Probing SNARE assembly and disassembly in vitro and in live cells

项目摘要/总结 胞内囊泡融合主要由SNARE（可溶性N-乙基马来酰亚胺敏感因子）介导 附着蛋白受体），其由囊泡膜蛋白小突触蛋白II（syb 2、VAMP 2或v- SNARE）和靶膜蛋白突触融合蛋白（syx）和SNAP-25（统称为t-SNARE）。为 当囊泡接近靶膜时，v-SNARE和t-SNARE以拉链样的方式组装， 一个囊泡融合机，能够释放分子货物。融合后，组装好的SNARE复合物 由NSF和α-SNAP组成的回收机器将其分解为单独的蛋白质， 一轮融合。由于囊泡融合是一个普遍存在的过程，并且对于细胞-细胞通讯至关重要，因此，突变 在SNARE中导致许多神经元和代谢疾病。在过去的几十年里，广泛的研究 揭示了陷阱周期中各个步骤的细节。然而， 每个步骤的顺序仍然不清楚。迫切需要探讨这种关系。这里我们 试图了解SNARE在体外和活细胞中的组装和拆卸。在目标1中，我们将开发 分析SNARE复合物形成的氢氘交换质谱法（HDX-MS） 和在生理相关条件下的体外变形。我们已经表明，序列覆盖率 在MS后收集的单个SNARE蛋白包括它们的SNARE基序，支持随后的SNARE序列。 HDX-MS将揭示其机制的细节。我们将使用这种方法来研究SNARE二元和三元 复杂的检查组装，然后引入回收蛋白质来评估复杂的拆卸。在目标2中， 我们的目标是了解时空调节的SNARE循环在囊泡胞吐作用， 开发用于SNARE组装和拆卸的遗传编码的基于强度的构象传感器 （icsenSNARE）.使用重构的测定，我们已经鉴定了第一种设计，icsenSNARE 000，其显示出 1.5-当SNARE复合物组装时，荧光增加倍数，并且可以通过拆卸来逆转 机械.最后，我们计划在活细胞中使用icsenSNARE来揭示细胞周期的时空调控。 SNARE循环与囊泡融合偶联。总之，拟议的研究将推进机械论 理解囊泡胞吐作用，为研究SNARE组装和拆卸开发新的方法， 并产生一个新的SNARE蛋白探针。这项工作的成果有可能有助于研究 SNARE相关的病理学，以改善人类健康。

PROJECT ABSTRACT/SUMMARY Intracellular vesicle fusion is primarily mediated by SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), which consist of vesicle membrane protein synaptobrevin II (syb2, VAMP2 or v- SNARE), and target membrane proteins syntaxin (syx) and SNAP-25 (collectively called t-SNAREs). As the vesicle approaches the target membrane, the v-SNARE and t-SNAREs assemble in a zipper-like manner to form a vesicle fusion machine, enabling release of the molecular cargo. After fusion, the assembled SNARE complex is disassembled by the recycling machinery, composed of NSF and α-SNAP, into individual proteins for the next round of fusion. Since vesicle fusion is a ubiquitous process and is critical for cell-cell communication, mutations in SNAREs lead to numerous neuronal and metabolic diseases. Over the past few decades, extensive studies have revealed great details of individual steps in the SNARE cycle. However, the molecular mechanism that orders the sequence of each step remains unclear. A pressing need exists to probe this relationship. Here, we seek to understand the assembly and disassembly of SNAREs in vitro and in live cells. In Aim 1, we will develop a hydrogen deuterium exchange-mass spectrometry (HDX-MS) approach to analyze SNARE complex formation and deformation in vitro under physiologically relevant conditions. We have shown that the sequence coverage collected after MS of the individual SNARE proteins includes their SNARE motifs, supporting that subsequent HDX-MS will reveal details into their mechanism. We will use this approach to study SNARE binary and ternary complex to examine assembly, then introduce the recycling proteins to assess complex disassembly. In Aim 2, our objective is to understand the spatiotemporal regulation of the SNARE cycle during vesicle exocytosis by developing a genetically encoded intensity-based conformational sensor for SNAREs assembly and disassembly (icsenSNARE). Using reconstituted assays, we have identified the first design, icsenSNARE000 that showed a 1.5-fold fluorescence increase upon assembly of the SNARE complex and could be reversed by the disassembly machinery. Finally, we plan to employ icsenSNARE in live cells to reveal how the spatiotemporal regulation of the SNARE cycle is coupled to vesicle fusion. Together, the proposed study will advance mechanistic understandings of vesicle exocytosis, develop a new approach for studying SNARE assembly and disassembly, and generate a new probe for the SNARE proteins. Achievement of this work has the potential to help study SNARE-related pathologies to improve human health.