High-performance affinity purification for identification of 15-deoxy-Δ 12,14 -PGJ₂ interacting factors using magnetic nanobeads

• Published in: Biomedical chromatography 2011-04, Vol.25 (4), p.466-471
• Main Authors: Maekawa, Naoya, Hiramoto, Masaki, Sakamoto, Satoshi, Azuma, Motoki, Ito, Takumi, Ikeda, Marie, Naitou, Michitaka, Acharya, Hukum P, Kobayashi, Yuichi, Suematsu, Makoto, Handa, Hiroshi, Imai, Takeshi
• Format: Article
• Language: English
• Subjects: Cell Growth Processes - physiology; Chromatography, Affinity - methods; HEK293 Cells; Humans; Ligands; Magnetite Nanoparticles - chemistry; Nitric Oxide Synthase Type II - metabolism; PPAR gamma - metabolism; Prostaglandin D2 - analogs & derivatives; Prostaglandin D2 - chemistry; Prostaglandin D2 - metabolism; Protein Binding; Voltage-Dependent Anion Channel 1 - metabolism
• ISSN: 1099-0801
• DOI: 10.1002/bmc.1469

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-Δ(12,14) -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.