神经递质和激素的囊泡释放依赖于胞吐作用/反式-SNARE复合物的动态组装,通过突触蛋白的顺序相互作用,Syntaxins,SNAP-25尽管SNARE介导的释放是所有可兴奋组织中细胞间通讯的基础,辅助蛋白调节储备囊泡向活性区的进口的作用,因此,缩放重复性胞吐作用仍较少探索。促分泌素是Ca2+传感器蛋白,SNAP-25是其唯一已知的相互作用伴侣。SNAP-25在活性区域内锚定容易释放的囊泡,因此有助于第一阶段的发布。然而,促分泌素的基因缺失反而阻碍了第二阶段的释放,呼吁替代蛋白质-蛋白质相互作用的存在。这里,我们筛选了大脑和胰腺中的促分泌素相互作用体,发现syntaxin-4严重超标。负载Ca2的促分泌素以纳摩尔的亲和力和1:1的化学计量与syntaxin-4相互作用。蛋白质复合物的晶体结构揭示了促分泌素中与syntaxin-4结合的疏水沟。该凹槽也用于结合SNAP-25。在等摩尔重组蛋白的混合物中,SNAP-25在与syntaxin-4的竞争中被促分泌素隔离。Kd差异表明促分泌素可以通过顺序相互作用形成单向的囊泡运动,一个由体外生物学数据支持的假设。这种机制可以促进运输囊泡向释放部位的运动,特别是在内分泌胰腺中,促分泌素,SNAP-25和syntaxin-4在α-和β-细胞中共存。因此,促分泌素可以通过差异蛋白质相互作用调节囊泡激素释放的速度和保真度。
Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of synaptobrevins, syntaxins, and SNAP-25. Despite SNARE-mediated release being fundamental for intercellular communication in all excitable tissues, the role of auxiliary proteins modulating the import of reserve vesicles to the active zone, and thus, scaling repetitive exocytosis remains less explored. Secretagogin is a Ca2+-sensor protein with SNAP-25 being its only known interacting partner. SNAP-25 anchors readily releasable vesicles within the active zone, thus being instrumental for 1st phase release. However, genetic deletion of secretagogin impedes 2nd phase release instead, calling for the existence of alternative protein-protein interactions. Here, we screened the secretagogin interactome in the brain and pancreas, and found syntaxin-4 grossly overrepresented. Ca2+-loaded secretagogin interacted with syntaxin-4 at nanomolar affinity and 1:1 stoichiometry. Crystal structures of the protein complexes revealed a hydrophobic groove in secretagogin for the binding of syntaxin-4. This groove was also used to bind SNAP-25. In mixtures of equimolar recombinant proteins, SNAP-25 was sequestered by secretagogin in competition with syntaxin-4. Kd differences suggested that secretagogin could shape unidirectional vesicle movement by sequential interactions, a hypothesis supported by in vitro biological data. This mechanism could facilitate the movement of transport vesicles toward release sites, particularly in the endocrine pancreas where secretagogin, SNAP-25, and syntaxin-4 coexist in both α- and β-cells. Thus, secretagogin could modulate the pace and fidelity of vesicular hormone release by differential protein interactions.