Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, 852-8521, Japan.
Department of Medical Pharmacology, Nagasaki University of Graduate School of Biomedical Sciences, Nagasaki, 852-8523, Japan.
Cell Mol Neurobiol. 2021 Nov;41(8):1817-1828. doi: 10.1007/s10571-020-00950-y. Epub 2020 Aug 27.
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 Ca-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 Ca-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca-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.
前胸腺素α（ProTα）参与多种细胞过程。在无血清应激下，缺乏信号肽序列的 ProTα 与 Ca 结合货物蛋白 S100A13 一起作为复合物从 C6 神经胶质瘤细胞中非经典释放。因此，ProTα 和 S100A13 被认为是损伤相关分子模式（DAMPs）/警报素的成员。然而，目前尚不确定应激诱导的 ProTα 和 S100A13 的释放是否涉及 SNARE 蛋白在多蛋白复合物的膜连接机制中。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。在下拉测定中，囊泡 SNARE p40 突触融合蛋白-1（Syt-1）以 Ca 依赖性方式与同源二聚体 S100A13 形成异源寡聚复合物。表面等离子体共振（SPR）中的 p40 Syt-1 与 S100A13 的相互作用也是 Ca 依赖性的。使用条件培养基（CM）进行免疫沉淀显示，无血清应激时，p40 Syt-1 与 ProTα 和 S100A13 一起被共释放。在原位邻近连接测定（PLA）中，无血清应激时，C6 神经胶质瘤细胞中 Syt-1 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。肉毒杆菌神经毒素/C1（BoNT/C1）显著阻断了无血清诱导的 ProTα-EGFP 释放，BoNT/C1 可切割靶 SNARE 突触融合蛋白-1（Stx-1）。在免疫细胞化学中，BoNT/C1 也阻断了 ProTα-EGFP、S100A13 和 Syt-1 的细胞内丢失。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。所有这些发现表明 SNARE 蛋白在应激诱导的 C6 神经胶质瘤细胞中非经典释放 DAMPs/警报素蛋白、ProTα 和 S100A13 中发挥作用。
