RESUMO
Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.
Assuntos
Toxinas Botulínicas Tipo A , Exocitose , Animais , Humanos , Ratos , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/isolamento & purificação , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Masculino , Sinaptossomos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/genética , Toxinas Botulínicas/química , Toxinas Botulínicas/isolamento & purificaçãoRESUMO
α-Synuclein (α-Syn) aggregation is closely associated with Parkinson's disease neuropathology. Physiologically, α-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of α-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)-a component of the SNARE complex that resides on SVs-directly interacts with the carboxy-terminal region of α-Syn through charged residues to regulate α-Syn's function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, α-Syn and other components. Moreover, VAMP2 binding protects α-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains α-Syn's function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson's disease.
Assuntos
Vesículas Sinápticas , Proteína 2 Associada à Membrana da Vesícula , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Vesículas Sinápticas/metabolismo , Animais , Humanos , Ligação Proteica , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Camundongos , Ratos , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Neurônios/metabolismo , Neurônios/patologia , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genéticaRESUMO
Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion.
Assuntos
Cálcio , Vesículas Sinápticas , Sinaptotagmina I , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Cálcio/metabolismo , Humanos , Animais , Vesículas Sinápticas/metabolismo , Multimerização Proteica , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Transição de Fase , Mutação/genética , Células HEK293 , Fusão de Membrana , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Separação de FasesRESUMO
By modulating stomatal opening and closure, plants control gas exchange, water loss, and photosynthesis in response to various environmental signals. During light-induced stomatal opening, the transport of ions and solutes across the plasma membrane (PM) of the surrounding guard cells results in an increase in turgor pressure, leading to cell swelling. Simultaneously, vesicles for exocytosis are delivered via membrane trafficking to compensate for the enlarged cell surface area and maintain an appropriate ion-channel density in the PM. In eukaryotic cells, soluble N-ethylmaleimide-sensitive factor adaptor protein receptors (SNAREs) mediate membrane fusion between vesicles and target compartments by pairing the cognate glutamine (Q)- and arginine (R)-SNAREs to form a core SNARE complex. Syntaxin of plants 121 (SYP121) is a known Q-SNARE involved in stomatal movement, which not only facilitates the recycling of K+ channels to the PM but also binds to the channels to regulate their activity. In this study, we found that the expression of a receptor-like cytoplasmic kinase, low-K+ sensitive 4/schengen 1 (LKS4/SGN1), was induced by light; it directly interacted with SYP121 and phosphorylated T270 within the SNARE motif. Further investigation revealed that LKS4-dependent phosphorylation of SYP121 facilitated the interaction between SYP121 and R-SNARE vesicle-associated membrane protein 722 (VAMP722), promoting the assembly of the SNARE complex. Our findings demonstrate that the phosphorylation of SNARE proteins is an important strategy adopted by plants to regulate the SNARE complex assembly as well as membrane fusion. Additionally, we discovered the function of LKS4/SGN1 in light-induced stomatal opening via the phosphorylation of SYP121.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Luz , Estômatos de Plantas , Proteínas Qa-SNARE , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Estômatos de Plantas/fisiologia , Estômatos de Plantas/metabolismo , Estômatos de Plantas/efeitos da radiação , Fosforilação , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Proteínas de Ciclo CelularRESUMO
Migrasomes, enriched with signaling molecules such as chemokines, cytokines and angiogenic factors, play a pivotal role in the spatially defined delivery of these molecules, influencing critical physiological processes including organ morphogenesis and angiogenesis. The mechanism governing the accumulation of signaling molecules in migrasomes has been elusive. In this study, we show that secretory proteins, including signaling proteins, are transported into migrasomes by secretory carriers via both the constitutive and regulated secretion pathways. During cell migration, a substantial portion of these carriers is redirected to the rear of the cell and actively transported into migrasomes, driven by the actin-dependent motor protein Myosin-5a. Once at the migrasomes, these carriers fuse with the migrasome membrane through SNARE-mediated mechanisms. Inhibiting migrasome formation significantly reduces secretion, suggesting migrasomes as a principal secretion route in migrating cells. Our findings reveal a specialized, highly localized secretion paradigm in migrating cells, conceptually paralleling the targeted neurotransmitter release observed in neuronal systems.
