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/总结 细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），由囊泡膜蛋白突触短蛋白 II（syb2、VAMP2 或 v- SNARE），以及靶膜蛋白突触蛋白（syx）和 SNAP-25（统称为 t-SNARE）。 当囊泡接近目标膜时，v-SNARE 和 t-SNARE 以拉链状方式组装形成 囊泡融合机，能够在融合后释放分子货物，组装成的 SNARE 复合体。 被由 NSF 和 α-SNAP 组成的回收机器分解成单独的蛋白质，用于下一步 由于囊泡融合是一个普遍存在的过程，并且对于细胞间的通讯至关重要，因此突变。 在过去的几十年里，广泛的研究表明 SNARE 会导致许多神经元和代谢疾病。 已经揭示了 SNARE 循环中各个步骤的详细信息，但是其分子机制。 顺序每个步骤的顺序仍不清楚。在这里，我们迫切需要探讨这种关系。 寻求了解 SNARE 在体外和活细胞中的组装和分解 在目标 1 中，我们将开发。 氢氘交换质谱 (HDX-MS) 方法分析 SNARE 复合物的形成 我们已经证明了序列覆盖度。 单个 SNARE 的 MS 后收集的蛋白质包含其 SNARE 基序，支持后续的 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。 在目标 2 中，对复合物进行组装检查，然后引入回收蛋白来评估复合物的分解。 我们的目标是了解囊泡胞吐作用期间 SNARE 循环的时空调节 开发用于 SNARE 组装和拆卸的基因编码的基于强度的构象传感器 (icsenSNARE)。使用重组分析，我们确定了第一个设计，icsenSNARE000，它显示了 SNARE 复合体组装后荧光增加 1.5 倍，并且可以通过拆卸来逆转 最后，我们计划在活细胞中使用 icsenSNARE 来揭示如何时空调节。 SNARE 循环与囊泡融合相结合，拟议的研究将推进机制。 对囊泡胞吐作用的理解，开发一种研究 SNARE 组装和拆卸的新方法， 并为 SNARE 蛋白生成新的探针，这项工作的成果有可能有助于研究。 改善人类健康的 SNARE 相关病理。