Mechanistic Principles of SNARE Disassembly in Neurotransmitter Release

神经递质释放中 SNARE 分解的机制原理

• 批准号: 10824093
• 负责人: Yousuf A Khan
• 金额: $ 4.09万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-11 至 2025-12-10
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10824093
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Ablation; Adaptor Signaling Protein; Amino Acids; Architecture; Behavior; Biochemical; Biological Assay; Biological Models; Biophysical Process; Biophysics; Brain; Cell membrane; Cell surface; Cells; Cognition; Complex; Cryoelectron Microscopy; Data; Development; Disease; Dissection; Electrophysiology (science); Enzymes; Fluorescence; Goals; Growth; Health; Hippocampus; Human; In Vitro; Individual; Infection; Link; Measures; Mediating; Membrane; Membrane Fusion; Mental disorders; Modeling; Molecular; Molecular Conformation; Monitor; Motivation; Mus; Mutagenesis; Mutagens; Mutation; N-ethylmaleimide-sensitive protein; Neurons; Neurotransmitters; Orthologous Gene; Perception; Play; Presynaptic Terminals; Process; Proteins; Recycling; Resolution; Role; SNAP receptor; Scientist; Signal Transduction; Statistical Data Interpretation; Synaptic Membranes; Synaptic Transmission; Synaptic Vesicles; System; Testing; Therapeutic; Time; Vesicle; Viral Packaging; Yeasts; biochemical tools; experimental study; fitness; high throughput screening; in vivo; machine learning framework; machine learning method; millisecond; mutant; neurotransmission; neurotransmitter release; next generation sequencing; patch clamp; presynaptic; presynaptic neurons; promoter; protein protein interaction; receptor; reconstruction; soluble NSF attachment protein; unsupervised learning; vector

PROJECT SUMMARY/ABSTRACT Complex behaviors of the brain, such as cognition, perception, motivation, and mental illness, still remain difficult to explain. To truly understand these processes, it is necessary to understand the basic mechanisms that underly them. Synaptic transmission, the release of neurotransmitters from the presynaptic neuron upon membrane fusion, relies on SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors). SNAREs on the neurotransmitter containing vesicles form a stable, trans SNARE complex with SNAREs on the presynaptic membrane. Once signaled, these SNAREs twist together to provide the energy necessary for membrane fusion. This cis SNARE complex, now a highly stable four helix bundle on one membrane, must be disassembled and recycled to allow further rounds of fusion. Without a pool of fusogenic SNAREs, synaptic transmission would cease. cis SNARE disassembly is accomplished by NSF (N- ethylmaleimide sensitive factor) and adaptor proteins called SNAPs (soluble NSF attachment proteins). Together, the three components form a 20S complex, in which NSF, upon ATP hydrolysis, disassembles SNARE complex and maintains a pool of fusogenic SNAREs. Yet the key dynamical processes and principles of this explosive disassembly step remain unknown. The overall goal of this project is to elucidate the fundamental mechanisms of synaptic transmission by understanding SNARE disassembly. To uncover the principles of SNARE disassembly, both NSF and its yeast ortholog Sec18 will be examined. Studying the dynamics of NSF in its neuronal context has proven difficult due to the complexity of the presynaptic system and the inability to investigate more than a handful of mutants at a time. Studying Sec18 and the yeast 20S (Y20S), in coordination with the neuronal 20S, will enable the use of a wide variety of molecular and biochemical tools that will allow for the dissection of NSF/Sec18 action. The high degree of orthology between the Y20S and 20S also means that observations and principles gained by studying the Y20S will directly transferrable to the neuronal 20S. The hypothesis is that disassembly of SNAREs by the Y20S is mediated by a conserved allosteric network that spans multiple promoters within the Y20S complex (and therefore the 20S complex as well), which play a key role in the modulation of neurotransmission. To test this hypothesis, CryoEM studies of Sec18 and the Y20S have already been completed. This has allowed for the determination of residues that correlate to differences in conformation, assisted by unsupervised machine learning methods. I propose saturation mutagenesis of every single residue in Sec18 in an in vivo assay tying Sec18 activity to survival that will reveal the fitness of each residue in its ability to mediate SNARE disassembly. Second, electrophysiology experiments on mutant NSF in key residues in this allosteric network will directly tie these biophysical mechanisms directly to synaptic transmission.

项目概要/摘要 大脑的复杂行为，如认知、知觉、动机和精神疾病，仍然 仍然难以解释。要真正理解这些过程，有必要了解基本原理 它们背后的机制。突触传递，突触前释放神经递质 膜融合后的神经元，依赖于 SNARE（可溶性 N-乙基马来酰亚胺敏感因子附着 蛋白质受体）。含有神经递质的囊泡上的 SNARE 形成稳定的反式 SNARE 复合物 突触前膜上有 SNARE。一旦发出信号，这些 SNARE 就会扭曲在一起以提供 膜融合所需的能量。这种顺式 SNARE 复合物，现在是一个高度稳定的四螺旋束 膜必须被拆卸和回收才能进行进一步的融合。无融合池 陷阱，突触传递将停止。 cis SNARE 拆卸由 NSF 完成（N- 乙基马来酰亚胺敏感因子）和称为 SNAP（可溶性 NSF 附着蛋白）的接头蛋白。 这三个成分一起形成 20S 复合物，其中 NSF 在 ATP 水解后分解 SNARE 复杂并维持着一个融合 SNARE 池。然而关键的动态过程和原理 这一爆炸性拆卸步骤的具体过程仍然未知。该项目的总体目标是阐明 通过理解 SNARE 分解来了解突触传递的基本机制。 为了揭示 SNARE 反汇编的原理，NSF 及其酵母直系同源物 Sec18 将被 检查了。由于神经元环境的复杂性，研究 NSF 在其神经元环境中的动态已被证明是困难的 突触前系统以及无法一次研究多个突变体。学习中 Sec18 和酵母 20S (Y20S) 与神经元 20S 配合，将能够使用多种 分子和生化工具将允许剖析 NSF/Sec18 行动。的高度 Y20S和20S之间的直系关系也意味着通过研究Y20S和20S所获得的观察结果和原理 Y20S将直接转移至神经元20S。假设是 SNARE 的拆卸 Y20S 由保守的变构网络介导，该网络跨越 Y20S 复合物内的多个启动子 （因此还有 20S 复合体），它们在神经传递的调节中发挥着关键作用。测试 根据这一假设，Sec18 和 Y20S 的 CryoEM 研究已经完成。这使得 在无人监督机器的辅助下确定与构象差异相关的残基 学习方法。我建议在体内试验中对 Sec18 中的每个残基进行饱和诱变 Sec18 的生存活性将揭示每个残基介导 SNARE 能力的适应性 拆卸。二、该变构网络中关键残基突变NSF的电生理学实验 将直接将这些生物物理机制与突触传递直接联系起来。