Assuntos
Movimento Celular , Humanos , Animais , Transdução de Sinais , Transporte Proteico , Miosina Tipo V/metabolismo , Proteínas SNARE/metabolismo , CamundongosRESUMO
A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.
Assuntos
Autofagossomos , Autofagia , Fusão de Membrana , Proteínas SNARE , Autofagia/fisiologia , Autofagossomos/metabolismo , Proteínas SNARE/metabolismo , Humanos , AnimaisRESUMO
Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.
Assuntos
Legionella pneumophila , Fagossomos , Proteínas SNARE , Ubiquitinação , Proteínas rab de Ligação ao GTP , Legionella pneumophila/metabolismo , Humanos , Fagossomos/metabolismo , Fagossomos/microbiologia , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Animais , Proteínas Qa-SNARE/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Vacúolos/metabolismo , Vacúolos/microbiologia , Células HEK293 , Camundongos , proteínas de unión al GTP Rab7/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Retículo Endoplasmático/metabolismoRESUMO
Anadromous Pacific salmon (genus Oncorhynchus) are known for homing behavior to their natal rivers based on olfactory imprinted memories during seaward migration. The SNARE complex is a regulator of vesicle exocytosis from the presynaptic membrane. Our previous study suggested that its component genes (Snap25, Stx1, and Vamp2) are more highly expressed in the olfactory nervous system (ONS) during the migration stages associated with olfactory imprinting in the evolutionary species of Pacific salmon, such as chum (O. keta) and pink (O. gorbuscha) salmon. Masu salmon (O. masou) has a significantly different life history from these species, living longer in rivers and being a more primitive Pacific salmon species. In this study, the transcription of snare mRNAs in the ONS was analyzed using mainly male wild masu salmon. Five cDNAs encoding masu salmon SNAREs, which are well conserved among vertebrates, were isolated and sequenced. Each snare mRNA was highly expressed in age 1+ (yearling) parr prior to smoltification, particularly in the olfactory bulb. Their transcription status was significantly different from that of chum and pink salmon, which showed high expression in earlier under-yearling juveniles. The present results and our previous studies indicate that snare mRNAs are highly transcripted until the seaward migration, reflecting neural development and neuroplasticity of the ONS for olfactory imprinting.
Assuntos
RNA Mensageiro , Animais , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Migração Animal/fisiologia , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Nervo Olfatório/metabolismo , Masculino , Oncorhynchus/genética , Oncorhynchus/metabolismo , Filogenia , Sequência de Aminoácidos , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismoRESUMO
Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.
Assuntos
Fusão de Membrana , Fosfatidilinositol 4,5-Difosfato , Eletricidade Estática , Água , Catálise , Lipossomos/química , Fusão de Membrana/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/farmacologia , Proteínas SNARE/metabolismo , Proteínas SNARE/química , Água/químicaRESUMO
Synaptic vesicle docking and priming are dynamic processes. At the molecular level, SNAREs (soluble NSF attachment protein receptors), synaptotagmins, and other factors are critical for Ca2+-triggered vesicle exocytosis, while disassembly factors, including NSF (N-ethylmaleimide-sensitive factor) and α-SNAP (soluble NSF attachment protein), disassemble and recycle SNAREs and antagonize fusion under some conditions. Here, we introduce a hybrid fusion assay that uses synaptic vesicles isolated from mouse brains and synthetic plasma membrane mimics. We included Munc18, Munc13, complexin, NSF, α-SNAP, and an ATP-regeneration system and maintained them continuously-as in the neuron-to investigate how these opposing processes yield fusogenic synaptic vesicles. In this setting, synaptic vesicle association is reversible, and the ATP-regeneration system produces the most synchronous Ca2+-triggered fusion, suggesting that disassembly factors perform quality control at the early stages of synaptic vesicle association to establish a highly fusogenic state. We uncovered a functional role for Munc13 ancillary to the MUN domain that alleviates an α-SNAP-dependent inhibition of Ca2+-triggered fusion.
