RESUMO
Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.
Assuntos
Diglicerídeos , Vesículas Sinápticas , Humanos , Exocitose , Transmissão Sináptica , Sinaptotagminas , VesículaRESUMO
Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from the endoplasmic reticulum to endosomes is a rate-determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin 5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 acts as an immunostimulant, we have identified the de novo RNA-binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAVL1 protein, Human antigen R. This activity ensures the export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.
Assuntos
Proteína Semelhante a ELAV 1 , Macrófagos , MicroRNAs , Proteínas Qa-SNARE , Animais , Humanos , Camundongos , Endossomos/metabolismo , Leishmania donovani/metabolismo , Leishmania donovani/genética , Ativação de Macrófagos , Macrófagos/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Transporte de RNA , Proteína Semelhante a ELAV 1/metabolismoRESUMO
Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca2+ influx. This Ca2+-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially "tethered" to the PM by a variable number of protein densities (â¼10 to 20 nm long) with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the "primed" vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca2+ influx.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Células Cultivadas , Microscopia Crioeletrônica , Hipocampo/citologia , Imageamento Tridimensional , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
Lipid accumulation in hepatocytes is the distinctive characteristic of nonalcoholic fatty liver disease. Serine/arginine-rich splicing factor 3 (SRSF3) is highly expressed in the liver and expression decreases in high-fat conditions. However, the role of SRSF3 in hepatic lipid metabolism needs to be clarified. Here, we showed that loss of SRSF3 was associated with lipid accumulation. We determined that SRSF3 regulated lipophagy, the process of selective degradation of lipid droplets by autophagy. Mechanistically, loss of SRSF3 impaired the fusion of the autophagosome and lysosome by promoting the proteasomal degradation of syntaxin 17 (STX17), a key autophagosomal SNARE protein. We found that ubiquitination of STX17 was increased and upregulation of seven in absentia homolog 1 was responsible for the increased posttranslational modification of STX17. Taken together, our data primarily demonstrate that loss of SRSF3 weakens the clearance of fatty acids by impairing lipophagy in the progression of nonalcoholic fatty liver disease, indicating a novel potential therapeutic target for fatty liver disease treatment.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Autofagia/genética , Ácidos Graxos/metabolismo , Hepatócitos/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Ubiquitinação , Proteínas Qa-SNARE/metabolismoRESUMO
To elucidate the efferent functions of sensory nerve endings, the distribution of calretinin and vesicular glutamate transporter 1 (VGLUT1) in laryngeal laminar nerve endings and the immunohistochemical distribution of proteins associated with synaptic vesicle release, i.e., t-SNARE (SNAP25 and syntaxin 1), v-SNARE (VAMP1 and VAMP2), synaptotagmin 1 (Syt1), bassoon, and piccolo, were examined. Subepithelial laminar nerve endings immunoreactive for Na+-K+-ATPase α3-subunit (NKAα3) were largely distributed in the whole-mount preparation of the epiglottic mucosa, and several endings were also immunoreactive for calretinin. VGLUT1 immunoreactivity was observed within terminal part near the outline of the small processes of NKAα3-immunoreactive nerve ending. SNAP25, syntaxin 1, and VAMP1 immunoreactivities were detected in terminal parts of calretinin-immunoreactive endings, whereas VAMP2 immunoreactivity was only observed in a few terminals. Terminal parts immunoreactive for calretinin and/or VGLUT1 also exhibited immunoreactivities for Syt1, Ca2+ sensor for membrane trafficking, and for bassoon and piccolo, presynaptic scaffold proteins. The presence of vesicular release-related proteins, including SNARE proteins, in the terminals of laryngeal laminar endings indicate that intrinsic glutamate modulates their afferent activity in an autocrine-like manner.
