Mechanistic Principles of SNARE Disassembly in Neurotransmitter Release

神经递质释放中 SNARE 分解的机制原理

基本信息

  • 批准号:
    10824093
  • 负责人:
  • 金额:
    $ 4.09万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
项目摘要/摘要 大脑的复杂行为,如认知、感知、动机和精神疾病,仍然是一个复杂的问题。 仍然难以解释。要真正理解这些过程,就必须了解基本的 它们背后的机制。突触传递,神经递质从突触前 神经元膜融合后,依赖于SNARE(可溶性N-乙基马来酰亚胺敏感因子附着 蛋白受体)。含有囊泡的神经递质上的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,这些陷阱就会扭曲在一起, 膜融合所需的能量。这个顺式陷阱复合物,现在是一个高度稳定的四螺旋束在一个 膜,必须拆卸和回收,以允许进一步的回合融合。如果没有融合剂 一旦发生SNARE,突触传递就会停止。顺式SNARE拆卸是由NSF(N- 乙基马来酰亚胺敏感因子)和称为SNAP(可溶性NSF附着蛋白)的衔接蛋白。 这三种成分一起形成20 S复合物,其中NSF在ATP水解时分解 SNARE复合物并维持融合SNARE池。然而,关键的动力学过程和原理 这个爆炸性的分解步骤仍然未知。本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分解的原理,NSF及其酵母直向同源物Sec 18将被 考察研究NSF在其神经元背景下的动力学已被证明是困难的,这是由于 突触前系统和无法同时研究多个突变体。研究 Sec 18和酵母20 S(Y20 S),与神经元20 S协调,将使得能够使用各种各样的神经元20 S。 分子和生物化学工具,将允许解剖NSF/Sec 18行动。的高度 Y20年代和20年代之间的同源性也意味着通过研究 Y20 S可直接转移到神经元20 S。这一假设是, Y20 S由跨越Y20 S复合物内多个启动子的保守变构网络介导 (and因此20 S复合物也是如此),其在神经传递的调节中起关键作用。测试 根据这一假设,Sec 18和Y20 S的CryoEM研究已经完成。这使得 在无人监督的机器的辅助下,确定与构象差异相关的残留物 学习方法我建议在体内试验中对Sec 18中的每一个残基进行饱和诱变, Sec 18活性对存活的影响,这将揭示每个残基在其介导SNARE的能力中的适应性 拆卸第二,电生理实验,在这个变构网络中的关键残基的突变NSF 将这些生物物理机制直接与突触传递联系起来。

项目成果

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