Ecto-F₀/F₁ ATPase as a novel candidate of prothymosin α receptor

ABSTRACT

Objectives: Prothymosin α (ProTα) was reported to inhibit the neuronal necrosis by facilitating the plasma membrane localization of endocytosed glucose transporter 1/4 through an activation of putative G_i-coupled receptor. The present study aims to identify a novel ProTα target, which may lead to an activation of G_i-coupled receptor.

Methods: We used G_i-rich lipid rafts fraction of retinal cell line N18-RE-105 cells for affinity cross-linking. The biological confirmation that F₀/F₁ ATPase is a target protein complex was performed by cell-free experiments using ELISA-based binding assay, surface plasmon resonance assay and quartz crystal microbalance assay, and cell-based experiments to measure extracellular ATP level in the HUVECs culture.

Results: From the cross-linking study and above-mentioned protein-protein interaction assays, ATP5A1 and ATP5B, F₁ ATPase subunits were found to ProTα binding target proteins. In the culture of HUVEC cells, furthermore, ProTα increased the extracellular ATP levels in a reversible manner by anti-ATP5A1- and ATP5B-antibodies.

Conclusion: The present study suggests that ProTα may activate ecto-F₀/F₁ ATPase and produced ATP. This study leads to next subjects whether produced ATP and its metabolites, ADP or adenosine may activate corresponding G_i-coupled receptors.

KEYWORDS:

• Prothymosin α
• ecto-F₀/F₁ ATPase
• N18-RE-105 cells
• HUVEC
• protein-protein interaction

Author contributions statement

H Ueda and H Matsunaga designed the whole study and wrote the draft manuscript. H Ueda, H Matsunaga, Y Matsushita, S Maeda, R Iwamoto were involved in the main study and gave substantial inputs to the plan for analysis. S Yokoyama and M Shirouzu prepared recombinant proteins. All authors read and approved the final manuscript.

Declaration of Interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This work was supported in part by Grants-in-Aid for the Platform for Drug Discovery, Informatics, and Structural Life Science [16am0101012j0005] (H. Ueda), [16am0101022j0005] (S. Yokoyama) from the Japan Agency for Medical Research and Development (AMED), Japan; and JSPS KAKENHI Grant Numbers JP14F04096 (H. Ueda), JP23659056, 25670061 (H. Ueda) and JP22657036 (H. Matsunaga). This paper has been published as part of a supplement issue covering the proceedings of the Fifth International Symposium on Thymosins in Health and Disease and is funded by SciClone Pharmaceuticals.