Assuntos
Epiglote , Ácido Glutâmico , Animais , Epiglote/metabolismo , Ácido Glutâmico/metabolismo , Terminações Nervosas/metabolismo , Ratos , Células Receptoras Sensoriais/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
Marchantia polymorpha is a model liverwort and its overall low genetic redundancy is advantageous for dissecting complex pathways. Proximity-dependent in vivo biotin-labelling methods have emerged as powerful interactomics tools in recent years. However, interactomics studies applying proximity labelling are currently limited to angiosperm species in plants. Here, we established and evaluated a miniTurbo-based interactomics method in M. polymorpha using MpSYP12A and MpSYP13B, two plasma membrane-localized SNARE proteins, as baits. We show that our method yields a manifold of potential interactors of MpSYP12A and MpSYP13B compared to a coimmunoprecipitation approach. Our method could capture specific candidates for each SNARE. We conclude that a miniTurbo-based method is a feasible tool for interactomics in M. polymorpha and potentially applicable to other model bryophytes. Our interactome dataset on MpSYP12A and MpSYP13B will be a useful resource to elucidate the evolution of SNARE functions.
Assuntos
Marchantia , Membrana Celular/metabolismo , Marchantia/genética , Marchantia/metabolismo , Proteínas SNARE/metabolismoRESUMO
Autophagy is a highly conserved intracellular degrading system and its dysfunction is considered related to the cause of neurodegenerative disorders. A previous study showed that the inhibition of endocytosis transport attenuates soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein transport to lysosomes and block autophagy. The other studies demonstrated oxidative stress, one of the inducers of neurodegenerative diseases inhibits endocytosis transport. Thus, we hypothesized that oxidative stress-induced endocytosis inhibition causes alteration of SNARE protein transport to lysosomes and impairs autophagy. Here, we demonstrated that oxidative stress inhibits endocytosis and decreased the lysosomal localization of VAMP8, one of the autophagy-related SNARE proteins in a human neuroblastoma cell line. Moreover, this oxidative stress induction blocked the autophagosome-lysosome fusion step. Since we also observed decreased lysosomal localization of VAMP8 and inhibition of autophagosome-lysosome fusion in endocytosis inhibitor-treated cells, oxidative stress may inhibit VAMP8 trafficking by suppressing endocytosis and impair autophagy. Our findings suggest that oxidative stress-induced inhibition of VAMP8 trafficking to lysosomes is associated with the development of neurodegenerative diseases due to the blocked autophagosome-lysosome fusion, and may provide a new therapeutic target for restoring the autophagic activity.
Assuntos
Autofagia , Lisossomos , Humanos , Autofagia/fisiologia , Lisossomos/metabolismo , Fusão de Membrana , Estresse Oxidativo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Transporte BiológicoRESUMO
BACKGROUND: Increasing evidence suggests that platelets play a central role in cancer progression, with altered storage and selective release from platelets of specific tumor-promoting proteins as a major mechanism. Fluorescence-based super-resolution microscopy (SRM) can resolve nanoscale spatial distribution patterns of such proteins, and how they are altered in platelets upon different activations. Analysing such alterations by SRM thus represents a promising, minimally invasive strategy for platelet-based diagnosis and monitoring of cancer progression. However, broader applicability beyond specialized research labs will require objective, more automated imaging procedures. Moreover, for statistically significant analyses many SRM platelet images are needed, of several different platelet proteins. Such proteins, showing alterations in their distributions upon cancer progression additionally need to be identified. RESULTS: A fast, streamlined and objective procedure for SRM platelet image acquisition, analysis and classification was developed to overcome these limitations. By stimulated emission depletion SRM we imaged nanoscale patterns of six different platelet proteins; four different SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) mediating protein secretion by membrane fusion of storage granules, and two angiogenesis regulating proteins, representing cargo proteins within these granules coupled to tumor progression. By a streamlined procedure, we recorded about 100 SRM images of platelets, for each of these six proteins, and for five different categories of platelets; incubated with cancer cells (MCF-7, MDA-MB-231, EFO-21), non-cancer cells (MCF-10A), or no cells at all. From these images, structural similarity and protein cluster parameters were determined, and probability functions of these parameters were generated for the different platelet categories. By comparing these probability functions between the categories, we could identify nanoscale alterations in the protein distributions, allowing us to classify the platelets into their correct categories, if they were co-incubated with cancer cells, non-cancer cells, or no cells at all. CONCLUSIONS: The fast, streamlined and objective acquisition and analysis procedure established in this work confirms the role of SNAREs and angiogenesis-regulating proteins in platelet-mediated cancer progression, provides additional fundamental knowledge on the interplay between tumor cells and platelets, and represent an important step towards using tumor-platelet interactions and redistribution of nanoscale protein patterns in platelets as a basis for cancer diagnostics.
