Mechanistic Principles of SNARE Disassembly in Neurotransmitter Release

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 仍然很难解释。为了真正理解这些过程，有必要了解基本的 不利于他们的机制。突触传递，突触前神经递质的释放 神经元在膜融合时依赖SNARS(可溶性N-乙基马来酰亚胺敏感因子附着 蛋白质受体)。含有囊泡的神经递质上的陷阱形成了一个稳定的反式陷阱复合体 突触前膜上有陷阱。一旦发出信号，这些陷阱就会扭曲在一起，提供 膜融合所需的能量。这个顺式陷阱复合体，现在是一个高度稳定的四股螺旋束 膜，必须拆卸和回收，以允许进一步的融合。没有一池融合子 陷阱，突触传输就会停止。顺式圈套拆卸由NSF(N- 乙基马来酰亚胺敏感因子)和称为SNAPs(可溶性NSF附着蛋白)的接头蛋白。 这三个组分一起形成了一个20S复合体，在这个复合体中，NSF在ATP水解后被分解 圈套复杂，并维持着一个融合诱捕池。然而，关键的动力学过程和原理 这一爆炸性拆解步骤的具体步骤尚不清楚。这个项目的总体目标是阐明 通过了解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及其酵母同源基因Sec18都将被 检查过了。由于NSF的复杂性，在其神经元环境中研究NSF的动力学被证明是困难的 突触前系统，以及无法同时调查几个以上的突变体。学习 Sec18和酵母20S(Y20S)与神经元20S协同工作，将使各种不同的 分子和生化工具，将允许剖析NSF/Sec18的作用。高度的 Y20s和20s之间的正射也意味着通过研究Y20s和20s获得的观察和原理 Y20S将直接移植到神经元20S。假设是陷阱的拆解是由 Y20S由一个保守的变构网络介导，该网络跨越Y20S复合体中的多个启动子 (因此，20s复合体也是如此)，它们在神经传递的调节中发挥着关键作用。为了测试 这一假设，对Sec18和Y20S的低温电子显微镜研究已经完成。这使得 在无监督机器的辅助下，测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中的每一个残基进行饱和突变 Sec18活性对生存的影响，将揭示每个残基在调节陷阱的能力方面的适合性 拆卸。第二，变构网络中关键残基突变型NSF的电生理实验 会将这些生物物理机制直接与突触传递联系起来。

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.