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Review
. 2023 Jun 6;12(12):1569.
doi: 10.3390/cells12121569.

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

Hiroshi Ueda  1   2
Affiliations
Review

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

Hiroshi Ueda. Cells. .

Abstract

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.

Keywords: DAMPs; GSDMD; MLKL; S100A13; SNARE complex; alarmins; exosomes; flippase; scramblase.

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Conflict of interest statement

The author Hiroshi Ueda declares that he has no conflict of interest.

Figures

Figure 1
Figure 1
Prothymosin α as endogenous necrosis inhibitor released from neurons in starving condition. (A). Preparation of conditioned medium (CM) from the primary culture of embryonic (E17) rat cortical neurons at 5 × 105 cells/cm2 (HD) cultured in the absence of serum. (B). Increase in survival activity of neurons cultured at a low-density (LD, 1 × 105 cells/cm2) by the addition of CM from HD culture [26]. (C). Schematic changes in cell death mode of cortical neurons by recombinant ProTα. In the absence of serum, freshly prepared cortical neurons showed features of necrosis, characterized by a decrease in electron density and ATP levels, in the cytosol and swollen mitochondria and by propidium iodide (PI) incorporation into the nucleus through the disrupted plasma membrane. The addition of ProTα converted the cell death mode into apoptosis at the time point of 12 h, which is characterized by nuclear fragmentation, annexin-V (ANX-V) flop-out and caspase 3 activation.
Figure 2
Figure 2
Serum deprivation-induced loss of ProTα in the nuclei of neurons and astrocytes. Results show that serum deprivation stress caused a loss of ProTα in the nuclei of cortical neurons and astrocytes in primary culture, while no significant ProTα immunoreactivity was observed in the cytosol. Details are described in a previous report [27].
Figure 3
Figure 3
Schematic illustration of stress-induced extracellular release of ProTα, S100A13 and p40 Syt-1 (working hypothesis). Upper panel: Under the normal condition, importin α transports ProTα possessing nuclear localization sequence into the nucleus. The loss of ATP by starving stress impairs the importin α action for the nuclear transport of ProTα, and existing ProTα in the nucleus is then diffused throughout the cell. The starving stress also causes Ca2+ influx, which triggers the formation of protein complex comprising ProTα, S100A13, Syt-1 and ANXA2 on filamentous F-actin network. Lower panel: The protein complex is tethered to the plasma membrane with the help of interaction between p40 Syt-1 and Stx-1 (stage 1). High levels of intracellular Ca2+ caused by CICR (stage 2) facilitate the tight binding of the protein complex to plasma membrane with the help of interaction between ANXA2 and acidic phospholipids (e.g., phosphatidylserine/PS) (stage 3), followed by the TMEM16F-mediated flop-out of ANXA2-PS complex (stage 4) and the extracellular release of protein complex (stage 5). Externalized ANXA2-PS will be flipped in by a force of ATP8A2, and the additional non-vesicular release of protein complex will be repeated (stage 6). Details are described in the text and have been reported previously [11].
Figure 4
Figure 4
Lack of stress-induced ProTα release in microglia and HeLa cells. (A–C). Representative pictures of ProTα immunocytochemistry in rat microglia (A), C6 glioma (B) and HeLa cells (C) cultured with various types of stress. (A). Lack of ProTα release from microglia by serum deprivation stress. ProTα is detected throughout the cell both in the presence or absence of serum. (B). ProTα is detected in the nucleus of C6 glioma cells in the presence of serum, while no ProTα is detected in the nucleus and cytosol after the treatment with oxygen glucose deprivation (OGD, glucose-free, 1% O2, 3 h) or heat shock (pre-heat treatment at 42 °C for 90 min, followed by incubation at 37 °C for 3 h). (C). Lack of ProTα release from HeLa cells by serum deprivation or heat shock stress. ProTα is detected throughout the cell in the presence of serum deprivation or heat shock stress.
Figure 5
Figure 5
Possible roles of stress-induced ANXA2 flop-out in the plasmin and MMP production. Upon ischemic stress, protein complex comprising ProTα and S100A13 is released to the extracellular space by the force of ANXA2 flop-out mechanism. Because of high levels of Ca2+, S100A13 is expected to keep binding to ANXA2. On the analogy of the previously reported model, in which S100A10 bound to ANXA2 could be a receptor for tPA [60], endogenous or exogenous tPA binds to externalized S100A13 on the vascular cells or other cells in vicinity and causes the production of plasmin and MMPs. Produced MMPs may cause a hemorrhage by degrading tight junction proteins. Endogenous ProTα released upon stress may bind to S100A13 and prevent the MMP production to some extent by the inhibition of tPA binding to S100A13. Exogenous tPA may produce large amounts of MMPs via binding to S100A13 and cause hemorrhages, which are suppressed by co-administration of ProTα.
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This work was supported by KAKENHI JP17H01586, JP19K21592 and JP21H03024 (HU) from the Japan Society for the Promotion of Science (JSPS) and the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) (16am0101012j0005) (HU) from the Japan Agency for Medical Research and Development (AMED).

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