Assuntos
Neoplasias , Proteínas SNARE , Plaquetas/metabolismo , Fusão de Membrana , Microscopia de Fluorescência/métodos , Neoplasias/metabolismo , Proteínas SNARE/metabolismoRESUMO
Atopic dermatitis (AD) is a chronic inflammatory dermal disease with symptoms that include inflammation, itching, and dry skin. 1-Iodohexadecane is known as a component of Chrysanthemum boreale essential oil that has an inhibitory effect on AD-like lesions. However, its effects on AD-related pathological events have not been investigated. Here, we explored the effects of 1-iodohexadecane on AD lesion-related in vitro and in vivo responses and the mechanism involved using human keratinocytes (HaCaT cells), mast cells (RBL-2H3 cells), and a 2,4-dinitrochlorobenzene (DNCB)-induced mouse model (male BALB/c) of AD. Protein analyses were performed by immunoblotting or immunohistochemistry. In RBL-2H3 cells, 1-iodohexadecane inhibited immunoglobulin E-induced releases of histamine and ß-hexosaminidase and the expression of VAMP8 protein (vesicle-associated membrane proteins 8; a soluble N-ethylmaleimide-sensitive factor attachment protein receptor [SNARE] protein). In HaCaT cells, 1-iodohexadecane enhanced filaggrin and loricrin expressions; in DNCB-treated mice, it improved AD-like skin lesions, reduced epidermal thickness, mast cell infiltration, and increased filaggrin and loricrin expressions (skin barrier proteins). In addition, 1-iodohexadecane reduced the ß-hexosaminidase level in the serum of DNCB-applied mice. These results suggest that 1-iodohexadecane may ameliorate AD lesion severity by disrupting SNARE protein-linked degranulation and/or by enhancing the expressions of skin barrier-related proteins, and that 1-iodohexadecane has therapeutic potential for the treatment of AD.
Assuntos
DinitroclorobenzenoRESUMO
Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1-SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery.
Assuntos
Modelos Moleculares , Proteínas SNARE/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Cristalografia por Raios X , Estrutura Quaternária de ProteínaRESUMO
Sialidosis is a neuropathic lysosomal storage disease caused by a deficiency in the NEU1 gene-encoding lysosomal neuraminidase and characterized by abnormal accumulation of undigested sialyl-oligoconjugates in systemic organs including brain. Although patients exhibit neurological symptoms, the underlying neuropathological mechanism remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts with sialidosis and induced the differentiation into neural progenitor cells (NPCs) and neurons. Sialidosis NPCs and neurons mimicked the disease-like phenotypes including reduced neuraminidase activity, accumulation of sialyl-oligoconjugates and lysosomal expansions. Functional analysis also revealed that sialidosis neurons displayed two distinct abnormalities, defective exocytotic glutamate release and augmented α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated Ca2+ influx. These abnormalities were restored by overexpression of the wild-type NEU1 gene, demonstrating causative role of neuraminidase deficiency in functional impairments of disease neurons. Comprehensive proteomics analysis revealed the significant reduction of SNARE proteins and glycolytic enzymes in synaptosomal fraction, with downregulation of ATP production. Bypassing the glycolysis by treatment of pyruvate, which is final metabolite of glycolysis pathway, improved both the synaptsomal ATP production and the exocytotic function. We also found that upregulation of AMPAR and L-type voltage dependent Ca2+ channel (VDCC) subunits in disease neurons, with the restoration of AMPAR-mediated Ca2+ over-load by treatment of antagonists for the AMPAR and L-type VDCC. Our present study provides new insights into both the neuronal pathophysiology and potential therapeutic strategy for sialidosis.
