The forkhead box O (FOXO) transcription factor belongs to the forakhead (FOX) protein family and controls diverse signaling pathways to regulate cell cycle, metabolism, differentiation, stress response, and apoptosis [1,2]. The FOXO subfamily has four members (FOXO1, FOXO3, FOXO4 and FOXO6) which share approximately 110 amino acid long DNA binding domain (called forkhead domain) throughout evolution [3,4]. They are expressed in a wide range of organisms (yeast to human) and their functional activity highly regulated by post-translational modification and interaction with other proteins including p53 [4,5]. The p53 protein acts as a central hub in the biological network which can modulate the activity of its interacting partners. In the past few years, the growing evidence suggests that both FOXO4 and p53 can interact with each other upon ionizing radiation which leads to cellular senescence [6]. An accumulation of senescent cell accelerates the aging process and other age-related diseases. However, the molecular mechanisms of this interaction remain unknown due to the lack of structural data. So our deep interest is to understand which regions of p53 and FOXO4 are involved in complex formation and what is the structure of the FOXO4-p53 complex. Therefore, pull-down assay, sedimentation velocity analytical ultracentrifugation (SV-AUC), 2D 1H-15N HSQC NMR spectroscopy was used to investigate the interaction between various constructs of FOXO4 and p53. Our preliminary data suggested that both FOXO4 and p53 are interacting with each other with the affinity (KD) in the micromolar range. HSQC NMR spectroscopy data reveals that the N-terminal segment as well as other regions of the FOXO4 DNA-binding domain is involved in p53 binding.
This work was supported by the Charles University Grant Agency (project: 251203).