神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

Ca2+和突触细胞融合

神经突触传递对于神经系统功能的实现具有十分重要的意义,而神经突触传递涉及到突触囊泡膜和突触前膜的融合,3种膜蛋白SNARE特异性识别并形成复合物,从而介导了神经递质的释放。Ca^2 通过其感受器突触结合蛋白而调节了突触细胞的融合过程,也最终影响了神经元的胞吐作用。     

蛋白磷酸化去磷酸化与突触囊泡循环

神经递质合成酶、胞吐相关蛋白、神经递质受体,以及离子通道等蛋白的磷酸化和去磷酸化对神经系统的功能具有重要作用。神经递质的释放往往伴随众多蛋白的磷酸化或去磷酸化过程,包括突触蛋白磷酸化引起突触囊泡从细胞骨架上解离、突触囊泡通过复合体SNARE和Ca2 的介导与突触前膜发生锚靠、融合和神经递质释放,以及以网格蛋白依赖的形式实现突触囊泡从突触前膜上内陷、出芽和缢缩后,从膜上裂解到胞浆中重新形成突触囊泡。因此,蛋白磷酸化和去磷酸化对于神经系统完成神经信号传递具有重要的意义。     

SNARE蛋白及其相关蛋白在精子顶体反应中的作用

精子顶体反应是指精子顶体外膜与精子质膜发生细胞内多点融合的反应,其过程属于特殊的胞吐过程。精子顶体反应是一个复杂严谨的生理过程,不仅需要诱导剂的诱导,还需要多种相关膜融合蛋白的参与。SNARE蛋白及其相关蛋白能够调控哺乳动物细胞内的融合,尤其在精子顶体反应过程中发挥着重要作用。其中SNARE蛋白是核心成分,与其相关蛋白相互作用,共同参与精子顶体反应过程。现主要对SNAREs、Rab、Munc-18、complexin、synaptotagmin和α-SNAP等蛋白质在精子顶体反应中的作用进行概述。     

Syntaxin-1蛋白的多重功能(英文)

Syntaxin-1是特异性地分布在神经细胞突触前质膜上的蛋白。它早期被作为分子量为35 kD的synaptotagmin-1结合蛋白,但很快就被认识到是细胞质膜融合的关键蛋白。Syntaxin-1通过与SNAP25和Synaptobrevin/VAMP蛋白聚合,进而形成被认为是神经突触囊泡融合必要因子的SNARE核心复合体。作为一个多结构域的蛋白,syntaxin-1与多个突触蛋白相互作用,其作用远超出了仅作为SNARE核心复合体中一个蛋白质成员的作用。本文着重介绍了有关syntaxin-1与其它SNARE组份蛋白、munc18蛋白和钙离子通道的相互作用及其功能的最新研究进展。全面揭示syntaxin-1作为SNARE核心复合体成员的功能以及超越这一功能的作用,还有待于对其结构以及与其它突触蛋白相互作用特性的进一步深刻理解。     

Synaptotagmin在神经递质释放过程中的作用

神经突触间递质的释放是神经系统完成其生理功能最重要的生物现象之一。在贮存递质的突触囊泡上存在一些神经细胞所特有的囊泡蛋白,如突触素（synapsin）、synaptobrevin和synaptotagmin等。其中synaptotagmins是膜转运蛋白中的一个家族,它们的特征是含有两个钙结合区：C2A和C2B。到目前为止,在哺乳动物中已经发现了15种synaptotagmin同形物（isoforms）。神经递质释放是由Ca^2＋内流以诱导突触囊泡发生胞吐而引起的,Ca^2＋需与细胞内部的Ca^2＋感受器相结合来协同控制囊泡胞吐释放,SynaptotagminⅠ可能作为快速同步释放的Ca^2＋感受器而发挥作用。现在已知synaptotagminⅠ在胞吐和胞吞两个过程中都扮演重要角色。     

Complexin 蛋白的研究进展

细胞中囊泡释放过程的SNARE假说认为SNARE(SolubleN ethyl maleimidesensitivefusionpro teinattachmentproteinreceptor)复合体是在突触囊泡和突触前膜融合过程中的基本元件。在这个过程中 ,还有许多蛋白质分子参与调控 ,如目前认为作为钙感受器的Synaptotagmin ,调节Syntaxin结合状态的nSec1,与NSF一起调节SNARE复合体解聚的alpha SNAP ,以及能与alpha SNAP竞争结合SNARE复合体的Complexin。本文就Complexin的研究状况作一介绍。     

拟南芥SNARE因子在膜泡运输中的功能

高等植物细胞含有复杂的内膜系统,通过其特有的膜泡运输机制来完成细胞内和细胞间的物质交流。膜泡运输主要包括运输囊泡的出芽、定向移动、拴留和膜融合4个过程。这4个过程受到许多因子的调控,如Coat、SM、Tether、SNARE和Rab蛋白等,其中SNARE因子在膜融合过程中发挥重要功能。SNARE因子是小分子跨膜蛋白,分为定位于运输囊泡上的v-SNARE和定位于靶位膜上的t-SNARE,两类SNARE结合形成SNARE复合体,促进膜融合的发生。SNARE蛋白在调控植物体生长发育以及对外界环境响应等生理过程中起重要作用。该文对模式植物拟南芥（Arabidopsis thaliana）SNARE因子的最新细胞内定位和功能分析等研究进展进行了概述。     

