【作者】 吴政星；

【导师】 徐涛； 周专； 瞿安连； Jens Rettig；

【作者基本信息】 华中科技大学 ， 生物物理， 2005， 博士

【摘要】 细胞分泌功能通过囊泡胞吐过程来完成。囊泡胞吐过程是一个涉及许多蛋白质、脂质分子等物质并有多个细胞器参与的复杂过程,由囊泡的生成、成熟、募集、拴系和锚定、囊泡的激活、最后囊泡与细胞膜融合及其内含物通过融合孔释放至胞外等步聚组成。SNARE 蛋白核心复合体是膜融合的分子基础,为膜融合提供能量。Ca²⁺是调节性分泌的触发信号,synaptotagmin 是囊泡膜融合过程的Ca²⁺感受蛋白,它与SNARE 蛋白一起组成膜融合的最小分子构件。细胞胞吐活动受到一些调节蛋白,如α-SNAP、NSF、Munc18/nSec1、Munc13、Rab 蛋白及效应物（rabphilin、Rim 和Noc2 等） 和钙结合蛋白（如calmodulin 和CAPS） 等的调控。囊泡的“激活”是囊泡获得（与靶膜） 融合能力的过程。它是低浓度钙（数百纳摩尔） 和磷脂酰肌醇代谢产物依赖性的耗能过程,需要ATP 水解提供自由能。囊泡激活过程受到Ca2+、二酰甘油、Munc13、蛋白激酶A 和蛋白激酶C、SV2A、NSF 以及Snapin 等信号分子和蛋白质的调控。在Ca2+依赖的调节性分泌活动中,囊泡的激活过程是限速性步骤。激活囊泡的数目代表可释放囊泡库的大小。可释放囊泡库的大小取决于未激活囊泡库的大小以及激活、去激活和消耗（即囊泡融合） 的速率。囊泡激活的分子机制目前还不是十分清楚,可能是SNARE 蛋白聚合形成的trans （异位）-SNARE 复合体或trans （同位）-SNARE 蛋白复合体与钙离子感受蛋白synaptotagmin 相互作用形成的复合物赋予了囊泡与靶膜融合的能力。Snapin 是新近发现的与SNAP-25 结合的小分子（15 kD） 蛋白,为普遍表达的胞浆蛋白。Snapin 与SNAP-25 结合并促进syanaptotagmin 与SNARE 复合体的结合。Snapin 通过超螺旋与SNAP-25、SNAP-23 和非神经元性的SNAP-25 同功型结合。也可能与RGS7、腺苷酸环化酶Ⅵ、EBAG9 产物和受体酪氨酸激酶MET 相互作用。在背根神经节细胞,Snapin 和synaptotagmin Ⅸ均与vanilloid 受体-1 （vanilloid receptor-1, TRPV1） 发生相互作用,调节PKA 介导的TRPV1 通道向细胞膜的募集过程,其调节作用可能通过调节SNARE 蛋白复合体依赖的胞吐过程来实现。Snapin也是溶酶体相关细胞器生物发生蛋白复合物（Biogenesis of Lysosome-related Organelles Comlex-1, BLOC-1） 的亚单位,因而也可能参与对细胞胞吞活动的调节。更多还原

