MOLECULAR CONTROL OF REGULATED EXOCYTOSIS

描述（由申请人提供）：在系统发育树中，许多分泌细胞类型都有受调节的胞吐作用。囊泡和质膜的融合由高度保守的分子机器介导，所述分子机器包括SNARE蛋白，其功能通过与其他蛋白的相互作用而微调。这些其他蛋白质之一是复合蛋白（CPX），一种约150个氨基酸的蛋白质，具有四种亚型（I-IV），化学计量结合SNARE复合物。复合蛋白作用的分子机制是有争议的，有一些证据表明它具有负面作用-“夹紧”SNARE复合物并抑制胞吐作用-还有其他证据表明它具有正面作用，导致胞吐作用增强。我们提出了一个系统的研究CPX在小鼠肾上腺嗜铬细胞和神经肌肉接头的功能。这两个模型系统，一个是突触分泌细胞，一个是突触制备，已被广泛用于研究钙依赖性胞吐。我们以前提出的数据支持的假设，CPXII在肾上腺嗜铬细胞胞吐中起着积极的调节作用，服务于总理囊泡。我们建议在我们以前的工作的基础上，通过在来自CPXII敲除（CPX KO）小鼠的嗜铬细胞中测试以下特定假设：1）CPXII必须结合SNARE复合物以促进引发。2)3）CPXII促进囊泡和钙通道的分子偶联。4)CPXII不影响胞吐的Ca敏感性。5)CPXII必须被磷酸化以促进引发。我们有初步证据表明，CPXI也在神经肌肉接头（NMJ）中发挥积极作用。我们将在小鼠CPXI KO模型中测试以下假设：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（Max Planck Institute，德国）提供。多学科团队包括Robert Chow博士，Jeannie Chen博士，Ralf Langen博士和Kien Ping Ko博士。了解CPX功能可能会导致新的策略，以改变分泌速率，以对抗疾病状态或改善分泌产物的生物技术生产。公共卫生相关性蛋白复合物控制细胞分泌的量。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速率，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.