MOLECULAR CONTROL OF REGULATED EXOCYTOSIS

描述（由申请人提供）：调节的胞吐作用在系统发育树上的许多分泌细胞类型中共享。囊泡和质膜的融合是由一个高度保守的分子机器介导的，其中包括SNARE蛋白，其功能通过与其他蛋白的相互作用而得到微调。其中一种蛋白质是络合蛋白（CPX），这是一种约150个氨基酸的蛋白质，具有四种异构体（I-IV），它以化学计量的方式与SNARE复合物结合。络合蛋白作用的分子机制是有争议的，一些证据表明其具有负面作用-“夹紧”SNARE复合物并抑制胞吐-而另一些证据表明其具有积极作用，导致胞吐增强。我们建议系统研究CPX在小鼠肾上腺染色质细胞和神经肌肉连接处的功能。这两个模型系统，一个是激素分泌细胞，一个是突触准备，已被广泛用于研究钙依赖性胞外分泌。我们之前提供的数据支持CPXII在肾上腺染色质细胞的胞外分泌中起积极调节作用的假设，为初始囊泡服务。我们建议在之前工作的基础上，在CPXII敲除（CPX KO）小鼠的染色质细胞中测试以下特定假设：1)CPXII必须结合SNARE复合体以促进启动。2)敲除细胞不增加自发胞外分泌3)CPXII促进囊泡和钙通道的分子偶联。4) CPXII不影响胞吐对Ca的敏感性。5) CPXII必须被磷酸化才能促进启动。我们有初步的证据表明CPXI在神经肌肉连接（NMJ）中也起着积极的作用。我们将在小鼠CPXI KO模型中验证以下假设：6)CPXI调节小鼠NMJ中的易释放池。敲除小鼠由我们的合作者Nils Brose（德国马克斯普朗克研究所）提供。多学科团队包括Robert Chow医生、Jeannie Chen医生、Ralf Langen医生和柯建平医生。了解CPX的功能可能会导致新的策略来改变分泌率，以对抗疾病状态或改善分泌产物的生物技术生产。与公共卫生相关的蛋白质复合物控制细胞的分泌量。了解它是如何做到这一点的，可能会导致新的策略来改变分泌率，以对抗疾病状态或改善分泌产物的生物技术生产。

DESCRIPTION (provided by applicant): Regulated exocytosis is shared among many secretory cell types across the phylogenetic tree. Fusion of vesicle and plasma membranes is mediated by a highly conserved molecular machine comprising the SNARE proteins, whose function is fine- tuned through interaction with other proteins. One of these other proteins is complexin (CPX), a ~150-amino acid protein having four isoforms (I-IV), that binds stoichiometrically to the SNARE complex. The molecular mechanism of complexin action is controversial, with some evidence for a negative role - "clamping" the SNARE complex and inhibiting exocytosis - and other evidence for a positive role, leading to enhanced exocytosis. We propose a systematic study of the function of CPX in mouse adrenal chromaffin cells and neuromuscular junction. These two model systems, one a hormone-secreting cell and one a synaptic preparation, have been used extensively to study calcium-dependent exocytosis. We have previously presented data supporting the hypothesis that CPXII plays a positive regulatory role in exocytosis in adrenal chromaffin cells, serving to prime vesicles. We propose to build on our previous work by testing the following specific hypotheses in chromaffin cells derived from CPXII knockout (CPX KO) mice: 1) CPXII must bind to the SNARE complex in order to facilitate priming. 2) Spontaneous exocytosis is not increased in knockout cells 3) CPXII facilitates molecular coupling of vesicles and calcium channels. 4) CPXII does not affect the Ca sensitivity of exocytosis. 5) CPXII must be phosphorylated to facilitate priming. We have preliminary evidence that CPXI also plays a positive role in the neuromuscular junction (NMJ). We will test the following hypothesis in a mouse CPXI KO model: 6) CPXI regulates the readily releasable pool in mouse NMJ. Knockout mice have been provided by our collaborator Nils Brose (Max Planck Institute, Germany). The multidisciplinary team includes Dr. Robert Chow, Dr. Jeannie Chen, Dr. Ralf Langen, and Dr. Chien-Ping Ko. Understanding CPX function could lead to new strategies to alter secretion rates to combat disease states or to improve biotechnological production of secreted products. PUBLIC HEALTH RELEVANCE The protein complexin controls how much cells can secrete. Understanding how it does this could lead to new strategies to alter secretion rates to combat disease states or to improve biotechnological production of secreted products.