驱动细胞膜融合的发动机——SNAREs蛋白

主要介绍了SNARE蛋在在膜融合过程中的核心驱动作用。自1980s后期SNARE蛋白被发现以后,SNAREs就作为细胞膜融合蛋白复合体的关键组分而获得普遍认同。尽管不同SNARE蛋白的基因组成序列存在差异,但它们的功能在进化上似乎是保守的,均涉及细胞生长、膜修复、细胞骨架动力学和突触传递等许多方面的细胞膜融合活动。从这些发现可以看到,膜融合机制展示了SNARE蛋白复合体作为一种超级微型机器工作的迷人画卷。     

神经突触化学传递中胞吐作用的分子机制

目　　录一、参与胞吐作用的相关蛋白　(一)突触囊泡膜蛋白　(二)突触前膜有关蛋白　(三)胞液可溶性蛋白质　(四)其他蛋白质二、突触囊泡泊靠和融合的分子机制突触传递是神经系统实现其功能的最基本方式。详细阐述突触传递的机制对人们理解神经信息传递的特异性、行为和可塑性以及学习和记忆等都是至关重要的。近年来,随着分子生物学的发展,在分子水平阐明突触传递的机制才有可能。神经末梢的突触前部分通常含有两类囊泡:一是透明的较小囊泡,含有乙酰胆碱、儿茶酚胺等经典递质;另一类是有致密核心的较大囊泡,含有神经肽类物质。迄今研究较深…     

Action of complexin on SNARE complex

Calcium-dependent synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: synaptobrevin/vesicle-associated membrane protein in the vesicular membrane and syntaxin and SNAP-25 in the presynaptic membrane. The SNAREs form a thermodynamically stable complex that is believed to drive fusion of vesicular and presynaptic membranes. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a positive regulator of synaptic vesicle exocytosis. Complexin binds selectively to the neuronal SNARE complex, but how this promotes exocytosis remains unknown. Here we used purified full-length and truncated SNARE proteins and a gel shift assay to show that the action of complexin on SNARE complex depends strictly on the transmembrane regions of syntaxin and synaptobrevin. By means of a preparative immunoaffinity procedure to achieve total extraction of SNARE complex from brain, we demonstrated that complexin is the only neuronal protein that tightly associates with it. Our data indicated that, in the presence of complexin, the neuronal SNARE proteins assemble directly into a complex in which the transmembrane regions interact. We propose that complexin facilitates neuronal exocytosis by promoting interaction between the complementary syntaxin and synaptobrevin transmembrane regions that reside in opposing membranes prior to fusion.     

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis.     

Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis

The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation.     

Accessory α-Helix of Complexin I Can Displace VAMP2 Locally in the Complexin-SNARE Quaternary Complex

The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory α-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory α-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory α-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.     

Role of lipid microdomains in P/Q-type calcium channel (Cav2.1) clustering and function in presynaptic membranes

Lipid microdomains can selectively include or exclude proteins and may be important in a variety of functions such as protein sorting, cell signaling, and synaptic transmission. The present study demonstrates that two different voltage-gated calcium channels, which both interact with soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins but have distinct subcellular distributions and roles in synaptic transmission, are differently distributed in lipid microdomains; presynaptic P/Q (Cav2.1) but not Lc (Cav1.2) calcium channel subtypes are mainly accumulated in detergent-insoluble complexes. The immunoisolation of multiprotein complexes from detergent-insoluble or detergent-soluble fractions shows that the alpha1A subunits of Cav2.1 colocalize and interact with SNARE complexes in lipid microdomains. The altered organization of these microdomains caused by saponin and methyl-beta-cyclodextrin treatment largely impairs the buoyancy and distribution of Cav2.1 channels and SNAREs in flotation gradients. On the other hand, cholesterol reloading partially reverses the drug effects. Methyl-beta-cyclodextrin treatment alters the colocalization of Cav2.1 with the proteins of the exocytic machinery and also impairs calcium influx in nerve terminals. These results show that lipid microdomains in presynaptic terminals are important in organizing membrane sites specialized for synaptic vesicle exocytosis. The cholesterol-enriched microdomains contribute to optimizing the compartmentalization of exocytic machinery and the calcium influx that triggers synaptic vesicle exocytosis.     

The t-SNARE syntaxin is sufficient for spontaneous fusion of synaptic vesicles to planar membranes

Vesicular trafficking and exocytosis are directed by the complementary interaction of membrane proteins that together form the SNARE complex. This complex is composed of proteins in the vesicle membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Here we show that modified synaptic vesicles (mSV), containing v-SNAREs, spontaneously fuse to planar membranes containing the t-SNARE, syntaxin 1A. Fusion was Ca(2+)-independent and did not occur with vesicles lacking v-SNAREs. Therefore, syntaxin alone forms a functional fusion complex with v-SNAREs. Our functional fusion assay uses synaptic vesicles that are modified, so each fusion event results in an observable transient current. The mSV do not fuse with protein-free membranes. Additionally, artificial vesicles lacking v-SNAREs do not fuse with membranes containing syntaxin. This technique can be adapted to measure fusion in other SNARE systems and should enable the identification of proteins critical to vesicle-membrane fusion. This will further our understanding of exocytosis and may improve targeting and delivery of therapeutic agents packaged in vesicles.     

Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes

The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex.     

Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone-shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

Calcium-dependent dissociation of synaptotagmin from synaptic SNARE complexes

The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+-dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+-dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30-300 microM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+ triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.