Assuntos
Proteínas do Tecido Nervoso , Vesículas Sinápticas , Vesículas Sinápticas/metabolismo , Animais , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos , Cálcio/metabolismo , Fusão de Membrana , Exocitose , Proteínas SNARE/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismoRESUMO
The regulated release of chemical messengers is crucial for cell-to-cell communication; abnormalities in which impact coordinated human body function. During vesicular secretion, multiple SNARE complexes assemble at the release site, leading to fusion pore opening. How membrane fusion regulators act on heterogeneous SNARE populations to assemble fusion pores in a timely and synchronized manner, is unknown. Here, we demonstrate the role of SNARE chaperones Munc13-1 and Munc18-1 in rescuing individual nascent fusion pores from their diacylglycerol lipid-mediated inhibitory states. At the onset of membrane fusion, Munc13-1 clusters multiple SNARE complexes at the release site and synchronizes release events, while Munc18-1 stoichiometrically interacts with trans-SNARE complexes to enhance N- to C-terminal zippering. When both Munc proteins are present simultaneously, they differentially access dynamic trans-SNARE complexes to regulate pore properties. Overall, Munc proteins' direct action on fusion pore assembly indicates their role in controlling quantal size during vesicular secretion.
Assuntos
Fusão de Membrana , Proteínas Munc18 , Proteínas do Tecido Nervoso , Proteínas SNARE , Animais , Ratos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Proteínas Munc18/metabolismo , Proteínas Munc18/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genéticaRESUMO
During neurotransmission, neurotransmitters are released less than a millisecond after the arrival of the action potential. To achieve this ultra-fast event, the synaptic vesicle must be pre-docked to the plasma membrane. In this primed state, SNAREpins, the protein-coiled coils whose assembly provides the energy to trigger fusion, are partly zippered and clamped like a hairpin and held open and ready to snap close when the clamp is released. Recently, it was suggested that three types of regulatory factors, synaptophysin, synaptotagmins, and complexins act cooperatively to organize two concentric rings, a central and a peripheral ring, containing up to six SNAREpins each. We used a mechanical model of the SNAREpins with two separate states, half-zippered and fully zippered, and determined the energy landscape according to the number of SNAREpins in each ring. We also performed simulations to estimate the fusion time in each case. The presence of the peripheral SNAREpins generally smoothens the energy landscape and accelerates the fusion time. With the predicted physiological numbers of six central and six peripheral SNAREpins, the fusion time is accelerated at least 100 times by the presence of the peripheral SNAREpins, and fusion occurs in less than 10 µs, which is well within the physiological requirements.
Assuntos
Fusão de Membrana , Proteínas SNARE , Vesículas Sinápticas , Vesículas Sinápticas/metabolismo , Proteínas SNARE/metabolismo , Transmissão Sináptica , Animais , HumanosRESUMO
General anesthetics disrupt brain network dynamics through multiple pathways, in part through postsynaptic potentiation of inhibitory ion channels as well as presynaptic inhibition of neuroexocytosis. Common clinical general anesthetic drugs, such as propofol and isoflurane, have been shown to interact and interfere with core components of the exocytic release machinery to cause impaired neurotransmitter release. Recent studies however suggest that these drugs do not affect all synapse subtypes equally. We investigated the role of the presynaptic release machinery in multiple neurotransmitter systems under isoflurane general anesthesia in the adult female Drosophila brain using live-cell super-resolution microscopy and optogenetic readouts of exocytosis and neural excitability. We activated neurotransmitter-specific mushroom body output neurons and imaged presynaptic function under isoflurane anesthesia. We found that isoflurane impaired synaptic release and presynaptic protein dynamics in excitatory cholinergic synapses. In contrast, isoflurane had little to no effect on inhibitory GABAergic or glutamatergic synapses. These results present a distinct inhibitory mechanism for general anesthesia, whereby neuroexocytosis is selectively impaired at excitatory synapses, while inhibitory synapses remain functional. This suggests a presynaptic inhibitory mechanism that complements the other inhibitory effects of these drugs.
