoxicidade Imunológica , Suscetibilidade a Doenças , Expressão Gênica , Estudos de Associação Genética , Deficiência de Glucosefosfato Desidrogenase/complicações , Humanos , Recém-Nascido , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfo-Histiocitose Hemofagocítica/complicações , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Conformação Proteica , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Relação Estrutura-Atividade , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM-Qa-SNARE complex, Vps45-Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45-Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.
Assuntos
Proteínas Munc18/química , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/metabolismo , Chaetomium/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Priming of synaptic vesicles involves Munc13-catalyzed transition of the Munc18-1/syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of syntaxin-1. This interaction ensures Munc18-1 to persistently associate with syntaxin-1 during the conformational change of syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.
Assuntos
Proteínas Munc18 , Proteínas do Tecido Nervoso , Proteínas SNARE , Membranas Sinápticas , Sintaxina 1 , Animais , Células HEK293 , Humanos , Camundongos , Proteínas Munc18/química , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos , Ratos , Proteínas SNARE/química , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Membranas Sinápticas/química , Membranas Sinápticas/genética , Membranas Sinápticas/metabolismo , Sintaxina 1/química , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMO
Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by â¼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.
Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/metabolismo , Exocitose/fisiologia , Cinética , Fusão de Membrana/fisiologia , Chaperonas Moleculares/química , Proteínas Munc18/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/química , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.
Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Fusão de Membrana , Proteínas Munc18 , Proteínas SNARE/química , Vesículas Transportadoras/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Membrana Celular/metabolismo , Drosophila melanogaster , Humanos , Bicamadas Lipídicas/metabolismo , Camundongos , Modelos Moleculares , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Proteínas SNARE/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Vesículas Transportadoras/metabolismo , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Xenopus laevisRESUMO
MUNC18-1 (also known as STXBP1) is an essential protein for docking and fusion of secretory vesicles. Mouse chromaffin cells (MCCs) lacking MUNC18-1 show impaired secretory vesicle docking, but also mistargeting of SNARE protein syntaxin1 and an abnormally dense submembrane F-actin network. Here, we tested the contribution of both these phenomena to docking and secretion defects in MUNC18-1-deficient MCCs. We show that an abnormal F-actin network and syntaxin1 targeting defects are not observed in Snap25- or Syt1-knockout (KO) MCCs, which are also secretion deficient. We identified a MUNC18-1 mutant (V263T in ß-sheet 10) that fully restores syntaxin1 targeting but not F-actin abnormalities in Munc18-1-KO cells. MUNC18-2 and -3 (also known as STXBP2 and STXBP3, respectively), which lack the hydrophobic residue at position 263, also did not restore a normal F-actin network in Munc18-1-KO cells. However, these proteins did restore the normal F-actin network when a hydrophobic residue was introduced at the corresponding position. Munc18-1-KO MCCs expressing MUNC18-1(V263T) showed normal vesicle docking and exocytosis. These results demonstrate that MUNC18-1 regulates the F-actin network independently of syntaxin1 targeting via hydrophobicity in ß-sheet 10. The abnormally dense F-actin network in Munc18-1-deficient cells is not a rate-limiting barrier in secretory vesicle docking or fusion.This article has an associated First Person interview with the first author of the paper.
