Probing SNARE assembly and disassembly in vitro and in live cells

在体外和活细胞中探测 SNARE 组装和拆卸

• 批准号: 10679644
• 负责人: Sarah Rose Dallo
• 金额: $ 3.47万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-14 至 2026-04-13
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679644
• 关键词: Achievement; Address; Area; Biological Assay; Biology; Cell Communication; Cells; Color; Complex; Coupled; Coupling; Deuterium; Disease; Engineering; Event; Exocytosis; Fluorescence; Goals; Green Fluorescent Proteins; Health; Human; Hydrogen; In Vitro; Individual; Lead; Link; Lipids; Maps; Mass Spectrum Analysis; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Metabolic Diseases; Molecular; Molecular Conformation; Monitor; Mutation; N-ethylmaleimide-sensitive protein; Neurons; Pathology; Pathway interactions; Performance; Physiologic Monitoring; Physiological; Physiological Processes; Positioning Attribute; Process; Proteins; Recycling; Regulation; Reporting; Resolution; Role; SNAP receptor; Solvents; Techniques; Technology; Testing; Time; Vesicle; Work; alpha-SNAP; biophysical analysis; conformational conversion; design; experimental study; genetic regulatory protein; improved; in vivo; insight; live cell imaging; nanodisk; neurotransmitter release; novel; novel strategies; prevent; protein reconstitution; receptor; reconstitution; sensor; soluble NSF attachment protein; spatiotemporal; success; syntaxin; tool; trafficking; vesicle-associated membrane protein; vesicular release

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.

项目摘要/摘要 细胞内囊泡融合主要由SNARE（可溶性N-乙基甲基酰亚胺敏感因子）介导 附着蛋白受体），由囊泡膜蛋白突触II（SYB2，VAMP2或V-组成） SNARE）和靶膜蛋白语法（SYX）和SNAP-25（统称为T-Snares）。作为 囊泡接近靶膜，以拉链方式组装的V-SNARE和T-SNARES形成 囊泡融合机，使得分子货物的释放。融合后，组装的军鼓复合物 由NSF和α-SNAP组成的回收机制将其分解为单个蛋白 融合弹。由于囊泡融合是一个普遍的过程，对于细胞 - 细胞通信至关重要，所以突变 在网罗中导致许多神经元和代谢疾病。在过去的几十年中，广泛的研究 在军鼓周期中揭示了各个步骤的细节。但是，分子机制 命令每个步骤的顺序尚不清楚。存在紧迫的需求来探究这种关系。在这里，我们 寻求在体外和活细胞中了解网罗的组装和拆卸。在AIM 1中，我们将发展 一种氢氘交换质量光谱法（HDX-MS）方法，用于分析SNARE复合物的形成 和体外变形在物理相关条件下。我们已经证明了序列覆盖范围 在单个圈圈蛋白的MS之后收集 HDX-MS将揭示其机制的细节。我们将使用这种方法来研究单圈和三元 复合物检查组装，然后引入回收蛋白来评估复杂的拆卸。在AIM 2中， 我们的目的是了解囊泡胞吐作用过程中的空间时间调节。 开发一个基于强度的一般编码的基于强度的构象传感器，用于蜗杆组装和拆卸 （Icsensnare）。使用重构的Assas，我们确定了第一个设计，Icsensnare000，该设计显示 军鼓复合物组装后的1.5倍荧光增加，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，以揭示如何进行空间时间调节 圈圈周期与囊泡融合耦合。拟议的研究将共同​​提高机理 对囊泡胞吐作用的理解，开发了一种新方法来研究军鼓组装和拆卸， 并生成新的探针对圈圈蛋白。这项工作的实现有可能帮助学习 与圈圈相关的病理学改善人类健康。