Involvement of SNARE Protein Interaction for Non-classical Release of DAMPs/Alarmins Proteins, Prothymosin Alpha and S100A13

Matsunaga, Hayato; Halder, Sebok Kumar; Ueda, Hiroshi

doi:10.1007/s10571-020-00950-y

Cited by 2 publications

(23 citation statements)

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“…According to previously described protocol [ 18 ], rat C6 glioma cells (American Type Culture Collection ® , CCL-1017 TM ; Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories GmbH, Pasching, Austria) and 1% penicillin-streptomycin (Sigma). ProTα-EGFP stably-expressing C6 glioma cells were prepared as described earlier [ 14 ].…”

Section: Methodsmentioning

confidence: 99%

“…According to previously described protocol [ 18 ], cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 30 min and then permeabilized with methanol for 10 min at room temperature. After washing with PBS, the fixed cells were incubated with blocking buffer (1% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS) for 3 h at 4 °C followed by incubation with a primary antibody (1:300 dilution in blocking buffer) overnight at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

“…This process is known as exocytosis [ 17 ]. In contrast, in the stress-induced non-classical and non-vesicular release of FGF-1 or ProTα, soluble p40 synaptotagmin-1 (p40 Syt-1) lacking the membrane-spanning domain of p65 Syt-1 is released together with S100A13, a cargo protein [ 15 , 18 ]. Our recent study further revealed that the intracellular delivery of anti-Stx-1 IgG or siRNA targeting Stx-1 blocked the serum-free release of Syt-1, S100A13, and ProTα [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, in the stress-induced non-classical and non-vesicular release of FGF-1 or ProTα, soluble p40 synaptotagmin-1 (p40 Syt-1) lacking the membrane-spanning domain of p65 Syt-1 is released together with S100A13, a cargo protein [ 15 , 18 ]. Our recent study further revealed that the intracellular delivery of anti-Stx-1 IgG or siRNA targeting Stx-1 blocked the serum-free release of Syt-1, S100A13, and ProTα [ 18 ]. These findings suggest that p40 Syt-1 may recruit the protein complex of S100A13 and ProTα to the target SNARE at the plasma membranes, analogous to p65 Syt-1 in the vesicular docking machinery in the case of classical exocytosis.…”

Section: Introductionmentioning

confidence: 99%

“…We speculate that annexin A2 (ANXA2) is another possible target protein for membrane tethering/docking since it is a Ca 2+ -dependent acidic phospholipid (e.g., phosphatidylserine/PS)-binding protein and flopped-out by plasma membrane phospholipid remodeling [ 19 ]. In addition, ANXA2 exhibits an affinity for S100A10 [ 20 , 21 , 22 ], a congener of S100A13, which is a cargo protein for the serum-free -and Ca 2+ -dependent non-classical release of ProTα and FGF-1 [ 11 , 18 ]. Here, we provide evidence for the role of ANXA2 in the non-classical release of the protein complex comprising ProTα, S100A13, and Syt-1.…”

Section: Introductionmentioning

confidence: 99%

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Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

Matsunaga

Halder

Ueda

2021

Cells

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Prothymosin alpha (ProTα) and S100A13 are released from C6 glioma cells under serum-free conditions via membrane tethering mediated by Ca2+-dependent interactions between S100A13 and p40 synaptotagmin-1 (Syt-1), which is further associated with plasma membrane syntaxin-1 (Stx-1). The present study revealed that S100A13 interacted with annexin A2 (ANXA2) and this interaction was enhanced by Ca2+ and p40 Syt-1. Amlexanox (Amx) inhibited the association between S100A13 and ANXA2 in C6 glioma cells cultured under serum-free conditions in the in situ proximity ligation assay. In the absence of Amx, however, the serum-free stress results in a flop-out of ANXA2 through the membrane, without the extracellular release. The intracellular delivery of anti-ANXA2 antibody blocked the serum-free stress-induced cellular loss of ProTα, S100A13, and Syt-1. The stress-induced externalization of ANXA2 was inhibited by pretreatment with siRNA for P4-ATPase, ATP8A2, under serum-free conditions, which ablates membrane lipid asymmetry. The stress-induced ProTα release via Stx-1A, ANXA2 and ATP8A2 was also evidenced by the knock-down strategy in the experiments using oxygen glucose deprivation-treated cultured neurons. These findings suggest that starvation stress-induced release of ProTα, S100A13, and p40 Syt-1 from C6 glioma cells is mediated by the ANXA2-flop-out via energy crisis-dependent recovery of membrane lipid asymmetry.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

Matsunaga

Halder

Ueda

2021

Cells

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Non-Vesicular Release of Alarmin Prothymosin α Complex Associated with Annexin-2 Flop-Out

Ueda

2023

Cells

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Nuclear protein prothymosin α (ProTα) is a unique member of damage-associated molecular patterns (DAMPs)/alarmins. ProTα prevents neuronal necrosis by causing a cell death mode switch in serum-starving or ischemic/reperfusion models in vitro and in vivo. Underlying receptor mechanisms include Toll-like receptor 4 (TLR4) and Gi-coupled receptor. Recent studies have revealed that the mode of the fatal stress-induced extracellular release of nuclear ProTα from cortical neurons in primary cultures, astrocytes and C6 glioma cells has two steps: ATP loss-induced nuclear release and the Ca2+-mediated formation of a multiple protein complex and its extracellular release. Under the serum-starving condition, ProTα is diffused from the nucleus throughout the cell due to the ATP loss-induced impairment of importin α–mediated nuclear transport. Subsequent mechanisms are all Ca2+-dependent. They include the formation of a protein complex with ProTα, S100A13, p40 Syt-1 and Annexin A2 (ANXA2); the fusion of the protein complex to the plasma membrane via p40 Syt-1–Stx-1 interaction; and TMEM16F scramblase-mediated ANXA2 flop-out. Subsequently, the protein complex is extracellularly released, leaving ANXA2 on the outer cell surface. The ANXA2 is then flipped in by a force of ATP8A2 activity, and the non-vesicular release of protein complex is repeated. Thus, the ANXA2 flop-out could play key roles in a new type of non-vesicular and non-classical release for DAMPs/alarmins, which is distinct from the modes conducted via gasdermin D or mixed-lineage kinase domain-like pseudokinase pores.

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Involvement of SNARE Protein Interaction for Non-classical Release of DAMPs/Alarmins Proteins, Prothymosin Alpha and S100A13

Cited by 2 publications

References 50 publications

Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

Non-Vesicular Release of Alarmin Prothymosin α Complex Associated with Annexin-2 Flop-Out

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