Assuntos
Encéfalo , Proteínas de Drosophila , Isoflurano , Proteínas SNARE , Sinapses , Animais , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/fisiologia , Feminino , Proteínas SNARE/metabolismo , Isoflurano/farmacologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila , Anestésicos Inalatórios/farmacologia , Transmissão Sináptica/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Corpos Pedunculados/efeitos dos fármacos , Corpos Pedunculados/metabolismo , Corpos Pedunculados/fisiologiaRESUMO
At the synapse, presynaptic neurotransmitter release is tightly controlled by release machinery, involving the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and Munc13. The Ca2+ sensor Doc2 cooperates with Munc13 to regulate neurotransmitter release, but the underlying mechanisms remain unclear. In our study, we have characterized the binding mode between Doc2 and Munc13 and found that Doc2 originally occludes Munc13 to inhibit SNARE complex assembly. Moreover, our investigation unveiled that EphB2, a presynaptic adhesion molecule (SAM) with inherent tyrosine kinase functionality, exhibits the capacity to phosphorylate Doc2. This phosphorylation attenuates Doc2 block on Munc13 to promote SNARE complex assembly, which functionally induces spontaneous release and synaptic augmentation. Consistently, application of a Doc2 peptide that interrupts Doc2-Munc13 interplay impairs excitatory synaptic transmission and leads to dysfunction in spatial learning and memory. These data provide evidence that SAMs modulate neurotransmitter release by controlling SNARE complex assembly.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas do Tecido Nervoso , Neurotransmissores , Receptor EphB2 , Proteínas SNARE , Transmissão Sináptica , Proteínas SNARE/metabolismo , Animais , Neurotransmissores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Receptor EphB2/metabolismo , Receptor EphB2/genética , Proteínas de Ligação ao Cálcio/metabolismo , Ligação Proteica , Humanos , Camundongos , RatosRESUMO
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.
Assuntos
Proteínas R-SNARE , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vacúolos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Vacúolos/genética , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Fusão de Membrana , Exocitose , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , MutaçãoRESUMO
BACKGROUND: Women with polycystic ovary syndrome (PCOS) have increased odds of concurrent depression, indicating that the relationship between PCOS and depression is more likely to be comorbid. However, the underlying mechanism remains unclear. Here, we aimed to use bioinformatic analysis to screen for the genetic elements shared between PCOS and depression. METHODS: Differentially expressed genes (DEGs) were screened out through GEO2R using the PCOS and depression datasets in NCBI. Protein-protein interaction (PPI) network analysis and enrichment analysis were performed to identify the potential hub genes. After verification using other PCOS and depression datasets, the associations between key gene polymorphism and comorbidity were further studied using data from the UK biobank (UKB) database. RESULTS: In this study, three key genes, namely, SNAP23, VTI1A, and PRKAR1A, and their related SNARE interactions in the vesicular transport pathway were identified in the comorbidity of PCOS and depression. The rs112568544 at SNAP23, rs11077579 and rs4458066 at PRKAR1A, and rs10885349 at VTI1A might be the genetic basis of this comorbidity. CONCLUSIONS: Our study suggests that the SNAP23, PRKAR1A, and VTI1A genes can directly or indirectly participate in the imbalanced assembly of SNAREs in the pathogenesis of the comorbidity of PCOS and depression. These findings may provide new strategies in diagnosis and therapy for this comorbidity.
