MOLECULAR CONTROL OF REGULATED EXOCYTOSIS

描述(由申请人提供)：在整个系统发育树中，许多分泌细胞类型共享调节的胞吐作用。囊泡和质膜的融合是由包含SNARE蛋白的高度保守的分子机器介导的，其功能通过与其他蛋白质的相互作用进行微调。另一种蛋白质是复合蛋白(CPX)，它是一种~150个氨基酸的蛋白质，有四种异构体(I-IV)，按化学计量比与SNARE复合体结合。络合蛋白作用的分子机制存在争议，有一些证据表明它具有负面作用--“夹住”SNARE复合体并抑制胞吐作用--以及其他一些积极作用的证据，从而导致胞吐作用增强。我们建议对环磷酰胺在小鼠肾上腺嗜铬细胞和神经肌肉连接中的功能进行系统的研究。这两个模型系统，一个是激素分泌细胞，另一个是突触准备，已被广泛用于研究钙依赖的胞吐作用。我们之前提供的数据支持CPXII在肾上腺嗜铬细胞胞吐作用中发挥积极调节作用的假设，为小泡提供基础。我们建议在我们以前工作的基础上，通过在CPXII基因敲除(CPX KO)小鼠的嗜铬细胞中测试以下特定假设：1)CPXII必须与SNARE复合体结合才能促进启动。2)基因敲除细胞的自发胞吐作用不增加。3)CPXII促进囊泡和钙通道的分子偶联。4)CPXII不影响胞吐的钙敏感性。5)CPXII必须被磷酸化以便于启动。我们有初步证据表明，CPXi在神经肌肉接头(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中容易释放的池。我们的合作者尼尔斯·布罗斯(德国马克斯·普朗克研究所)提供了基因敲除小鼠。这个多学科团队包括罗伯特·周博士、陈珍妮博士、拉尔夫·兰根博士和高建平博士。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，或改善分泌产品的生物技术生产。与公共健康相关蛋白质复合素控制着细胞的分泌量。了解它是如何做到这一点的，可能会导致新的战略，以改变分泌率，以对抗疾病状态，或改善分泌产品的生物技术生产。

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.