Prothymosin alpha (ProTα) is involved in multiple cellular processes. Upon serum-free stress, ProTα lacking a signal peptide sequence is non-classically released from C6 glioma cells as a complex with Ca²⁺-binding cargo protein S100A13. Thus, ProTα and S100A13 are conceived to be members of damage-associated molecular patterns (DAMPs)/alarmins. However, it remains to be determined whether stress-induced release of ProTα and S100A13 involves SNARE proteins in the mechanisms underlying membrane tethering of the multiprotein complex. In the present study, we used C6 glioma cells as a model of ProTα release. In pull-down assay, p40 synaptotagmin-1 (Syt-1), a vesicular SNARE, formed a hetero-oligomeric complex with homodimeric S100A13 in a Ca²⁺-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca²⁺-dependent in surface plasmon resonance (SPR). Immunoprecipitation using conditioned medium (CM) revealed that p40 Syt-1 was co-released with ProTα and S100A13 upon serum-free stress. In in situ proximity ligation assay (PLA), Syt-1 interacted with S100A13 upon serum-free stress in C6 glioma cells. The intracellular delivery of anti-Syt-1 IgG blocked serum free-induced release of ProTα and S100A13. Serum free-induced ProTα-EGFP release was significantly blocked by botulinum neurotoxin/C1 (BoNT/C1), which cleaves target SNARE syntaxin-1 (Stx-1). In immunocytochemistry, the cellular loss of ProTα-EGFP, S100A13, and Syt-1 was also blocked by BoNT/C1. Furthermore, the intracellular delivery of anti-Stx-1 IgG or Stx-1 siRNA treatment blocked Syt-1, S100A13 and ProTα release from C6 glioma cells. All these findings suggest that SNARE proteins play roles in stress-induced non-classical release of DAMPs/alarmins proteins, ProTα and S100A13 from C6 glioma cells.

胸腺肽原 (ProTα) 参与多个细胞过程。^{在无血清应激时，缺乏信号肽序列的 ProTα 作为与 Ca 2+}结合货物蛋白 S100A13的复合物非经典地从 C6 神经胶质瘤细胞释放。因此，ProTα 和 S100A13 被认为是损伤相关分子模式 (DAMP)/警报的成员。然而，应激诱导的 ProTα 和 S100A13 释放是否涉及 SNARE 蛋白在多蛋白复合物膜束缚机制中的作用仍有待确定。在本研究中，我们使用 C6 神经胶质瘤细胞作为 ProTα 释放的模型。在下拉试验中，p40 synaptotagmin-1 (Syt-1)，一种囊泡 SNARE，与 Ca ^{2+中的同二聚体 S100A13 形成异寡聚体复合物}依赖方式。p40 Syt-1 和 S100A13 的相互作用也是 Ca ²⁺-依赖于表面等离子共振（SPR）。使用条件培养基 (CM) 的免疫沉淀显示 p40 Syt-1 在无血清应激时与 ProTα 和 S100A13 共同释放。在原位邻近连接试验 (PLA) 中，Syt-1 在 C6 神经胶质瘤细胞的无血清应激下与 S100A13 相互作用。抗 Syt-1 IgG 的细胞内递送阻断了无血清诱导的 ProTα 和 S100A13 的释放。血清游离诱导的 ProTα-EGFP 释放被肉毒杆菌神经毒素/C1 (BoNT/C1) 显着阻断，肉毒杆菌神经毒素/C1 (BoNT/C1) 切割靶标 SNARE syntaxin-1 (Stx-1)。在免疫细胞化学中，ProTα-EGFP、S100A13 和 Syt-1 的细胞丢失也被 BoNT/C1 阻断。此外，抗 Stx-1 IgG 或 Stx-1 siRNA 处理的细胞内递送阻断了 C6 神经胶质瘤细胞中 Syt-1、S100A13 和 ProTα 的释放。