DataSheet6_Screening of Hydrocarbon-Stapled Peptides for Inhibition of Calcium-Triggered Exocytosis.PDF

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）之间的核心相互作用界面，对于神经元突触内钙离子触发的神经递质释放至关重要。该核心界面的相互作用残基，在参与快速同步神经递质释放的突触结合蛋白家族（synaptotagmins, Syt1、Syt2、Syt9）、SNAP-25以及语法素-1A同源蛋白中跨物种保守。这种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。该核心相互作用界面在介导黏蛋白颗粒膜融合的融合复合体中同样具有保守性。钙离子刺激的黏蛋白分泌过程，由SNARE蛋白语法素-3（syntaxin-3）、SNAP-23、VAMP8、Syt2及其他多种蛋白共同介导。本研究设计并筛选了一系列由SNAP-25片段构成的烃链 stapled 肽（hydrocarbon-stapled peptides），这些片段包含了高分辨率晶体结构中观测到的核心界面关键残基。我们筛选得到4个经圆二色光谱法（circular dichroism）验证具备高α螺旋结构含量的stapled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。在使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检测实验中，筛选得到的肽段同样能够有效抑制钙离子触发的膜融合过程。综上，靶向干扰该核心相互作用界面的烃链 stapled 肽可显著抑制钙离子触发的胞吐作用。本抑制剂筛选实验表明，这类化合物或可用于对抗黏液过度分泌，而黏液过度分泌是慢性阻塞性肺疾病（COPD）、哮喘及囊性纤维化病理生理过程中气道阻塞的主要诱因。