Assuntos
Sinalização do Cálcio/fisiologia , Mucolipidoses/fisiopatologia , Neurônios/patologia , Neurônios/fisiologia , Exocitose/fisiologia , Glicólise/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas , Sinapses/patologia , Sinapses/fisiologiaRESUMO
Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+ Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.
Assuntos
Cálcio/metabolismo , Exocitose , Fusão de Membrana/fisiologia , Multimerização Proteica/fisiologia , Sinaptotagmina I/metabolismo , Animais , Cátions Bivalentes/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Imunofluorescência , Proteínas de Fluorescência Verde/química , Microscopia Eletrônica , Mutação , Células PC12 , Ligação Proteica/fisiologia , Ratos , Proteínas Recombinantes/metabolismo , Sinaptotagmina I/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
SNAREs (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) help intracellular vesicle trafficking and membrane fusion among eukaryotes. They are vital for growth and development of phyto-pathogenic fungi such as Fusarium graminearum which causes Fusarium Head Blight (FHB) of wheat and barley. The SNARE protein Syn8 and its homologues play many roles among different organisms. Here, we have characterized FgSyn8 in F. graminearum as a homologue of Syn8. We have integrated biochemical, microbiological and molecular genetic approaches to investigate the roles of this protein. Our results reveal that FgSyn8 is indispensable for normal vegetative growth, conidiation, conidial morphology and pathogenicity of F. graminearum. Deoxynivalenol (DON) biochemical assay reveals active participation of this protein in DON production of F. graminearum. This has further been confirmed by the production of bulbous structures among the intercalary hyphae. FgSyn8 mutant strain produced defects in perithecia formation which portrays its role in sexual reproduction. In summary, our results support that the SNARE protein FgSyn8 is required for vegetative growth, sexual reproduction, DON production and pathogenicity of F. graminearum.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/metabolismo , Proteínas Q-SNARE/metabolismo , Tricotecenos/metabolismo , Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Hifas/genética , Hifas/metabolismo , Hifas/patogenicidade , Doenças das Plantas/microbiologia , Proteínas Q-SNARE/genética , Triticum/microbiologia , VirulênciaRESUMO
OBJECTIVE: Endothelial cells store VWF (von Willebrand factor) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab effectors, and SNARE (soluble NSF attachment protein receptor) proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study, we investigate the function of syntaxin-3 in VWF secretion. APPROACH AND RESULTS: In human umbilical vein endothelial cells and in blood outgrowth endothelial cells (BOECs) from healthy controls, endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease, carrying a homozygous mutation in STX3(STX3-/-), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3-/- BOECs. VWF multimer analysis showed normal patterns in plasma of the microvillus inclusion disease patient, and media from STX3-/- BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a defect in basal as well as Ca2+- and cAMP-mediated VWF secretion was found in the STX3-/- BOECs. We also show that syntaxin-3 interacts with the WPB-associated SNARE protein VAMP8 (vesicle-associated membrane protein-8). CONCLUSIONS: Our data reveal syntaxin-3 as a novel WPB-associated SNARE protein that controls WPB exocytosis.
