High-performance affinity purification for identification of 15-deoxy-Δ12,14-PGJ2 interacting factors using magnetic nanobeads

Naoya Maekawa, Masaki Hiramoto, Satoshi Sakamoto, Motoki Azuma, Takumi Ito, Marie Ikeda, Michitaka Naitou, Hukum P. Acharya, Yuichi Kobayashi, Makoto Suematsu, Hiroshi Handa, Takeshi Imai

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Abstract

Prostaglandin J2 (PGJ2) family have been reported to show various kinds of biological activities. Considerable progress has been made toward understanding the mechanism of adipogenesis, however, the mechanisms of other actions of PGJ2 family remain controversial. The 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) is one of the members of PGJ2 family, and is known as a ligand for peroxisome proliferator-activated receptor γ (PPARγ), which promotes the expression of the crucial genes for adipogenesis. In this study, we found that 15d-PGJ2 did not stimulate PPARγ-mediated gene expression in HEK293 cells whereas 15d-PGJ2 transactivated PPARγ-dependent transcription in other cell lines. Moreover, we confirmed that 15d-PGJ2 suppressed the growth of HEK293 cells. These observations suggest that 15d-PGJ2 shows another biological activity e.g. growth inhibition in HEK293 cells via unknown receptor for 15d-PGJ2. The aim of this study is to develop and validate effective purification system for PGJ2 interacting factors (PGJIFs). We have recently developed high performance magnetic nanobeads. In this study, we have newly developed 15d-PGJ2-immobilized beads by conjugating 15d-PGJ2 to the surface of these nanobeads. Firstly, we showed that PPARγ specifically bound to 15d-PGJ2-immobilized beads. Secondly, we newly identified voltage dependent anionic channel 1 (VDAC1) as new PGJIF from crude extracts of HEK293 cells using this affinity purification system. These data presented here demonstrate that 15d-PGJ2-immobilized beads are effective tool for purification of PGJIFs directly from crude cell extracts.

Original languageEnglish
Pages (from-to)466-471
Number of pages6
JournalBiomedical Chromatography
Volume25
Issue number4
DOIs
Publication statusPublished - 2011 Apr 1

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Keywords

  • Affinity purification
  • Magnetic nanobeads
  • Prostaglandin J
  • Prostaglandin J interacting factors

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

Cite this

Maekawa, N., Hiramoto, M., Sakamoto, S., Azuma, M., Ito, T., Ikeda, M., Naitou, M., Acharya, H. P., Kobayashi, Y., Suematsu, M., Handa, H., & Imai, T. (2011). High-performance affinity purification for identification of 15-deoxy-Δ12,14-PGJ2 interacting factors using magnetic nanobeads. Biomedical Chromatography, 25(4), 466-471. https://doi.org/10.1002/bmc.1469