Table1_Screening of Hydrocarbon-Stapled Peptides for Inhibition of Calcium-Triggered Exocytosis.docx

The so-called primary interface between the SNARE complex and synaptotagmin-1 (Syt1) is essential for Ca2+-triggered neurotransmitter release in neuronal synapses. The interacting residues of the primary interface are conserved across different species for synaptotagmins (Syt1, Syt2, Syt9), SNAP-25, and syntaxin-1A homologs involved in fast synchronous release. This Ca2+-independent interface forms prior to Ca2+-triggering and plays a role in synaptic vesicle priming. This primary interface is also conserved in the fusion machinery that is responsible for mucin granule membrane fusion. Ca2+-stimulated mucin secretion is mediated by the SNAREs syntaxin-3, SNAP-23, VAMP8, Syt2, and other proteins. Here, we designed and screened a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface as observed in high-resolution crystal structures. We selected a subset of four stapled peptides that were highly α-helical as assessed by circular dichroism and that inhibited both Ca2+-independent and Ca2+-triggered ensemble lipid-mixing with neuronal SNAREs and Syt1. In a single-vesicle content-mixing assay with reconstituted neuronal SNAREs and Syt1 or with reconstituted airway SNAREs and Syt2, the selected peptides also suppressed Ca2+-triggered fusion. Taken together, hydrocarbon-stapled peptides that interfere with the primary interface consequently inhibit Ca2+-triggered exocytosis. Our inhibitor screen suggests that these compounds may be useful to combat mucus hypersecretion, which is a major cause of airway obstruction in the pathophysiology of COPD, asthma, and cystic fibrosis.

所谓的SNARE复合体（SNARE complex）与突触结合蛋白-1（synaptotagmin-1，Syt1）之间的初级相互作用界面，对于神经元突触内钙离子触发的神经递质释放至关重要。该初级界面的相互作用残基，在参与快速同步释放的突触结合蛋白家族（Syt1、Syt2、Syt9）、突触体相关蛋白25（SNAP-25）以及突触融合蛋白1A（syntaxin-1A）同源蛋白中，于不同物种间均保持保守。
这一不依赖钙离子的界面在钙离子触发事件发生前便已形成，在突触囊泡启动过程中发挥关键作用。该初级界面同样保守存在于介导黏蛋白颗粒膜融合的膜融合装置中。钙离子刺激的黏蛋白分泌过程由SNARE蛋白突触融合蛋白3（syntaxin-3）、突触体相关蛋白23（SNAP-23）、囊泡相关膜蛋白8（VAMP8）、Syt2及其他蛋白共同介导。
本研究设计并筛选了一系列由SNAP-25片段构成的烃交联肽（hydrocarbon-stapled peptides），这些片段包含了高分辨率晶体结构中观测到的初级界面关键残基。我们筛选出4种经圆二色谱（circular dichroism）验证具有高α螺旋含量的交联肽，它们可同时抑制神经元SNARE复合体与Syt1介导的不依赖钙离子及依赖钙离子的群体脂质混合反应。
在采用重组重构的神经元SNARE复合体与Syt1，或重组重构的气道SNARE复合体与Syt2构建的单囊泡内容物混合实验中，上述筛选得到的肽类同样可抑制钙离子触发的膜融合过程。
综上，可靶向干扰该初级相互作用界面的烃交联肽，能够有效抑制钙离子触发的胞吐作用。本研究的抑制剂筛选结果表明，这类化合物或可用于治疗黏液过度分泌——而黏液过度分泌是慢性阻塞性肺疾病（COPD）、哮喘及囊性纤维化等疾病病理生理过程中气道阻塞的主要诱因。