Assuntos
Células Endoteliais/metabolismo , Exocitose , Síndromes de Malabsorção/metabolismo , Microvilosidades/patologia , Mucolipidoses/metabolismo , Proteínas Qa-SNARE/metabolismo , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo , Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Células Endoteliais/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Síndromes de Malabsorção/diagnóstico , Síndromes de Malabsorção/genética , Microvilosidades/genética , Microvilosidades/metabolismo , Mucolipidoses/diagnóstico , Mucolipidoses/genética , Mutação , Proteínas Qa-SNARE/genética , Proteínas R-SNARE/metabolismo , Via Secretória , Transdução de Sinais , Corpos de Weibel-Palade/ultraestruturaRESUMO
The cell surface delivery of extracellular matrix (ECM) and integrins is fundamental for cell migration in wound healing and during cancer cell metastasis. This process is not only driven by several soluble NSF attachment protein (SNAP) receptor (SNARE) proteins, which are key players in vesicle transport at the cell surface and intracellular compartments, but is also tightly modulated by cholesterol. Cholesterol-sensitive SNAREs at the cell surface are relatively well characterized, but it is less well understood how altered cholesterol levels in intracellular compartments impact on SNARE localization and function. Recent insights from structural biology, protein chemistry and cell microscopy have suggested that a subset of the SNAREs engaged in exocytic and retrograde pathways dynamically 'sense' cholesterol levels in the Golgi and endosomal membranes. Hence, the transport routes that modulate cellular cholesterol distribution appear to trigger not only a change in the location and functioning of SNAREs at the cell surface but also in endomembranes. In this Commentary, we will discuss how disrupted cholesterol transport through the Golgi and endosomal compartments ultimately controls SNARE-mediated delivery of ECM and integrins to the cell surface and, consequently, cell migration.
Assuntos
Colesterol/metabolismo , Membranas Intracelulares/metabolismo , Proteínas SNARE/metabolismo , Animais , Humanos , Transporte ProteicoRESUMO
Overexposure to manganese (Mn) has been known to disrupt neurotransmitter release in the brain. However, the underlying mechanisms of Mn exposure on neurotransmitter vesicle release are still unclear. The current study investigated whether the protein expression and their interaction of SNARE complex associated proteins were the media between Mn exposure and neurotransmitter vesicle fusion disorders. After the neurons were respectively exposed to Mn (0-200 µM) for 0, 6, 12, 18, 24 h, there were different degrees of cell injury in neurons. According to the results, Mn exposures in subsequent experiments were restricted to concentrations of 100 µM for 0, 6, 12, 18, 24 h. Mn was found to down-regulate the expression of SNAP-25 and up-regulate the expression of VAMP-2 in cultured neurons. Moreover, the interaction of Munc 18 and Syntaxin increased significantly in response to Mn treatment for 18-24h, and the interaction of VAMP-2 and Synaptophysin increased first and then decreased. FM1-43-labeled synaptic vesicles also provided evidence that the treatment with Mn resulted in neurotransmitter vesicle fusion increasing first and then decreasing, which was consistent with the 80 kDa protein levels of SNARE complexes. The findings clearly demonstrated that Mn induced the disorders of neurotransmitter vesicle release via disturbing the protein expression and their interaction of SNARE complex associated proteins. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 705-716, 2017.
Assuntos
Manganês/toxicidade , Fusão de Membrana/efeitos dos fármacos , Proteínas SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Exocitose , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Cultura Primária de Células , Proteínas Qa-SNARE/metabolismo , Ratos Wistar , Transmissão SinápticaRESUMO
PI(4,5)P2participates directly in priming and possibly in fusion steps of Ca²âº-triggered vesicle exocytosis. High concentration nanodomains of PI(4,5)P2reside on the plasma membrane of neuroendocrine cells. A subset of vesicles that co-localize with PI(4,5)P2 domains appear to undergo preferential exocytosis in stimulated cells. PI(4,5)P2directly regulates vesicle exocytosis by recruiting and activating PI(4,5)P2-binding proteins that regulate SNARE protein function including CAPS, Munc13-1/2, synaptotagmin-1, and other C2 domain-containing proteins. These PI(4,5)P2effector proteins are coincidence detectors that engage in multiple interactions at vesicle exocytic sites. The SNARE protein syntaxin-1 also binds to PI(4,5)P2, which promotes clustering, but an activating role for PI(4,5)P2in syntaxin-1 function remains to be fully characterized. Similar principles underlie polarized constitutive vesicle fusion mediated in part by the PI(4,5)P2-binding subunits of the exocyst complex (Sec3, Exo70). Overall, focal vesicle exocytosis occurs at sites landmarked by PI(4,5)P2, which serves to recruit and/or activate multifunctional PI(4,5)P2-binding proteins. This article is part of a Special Issue entitled Phosphoinositides.