【Abstract】 Neurotransmitters and hormones are released through exocytosis of vesicles. The vesicular exocytosis consists of complex serial events of trafficking, tethering, docking, priming, fusing with cell membrane. SNARE protein complex is essential molecular machinery in most, if not all, exocytosis events studied so far, and supply the energy for vesicle fusion with their target membrane. Synaptotagmins are Ca2+ sensors in rgulative exocytosis molecular machinery. Its association with SNARE complex is essential for membrane fusion, and the complex of synaptotagmin with SNARE proteins is basic molecular machinery for vesicle fusion. Exocytosis is controlled and regulated strickly by regulative proteins, such as α-SNAP, NSF, Munc18/nSec1, Munc13, Rab, Rab effectors （including rabphilin, Rim, and Noc2）, and calcium-binding proteins. Priming of viscle is a biochemical process which prepares membranes for fusion. It is a speed-limited step in exocytosis, and a low level calcium-, ATP-, and phosphatidyl inositol metabolites-depended process. The primed vesicles constitute releasable pool （RP）. The size of releasable pool is depended on the size of un-primed pool, and rate of pimimg, un-priming, and exocytosis. The priming step maybe corresponds to the assembly of the SNARE complex, in which the vesicle-associated v-SNARE protein synaptobrevin （VAMP） interacts with two plasma membrane-associated t-SNARE proteins, SNAP-25 and syntaxin, to form a stable SNARE complex. The priming process is most likely aided by the action of Munc13. Furthermore, maturation into a release-ready vesicle requires synaptotagmin. Synaptotagmins, integral Ca²⁺-binding proteins and Ca2+ sensors of vesicle membrane, provide Ca2+-dependent regulation of the fusion machinery. However, the molecular mechanisms underlying regulation of the structural and functional coupling of the calcium sensor with the SNARE-based fusion machinery during priming and maturation remains unclear. Snapin was first identified as a SNAP-25 binding protein that associates with the SNARE complex and enhances the association of synaptotagmin with the SNARE complex. Snapin is a ubiquitously expressed soluble protein that is present in both cytosol and peripheral membrane-associated fractions. In addition to binding to SNAP-25, Snapin can form a complex with SNAP-23, the nonneuronal isoform of SNAP-25. Both Snapin and synaptotagmin IX interact in vitro and in vivo with the vanilloid receptor-1 （TRPV1） and consequently modulate PKA-mediated recruitment of the functional TRPV1 channel to the plasma membrane in dorsal root ganglion neurons via SNARE-dependent exocytosis. Snapin was also identified as a subunit of biogenesis of lysosome-related organelles Complex-1 （BLOC-1）, suggesting its potential role in the endocytic pathway. Snapin is an important target of PKA, and phosphorylation modulates the efficacy of Snapin action on release. In the present study, we generated null mutations in the mouse snapin gene via homologous recombination to abolished snapin expression in mice, and functionally evaluated Snapin’s role in neuroexocytosis by state-of-the-art biochemical and biophysical techniques. We found that the absence of Snapin expression did not affect the expression level of a large variety of proteins involved in synaptic vesicle exocytosis, indicates that snapin deletion does not result in any apparent compensatory changes in the expression of known proteins that are required for presynaptic function. Furthermore, our biochemical data demonstrate that the formation of the SNARE complex is not affected by deletion of snapin. We detected a marked reduction （34 ±3%, from 11 littermates, p<0.01） in the association of SNAP-25 and synaptotagmin in brain homogenates of snapin KO mice when compared to wild-type littermates. Using flash photolysis of caged calcium as a fast stimulus and high-temporal-resolution measurement of membrane capacitance and catecholamine release, we found that deletion of snapin in chromaffin cells led to a 45 % reduction of calcium-dependent burst exocytosis, while the sustained release phase remained unaffected. Furthermore, reintroduction of Snapin into the mutant cells could fully rescue this inhibitory effect within hours, indicating the specificity of the observed burst reduction upon snapin deletion. Our electron microscopy analysis demonstrated no significant differences between these groups either in the distribution of the vesicles relative to the plasma membrane or in the total number of vesicles per cell section, suggesting that Snapin is not directly involved in morphological docking of LDCVs in chromaffin cells. Furthermore, our calcium ramp experiments showed that neither the Ca2+ sensitivity nor the Ca2+ cooperativity of secretion is changed in the absence of Snapin, excluding the possibility that Snapinis directly involved in the final Ca2+-triggered fusion reaction. Thus, our biochemical and electrophysiological studies using snapin knockout mice indicate that Snapin plays a critical role in modulating neurosecretion, likely by stabilizing synaptotagmin-SNARE complex, decreasing the rate of de-priming, and thus stabilizing the release-ready vesicles. We presented a short introduction of biophysics techniques frequently used for study of exocytosis. We investigated the docking, undocking, and fusing with plasma membrane process of vesicle in PC12 cells by total internal reflection fluorescence microscopy, our data strongly support the original hypothesis that vesicle docking was a reversible process.更多还原