Assuntos
Actinas/metabolismo , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Sintaxina 1/metabolismo , Actinas/genética , Animais , Western Blotting , Células Cromafins/metabolismo , Eletrofisiologia , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imuno-Histoquímica , Fusão de Membrana/fisiologia , Camundongos , Camundongos Knockout , Proteínas Munc18/genética , Vesículas Secretórias/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Sintaxina 1/químicaRESUMO
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
Ulcers due to infections with Mycobacterium ulcerans are characterized by complete lack of wound healing processes, painless, an underlying bed of host dead cells and undermined edges due to necrosis. Mycolactone, a macrolide produced by the mycobacterium, is believed to be the toxin responsible. Of interest and relevance is the knowledge that Buruli ulcer (BU) patients remember experiencing trauma previously at the site of the ulcers, suggesting an impairment of wound healing processes, the plausible effect due to the toxin. Wound healing processes involve activation of the blood platelets to release the contents of the dense granules mainly serotonin, calcium ions, and ADP/ATP by exocytosis into the bloodstream. The serotonin release results in attracting more platelets and mast cells to the wound site, with the mast cells also undergoing degranulation, releasing compounds into the bloodstream by exocytosis. Recent work has identified interference in the co-translational translocation of many secreted proteins via the endoplasmic reticulum and cell death involving Wiskott-Aldrich syndrome protein (WASP), Sec61, and angiotensin II receptors (AT2R). We hypothesized that mycolactone by being lipophilic, passively crosses cell membranes and binds to key proteins that are involved in exocytosis by platelets and mast cells, thus inhibiting the initiation of wound healing processes. Based on this, molecular docking studies were performed with mycolactone against key soluble n-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and regulators, namely Vesicle-associated membrane protein (VAMP8), Synaptosomal-associated protein (SNAP23, syntaxin 11, Munc13-4 (its isoform Munc13-1 was used), and Munc18b; and also against known mycolactone targets (Sec61, AT2R, and WASP). Munc18b was shown to be a plausible mycolactone target after the molecular docking studies with binding affinity of -8.5 kcal/mol. Structural studies and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding energy calculations of the mycolactone and Munc18b complex was done with 100 ns molecular dynamics simulations using GROMACS. Mycolactone binds strongly to Munc18b with an average binding energy of -247.571 ± 37.471 kJ/mol, and its presence elicits changes in the structural conformation of the protein. Analysis of the binding interactions also shows that mycolactone interacts with Arg405, which is an important residue of Munc18b, whose mutation could result in impaired granule exocytosis. These findings consolidate the possibility that Munc18b could be a target of mycolactone. The implication of the interaction can be experimentally evaluated to further understand its role in granule exocytosis impairment in Buruli ulcer.
Assuntos
Macrolídeos/metabolismo , Proteínas Munc18/metabolismo , Plaquetas/metabolismo , Úlcera de Buruli , Exocitose , Humanos , Macrolídeos/química , Mastócitos/metabolismo , Simulação de Acoplamento Molecular , Proteínas Munc18/química , Ligação ProteicaRESUMO
Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Munc18/química , Proteínas Sensíveis a N-Etilmaleimida/química , Proteínas do Tecido Nervoso/química , Proteína 25 Associada a Sinaptossoma/química , Sinaptotagminas/química , Animais , Células CHO , Cálcio/química , Cricetinae , Cricetulus , Microscopia Crioeletrônica , Citoplasma/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Mutação , Proteínas R-SNARE/química , Ratos , Sintaxina 1/químicaRESUMO
The interaction between the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (Sx) and regulatory partner Sec/Munc18 (SM) protein is a critical step in vesicle fusion. The exact role played by SM proteins, whether positive or negative, has been the topic of much debate. High-resolution structures of the SM:Sx complex have shown that SM proteins can bind syntaxin in a closed fusion incompetent state. However, in vitro and in vivo experiments also point to a positive regulatory role for SM proteins that is inconsistent with binding syntaxin in a closed conformation. Here we present protocols we used for the expression and purification of the SM proteins Munc18a and Munc18c and syntaxins 1 and 4 along with procedures used for small-angle X-ray and neutron scattering that showed that syntaxins can bind in an open conformation to SM proteins. We also describe methods for chemical cross-linking experiments and detail how this information can be combined with scattering data to obtain low-resolution structural models for SM:Sx protein complexes.
Assuntos
Proteínas Munc18/metabolismo , Ligação Proteica , Proteínas Qa-SNARE/metabolismo , Espalhamento a Baixo Ângulo , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Deutério/química , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Fusão de Membrana , Proteínas Munc18/química , Proteínas Munc18/isolamento & purificação , Difração de Nêutrons , Estrutura Terciária de Proteína , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Difração de Raios XRESUMO
Early Infantile Epileptic Encephalopathy (known as Ohtahara Syndrome) is one of the most severe and earliest forms of epilepsy, characterized by early seizures onset. It affects newborns and children between two and six years old. Among the genes that have been associated with early infantile epileptic encephalopathy, the STXBP1 gene, which encodes the Syntaxin binding protein1a that is involved in SNARE complex formation, contributes to synaptic vesicles exocytosis. The aim of this study was to identify the most pathogenic polymorphisms of STXBP1 gene and determine their impact on the structure and stability of Stxbp1 protein. The high-risk nonsynonymous single nucleotide polymorphisms (nsSNPs) in the STXBP1 gene were predicted using 13 bioinformatics tools. The conservation analysis was realized by CONSURF web server. The analysis of the impact of the pathogenic SNPs on the structure of Stxbp1 protein was realized using YASARA software, and the molecular dynamics simulation was performed using GROMACS software. Out of 245 nsSNPs, we identified 11 (S42P, H103D R190W, R235G, D238E, L256P, P335S, C354Y, L365V, R406C, and G544D) as deleterious using in silico prediction tools. Conservation analysis results revealed that all these nsSNPs were located in conserved regions. The comparison of the hydrogen and hydrophobic interactions in the wild type Stxbp1 structure and its mutant forms showed that all these nsSNPs affect the protein structure on different levels. The molecular dynamics simulations revealed that the total of nsSNPs affect the protein stability, residual fluctuation, and the compaction at different levels. This study provides helpful information on high risk nsSNPs that may affect the Stxbp1 protein structure and function. Thus, these variants should be taken into consideration during the genetic screening of patients suffering from early infantile epileptic encephalopathy.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Munc18/química , Proteínas Munc18/genética , Polimorfismo de Nucleotídeo Único , Espasmos Infantis/genética , Biologia Computacional , Bases de Dados Factuais , Epilepsia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lactente , Recém-Nascido , Mutação , Conformação Proteica , Análise de Sequência de ProteínaRESUMO
Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.
