Regulation of exocytosis studies with flipped SNAREs

描述（由申请人提供）：最近，我们建立了一种新的膜融合系统，在该系统中，胞吐所需的“翻转”v-和t-SNAREs在两个细胞群体的表面表达，驱动细胞间融合，从而证明SNAREs足以融合生物膜。在这里，我们建议利用这一发展来询问有关依赖于snare的融合的关键机制问题，特别是关于调节蛋白（已知具有生理功能）如何在分子水平上单独或协同作用来控制胞吸的问题。对这些问题的严谨研究需要一种简化的系统，在这种系统中，蛋白质组成和拓扑结构可以在生物相关的环境中进行控制，以便在将每个调节器（单独或组合）添加到SNAREs的核心融合机制中时，可以评估其动力学效应。调节蛋白将通过加入信号序列翻转，并与细胞表面的v-或t-SNAREs共表达，或作为纯重组蛋白添加到培养基中。融合动力学和过渡状态将使用最初为病毒融合蛋白开发的既定技术进行测量。我们将首先研究一组已知在整个细胞和生物体中调节胞外分泌的蛋白质：synaptotagmins， Sec/Munc蛋白，complexins，和tomosyns，以及NSF和SNAP。虽然它们的一般生理重要性是明确的，但由于缺乏对最小功能融合系统的机制研究，其作用的分子机制以及它们之间的功能相互作用尚不清楚。我们的长期愿景是不断努力，一个蛋白质一个蛋白质，直到我们能够重建基本特性和调节胞吐作用的微调。胞吐和相关过程的失衡是糖尿病和肥胖的主要形式，并且可能在学习、情绪和炎症性疾病中起重要作用。了解监管机构的运作方式，可能会确定新的干预目标。

DESCRIPTION (provided by applicant): Recently, we established a novel membrane fusion system in which "flipped" v- and t-SNAREs needed in exocytosis are expressed on the surface of two cell populations, driving cell-cell fusion thereby demonstrating that SNAREs are sufficient to fuse biological membranes. Here, we propose to capitalize on this development to ask key mechanistic questions about SNARE-dependent fusion, especially questions concerning precisely how regulatory proteins - known to function physiologically - act alone and in concert to control excocytosis at the molecular level. Rigorous studies of these questions require a simplified system of this kind in which protein composition and topology can be controlled in a biologically-relevant environment so that the kinetic effect of each regulator can be assessed when it is added (alone or in combination) to the core fusion machinery of SNAREs. Regulatory proteins will be flipped by adding signal sequences and co-expressed with v- or t-SNAREs on the surface of cells, or added as pure recombinant proteins to the medium. Fusion kinetics and transition states will be measured using established techniques originally developed for viral fusion proteins. We will initially study a well-established group of proteins known to regulate exocytosis in whole cells and organisms: synaptotagmins, Sec/Munc proteins, complexins, and tomosyns, as well as NSF and SNAP. While their general physiologic importance is clear, the molecular mechanism of action - and functional interactions among themselves - are not clear due to the dearth of mechanistic studies in minimal functional fusion systems. The long-term vision is to work our way up - protein by protein - until we can reconstitute the basic properties and fine-tuning of regulated exocytosis. Imbalances in exocytosis and related processes underly major forms of diabetes and obesity, and are likely important in learning, mood, and inflammatory disorders. Knowledge of how the regulators work will likely identify novel targets for intervention.