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 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.
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Abbreviations
- Amx:
-
Amlexanox
- BoNT:
-
Botulinum neurotoxin
- CM:
-
Conditioned medium
- DAMPs:
-
Damage-associated molecular pattern molecules
- 2-DG:
-
2-Deoxy-d-glucose
- EGTA:
-
Ethylene glycol tetraacetic acid
- In situ PLA:
-
In situ proximity ligation assay
- NLS:
-
Nuclear localization signal
- PEA:
-
Proximity extension assay
- ProTα:
-
Prothymosin alpha
- RU:
-
RESONANCE unit
- SNARE:
-
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor
- Stx-1:
-
Syntaxin-1
- Syt-1:
-
Synaptotagmin-1
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Acknowledgements
We thank Y. Matsushita, T. Sakamoto, T. Kubo, N. Matsunaga, M. Murata, and Y. Yoshizaki for technical assistance. Parts of this study were supported by KAKENHI (Scientific Research B: 13470490 and 15390028 from JSPS to HU, 17025031, 18023028, and 20023022 from MEXT to HU, H20-Research on Biological Resources and Animal Models for Drug Development-003 from MHLW to HU, Young Scientists B: 20770105 from JSPS to HM, Exploratory Research 22657036 from JSPS to HM, and project grants the Platform for Drug Discovery, Informatics, and Structural Life Science from Japan Agency for Medical Research and Development (16am0101012j0005) to HU and “Therapeutic Innovation Center” from the Nagasaki University Major Research Project to HU.
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H.M. contributed to manuscript writing, experimental design, and performance. S.K.H. conducted data analysis and manuscript writing. H.U. wrote the manuscript and designed the experiments. All authors read and approved the final manuscript.
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Hayato Matsunaga and Sebok Kumar Halder have contributed equally to this work.
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Matsunaga, H., Halder, S.K. & Ueda, H. Involvement of SNARE Protein Interaction for Non-classical Release of DAMPs/Alarmins Proteins, Prothymosin Alpha and S100A13. Cell Mol Neurobiol 41, 1817–1828 (2021). https://doi.org/10.1007/s10571-020-00950-y
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DOI: https://doi.org/10.1007/s10571-020-00950-y