Assuntos
Exocitose , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Peptídeos/metabolismo , Sinapses/metabolismo , Motivos de Aminoácidos , Humanos , Proteínas Munc18/genética , Neurônios/química , Neurônios/metabolismo , Peptídeos/química , Ligação Proteica , Transporte Proteico , Transdução de Sinais , Sinapses/química , Sinapses/genética , Transmissão Sináptica , Sintaxina 1/química , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMO
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE-SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE-SLP intermediate must form before SNAREs can drive efficient vesicle fusion.
Assuntos
Exocitose/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Proteínas Munc18 , Peptídeos , Proteínas SNARE , Animais , Células COS , Chlorocebus aethiops , Cinética , Camundongos , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Ratos , Proteínas SNARE/química , Proteínas SNARE/metabolismoRESUMO
As a SNARE binding protein, tomosyn has been reported to negatively regulate synaptic exocytosis via arresting syntaxin-1 and SNAP-25 into a nonfusogenic product that precludes synaptobrevin-2 entry, raising the question how the assembly of the SNARE complex is achieved. Here, we have investigated new functions of tomosyn in SNARE complex formation and SNARE-mediated vesicle fusion. Assisted by NSF/α-SNAP, syntaxin-1 escapes tomosyn arrest and assembles into the Munc18-1/syntaxin-1 complex. Munc13-1 then catalyzes the transit of syntaxin-1 from the Munc18-1/syntaxin-1 complex to the SNARE complex in a manner specific to synaptobrevin-2 but resistant to tomosyn. Our data suggest that tomosyn ensures SNARE assembly in a way amenable to tight regulation by Munc18-1 and Munc13-1.
Assuntos
Complexos Multiproteicos/química , Proteínas Munc18/química , Proteínas do Tecido Nervoso/química , Proteínas R-SNARE/química , Proteína 25 Associada a Sinaptossoma/química , Humanos , Complexos Multiproteicos/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas R-SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The membrane fusion necessary for vesicle trafficking is driven by the assembly of heterologous SNARE proteins orchestrated by the binding of Sec1/Munc18 (SM) proteins to specific syntaxin SNARE proteins. However, the precise mode of interaction between SM proteins and SNAREs is debated, as contrasting binding modes have been found for different members of the SM protein family, including the three vertebrate Munc18 isoforms. While different binding modes could be necessary, given their roles in different secretory processes in different tissues, the structural similarity of the three isoforms makes this divergence perplexing. Although the neuronal isoform Munc18a is well-established to bind tightly to both the closed conformation and the N-peptide of syntaxin 1a, thereby inhibiting SNARE complex formation, Munc18b and -c, which have a more widespread distribution, are reported to mainly interact with the N-peptide of their partnering syntaxins and are thought to instead promote SNARE complex formation. We have reinvestigated the interaction between Munc18c and syntaxin 4 (Syx4). Using isothermal titration calorimetry, we found that Munc18c, like Munc18a, binds to both the closed conformation and the N-peptide of Syx4. Furthermore, using a novel kinetic approach, we found that Munc18c, like Munc18a, slows down SNARE complex formation through high-affinity binding to syntaxin. This strongly suggests that secretory Munc18s in general control the accessibility of the bound syntaxin, probably preparing it for SNARE complex assembly.