Synapsin1的棕榈酰化修饰对突触囊泡聚集的调节作用及其分子机制研究

Synapsin1 (SYN1) was known as one of the key regulators in controlling the SVs clustering. Depletion of SYN1 block SVs clustering, inhibit neurotransmitters release, and cause similar pathological features of epilepsy. The clustering of SVs in synapse are dynamic and reversible processes, and its regulation mechanism is far from illumination. Protein palmitoylation is the most common form of lipid modifications in brain, which can regulate the trafficking and localization of proteins to cell membranes as well as protein-protein interactions. Our preliminary data demonstrated that SYN1 was palmitoylated at Cys223, Cys360, and Cys370, and the non-palmitoylated SYN1 could not sustain the SVs clusters at synapses, indicating that palmitoylated-SYN1 might be involved in the regulation of SVs clustering and releasing behavior. Therefore, our current study will focus on the following points: 1) confirm the enzymes responsible for SYN1-palmitoylation, 2) clarify the roles of SYN1-palmitoylation in the controlling of SVs clustering, 3) explore the molecular mechanism of SYN1-palmitoylation on SVs clustering. The understanding of these questions would shed light on the dynamic nature of SVs cluster at synapses, which might help us to peek into the pathological mechanisms of epilepsy from a brand-new angle.

Synapsin1（SYN1）是决定突触囊泡聚集的关键蛋白，敲除SYN1导致突触囊泡聚集受阻，GABA释放减少，并引起癫痫发作。SYN1对突触囊泡聚集的调控是一个动态可逆的生命过程，其具体的调节机制仍待完善。棕榈酰化是脑中最常见的脂肪酸化修饰，能够动态可逆的调节蛋白质在细胞内膜结构上的定位以及蛋白分子间的相互作用。申请人前期工作发现在脑中SYN1被棕榈酰化，Cys223、Cys360和Cys370是其棕榈酰化修饰位点，阻断SYN1的棕榈酰化影响了突触囊泡的聚集，暗示SYN1的棕榈酰化可能参与调控了突触囊泡的聚集和释放。基于此，本课题拟开展以下研究：①鉴定催化SYN1棕榈酰化和去棕榈酰化修饰的酶；②明确SYN1的棕榈酰化对突触囊泡聚集的影响；③探索SYN1的棕榈酰化调节突触囊泡聚集的具体分子机制。本课题的开展有助于进一步阐明突触囊泡聚集的动态调节机制，并为认识癫痫的发病机理提供新的视角。

Synapsin1是决定突触前区域中囊泡聚集的关键蛋白。已知磷酸化修饰通过影响该蛋白与囊泡膜和F-actin的结合能力调节突触囊泡聚集和释放的动态过程，但具体的分子机制仍不清楚。棕榈酰化是脑中最常见的脂肪酸化修饰，能够动态可逆的调节蛋白质在细胞内膜结构上的定位以及蛋白分子间的相互作用。本研究中我们发现Synapsin1被棕榈酰化，该修饰发生在其Cys223、Cys360和Cys370上；脑中ZDHHC5催化Synapsin1的棕榈酰化，ABHD17a催化其去棕榈酰化；该翻译后修饰对Synapsin1维持突触囊泡的聚集十分重要，突变其棕榈酰化修饰位点或敲除ZDHHC5均导致突触囊泡聚集异常；进一步研究发现Synapsin1的棕榈酰化通过促进其与F-actin的结合稳定突触囊泡的聚集，并不影响其与囊泡膜的直接结合能力。更有趣的是，Synapsin1的磷酸化修饰能够负调控其棕榈酰化水平。当敲除ABHD17a或用PalmB处理抑制Synapsin1棕榈酰化水平降低后，Forskolin虽然可以激活Synapsin1的磷酸化，但该刺激引起的突触囊泡释放却被部分抑制，说明Synapsin1的磷酸化修饰通过负调控其棕榈酰化调节突触囊泡的聚集。总之，本研究发现了Synapsin1的磷酸化修饰与棕榈酰化修饰共同调节突触前囊泡的循环，神经元兴奋后Synapsin1的磷酸化修饰被激活，进而降低其棕榈酰化修饰水平，促使Synapsin1与F-actin的结合能力减弱以及后续的囊泡释放，为神经元调节突触囊泡动态变化提出了一种新机制。主要研究结果已整理并以第一作者身份发表在Cell Death and Disease上。