Assuntos
Exocitose/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Transdução de Sinais , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , Vesículas Transportadoras/química , Proteínas de Transporte Vesicular/genéticaRESUMO
HPC-1/syntaxin1A (STX1A), a neuronal soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, contributes to neural function in the CNS by regulating transmitter release. Recent studies reported that STX1A is associated with human neuropsychological disorders, such as autism spectrum disorder and attention deficit hyperactivity disorder. Previously, we showed that STX1A null mutant mice (STX1A KO) exhibit neuropsychological abnormalities, such as fear memory deficits, attenuation of latent inhibition, and unusual social behavior. These observations suggested that STX1A may be involved in the neuropsychological basis of these abnormalities. Here, to study the neural basis of social behavior, we analyzed the profile of unusual social behavior in STX1A KO with a social novelty preference test, which is a useful method for quantification of social behavior. Interestingly, the unusual social behavior in STX1A KO was partially rescued by intracerebroventricular administration of oxytocin (OXT). In vivo microdialysis studies revealed that the extracellular OXT concentration in the CNS of STX1A KO was significantly lower compared with wild-type mice. Furthermore, dopamine-induced OXT release was reduced in STX1A KO. These results suggested that STX1A plays an important role in social behavior through regulation of the OXTergic neural system. Dopamine (DA) release is reduced in CNS of syntaxin1A null mutant mice (STX1A KO). Unusual social behavior was observed in STX1A KO. We found that oxytocin (OXT) release, which was stimulated by DA, was reduced and was rescued the unusual social behavior in STX1A KO was rescued by OXT. These results indicated that STX1A plays an important role in promoting social behavior through regulation of DA-induced OXT release in amygdala.
Assuntos
Tonsila do Cerebelo/metabolismo , Ocitocina/metabolismo , Transtornos do Comportamento Social/genética , Transtornos do Comportamento Social/patologia , Sintaxina 1/deficiência , Tonsila do Cerebelo/efeitos dos fármacos , Análise de Variância , Animais , Benzoxazinas/farmacologia , Modelos Animais de Doenças , Dopamina/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Comportamento Exploratório/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdiálise , Ovariectomia , Ocitocina/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Sintaxina 1/genéticaRESUMO
Complexin, a presynaptic protein that avidly binds to assembled SNARE complexes, is widely acknowledged to activate Ca(2+)-triggered exocytosis. In addition, studies of invertebrate complexin mutants and of mouse neurons with a double knockdown (DKD) of complexin-1 and -2 suggested that complexin maintains the readily releasable pool (RRP) of vesicles and clamps spontaneous exocytosis. In contrast, studies of mouse neurons with a double knockout (DKO) of complexin-1 and -2, largely carried out in hippocampal autapses, did not detect changes in the RRP size or in spontaneous exocytosis. To clarify complexin function, we here directly compared in two different preparations, cultured cortical and olfactory bulb neurons, the phenotypes of complexin DKD and DKO neurons. We find that complexin-deficient DKD and DKO neurons invariably exhibit a ~50% decrease in vesicle priming. Moreover, the DKD consistently increased spontaneous exocytosis, but the DKO did so in cortical but not olfactory bulb neurons. Furthermore, the complexin DKD but not the complexin DKO caused a compensatory increase in complexin-3 and -4 mRNA levels; overexpression of complexin-3 but not complexin-1 increased spontaneous exocytosis. Complexin-3 but not complexin-1 contains a C-terminal lipid anchor attaching it to the plasma membrane; addition of a similar lipid anchor to complexin-1 converted complexin-1 from a clamp into an activator of spontaneous exocytosis. Viewed together, our data suggest that complexin generally functions in priming and Ca(2+) triggering of exocytosis, and additionally contributes to the control of spontaneous exocytosis dependent on the developmental history of a neuron and on the subcellular localization of the complexin.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cálcio/metabolismo , Córtex Cerebral/metabolismo , Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Fenótipo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Córtex Cerebral/citologia , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Bulbo Olfatório/citologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a â¼ 100 kDa heavy chain and a â¼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter.