Involvement of SNARE Protein Interaction for Non-classical Release of DAMPs/Alarmins Proteins, Prothymosin Alpha and S10
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ORIGINAL RESEARCH
Involvement of SNARE Protein Interaction for Non‑classical Release of DAMPs/Alarmins Proteins, Prothymosin Alpha and S100A13 Hayato Matsunaga1,2 · Sebok Kumar Halder1,3 · Hiroshi Ueda1,4 Received: 15 May 2020 / Accepted: 19 August 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract 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 C a2+-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 C a2+-dependent manner. The interaction between p40 Syt-1 and S100A13 was also 2+ 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. Keywords Non-vesicular release · Snares · Damps/alarmins · Prothymosin alpha · S100A13 Abbreviations Amx Amlexanox BoNT Botulinum neurotoxin Hayato Matsunaga and Sebok Kumar Halder have contributed equally to this work. * Hiroshi Ueda ueda.hiroshi.8e@kyoto‑u.ac.jp 1
Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852‑8521, Japan
2
Present Address: Department of Medical Pharmacology, Nagasaki University of Graduate School of Biomedical Sciences, Nagasaki 852‑8523, Japan
3
Present Address: San Diego Biomedical Research Institute, San Diego, CA 92121, USA
4
Present Address: Department of Molecular Pharmacology, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto 606‑8501, Japan
CM Conditioned medium DAMPs Damage-associated molecular pattern molecules 2-DG 2-Deoxy-d-glucose EGTA Ethylene glycol tetraace
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