Prolyl oligopeptidase participates in the cytosine arabinoside-induced nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase in a human neuroblastoma cell line
Introduction
Prolyl oligopeptidase (POP) is a post-proline cleaving serine peptidase that is highly similar among several species [1,2]. We searched for proteins that bind to POP and identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [3]. In human neuroblastoma NB-1 cells, cytosine arabinoside (Ara-C) was found to induce cell death by translocating GAPDH into the nucleus. Further, an interaction between POP and GAPDH during this process may occur [3].
GAPDH is widely regarded as a ubiquitous glycolytic enzyme and is a simple ‘housekeeping’ protein. However, this enzyme has been shown to be involved in many cellular processes. Due to the non-glycolytic roles of GAPDH, this enzyme is considered a moonlight protein in cells. GAPDH plays an important role in stress response leading to apoptosis [4], with the cytoplasmic to nuclear translocation of GAPDH preceding the onset of apoptosis [5]. A general mechanism of Ara-C cytotoxicity is the incorporation of nucleoside analogs into DNA, catalyzed by DNA polymerase [6]. However, Ara-C has been reported to induce apoptosis in postmitotic cerebellar granular cells cultured from postnatal rats [6,7]. Further, GAPDH nuclear translocation has been demonstrated to be involved in the apoptosis of non-neuronal and neuronal cells [8]. However, the mechanism of nuclear translocation of GAPDH induced by Ara-C has not been elucidated. Interestingly, the POP inhibitor, ONO-1603, was found to suppress the overexpression of GAPDH mRNA in cultured central nervous system neurons undergoing age-induced apoptosis and prevented GAPDH nuclear translocation [9]. Puttonen et al. [10] also reported that another POP inhibitor, Z-Pro-Prolinal, inhibited 6-hydroxydopamine-induced GAPDH nuclear translocation in monkey fibroblast CV1–P cells. The aim of this study was to compare POP-knockout (POP–KO) cells with wild-type cells and to demonstrate that the interaction between POP and GAPDH is involved in Ara-C-induced GAPDH nuclear translocation and cell death. Our results show that the interaction between POP and GAPDH is required for Ara-C-induced GAPDH nuclear translocation and cell death.
Section snippets
Cell culture and reagents
NB-1 (IFO 50295) human neuroblastoma cells were obtained from the Health Science Research Resources Bank (HSRRB, Osaka, Japan) and maintained in RPMI-1640 (Sigma-Aldrich) containing 10% fetal calf serum (FCS, Gibco) and 50 μg/ml kanamycin at 37 °C in a humidified atmosphere of 5% CO2/95% air. POP-KO NB-1 cells were generated using CRISPR/Cas9 technology through the guide-it CRISPR/Cas9 system (Takara Bio Inc.), in accordance with the manufacturer's instructions. sgRNAs were designed using
Ara-C-induced nuclear translocation of GAPDH involves its interaction with POP in NB-1 cells
Using confocal microscopy, we monitored the distribution of GAPDH between cellular compartments in NB-1 cells before and after Ara-C treatment. Fluorescence immunostaining showed that GAPDH immunoreactivity primarily displayed cytosolic localization and was excluded from the nucleus in untreated control cells; however, after exposure to 20 μM Ara-C for 24 h, GAPDH immunoreactivity was significantly accumulated in the nucleus. Furthermore, pre-treatment with 40 μM SUAM-14746 was found to
Discussion
Ara-C induces the nuclear translocation of GAPDH and causes cell death [8]. Previously, we reported that GAPDH is a binding partner of POP, SUAM-14746 protects against Ara-C-induced cytotoxicity but does not affect the cytotoxicity of doxorubicin, and SUAM-14746 enhances the interaction between POP and GAPDH in the cytoplasm and inhibits the nuclear translocation of GAPDH [3]. In the present study, we generated POP-KO cells to demonstrate the involvement of POP in the Ara-C-induced nuclear
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
We would like to thank Editage (www.editage.com) for English language editing.
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