Tandem Affinity Purification and Nano HPLC-ESI-MS/MS Reveal Binding of Vitamin D Receptor to p53 and other New Interaction Partners in HEK 293T Cells

• Published in: Anticancer research Vol. 38; no. 2; pp. 1209 - 1216
• Main Authors: Pemsel, Anja, Rumpf, Saskia, Roemer, Klaus, Heyne, Kristina, Vogt, Thomas, Reichrath, Joerg
• Format: Journal Article
• Language: English
• Published: Greece International Institute of Anticancer Research 01.02.2018
• Subjects: Affinity; Binding; Calcitriol; Cofactors; High performance liquid chromatography; Ionization; Liquid chromatography; p53 Protein; Protein purification; Proteins; Purification; Receptors; Repressors; Retinoid X receptors; Spectrometry; Transcription; Vitamin D; Vitamin D receptors; Vitamin D3; tandem affinity purification; Vitamin D; Vitamin D receptor
• ISSN: 0250-7005; 1791-7530
• DOI: 10.21873/anticanres.12341

While nuclear cofactors that contribute to vitamin D receptor (VDR)-mediated gene transcription, including retinoid X receptors, nuclear co-activators and co-repressors, have been extensively investigated, little is known about cytoplasmic VDR-binding partners and the physiological relevance of their interaction. To gain new insight into this topic, we isolated whole-cell protein extracts of 1,25-dihydroxyvitamin D stimulated and UV-B-irradiated vs. non-irradiated HEK 293T cells transfected with a plasmid called pURB VDR C-Term TAP tag. VDR complex was purified by tandem affinity purification (TAP). The nuclear tumor-suppressor protein p53 and its negative regulator novel INHAT repressor (NIR), in addition to 43 other nuclear or cytoplasmatic VDR binding partners, were identified using nano high-performance liquid chromatography-electrospray ionization tandem mass spectrometric analysis. VDR binding to p53 was confirmed by western blot analysis. Future studies are required to further elucidate the functional significance of these interactions.