Assuntos
Depressão , Síndrome do Ovário Policístico , Mapas de Interação de Proteínas , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/epidemiologia , Humanos , Feminino , Depressão/genética , Depressão/epidemiologia , Mapas de Interação de Proteínas/genética , Proteínas Qb-SNARE/genética , Comorbidade , Proteínas Qc-SNARE/genética , Polimorfismo de Nucleotídeo Único , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Biologia Computacional/métodos , Predisposição Genética para DoençaRESUMO
Multiple sclerosis (MS) is a multifactorial polygenic disease; results from autoimmune and neurodegenerative processes which lead to multifocal lesions of the central nervous system. Axonal degeneration was found to be prominent in the inflammation period of MS and contribute to the progression of disability. Soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) complex plays a vital role in the release of neurotransmitter by synaptic vesicle fusion. Stx-1A protein (Stx-1A), a major component of the SNARE complex, is widely expressed in brain tissue. This study intended to evaluate the prevalence of the Stx-1A gene polymorphism (rs1569061) in the Egyptian population with MS and to investigate its association with various clinical factors. This study included 65 adult Egyptian MS patients and 35 age- and sex-matched normal control subjects. Diagnosis of MS was made by an experienced neurologist according to revised McDonald criteria. All Patients underwent full history taking, included Age of onset of MS, disease duration, disease course and degree of disability according to the Expanded Disability Status Scale (EDSS) and family history of neurological diseases. Stx-1A gene polymorphism (rs1569061) genotyping was performed by TaqMan assay based quantitative real time (qPCR) and verified by sanger sequencer. Genotype and allele frequencies of (rs1569061) did not differ significantly between case and control groups. No difference was detected when comparing the genotype frequency and the allele frequency to different disease parameters. Discrepancy of the minor allele frequency (MAF) of Stx-1A gene (rs1569061) between different populations was noted. In conclusion, our study in Stx-1A gene polymorphism (rs1569061) and MS showed that no difference between the patient and control as regards gene frequency and allele frequency. Predicting no association between the studied polymorphism and MS in the Egyptian population. However, discrepancy between different population was noted as regards the MAF for Stx-1A gene (rs1569061).
Assuntos
Esclerose Múltipla , Sintaxina 1 , Adulto , Humanos , Egito/epidemiologia , Frequência do Gene , Esclerose Múltipla/genética , Polimorfismo Genético , Proteínas SNARE , Sintaxina 1/genética , População do Norte da África/genéticaRESUMO
The SNAP receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin mediate neurotransmitter release by forming tight SNARE complexes that fuse synaptic vesicles with the plasma membranes in microseconds. Membrane fusion is generally explained by the action of proteins on macroscopic membrane properties such as curvature, elastic modulus, and tension, and a widespread model envisions that the SNARE motifs, juxtamembrane linkers, and C-terminal transmembrane regions of synaptobrevin and syntaxin-1 form continuous helices that act mechanically as semirigid rods, squeezing the membranes together as they assemble ("zipper") from the N to the C termini. However, the mechanism underlying fast SNARE-induced membrane fusion remains unknown. We have used all-atom molecular dynamics simulations to investigate this mechanism. Our results need to be interpreted with caution because of the limited number and length of the simulations, but they suggest a model of membrane fusion that has a natural physicochemical basis, emphasizes local molecular events over general membrane properties, and explains extensive experimental data. In this model, the central event that initiates fast (microsecond scale) membrane fusion occurs when the SNARE helices zipper into the juxtamembrane linkers which, together with the adjacent transmembrane regions, promote encounters of acyl chains from both bilayers at the polar interface. The resulting hydrophobic nucleus rapidly expands into stalk-like structures that gradually progress to form a fusion pore, aided by the SNARE transmembrane regions and without clearly discernible intermediates. The propensity of polyunsaturated lipids to participate in encounters that initiate fusion suggests that these lipids may be important for the high speed of neurotransmitter release.
Assuntos
Fusão de Membrana , Proteínas SNARE , Proteínas SNARE/metabolismo , Simulação de Dinâmica Molecular , Proteínas R-SNARE , Sintaxina 1 , Neurotransmissores , LipídeosRESUMO
The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.
Assuntos
Sinapses , Vesículas Sinápticas , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Potenciais de Ação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.